Comparative in vitro analyses of the effect of immunoglobulin light chain and fatty acid free

Comparative in vitro analyses of the effect

of immunoglobulin 𝝀 light chain and fatty

acid free albumin on proximal tubular

epithelial cells-involvement of megalin

phosphorylation

Thesis submitted for the degree of Doctor of Philosophy at the

University of Leicester

By

Dalia Muhammed Alammari

(BSc, MSc)

Department of Infection, Immunity and Inflammation

University of Leicester

December 2015

I

ABSTRACT

Comparative in vitro analyses of the effect of immunoglobulin λ light chain and fatty

acid free albumin on proximal tubular epithelial cells-involvement of megalin

phosphorylation

Dalia Alammari

Kidney disease is a major challenge for health care systems, and the prevalence is

increasing. Proteinuria is a hallmark of progressive renal dysfunction and describes the

pathological excess of plasma proteins in urine, mainly albumin.

Multiple Myeloma is a cancer of plasma cells that leads to excessive presence of free

light chain protein (FLC) in blood. Renal failure due to overproduction of FLC and the

associated light chain proteinuria occurs as a result of decreased renal function or as a

direct toxic effect on the proximal tubular cells (PTCs) by excessive protein. Proteins

are normally reabsorbed by endocytosis via megalin receptor that binds proteins and

mediates their uptake. Exceeding the proximal tubular epithelial cells (PTECs)

reabsorption capacity might trigger inflammation detrimental to the kidney. In

proteinuric nephropathy the cytoplasmic tail of megalin (MegCT) is phosphorylated

after interaction between proteins and megalin on the PTECs, which activates signalling

cascades that regulate the phosphorylation.

An in vitro proteinuric model was established using HK2 cells (a proximal tubular

epithelial cell line derived from normal human kidney) treated with high concentrations

of essentially fatty acid free human serum albumin (FAF-HSA) or lambda light chain

(𝜆-LC) isolated and purified from the urine of a myeloma patient, to induce cellular

damage. The potential pathogenic role for FAF-HSA and 𝜆 -LC on HK2 cells was

examined. Also, renal toxicity that comes from the intracellular signalling through

phosphorylation of MegCT was addressed by utilising antibodies directed against

specific phosphorylation site (PPPSP) of the intracellular portion of megalin in HK2

cells stimulated with different concentrations of FAF-HSA and 𝜆-LC, so-called pre-

stimulated HK2.

In vitro analyses showed (i) a detrimental effect of FAF-HSA and 𝜆-LC on viability of

HK2, (ii) phosphorylation of the cytoplasmic tail of megalin in pre-stimulated HK2

cells. (iii) Production of inflammatory cytokines and H2O2 generation, activation of

autophagy process and increase in several kidney biomarkers/ injury mediators, which

are involved in different pathways in response to protein overload. All these reasons are

likely to contribute to direct PTECs injury and kidney failure in patients.

Potentially these mechanisms may be attractive for drug development to benefit patients

with kidney failure and help to inhibit the progression of proteinuric nephropathy and as

such may save lives.

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/Urine

II

STATEMENT

This thesis results from work undertaken at the University

of Leicester during the period of registration.

III

DEDICATE

To My….

Husband Mohammed

Mother Heyam,

Brother Hamzah

&

To the spirit of my Father (God mercy be

upon him)

IV

ACKNOWLEDGMENT

‘For his mercy and blessing all praise and gratitude goes to the Almighty God’

Writing of this thesis has not been an easy process. It required a lot of work, time and

patience. I could have never been able to write this thesis without guidance and support

from others.

I would like to take this opportunity to offer my deepest thanks, respect and gratitude to

my supervisor Dr. Cordula Stover. I am extremely grateful for her constant

encouragement, support, guidance, advice and patience throughout this research. I could

not have asked for a better mentor as I am blessed I got the best one.

I would like to thank my second supervisor Dr. Alan Bevington for his help and advice.

Also, I would thank my progress review panel members, Professor Nigel Brunskill and

Dr. Primrose Freestone for their positive feedback and advice.

It is a pleasure to acknowledge with sincere thanks to Dr. Simon Byrne for his time,

advice and help with many different methods in this project. I would like to express my

deep gratitude to Dr. Ravinder Chana for his time and help with the phosphorylation

work I really appreciate that. My thanks would be extended to Dr. Mike Browning for

his help to get the myeloma patient urine sample and to Professor Russell Wallis and

Dr. Chris Furze for their help in the purification work. I would like to thank the late

Stefan Hyman and Natalie Allcock of the electron microscopy laboratory for processing

my samples for scanning and transmission electron microscopic and for providing me

with such lovely images. It is a pleasure to extend my thanks to all my friends either in

the UK or in my country or anywhere (Amira, Sara and Nawal) and all people in lab

211B for their help, support and encouragement.

My thanks and appreciations are also offered to the Saudi Arabian Cultural Bureau in

London for their help. Also, I would like to thank all the staff at the University of

Leicester who was very kind and supportive. My thanks are also offered for any kind of

help, which I received from anybody and by any means.

Finally, a very special thanks to my lovely husband Mohammed. This degree cannot be

finish without his love, help, support and patience in this long journey. My special

gratitude to my mother, brother, and my family for giving me all the help and support I

needed.

Dalia Alammari

V

PUBLICATIONS ARISING FROM THIS THESIS

Dalia Alammari, Alan Bevington and Cordula Stover. Is Megalin Phosphorylation the

Reason for Kidney Damage in Myeloma?. Poster Presentation. The 7th Saudi Student

conference, Edinburgh, UK, February 2014, (Appendix I).

Dalia Alammari, Alan Bevington and Cordula Stover. Monoclonal Light Chain

mediated damage of Proximal Tubular Epithelial Cells – a mechanism of renal

pathology in Multiple Myeloma involving Megalin. Presentation (Talk). The 6th

Annual Postgraduate Student Conference, University of Leicester, Leicester, UK, April

2014.

Dalia Alammari, Alan Bevington and Cordula Stover. Proteinuria mediated damage of

Proximal Tubular Epithelial Cells (PTECs) - a mechanism of renal pathology in

Multiple Myeloma involving Megalin. Poster Presentation. Kidney Week 2014

conference, Glasgow, UK, April 2014, (Appendix II).

Dalia Alammari, Alan Bevington and Cordula Stover. How can we help blood cancer

patients reduce their kidney damage?. Presentation (Talk). Manchester Life Sciences

Ph.D. Conference in University of Manchester, University of Manchester, Manchester

UK, May 2014.

Dalia Alammari, Alan Bevington and Cordula Stover. How can we help blood cancer

patients reduce their kidney damage?. Poster Presentation. University of Leicester

Postgraduate Research Festival, selected as one of the 50 most promising researchers

among 1500 postgraduates at the University, University of Leicester, Leicester, UK,

June 2014, (Appendix III).

Dalia Alammari, Alan Bevington and Cordula Stover. Can Myeloma light chain

activate kidney proximal tubular cells to become pro-inflammatory cells?.

Presentation (Talk). The 7th Annual Postgraduate Student Conference, selected as one of

the best presentation, University of Leicester, Leicester, UK, April 2015.

Zwaini, Z., Alammari, D., Byrne, S., Stover, C., 2016. Mode of proximal tubule

damage: differential cause for the release of TFF3? Frontiers in Immunology. 7, 122.

Dalia M. Alammari, Ravinder S. Chana, Christopher Furze, Russell Wallis, Alan

Bevington, Nigel J. Brunskill, Richard J. Baines and Cordula M. Stover. Induction of

megalin phosphorylation at PPPSP motif by light chains (In preparation).

VI

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. XI

LIST OF FIGURES .......................................................................................................... XII

LIST OF ABBREVIATIONS ..................................................................................... XVIII

Chapter One-Introduction ..................................................................................................... 1

1. Why is my urine foamy? ................................................................................................. 2

1.1 The Epidemiology of Renal Failure: ......................................................................... 2

1.2 Kidney Diseases: ....................................................................................................... 3

1.2.1 Acute and Chronic Kidney Diseases: ................................................................. 3

1.2.2 Nephropathies of Systemic Diseases: ................................................................. 4

1.2.3 Dysproteinemic: .................................................................................................. 5

1.3 Multiple Myeloma: .................................................................................................... 6

1.3.1 Myeloma Nephropathy: ...................................................................................... 7

1.3.2 Cast nephropathy: ............................................................................................... 7

1.3.3 Fanconi syndrome proximal tubulopathies: ........................................................ 8

1.3.4 Amyloidosis: ....................................................................................................... 8

1.3.5 Light-chain deposition disease: .......................................................................... 8

1.4 Monoclonal FLC and Myeloma Kidney: .................................................................. 9

1.4.1 Immunoglobulin and Light Chain Structure: ...................................................... 9

1.5 Kidney, anatomy and function: ............................................................................... 10

1.6 Protein uptake and Endocytosis: ............................................................................. 11

1.6.1 Proteinuria: ........................................................................................................ 11

1.6.2 PTCs and Endocytosis process: ........................................................................ 13

1.6.3 Megalin: ............................................................................................................ 15

1.6.3.1 Megalin Signalling Functions: ................................................................... 15

1.6.3.2 Megalin mutation and human diseases: ..................................................... 16

1.6.3.3 Description of megalin transgenic mouse: ................................................. 16

1.6.4 Megalin-Associated Molecules: ....................................................................... 16

1.6.4.1 Cubilin: ....................................................................................................... 16

1.6.5 Non Megalin/cubilin mediated up take of protein in PTCs: ............................. 18

1.6.5.1 Cluster of differentiation 36 (CD36): ......................................................... 18

1.7 Signalling role of megalin in PTCs toxicity: ........................................................... 21

1.7.1 Signalling pathway regulates protein endocytosis in PTCs: ............................. 21

1.7.2 Megalin phosphorylation and PTCs toxicity: ................................................... 23

1.8 Proteinuria and Proximal Tubular cells Toxicity: ................................................... 23

1.8.1 The effect of proteinuria on PTCs viability: ..................................................... 24

VII

1.8.2 Inflammatory cytokines/chemokines and fibrogenic mediators: ...................... 24

1.8.2.1 Interlukin-6 (IL6): ...................................................................................... 24

1.8.2.2 Interlukin-8 (IL-8): ..................................................................................... 25

1.8.2.3 Monocyte Chemoattractant Protein-1 (MCP-1): ........................................ 26

1.8.3 Complement components as pathogenic mediators of tubular toxicity in

proteinuria: ................................................................................................................. 27

1.8.3.1 Mechanism of alternative pathway activation and regulation: .................. 28

1.8.3.2 Evidence of complement pathway contributes to induce renal injury: ...... 28

1.8.4 Reactive oxygen species (ROS) and renal tubular injury: ................................ 30

1.8.5 Autophagy in renal tubular injury: .................................................................... 32

1.8.5.1 Evidence of autophagy induces protection/injury in PTCs in kidney

diseases: .................................................................................................................. 33

1.8.6 Apoptosis in proximal tubular injury: ............................................................... 36

1.8.6.1 Proteinuria and renal tubular apoptosis: ..................................................... 37

1.8.7 Evidence for toxicity of excess proteins to PTCs: ............................................ 38

1.8.7.1 Novel biomarkers/mediators of PTCs toxicity: .......................................... 38

1.8.7.2 Kidney injury molecule-1 (Kim-1): ........................................................... 39

1.8.7.3 Transforming growth factor beta (TGF-β): ................................................ 40

1.8.7.4 Tumor necrosis factor alpha (TNF-α): ....................................................... 41

1.9 Model for Proximal Tubular Epithelial Cells (PTECs): .......................................... 43

1.9.1 Human Embryonic Kidney Cells 293 (HEK293): ............................................ 45

1.10 Hypothesis: ............................................................................................................ 46

1.11 Aims: ..................................................................................................................... 46

Chapter Two-Materials and Methods ................................................................................. 48

2. General Methods ........................................................................................................... 49

2.1 Tissue culture: ......................................................................................................... 49

2.2 Scanning Electron Microscopy (SEM): ................................................................... 51

2.3 Histology: ................................................................................................................ 52

2.3.1 Immunohistochemistry staining protocol for endogenous Alkaline

Phosphatase: ............................................................................................................... 52

2.3.2 Immunocytochemistry: ..................................................................................... 53

2.3.3 Mouse PTECs preparation: ............................................................................... 53

2.4 MTT assay: .............................................................................................................. 54

2.5 LDH assay: .............................................................................................................. 54

2.6 Crystal Violet assay: ................................................................................................ 56

2.7 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR):.............................. 56

2.7.1 Preparation of Ribonucleic Acid (RNA): ......................................................... 56

2.7.2 Preparation of Complementary Deoxyribonucleic Acid (cDNA): ................... 57

2.7.3 PCR Protocol: ................................................................................................... 57

VIII

2.7.4 Human Primers ................................................................................................. 59

2.8 Real-Time Quantitative polymerase chain reaction (RT-qPCR): ........................... 61

2.8.1 RT-qPCR analysis and calculation: .................................................................. 63

2.8.2 Primer Efficiency: ............................................................................................. 64

2.9 Protein Immunoblot (Western blot): ....................................................................... 66

2.9.1 Preparation of cell lysate: ................................................................................. 66

2.9.2 Protein Assay: ................................................................................................... 66

2.9.3 Western Blot Protocol: ...................................................................................... 67

2.9.4 Preparing the Maxi Gradient Gel for the Megalin Western blot: ..................... 72

2.9.5 Preparing the 4% Gel for the Megalin Western blot: ....................................... 74

2.10 Ultra structural analysis by transmission electron microscopy (TEM): ................ 74

2.11 Detection of Apoptosis: ......................................................................................... 75

2.12 Quantitative measurement by ELISA: ................................................................... 77

2.12.1 Mini Elisa for candidate cytokines and chemokine from HK2 (+/-GF)

cells: ........................................................................................................................... 77

2.12.2 Human TFF3 Immunoassay: .......................................................................... 78

2.13 Determination of H2O2 Production by DCFDA: ................................................... 79

2.14 Measuring Hydrogen Peroxide Production (H2O2) Production by Amplex®

Red: ................................................................................................................................ 82

2.15 Proteome Profile® analysis of HK2-GF cells: ....................................................... 83

2.15.1 Proteome profile Data Analysis: ..................................................................... 84

2.16 Phosphorylation of the recombinant expressed cytoplasmic tail of Megalin

receptor: ......................................................................................................................... 88

2.16.1 Rationale for use of pGEX-4T1 plasmid and MegCT-fusion protein: ........... 88

2.16.2 Preparation and collection of MegCT-GST Fusion protein: .......................... 89

2.16.3 Stimulation of MegCT-GST fusion protein phosphorylation in vitro: ........... 91

2.17 Statistics and Data analysis: .................................................................................. 96

Chapter Three-FLC Purification ........................................................................................ 97

3. Purification of monoclonal Free Light Chain (FLC): ................................................... 98

3.1 Source of Free Light Chain: .................................................................................... 98

3.2 Urine Sample analyses: ........................................................................................... 98

3.3 Dipstick test: .......................................................................................................... 100

3.4 Albumin Excretion: ............................................................................................... 102

3.5 Protein sequencing: ............................................................................................... 104

3.5.1 Gel Digestion Protocol (Protein Digestion): ................................................... 104

3.5.2 Matrix Assisted Laser Desorption/Ionization - Time-of-Flight (MALDI-

TOF): ....................................................................................................................... 105

3.5.3 Results of MALDI-TOF MS analyses: ........................................................... 105

3.6 Extraction of protein from the urine sample: ........................................................ 105

IX

3.7 Purified FLC Protein Sequencing: ......................................................................... 115

3.8 RSLCnano HPLC System: .................................................................................... 115

3.9 LTQ-Orbitrap-Velos Mass Spectrometer: ............................................................. 115

3.10 LC-MS/MS Sequance Results: ............................................................................ 116

3.11 Protein Concentrate column: ............................................................................... 116

3.12 Endotoxin Measurement: ..................................................................................... 117

3.12.1 Endotoxin Removal: ......................................................................................... 118

3.13 Human Serum Albumin Devoid of Fatty Acids: ................................................. 118

3.14 Calculation of protein load: ................................................................................. 119

Chapter Four- Characterisation of Renal Proximal Tubular Epithelial Cells ............... 121

4. Introduction ................................................................................................................. 122

4.1 Aim ........................................................................................................................ 122

4.2 Results ................................................................................................................... 123

4.2.1 Culture characteristics of Human Renal Proximal Tubular Epithelial Cell

line (HK2): ............................................................................................................... 123

4.2.2 Transmission Electron Microscopy analysis (TEM) for HK2 cell line: ......... 125

4.2.3 Scanning Electron Microscopy (SEM) analysis for brush border of HK2

(+/-GF) cells:............................................................................................................ 127

4.2.4 Villin-1 – A marker of brush border differentiation in renal cells: ................ 130

4.2.5 Mouse Proximal Tubular Epithelial Cells (mPTEC) preparation: .................. 131

4.2.6 Alkaline phosphatase enzyme marker of proximal tubular cells: ................... 133

4.2.7 Human Proximal Tubule Epithelial cell culture: ............................................ 135

4.3 Discussion .............................................................................................................. 139

Chapter Five- Establishing an in vitro Model of Protein-Induced Epithelial Cell

Damage ............................................................................................................................... 144

5. Introduction: ................................................................................................................ 145

5.1 Aim: ....................................................................................................................... 147

5.2 Results: .................................................................................................................. 148

5.2.1 Dose and time dependent influence of FAF-HSA and 𝛌-LC on cell

viability: ................................................................................................................... 148

5.2.1.1 MTT Assay: ............................................................................................. 148

5.2.1.2 LDH Activity Assay: ................................................................................ 153

5.2.1.2.1 PTECs Protein Overload Model: .......................................................... 154

5.2.2 Autophagy as a response of HK2 (+/- GF) to cell damage by protein

overload: .................................................................................................................. 161

5.2.3 Effect of overload protein concentration to induce apoptosis: ....................... 175

5.2.4 Effect of FAF-HSA and 𝛌-LC on mediators of inflammation in kidney

damage: .................................................................................................................... 184

X

5.3 Discussion .............................................................................................................. 191

Chapter Six-Megalin Phosphorylation in Renal Proximal Tubular Epithelial Cells ..... 199

6. Introduction ................................................................................................................. 200

6.1 Aim ........................................................................................................................ 203

6.2 Results ................................................................................................................... 205

6.2.1 Expression of mRNA and protein of Megalin in HK2 (+/-GF) cells: ............ 205

6.2.2 MegCT-GST fusion protein phosphorylation in HK2 (+/-GF) cells: ............. 211

6.2.3 Effects of protein overload on mRNA expression for Megalin and CD36 by

HK2 (+/-GF) cells: ................................................................................................... 217

6.3 Discussion .............................................................................................................. 220

Chapter Seven- Effects of Protein Overload on Proximal Tubular Cells in the

Progression of Damage In Vitro ....................................................................................... 227

7. Introduction ................................................................................................................. 228

7.1 Aim ........................................................................................................................ 229

7.2 Results ................................................................................................................... 230

7.2.1 Evaluation the effect of FAF-HSA and 𝛌-LC on cytokine production in

HK2 (+/-GF) cells: ................................................................................................... 230

7.2.2 Complement component production by HK2 (+/-GF) cells: .......................... 238

7.2.3 Effect of Protein Overload on H2O2 production: ............................................ 249

7.2.4 Study of potential biomarkers of human kidney injury using an in vitro

protein stimulation model: ....................................................................................... 255

7.3 Discussion .............................................................................................................. 267

8. Summary: ................................................................................................................ 283

Conclusion .......................................................................................................................... 290

Findings and Future work................................................................................................. 291

Appendix ............................................................................................................................. 293

References .......................................................................................................................... 302

XI

LIST OF TABLES

Table (1.1): Classification of systemic diseases that have adverse renal effects. ............. 5

Table (1.2): Main types of dysproteinemia diseases and renal complications. ................ 6

Table (1.3): Biomarkers of kidney injury. ...................................................................... 39

Table (2.1): Mammalian Ringer Fixation for SEM solution .......................................... 52

Table (2.2): Preparation of 5 x Tris/Borate/EDTA solution for electrophoresis. ........... 58

Table (2.3): Preparation of qRT-PCR reaction. ............................................................. 62

Table (2.4): The temperature cycling conditions of qRT-PCR. ..................................... 62

Table (2.5): Preparation of 10 ml Lysis Buffer .............................................................. 69

Table (2.6): Preparation of buffers for Western blot. ..................................................... 70

Table (2.7): Primary antibodies. ..................................................................................... 71

Table (2.8): Secondary antibodies. ................................................................................. 71

Table (2.9): Preparation for Gradient Gel. ...................................................................... 73

Table (2.10): Preparation of 4% gel. ............................................................................... 74

Table (2.11): Preparation of Paraformaldehyde and Methyl green. ............................... 76

Table (2.12): Luria Broth media and Glutathione Sepharose 4B preparation. .............. 90

Table (2.13): Table of agents used in the studies of MegCT phosphorylation.. ............ 93

Table (2.14): JNK and Kinase buffers preparations. ...................................................... 94

Table (3.1): Coomassie Blue Preparation. ...................................................................... 99

Table (3.2): The classification of urinary albumin excretion. ...................................... 103

Table (3.3): preparation of 1M Sodium Phosphate Buffer pH 9.0. .............................. 106

XII

LIST OF FIGURES

Figure (1.1): The basic structure of immunoglobulin......................................................10

Figure (1.2): Megalin-mediated endocytosis and recycling to the cell surface..............14

Figure (1.3): Schematic structures of megalin and cubilin receptors in relation to the

plasma membrane............................................................................................................18

Figure (1.4): Mechanism of multiple myeloma and kidney failure.................................20

Figure (1.5): Mechanism of activation and regulation of complement alternative

pathway............................................................................................................................30

Figure (1.6): H2O2 generation by NADPH......................................................................32

Figure (1.7): Diagram of the steps of autophagy.............................................................35

Figure (1.8): Light microscopic appearance of Human Kidney (HK2) cells…………..44

Figure (2.1): Light microscopic appearance of human embryonic kidney 293 (HEK 293)

cells……………………………………………………………………………………..50

Figure (2.2): Lactate Dehydrogenase (LDH) detection mechanism…………………....55

Figure (2.3): 1 Kb Plus DNA Ladder in 1% agarose gel electrophoresis........................59

Figure (2.4): Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) for β-

actin (Housekeeping gene) expression in HK2 cells.......................................................65

Figure (2.5): Example for the standard curve showing the absorbance of different

concentrations of BSA by Pierce 660nm Protein assay..................................................67

Figure (2.6): Protein marker (Precision Plus ProteinTM Standards, BIO RAD (10-250

kDa))……………………………………………………………………………………72

Figure (2.7): Examples for Standard curves for (A) IL-6, MCP-1 (B) and (C) IL-8 Mini

ELISA..............................................................................................................................78

Figure (2.8): Formation of fluorescent DCF by ROS activity.........................................81

Figure (2.9): Conversion of Amplex Red to resorufin by HRP using H2O2...................82

Figure (2.10): Standard curve for Measuring Hydrogen Peroxide Production (H2O2)

Production by Amplex® Red..........................................................................................83

XIII

Figure (2.11): Example of the Human Kidney Biomarker Array result for HK2-GF

control sample..................................................................................................................85

Figure (2.12): (A) The Human Kidney Biomarker Array coordinates. (B) The table

shows the reference of the 38 proteins............................................................................87

Figure (2.13): Preparations steps of MegCT-GST Fusion protein..................................91

Figure (2.14): Phosphorylation of Meg-CT.....................................................................96

Figure (3.1): Characterisation of urinary protein profile from a multiple myeloma

patient............................................................................................................................100

Figure (3.2): Comparing the urinary proteins in the patient (P) and the control (C)

sample…………………………………………………………………………………101

Figure (3.3): Estimation of albumin concentration in MM patient urine sample..........103

Figure (3.4): The purified free light chain…………………………………………....106

Figure (3.5): Comparison of FLC binding to different resin types using dialysis urine

sample with pH 7.6 sodium phosphate buffer in 12 % SDS-PAGE stained with

Coomassie Blue reduced condition...............................................................................108

Figure (3.6): Optimizing the volumes of dialysed protein sample loaded in 1ml Q

Sepharose Fast Flow column for better binding............................................................109

Figure (3.7): Electrophoretic (SDS-PAGE) final analysis of FLC purification............110

Figure (3.8): Chromatogram of Ion exchange column High-performance liquid

chromatography (HPLC) system...................................................................................112

Figure (3.9): Chromatogram of removal of impurities from FLC sample by size

exclusion High-performance liquid chromatography (HPLC) system..........................113

Figure (3.10): SDS-PAGE analysis for fractions eluted from Ion exchange column

1.5ml/min.......................................................................................................................114

Figure (3.11): Protein Sequence Coverage, 87/106 amino acid (~82%), (covered

locations were indicated in yellow)...............................................................................116

Figure (3.12): Methodology of Monoclonal Free Light Chain Protein Purification.....120

Figure (4.1): Microscopic appearance of HK2 cells......................................................124

Figure (4.2): Transmission electron microscopy (TEM) for HK2 (+/-GF) cells...........126

Figure (4.3): Typical scanning electron micrographs showing microvilli on the surface

of HK2 cells...................................................................................................................130

XIV

Figure (4.4): Western blot analysis of Villin-1 as a marker of brush border

differentiation................................................................................................................131

Figure (4.5): Microscopic documentation of isolation of murine renal tubules............132

Figure (4.6): Histochemical alkaline phosphatase staining (ALP)................................135

Figure (4.7): Differences in proliferation in HK2 and HK2-GF cells...........................138

Figure (5.1): Effects of FAF-HSA overload on cell viability measured using MTT assay

in HK2 (+/-GF) cells for (2, 4 and 6h)………………………………………………..150

Figure (5.2): Effects of FAF-HSA overload on cell viability measured using MTT assay

in HK2 (+/-GF) cells for (24, 48 and 72h)……………………………………………151

Figure (5.3): Effects of 𝜆-LC on cell viability measured using MTT assay in HK2 (+/-

GF) cells for (24, 48 and 72h)………………………………………………………...152

Figure (5.4): Impact of FAF-HSA on HK2 (+/-GF) cells viability, assessed by

measurement of LDH (2,500 cells/well)……………………………………………...156


measurement of LDH (5,000 cells/well)…………………………………….………..157


measurement of LDH (10,000 cells/well)…………………………………………….158

Figure (5.7): Impact of 𝜆-LC on HK2 (+/-GF) cells viability, assessed by measurement

of LDH (10,000 cells/well)………………………………...………………………….159

Figure (5.8): Impact of 24 and 72h incubation of HK2 (+/-GF) cells with excess

amounts of FAF-HSA or 𝜆-LC on LDH release into the medium……………………160

Figure (5.9): Representative transmission electron micrographs (TEM) showing

different stages of autophagocytosis in cells exposed to (0.2 μM) tunicamycin ……..164

Figure (5.10): Representative transmission electron micrographs (TEM) of HK2-GF

cells incubated in serum free media for 24 and 72h (control cells)...............................165


cells treated with (5mg/ml) FAF-HSA for 24h.............................................................166


cells treated with (5mg/ml) FAF-HSA for 72h.............................................................167

Figure (5.13): Representative transmission electron micrographs (TEM) of HK2 cells

incubated in serum free media for 24 and 72h (control cells).......................................168

XV


treated with (5mg/ml) FAF-HSA for 24h……………………………………………..169


treated with (5mg/ml) FAF-HSA for 72h……………………………………………..170


cells treated with (5mg/ml) 𝜆-LC for 24h……………………………………………..171


cells treated with (5mg/ml) 𝜆-LC for 72h……………………………………………..172


treated with (5mg/ml) 𝜆-LC for 24h…………………………………………………..173

Figure (5.19): Representative transmission electron micrographs (TEM) of HK2 treated

with (5mg/ml) 𝜆-LC for 72h…………………………………………………………..174

Figure (5.20): Specific staining of DNA fragmentation associated with apoptosis

(ApopTag®) in stimulated HK2 (+/-GF) cells with overload proteins.........................177

Figure (5.21): Western Blot analysis of Caspase-3 from stimulated HK2 (+/-GF) cells

with overload proteins……………………………………………………………….. 182

Figure (5.22): Effects of FAF-HSA or 𝜆-LC on Caspase-3 mRNA expression from HK2

(+/- GF)..........................................................................................................................183

Figure (5.23): mRNA expression of TGF-𝛽 from stimulated HK2 (+/-GF) cells with

overload proteins...........................................................................................................187

Figure (5.24): mRNA expression of TNF-𝛼 from stimulated HK2 cells (+/-GF) with

overload proteins……………………………………………………………………...188

Figure (5.25): mRNA expression of KIM-1 from stimulated HK2 (+/-GF) cells with

overload proteins……………………………………………………………………...189

Figure (5.26): Semi-quantitative analysis of clusterin mRNA expression from HK2-GF

cells after stimulating with 5mg/ml FAF-HSA or 𝜆-LC for 24 and 72h……………...190

Figure (6.1): The Megalin receptor…………………………………………………...204

Figure (6.2): Analysis of megalin mRNA expression in HK2, HK2-GF and HEK 293

cells. .………………………………………………………………………………….205

Figure (6.3): RT-qPCR efficiency for megalin and β-actin gene expression in HEK293

cells................................................................................................................................207

Figure (6.4): RT-qPCR for mRNA megalin and β-actin gene expression in (5 𝜇𝑔

mRNA) HK2 (+/-GF) cells……………………………………………………………208

XVI

Figure (6.5): Gel electrophoresis analysis for RT-qPCR product (megalin receptor)...209

Figure (6.6): Western blot analyses of megalin……………………………………… 211

Figure (6.7): Time course of PDBU stimulated phosphorylation of MegCT-GST fusion

protein by HEK2293………………………………………………………….......…...213

Figure (6.8): Time course of PDBU stimulated phosphorylation of MegCT-GST fusion

protein by HK2 (+/-GF) cells…………………………………………………………214

Figure (6.9): Effect of FAF-HSA on phosphorylation of MegCT-GST fusion

protein…………………………………………………………………………………215

Figure (6.10): Effect of 𝜆-LC on phosphorylation of MegCT-GST fusion protein…..216

Figure (6.11): Effect of FAF-HSA or 𝜆-LC on megalin and CD36 mRNA expression

from HK2 (+/- GF) cells………………………………………………………………218

Figure (6.12): Interactions of Albumin or 𝝀-LC with PTECs and different signalling

kinase pathways that regulate MegCT phosphorylation………………………………224

Figure (7.1): The effects of 𝜆-LC and FAF-HSA on IL-6 protein production and mRNA

expression by HK2 (+/-GF) cells……………………………………………………...232

Figure (7.2): The impacts of 𝜆-LC or FAF-HSA on IL-8 protein production and mRNA

expression by HK2 (+/-GF) cells……………………………………………………...234

Figure (7.3): The effects of 𝜆 -LC or FAF-HSA on MCP-1 protein production and

mRNA expression from HK2 cells (+/-GF)…………………………………………..237

Figure (7.4): Effects of FAF-HSA or 𝜆-LC on C3 mRNA expression from HK2 (+/-

GF)…………………………………………………………………………………….240

Figure (7.5): Impacts of FAF-HSA or 𝜆-LC on FB mRNA expression from HK2 (+/-

GF)…………………………………………………………………………………….241

Figure (7.6): Effects of FAF-HSA or 𝜆-LC on FH mRNA expression from HK2 (+/-

GF)…………………………………………………………………………………….242

Figure (7.7): Effects of FAF-HSA or 𝜆-LC on Properdin mRNA expression from HK2

(+/-GF)………………………………………………………………………………...243

Figure (7.8): Effects of exposure to FAF-HSA or 𝜆-LC on C3 protein production from

HK2 (+/-GF) cells……………………………………………………………………..245

Figure (7.9): Effects of exposure to FAF-HSA or 𝜆-LC on FB protein production from

HK2 (+/-GF) cells……………………………………………………………………..246

XVII

Figure (7.10): Juxtaposition of mRNA and protein for AP components (C3, FB, P and

FH) from HK2 (+/-GF) stimulated with FAF-HSA and 𝜆-LC for 24 and 72h………..248

Figure (7.11): Time course of H2O2 generation in HK2 (+/-GF) cells………………..253

Figure (7.12): Effect of FAF-HSA or 𝜆 -LC on H2O2 production by HK2 (+/-GF)

cells……………………………………………………………………………………254

Figure (7.13): The human kidney biomarker array detects multiple analyses cell culture

lysates samples………………………………………………………………………..262

Figure (7.14): Densitometric intensity for Western blot semi-quantitative analysis of

TFF3 production from stimulated HK2-GF cell lysates; cells stimulated with FAF-HSA

(5mg/ml) or 𝜆-LC (1 or 5 mg/ml) for 72h.……………………………………………264

Figure (7.15): The effects of 𝜆-LC and FAF-HSA on TFF3 production by HK2 cells

(+/-GF)………………………………………………………………………………...265

Figure (7.16): Compilation of proteins detected by proteome profile human kidney

array, which are differentially influenced by 𝜆-LC and FAF-HSA…………………...266

Figure (7.17): The biomarkers were measured by proteomic profile assay that might

have a role in the process of PTECs injury in our protein overload condition………..282

Figure (8.1): Hypothetic signal pathways of proximal tubule cells in response to

overload 𝜆-LC…………………………………………………………………………287

Figure (8.2): Hypothetic signal pathways of proximal tubule cells in response to

overload FAF-HSA……………………………………………………………………289

XVIII

LIST OF ABBREVIATIONS

AKD Acute Kidney Disease

AKI Acute Kidney Injury

ATP Adenosine Triphosphate

AP Alternative Pathway

𝛽2m Beta 2 Microglobulin

bp base pair

CKD Chronic Kidney Disease

C3 Complement component 3

CYR61 Cysteine-rich angiogenic inducer 61

dH2O Distilled water

ELISA Enzyme Linked Immunosorbent Assay

EGF Epidermal Growth Factor

EGFR Epidermal Growth Factor Receptor

FAF-HSA Fatty Acid Free- Human Serum Albumin

FB Factor B

FH Factor H

FP Factor P

FLC Free Light Chain

GST Glutathione-S-transferase

GSK-3 Glycogen synthase kinase-3

g Gram/gravity

h Hour

HBSS Hank's Balanced Salt Solution

HEK293 Human Embryonic Kidney 293

XIX

HK2 Human Proximal Tubular Cells without EGF cocktail

HK2-GF Human Proximal Tubular Cells with EGF cocktail

HK2 (+/-GF) Human Proximal Tubular Cells with and without EGF cocktail

H2O2 Hydrogen Peroxide

Ig Immunoglobulin

IL-6 Interleukin-6

IL-8 Interleukin-8

IL-10 Interleukin-10

IPTG Isopropyl 𝛽-D-thigalactpyranoside

𝜅 Kappa

KIM-1 Kidney Injury Molecule-1

kDa Kilo Dalton

LDH Lactate Dehydrogenase

𝜆-LC Lambda Light Chain

LDL-R Low-Density Lipoprotein Receptor

LRP-1 Low-Density Lipoprotein like Receptor Protein-1

LB Luria Broth

MMP-9 Matrix MetalloProteinase-9

Meg-CT Megalin Cytoplasmic Tail

mRNA messenger Ribonucleic Acid

𝜇l microliter

mg milligram

ml milliliter

min minutes

MAPK Mitogen-Activated Protein Kinases

MCP-1 Monocyte Chemoattractant Protein-1

mPTEC mouse Proximal Tubular Epithelial Cells

XX

MM Multiple Myeloma

ng nanogram

NGAL Neutrophil Gelatinase-Associated Lipocalin

OK cells Opossum Kidney

OD Optical Density

PI3K Phosphoinositide 3-Kinase

PBS Phosphate Buffered Saline

PDBU Phorbol ester 12,13-dibutyrate

PKB Protein Kinase B

PKC Protein Kinase C

PTEC Proximal Tubular Epithelial Cells

ROS Reactive Oxygen Species

RT-PCR Reverse Transcription Polymerase Chain Reaction

RT-qPCR Reverse Transcription Quantitative Polymerase Chain Reaction

rpm revolutions per minute

RT Room Temperature

sec second

TSP-1 Thrombospondin-1

TGF-𝛽 Transforming Growth Factor-beta

TFF3 Trefoil Factor 3

TNF-𝛼 Tumor Necrosis Factors-alpha

TNF-R Tumor Necrosis Factors Receptor

VCAM-1 Vascular Cell Adhesion Molecule-1

V Volts

v/v Volume/Volume

w/v Weight/Volume

1

Chapter One-Introduction

2

1. Why is my urine foamy?

It is one of the questions that people often ask; normally, urine does not appear foamy.

Foamy or bubbly urine could be normal or abnormal. There are several causes for normal

foamy urine such as rapid urination, concentrated urine and dehydration. However, it could

be proteinuria (proteins in urine), which is abnormal and a sign of kidney problems (New

Health Guide, 2014).

1.1 The Epidemiology of Renal Failure:

Kidney diseases are a worldwide health problem (Levey et al., 2007) and cause significant

morbidity and mortality (Fearn & Sheerin, 2015). 8-16% of the population worldwide is

affected by chronic kidney disease (CKD) (Jha et al., 2013). In 1990 CKD was ranked 27th

in the list of causes of total number of deaths worldwide according to a Global Burden of

Disease study (2010), however, it rose to 18th in 2010. It is estimated that one in four

women and one in five men have CKD in people aged between 65 and 74 worldwide

(National Kidney Foundation, 2015).

The early diagnosis of CKD can lead to slow, stop or treat the progression of kidney

disease. However, if the patient is in the end-stage kidney disease (ESKD), that means the

patient need kidney dialysis or kidney transplantation, which is called renal replacement

therapy (RRT), but these RRT are not available for all of the patients (Coresh & Jafar,

2015). This is because the treatment of kidney disease is costly and expensive. The NHS

Kidney Care in England costs more on CKD than cancer disease like breast and lung

(National Kidney Foundation, 2015). For example, NHS in England spends ~£1.44 billion,

which is (~1.3%) of the total NHS budget on CKD treatment (Evans & Taal, 2011).

3

Worldwide, only a quarter to a half of CKD patients needing RRT received it in 2010, and

~92% of them resided in high to high-middle income countries like the USA and Japan, and

just ~7% in low to low-middle income countries such as Asia and Africa (China, Indonesia

and India), which are home to half of the world’s population (Coresh & Jafar, 2015).

To minimise the development and progression of kidney disease, and also to reduce the

high amount that countries spend on CKD treatment, focus is on the risk factors that are

associated with increasing the rate of CKD progression. In addition, the goal is to decrease

the number of annual deaths caused by CKD, in 2010 the rate was 16.3 per 100,000 (Jha et

al., 2013). Risk factors for CKD development can be divided into two groups: initiating

factors and perpetuating factors. The initiating factors lead to increasing the risk of

developing CKD, such as high normal urinary albumin, nephrotoxins and diabetes, but the

perpetuating factors increase the risk of CKD and lead to ESKD like proteinuria,

nephrotoxins, cardiovascular disease, hypertension and acute kidney injury (AKI) (Evans &

Taal, 2011). The reduction of CKD risk factors has a positive impact on slowing the

progression of the kidney damage that leads to kidney failure and death (Levey & Coresh,

2012).

1.2 Kidney Diseases:

1.2.1 Acute and Chronic Kidney Diseases:

There are two main causes of elevated urinary protein excretion in patients with kidney

damage: the primary renal diseases affect the renal function and cause kidney failure, such

as membranous nephropathy and focal glomerulosclerosis diseases. However, the

secondary renal diseases (systemic diseases) are diseases leading to develop renal damage

in several weeks or several years, so, the kidney diseases come from and following the

systemic diseases such as diabetes, HIV-associated nephropathy and

immunoglobulinpathay (Schena et al., 2001).

There are two types of kidney diseases, chronic and acute. Around 13-18% of people suffer

from AKI. It is rapid damage and loss of kidney function, which occurs as result of another

4

serious diseases such as diabetes, dehydration and infection. In addition, patients with long

time CKD can develop AKI (NHS, 2014).

The definition of CKD means disorders in kidney structure and function. The important

sign of the presence of kidney damage is proteinuria, and for the decrease of kidney

function it is the glomerular filtration rate (GFR) for 3 months. Depending on the GFR, the

CKD is classified into 5 stages (Levely & Coresh, 2012). The stages go from G1 to G5. G1

and G2 with a normal to mild decrease in GFR indicate the patient is at low risk of CKD;

G3 with a moderate to severe decrease in GFR will increase the risk of CKD; G4 with a

severe decrease in GFR indicates the patient will be at high risk of CKD and finally, G5

patients have a very high risk of CKD and kidney failure (Said et al., 2015). Standard

determination of GFR by creatinine clearance becomes unreliable as CKD progresses.

Also, albuminuria is classified into 3 stages from A1 to A3. A1 has a normal to mild

increase in albuminuria with a low risk of CKD; A2 shows a moderate increase with a

higher risk of CKD, and lastly A3 has a severe increase with a very high risk of CKD and

kidney failure (Said et al., 2015). Several experiments and clinical studies found that

proteinuria was involved in the pathogenesis of kidney disease progression. Population

epidemiology studies discovered that there was a relation between the increase in

albuminuria and mortality with kidney disease (Levely & Coresh, 2012).

1.2.2 Nephropathies of Systemic Diseases:

The nephropathies of systemic diseases, classified in six groups, have adverse renal effects

(Williams & Mallick, 1994) (Table 1.1).

5

Groups

A. Metabolic (ex. Diabetes mellitus).

B. Vasculitic (ex. Systemic lupus erthematosus (SLE)).

C. Dysproteinaemic (ex. Multiple Myeloma).

D. Haematuric (ex. IgA nephropathy).

E. Thrombomicroangiopathic (ex. Hemolitic uraemic syndrome (HUS).

F. Miscellaneous (ex. Infectious bacterial endocarditis hepatitis B).

Table (1.1): Classification of systemic diseases that have adverse renal effects.

1.2.3 Dysproteinemic:

Dysproteinemia is abnormal excessive production of immunoglobulin (Ig) molecules by

plasma B-cells. The common Ig fragment produced is a free monoclonal light chain that

might be pathogenic and associated with kidney damage development (Markowitz, 2004

and Williams & Mallick, 1994).

There are three main dysproteinemia diseases: Multiple Myeloma, Waldenstrom

macroglobulinemia and Cryoglobulinamia (Williams & Mallick, 1994) (Table 1.2).

6

Dysproteinemia Diseases Renal complication

(A) Multiple Myeloma involves

the production of any

monoclonal Ig (IgG, IgA,

IgD and IgE) or free light

chain (FLC).

(B) Waldenstrom’s

macroglobulinemia involves

the production of

monoclonal IgM.

(C) Cryoglobulinamia, has three

types: (I) Any monoclonal

Ig (IgG, IgA, IgD and IgE)

or free light chain (FLC),

(II) Essential mix polyclonal

IgG and monoclonal IgM,

(III) Polyclonal IgG or IgM.

(A) Myeloma

nephropathy, Light

chain deposition

(LCD), Amyloidosis,

or Fanconi syndrome.

(B) Glomerular hyaline

thrombi or Hyper

viscosity syndrome.

(C) Proliferative

glomerulonephritis and

Glomerular hyaline

thrombi.

Table (1.2): Main types of dysproteinemia diseases and renal complications.

1.3 Multiple Myeloma:

Multiple Myeloma (MM) is a blood cancer disease that leads to dividing plasma B cells

over and over in unregulated way. Normally, the bone marrow produces plasma B

7

lymphocyte cells, which is type of white blood cells produce normal antibody Ig to help in

fighting infection (Male, 2004). As shown in (Table 1.2) in MM, plasma B cells (myeloma

cells) produce a high concentration of monoclonal Igs or free light chains in Kappa (𝜅) or

Lambda (𝜆) forms (Williams & Mallick, 1994 and Kuby, 1997).

According to Cancer Research UK in 2012 ~ 114,000 patients were diagnosed with MM

worldwide, and 80,000 died. Kidney injury is a common complication of patients with MM

and causes an increase in mortality (Heher et al., 2013).

1.3.1 Myeloma Nephropathy:

The presence of light chains in urine is a sign of myeloma kidney as in Bence-Jones

Proteins. The myeloma kidney results from tubulointerstitial damage caused by monoclonal

of light chains (Williams & Mallick, 1994), cast nephropathy or a combination of both

(Hutchison et al., 2012).

1.3.2 Cast nephropathy:

Cast nephropathy is cast formation by precipitation of FLCs in the lumen of the distal

nephron, which leads to an increase in the pressure on PTCs and reduces the glomerular

blood flow to nephrons and causes inflammation and fibrosis (Hutchison et al., 2012). Cast

generates with excessive or very low concentrations of FLCs conditions (Kapoulas et al.,

2015). Patients with cast nephropathy have advanced light chain myeloma. The factors

leading and contributing to cast formation are many, including the type and the

concentration of light chains, dehydration and tubular flow rate (Korbet & Schwartz, 2006).

Cast nephropathy in acute kidney injury can be precipitated by dehydration and

hypercalcemia; acute tubular necrosis and tubular atrophy might also exist (Kapoulas et al.,

2015).

8

1.3.3 Fanconi syndrome proximal tubulopathies:

Tubular injury is one of the results of light chain type and dose dependent toxicity to PTCs

(Korbet & Schwartz, 2006). The direct toxic effect of monoclonal-FLC causes proximal

tubule fanconi syndrome (FS), which leads to aminoaciduria, glycosuria and bicarbonate

wasting (Williams & Mallick, 1994). FS is associated with crystalline deposits intracellular

in PTCs by the deposit of 𝜅-LC (Kapoulas et al., 2015).

1.3.4 Amyloidosis:

When proteins accumulate in organs like kidney or liver, amyloidosis occurs. It occurs in

three different types: primary, secondary, and familial forms. The type depends on the

cause of the amyloidosis and the type of the protein that accumulated in the organ. The

most common form of amyloidosis is the primary, which is related to MM. It is caused

when FLCs accumulate in the kidney. Secondary amyloidosis is associated with chronic

inflammatory diseases like rheumatoid arthritis. Treating patients with this type of

amyloidosis can stop or slow the progression of amyloidosis. The last type, hereditary or

familial amyloidosis, is rare. It occurs when the patient has a mutation of a protein, which

leads to a life-long production of this protein, and the consequence of this is the protein

accumulates in the organ (Williams & Mallick, 1994 and Schena et al., 2001).

1.3.5 Light-chain deposition disease:

Light-chain deposition disease (LCDD) is plasma cell dyscrasia. It is a monoclonal LC

deposition disease in an organ. It is a rare disease. 10% of MM patients might have LCDD

in kidney glomerular and tubular (Williams & Mallick, 1994 and Leung, 2007).

9

1.4 Monoclonal FLC and Myeloma Kidney:

Kidney failure is a consequence of MM diseases. Between 20-40% of MM patients have

kidney injury (Dimopoulos et al., 2008). However, from a clinical point of view, not all

monoclonal FLCs are nephrotoxic because a number of MM patients with high

concentration of urinary FLCs (proteinuria) do not develop kidney damage, and they

explained that the toxicity of FLC to induce kidney injury depends on FLC structure

specifically the V region and the environmental factors like pH and urea concentration

(Mussap & Merlini, 2014).

1.4.1 Immunoglobulin and Light Chain Structure:

Igs are glycoproteins with a Y-shaped structure. The basic structure of Ig consists of two

identical heavy polypeptide chains, each one (50 kDa), and two identical light chains each

one (25 kDa). The heavy chains form the Fc fragment and the light chains contribute to the

Fab fragment (Male, 2004 and Kuby, 1997). Fab is the fragment antigen-binding site in Ig,

which the antigen binds to it, and Fc is the fragment crystallizable region that is the tail

region of the antibody, which interacts with the cell surface (Male, 2004).

Each Ig antibody has a 𝜅 or 𝜆 light chains isotype: in human 40% of light chains are 𝜆 and

60% are 𝜅, and there are four types of 𝜆 light chain (𝜆1, 𝜆2, 𝜆3 and 𝜆4) depending on a

minor difference in the amino acid sequences. There are five main heavy chains (isotypes)

(𝜇, 𝛿, 𝛾, 휀 and 𝛼). Each heavy and light chain consists of a variable (V) and constant (C)

region and each region consist of 100-110 amino acids. The constant region is responsible

for the type of the antibody, such as when heavy chain is the class of antibody IgG (Kuby,

1997) (figure 1.1).

10

1.5 Kidney, anatomy and function:

The kidneys are two bean shaped organs located at the rear of the abdominal cavity. In

humans, each kidney is approximately 12 cm long, 7 cm wide, 4 cm thick and weight about

115-170g (Lote, 1994). Each kidney consists of a renal capsule, cortex and medulla. The

cortex and medulla contain millions of tiny structures called nephrons, which are the basic

units of operation of the kidney. The nephron consists of the renal corpuscle, which

consists of glomerular capillaries and bowman’s capsule, proximal tubules and distal

tubules in the cortex and the loop of henle and collecting duct in the medulla (Koeppen &

Stanton, 2012).

HC

LC

Fab

Fc

Coo- Coo-

NH3+

NH3+

NH3+

NH3+

VH VH

VL VL

CL

CL

CH

CH

CH

CH

CH

CH

𝝀 or 𝜿

𝝁, 𝜹, 𝜸, 𝜺 𝐨𝐫 𝜶

Figure (1.1): The basic structure of immunoglobulin. LC: Light chain, HC: Heavy

chain, V: Variable, C: Constant, Fab: Fragment antigen binding and Fc: Fragment

crystallizable.

11

The kidney plays essential regulatory roles such as regulation of blood pressure and

elimination of waste substances like urea. It has an important role in the reabsorption of

water, glucose and many other nutrients. In 24h ~180 L of plasma is filtered by the kidney

(Koeppen & Stanton, 2012).

The proteins and other large molecules with a molecular-weight ≥ 70 kDa cannot pass

through the glomerular filter into bowman's capsule and then to the tubular lumen, they are

filtered out of the glomerulus by an ultrafiltration process that returns molecules like

albumin (69 kDa) to the capillaries. However, all other molecules with low molecular

weight (<70 kDa) pass into the nephrons. The substances, which the body needs to retain

are filtered and reabsorbed in the proximal tubular cells and returns into the bloodstream by

the endocytosis process (Lote, 1994).

1.6 Protein uptake and Endocytosis:

The mechanisms of proteins to induce kidney damage (toxic nephropathy) are different;

kidney damage might occur due to glomerular injury, increasing the quantity of proteins in

blood and increasing the reabsorption in the kidney, low reabsorption proteins at proximal

tubule cells or toxicity of filtrated proteins. All of these reasons may affect the kidney

functioning and lead to presenting a cocktail of macromolecules that are abnormally filtered

by the glomerulus and present in the urine as proteinuria like albumin or light chain

(Abbate et al., 2006 and Caruso-Neves et al., 2005 and Toblli et al., 2012) .

1.6.1 Proteinuria:

Proteinuria is a sensitive marker for kidney progression function. It is one of the major

health care problems, as hundreds of people world wiled suffer from proteinuria (Toblli et

al., 2012). Normally, more than 0.01g/100ml proteins in urine are a hallmark of progressive

renal dysfunction (Lote, 1994).

12

In normal condition, the permeable capillaries in glomerulus are the effective barriers for

protein filtration with high molecular weight like albumin (65kDa) and the smaller proteins

such as immunoglobulin (25kDa) are reabsorbed at the proximal tubule. The

pathophysiological mechanism for proteinuria can be classified to glomerular proteinuria

and tubular proteinuria (Carroll, 2000).

Albumin is the most abundant plasma protein: it constitutes of 60% of the total. It is

synthesized in the liver. It is an anionic, flexible, heart-shaped molecule (Lote, 1994). There

are several functions that have been ascribed to this protein, including the maintenance of

the blood volume, acid/ base buffer functions, antioxidant functions and transportation of a

number of different substances like fatty acids, ions, hormones, and vitamins. Both serum

and urinary albumin levels are important prognostic indicators in renal disease (Birn &

Christensen, 2006). Approximately 3.3g of albumin are filtered daily in human kidneys.

The proximal convoluted tubule reabsorbs 71%, the loop of henle and distal tubule 23%,

and the collecting duct 3% of the glomerular filtered albumin (Tojo & Kinugasa, 2012).

The urinary albumin excretion classified as normal is (< 15mg/24h), high normal is (15-

30mg/24h), microalbuminuria is (30-300mg/24h) and macroalbuminuria is (>300mg/24h)

(de Jong & Curhan, 2006). Appearing high concentration of albumin in urine is mostly

because of glomerular abnormalities alter the permeability of the glomerular basement

membrane (Carroll, 2000).

However, the present of low molecular weight proteins in urine is commonly occurring

with tubulointerstitial disease, which prevents the proximal tubule to reabsorb the proteins.

Also, it can be because of protein overflow exceed the ability of the proximal tubules to

reabsorb filtered proteins (Carroll, 2000).

Most often, the tubular proteinuria occurs in multiple myeloma as a result of the FLC

immunoglobulin fragments (Bence Jones Proteins) overproduction.

FLC proteinuria is a sign in MM patients for kidney dysfunction. In a healthy person,

~500mg of FLC is produced daily and most of that is reabsorbed by the kidneys, with only

1 to 10mg/day appearing in the urine. However, in MM, the level of FLC raise sometimes

to 100,000 mg/l serum concentration (Basnayake et al., 2010). In MM patients with light

13

chain proteinuria 16% (<1g/day), 47% (1-10g/day) and 63% (>10g/day) appear in urine

(Korbet & Schwartz, 2006).

Increasing the level of FLC in serum leads to an increased burden of kidney reabsorption,

allowing FLC to travel to distal nephron to complete the journey and appear in urine

(proteinuria) and end up in kidney failure (figure 1.4) (Basnayake et al., 2010).

1.6.2 PTCs and Endocytosis process:

Proximal tubular cells (PTCs) are the most abundant cell type in the kidney (Hutchison et

al., 2012) . They consist of two parts, the convoluted and straight part, and brush border,

which consist of millions of microvilli to increase the availability of the luminal surface

area for absorption of the tubular fluid. Proteins are essentially reabsorbed ~30g/day of

proteins enter the kidneys and most of them are reabsorbed in the proximal tubules by

receptor mediated endocytosis (Lote, 1994). Clathrin mediated endocytosis is a process

involves ligand like (albumin or light chain) that bind to a specific binding site in a receptor

such as megalin in the apical membrane in the proximal tubules cells; this complex is

internalized by invagination of the plasma membrane caused by adaptor molecule-mediated

formation of a cytoplasmic coat. The internalization is followed by cytoplasmic coat

dissociation of the invaginations from the plasma membrane, forming vesicles, followed by

acidification of the intravesicular lumen and the dissociation of the ligand from the

receptor. Finally, the receptor is recycled back to the luminal membranes through dense

apical tubules, and the ligand is delivered to lysosomes and cleaved to amino acids (figure

1.2) (Lote, 1994 and Birn & Christensen, 2006).

14

Figure (1.2): Megalin-mediated endocytosis and recycling to the cell surface.

Ligand binds to receptor Ligand

Megalin Receptor Endocytosis

Ligand and receptor separate

Receptor in vesicle

moves

to the cell

membrane

Recycled receptor 8

5

4

3 2

1

7

6

Endocytic vesicle

Ligand goes to lysosome degradation

to amino acid and reabsorbed

Clathrin- coated pit

15

1.6.3 Megalin:

Megalin, initially described as gp330 (Christensen & Birn, 2001), is a 600kDa glycoprotein

and belongs structurally to the low-density-lipoprotein-receptor (LDL-R) family, consisting

of three major domains: a large extracellular domain, small transmembrane domain, and

intracellular domain (cytoplasmic tail) (figure 1.3) (De et al., 2014). The megalin structure

is described in chapter 6.

It scavenges filtered proteins and transports them by endocytosis process. It is expressed in

epithelial cells of the small intestine in the visceral yolk sac, and the cytotrophoblast of the

placenta and is abundantly expressed in the apical membrane of proximal tubule cells

(PTCs). In the kidney proximal tubule, megalin is localised to the brush border, coated pits

and endocytic vesicles. The complete cDNA sequence was characterised for human

megalin and the gene located to chromosome 2q24-q31 (Christensen & Birn, 2001 and

Verroust & Christensen, 2002).

It is a multiligand endocytic receptor that mediates uptake of extracellular ligands like

vitamins-binding protein (Vitamin D-binding protein), Apolipoproteins (Apolipoproteins

B), low-molecular-weight peptides and hormones (Insulin, β 2M and EGF), drugs

(Aminoglycosides), enzymes and enzyme inhibitors (Lipoprotein Lipase) and proteins such

as Ig light chain and albumin (Christensen & Birn, 2001 and Verroust & Christensen,

2002).

1.6.3.1 Megalin Signalling Functions:

The megalin cytoplasmic domain contains several regions, including many Src homology 3

and one Src homology 2 recognition sites and also NPXY motifs. These regions suggest

possible signalling function and are involved in the endocytosis process (Christensen &

Birn, 2001).

16

1.6.3.2 Megalin mutation and human diseases:

There are two rare diseases that are associated with megalin mutation, Donnai-Barrow

syndrome (DBS) and facio-oculo-acoustico-renal syndrome (FOAR). They are inherited

disorders affect in several organs in the body. Patients with either of these two diseases will

suffer from developmental delay, proteinuria, hearing loss and ocular abnormalities. This

reflects the important role of megalin in organs development like brain, eye, ear and kidney

(Marzolo & Farfán, 2011 and Pober et al., 2009).

1.6.3.3 Description of megalin transgenic mouse:

The functions of megalin are deduced from the study of megalin-deficient mice. Megalin-

deficient mice generated by gene targeting exhibit severe forebrain abnormalities and lung

defects. Most of them die perinatally, and only 1 out of 50 survives to adulthood. In

general, they had normal kidneys; however, ultra-structurally the proximal tubule cells are

characterized by a loss of apical endosomes, coated pits, and recycling dense apical tubules.

This supports the understanding that megalin is essential for these processes.

In addition, the megalin-deficient mice excrete an increased amount of low-molecular-

weight plasma proteins, such as light chain in urine; this is a result of defective tubular

reabsorption, due to the absence of immune detectable protein in the proximal tubule cells

of deficient mice. No changes in water, glucose and amino acids transport have been found

in these mice (Christensen & Birn, 2001).

1.6.4 Megalin-Associated Molecules:

1.6.4.1 Cubilin:

Cubilin multiligands glycoprotein receptor, also named gp280. It is a 460 kDa and consist

of ~3,600-amino acid protein consisting of a 110 amino acid N‐ terminal stretch, followed

by 8 EGF and 27 CUB (Complement C1r/C1s, Uegf and Bone morphogenic protein‐ 1)

17

domains, with no apparent cytoplasmic domain (figure 1.3) (Verroust & Christensen,

2002). It is highly expressed in the visceral yolk sac, the epithelium of the small intestine

and in the PTCs for normal reabsorption of proteins. Albumin and light chain are ligands to

cubilin and made cubilin–ligand complexe to be reabsorbed in PTCs but this complex

needs megalin (megalin-cubilin complex) to be internalised and also to recycle cubilin

because cubilin does not have a transmembrane domain to engage in endocytosis

(Christensen & Birn, 2001). Immunomorphological and biochemical data suggest that the

internalisation of cubilin is, at least in part, carried out by megalin (Verroust & Christensen,

2002).

Although it has been identified that fewer ligands bind to cubilin compared with megalin,

still it was identified as an important receptor in tubular protein reabsorption. This is due to

some cases with intense proteinuria that were the result of mutations in the cubilin gene.

For example, in human mutations of cubilin lead to Imerslund-Gräsbeck syndrome, which

is characterised by vitamin B12 deficiency and significant proteinuria. Also, in animal

studies with a lack of cubilin expression on apical in PTC, appreciable proteinuria (Baines,

2010).

18

1.6.5 Non Megalin/Cubilin mediated up take of protein in PTCs:

1.6.5.1 Cluster of differentiation 36 (CD36):

Another receptor, CD36, exists for protein handling in PTCs and different in vivo, in vitro

and clinical samples were examined to investigate if CD36 binding protein and

reabsorption in PTCs.

CD36 is an (88kDa) glycoprotein. It is a class B scavenger receptor, present in glomerular

cells, endothelial cells and PTCs (Yang et al., 2007 and Baines et al., 2012).

Renal biopsy tissue obtained from adult patients with membranous nephropathy or focal

segmental glomerulosclerosis with heavy proteinuria showed a significant increase in

CD36 expression in PTCs. In vitro, exposure of PTCs to several albumin concentrations

Figure (1.3): Schematic structures of megalin and cubilin receptors in relation to

the plasma membrane

Luminal

Plasmamembrane

COOH

Megalin

Transmembrane

domain

NH2

Cubilin

CUB

domain

EGF-type

repeat

Cytoplasmic tail

NH2

Extra

cellular

Domain

19

showed an increase in CD36 expression. Also, CD36-transfected PTCs showed enhanced

the binding and uptake of albumin, and using a CD36 inhibitor abrogated this effect.

However, blocking megalin did not. In vivo, CD36 null mice showed significant increase

in urinary protein-to-creatinine ratio and albumin-to-creatinine ratio. Although, mouse

PTCs from WT and CD36 null mouse showed comparable megalin expression, and CD36

expression from WT and, as expected, absence in CD36 null mice (Baines et al., 2012).

Thus, CD36 is involved in albumin uptake in PTCs and might have a potential role in

proteinuric nephropathy.

20

Figure (1.4): Mechanism of multiple myeloma and kidney failure. FLC: Free light chain and PTCs: Proximal tubular cells.

Multiple Myeloma

Patient

Bone marrow

Abnormal Plasma cells

(myeloma cells)

Increase the endocytosis via Megalin

receptor

Damage PTCs

Cells

PTCs

Over filtration in Kidney

Glomerular

Proteinuria and Kidney failure

FLC

Megalin

Bruch Border

21

1.7 Signalling role of megalin in PTCs toxicity:

1.7.1 Signalling pathway regulates protein endocytosis in PTCs:

Different glomerular filtrated proteins are reabsorbed in PTCs by endocytosis process, with

receptor-mediated mechanism involves clathrin. Receptors mediated endocytosis like

megalin and cubilin. Many components are involved in endocytosis to regulate the process

such as G-proteins that are abundantly expressed in PTCs (Caruso-Neves et al., 2005 and

Baines, 2010).

There are two classes of G proteins depending on the function: the monomeric small

GTPases and heterotrimeric G protein complex. The heterotrimeric G protein complex is

activated by G protein-coupled receptors. Heterotrimeric G protein complex consists of two

protein subunits 𝛼 and 𝛽𝛾 complex. G𝛼 has been grouped into four classes Gs, Gi, Gq, and

G12. They are grouped depending on their sequence and gene structure (Hurowitz et al.,

2000). PTCs have a large amount of G𝛼i3 in their apical membranes when the endocytosis

process occurs, which could play a role in the regulation of endocytosis of macromolecules

like albumin. For example, albumin uptake was increased with over-expression of G𝛼i3,

however, inhibiting G𝛼i3 showed a decrease in the process in OK cells (Brunskill et al.,

1996).

Endocytosis activates the signalling pathway that is controlled by different kinases

(Caruso-Neves et al., 2005). Phosphoinositide 3-kinase (PI3K) is a lipid kinase that plays a

role in many signalling pathways including regulating cell survival, intracellular trafficking

and cell growth. It is involved in the endocytosis process in generating vesicles from

plasma membrane (Sauvonnet, 2013) PI3K has an important role in receptor-mediated

endocytosis process. PI3K is composed of a catalytic p110 subunit and a regulatory p85.

The activation of PI3K occurs via many receptors tyrosine kinases like EGFR, which

augments macromolecules endocytosis. Inhibiting PI3K by wortmannin and LY294002

inhibited the receptor-mediated endocytosis of albumin in PTCs, which means PI3K has a

regulation role (Brunskill et al., 1998). Activation of PI3K leads to Akt/PKB (Sauvonnet,

2013).

22

Protein kinase B (PKB)/Akt belongs to the ACG family that is a serine/threonine specific

protein kinase (Caruso-Neves et al., 2005). PKB/Akt has a role in several processes such

as cell proliferation, apoptosis and cell survival (Sauvonnet, 2013). Both serine and

threonine phosphorylation are required for PKB/Akt activation Both PI3K and PKB/Akt

are regulators for endocytosis (Caruso-Neves et al., 2005). PKB/Akt activates MAPKs

(Baines, 2010).

Mitogen-activated protein kinases (MAPKs) are components that have a role in cell growth,

proliferation and transformation. This kinase cascade consists of three-kinases, which are

MEK kinase that is phosphorylated MAPK and leads to extracellular-signal-regulated

protein kinases (ERKs) activation. Albumin activated ERKs, for example, stimulating

PTCs with different concentration of albumin (1-10mg/ml) showed dose dependence in

EKR activation. This finding suggests a correlation between albuminuria and cell growth

due to at the same time the ERK activation leads to inhibit c-Jun N-terminal kinases (JNK),

which inhibits cell apoptosis (Dixon & Brunskill, 2000 and Baines, 2010). MAPK

activation leads to inhibit glycogen synthase kinase 3 (GSK-3).

Other proteins kinases regulating the endocytosis process are protein kinase A (PKA) and

protein kinase C (PKC). However, they have opposite role by inhibiting endocytosis

(Caruso-Neves et al., 2005). They are serine/threonine kinases (Baines, 2010). Stimulating

PTCs with albumin stimulated PKC activity and inflammatory cytokines; so, inhibiting

PKC by activated MAPK shows the opposite effect (Morigi et al., 2002) and Cabezas et

al., 2011).

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase. It has a role in

several signalling pathways such as gene cell cycle progression, cell differentiation and

epithelial cell function and survival. GSK-3 exists in two isoforms: GSK-3α and GSK-3β.

Previous studies have found that GSK-3 inhibition has a protective role in acute kidney

injury. GSK-3 is inhibited by phosphorylation of serine 9 of GSK-3β and serine 21 of

GSK-3α (Rao, 2012). GSK-3 inhibitor have been successfully trialled in renal diseases

(Soos et al., 2006) and shown to improve a range of renal conditions, which have a

component of proteinuria (Obligado et al., 2008).

https://en.wikipedia.org/wiki/Serine/threonine_protein_kinase

23

1.7.2 Megalin phosphorylation and PTCs toxicity:

Megalin receptor is a mediator of the endocytosis process and has a signalling function that

might be involved in tubular toxicity in proteinuric condition. The megalin cytoplasmic tail

(Meg-CT) is longer than other LDL-R receptors. Megalin is involved in signalling and is

phosphorylated by different kinases like PKC. The phosphorylation occurs in serine, and

this phosphorylation has an effect on regulation megalin receptor recycling to the cell

surface. For example, high albumin concentration decreased megalin expression in PTECs,

PKC levels were reduced and cells were apoptotic (Baines & Brunskill, 2008).

Previous studies showed that Meg-CT is phosphorylated in PPPSP motif by GSK-3; the

function of this motif is related to control megalin recycling from the endocytosis. The

phosphorylation occurs independent of the type of ligand binding to megalin (Yuseff et al.,

2007).

Dab2 is cytosolic adaptor protein that binds to Meg-CT. It has an important role in megalin

expression and function (Marzolo & Farfán, 2011). Losing Dab2 leads to loss of the cell

surface polarity of megalin (Yang et al., 2007). For example, in vivo study Dab2 knockout

mice showed the expression of protein in urine, and reduction in megalin expression to the

apical surface of PTCs (Morris et al., 2002).

In summary, PTCs toxicity in proteinuric conditions might come from the phosphorylation

of Meg-CT and reduction of megalin recycling to the cell surface.

1.8 Proteinuria and Proximal Tubular cells Toxicity:

Dysfunction in filtration or reabsorbed processes in kidneys for many different reasons are

leading to proteinuria by three mechanisms contributing to tubular damage and initiating

interstitial pathological changes due to PTCs being the main cell type in kidneys, also, their

unique position between the tubular lumen and surrounding interstitium (Wang et al.,

1997): (i) Excess endocytosis of filtered protein could stimulate cytokine and chemokine

24

secretion by PTC, and that initiates inflammation and fibrosis, (ii) The toxic effect on PTC

might present in the proximal tubular filtrate proteins like complement components, (iii)

The excessive reabsorption of filtered proteins by PTC as a result of glomerular damage

may result in cell stress and generate reactive oxygen species (Baines, 2010 and Hutchison

et al., 2012).

1.8.1 The effect of proteinuria on PTCs viability:

Previous studies investigated the effect of protein overload on PTCs growth and viability

like albumin because it is the main filtered protein in urine of proteinuric patients. For

example, PTCs stimulated with albumin concentration between 10-100 mg/ml showed a

decrease in cells viability and induced apoptosis (Caruso-Neves et al., 2005) . In another

example, exposure of OK cells to high concentration of protein has a direct toxic effect on

PTCs viability (Baines, 2010).

1.8.2 Inflammatory cytokines/chemokines and fibrogenic mediators:

The toxicity of protein overload on PTCs seems to be occurring after endocytosis process

through the endocytic receptor megalin leading to inflammatory cytokines and chemokines

production from PTC. Increase the cytokines such as interleukin-6 (IL-6) and chemokines

like interleukin-8 (IL-8) and monocyte chemo attractant protein (MCP-1) production in

PTCs in patients with proteinuria has been proposed as one of the major mechanisms

contributing to tubular injury and progressive kidney disease (Sengul et al., 2002).

1.8.2.1 Interlukin-6 (IL6):

IL-6 is glycoprotein (gp130) is one of the main pro-inflammatory cytokines (Scheller et al.,

2011). It is a (26kDa) protein, produced in many kidney cells including glomerular,

mesangial and PTCs (Sengul et al., 2002). IL-6 appears to have as its main function that

25

inducing of acute inflammatory responses (Mäkelä et al., 2004) contributes to proximal

tubular injury after excessive protein endocytosis such as FLCs in MM (Hutchison et al.,

2012) . High IL-6 was detected in plasma and urine in patients with MM disease and renal

transplant rejection (Sengul et al., 2002). Another study was conducted in hospitalised

patients with nephropathia epidemica (NE), one of the most common causes of acute renal

failure and proteinuria with loss of low-molecular-weight proteins like 𝛽2-microglobulin,

indicating tubular injury. The data showed that an increase in urinary and plasma IL-6

excretion, and urinary IL-6 excretion correlated with albumin, IgG, and protein excretion.

The urinary IL-6 excretion level did not correlate with plasma IL-6 levels, which means the

increases in urinary IL-6 excretion, is not caused by the filtration of plasma IL-6 but reflect

the production of IL-6 in dysfunction tubules in kidneys (Mäkelä et al., 2004). Also, the

urinary IL-6 level was measured in patients with IgA nephropathy disease and it was

concluded that the level of urinary IL-6 excretion could reflect the inflammation and

tubular dysfunction (Nakamura, et al., 1995).

In vivo study, transgenic mice with membrane proliferative glomerulonephritis and massive

bone marrow infiltration, they showed many similarities to MM, the IL6 expression level

was increased highly in these mice (Sengul et al., 2002).

The increase proximal tubular production of IL-6 might contribute to the pathogenesis of

tubulointerstitial disease.

1.8.2.2 Interlukin-8 (IL-8):

IL-8 is a pro-inflammatory chemokine responsible for activating T cells and monocytes in

the inflammation site (Tang et al., 2003). The massive reabsorption of proteins like FLCs

and albumin in PTCs induces the release of IL-8 in renal tubular inflammation (Hutchison

et al., 2012). The relation between IL-8 and renal tubular inflammation comes from these

observations: (i) IL-8 made by PTCs (Stadnyk, 1994), (ii) The production of IL-8 in PTCs

can be regulated by several pro-inflammatory cytokines such as TNF-𝛼, (iii) IL-8 urinary

level increase in patients with kidney diseases like IgA nephropathy (Tang et al., 2003).

26

In vivo, the repeated injection of rabbits with bovine serum albumin (BSA) caused

deposition of immune complexes consisting of BSA and rabbit IgG in glomeruli, and the

urinary levels of protein and albumin increased markedly compared with untreated animals.

Injecting rabbits with anti-IL-8 antibody led to normalised urinary levels of protein and

albumin that means IL-8 has a role in progression of proteinuria and kidney nephropathy

(Wada et al., 1994).

Also, immunohistochemistry of IL-8 on renal biopsy derived from human nephrotic

patients with minimal change nephrotic syndrome and hypertensive nephrosclerosis with

proteinuria showed strong IL-8 staining compared with minimal proteinuric control (Tang

et al., 2003 and Abbate et al., 2006) ). Thus, IL-8 has a role in proteinuria and tubular

inflammation.

1.8.2.3 Monocyte Chemoattractant Protein-1 (MCP-1):

MCP-1 is a prototype of the 𝜷 chemokine family. It is chemoattractant for macrophages

and T lymphocytes, which are the predominant inflammatory cells (Wang et al., 1999).

Many evidences suggest that MCP-1 has a role in tubulointestinal inflammation. The

expression of MCP-1 was elevated in renal tubular cells, such as in ischemia, and might

perform as a mediator for inflammation (Wang et al., 1997).

More evidence shows the correlation between proteinuria and the overexpression of

chemokines is available. In vivo, rats with protein-overload proteinuria models with

interstitial inflammation and tubular up-regulated MCP-1. To support the possibility that

MCP-1 is important and has a role in tubularinterstitial inflammation, anti-MCP-1 antibody

therapy to rats with tubulointerstitial nephritis led to reduced macrophage infiltration

significantly and interstitial inflammation and fibrosis and tubular damage (Zoja et al.,

2003 and Abbate et al., 2006) .

Albuminuria, urinary MCP-1 and interstitial macrophage infiltration were measured in

patients with CKD such as light chain nephropathy, IgA nephropathy,

27

ischaemic/hypertensive nephropathy and diabetic nephropathy, the data showed a

correlation between albuminuria, urinary MCP-1, interstitial macrophage infiltration. The

findings support the hypothesis that albuminuria triggers tubular MCP-1 expression and is

one of the important disease progressions in human chronic kidney disease (Eardley et al.,

2006).

From several studies it can be concluded that there was a concomitant up-regulation of pro-

inflammatory MCP-1in tubular epithelial cells with a progressive kidney disease.

1.8.3 Complement components as pathogenic mediators of tubular

toxicity in proteinuria:

Complement system is part of innate immunity. The main functions for the complement

system include protection from foreign pathogens; it also plays immune regulatory roles

like enhancing humoral immunity and modifying T cell immunity. Several studies showed

that the complement system has a pathogenic role in different diseases such as kidney,

inflammatory and autoimmune diseases (Thurman & Holers, 2006). The activation of the

complement system contributes to kidney tubular and interstitial injury through cytotoxic,

pro-inflammatory, and fibrogenic effects.

There are three pathways responsible for complement activation: classical, lectin and

alternative pathways. Several studies of kidney suggested a strong association between

proteinuria and alternative pathway as a mediator of progressive dysfunction, interstitial

fibrosis and tubular damage. In patients with kidney disease and proteinuria, the

complement components can be found in their urine such as diabetic nephropathy, IgA

nephropathy and focal segmental glomerulosclerosis (FSGS) (Fearn & Sheerin, 2015).

This might be because the complement proteins filter in glomerular and enter the tubule, or

it could be due to spill over of complement activated in the glomerulus (Fearn & Sheerin,

2015). Tubular cells produce the major components of alternative pathway, which are

complement component C3, Factor B (FB), Factor H (FH) and Properdin or Factor P (FP)

28

(Peake et al., 1999). Complement activation via alternative pathway has an additional

mechanism, which might contribute to the progression of renal disease. Activating the

alternative pathway on renal tubular epithelial cells led to stimulate pro-inflammatory

cytokines like tumor necrosis factor (TNF-𝛼) and IL-6, and reactive oxygen species, which

led to inflammation (Fearn & Sheerin, 2015). In addition, the local production of C3 by

renal tubule themselves may contribute to tubule injury (Zhou et al., 2001). Uncontrolled

complement activation might contribute to tissue injury (Lenderink et al., 2007).

1.8.3.1 Mechanism of alternative pathway activation and regulation:

The alternative pathway initiates when C3 binds to FB, which is activated by factor D (FD),

forming C3 convertase and generate C3a and C3b. FB cleaves and generates Ba and Bb.

C3b and Bb bind to generating unstable C3bBb.

Properdin of FP has the role to stabilizing C3bBb during the process and generates C3bBb-

P (C3 convertase). The C3bBb-p binds to C3b and generates C3bC3bBb-P (C5 convertase),

then binds to C5b and generates C5b-9 membrane attach complex (MAC), which generate

pores on cell surface and lysis the cell (Thurman & Holers, 2006). The activation of

alternative pathway controls by FH inhibits the alternative pathway C3 convertase. Also,

FH is a cofactor for Factor I (FI), which inactivating cleavage of C3b by decay acceleration

of the C3 convertase (Thurman & Renner, 2011) (figure 1.5).

1.8.3.2 Evidence of complement pathway contributes to induce renal injury:

The complement system plays a role as a pathophysiologic mediator of multiple kidney

diseases in humans (Mathern & Heeger, 2015).

Membranous nephropathy (MN) is a disease causing nephrotic syndrome in adults, and

characterized by a fine granular deposit of IgG with C3 in the peripheral capillary loops. In

MN the data showed complement activation via lectin pathway and MACs were detected in

MN patient’s urine and considered as marker of ongoing injury (Mathern & Heeger, 2015).

29

Another glomerular disease is IgA nephropathy, characterised by IgA deposition. It is

likely mediated by alternative pathway-dependent, antibody initiated complement

activation. Deposits of C3 and C5b-9 were detected in the glomeruli and correlate with

kidney disease severity and prognosis (Mathern & Heeger, 2015).

Monoclonal gammopathy has been associated with the activation of the alternative

pathway. Circulating light chain binds to FH that is responsible for alternative pathway

inhibition, thus over activation of the alternative pathway leads to cells inflammation

(Mathern & Heeger, 2015).

Ischemia-reperfusion injury results from mitochondrial damage or tissue hypoxia leading to

the generation of free oxygen radicals on reperfusion that initiates damage. Ischemia-

reperfusion injury up-regulated the production of complement components in tubular cells;

the local activation via alternative pathway led to inflammation (Mathern & Heeger, 2015).

30

1.8.4 Reactive oxygen species (ROS) and renal tubular injury:

ROS are chemical molecules formed as a natural product of the normal metabolism of

oxygen (Devasagayam et al., 2004). ROS has a role in many physiologic and

pathophysiologic processes. ROS is produced by nicotinamide adenine dinucleotide

phosphate (NADPH) oxidases by catalyzing the transfer of electrons from NADPH to

molecular oxygen via Nox subunit to produce ROS (figure 1.6). ROS has functions in

normal cellular physiology, regulating different biologic processes like cell defense, cell

signalling as second messengers, hormone synthesis, gene expression, cell growth and

apoptosis (Sedeek et al., 2013).

Figure (1.5): Mechanism of activation and regulation of complement alternative

pathway. C3: Complement component C3, FB: Factor B, FD: Factor D, FH:

Factor H, FI: Factor I and MAC: Membrane Attach Complex.

C3

FB FD

C3bB

C3a

C3b-FI

Ba

FP FH

C3bBb-P (C3 Convertase)

C3bC3bBb-P (C5 Convertase)

C3

C3a

C5b-9 (MAC)

C5a

FI

31

ROS plays a role in the pathophysiology of many systemic diseases including hypertension,

diabetes mellitus, infection, environmental toxins; smoking and alcohol consumption

induce oxidative stress in kidney (Ozbek, 2012). In addition, ROS-mediated stresses in a

number of diseases like cardiovascular pathology and immunodeficiency and kidney

diseases. Also, it is an inflammatory mediator (Panday et al., 2014).

In normal conditions ROS produce inactivate. Normally, there is a balance between oxidant

(ROS) and antioxidant defence systems. The antioxidant can be divided into enzymatic

types like glutathione peroxidase and nonenzymatic such as vitamin A, C and E. However,

in pathological conditions, increased the generation of ROS and/or reduction of the

antioxidant defence system leads to tissue damage (Birben et al., 2012).

ROS plays several roles in kidney physiologic processes such as glucose transport;

tubuloglomerular feedback that is a kidney mechanism regulating the glomerular filtration

rate (GFR) and electrolyte transport (Sedeek et al., 2013). However, ROS mediates

complications in kidney diseases by oxidative stress, oxidative stress-related mediators and

inflammation (Ozbek, 2012).

Many evidences demonstrated that renal tubular epithelial cells are capable to produce ROS

and particularly hydrogen peroxide (H2O2) under toxic conditions; hypoxia and protein

overload (Morigi et al., 2002). In protein overload conditions NADPH oxidase is

responsible and required for intracellular H2O2 generation in PTCs because inhibiting

NADPH in PTCs protein overload model by (albumin or IgG) showed reduction in H2O2

generation (Morigi et al., 2002).

In vitro, opossum kidney PTCs exposed to bovine serum albumin led to increased ROS

generation and they concluded that increased formation of ROS as a response to

albuminuria might consequently cause tubulointerstitial damage (Hodgkins & Schnaper,

2012).

32

1.8.5 Autophagy in renal tubular injury:

Autophagy is a Greek word ‘auto’ means self and ‘phagos’ means eat (Takabatake et al.,

2014). Autophagy is a catabolic process. Three types of autophagy have been identified:

macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy is

the major type of autophagy. It induces in response to stress conditions such as cell

starvation, hypoxia, and oxidant injury as an adaptive and protective mechanism for cell

survival (Kaushal, 2012 and Takabatake et al., 2014), but autophagy is able to contribute to

cell death when deregulated such as in cancer diseases (Jiang et al., 2012).

Autophagy formation is divided into four steps. First one is the initiation step that is

initiated by different autophagic protein complexes such as kinase 1 or 2 (ULK1 or ULK2)

complex, the class III phosphatidylinositol 3-kinase complex, Atg12–Atg5–Atg16

conjugation, and lipidation of microtubule-associated protein 1 light chain 3 (LC3). After

that is the nucleation step, which is the formation of the phagophore membrane for

phagophor expansion. Then, the maturation step (elongation and closure) that completes the

NADPH NADP+

O2

H2O2

O2

-

Figure (1.6): H2O2 generation by NADPH.

33

formation of autophagosomes (autophagic vesicles or vacuoles) with the double membrane

being generated. Finally, the autophagosome fuses with lysosome and forms an

autophagolysosome and free amino acid and fatty acid generates. The generated free amino

acids and fatty acids are recycled to synthesize new proteins and bio-energetic supplies of

the cell (figure 1.7) (Benbrook & Long, 2012).

Interestingly, accumulation of evidences from in vivo and in vitro studies demonstrated that

macroautophagy is one of the stress responses of renal tubular cells to acute injury.

Whether autophagy plays a pro-survival or a pro-death role remains very controversial (Liu

et al., 2014).

Autophagy activation has an important role in PTCs, possibly because these tubules contain

an enormous number of mitochondria that provide the energy for the reabsorption

processes that occur in these cells, and lysosomal machinery has an essential role in the

reabsorption and degradation of glomerular filtrate plasma proteins (Takabatake et al.,

2014).

1.8.5.1 Evidence of autophagy induces protection/injury in PTCs in kidney diseases:

To examine the role of autophagy in kidney disease, investigators usually use autophagy-

deficient mice by knocking out a specific Atg that is responsible for initiating the

autophagy, which leads to inhibiting the autophagy event, or by using inhibitors for

autophagy process (Liu et al., 2014). The role of autophagy was investigated in renal

tubular cells in different in vivo and in vitro models.

In vitro study, the role of autophagy in human PTECs was investigated under hypoxia

condition, as one of the ischemia injury causes. The results showed increased in autophagic

vacuoles and lysosomes in 1% O2 and 24h hypoxic cells compared to normoxic control and

by using anti-LC3 and anti-LAMP antibodies, which are markers of autophagosomes and

lysosomes, respectively. By using lysosome inhibitor the autophagosomal vacuoles sill

increased after 6 and 24h of hypoxia. They concluded that a high turnover of autophagy

might lead to autophagic cell death (Suzuki et al., 2008).

34

In vivo study, the role of autophagy was investigated in mice with severe proteinuria

induced by intraperitoneal albumin (free fatty acid) overload that led to tubular proximal

tubular cell damage, apoptosis, and activated autophagy, and in proximal tubule specific

autophagy-deficient mice, resulting from an Atg5 gene deletion. The mice, also developed

severe proteinuria, induced proximal tubular cell damage autophagy was activated after

intraperitoneal albumin (free fatty acid) overload. From these results they suggest that

proteinuria-induced autophagy has a renoprotective role (Yamahara et al., 2013).

35

Figure (1.7): Diagram of the steps of autophagy.

Induction/Initiation

Nucleation/ Elongation

Phagophore membrane

Autophagosome

Lysosome

Fusion

Autophagolysosome

5

3

4

2

1

Autophagic

protein

complexes

36

1.8.6 Apoptosis in proximal tubular injury:

Apoptosis or programmed cell death is a way of cell death. During apoptosis the

morphological and biochemical characteristics of cells changes: for example, the size of

cells decrease due to the reduction in cytosolic volume and condensation of nuclear

chromatin, round up, internucleosomal DNA fragmentation and the cell breaks into many

parts called apoptotic bodies, which contain fragments like mitochondria. Also, cell

detachment is one of the characteristics of apoptotic cells (Lieberthal & Levine, 1996 and

Erkan et al., 2001).

The morphological and biochemical changes in apoptotic cells correlate with caspase

activation. Caspases are complex cascades of protein that cleave enzymes. In mammals, 14

caspases were identified, which lead to apoptotic cell death (Kwak, 2013). These caspases

are present as in inactive forms in all animal cells (Reed, 2000). Apoptosis signalling

induces through three pathways, including cytokines pathway like TNF-𝛼, mitochondrial

pathway, and endoplasmic reticulum (ER) pathway (Kwak, 2013).

Cytokines pathway activates caspase8; ER activates capase12 and mitochondrial pathway

activates caspase-9; all three pathways subsequently activate caspase3, resulting in

apoptosis through DNA fragmentation (Kwak, 2013).

Apoptosis plays a role in pathogenesis of acute and chronic renal disease, specifically in

renal tubular epithelial cells injury. There are several triggers for apoptosis in renal tubular

cells include physiological activators such as TNF-𝛼 and TGF-𝛽 and cytotoxic stimuli like

ischemia and nephrotoxins (Lieberthal & Levine, 1996).

Several in vitro and in vivo studies have examined the apoptosis in many kidney diseases

that showed injury in renal tubular cells. For example, in vivo apoptotic bodies were

detected in tubular cells in kidney of ischemia/reperfusion (for 12-48h) rat model

(Lieberthal & Levine, 1996).

37

1.8.6.1 Proteinuria and renal tubular apoptosis:

Previous study reported that excess endocytosis in PTCs trigger the apoptotic pathway and

many evidences suggested that proteinuria causes tubule cells apoptosis (Hutchison et al.,

2012).

The references listed here after deal with the interaction of high protein and kidney cell

response in different setting, there by supporting a fundamental relationship between

proteinuria and tubular apoptosis.

The settings are: stimulation of HK2 cells and primary PTECs with albumin and

immunoglobulin light chain in vitro and in vivo rat model of protein overload and a clinical

histopathological study.

Here are the details:

In vitro, exposure of cultured PTCs to bovine serum albumin (BSA) with or without fatty

acid (0, 5, 10, or 20mg/ml) for 24, 48 and 72h induced apoptosis in a dose-and duration-

dependent manner. Cells stained with annexin, which is an early marker for apoptosis

and showed positive staining compared to negative staining in control cells (Erkan et

al., 2001).

Another in vitro study detects apoptotic cells after exposure of human proximal tubular

cells (SV40) to immunoglobulin light chain (200 𝜇mol/L) for 24h using TUNEL assay

using the ApopTag kit (Pote et al., 2000).

In vivo study also showed evidence that proteinuria induced apoptosis in kidney or

proteinuria rat model; they injected them with 0.5g of BSA /100g body weight. The number

of apoptotic cells was in tubular cells compared with the control. Apoptosis was detected

by detecting DNA fragmentation using (TUNEL) assay commercial kit (Tejera et al.,

2004).

Previous study detected apoptotic cells in PTECs in biopsy specimens of 30 patients with

focal glomerulosclerosis (FSGS) and proteinuria. Apoptosis was detected by morphology

38

and DNA fragmentation was detected using (TUNEL) assay commercial kit. However, the

normal kidney tissues were devoid of tubular cell apoptosis (Erkan et al., 2005).

Thus, previous studies provided evidence for a strong correlation between proteinuria and

renal tubule cells apoptosis. Apoptotic cell death initiated by proteinuria induced proximal

tubular cells injury may be an important mechanism of renal disease.

1.8.7 Evidence for toxicity of excess proteins to PTCs:

1.8.7.1 Novel biomarkers/mediators of PTCs toxicity:

AKI is one of the high-risk conditions lead to death; early kidney injury diagnosis might

help to prevent or delay more kidney damage. Several specific, sensitive and reliable

clinical biomarkers were established and used as evidences for renal damage induced by

proteinuria (protein overload). Several of these biomarkers have been used in vitro and in

vivo studies as early markers for renal injury.

These markers can be measured in different patient samples like serum, urine or biopsy, but

urine is the sample that provides the most promising markers for early detection (Vaidya et

al., 2008). There are traditional ways for AKI diagnosis including measuring serum

creatinine, GFR, urea, fractional excretion of sodium and proteinuria. However, traditional

ways of measuring have many disadvantages. For example, the serum creatinine’s

concentrations changes depending on gender, age, body weight and muscle metabolism,

and the serum creatinine concentrations change when advanced kidney injury already

occurred and 30% of GFR is reduced, so, kidney injury in the short term cannot be

diagnosed by these traditional ways (Peres et al., 2013).

39

A single biomarker may not be accurate to diagnose kidney injury occurs. Several methods

use to quantify these markers such as ELISA, which detects the antigen using two

antibodies. With nanotechnology it is possible to detect antigens using one capture antibody

with a read-out that is based on the principle of a change in conductance owing to an

antigen binding to the antibody (Vaidya et al., 2008).

More than 20 biomarkers were established and studied to diagnose kidney injury

(Table 1.3). These markers appeared to be stable in collected samples to be measured

(Pacific Biomarkers, 2012).

Some of these protein markers are up-regulated when kidney is damaged and appear in

plasma and urine (de Geus et al., 2012). The effect of proteinuria over load (like albumin

and light chain in MM) on PTCs and several biomarkers were investigated and discussed in

chapter 7.

Glomerular

Total protein, Cystatin C (urinary), 𝛽 2

microglobulin, 𝛼1 microglobulin and Albumin.

Proximal Tubules

Clusterin, NGAL, Cystatin C, IL-18, Cyr61,

Kim-1, fetuin-A, 𝛽 2 microglobulin and

Albumin.

Table (1.3): Biomarkers of kidney injury.

1.8.7.2 Kidney injury molecule-1 (KIM-1):

Recently, KIM-1 was discovered as one of the best urinary biomarkers for human and

animal kidney diseases because the ectodomain of KIM-1 is stable at RT and can be

measured in a 24h urine sample in a very small volume of sample (30𝜇 l). KIM-1 is

implicated in damage/repair processes (van Timmeren et al., 2006). It plays different

functions such as phosphatidylserine receptor to recognize apoptotic cells, scavenger

receptor and mediate uptake of necrotic-cell debris (Ichimura et al., 2012).

40

Normally, it is undetectable in kidney but the expression increases significantly in renal

injury (Lock, 2010 and van Timmeren et al., 2006). For example, it was induced in

proximal tubular injury after ischaemia-reperfusion and nephrotoxic injury (Baines, 2010).

KIM-1 can be assessed in renal biopsy specimens by immunohistochemistry or by

measuring KIM-1 excretion in the urine. Urinary Kim-1 detection could be used as an early

marker for renal damage (Vaidya et al., 2008).

Detection of the expression of urinary KIM-1 is more sensitive than histology for early

tubular injury. For example, urinary KIM-1 in 201 patients with acute kidney injury was

detected, but patients with the highest KIM-1 had a 3.2-fold odds ratio for hospital death

compared to patients with a lower amount (Lock, 2010).

High KIM-1 was detected after proteinuria-induced renal damage. A rat proteinuria model

to induce tubular damage by injecting with 2g of BSA for 3 weeks showed KIM-1 protein

expression was significantly increased in rats with proteinuria compared to the control rats

that were injected with saline instead of albumin. Also, the urinary excretion of KIM-1 was

markedly elevated in rat-proteinuria. The urinary KIM-1 levels elevation in proteinuria

correlated with KIM-1 tissue expression (van Timmeren et al., 2006). Thus, there are many

evidences showing the relation between KIM-1 expression (promising biomarker) and

tubular pathology/damage in kidney diseases.

1.8.7.3 Transforming growth factor beta (TGF-𝜷):

TGF-𝛽 is (25kDa) protein, and a member of the polypeptide growth factor family. All body

cells produce TGF-𝛽 like epithelial, endothelial and connective tissue cells. There are three

isoforms of TGF-𝛽: TGF-𝛽1, TGF-𝛽2 and TGF-𝛽3; each one produces in a different

tissue. TGF-𝛽1 is the most important one in humans. It plays many functions including

regulating the proliferation and differentiation of cells, embryonic development and wound

healing; also, it has an important role in signalling pathway in these processes (Blobe et al.,

2000 and Goumenos et al., 2002).

41

Elevation or reduction in TGF-𝛽 production is linked to several diseases specifically

fibrotic diseases in kidney, liver or lung, and can be used as a diagnostic marker for human

disease. For example, TGF-𝛽 protein and mRNA levels were elevated in patients with

fibrotic kidney diseases like IgA nephropathy, focal glomerulonephritis, lupus nephritis,

and diabetic nephropathy (Blobe et al., 2000). TGF-𝛽1 is one of the most fibrogenic

growth factors linked with renal fibrosis (Goumenos et al., 2002).

Previous studies showed a correlation between TGF-𝛽1 renal expression and progression of

human kidney diseases in patients with proteinuria. One study examined the TGF-𝛽1

production and urinary excretions in 25 patients with glomerular diseases and heavy

proteinuria. The data showed strong TGF-𝛽1 immunostaining in the tubule area in the renal

sections, and the TGF-𝛽1 urinary excretion was increased in patients with proteinuria

compared to healthy and glomerular disease without proteinuria patients. From these results

they concluded that the severity of TGF-𝛽1 production and urinary excretion in patients

with glomerular diseases and heavy proteinuria is correlated to the degree of tubular

epithelial cells injury by filtered protein (Goumenos et al., 2002).

In vitro study, exposure cultured PTECs to several concentrations of HSA for 8h showed a

significant increase in TGF- 𝛽 1 protein secretion (albumin concentration-dependent)

(Diwakar et al., 2007). From different studies it can be confirmed that TGF-𝛽1 plays a role

in developing fibrotic disease in kidney.

1.8.7.4 Tumor necrosis factor alpha (TNF-𝜶):

TNF-𝛼 cytokine is a (26kDa) trans-membrane protein. It has properties that play a role as a

pro-inflammatory cytokine that leads to activating inflammatory cells at the site of injury,

or an immunoregulatory role. Also, it can stimulate the release of other cytokines and

chemokines such as MCP-1, IL-6 and IL-8 (Ernandez & Mayadas, 2009).

42

Commonly, inflammatory diseases affect kidney, and TNF-𝛼 is mainly associated with an

inflammatory cascade that ends up with renal injury. On the one hand, TNF-𝛼 is not

detectable in normal kidney; on the other hand, most renal cells produce TNF-𝛼 but in

diseases and different inflammatory conditions (Ernandez & Mayadas, 2009).

For example, in vivo rat model of ischemia-reperfusion injury showed an increase in TNF-

𝛼 production localized in tubular cells in sections of renal samples stained for TNF-𝛼 by

immunohistochemical techniques (Donnahoo et al., 2001).

In vitro study, stimulating human PTCs with κ-LC (50μM) for 72h (proteinuria model)

showed a significant increase in TNF-𝛼 production (Arimura et al., 2006).

The upregulation of renal TNF- 𝛼 expression is correlated with urinary excretion in

glomerulonephritis diseases and proteinuria. Thus, TNF-𝛼 might implicate in glomerular

inflammation (Ernandez & Mayadas, 2009).

TNF-𝛼 is cytotoxic to renal epithelial cells, resulting in significant glomerular injury. In 24

diabetic patients with renal failure, the TNF- 𝛼 plasma concentrations were markedly

increased and correlated with urinary TNF- 𝛼 excretion. From these results it can be

suggested that this TNF-𝛼 plays a role in the pathogenesis of proteinuria and tubular renal

damage (Navarro et al., 1999).

TNF-𝛼 pro-inflammatory might be involved in renal injury because TNF-𝛼 deficient mice

were partially protected from glomerular injury (proteinuria) and using TNF-𝛼 blockade

treatment clearly offers protection against glomerular injury (Ernandez & Mayadas, 2009).

In conclusion, TNF-𝛼 has a strong correlation with the progression kidney injury.

43

1.9 Model for Proximal Tubular Epithelial Cells (PTECs):

In kidney, the PTECs are responsible for reabsorption of filtered solutes and excretion of

waste products. Some solutes, like phosphate and amino acids, are filtered in the

glomerulus and reabsorbed in the PTECs by active sodium-coupled transport. However,

other filtrates such as albumin and low-molecular-weight proteins like FLC are reabsorbed

by receptor-mediated endocytosis as described previously (See1.6.2). Currently, a variety

of primary and commercially cell line of human and animal PTECs sources are available

for researching purposes (Wilmer et al., 2010) such as investigating the role of PTECs in

different kidney failure diseases. Primary PTECs were isolated from either human or

animal kidney material like mouse and rabbit. However, the primary human renal proximal

tubule epithelial cells are limited use in clinical and basic research because of their limited

lifespan in culture (Kowolik et al., 2004). Many other reasons are limited the use of

primary PTECs in researches and applications will be discussed in chapter 4.

Cell lines, due to the immortalization process that generated them can be grown for

prolonged periods in vitro. For example, TH1 cells are derived from primary human renal

proximal tubule epithelial cells immortalized by two lentiviral vectors carrying the human

telomerase (hTERT) and the SV40 T antigen (Tag) to increase the number of cell divisions

and they were characterised and showed similar features to human primary PTECs, so they

potentially can be used in basic research, applications like drug toxicity screening and

tissue regeneration (Kowolik et al., 2004).

Also, SV40 immortalized human proximal tubule cell was used to study the effect of FLC

on the endocytosis process and the production of cytokines in proteinuric state (Sengul et

al., 2002). Another example, Opossum Kidney (OK) cells are human cortex proximal

tubule epithelial cell line dived from normal adult female, they were used in albumin

uptake (endocytosis process) in proteinuric nephropathies (Baines et al., 2012).

https://en.wikipedia.org/wiki/In_vitro

44

Human Kidney 2 (HK2) cell line also is one of the commercial PTECs that obtained from

normal adult renal cortex and transfected with recombinant HPV16 E6/E7 genes (figure

1.8). These commercial cell lines have been characterised in detail. However, few of them

were characterized in terms of transporter expression like HK2 cells, for example, in this

study they concluded that HK2 cells are of limited value as an in vitro model of drug

transporter expression (Jenkinson et al., 2012). Although, HK2 cell has been extensively

used in nephrotoxic studies as renal pathophysiological modal in vitro (Jenkinson et al.,

2012). Also, these cells were widely utilised in proteinuria studies in vitro. For example,

HK2 cells were used as a proteinuria vitro model system by stimulated with albumin to

evaluate the potential significance of different markers of tubular damage (Newman et al.,

2000). Also, they used as protein overload model by treated with albumin and IgG to

measure the cells H2O2 production (Morigi et al., 2002). In addition, previously HK2 cells

were used in megalin cytoplasmic tail phosphorylation by albumin as a source of human

PTECs, which is the main point that will be investigated in this project with FLC and

albumin (as positive control) proteins in proteinuria conditions. The advantages for using

HK2 cells as a source of human PTECs in the project will be discussed in chapter 4.

Figure (1.8): Light microscopic appearance of Human Kidney (HK2) cells.

10 X

45

1.9.1 Human Embryonic Kidney Cells 293 (HEK293):

The HEK293 cell line is one of the most cell lines that frequently use in cell biology. It

derived by transformation of primary cultures of human embryonic kidney cells with

sheared adenovirus 5 DNA (Kavsan et al., 2011). They are easy to grow in culture,

transfect, and they widely used as hosts for gene expression. HEK 293 cells have a very

complex karyotype, with two or more copies of each chromosome, including three copies

of the X chromosome (Lin et al., 2014).

For many years it was assumed that HEK 293 cells were generated by transformation of

either a fibroblastic, endothelial or epithelial cell that are all abundant in kidneys. HEK 293

cells were compared with human kidney, adrenal and central nervous tissue. The results

showed that HEK 293 closely resembled adrenal cells. So, likely embryonic adrenal cell

seems to be the most cell of origin of HEK 293 line. As a consequence, HEK 293 cells

should not be used as an in vitro model of typical kidney cells or kidney function (Shaw et

al., 2002). In addition, due to it is a transformed cell line; they cannot be used as model for

normal cells in an in vitro. For all of these reasons the HEK293 (embryonic origin) were

used in this project as appositive control in some of the experiments.

https://en.wikipedia.org/wiki/Adenovirus

https://en.wikipedia.org/wiki/DNA

https://en.wikipedia.org/wiki/Cell_culture

https://en.wikipedia.org/wiki/Karyotype

https://en.wikipedia.org/wiki/Fibroblast

https://en.wikipedia.org/wiki/Endothelial_cell

https://en.wikipedia.org/wiki/Epithelial_cell

46

1.10 Hypothesis:

The hypothesis of this thesis is that in kidney failure, monoclonal FLC (MM) is able to

phosphorylate Meg-CT and exert pro-inflammatory effects on PTCs damage.

1.11 Aims:

Proteinuria is a hallmark of nephropathy in many diseases such as diabetes, multiple

myeloma and nephrotoxins. The treatment for renal dysfunction depends on the cause and

the severity of the kidney’s condition. Untreated patients with acute or chronic kidney

failure might need kidney dialysis or in some cases they need renal replacement

transplantation (RRT).

To decrease the number of morbidity and mortality caused by renal failure, to give more

patients the chance to save their lives and to develop treatment strategies, we need to

understand the mechanisms of progressive renal damage.

Glomerular filtered proteins including albumin and free light chain in multiple myeloma

patients that are reabsorbed in PTECs via receptor-mediated endocytosis process like

megalin might have (a) direct toxic effect on cells by exceeded the capacity on PTCs to

reabsorb, (b) megalin could be more than an endocytic receptor for filtered protein,

stimulating signalling pathway and ending up with Meg-CT phosphorylation (c) production

of inflammatory cytokines, generating reactive oxidase species and activating the

complement system. All of these reasons might lead to or contribute to PTECs damage and

loss of renal function. Early diagnosis of renal dysfunction could help to save patients’

kidneys from additional damage.

47

The aims of this project:

To analyse a proximal tubular cell line for relevant characteristics.

To establish an in vitro model of protein overload using HK2 cells to study the

effect of protein overload.

To determine Meg-CT phosphorylation in stimulated HK2 (+/-GF) cells with FLC

in PPPSP motif.

To determine the response of HK2 (+/-GF) cells stimulated with FLC.

The focus of the thesis is mainly on the role of monoclonal light chains isolated from a

patient with multiple myeloma, and FAF-HSA is used as a positive control.

48

Chapter Two-Materials and Methods

49

2. General Methods

This chapter presents the materials and methods that were utilised for the experiments in

this project.

2.1 Tissue culture:

Two different types of kidney cells were used. The HK2 cell line (human kidney 2)

representing proximal tubular epithelial cells (PTECs) is derived from the normal

kidney of an adult male, and is human papillomavirus 16 (HPV- 16) transformed. It was

obtained from the American Type Culture Collection (CRL-190, Manassas, VA).

Human Embryonic Kidney 293 (HEK 293) cell line (figure 2.1) is often used for

transfection and was supplied by Dr C. Erridge (Cardiovascular Department, University

of Leicester). The cells were grown in areas approved for biohazardous work.

The standard culture medium utilised to grow (HK2 and HEK293 cells) was 500ml of

Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (Ham's) (1:1) (DMEM:

F12), L-Glutamine and 15mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid) (GIBCO, 31330) supplemented with 5ml penicillin (100 IU/ ml)/streptomycin

(100 μg/ ml) (Sigma, P4333) and 10% foetal calf serum (FCS).

However, the standard culture medium used to grow (HK2-GF) was 500ml of

Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (Ham's) (1:1) (DMEM:

F12), L-Glutamine and 15mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid)(GIBCO, 31330) supplemented with 5ml penicillin (100 IU/ ml)/streptomycin (100

μg/ ml) (Sigma, P4333), 10% foetal calf serum (FCS), 25 𝜇𝑙 recombinant human

epidermal growth factor (EGF) of (200μg/ml (Sigma E 9644)), 500 𝜇𝑙 of (Insulin-

Transferrin-Sodium selenite) (ITS) (Sigma I1884), 1𝜇𝑙 Triidothyronine (4pg/ml) (Sigma

T5516) and 180 𝜇𝑙 Hydrocortisone of (100μg/ml) (Sigma H 0135)).

Cells were grown to confluence in a humidified atmosphere with 5% carbon dioxide

(CO2), at 37C˚, and passaged using trypsin/EDTA (Sigma T4174). After between 6 and

10 days, spent medium was discarded from a 75cm2 flask (Nunclon), followed by

rinsing 5ml of 1x trypsin/EDTA (Sigma) diluted in phosphate buffer saline (PBS) for

50

5min in 37C˚, then the cells were transferred to a 50ml reaction tube with 10ml medium

and centrifuged for 5min at 200 x g. The supernatant was then discarded and fresh

medium was added to the cells, transferred to a vented cell culture flask and left to grow

in the incubator.

After culturing the cells (HK2, HEK293 and HK2-GF) for experiments for 24h to be

adherent, in the next day the supplemented standard medium was change to DMEM:F12

serum free media for 24h before experiments carried out to reduce the possibility of

carry offer the effects of FCS on cultured cells.

10 X

Figure (2.1): Light microscopic appearance of human embryonic kidney 293

(HEK 293) cells. They are polygonal and appear as flat, pavement-like epithelial

cells.

51

2.2 Scanning Electron Microscopy (SEM):

SEM was employed to visualise evidence of brush border characteristics of the cell

surface (villous protrusions). Two samples were inspected, namely HK2 and HK2-GF

cells, and were cultured to confluence on 12 well plates that had glass cover slips

(diameter 13mm).

The samples were rinsed twice in a fixation buffer for SEM (Hayat mammalian ringer,

Table 2.1) at 37C˚ to remove medium and then fixed in 3% glutaraldehyde in 0.1M

Sørensen's phosphate buffer (pH 7.2) for 2h in the fume hood at RT. The subsequent

procedure was carried out by Natalie Allcock of the Electron Microscopy Laboratory,

University of Leicester.

Cells were washed 3x in 0.1M Sørensen's phosphate buffer for 10min each. Then, they

were fixed in 1% osmium tetroxide/0.1M Sörensens phosphate buffer for 45min,

followed by washing 3x for 10min in double distilled water (DDW). Next, the

coverslips were transferred into the Critical Point Drier (CPD) holder in DDW and

placed in 30, 50 and 70% ethanol for 15min each and stored in fresh 70% ethanol at RT.

After that, they were placed in 90% ethanol for 30min and in 100% Analytical grade

ethanol 3x for 30min each. Then, they were transferred again into the CPD. Finally;

they were mounted onto aluminium stubs using carbon sticky tabs and sputter coated for

90sec 2.2 kilo Volts (kV) with Au (gold). Samples were viewed in the Hitachi S3000H

Scanning Electron Microscope with an accelerating voltage of 10kV.

52

2.3 Histology:

2.3.1 Immunohistochemistry staining protocol for endogenous Alkaline

Phosphatase:

Alkaline phosphatase is a hydrolase produced by the PTECs brush-border membrane (in

the cortex part of the kidney) (Terryn et al., 2007). In the following methodology

developed and done by Dr. Simon Byrne, kidney sections (5micron) from C57BC/6

mice were fixed in 10% of Neutral-buffered formalin (3.7% formaldehyde in PBS) for

5min. Then, the slides were washed 3x in tap water and placed briefly in 200mM

Tris/HCL buffer (pH 9-9.5) to raise the pH within the tissue. After that, the slides were

incubated with the substrate BCIP/NBT (5-Bromo-4-Chloro-3-indolyl phosphate/Nitro

blue tetrazolium) for 2min to detect the alkaline phosphatase in the cortex part. Next,

the substrate was washed off by tap water and the nuclear counterstain 1% Neutral red

was applied for 2min. Finally, the slides were washed in tap water, rinsed in dH2O,

dehydrated in 100% IMS, cleared in Xylene and cover slipped in DPX mountant.

Table (2.1): Mammalian Ringer Fixation for SEM solution (Provided

from Natalie Allcock of the Electron Microscopy Laboratory,

University of Leicester).

Solutions 1L dH2O

NaCl 10g

KCL 4.67g

CaCl * 6H2O 2.67g

NaHCO3 0.55g

Glucose 0.55g

MgCl2 * 6H2O 0.27g

53

2.3.2 Immunocytochemistry:

Immunocytochemistry (ICC) is a common technique that is used to detect the presence

of specific proteins in cells. The Alkaline Phosphatase Magenta Immunohistochemical

Substrate Solution Kit (Sigma AM0100-1KT) was chosen due to this substrate being

usable for histochemical and membrane-type to detect the alkaline phosphatase activity

in the HK2 cells and mouse PTECs (mPTECs preparation, see 2.3.3). The resulting

magenta precipitate at the site of alkaline phosphatase activity comes from the

combination of Gormori’s Tris AzoCoupling (TAC) buffer with naphthol after

hydrolysis by the alkaline phosphatase. The cells were sub cultured in 6 well plates to

confluence, and then the kit was used according to manufacturer’s instructions. Methyl

green was used as nuclear counter stain (Methyl green stain was prepared by Dr. Simon

Byrne).

2.3.3 Mouse PTECs preparation:

The C57BC/6 mouse was culled by cervical dislocation by experienced animal

technicians. The dead mouse was placed on its back on a clean surface in the tissue

culture cabinet. The mouse was sprayed with alcohol (70% IMS), then cut and opened

by sterile scissors and forceps. The first kidney was located by gently pushing the

stomach to the animal's right side by using the sterile forceps. The kidney appeared as a

dark red. It was held with the sterile forceps and cut free by the sterile scissors. They

were placed in a sterile glass petri dish containing HBSS.

The capsule was removed from the kidneys with a sterile scalpel blade and forceps. The

kidneys were moved to a new petri dish containing fresh HBSS, and small pieces of

cortex were sliced away from the medulla. Next, the pieces were collected in a tube

containing 20ml of HBSS; this step was repeated 3x for washing. Then, the pieces were

poured into 40µm sterile plastic sieves, and pushed and mashed with the plunger of a

1ml syringe with occasional HBSS washing. The tissues that were passed through were

the PTECs. The cells centrifuged for 5min at 200 x g, the supernatant was removed and

then a fresh 20ml of HBSS were added; this step was repeated 3x for washing. After

that the pellets were re-suspended with 9.5ml fresh HBSS and 0.5ml of prepared

54

collagenase type II solution (Enzyme) diluted in acetic acid (0.1M) (final concentration

is 0.05mg/ml). The tube was incubated for 2min at 37C˚. Then, 10ml of DMEM: F12

medium were added to the tube and centrifuged for 5min at 200 x g. Finally, the

supernatants were removed and 3ml from DMEM: F12 medium (the same medium was

used to culture HK2-GF cells) were added, re-suspended and placed in 25cm² flasks or

6 well plates depending on the experiment they were to be used for; they were incubated

in a 37C˚ incubator for four days or to be confluent (Terryn et al., 2007).

2.4 MTT assay:

Thiazolyl Blue Tetrazolium Bromide or Methylthiazolyldiphenyl-tetrazolium bromide

(MTT) is a colorimetric assay measuring the cellular metabolic activity of cells; the

yellow substance is reduced to an insoluble and coloured (purple) formazan product by

mitochondria (Zhu et al., 2012). The cells are then solubilised with an organic solvent

Dimethyl Sulfoxide (DMSO) (Fisher Chemicals) and the released solubilised formazan

reagent is measured spectrophotometrically.

The cells were sub cultured in 96 well plates, the density depending on the experiment,

and treated with proteins or controls (un-treated cells) for different time points in

triplicate. After each time point 20μl of (5mg/ml) MTT solution (Sigma M5655)

dissolved in PBS (w/v) was added to each well and incubated in 5% CO2, 37C˚. The

MTT solution was discarded after 4h incubation and 200μl of DMSO were added for

10min to let the precipitate dissolve. The absorbance of the violet solution was

measured at 550nm by spectrophotometer (Thermo Scientific, MULTISKAN FC), (Zhu

et al., 2012).

2.5 LDH assay:

Lactate dehydrogenase (LDH) is a photometrical assay that detects the release of

cytoplasmic LDH that occurs when cells are injured and the membrane integrity is

compromised (Kendig & Tarloff, 2007). The LDH release is frequently used as an end-

point for cytotoxicity and a marker for cell death or damage (Kendig & Tarloff, 2007).

LDH activity was measured using a commercial assay (CytoTox-ONE Non Radio,

55

Cytotoxicity Assay, Promega G1781 and G1782). First, the LDH released in

supernatant could be measured by the two steps of enzymatic reaction. The LDH

catalysed the transformation of lactate to pyruvate by reduction of NAD+ to NADH.

Then, diaphorase used NADH to reduce a tetrazolium salt (INT) to a red formazan

product (figure 2.2). Thus, the formazan amount directly reflects the released LDH

amount in the supernatant. After sub culturing and incubating cells containing protein or

controls for different time points, 50μl of supernatant from each sample was transferred

in triplicate to a new 96 well plates. Fresh media was also loaded into the plate as a

control for background absorbance. The substrate mix was prepared and 50μl added to

each well; the plate was then covered with foil to protect it from light and incubated for

30min at RT according to manufacturer instructions. Stop solution was added, and

absorbance measured at 490nm using a plate reader (Thermo Scientific, MULTISKAN

FC).

Figure (2.2): Lactate Dehydrogenase (LDH) detection mechanism (Chemical

reaction).

Damaged

Cell

Lactate

Dehydrogenase

(LDH)

Lactate Pyruvate

NAD+ NADH

Tetrazolium salt

(INT)

Formazan

56

2.6 Crystal Violet assay:

Crystal violet (CV) or Gentian violet is a triphenylmethane dye also known as

hexamethyl pararosaniline chloride. CV is a simple assay originally used to quantify

cell viability by measuring the absorbance of the dye that is taken up by live cells. This

dye stains the cell’s DNA (Vega-Avila & Pugsley, 2011)

The cells were seeded in a 96 well plates and incubated for different time periods (24,

48, 72 and 96h); the supernatants were discarded and the cells were stained with 0.5%

filtered crystal violet 50μl/well, which dissolved in 20% (v/v) methanol in H2O, for 3-

4min at RT. Then, the dye was removed and the plate was washed 3x under the tap and

dried by inverting the plat to remove excess water and blotted on a paper towel. Finally,

100μl of 20% (v/v) acetic acid in H2O was added in each well to solubilize the

accumulated dye in the cell’s nucleus and absorbance measured at 540nm using a plate

reader (Thermo Scientific, MULTISKAN FC), (Protocol from Lab 211B).

2.7 Reverse Transcriptase Polymerase Chain Reaction (RT-

PCR):

2.7.1 Preparation of Ribonucleic Acid (RNA):

Total RNA was extracted by Trizol Reagent (Ambion) following the manufacturer’s

instruction. 1ml of Tri-reagent was added for each sample to lyse the cells. The lysed

cells were transferred to 1.5ml fresh reaction tubes to prepare the total RNA. The

samples were incubated for 5-15min at RT after adding 100μL of chloroform to each

sample and centrifuged at 12,000 x g for 15min. Three layers were formed in the tubes,

however, just the aqueous phase, which is the top layer, was collected in a new reaction

tube. Then, 500μL of Isopropanol was added to each sample and shaken for 5sec

followed by incubating for 5-10min at RT to precipitate the total RNA, and then

centrifuged at 12,000 x g for 8min; the supernatants were then discarded. Then, 1ml of

75% (v/v) ethanol in H2O was added as a washing step and centrifuged at 7,500 x g for

5min. Finally, the ethanol was removed and the RNA pellets left to dry. The RNA

pellets were re-suspended in 10-20μL of 0.02% (v/v) Diethylpyrocarbonate (DEPC)

57

(Sigma D-5758) treated water. The Nanodrop machine (Thermo Scientific) was used to

measure the amount of the total RNA in ng/μl in each sample. 2μg of the total RNA was

used for cDNA synthesis.

2.7.2 Preparation of Complementary Deoxyribonucleic Acid (cDNA):

From the total RNA, first strand cDNA was synthesised by using the Thermo Scientific

RevertAid H Minus First Strand cDNA Synthesis Kit (K1639) following the

manufacturer’s instructions. 2μg of the total RNA with 2μL of random hexamer primer

and water (nuclease-free) were added up to a final volume of 12μl per one reaction tube

(must be in ice). The samples were incubated for 5min at 65 C˚ to denature the total

RNA, after which they were chilled on ice. Then, 4μl of 5xReaction buffer, 1μl of

RiboLock RNase inhibitor (20U/μl), 2μl of dNTP Mix (10mM) and 1μL of RevertAid

H Minus M-MuL Revers Transcripase (200U/μL) was added for a final volume of 20μl

for each reaction. Afterward, the sample tubes were incubated at 25C˚ for 5min

followed by 60min at 42C˚, and the reaction was stopped by heating at 70C˚ for 5min in

the PCR machine (TECHNE, TC-3000).

2.7.3 PCR Protocol:

PCR was used for amplification of many target genes. The reaction was performed by

mixing 10x Reaction Buffer (2.5μl) (Thermo), MgCl2 (25mM) (1.5μl), dNTPs

(1.25mM) (4μl), Thermoprime Enzyme (5U/μl) (0.2μl), PCR water (10.8μl), forward

primer (5μM) (2μl), reverse Primer (5μM) (2μl) and from cDNA template (2μl) in 0.2μl

Eppendorf tubes. Cyclic amplification was performed in the (TECHNE, TC-512) PCR

machine. The program started with 95C˚ for 2min. Next, the reaction was cycled up to

30 times including denaturation (94C˚ for 1min), annealing (53-60C˚, depending on the

primer sequences, for 1min), and elongation (72C˚ for 1min, depending on the expected

product size) steps. Finally, the temperature was kept at 72C˚ for 10min for final

extension and then the reaction stopped and stabilized at 4C˚.

58

An Example for Program of the Amplification in (TECHNE, TC-512) PCR machine:

94C˚ 2min x1 cycle Initial denaturation

94C˚ 1min Denaturation

55C˚ 1min x30 cycles Annealing

68C˚ 1min Extension /Elongation

68C˚ 10min x1 cycle Final Elongation

4C˚ Hold

To analyse the RT-PCR samples, 2μl of loading dye was added (10x DNA gel loading

dye, 15% (w/v) BP blue, 50% (v/v) glycerol and 0.5mM EDTA) to the product and 10μl

loaded in each well of a 1% (w/v) agarose/1x TBE buffer gel (the electrophoresis step

for 60min at 90V in 1X TBE buffer) (Table 2.2). 1% (w/v) agarose was prepared by

mixing 1g of agarose and 100ml of 1xTBE (Tris base, boric acid, EDTA), heating in a

microwave for 3min, and letting the mixture cool to 60C˚. 20μl of Ethidiume Bromide

(0.5mg/ml) (Sigma) was added to make the samples visible under the Ultra Violet light.

Finally, the molten agarose was poured in the gel-assembled tray with comb.

5X TBE

54g Tris Base (Sigma)

27.5g Boric Acid (FISONS)

20ml Ethylenediaminetetraacetic acid

(EDTA) (0.5M), pH8.0

Table (2.2): Preparation of 5 x Tris/Borate/EDTA solution for electrophoresis.

59

In addition, 2μl of the DNA Ladder was run with the samples (Invitrogen by life

technologiesTM, TrackIt™ 1 Kb Plus DNA Ladder, 10488-085) (figure 2.3). Finally, the

gel was visualised using Imagequant 100 (UV trans-illuminator) and the picture of the

gel was taken by the DC120 digital camera.

2.7.4 Human Primers

Gene Primer sequence/

References

Annealing

(Tm)

Size (NCBI)

β-actin

5`CACCAACTGGGACGACAT-3`

5`ACAGCCTGGATAGCAACG-3`

(Izaguirre-Carbonell et al., 2015)

55C˚

~160bp

X00351.1

C3

5`GCTGAAGCACCTCATTGTGA-3`

5`CTGGGTGTACCCCTTCTTGA-3`

(Wan et al., 2007)

58C˚

~168bp

NM_000064

2000

1650

1000

850

650

500

400

300

200

100

bp

Figure (2.3): 1 Kb Plus DNA Ladder in 1% agarose gel electrophoresis. Semi-

quantitative RT-PCR for mRNA 𝛽-actin gene expression from HK2 cells. 𝛽-actin

(~ 200 bp).

60

FH

5`GGAACCACCTCAATGCAAAG-3`

5ÀAGCTTCTGTTTGGCTGTCC-3`

(Mandal & Ayyagari, 2006)

55C˚

~276bp

Y00716.1

FB

5`GTTGAAGTCAGGGACTAACACC-3`

5`CCACAGTGAAACAATGTGC-3`

(Peake et al., 1999)

60C˚

~548bp

AF349679.1

IL-6

5`TCCTGCAGAAAAAGGCAAAG-3`

5`GCCCAGTGGACAGGTTTCT-3`

(Villeneuve et al., 2008)

60C˚

~250bp

NM_000600

IL-8

5ÀCTGAGAGTGATTGAGAGTGGAC-3`

5ÀACCCTCTGCACCCAGTTTTC-3`

(Mehdad et al., 2016)

58C˚

~111bp

BC013615.1

MCP-1

5`TGCGCAGAATGAGATGAGTTG-3`

5`GTGAGGAACAAGCCAGAGCTG-3`

(Villeneuve et al., 2008)

55C˚

~177bp

S71513

TGF-β

5`CCCAGCATCTGCAAAGCTC-3`

5`GTCAATGTACAGCTGCCGCA-3`

(Kwon et al., 2012)

60C˚

~157bp

NM_000660

TNF-α

5`CTTCTCCTTCCTGATCGTGG-3`

5`GCTGGTTATCTCTCAGCTCCA-3`

(Lab 211B)

55C˚

~78bp

NM_000594.

2

KIM-1

5`CTGCAGGGAGCAATAAGGAG-3`

5ÀCCCAAAAGAGCAAGAAGCA-3`

(Lab 211B)

54C˚

~210bp

NM_001302

1J6

61

2.8 Real-Time Quantitative polymerase chain reaction (RT-

qPCR):

To quantify many gene expressions in different samples, the RT-qPCR technique using

SYBR Green I dye (SensiMixTM SYBR Kit, Cat.QT605) was used, which is a high-

performance reagent designed for superior sensitivity and specificity on various real-

time instruments. The master mix provided contains all the components for real-time

PCR including the SYBR Green I dye. The cDNA samples synthesised from extracted

total RNA from each sample was used as templates in RT-qPCR. The RT-qPCR

reactions started with diluting each cDNA sample 1:4 in autoclaved nanopure H2O.

Then, the RT-qPCR samples were prepared as shown in (Table 2.3). Additionally,

negative control (non-template control) prepared for each gene by utilising autoclaved

nanopure H2Oinstead of the cDNA template. Afterward, the qPCR samples were run on

the Corbett: Rotor-GeneTM 6000 machines and software (Table 2.4). Each sample was

prepared in duplicate and strip tubes and (0.1 ml caps) were used (QIAGEN, 981103).

Caspase-3

5`GCTTGTCGGCATACTGTTTCAG-3`

5`AGAACTGGACTGTGGCATTGAG-3`

(Ye et al., 2012)

60C˚

~190bp

NM_004346

Megalin

5`TAAGTCAGTGCCCAACCTTT-3`

5`GCGGTTGTTCCTGGAG-3`

(Tsaroucha et al., 2008)

53-58C˚

~300bp

NM_004525

Properdin

5`GGCACGGGTAGGATTAGGTCCACA-3`

5`GCATCCAGCACTGCCCCTTGAAA-3`

(Nagamachi et al., 2014)

55C˚

___

NM_002621

CD36

5`GTCGCAGTGACTTTCCCAAT-3`

5`ATGTAAACCCAGGACGCTGAG-3`

(Lab 211B)

55C˚

___

L06850

62

Reagent Volume

2x SensiMixTM SYBR 10μl

5μM Forward primer 2μl

5μM Reverse primer 2μl

Autoclaved Nanopure H2O 3μl

cDNA template 3μl

Total volume 20μl/ Reaction

Table (2.3): Preparation of RT-qPCR reaction.

Cycles Temperature Time

1

95 C˚

10 min

40

95 C˚

55-60 C˚

72 C˚

15 sec

15 sec

15 sec

Table (2.4): The temperature cycling conditions of qRT-PCR.

63

The amplification of each sample is tracked in real time and the machine calculates the

cycle threshold (CT) value for each sample. In RT-qPCR, a positive reaction is detected

by accumulation of a fluorescent signal. The CT is defined as the number of cycles

required for the fluorescent signal to cross the threshold. The relative position of the

crossing of this CT line with the threshold against the cycle numbers (on the x-axis)

gives an indication of the abundance of gene expression. So, when CT < 29, that means

abundant amount of the target gene; CT between 30-37 means indicative of moderate

amounts of target, however, if CT is 38-40 are likely to be negligible amounts of target

and that the amplification may have occurred because of some environmental

contamination. The threshold of each target gene product was set manually and CT

under the threshold through all 40 cycles means a negative result, or the expression of

the target gene is very low or an undetectable (figure 2.4 A).

The melting curve program consisted of temperatures between 55C˚ to 99C˚. The

melting curve results were used as evidence that only one product was amplified and

there were no contaminants or primer dimer products. The light cycler software plotted

the single peak (Tm) at around an 80-90C˚ (figure 2.4 C).

2.8.1 RT-qPCR analysis and calculation:

The RT-qPCR was chosen as the best approach of RT-qPCR for gene expression studies

to compare changes in gene expression. In relative RT-qPCR, the quantification of the

target gene normalised against a reference gene.

The ∆CT method was used to calculate the relative expression target gene in each

sample (Livak & Schmittgen, 2001):

2^-∆∆CT

∆∆CT =∆CT (test)-∆CT (calibrator)

∆CT (test) = CT (target gene) - CT (ref gene)

∆CT (calibrator) = CT (target gene) - CT (ref gene)

CT: Cycle number at which detectable signal is achieved.

64

Calibrator: The control sample, meaning an un-treated sample.

Test: Test sample means treated.

Reference (ref): The reference gene is one that expressed at a constant level in all test

and control samples without being affected by the experiment treatment in the study.

2.8.2 Primer Efficiency:

This experiment was done to determine the efficiency of the primer that was used in

RT-qPCR experiments by running a standard curve. The slope of the standard curve (Ct

vs. cDNA Concentration) is related to the efficiency, and should be close to 0.99.

The standard curve is formed by using a serial dilution of cDNA (template) prepared

from cells that are going to be examined or are known to express the examined gene.

Ideally, the efficiency curve should be determined for samples that are going to be

examined. Due to the cDNA concentration being unknown, a 1:10 dilution was used to

create 1, 0.1, 0.001, 0.0001, and 0.00001 cDNA concentrations in dH2O. Then each

concentration was used as a sample and different genes were tested by RT-qPCR as

described in (2.8) (figure 2.2 B). (Most of the primer efficiencies were tested by Lab

211b and available for this project).

Threshold Line

CT

Amplification

NTC

65

Figure (2.4): Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) for

β-actin (Housekeeping gene) expression in HK2 cells. (A) Shows the

amplification plot of β-actin serial dilution of cDNA (1:10). (B) The standard

curve of β-actin (efficiency = 0.96). (C) Shows the melting curves analysis for β-

actin to determine specificity of the products. (NTC: Non-template control or

negative control) (Tm: Melting temperature).

cDNA

Slope

Tm

66

2.9 Protein Immunoblot (Western blot):

This technique is used to detect specific protein expression from samples. After sub

culturing cells in 6 well plates to 80% confluence, they were stimulated with protein for

the experiment. The supernatants were discarded and the cells washed 2x with PBS to

remove any remaining exogenously added protein.

2.9.1 Preparation of cell lysate:

200-500μl of lysis buffer (Table 2.5) was added in each well; the cells were then

scraped and transferred to fresh eppendorf tubes and the samples were incubated for

30min on ice. They were centrifuged at 14,000 x g for 5min and the supernatants were

transferred into new fresh reaction tubes.

2.9.2 Protein Assay:

The protein concentration of the lysates was determined by Pierce 660nm Protein assay

(Thermo Scientific prod (22660)) according to the manufacturer’s protocol.

First, a range of bovine serum albumin (BSA) (PAA, K45-001) concentrations (as a

source of protein) were prepared for the standard curve (2000, 1500, 1000, 750, 500,

250, 125, 50, 25 and 0 μg/ml) by dissolving in PBS. 10μl from each concentration were

added in each well of a 96 well plate in triplicate, and then 150μl of the protein assay

reagent were added for each well and incubated at the RT for 5min (the colour changed

from yellow to blue proportional to the concentration of the protein). After the

incubation time the plate was read spectrophotometrically (Thermo Scientific,

MULTISKAN FC) at 650nm (figure 2.5).

67

2.9.3 Western Blot Protocol:

With this information, the protein samples were prepared for SDS-PAGE analysis by

mixing 20μg from each sample with 2xloading buffer (Table 2.6) 1:2 dilution into new

reaction tubes and heated at 95C˚ for 5min to denature the protein to the primary

structure for protein, by breaking down the disulphide bonds. Then the samples were

centrifuged in 16,000 x g for 5sec to remove insoluble debris and the supernatants were

used. After that the protein samples were run in 12% SDS-PAGE gel with a 5μl protein

marker (figure 2.6) in 1X running buffer 60mA for 1-1.5h (Table 2.6).

The separated proteins on the gel were electrophoretically transferred to a methanol-

activated (submerge for 30sec) PVDF membrane (GF Heath care Life Science,

AmershamTmHybondTm 0.2μmPVDF, 1060006), using 1xblotting buffer (transfer

buffer). After blotting at 250mA for 1h, the membrane was washed quickly in PBS and

blocked with 20ml of 5% dried skimmed milk in PBS (w/v) for 2h on the shaker at RT

or overnight at 4C˚. Then the membrane was washed in PBS-0.05% Tween (v/v) 3x for

10min each with shaking. After that, the membrane was probed with the primary

antibody (Table 2.7) diluted in 5% skimmed milk in PBS (w/v) overnight at 4C˚,

washed as above and probed with a second antibody (Table 2.8) diluted in 5% skimmed

milk in PBS (w/v) for 2h maximum at RT with shaking. Finally, the membrane was

washed as described and the face of the membrane exposed to Enhanced-chemi

luminescence (ECL) (Pierce™ ECL Western Blotting Substrate, 32106) reagents

consisting of 1ml reagent A and 1ml reagent B, wrapped in cling film, and exposed to

film (Bio-Max Light, Sigma) in a light-tight cassette. Then the film was submerged in

Figure (2.5): Example for the standard curve showing the absorbance of different

concentrations of BSA by Pierce 660nm Protein assay.

68

developing solution for 2min until antibody-reactive bands appeared, then were washed

in water, then submerged in a Fixer solution, washed in water again and then the film

was left to dry.

Solutions μl

1M pH7.5 β-glycerophosphate (w/v) in H2O

(Sigma G-5422)

100

0.5m pH 8.0 Ethylenedi-aminetetra-acetic acid

(EDTA) (w/v) in H2O (ALDRICH)

20

40mM pH 7.5 Ethylene glycol tetra acetic acid

(EGTA) (w/v) in H2O

250

1M pH7.5 Tris-HCL (w/v) in H2O

500

100mM Na Orthovanadate (ALDRICH, 450243)

100

1M Benzamidine (w/v) in H2O

10

100mM Phenylmethanesulfonylfluoride (PMSF)

(w/v) in Ethanol

20

http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web&cd=1&cad=rja&uact=8&sqi=2&ved=0CCEQFjAA&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FEthylenediaminetetraacetic_acid&ei=kWjSVO77CcbD7gbk_YCABg&usg=AFQjCNFpeOGTYpsUVrqBdb_6xsXoqNh15g&bvm=bv.85142067,bs.1,d.bGQ

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69

β-Mercaptoethanol (Sigma M-7154)

10

10% Triton X-100(v/v) in H2O (BDH, 30632)

1000

500mM Na Fluoride (w/v) in H2O

1000

Protease Inhibitors (Sigma P8340)

100

Autoclaved H2O

6970

Table (2.5): Preparation of 10 ml Lysis Buffer (should be in ice), (Source: Dr. Alan

Bevington, University of Leicester).

70

Solutions Preparation

2x Loading Buffer

4ml dH2O, (1ml) 0.5M pH 6.8 Tris-HCL, 800μl

Glycerol, 1.6ml 10% sodium dodecyl sulfate

(SDS) (Fisher) (w/v) in H2O, Bromophenol

blue and 200mMDithiothreitol (DTT) (Sigma)

SDS-PAGE gel

12% Resolving Buffer: H2O, 30%

Acrylamide/Bis solution, 1.5M pH 8.8 Tris,

10% SDS, 10% Ammonium persulfate (APS)

(Sigma A-3678) and 10𝜇𝑙TEMED (Sigma T-

9281).

5% Stacking Buffer: H2O, 30% Acrylamide/Bis

solution, 1M pH 6.8 Tris, 10%SDS, 10% APS

and 6𝜇𝑙 TEMED.

10 x Running buffer

30g Tris-Base, 144g Glycine (Fisher) and 10g

SDS in 1L dH2O (for 1x Running buffer diluted

1:10 in dH2O).

1 x Blotting Buffer

5.9g Tris-Base, 2.9g Glycine, 100ml Methanol

and 3.4ml 10%SDS and 1L dH2O.

Table (2.6): Preparation of buffers for Western blot.

71

Primary Antibodies Dilution Company

Rabbit polyclonal-anti Villin-1 1:1000 Cell Signalling

(R814)

Mouse monoclonal-anti Caspase-3 1:1000 Santa Cruse

(sc-56053)

Goat polyclonal-anti Megalin 1:1000 Santa Cruse

(sc-16476)

Mouse monoclonal-anti C3 1:1000 Abcam (ab11874)

Goat polyclonal-anti FB 1:1000 Santa Cruse

(sc-34888)

Rabbit polyclonal- anti FH 1:500 Santa Cruse

(sc-33156)

Goat polyclonal-anti Properdin 1:500 Santa Cruse

(sc-67794)

Mouse monoclonal-anti TFF3 1:500 Biorbyt (orb243923)

Mouse monoclonal-anti 𝜷-actin 1:2000 Sigma (A5316)

Table (2.7): Primary antibodies.

(Conjugated HRP) Dilution Company

Swine anti-rabbit 1:2000 Dako (K5007)

Goat anti -mouse 1:2000 1 Dako (P0447)

Donkey anti -goat 1:2000 Santacruz (sc-2056)

Table (2.8): Secondary antibodies.

72

Dual Color (Catalog #161-0374) All Blue (Catalog #161-0373)

2.9.4 Preparing the Maxi Gradient Gel for the Megalin Western blot:

This type of gel is normally used to separate high molecular weight proteins like

megalin (~ 600 kDa). 3 gels are prepared separately with different concentrations (4, 15

and 4 %) (Table 2.9). After the 3 solutions were prepared, the 4% (Low concentration

(L)) and the 15% (High concentration (H)) were poured in the gradient gel maker, and

then a magnetic stirrer was used for mixing. Then, the power supply (Pharmacia LKB-

PUMP) was opened to encourage the (L) solution to move to the (H) in the gradient gel

maker, and the gradient gel maker should be on stir to let the magnetic stirrer to mix the

two concentrations together before transferring to the gel caster. Next, the gel was left

to dry, and the third solution 4% (stack) was poured. After the gel had polymerised, the

comb was taken out and the caster was put in the tank. The protein samples were

denatured as described previously and loaded. The marker was pre-stained with a

protein ladder, broad range 10-230KDa (Biolabs P7710S) and Hi Mark High Molecular

Figure (2.6): Protein marker (Precision Plus ProteinTM Standards, BIO RAD

(10-250 kDa)).

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73

Weight protein standard (novex), then a running buffer (1x SDS) was poured in the tank

before running the gel in 300V/ 60-56 AM for 5h, then the normal immunoblot steps

were followed as described in (2.9.3).

Solution 4% 15% 4%

(Stack)

10% SDS (Sodium dodecyl sulphate) (Sigma L-

4509 nano pure)

250µl 250µl 100µl

Plus one Acrylamide PAGE (40%) 2.5ml 9.375ml 1ml

Resolving Buffer (1.5M Tris/HCL pH8.8) 6.25ml 6.25ml -

Stacking Buffer (1M Tris/HCL pH6.8) - - 2.5ml

Glycerol - 6.25µl -

dH2O 16ml 8.5ml 6.4ml

TEMED (Tetramethylethylenediamine) (Sigma) 25 µl 25 µl 12 µl

10% (w/v) APS (Ammonium persulphate) to be

added just before the gel is cast (initiates

polymerisation).

80µl 80µl 40µl

Table (2.9): Preparation for Gradient Gel.

74

2.9.5 Preparing the 4% Gel for the Megalin Western blot:

The same immunoblot technique was used as described in (2.9.3) but the gel preparation

was different; only a 4% resolving gel was used (Table 2.10).

2.10 Ultra structural analysis by transmission electron

microscopy (TEM):

A number of recent studies demonstrated that the induction of autophagy serves a

crucial role in protecting renal proximal tubular cells from many stresses (Inoki, 2013).

Based on this observation, the project analysed the presence of autophagic vacuoles in

HK2 (+/-GF) cells exposed to FAF-HSA and 𝜆-LC by TEM.

The HK2 (+/-GF) cells were sub cultured at (1x106 cells/well) density in a 6 well plates

for 24h to be adherent. The next day, the medium was harvested and the cells were

exposed to (5mg/ml) of FAF-HSA and 𝜆-LC in a serum free medium for 24 and 72h in

the incubator at 37C˚, 5% CO2. After each time point, the cells were washed twice with

PBS and trypsinized for 2min at 37C˚, collected with 2ml of serum free medium and

centrifuged for 5min at 200 x g. The supernatants were discarded and each pellet was

primary fixed in 2.5% glutaraldehyde in 0.1M Sørensen's phosphate buffer pH 7.3 for

24h.

The pellets from the centrifuged samples at 16,000 rpm were then washed in de-ionised

water 3x for 10min and 1% aqueous Osmium Tetroxide / 1.5% Potassium Ferricyanide

was added to each sample for post fixation for 1h. The samples were washed as before

4% Resolving Buffer ml

H2O 8.9

40% Acrylamide/Bis solution 2

Resolving buffer 3.8

20% SDS 0.15

10% Ammonium sulphate 0.15

TEMED 0.006

Table (2.10): Preparation of 4% gel.

75

and stained with 1% aqueous uranyl acetate for 1h at 4C˚, then the samples were

washed again as described previously. Next, the samples were embedded in Spurr’s

modified resin.

After polymerisation at 60C˚ for 16h, the embedded samples were sectioned using a

Reichert Ultracut E ultramicrotome. Ultrathin sections of ~ 80nm thickness were cut

from each sample and collected onto copper mesh grids. The sections were

counterstained for 2min in Reynold’s Lead citrate. The samples were viewed on the

JEOL 1400 TEM with an accelerating voltage of 80kV. The images were captured

using Megaview III digital camera with iTEM software. The subsequent procedure was

carried out by Natalie Allcock of the Electron Microscopy Laboratory, University of

Leicester.

2.11 Detection of Apoptosis:

Apoptosis, programmed cell death, can occur when disease or toxic agents damage

cells. Because albumin-induced apoptosis has been described in HK2 cells (Erkan et al.,

2007), this project analysed HK2 (+/-GF) cells after 24 and 72h protein stimulation

occurred using ApopTag® In Situ Apoptosis Detection Kits (Millipore, Cat no. S7100).

This method labels apoptotic cells by modifying fragmented DNA with free 3’-OH

groups using terminal deoxynucleotidyl transferase (TdT).

HK2 (+/-GF) cells (1x 106 cells/well) were cultured in two separate 6 well plates with

autoclaved glass cover slips (22x22mm) (ACADEMY, Cat no. 1200-03-14) for 24h to

be adherent. The following day the medium was replaced with a serum-free medium

with 𝜆-LC or FAF-HSA (5mg/ml) for 24 and 72h. After each time period the medium

was harvested and the wells were washed 2x with 2ml of PBS, and fixed with 1%

Paraformaldehyde (Agar, Cat no. R1018) in PBS (w/v) pH 7.4 for 10min at RT (Table

2.11). The wells were washed in two changes of PBS for 5min each. Then, the samples

were post-fixed for 5min in pre-cold Ethanol: Acetic acid (2:1) at -20C˚, then the cells

were washed as described previously. Next, freshly prepared 3.0% hydrogen peroxide

(Sigma, Cat no. 216763) in PBS (v/v) was added to the wells for 5min at RT and

washed. Immediately, 75μL of equilibration buffer was added to each well and

76

incubated for at least 10sec at RT. Subsequently, the equilibration buffer was discarded

and 55μL working strength TdT enzyme was added to each well for 1h and incubated at

37C˚ in the incubator. After 1h the working strength stop/wash buffer was added to the

samples at RT. Next, the samples were washed in 3 changes of PBS for 1min each and

65μL of anti-digoxigenin was added to each well and incubated at 37C˚ for 30min. The

samples were washed in 4 changes of PBS for 2min per wash at RT. Then, 75μL of

0.05% DAB (3, 3’-diaminobenzidine) (Sigma, Cat no. D-5637) (working strength

peroxidase substrate) in PBS (w/v) was added to each sample for 6min at RT, and

washed 3x with dH2O for 1min each.

Finally, the cells were stained with 0.5% Methyl green (Counterstain) for 10min at RT

(Table 2.11) and washed in 3 changes of dH2O, dehydrated in 100% isopropanol and

mounted in Xylene. Gently, each coverslip was removed from each well and placed

upside down on a slide prepared with one drop of D.P.X mountant (BDH, Cat no.

36029) and left to dry. The slides were examined under the light microscope.

Methyl Green

Acetate Buffer (0.133M), pH 4.0.

The 2% of methyl green was prepared

in dH2O (w/v) (contaminated with

crystal violet), and then washed with

chloroform to extract crystal violet.

The 2% methyl green was diluted with

acetate buffer 1:4 for 0.5% final

concentration.

1% Paraformaldehyde

100 ml dH2O boiled with 4μl of

10M NaOH

Then, in the fume hood 1g

paraformaldehyde was added and

shaken carefully.

Finally, 1 PBS tablet was added.

Table (2.11): Preparation of Paraformaldehyde and Methyl green

(They were prepared by Dr.Simon Byrne).

77

2.12 Quantitative measurement by ELISA:

2.12.1 Mini Elisa for candidate cytokines and chemokine from HK2

(+/-GF) cells:

Interleukin 6 (IL-6), Interleukin 8 (IL-8) and Monocyte Chemo-attractant Protein-1

(MCP-1) are important mediators released from epithelial cells (Stadnyk, 1994).

ELISA was used to quantitatively measure these inflammatory products in the sample

supernatant (Human IL-6, IL-8 and MCP-1 Mini ELISA Development Kit, 900-M16,

Peprotech, 900-M18 and 900-M31, respectively), the manufacturer’s protocol was

followed.

ELISA 96 well microplates (Nunc Maxisorp) were used. Firstly, the plates were coated

with capture antibodies that were diluted with PBS to a concentration of (1μg/ml for IL-

6, 0.5μg/ml for IL-8 and 0.25μg/ml for MCP-1) and immediately 100μl were added to

each well. The plates were sealed and incubated overnight at RT. On the next day, the

plates were aspirated to remove liquid and washed 4x with washing buffer (0.05% (v/v)

Tween-20 in PBS) 300μl/well. After the last wash the plates were inverted to remove

the remaining buffer and blotted on paper towels. Then, they were blocked with

300μl/well with 1% (w/v) BSA in PBS and incubated for 2h at RT; afterward the plates

were aspirated and washed 4x as described.

The IL-6, IL-8 and MCP-1 standards (figure 2.7) were diluted starting from (1.5 for IL-

6 and 1 for IL-8 and MCP-1 ng/ml to zero) in diluent (0.05% (v/v) Tween-20, 0.1%

BSA in PBS). Next, 100μl of standards and samples (supernatants) were added to each

well in triplicate and incubated at RT for at least 2h. The plates were aspirated and

washed 4 times. Subsequently, 100μl/well was added from detection antibodies that

were diluted to a concentration of 0.5μg/ml for all three ELISAs, and incubated for 2h

at RT. After 2h the plates were aspirated and washed 4x as described. Afterwards,

100μl/well from 5.5μl of Avidin-HRP conjugate, which was diluted 1:2000 for a total

volume of 11ml, was added and incubated for 30min at RT. Finally, the plates were

aspirated, washed 4x and 100μl/well of ABTS substrate solution was added to each well

(1-STEPTMABTS, Thermo scientific Cat No.37615), then incubated for 30min

maximum at RT for colour development. The colour development was monitored using

http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web&cd=1&cad=rja&uact=8&sqi=2&ved=0CCEQFjAA&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2755091%2F&ei=UozbVOG9De2t7Aa_l4GQBQ&usg=AFQjCNH9rL5TWl8mnrERGPOHiaDBGOJoSA&bvm=bv.85761416,d.d2s

78

a plate reader (infinite f50, TECAN) at 405nm with wavelength correction set at 650nm.

The sensitivity of the ELISAs kits is less than 1.5ng.

2.12.2 Human TFF3 Immunoassay:

Trefoil Factor 3 (TFF3) release in supernatants from HK2 (+/-GF) cells stimulated with

FAF-HSA and 𝜆-LC (5mg/ml) for 24 and 72h was measured by TFF3 immunoassay

(QuantikineELISA, Human TFF3 Immunoassay (Cat no. DTFF30)) (done by Dr. Zina

Zwaini, Lab 211B, University of Leicester).

This is a quantitative sandwich enzyme immunoassay technique. Human TFF3

monoclonal antibody has been pre-coated onto a micro plate. At the beginning 50μL of

standards and samples, which were prepared as descried in the kit, were pipetted into

the wells in triplicate and incubated for 2h on shaker at RT. After 2h the plate was

washed 4x with 400μL of washing buffer (provided with the kit). Then 200μL of human

0 5 0 0 1 0 0 0 1 5 0 0

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

H u m a n I L 6 P g /m l

OD

(4

05

nm

)

0 5 0 0 1 0 0 0 1 5 0 0

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

H u m a n M C P -1 P g /m l

OD

(4

05

nm

)

0 5 0 0 1 0 0 0 1 5 0 0

0 .0

0 .2

0 .4

0 .6

0 .8

H u m a n I L 8 P g /m l

OD

(4

05

nm

)

Figure (2.7): Examples for Standard curves for (A) IL-6, MCP-1 (B) and (C) IL-8

Mini ELISA.

79

TFF3 conjugate was added to each well for 2h on shaker at RT. Afterwards, the plate

was washed as before and 200μL of substrate solution was added to each well and

incubated for 30min at RT (protected from light). A color developed in proportion to the

amount of TFF3 bound in the initial step. 50μL of stop solution was added to each well,

and the color in the wells changed from blue to yellow. The intensity of the color was

measured within 30min using a micro plate reader at 450nm and 540nm. The readings

at 540nm were subtracted from the readings at 450nm. The aim for this subtraction is to

correct the optical imperfections in the plate because the direct reading at 450nm

without correction may be higher and less accurate.

2.13 Determination of H2O2 Production by DCFDA:

H2O2 is one of the major reactive oxygen species (ROS) in the cell. This project chose to

measure H2O2 production by exposing the experimental cells to a cell permeable form

of fluorescein, DCFDA (2′, 7′-dichlorodihydrofluorescein diacetate). DCFDA

sensitively detects intracellular oxidants and can be used to follow changes in ROS

production over time (Eruslanov & Kusmartsev, 2010) (figure 2.8). A DCFDA Cellular

ROS Detection Assay Kit was used (Abcam, Cat no. ab113851).The HK2 (+/-GF) were

seeded with 10,000 cells/well density in a dark, clear-bottomed 96 well microplate

(PerkinElmer, Cat no.6005182) in standard medium and incubated overnight to be

adherent at 5% CO2, 37C˚. After that, the medium was removed and 100μL/well of 1x

buffer (supplied with the kit) was added to wash the cells. Next, the cells were stained

with 100μL/well of 25𝜇M DCFDA solution diluted in 1xbuffer for 45min at 5% CO2,

37C˚ in the dark. Then, the DCFDA solution was harvested and the cells were washed

again as described. Afterward the cells were treated and incubated with 100μL/well of

(5mg/ml) FAF-HSA or 𝜆-LC in free DMEM-F12 mediums for different time periods (5,

10, 20, 30, 60 and 360min) at 5% CO2, 37C˚ in the dark. After each time point the

100μL supernatants were transferred to a new dark 96 well microplate to measure the

conversion of DCFDA to DCF as indicator of the H2O2 released in the supernatant and

100μL of fresh free DMEM-F12 mediums were added to each well to measure the H2O2

production inside the cells.

80

Finally, the plates were measured using a fluorescent plate reader (Packard

FluoroCountTM microplate reader) with an excitation wavelength at 485nm and an

emission wavelength at 535nm.

81

Figure (2.8): Formation of fluorescent DCF by ROS activity: DCFDA is a

fluorogenic (non-fluorescent) dye. After diffusion in to the cell the DCFDA is

deacetylated by cellular esterase, which is later oxidized by ROS into highly

fluorescent compound DCF (2’, 7’ –dichlorofluorescein) that can be detected by

fluorescence spectroscopy.

OH HO

O-

DCFDA uptake by

cell

(Non-fluorescent)

DCF de-acetylation by

esterases

(Non-fluorescent)

DCF

(Fluorescent)

HO

O-

Cell membrane

Oxidation

Diffusion in to the cell

H2O2

H2O

ROS

82

2.14 Measuring Hydrogen Peroxide Production (H2O2)

Production by Amplex® Red:

H2O2 is a cell-damaging agent. The production of H2O2 from cells could lead to

oxidative stress and disease (Eruslanov & Kusmartsev, 2010). The H2O2 concentration

in cell culture supernatant was measured by Amplex® Red Hydrogen

Peroxide/Peroxidase Assay Kit (Invitrogen, A22188) according to the manufacturer’s

protocol. The Amplex red substrate (the hydrogen donor) is oxidised by H2O2 in the

presence of Hydrogen Peroxide (HRP) to create a coloured compound called resorufin

that can be detected (figure 2.9). So, increasing amounts of H2O2 in the sample will

form increasing amounts of fluorescent product.

After sub culturing cells in 6 well plates and incubating cells containing protein or

controls (un-stimulated cells) for different time periods, 50μl from each sample

supernatant and serial dilution (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15 and 0μM) of H2O2

standards (figure 2.10) as described in the kit and loaded into a 96 well plates in

triplicate. Then, 50μl was added from the Amplex red solution and horseradish

peroxidase (HRP) mixture to each sample and incubated for 30min at RT, protected

from light. The H2O2 concentration was measured in free medium as a control for

background absorbance. The plate was read spectrophotometrically at 560nm.

Figure (2.9): Conversion of Amplex Red to resorufin by HRP using H2O2

(Chemical reaction).

Amplex Red

Resorufin

H2O2 H2O

Hydrogen

peroxide (HRP)

83

2.15 Proteome Profile® analysis of HK2-GF cells:

In order to globally detect protein expression changes in HK2-GF cells before and after

protein stimulation, lysates were hybridised to a protein array, which is specifically

designed to detect proteins that have a role in renal cell and kidney function (Human

Kidney Biomarker Array kit, R&D Systems, Cat no. ARY019).

Four samples in a 6 well plate (1x 106 cells/well) of HK2-GF cells were sub cultured for

24h to be adherent. The next day the supernatants were discarded and three wells were

stimulated with 𝜆-LC (1 and 5 mg/ml) and FAF-HSA (5mg/ml) for 72h. The type of the

cells (HK2-GF) and the stimulation time (72h) were chosen depending on the indirect

cytotoxicity results (LDH) (See 5.2.1.2.1) and the ELISA results of IL-6, IL-8 and

MCP-1 (See 7.2.1). In addition, the HK2-GF cells were grown in the same medium that

primary proximal tubular cells grow in. However, 𝜆-LC (1 and 5mg/ml) concentrations

were decided on to examine the dose effects on the cells and the (5mg/ml) FAF-HSA to

examine the mechanism of damage between the 𝜆-LC and the FAF-HSA by using the

same concentration.

After 72h, the supernatants were harvested and the samples were washed with PBS.

After that, cell lysates were prepared by adding 200𝜇l of lysis buffer (Table 2.5) to each

well. The cells were scraped, collected in tubes and incubated for 30min on ice. Next,

the samples were centrifuged at 14,000 x g for 5min to remove cell debris. The

supernatants were collected in fresh reaction tubes. The protein concentration for each

Figure (2.10): Standard curve for Measuring Hydrogen Peroxide Production

(H2O2) Production by Amplex® Red.

0 5 10 150.00

0.05

0.10

0.15

0.20

H2O2 mMO

D (

560)

84

sample was determined as mg/ml using the Nanodrop machine (Thermo Scientific).

200μg of protein was used from each sample, according to manufacturer’s

recommendations.

The Human Kidney Biomarker Array kit had four nitrocellulose membranes, each

containing 38 different capture antibodies for the 38 different proteins printed in

duplicate. The membranes were blocked with 2ml blocking buffer (provided with the

kit) for 1h at RT with shaking. Then each sample was mixed with 15𝜇𝑙 of reconstitution

detection antibody cocktail and incubated for 1h at RT. The blocking buffer was

aspirated from the membranes, and then the samples were added to each membrane and

incubated overnight at 4C˚ on the shaker. The next day the membranes were washed

with the washing buffer 3x each for 10min and then each membrane was incubated for

30min at RT with 2ml of Streptavidin-HRP. Finally, the membranes were washed as

described previously and the face of the membrane exposed to Chemi Reagent Mix,

consisting of 1ml Chemi Reagent A and 1ml of B, wrapped in cling film, and exposed

to film for 10min in a light-tight cassette. Then the film was submerged in Develop

solution for 2min until antibody-reactive spots appeared, washed in water, and

submerged in solution, then washed in water and left to dry.

2.15.1 Proteome profile Data Analysis:

ImageJ2 software was used to analyse the data by measuring the pixel densities on

developed X-ray film for each spot that reflects the 38 different protein expression

levels (figure 2.11 A). The density of each protein, reference spots (positive control),

negative control (PBS) and background signal (clear area of the array (no antibody))

spots were measured in duplicate and the mean average was calculated (figure 2.11 B).

Then the average of the background density was subtracted from each spot (figure 2.11

C). Lastly, each spot was normalised with the reference spot and the graphs were

plotted to compare the signal of the 38 proteins in the four samples (figure 2.12).

The mean of each spot

The mean of the reference spots (positive control)

85

Figure (2.11): Example of the Human Kidney Biomarker Array result for HK2-

GF control sample. (A) Autoradiography film for the control sample shows the

positive signals on developed film. (B) Autoradiography film with light

background to measure the density of each signal. (C) The areas that were

measured by Image J2 software in fix circle size (13 diameters). The background

signal (clear area of the array (no antibody)).

C

Background

signal

B Positive signal

Negative signal

A

87

Figure (2.12): (A) The Human Kidney Biomarker Array coordinates. (B) The

table shows the reference of the 38 proteins, (Human Kidney Biomarker Array

kit, R&D Systems, Cat no. ARY019).

88

2.16 Phosphorylation of the recombinant expressed

cytoplasmic tail of Megalin receptor:

2.16.1 Rationale for use of pGEX-4T1 plasmid and MegCT-fusion

protein:

Meg-CT cDNA was ligated between the EcoRI/Xhol sites of pGEX-4T1 plasmid

(figure 2.14 A). The recombinant plasmid was used to transform E.coli (DH5α) to

induce a high-level expression of genes as fusions with Schistosoma japonicum

Glutathione-S-transferase (GST). Fragments of proteins fused to GST are easily and

rapidly purified. GST Fusion proteins have been widely used in protein/protein

interaction detecting methods in vitro because it easily binds to glutathione sepharose

beads and can be collected by centrifugation from the reaction mixture.

Competent cells (E.coli) 1ng were added to 100µl of plasmid DNA and mixed; the

mixture was incubated on ice for 30min before being heat shocked for 45sec at 42C˚.

After that, 900µl of SOC media were added and the mixture was incubated on ice for

2min. Then, the transfection was incubated horizontally in a shaking incubator at

225rpm at 37C˚ for 1h. The cells were grown by spreading 100µl of the transfection

mixture on Luria broth (LB) agar plates (containing 50µg/ml ampicillin) and incubated

overnight at 37C˚. The next day, one colony was used to inoculate 100ml of LB media

and grown overnight at 37C˚ with shaking. Finally, glycerol stocks of transformed

bacteria were made by mixing 10ml of the overnight culture with 4.5ml of a 50:50

mixture of sterile culture media and glycerol, stored at -80C˚. (The plasmid

construction and stocks preparation were prepared by Dr. Richard Baines, PhD

thesis, University of Leicester, 2010).

89

2.16.2 Preparation and collection of MegCT-GST Fusion protein:

The frozen glycerol stock in -80 (pGEX-4T1-MegCT transfected DH5α E. coli) was

scraped, inoculated in 20ml of LB with 20𝜇l of filter sterilised ampicillin diluted in

nanopure H2O (100 mg/ml stock) (w/v) and incubated at 37C˚ with shaking overnight.

The next day, 200-250ml of fresh LB broth (Table 2.12) were poured in a sterile flask

and 2.5ml of the overnight culture were added to a dilution of 1:100 and incubated at

30C˚ for 4h with shaking. After 4h 250𝜇𝑙 of filter sterile IPTG (1M stock) in dH2O

(w/v) (Isopropyl β-D-1-thiogalactopyranoside (Sigma, Cat no. I-6758)) was added to a

final concentration of 1mM to induce the expression of the fusion protein, and

incubation continued for additional 2h at 30C˚ with shaking. The bacterial pellet was

collected by centrifugation at 8000rpm (Beckman coulter Avanti j-e centrifuge) for

10min at 4C˚. The supernatant was discarded and the pellet was re-suspended in 19ml

of ice cold PBS. The cell wall of the bacteria was disrupted by brief sonication (MSE

Sanyo Soniprep 150) about 6 times, each time for 5sec, while on ice, until partial

clearing of the cell suspension was observed. Then the fusion protein was solubilised

with 1ml of 20% (v/v) of Triton X-100 (BDH chemicals, Cat no. 30632 4N) in dH2O to

a final concentration 1% and was gently shaken on ice for 30min. The pellet was

collected after 10min centrifuging at 12,000 x g at 4C˚ and the supernatant was

decanted. Afterward, 2ml of 50% slurry of glutathione sepharose 4B (52-2303-00 GE

Healthcare) (Table 2.12) in PBS were added to the supernatant, followed by mixing for

1h at 4C˚ on the tube rotor. The fusion protein bound to the glutathione sepharose bead

pellet was collected by centrifugation at 500 x g for 5min at 4C˚, followed by washing

the pellet 3x with 10ml of ice cold PBS and finally re-suspending in 2ml of PBS and

75µl of protease inhibitor cocktail was added (Sigma, Cat no. P8340) (figure 2.13).

90

Luria Broth (LB)

For 1L: 16g Trypton, 10g Yeast and 5g NACL.

After autoclaving, for each 1L of media 1ml of 100mg/ml stock

filter sterilised ampicillin sodium (Melford Laboratories LTD

A0104) diluted in nanopure water was added (100µg/ml).

Glutathione Sepharose 4B Preparation

Glutathione sepharose stock in 20% ethanol. 1.25 ml of the stock was

washed twice with 5ml PBS and centrifuged at 500g for 5min to

remove the ethanol. Then the 5ml of PBS was discarded and the

slurry was re-suspended in 1 ml fresh PBS to end up with 50% slurry.

Table (2.12): Luria Broth media and Glutathione Sepharose 4B

preparation

91

2.16.3 Stimulation of MegCT-GST fusion protein phosphorylation in

vitro:

There is a composite overview of this part in (figure 2.14). In 6 well plates, 500,000

cells/well were seeded and allowed to attach to be confluent in a standard culture

medium in the incubator, 5% CO2 at 37C˚. The confluent monolayers of HEK293 or

HK2 (+/-GF) cells were quiesced overnight in a serum free medium. The next day, the

medium was aspirated and freshly prepared agonists (Table 2.13). Each agonist was

diluted in 1ml of serum free medium and added to each well, and then the plates were

returned to the incubator for different times of incubation depending on the experiment

to investigate signalling kinetics. To halt the stimulation, the 6 well plates were put on

ice, the supernatants were harvested and each well was washed 2x with 2ml of ice cold

PBS. The cells in each well were lysed with 200µl of JNK lysis buffer for 10min (Table

2.14) while on a shaking rotator on ice. The samples were collected and centrifuged at

10,000 x g for 10min at 4C˚. The supernatants were transferred to new fresh reaction

tubes and 50µl from the MegCT-GST beads were added to each sample. The samples

were incubated for 1-2h at 4C˚ on an end-over-end rotator. Then, the beads were

washed once in 1ml of JNK lysis buffer and twice with 1ml of kinase buffer (Table

Figure (2.13): Preparations steps of MegCT-GST Fusion protein. (1) Bacteria

lysate from 24h growth (2) Bacteria lysate with IPTG for 2h (3) Bacteria after

sonication step (4) elate from beads.

4 3 2 1

50kDa

92

2.14). Afterward, they were collected by centrifugation at 3000 x g for 5min and 50µl

from 100µM Adenosine triphosphate (ATP) (Sigma, Cat no. A7669) (1mM ATP stock

diluted in H2O, then diluted with Kinase buffer to give a final concentration of 100µM

for each reaction) as a source of the phosphate and incubated for 30min at 30C˚. Finally,

the beads were collected after centrifugation for 5min at 3000 x g and washed 2x with

500µl of ice cold PBS. Thereafter, the samples were run on 12% SDS-PAGE after

solubilisation and denaturation in the loading buffer (ratio 1:2 for each sample)

followed by boiling at 95C˚ for 10min. Proteins were electrophoretically transferred to

methanol-activated PVDF membrane, blocked in 5% skimmed milk in PBS (w/v)

overnight at 4C˚ and then incubated with primary antibody, namely the antibody

generated in rabbit against the phospho relevant site (1:1000 dilution) (See 6) in fresh

5% skimmed milk, overnight at 4C˚.The blot was washed, incubated with HRP

conjugate and Swine-anti rabbit secondary antibody (1:2000) in 5% skimmed milk in

PBS (w/v) for 2h at RT and then reactive protein bands were visualised by incubating

with ECL Western Blotting Detection System (see 2.9.3).

93

Agonist Rationale

FAF-HSA (5 or 30mg/ml)

A major component of proteinuric

urine.

A ligand of Megalin receptor.

Phorbol 12,13-dibutyrate

(PDBU) (CALBIOCHEM,

Cat no. 524390) (positive

control) – a stimulator of

PKC (10µM final

concentration)

Protein kinase C activity stimulated by

HSA in PTC.

Protein Kinase C activity regulates

albumin endocytosis by PTECs in

vitro.

𝝀-LC (1 or 5mg/ml)

A major component of proteinuric

urine in Multiple Myeloma disease.

A ligand of Megalin receptor.

Table (2.13): Table of agents used in the studies of MegCT phosphorylation and

rationale for their use.

94

JNK Lysis Buffer Kinase Buffer

20mM Tris-HCL (Sigma)

0.5% Tergitol-type NP-40 (BDH,

Cat no.56009)

250mM Sodium chloride (NaCl in

dH2O) (Sigma)

-3mM Ethylenediaminetetraacetic

acid (EDTA pH 8.0 in dH2O)

(ALDRICH)

3mM Ethyleneglycoltetraacetic

acid (EGTA pH 8.0 in dH2O)

1mM PMSF in ethanol.

2mM Sodium orthovanadate

(Na3Vo4 pH 10.0 in dH2O)

(AlDRIH, Cat no. 450243)

1mM DTT (Sigma)

Protease Inhibitor Cocktail III

diluted 1:25

50mM 4-(2-

hydroxyethyl)-1-

piperazineethanesulfonic

acid (HEPES in dH2O)

(Sigma)

20mM β-

glycerophosphate

(Sigma, Cat no. G5422)

10mM Magnesium

chloride (MgCl2)

(FISONS reagent)

1mM DTT

50µM Na3Vo4 pH 10.0

Table (2.14): JNK and Kinase buffers preparations.

95

GST

pGEX

4T1

cDNA Megalin -CT

- Cloning of Megalin CT-GST in

prokaryotic expression vector.

- Production of MegCT-GST fusion

protein in E.coli (IPTG induction)

Coomassie blue gel of MegCT – GST

fusion protein

Albumin or LC

Activate

Kinase

pathway and

phosphorylate

MegCT in

PPPSP site

Cell

Megalin

Cells were stimulated and lysates were incubated with Meg-CT-GST bound to

glutathione beads for 2h at 4ºC, collected beads were incubated with ATP for 30

minutes at 30ºC. Finally; antibodies specifically raised against predicted

phosphorylation sites were used for detection of activation.

A B

96

2.17 Statistics and Data analysis:

The experiment’s conditions were kept as standard as possible and consistent replicates

results were analysed. Non-reproducible results for replicates of tested sample in the

experiment was not accepted, the experiment was excluded and should be repeated.

Data were expressed as Means ± SD for all experiments (n= the total number of

independent experiments, biological replicates). Statistical analysis was performed

using Graph Pad Prism 6 (Graph Pad, San Diego California, USA). For comparison of

one data set with a control data set, an unpaired t-test was performed. The values p<0.05

were deemed as significant. The standard curves were plotted by Graph prism 6

software, then the unknown samples were calculated. The density of Western blot and

PCR bands and the statistical analysis was measured by ImageJ2 software.

Figure (2.14): Phosphorylation of Meg-CT. (A) Production of construct,

purification of recombinant Meg-CT and (B) Detection of phosphorylation

signalling Meg-CT by utilising antibodies directed against specific activation sites

of the intracellular portion of megalin using stimulated cell lysate.

97

Chapter Three-FLC Purification

98

3. Purification of monoclonal Free Light Chain (FLC):

This chapter presents the development and optimisation of methodology for the

purification of FLC from urine. This was necessary in order to have purified

monoclonal light chains from a relevant source (multiple myeloma urine) with which to

assess their biological effects directly on proximal tubular epithelial cells in vitro. It was

not the scope of this work to test FLC from more than one patient or to include non-

monoclonal FLC in the analyses. The purpose of using monoclonal myeloma produced

FLC was to have a high concentration of uniform proteins with which high

concentration of uniform FAF-HSA would be compared. Non-monoclonal FLC that

might be considered as possible vehicle control of polyclonal would induce

heterogeneity in sample used for stimulation.

3.1 Source of Free Light Chain:

In multiple myeloma patients, the concentration of only one type of immunoglobulin

that is overproduced is very high in urine. In addition, it is often the main protein in the

urine, which aids in the purification process. Others have used urine from myeloma

patients as a convenient source of light chains (Sengul et al., 2002 and Li et al., 2008).

In this project, monoclonal FLC was purified from the urine of a patient with multiple

myeloma (MM) who had light chain proteinuria of ~5g/L. The light chain concentration

was measured by nephelometry at Leicester Royal Infirmary Hospital.

3.2 Urine Sample analyses:

The urine sample from the MM patient was a 24-hour collection sample (~1.5L) in a

plastic container (The urine sample was supplied by Dr. Mike Browning, Leicester

Royal Infirmary Hospital); from the physical examination the sample was foamy

(indicative of high protein content) and dark yellow in colour. In addition, in

microscopic examination the patient sample some crystal formation and casts showed.

The monoclonal FLC circulate as monomers (~25kD) and dimers (~50kD) (Wang &

Sanders, 2007). A denatured (5min in 95C˚) 20µl urine sample was separated using

99

12% SDS-PAGE gel and stained with coomassie blue (Table 3.1) by heating the gel

with the stain in the microwave for 5sec or leaving it to stain overnight on shaker at RT.

Then the gel was de-stained (Table 3.1) for at least 2h on shaker at RT or until the

protein bands are distinctly visible. A predominant band at (25kDa) is likely to be the

monomeric light chains (figure 3.1).

Coomassie Blue Stain

De-stain

For 1L:

1g Coomassie Brilliant Blue R250

(BIO-RAD, Cat.161-0400)

100ml Acetic Acid

400ml Methanol

500ml H2O

For 5L:

1.25L Methanol

0.5L Acetic Acid

3.25L H2O

Table (3.1): Coomassie Blue stain Preparation.

100

3.3 Dipstick test:

Typically, urinary analysis starts with a quick semi-quantitative chemical test called a

dipstick (Combur 10Test, Cobas, Cat no. 04510062). It screens for the presence of

proteins, blood and glucose in urine samples (de Jong & Curhan, 2006).

A control sample from normal patient (female) was used to compare with the patient

sample. It was a first-morning collection sample, which is suitable in the diagnosis of

the proteinuria (de Jong & Curhan, 2006). The dipsticks were dipped directly in the

non-centrifuged urine samples for 1sec in both the control urine sample ( C ) and in the

MM patient urine sample ( P ). The P sample was alkaline (pH 9.0) compared to the C

sample, which was acidic (pH 5.0). Urine is commonly acidic, with a pH (5.5-6.5),

however, alkaline urine may be seen in proximal renal tubular acidosis caused by a

failure of the proximal tubular cells to reabsorb bicarbonate from the urine due to

multiple myeloma disease (Patient, 2014).

Figure (3.1): Characterisation of urinary protein profile from a multiple myeloma patient.

20µl were run on 12% SDS-PAGE and coomassie blue stained (20µl of neat sample were

loaded).

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/Glucose

http://en.wikipedia.org/wiki/Proximal_tubular

http://en.wikipedia.org/wiki/Bicarbonate

101

Additionally, proteins were detected in the P sample by ~ +2 (1g/L) (the colour change

from yellow to the dark green colour and negative result for C (the colour did not

change). In addition, for further investigation the two samples were runs using SDS-

PAGE The gel shows the presence of a high amount of FLC protein and some other

high and low molecular weight proteins in the P sample, however, the C sample showed

significantly fewer proteins (figure 3.2).

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

C P

Figure (3.2): Comparing the urinary proteins in the patient (P) and the control (C)

sample, 20µl was run in 12% SDS-PAGE and coomassie blue stained (reduced

condition).

102

3.4 Albumin Excretion:

Albuminuria is a sign of renal damage. The albumin concentration was measured in the

myeloma patient sample by generating a standard curve from the known concentration

of BSA (PAA, Cat no. K41-001) from 5-200mg/L diluted in dH2O, and comparing to

the band of the size of albumin (~68kDa) obtained from the serially diluted BSA

samples run on a gel in parallel.

The classification of urinary albumin excretion in a male and female was demonstrated

in (Table 3.2) (de Jong & Curhan, 2006). 10μl from each reduced BSA sample and the

P sample were run in 12% SDS-PAGE gel and stained with coomassie blue stain (figure

3.3 A).

The gel was scanned, the band density areas were measured by using ImageJ2 software

and the standard curve was plotted by Graph prism 6 software (figure 3.3 B). Then, a

formulated standard curve was used to determine the albumin concentration of the P

sample. The P sample had ~ 12.7822 mg/L (high normal) by calculating the albumin

concentration from the standard curve. So, the patient had no albuminuria.

Previous studies such as (Li et al., 2008) purified LCs to use in their studies from the

urine of multiple myeloma patients with myeloma kidney, but with minimal or no

albuminuria to exclude patients with significant glomerulopathy and all LCs considered

tubulopathic. That means they affected the tubular cells, and that was what we focused

on in this project.

103

Albumin (mg/L)

Normal <10

High normal 10 to < 20

Micro albuminuria 20 to < 200

Macro albuminuria > 200

Table (3.2): The classification of urinary albumin excretion.

0 50 100 150 200 2500

1000

2000

3000

BSA

Conc mg/L

Area o

f D

en

sit

y

1 2 3 4 5 P

B A

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

Figure (3.3): Estimation of albumin concentration in MM patient urine sample.

(A) Coomassie blue 12% SDS-PAGE gel shows the known BSA concentration

samples with molecular weight ~68kDa from 1 to 5 (5, 10, 20, 100 and 200

mg/L), (P) the patient sample (10μl reduced condition) with the albumin band

appear in the same size of the BSA bands. (B) The plotted BSA standard curve

involving the five BSA points. The orange circles indicate pipette excision of

protein samples that were sequenced by the MALDI-TOF method.

104

3.5 Protein sequencing:

The high molecular weight proteins bands in the P sample were sequenced to identify

the type of proteins that were excreted in the urine of the myeloma patient with the light

chain (figure 3.3 A). Protein sequencings were performed in the University of Leicester

Protein Nucleic Acid Chemistry Laboratory (PNACL) by MALDI-TOF technique.

3.5.1 Gel Digestion Protocol (Protein Digestion):

The gel plug was placed into a 200μl reaction tube and washed with 100μl of

ammonium bicarbonate (400mM): 100% acetonitrile in a ratio of 1:1 twice for 20min

each to equilibrate (pH 8.0) and to remove the coomassie blue staining. After that, it

was washed briefly with 100μl acetonitrile to remove aqueous solution, and then the

plug was shrunk by washing in 100μl acetonitrile for 10-15min and dried for 10min.

Next, the 100μl of dithiothreitol solution (10mM) in ammonium bicarbonate (50mM)

(w/v) (DTT, Calbiochem) was added for 30min at 60C˚ in a heat block. The liquid was

removed without shrinking the gel and 100μl of iodoacetamide solution (100mM) in

ammonium bicarbonate (50mM) (w/v) was added for 30min at RT in the dark (these

two steps were repeated twice).

Afterward, the sample was washed briefly with 100μl acetonitrile twice for 10-15min

and left to dry for 10min to remove all of the acetonitrile. 10μl of trypsin solution

(containing 50mM ammonium bicarbonate) (v/v) was added (Modified Porcine Trypsin,

Promega) and the sample was then incubated at 37C˚ in a heating block for 3h. Then,

2μl of neat formic acid was added, mixed and left for 20min. Finally, the sample was

sonicated in an Ultrasonic Bath for 10min (optional). A 0.5𝜇𝑙 spot was washed with

ammonium phosphate (10mM) and left to dry. The sample is ready for the MALDI-

TOF or LC-MS/MS technique.

105

3.5.2 Matrix Assisted Laser Desorption/Ionization - Time-of-Flight

(MALDI-TOF):

The digested peptides were analysed using MALDI-TOF mass spectrometry. The

sample peptides were mixed 1:1 with the matrix mixture (α-cyano-4-hydroxycinnamic

acid (sigma) (10mg/mL in 0.1% Trifluoroacetic acid (TFA)/50% acetonitrile), and were

spotted onto a 96 well hydrophobic coated MALDI target plate using the drying droplet

method. The peptides were analysed using a Voyager DE-STR MALDI-TOF mass

spectrometer (Applied Biosystems). Spectra were acquired in the reflector mode, with

positive polarity between the mass ranges of 700 - 4000Da; an average of at least 500

laser shots were acquired. The spectra were analysed using Data Explorer software

(Applied Biosystems). Peptide Mass Fingerprint Database Search was carried out using

Mascot database search.

3.5.3 Results of MALDI-TOF MS analyses:

The protein samples from figure 3.3 were both identified as Serum Albumin ~68kDa, as

expected.

3.6 Extraction of protein from the urine sample:

The methodology of light chain purification overview presented in (figure 3.12).

Following methodology, standard proteins were initially precipitated with 70% (w/v)

saturated ammonium sulphate (MELFORD, A0502) in nanopure H2O overnight at 4C˚.

Afterward, the precipitated proteins were collected in 50ml tubes and centrifuged for

30min at ~1800 x g; the supernatants were then discarded and the pellets were collected

for dialysis against dH2O for 24h at 4C˚ with stirring. Cellulose dialysis membrane

tubes with a cut off of 12,000 to 14,000 Daltons were used (MEDICELL international

Ltd Dialysis Tubing-Visking, Cat no. DTV.12000.01), they were boiled in 50mM

EDTA pH 8.0 for 2min, and subsequently rinsed with dH2O. After the first 24h of

dialysis, the proteins were dialysed against 0.01M sodium phosphate buffer pH 9.0

(Table 3.3) for 24h at 4C˚ with stirring. Next, the protein solutions in dialysis tubes

http://en.wikipedia.org/wiki/Time-of-flight

106

were collected in fresh tubes, and 20µl from the urine sample before and after dialysis

were run in 12% SDS-PAGE and stained with coomassie blue to be compared (figure

3.4 A and B).

Table (3.3): preparation of 1M Sodium Phosphate Buffer pH 9.0.

1M Sodium Phosphate Buffer pH 9.0

178g of Na2HPO4

I L dH2O

The pH was adjusted with HCL

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

Figure (3.4): The purified free light chain (A) Original proteinuria

sample and (B) protein sample after dialysis separated in 12% SDS-

PAGE and stained with coomassie blue (20µl reduced condition).

Major Free Light Chain protein at 25kDa is clearly visible.

107

An ion exchange chromatography column was utilised to separate the proteins

according to the interactions between the charge of protein and the charge on the resin;

the monoclonal FLC is likely to be a negatively charged protein (cation).

To pilot binding different sample types (urine, protein after precipitation with 70%

saturated ammonium sulphate and protein after dialysis) were used with different ion

exchange anion resin, such as Diethylaminoethyl-cellulose (DEAE-cellulose) (Sigma D-

8382), Sepharose 4B (Sigma) and P Sepharoes with sodium phosphate buffer of

different pH 7.6, 8.0 and 8.5.

1ml of resin in column (BIORAD, Poly-Prep Chromatography column, Cat no. 731-

1550) was washed and equilibrated with 10ml of (100mM then 10mM) sodium

phosphate buffer for each resin with different a pH level starting with 7.6, 8 and 8.5.

Then, 2ml, 0.5ml and 0.5ml different pH protein samples, diluted 1:4 with (10mM)

sodium phosphate buffer, were loaded in the column. The flow through was collected

and the column was washed with 10ml of (10mM) sodium phosphate buffer to remove

the remaining non-bound proteins. After that, the column was eluted with 10ml of (1M)

NaCl in sodium phosphate buffer (w/v) (ACROS). The flow through and eluted samples

were run in 12% SDS-PAGE gel and stained with coomassie blue as described. The

protein of interest (FLC) did not bind to any resin (figure 3.5).

In 1989, Norden and his group demonstrated that the isoelectric point (pI) of LC from

MM patients was in the range of 5.0-7.5. Due to this, protein separation can be achieved

based on the pI of the protein. The pH of the sodium phosphate buffer was increased to

pH 9.0; the volumes of protein samples that loaded in the column were optimised

(figure 3.6), achieving around 99% of FLC binding to Q Saphrose Fast Flow beads (GE

Health Care Life Science 17-0510-01) and eluted with 10ml of 1M NaCl with 1:4

diluted sample in 0.01M sodium phosphate buffer (figure 3.7).

108

Figure (3.5): Comparison of FLC binding to different resin types using dialysis

urine sample with pH 7.6 sodium phosphate buffer in 12 % SDS-PAGE stained

with coomassie Blue reduced condition. (1) Original proteinuria sample, (2, 3 and

4) The flow throw the column after loading proteinuria sample (unbound proteins)

in P Sepharose, Sepharose 4B and DEAE cellulose, respectively. (5, 6 and 7) the

eluted (bound proteins) with 1M NaCl to the resin P Sephrose, Sepharose 4B and

DEAE cellulose, respectively.

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

1 2 3 4 5 6 7

2 5k Da

FL C

109

C A B

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

1 2 1 2 1 2

2 5k Da

FL C

Figure (3.6): Optimizing the volumes of dialysed protein sample loaded in 1ml Q

Sepharose Fast Flow column for better binding. (A) 2ml, (B) 0.5ml, (C) 0.5ml

diluted 1:4 in 0.01M sodium phosphate buffer pH 9.0. (A1, B1 and C1) show the

unbound proteins. (A2, B2 and C2) show the bound proteins (FLC) to the resin

and eluted with 1M NaCl. 12% SDS-PAGE analysis and coomassie blue staining

for (20 μl from each sample in reduced condition).

110

Figure (3.7): Electrophoretic (SDS-PAGE) final analysis of FLC purification.

(1) The original urine sample. (2) The dialysed sample. (3) The 1:4 diluted sample

in 0.01M sodium phosphate buffer pH 9.0. (4) The unbound proteins to the Q

Sapharose Fast Flow beads column. (5) The FLC bound to the column and eluted

with 1M NaCL.

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

1 2 3 4 5

2 5k Da

FL C

111

Next, a high-performance liquid chromatography (HPLC) system was used to purify

FLC from the mixture of proteins in the urine sample. The sample (dialysed proteins)

was diluted 1:2 in sodium phosphate buffer pH 9.0 and loaded by the pump to the

column (GE Healthcare Bio-Science AB) that contained 20-30ml Q Saphrose Fast Flow

beads. The column was washed with 40ml 0.01M sodium phosphate buffer pH 9.0 (low

salt buffer), 40ml 1M NaCl in 0.01M sodium phosphate buffer pH 9.0 (high salt buffer)

then low salt buffer again to be equilibrated and be ready to use (buffers should be

filtered to be degased). After loading the sample in the column (50ml in each

purification run) 40ml of 0.01M sodium phosphate buffer pH 9.0 was loaded to wash

and remove the unbound proteins. The bound proteins were collected as a 1.5ml/min

fraction in 1.5ml fresh reaction tubes by eluting with increasing NaCl concentration for

45min from 0 to 100%. Elution was monitored by measuring the absorbance at 2400

mAU (figure 3.8). The collected fractions were run in 12% SDS-PAGE gel (in reduced

condition) and stained with coomassie blue. The protein samples were contaminated

with high and low molecular weight proteins (figure 3.10 A). The fractions, which had a

very high concentration of FLC, were collected and pooled together (figure 3.10 A from

fraction 1 to fraction 12). The Superdex 200 16/60 column was used to purify the target

protein (FLC) by size (GE Healthcare Life Science, HiLoad 16/600 Superdex 200 pg).

This type of column is designed for a high-resolution preparative gel filtration

chromatography (figure 3.9). The column was washed with filtered PBS, and then 5ml

of the sample pooled from fractions was loaded each time in the column due to the

limited capacity of the column. Then, again the samples were collected as 1.5ml/min

fractions in 1.5 ml eppendorf tubes (figure 3.9) and run in 12% SDS-PAGE gel and

stained with coomassie blue. Figure 3.10 C shows FLC purified by ~95% from low and

high molecular weight proteins. (Thanks for Professor Russell Wallis and Dr. Chris

Furze for their help in HPLC).

112

Figure (3.8): Chromatogram of Ion exchange column High-performance liquid chromatography (HPLC) system. Fifty ml of 1:2 diluted

FLC sample in sodium phosphate buffer pH 9.0 was run through the Ion exchange column (Q Sepharose Fast Flow resin). Brown line is

conductivity; Blue line UV absorbance (mill absorbance units (mAU)); Green line % elution buffer (1M NaCl).

113

Figure (3.9): Chromatogram of removal of impurities from FLC sample by size exclusion High-performance liquid chromatography

(HPLC) system. Collected and mixed FLC fractions from the Ion exchange column were loaded in Superdex 200 16/60 column. The first

two small peaks separate the high molecular weight proteins. Fractions from 35-45 contain FLC.

114

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

1 2 3 4 5 6 7 8 9

( k D a )

2 5 0

1 5 0

1 0 0

7 5

5 0

3 7

2 5

2 0

1 5

1 0

1 2 3 4 5 6

C B

Figure (3.10): SDS-PAGE analysis for fractions

eluted from Ion exchange column 1.5ml/min. (A)

2µl from each fraction appears as a peak in figure

(3.11) the FLC protein is co-purified with unknown

higher and lower molecular weight protein.

Fractions 1-12 have high concentration of FLC

(they were collected and mixed for the Superdex

200 16/60 column. (B) 2µl from each fraction

appears as a peak in figure in non-reduced

condition. (C) 2µl from the same fractions in B in

reduced condition shows ~95% purified FLC.

Reduced: the sample boiled for 5mints at 95C ̊ with

loading dye has DTT to breakdown the sulphate

bound. The orange circles indicate the samples

taken to sequence analysis.

115

3.7 Purified FLC Protein Sequencing:

From the ~95% purified FLC that was run in 12% SDS-PAGE gel (non-reduced

condition) and stained with coomassie blue two spots were sequenced; the first one

from the band ~ 50kDa and the second one from the band (~ 25kDa). This was done by

the liquid chromatography–mass spectrometry (LC-MS/MS) technique (figure 3.10 B).

At the beginning the spot samples were digested as described previously (See 3.5.1).

Afterward, the LC-MS/MS was performed by using an RSLCnano HPLC system

(Dionex, UK) and LTQ-Orbitrap-Velos Mass Spectrometer (Thermo Scientific).

3.8 RSLCnano HPLC System:

To separate the protein peptides mixture, samples were loaded onto a Reverse-Phase

Trap Column (0.3mm ID x 1mm) (5μm particle size, 300 Å pore size, Acclaim PepMap

C18 media (peptide) (Dionex) and maintained at a temperature of 37C˚). The loading

buffer was (0.1% formic acid / 0.05% trifluoroacetic acid / 2% acetonitrile).

The protein peptides were eluted from the trap column at a flow rate of 0.3µl/min and

through a Reverse-Phase PicoFrit Capillary Column (75μm ID x 400mm) (Symmetry

C18 media, 100 Å pore size (Waters, UK)) that was packed in-house using a high-

pressure device (Proxeon Biosystems, Denmark). Peptides were eluted in 2h.

3.9 LTQ-Orbitrap-Velos Mass Spectrometer:

After peptide separation, they were characterised and identified using mass

spectrometry, which is a technique where the ratio between the mass and the charge

(m/z) of ionized molecules in the gas phase is measured. Generally, a mass spectrometer

consists of an ionization source, mass analyzer and detector (Gris & Baldoni, 2013).

116

The separated peptides from the column were sprayed directly into the nanospray ion

source of the LTQ-Orbitrap-Velos mass spectrometer. In this stage the molecules were

ionized and transferred to the gas phase. Then they were moved to the mass analyzer,

where the ions formed were separated with their m/z ratios and later detected.

3.10 LC-MS/MS Sequance Results:

The data was analysed using Scaffold Q+S4 (version 4.0.5, Proteome Software). The

two sequenced bands in (figure 3.10 B) were identified as a dimer (~50kDa) and

monomer (~25kDa) shapes for Ig Lambda-Light Chain-2 constant region (𝜆-LC-2).

The protein sequence and the number of peptides matching for both bands based on

(~82 %) identity match (figure 3.11) (highlighted in yellow).

3.11 Protein Concentrate column:

The protein samples were concentrated using Millipore's Amicon® Ultra-15 centrifugal

filter devices column for volumes up to 15ml with a 10K membrane cut off. The

purified protein fractions from the Superdex 200 16/60 column were each collected in

15ml amounts together as one sample. Then, the concentration of each sample was

measured by Nano-drop machine (Thermo Scientific) at A280 nm absorbance and stock

concentration was decided to be 100mg/ml for simple dilution calculations in the

experiment later.

G Q P K A A P S V T L F P P S S E E L Q A N K A T L V C L I S D F Y P G A V T V A W K A D S S

P V K A G V E T T T P S K Q S N N K Y A A S S Y L S L T P E Q W K S H R S Y S C Q V T H E G S T

V E KT V APTECS

Figure (3.11): Protein Sequence Coverage , 87/106 amino acid (~82%), (covered

locations were indicated in yellow).

117

The volume of the 𝜆-LC sample with 100mg/ml concentration was loaded to the column

and centrifuged at 3,000 × g for 30-45min. Once the desired concentration was achieved

(100mg/ml) the protein was recovered from the bottom of the unit in eppendorf tubes.

3.12 Endotoxin Measurement:

Endotoxin is a lipopolysaccharide (LPS) found in the outer cell wall of all gram-

negative bacteria. Even a small amount of endotoxin can cause interference in vitro

stimulation with purified proteins (activation, toxicity). As the monoclonal 𝜆-LC protein

will be used in tissue culture experiments, it was critical that the 𝜆-LC should be free of

endotoxin. A quantitative, chromogenic Limulus amoebocyte lysate (LAL) assay kit

(PierceLAL Chromogenic Endotoxin Quantitation Kit, Thermo Scientific, Cat

no.88282) was used to test and measure the presence of endotoxin in the FLC purified

sample. This assay uses the reaction of endotoxin with LAL to activate protease enzyme

that acts on a synthetic substrate to produce a yellow colour.

A 96 well plate was warmed for 10min at 37C˚. A standard was prepared in duplicate,

ranging from 1.0 - 0.1 Endotoxin Units (EU)/ml. 50 𝜇𝑙 of the 𝜆 -LC were tested.

Endotoxin free water was the negative control. After 5min at 37C˚, 50μl of LAL was

added to each well and incubated for 10min at 37C˚. Then 100μl/ well of substrate

solution was added and the plate was incubated for 6min at 37C˚. Finally, 100μl/well of

stop solution (25% acetic acid in dH2O (v/v)) was added. The plate was measured in the

plate reader at 405nm. The Endotoxin level in the 𝜆-LC was shown to exceed 1.0

EU/ml. One EU being equal to ~0.2ng endotoxin/ml; an approximation for the tested

samples is at least 0.4ng/ml endotoxin (Done by Dr. Cordula Stover).

(Sigma, http://www.sigmaaldrich.com/life-science/stem-cell-biology/3d-stem-cell

culture/learning-center/what-is-endotoxin.html).

http://www.sigmaaldrich.com/life-science/stem-cell-biology/3d-stem-cell%20culture/learning-center/what-is-endotoxin.html

http://www.sigmaaldrich.com/life-science/stem-cell-biology/3d-stem-cell%20culture/learning-center/what-is-endotoxin.html

118

3.12.1 Endotoxin Removal:

Endotoxin was removed from the 𝜆 -LC by using Pierce High-Capacity Endotoxin

Removal Spin Column 1.0ml (Thermo SCIENTIFIC, Cat no. 88276).

The column was equilibrated to RT and centrifuged for 1min at 500 x g to remove the

storage solution. Then, the column was regenerated by adding 8ml of 0.2N NaOH in

95% ethanol, incubated for 2h at RT and centrifuged at 500 × g for 1min to remove the

NaOH. After that, 8ml of 2M NaCl in endotoxin free water (w/v) (Sigma, Cat no.

95289), was added to the column, suspended and centrifuged. Next, 8ml of endotoxin

free water was added, suspended in the column and centrifuged. Afterward, 8ml of

150mM NaCl in PBS (w/v) was added to the column, suspended and centrifuged. Up to

10ml of the sample was added to the resin. After 1h of gentle mixing at 4C˚ the column

was centrifuged at 500 × g for 1min to collect the sample and stored in -20. All the

equipment that used was endotoxin free. The Endotoxin level in the 𝜆-LC was measured

after endotoxin removal and the result showed very low endotoxin level below 0.1

EU/ml (lower than ~ 0.02 ng endotoxin/ml).

3.13 Human Serum Albumin Devoid of Fatty Acids:

Albumin is the most extensively studied protein as it is by far the most abundant protein

in nephrotic urine. This project will examine the role of albumin overloading and

PTECs damage. In progressive renal disease, PTECs are exposed to high levels of

filtrated albumin. The effect of overloading albumin on the viability of HK2 (+/-GF)

cells will be measured by different assays in order to establish a model and then to study

disease mechanisms using this in vitro model. Albumin that is essentially free of fatty

acid will be used to exclude toxic effects of fatty acid. Albumin, normally, is a carrier

for fatty acids (FA) and previous studies have documented a tubule toxic effect of FA

carrying albumin in vitro at 24h of stimulation using primary human PTC (Arici et al.,

2003) .

119

Newman et al., 2000 used human serum albumin 95% pure and free of fatty acid in

concentrations from (0.05 to 5mg/ml) to cover the range of the normal and the

pathological condition in kidneys according to their calculation. However, they failed to

detect any significant effect from the albumin on HK2 cell proliferation after 8h

stimulation time. They suggested to use higher concentrations of albumin to inhibit

HK2 cells proliferation. Based on these studies, the concentrations of the albumin used

in our studies were chosen to target the pathological condition in kidneys and

inflammatory response.

3.14 Calculation of protein load:

The human serum albumin utilised in this project is more than 95% pure and essentially

fatty acid free (Sigma, Cat no. A1887). The stock concentration was 500mg/ml, using

endotoxin free water. After filter sterilisation, aliquots (500mg/ml) were stored at -20C˚.

The (5mg/ml) LC concentration chose depend on the LC concentration in the urine of

MM patients.

120

Urine sample from

MM patient had

(5g/L) free light chain

The sample was precipitated

in 70% saturated ammonium

sulphate for 24h at 4C˚

Precipitated proteins were dialysed

against 0.01M Sodium phosphate

buffer pH 9.0 for 24h at 4C˚

HPLC

was used

to purify

FLC (Ion

exchange

column)

The Superdex 200 16/60

column

The purified FLC was concentrated by

centrifugal filter devices column

The endotoxin (LPS) was

measured and removed from

the concentrated sample

The sample ready to use in the

tissue culture after filtration

(stored at -20C˚)

Figure (3.12): Methodology of Monoclonal Free Light Chain Protein Purification.

121

Chapter Four- Characterisation of Renal

Proximal Tubular Epithelial Cells

122

4. Introduction:

The kidney is key in cleansing the blood of otherwise toxic substances and producing

hormones such as erythropoietin and vitamin D metabolite (Van der Hauwaert et al.,

2013) Proximal tubules form a significant portion of the kidneys, and proximal tubule

epithelial cells are the most abundant cell type in kidneys (Nakhoul & Batuman, 2011)

and play a major role in the reabsorption of larger and smaller proteins such as albumin

(68kDa) and free light chain (FLC, 25kDa). Additionally, proximal tubule cells are

specifically sensitive and represent a primary target for toxins that effects can extend to

kidney failure (Van der Hauwaert et al., 2013). To study cellular effects of protein

uptake, human proximal tubular cells (HK2) were used.

This project has modeled the experimental designs on published work: a cell line HK2

was established and studied for the effects of different proteins overload such as FLC

and albumin.

As described in material and method (2.1), HK2 cells were grown in a medium

supplemented with an EGF cocktail that is favourable for cell differentiation, to study

the effects of the EGF cocktail on the HK2 cells proliferation and their response after

being exposed to pathological concentrations of FAF-HSA and 𝜆-LC proteins. They

were compared to HK2 grown in the absence of the EGF cocktail in their growth

medium. Also, mouse primary proximal tubule epithelial cell cultures were grown in a

medium with the EGF cocktail as a control for several experiments.

4.1 Aim:

The goal of the present study was initially to characterise features of the human

proximal tubule epithelial cell line (HK2) and mouse primary proximal tubule epithelial

cell (mPTEC) and to determine the effect of EGF cocktail on HK2 cells and their

differentiation.

123

4.2 Results:

4.2.1 Culture characteristics of Human Renal Proximal Tubular

Epithelial Cell line (HK2):

A sub confluent monolayer of HK2 cells was achieved in approximately one week when

seeded in a 75cm2 flask (figure 4.1 A). After 10 days of culturing the cells in (figure 4.1

B) show confluency, high cell density, and formation of typical domes (arrows). The

presence of domes is a routine feature of HK2 cells and is a hallmark of cultured renal

epithelial cells.

Domes are out-of-focus areas of the cell monolayer seen upon light microscopy

analysis. They are formed where fluid has become trapped underneath the monolayer

because of the active transport of ions and water across the cell monolayer. This is

thought to occur because of the typical apical to basolateral transport found in epithelial

cells. In a culture, this leads to local detachment of the monolayer from the plastic flask

surface, forming a raised area with an underneath reservoir of accumulated fluid (Kim

et al., 2002). The HK2-GF cells have the same culture morphology and dome

formations of HK2 features (data not shown).

Cells seem metabolically very active, as the phenol red containing medium acidifies

quicker than in other fast growing cultures such as J774 or HepG2.

124

10 X

Domes

Figure (4.1): Microscopic appearance of HK2 cells. (A) Shows HK2 cells grown

at low and (B) at high density. Cells are polygonal, sometimes spindle shaped (A)

and produce three-dimensional outgrowths, so-called domes (B) after about 10

days.

10 X 40 X

125

4.2.2 Transmission Electron Microscopy analysis (TEM) for HK2 cell

line:

For TEM, HK2 (+/-GF) cells were harvested by trypsin and fixed. The process carried

out by Natalie Allcock of the Electron Microscopy Laboratory, University of Leicester.

Figure (4.2) demonstrates the control HK2-GF cells; (A1) shows ultrastructural

evidence of microvilli reminiscent of brush border and (A2) nucleus in early prophase

of mitosis, because the chromosomes are visible. Numerous mitochondria are present in

figure 4.2 (B1), consistent with a metabolically active cell. Additionally, vacuoles are

typically found (figure 4.2 (B2)) (Liu et al., 2009). Figure 4.2 (C) shows the image of

the HK2 cell line, which has the same HK2-GF cell features that are explained in

figures 4.2 (A and B).

5 𝝁𝑴

1

2

126

5 𝝁𝑴

Figure (4.2): Transmission electron microscopy (TEM) for HK2 (+/-GF) cells.

They were harvested by trypsin, spun, and fixed. (A) HK2-GF and (C) HK2 cell

line. (A) Shows ultrastructural evidence of microvilli (1), nucleus (2) and (B)

Shows numerous mitochondria, consistent with a metabolically active cell (1) and

vacuoles of varying electron densities (2).

1𝝁𝑴

1

2

127

4.2.3 Scanning Electron Microscopy (SEM) analysis for brush border

of HK2 (+/-GF) cells:

SEM was used to visualise the basic characteristics of HK2 cells and in comparing the

cell surface of HK2 and HK2-GF cells; also, to evaluate the brush border that was made

by thousands of microvilli on the apical surface of epithelial cells.

All cells were cultured to confluence on glass cover slips in 12 well plates and fixed.

Then they used for electron microscopy, the subsequent procedure was carried out by

Natalie Allcock of the Electron Microscopy Laboratory, University of Leicester.

Images in (figure 4.3) characterise the observed morphology of HK2 cells. The brush

border is a typical differentiation of proximal tubule cells and microvilli constituents

appear clearly in HK2 cells (figure 4.3 (A2)). As well, (figure 4.3 (A2)) shows a

connecting net between the cells. Moreover, HK2 cells at 10μm magnification show

some ruffles and protrusions (figure 4.3 (A3)) and at higher magnifications (5 and 3μm)

typical microvilli appear on the cell surface (figure 4.3 (A4 and 5)).

On the other hand, images of HK2-GF first appear to be densely grown in (figure 4.3

(B2)) and in a possible dome formation (figure 4.3 (B3)). At 20μm magnification, cells

show ruffles, protrusions and densely packed microvilli (figure 4.3 (B4, B5 and B6)).

There was a difference in microvilli density between the HK2 and HK2-GF cells. The

HK2-GF showed densely packed microvilli compared to HK2 cells when focusing at

5μm magnification. This difference appears clearly by comparing (A5 to B6) in figure

4.3.

128

Microvilli

HK2 Cells

1

5

4 3

2

129

Microvilli

HK2-GF Cells

6 5

4 3

2 1

130

4.2.4 Villin-1 – A marker of brush border differentiation in renal cells:

Villin-1 is a specific protein marker for microvilli in tubular brush border cells. It

belongs to the actin-binding protein family. Villin-1 plays a role in the initiation,

organization, and formation of microvilli at the apical membranes of cells in developing

proximal tubules (Kang & Lee, 2014). Western blot was used to detect Villin-1 protein

expression from HK2 (+/-GF) cells. The cells were sub cultured in 6 well plates till 80%

confluence; cells were lysed and separated by SDS-PAGE as described in materials and

methods (see 2.9.3). Fresh mouse cortex and HEK293 cell lysates were used as positive

controls for mouse and human Villin-1 expression. A Villin-1 reactive band was

detected in the HK2-GF lysate and a fainter band in the HK2 cell lysate at the same size

of 95kDa, compared to the positive control HEK293 cells. Additionally, a clear band

was detected from the mouse cortex lysate. The same membrane was re-probed with β-

actin antibody to show protein loading (figure 4.4).

HK2-GF HK2 HEK293 Cortex

Villin-1

95kDa

𝜷-Actin

42 kDa

Positive controls

Figure (4.3): Typical scanning electron micrographs showing microvilli on the

surface of HK2 cells grown in a standard medium (A) or in cells in medium

supplemented with EGF (B). The cells were cultured in 12 well plates to achieve

80% confluency before fixing and processing of coverslips for SEM. Dome

formation is captured for cells grown in the presence of the EGF cocktail. In this

area, microvilli formation is particularly dense.

131

4.2.5 Mouse Proximal Tubular Epithelial Cells (mPTEC) preparation:

In some studies, to investigate or assess mechanisms of PTC physiology and

pathophysiology, primary cell culture systems can be helpful and are normally utilised

to avoid the complexity of whole organ or whole animal experiments. However,

isolation and preparation of primary cell cultures like mice has many disadvantages

such as a modest yield of PTC populations, contamination with other cell types (impure

culture) and they take a long time to grow. So, mouse PTCs were prepared to use as

positive control for some experiments in this project, and the initial idea to stimulate

them with albumin was abandoned.

Figure 4.5 illustrates the steps of isolation of the mPTEC culture obtained from the renal

cortex of C57BC/6 mice and was successfully grown to confluence. Figure 4.5 shows

(1) the morphology of isolated mouse renal tubules after sieving, (2) adherent polygonal

cells after 5 days plating the collagenase digest (3) confluent monolayer mPTECs

culture at days 7-10 and (4) cobblestone morphology with domes that have been

reported as a hallmark of mPTECs, which are partly a consequence of high cell density.

Figure (4.4): Western blot analysis of Villin-1 as a marker of brush border

differentiation. 20𝜇g of HK2 (+/-GF) was loaded in 12% SDS-PAGE gel. In

addition, mouse cortex and HEK 293 cell lysates were used as positive controls.

HK2-GF cells show more Villin-1 expression compared to HK2 cells. This

antibody crosses react (mouse and human). These Western blots are

representative of two individually performed experiments. The size of the Villin-1

band is as expected (same in the manufacture data sheet). The same protein

concentration was loaded from each sample (20𝜇𝑔) and β-actin was used to show

protein loading.

132

10 X

40 X

20 X 20 X

10 X

Tubule

Dome

1

2 3

4

Figure (4.5): Microscopic documentation of isolation of murine renal tubules (1),

outgrowth of adherent polygonal cells after 5 days (2), (3) confluency of cells

showing vacuolar inclusions after 7-10 days of incubating, evidence of lack of

contact inhibition at high cell densities after 10-14 days, dome formation (4).

133

4.2.6 Alkaline phosphatase enzyme marker of proximal tubular cells:

Alkaline phosphatase (ALP) is a normal constituent of the proximal tubular brush

border membrane. Kidneys were removed from C57BC/6 mouse, sectioned and reacted

with BCIP/NBT substrate to detect ALP in the cortex.

The representative image in figure 4.6 (A1) shows a positive blue area in the cortex of

the kidney section, which contains the renal tubules, indicative of ALP detection,

compared to the area of kidney medulla in the section with no blue staining. The area

with ALP detection appeared clear in higher magnifications 10X (A2) and 40X (A3),

respectively. 1% Neutral Red was used as a counterstain.

Figure 4.6 (B) demonstrates mPETCs, which were used as positive control for ALP

expression. They were prepared from C57BC/6 mouse kidneys as described (material

and methods, see 2.3.3). When mPETCs and HK2 cells in figure 4.6 (B and C) became

confluent in the 6 well plates, they were fixed and stained for ALP activity with a

Magenta substrate solution.

As shown in figure 4.6 (B1) the intensity of the staining was varied, with areas of

intense red or purple staining being separated by areas of less intense or no staining.

The amount of alkaline phosphatase positive cells varied and this may be due to the age

and confluency of the culture (Ryan et al., 1994). In contrast, all HK2 cells consistently

showed uniform staining for ALP (figure 4.6 (C)). Methyl green stain (Green colour

area) was used as a nuclear counterstain.

134

40 X

4 X 10 X

1

3

2

Cortex

Medulla

4 X 10 X

1 2

135

4.2.7 Human Proximal Tubule Epithelial cell culture:

The HK2 cell line was maintained as individually adapted sub-lines. One type was

grown in the medium supplemented with an EGF cocktail (HK2-GF), which was used

for primary proximal tubule epithelial cell culture, and the second type without an EGF

cocktail (materials and methods, see 2.1). HK2-GF cells grew and divided quicker than

HK2 cells. Also, they become confluent and formed domes faster. To study the HK2

(+/-GF) cells proliferation, both cell types were sub cultured with (2,500 cells/well)

density, incubated for different times, and the cell viability was indirectly determined by

MTT, crystal violet and LDH assays. Both cell types were sub cultured and analysed for

the proliferation experiments at the same time. HK2 (+/-GF) cells were initially

measured by adding the MTT solution to the cells after each time point figure 4.7 (A).

Figure (4.6): Histochemical alkaline phosphatase staining (ALP). (A) Paraffin

kidney section (5 micron) from wild type mice for brush-border ALP detection by

using (BCIP/NPT) substrate, which reacts with ALP, a marker enzyme for brush-

border, and become insoluble blue or black precipitate (the red arrow indicates the

area in (A1)). For the nuclear counterstain 1% Neutral Red stain was used (the red

area). Cytochemical staining for ALP activity, which is typically found in brush

border of epithelial cells with microvilli like (B) mPTECs and (C) HK2 cells. The

red or pink areas indicate the ALP activity. Methyl green stain (Green colour) was

used as a nuclear counterstain. There is a mixed population of cells positive for

ALP activity (A and B), with marked extent of enzymatic activity where cell

densities are high (B).

20 X

136

The mitochondrial activity of HK2-GF was increased significantly, double than of the

HK2 cells from the first 24h incubation, and this cell mitochondrial activity increased

with an increase in cell numbers; with HK2 (+/-GF) the mitochondrial activity increases

steadily with the time, but significantly more in HK2-GF. Similarly, cell viability was

determined by staining the cells after each time point, using crystal violet, which stains

the DNA of the adherent live cell.

Figure 4.7 (B) shows the number of the HK2-GF cells elevated gradually and

significantly more with time, compared with HK2 cells that increased and divided more

slowly. Lastly, cell proliferation was indicated by the LDH biomarker that can be

quantified in cell supernatant, which is normally released from injured or dead cells, for

the same experiment that was used to detect the cell viability indirectly by the MTT and

crystal violet to be comparable. As shown in figure 4.7 (C) by measuring the LDH

release from HK2 (+/-GF) cells after each time point, the LDH level increased

significantly with time in the HK2-GF cells compared to the HK2 cells after 96h.

Additionally, LDH released from the HK2 cells did not change in the first three days,

compared to the HK2-GF cells that showed more of an increase over the same three

days. For future stimulation work, the MTT and LDH release assays were chosen to

measured mitochondrial activity and cytoplasmic leakage, respectively.

In summary, by characterising the morphology and enzyme markers of HK2 (+/-GF)

cell line in this chapter, they appeared to be appropriate tool to study the cell injury and

repair in this project to establish the protein overload model in vitro and to study the

effect of proteinuria conditions on proximal tubular cells in kidney diseases.

137

Crystal Violet

24

h

48

h

72

h

96

h0.0

0.1

0.2

0.3

0.4

0.5

OD

(520 n

m)

HK2-GF

HK2

**

**

*

MTT assay

24h

48h

72h

96h

0.0

0.2

0.4

0.6

0.8O

D (

550

nm)

HK2-GF

HK2

***

**

*

**

138

Figure (4.7): Differences in proliferation in HK2 and HK2-GF cells. Cells were

grown for 24, 48, 72 and 96h at a density of 2500 cells/well in their respective

medium and different measures of proliferation were determined after each time

point using (A) MTT assay (Mitochondrial cell activity), (B) Crystal Violet (Cell

adherence) and (C) LDH assay (Cytoplasmic leakage). For LDH assay (C) the

supernatants were collected from the same cell experiments that were used to

detect the cell viability by MTT assay (A) and crystal violet (B) to be comparable.

The data are represented as means of triplicates ± SD (n = 2) (Unpaired t test P <

0.05).

C LDH assay

139

4.3 Discussion:

Proximal tubule epithelial cells (PTECs) are distinctly involved in renal disease because

of their sensitivity to injury (Liu et al., 2009). To create a protein overload model for

this project, which would allow for studying the PTECs physiology, injury and repair,

an isolated cell system was exposed to proteins PTECs might normally find themselves

exposed to.

Nowadays, there are many sources for human renal proximal tubule epithelium cells,

such as renal cell lines and primary cultures that are used for renal physiology and

nephrotoxicity studies (Van der Hauwaert et al., 2013). However, each source has

many advantages and disadvantages.

There are common problems with primary PTECs cultures such as the difficulty in

establishing pure PTECs cultures; the culture may be contaminated with different types

of kidney cells like distal nephron. Additionally, it is difficult to have high cell density

and reproducible characteristics from human primary PTECs cultures (Qi et al., 2007).

Also, commercial human PTECs cultures are very expensive and limited passage

numbers necessarily have the consequence of limiting the number of experiments.

The laboriousness of repeated isolation and confirmation of preparation uniformity

(Ryan et al., 1994) and inter individual differences adding to the inherent variation

when using this source (Van der Hauwaert et al., 2013) make alternatives attractive.

One of the advantages of using HK2 and HEK 293 cell lines is that they are easy to

grow for in vitro studies (Van der Hauwaert et al., 2013). However, in 1994 Ryan et al.

reported that as the HEK 293 cell line was transformed from original embryonic tissues,

they might not be ideal for mechanism studies of adult PTECs in physiology and

disease; furthermore, a systematic analysis of HEK293 cast doubt the stability of these

cells to study kidney epithelial cells become of their expression of neuunal protein

(Shaw et al., 2002). Because of these reasons, HEK293 was used in this project merely

as a positive control in some of the experiments.

Given the above considerations, the primary goal was to characterise the HK2 cells to

ensure their phenotypic purity, confirm the published inflammatory phenotype and

investigate other features that make HK2 cells comparable to primary cultured cells.

140

This was so much more important as Van der Hauwaert et al. (2013) assumed that the

HK2 cell line, because of long-term culture and multiple passaging, would gradually

lose its specific functions, epithelial phenotype and characteristics. In contrast, cultured

primary PTECs retain their phenotypic characteristics and specific functions, like brush

border enzymatic activity.

Ryan et al. (1994) prepared primary PTECs from adult male human kidney tissue and

because of the disadvantages of using primary PTECs; they transformed the primary

cells to establish an immortalised adult human kidney proximal tubular cell line

retaining the normal phenotypic expression. Transforming the cells with HPV-16 gene

resulted in a cell line that survived for more than one year in culture and can be frozen

in liquid nitrogen and thawed to reuse.

The cells reportedly showed clear microvilli that made the brush border, which is

observed in the primary culture. The transformed HK2 cells possessed several typical

brush border enzymes, like alkaline phosphatase and gamma glutamyl transpeptidase

that suggest normal phenotypic expression. Furthermore, by immunochemistry the cells

stained positively with cytokeratin, which is characteristic of epithelial cells. By

contrast, they did not stain for Factor VIII-related antigen or with antibodies for

CALLA endopeptidase, which are endothelial cell markers. Finally, a cell injury

experiment (provoked by exposure of the cells to H2O2) showed that HK2 cells

proliferation was markedly inhibited, consistent with the effect of H2O2 on freshly

isolated proximal tubular cells. Taken together, the cells are good model for cell

cytotoxicity studies.

The HK2 cell line in this project was prepared from adult male human kidney (HPV-16

transformed). Different studies that use primary human PTCs grow these cells in a

medium with EGF as a required supplement. In addition, the source of HK2 cells that

were used in this project recommends adding EGF to the culture medium. EGF is one of

EGFR ligands in kidney cells. EGFR can be activated under physiological or

pathophysiological conditions by several mechanisms. Depending on the activating

ligand, EGFR has different biological effects such as cell proliferation, differentiation

and survival (Zhuang & Liu, 2014).

However, some studies on HK2 cells avoid adding

EGF in culture media for two reasons. Firstly, to avoid a confounding effect of EGF on

cells when they are treated with excess proteins, as in this project. Secondly, although

141

EGFR/EGF contributes in renal repair, it is also involved in the development and

progression of renal fibrosis. One study showed decrease in renal fibrosis in mice with

deletion of EGFR in proximal renal tubular cells after angiotensin II infusion (Tang et

al., 2013). So, the HK2 cells in this study were grown in the same medium (DMEM-

F12) but with different supplements, namely with and without EGF cocktail, which was

used to allow differentiation in primary PTCs cultures.

The HK2 (+/-GF) cells behaved similarly; they showed typical epithelial morphology

with high cell density and dome formation after 10 days of confluency. Van Der Biest et

al., 1994 showed dome formation in primary human PTCs after twelve days of

confluency, dome formation being a routine feature of tubular cells.

Ultrastuctural analysis of HK2 (+/-GF) cells showed clear evidence of microvilli and

noticeably numerous mitochondria. Qi et al. (2007) demonstrated the cellular

ultrastructure for human PTC monolayer using TEM grown in a medium supplemented

with EGF. These primary cells are characterised by apical microvilli and close

association of mitochondrial rich cells.

HK2 cells grown without EGF had also been shown to be rich in microvilli, rough

endoplasmic reticulum and mitochondria (Liu et al., 2009). The brush border and

microvilli on the HK2 cells’ surface were evaluated clearly with SEM. The HK2-GF

cells showed densely grown cells with possible dome formation compared with HK2

cells at the same time of growing. In addition, on the HK2 cells’ surface typical

microvilli appeared with some ruffles, however, microvilli were densely packed on the

HK2-GFs’ surface. In 2008, Wieser and his group, who investigated HK2 cells that

were grown in media supplemented with EGF, showed densely packed microvilli and

solitary cilia by SEM.

Villin-1 is a specific protein marker for microvilli and is required for the assembly of

microvilli at the apical membranes of cells in developing proximal tubules (Ongeri et

al., 2011). In 1986, Grone and his group showed that the brush border of the proximal

tubular epithelium in sections of normal human kidney was stained strongly positive

with the Villin-1 antibody, compared with negative staining for Villin-1 in distal tubules

and medullary tubular parts in the kidney. Similarly, our results showed the presence of

Villin-1 in HK2 (+/-GF) cells and also HEK293 cells, which were used as a positive

142

control. Additionally, cortex from C57BC/6 mouse tested positive for Villin-1

expression. However, the product size for Villin-1 was different between the HK2 (+/-

GF) cells and the mouse cortex samples, and this could be due to post translation

modification, such as glycosylation. The result sizes for mouse and human Villin-1

were consistent with manufacturer’s information.

To further characterise HK2 (+/-GF) cells, cell functionality of alkaline phosphatase,

which is one of the enzyme markers and a normal constituent expressed by proximal

tubular brush border membrane, was tested. According to Van Der Biest and his group

in 1994, who reported a lower activity of alkaline phosphatase in the primary human

PTCs compared to the value of fresh renal tissue from medulla, this could be due to a

loss of alkaline phosphatase during the preparation of PTCs from kidney tissue. In

contrast, our results showed strongly positive alkaline phosphatase in the cortex of the

mouse kidney sections, which contains the renal tubules, compared to the area of kidney

medulla.

In 2012, Kamiyama et al. reported that alkaline phosphatase activity was high in

purified mouse proximal tubules; similarly, the primary human PTCs contained high

alkaline phosphatase activity (Qi et al., 2007).

Comparably, the mPTECs that were used as positive control for alkaline phosphatase

staining in vitro showed that the intensity of the staining was variable; the proportion of

alkaline phosphatase positive cells varied with confluency of the culture. In contrast, the

HK2 (+/-GF) cells consistently demonstrated uniform staining for alkaline phosphatase,

so the HK2 (+/-GF) cell line showed similar alkaline phosphatase expression to the

primary proximal tubules.

Lastly, it was noticed that the proliferation was different between the HK2-GF and HK2

cells. The HK2-GF grew, divided and became confluent faster than HK2 cells, which

were measured in several ways (viability and activity assays). The EGF cocktail that

was added to the medium for cell differentiation is likely to have had a role in the

proliferation of HK2-GF cells.

In summary, the present chapter describes that an immortalized adult human proximal

tubular cell line, HK2 (+/-GF), has similar functional and morphologic characteristics to

normal primary adult human proximal tubular epithelium cell culture, as available from

143

the literature. In addition, HK2 (+/-GF) cells could prove to be a powerful and positive

choice for the study of the proteinuria model for this project, including physiology,

pathophysiology and mechanisms of cell injury and repair.

144

Chapter Five- Establishing an in vitro

Model of Protein-Induced Epithelial Cell

Damag

145

5. Introduction:

The studies in this chapter examine the effect of protein overload on the cellular integrity of

PTECs. In progressive renal disease, PTECs are exposed to high levels of protein like

albumin or LC in MM patients. Several investigators have examined the ability of large

quantities of albumin and LC to cause damage in HK2 cells, but data on this issue is

conflicting.

Albumin is the most abundant protein in proteinuric tubular fluid. Several investigators

have examined the ability of large quantities of albumin to cause cytotoxicity in PTCs

(Arici et al., 2003). Also, recent studies have shown that a significant amount of LC, which

is a low molecular weight protein to be endocytosed into PTECs, is capable of causing cell

damage and may lead to kidney disease (Wang & Sanders, 2007). A series of experiments

designed in this chapter to pursue the hypothesis that overload albumin and LC proteins

have a toxic effect on the HK2 (+/-GF) cells.

The cytotoxic effect of overload proteins (FAF-HSA and 𝜆-LC) was measured indirectly by

MTT assay to capture cellular metabolic activity and cell viability. In addition, to support

the MTT results, the LDH release was measured as well, which is a marker for lethal cell

injury in vitro because the enzymatic activity is relatively stable in a cell culture medium

and can be measured easily after leakage out of cells with a compromised membrane (Riss

& Moravec, 2005).

In addition, one of the effects of protein overload includes probably triggering the

autophagy pathway. Takabatake et al., 2014 demonstrated that proximal tubules expend a

large amount of energy through the reabsorption process and these tubules contain large

number of mitochondria that provide the energy for this reabsorption. The lysosomal

machinery plays a role in the reabsorption and degradation of albumin and low molecular

weight proteins like LC from the glomerular filtrate. Consequently, autophagy possibly

plays an important role in proximal tubules. Therefore, the intracellular response of HK2

(+/-GF) cells stimulated with FAF-HSA or 𝜆 -LC was investigated by TEM, which

specifically evaluates autophagy.

146

In pathological conditions like protein overload may contribute to progressive renal damage

by inducing apoptosis, tubular cell injury and death. Apoptosis is a physiologic mechanism

for eliminating unwanted cells like ischemia and nephrotoxicity injury. Several caspases

are involved in the apoptotic process, and they play an essential role during the initiation of

apoptotic cell death. Caspase-3 is an important protease in the execution of apoptosis (Lee

et al., 2013). So, apoptosis and caspase-3 were measured in HK2 (+/-GF) cells stimulated

with FAF-HSA or 𝜆-LC.

Lastly, several kidney injury markers were measured in mRNA levels, due to their allowing

for monitoring the disease progression at initial and early stages such as TGF-𝛽1 cytokine.

It is one of the critical genes in the pathogenesis of kidney disease and promotes cell

apoptosis via the activation of caspases that cause renal fibrosis (Hsieh et al., 2012). In

addition, it is involved in widely different biological processes like cell

growth/proliferation, differentiation, and regulation of the immune system (Sullivan et al.,

2009). Also, it was reported that the up-regulation of TGF-𝛽1 expression is consisted in

most fibrotic disease (Sullivan et al., 2009). Furthermore, TNF-𝛼 is one of the cytokines

produced by many cell types in response to infection or injury, and it may modulate the

cellular differentiation, proliferation and apoptosis (Sullivan et al., 2009). Also, KIM-1 is

markedly expressed by PTECs but plays a conflicting role in kidney injury and healing

(Lim et al., 2014). The role of KIM-1 in our condition was discussed in this chapter.

Finally, clusterin is a further kidney injury marker (Vaidya et al., 2008); clusterin protein

translated in the renal tubular epithelium cells after expression of clusterin mRNA

following the kidney injury (Khan & Pandey, 2014).

147

5.1 Aim:

The aim of these experiments was to determine and quantify the effects of protein overload

on the HK2 (+/-GF) cells in order to establish a model of protein induced epithelial cell

damage, study disease mechanisms using this model in vitro and to monitor the response

and health of cells in culture after treatment with various stimuli. To do this, commercially

available human serum albumin devoid of fatty acid and purified immunoglobulin light

chains were used in parallel.

148

5.2 Results:

5.2.1 Dose and time dependent influence of FAF-HSA and 𝝀-LC on cell

viability:

5.2.1.1 MTT Assay:

The effect of high dose FAF-HSA and 𝜆-LC on HK2 cells grown in different media (+/-

GF) was analysed using the photometric MTT assay, which detects changes in

mitochondrial activity.

HK2 (+/-GF) cells were stimulated with FAF-HSA (5, 10, 20 and 30mg/ml in serum free

medium) for different time periods and un-stimulated cells in serum free medium served as

a control for each time point.

At early time points (2, 4 and 6h stimulation) over a range of FAF-HSA concentrations,

there was no evidence of a toxic effect on both types of HK2 (+/-GF) cells (figure 5.1).

Also, when stimulating the HK2-GF cells with the same concentrations of FAF-HSA for

longer periods, a distinctly different observation was made: after the 24 and 48h incubation

period, there was no significant change in cell viability compared to the control for each

time point separately. In contrast, at 72h, there was a major decrease in the number of the

cells after stimulation with FAF-HSA compared to the control, and this gradually decreased

as the concentration of FAF-HSA increased (figure 5.2 A). Thus, there is an inverse

relation between the viability of the cells and the concentration of FAF-HSA at this time

point.

However, HK2 cells showed reduced cell viability with all different FAF-HSA

concentrations compared to the un-stimulated control from the first 24h of stimulation and

even more pronounced at 48 and 72h. There appears to be an inverse relationship between

cell viability and the concentration of FAF-HSA: The greater the concentration of FAF-

HSA, the lower the reading of mitochondria activity (figure 5.2 B).

149

While activity levels of control cells remain stable for 24 and 48h, there is an increase of

activity for HK2 (+/-GF) at 72h, suggesting increased proliferation at this time point in the

presence of the EGF cocktail. The effect, however, is not sufficient to inhibit the toxic

effect of high doses of FAF-HSA. In addition, HK2-GF shows a significant protection from

the FAF-HSA induced toxic effect compared to HK2 at all-time points, with no decrease in

the number of the cells in the first 24 and 48h and a significant decrease at 72h with the

higher concentration of FAF-HSA. In contrast, the HK2 cells show a significant decrease

from the first 24h with the higher concentration of FAF-HSA. For example, with

(30mg/ml) FAF-HSA the HK2 cells showed significant reduction from the initial 24h

incubation up to 72h, however, FAF-HSA with the same concentration had no effect on

HK2-GF cells at 24 and 48h but a significant decrease at 72h.

Next, HK2 (+/-GF) cells were sub-cultured and stimulated with 𝜆-LC (1, 5 and 10mg/ml in

a serum free medium) for 24, 48 and 72h. Un-stimulated cells in a serum-free medium were

used as controls for each time point. 𝜆-LC at 10mg/ml was found to significantly impair

HK2-GF cell viability after 24h of incubation when compared with un-stimulated cells. By

contrast, after 48 and 72h of stimulation with a range of 𝜆-LC between (1 and 10 mg/ml),

cell viability decreased compared to the control (figure 5.3 A).

Also, 𝜆-LC showed evidence of a toxic effect in the first 24h incubation time of HK2 cells,

however, there was a significant decrease in cell viability after 48 and 72h of incubation

time with all 𝜆-LC concentrations (1-10 mg/ml) compared with the control (figure 5.3 B).

150

Figure (5.1): Effects of FAF-HSA overload on cell viability measured using MTT

assay in HK2 (+/-GF) cells. The HK2 (+/-GF) cells were cultured at density

(2,500 cells/well) for 24h and treated with ascending concentrations of FAF-HSA

(5, 10, 20 and 30mg/ml) in serum free media. After incubation for 2, 4 and 6h

mitochondrial activity, indicative of cell viability, was determined and expressed

as photometrical densities of the solubilised end product, formazan. Exposure to

FAF-HSA showed on change in cell viability for HK2 (+/-GF) cells. Un-treated

cells were used as control. The data are represented as means of triplicates ± SD

(n = 3) (Unpaired t test p < 0.05vs. control), C: control sample.

2h

4h

6h

C 5

10

20

30

C 5

10

20

300.0

0.1

0.2

0.3HK2-GF

HK2

OD

(55

0 nm

)mg/ml

c 5

10

20

30 c 5

10

20

300.0

0.1

0.2

0.3HK2-GF

HK2

OD

(55

0 nm

)

mg/ml

c 5

10

20

30 c 5

10

20

300.0

0.1

0.2

0.3HK2-GF

HK2

OD

(550 n

m)

mg/ml

151

C 510

20

30 C 5

10

20

30 C 5

10

20

300.0

0.5

1.0

1.5

OD

(55

0nm

)

24h

48h

72h

mg/ml

*

HK2-GF Cells

C 51

02

03

0 C 51

02

03

0 C 51

02

03

00.0

0.5

1.0

1.5

OD

(550nm

)

24h

48h

72h

*** ** ***

mg/ml

HK2 Cells

Figure (5.2): Effects of FAF-HSA overload on cell viability measured using MTT

assay in HK2 (+/-GF) cells. The HK2 (+/-GF) cells were cultured at density

(2,500 cells/well) for 24h and treated with ascending concentrations of FAF-HSA

(5, 10, 20 and 30mg/ml) in serum free media. After incubation for 24, 48 and 72h

mitochondrial activity, indicative of cell viability, was determined and expressed

as photometrical densities of the solubilised end product, formazan (A) HK2-GF

and (B) HK2 cells. Exposure to FAF-HSA showed a dose and time-dependent

decrease in cell viability for HK2 cells, however, a significant decrease in cell

viability after 72h with the highest concentration of stimulant in HK2-GF cells.

Un-treated cells were used as control. The data are represented as means of

triplicates ± SD (n = 3) (Unpaired t test p < 0.05vs. control), C: control sample.

152

Figure (5.3): Effects of 𝜆-LC on cell viability measured using MTT assay in HK2

(+/-GF) cells. The HK2 (+/-GF) cells were cultured for 24h and treated with

ascending concentrations of 𝜆-LC (1, 5 and 10mg/ml) in serum free media. After

incubation for 24, 48 and 72h mitochondrial activity, indicative of cell viability,

was determined and expressed as photometrical densities of the solubilised end

product, formazan. (A) HK2-GF and (B) HK2 cells exposure to 𝜆-LC showed

decrease in cell viability after 48 and 72h time points in relation to ascending 𝜆-

LC concentration compared to the control. Un-treated cells were used as control.

The data are represented as means of triplicates ± SD (n = 3) (Unpaired t test p <

0.05 vs. control), C: control sample.

c 1 5

10 c 1 5

10 c 1 5

100.0

0.1

0.2

0.3

0.4

OD

(550

nm)

mg/ml

** ***

*

**

c 1 5 10

c 1 5 10

c 1 5 10

0.0

0.1

0.2

0.3

0.4

OD

(59

5nm

)

24h

48h

72h

mg/ml

** ***

*

HK2-GF Cells

HK2 Cells

c 1 5

10 c 1 5

10 c 1 5

100.0

0.1

0.2

0.3

0.4

OD

(550nm

)

Comparing GF LC MTT

24h

48h

72h

mg/ml

*

**

*

153

5.2.1.2 LDH Activity Assay:

To further quantify cytotoxicity of FAF-HSA and 𝜆-LCon HK2 (+/-GF) cells, the Lactate

Dehydrogenase assay (LDH) was used to capture cell membrane damage. While a decrease

in optical density in the MTT assay signifies decreased metabolic activity or cell numbers,

an increase in optical density in the LDH assay quantifies the extent of cytoplasmic

leakage.

Integrity of HK2 (+/-GF) cells was analysed by LDH release into the culture media after

stimulation with FAF-HSA (5, 10, 20 and 30mg/ml) in serum free media for 24, 48 and

72h. Un-stimulated cells in serum-free media served as controls and medium alone for

background.

At the beginning HK2 (+/-GF) cells were seeded (2,500 cells/well) in 96 well plates for

each time point separately, stimulated, and then the activity of LDH released in the media

was measured at the end of the stimulation. At 24 and 48h of stimulation over a range of

FAF-HSA concentrations, there was a significant increase in LDH media release, however,

after 72h the LDH activity was significantly reduced compared with activity in the previous

time points on both types of HK2 (+/-GF) cells (figure 5.4 (A and B)). This was

unexpected. Therefore, the experiments were repeated with increased cell numbers. The

HK2 cells (+/-GF) were seeded (5,000 and 10,000 cells/well) and the experiments were

performed as described. It was found that the LDH activity was significantly increased at

higher concentrations of FAF-HSA compared to the un-stimulated control. There was an

incremental increase of LDH release from cells stimulated with increasing concentration of

FAF-HSA over 24, 48 and 72h. The greatest cytoplasmic leakage was observed for all test

samples at 72h. Control cells showed increased LDH release at 48 up to 72h, probably due

to an increase in density (figure 5.5 and 5.6).

Comparing the LDH release after 72h of stimulation in HK2-GF and HK2 cells, it is

notable that the maximum LDH activity was achieved in HK2 cells, suggesting a possibly

protective effect of the EGF cocktail in the presence of high albumin concentration (20 and

30mg/ml) with HK2-GF cells (figure 5.6).

154

From the pilot experiments were conducted in order to establish the optimal cell number

needed to analyse protein induced cell damage at different time points using different FAF-

HSA concentrations. From this, it was concluded that (10,000 cells /well) was optimal to

analyse the impact of 𝜆-LC on HK2 (+/- GF) cell viability.

HK2 (+/-GF) cells were incubated with 𝜆-LC (1, 5 and 10mg/ml) in a serum free medium;

un-stimulated cells in serum free media were controls and medium alone was used for

background measurement. There was no significant difference between LDH releases from

HK2 (+/-GF) cells treated with different 𝜆-LC concentration at 24h (figure 5.7), while, after

48h there was a statistically significant increase in the supernatant LDH presence with (5

and 10mg/ml) 𝜆-LC concentrations from HK2-GF (figure 5.7 A) and with (10mg/ml) from

HK2 cells (figure 5.7 B). At 72 h, treating HK2 (+/-) GF with 𝜆-LC caused the largest LDH

release compared to the un-stimulated control (figure 5.7 (A and B)).

In summary, from MTT and LDH assays results suggest that high FAF-HSA and 𝜆-LC

concentrations cause reduction in cell viability and elevation in LDH released in the

supernatants of HK2 (+/-GF) with a long time of incubation.

5.2.1.2.1 PTECs Protein Overload Model:

In order to have sufficient numbers of cells for several analyses in this project, it was

necessary to provide evidence that the stimulation model performed so far in a 96 well

plate’s format (see 5.2.1) was transferable to a 6 well plate format. A photometric LDH

activity assay was used to assess whether FAF-HSA or 𝜆-LC overload causes cell injury.

This was confirmed successfully by measuring the LDH leakage as a marker of damage

induced by FAF-HSA and 𝜆-LC, therefore providing cells and supernatants for subsequent

analyses in (chapter 7).

HK2 (+/-GF) cells were transferred into 6 well plates with density (5 x 105 cells/well),

incubated for 24 and 72h with (5mg/ml) FAF-HSA or 𝜆-LC, and cells with no added

protein in the medium were used as controls. Figure 5.8 (A) shows a significant rise in the

LDH release in supernatants from HK2 (+/-GF) cells treated with FAF-HSA at 72h

155

compared to the control. In contrast, released LDH from HK2 (+/-GF) cells after 𝜆-LC

stimulation was increased significantly after the first 24h of incubation. While after 72h of

incubation the LDH level was elevated more compared with the 24h level from both HK2

(+/-GF) cells, by comparing both cellular medium conditions, HK2 cells showed a much

higher LDH release than HK2-GF at 72h (figure 5.8 (A)). The addition of the EGF cocktail

appears to exert a protective effect when 𝜆-LC induced cell damage. This effect is not

pronounced when incubating the cells with FAF-HSA. In figure 5.8 (B) the red colour

reflects the amount of the LDH activity in the sample, and it can be seen that HK2 (+/-GF)

cells showed more intense colour with longer time stimulation with HSA-FFA and 𝜆-LC at

72h compared to the faint reaction for the control or low LDH activity sample; however,

maximum intensity appeared with 𝜆 -LC stimulation. Thus, as with the previous

experiments in (see 5.2.1.2), it can be conclusively proven that FAF-HSA and 𝜆-LC exert

cytotoxic effects on HK2 (+/-GF) cells.

156


measurement of LDH. HK2 (+/-GF) cells were cultured overnight (2,500

cells/well) in 96 well plates. Following this, the cells were treated with (5, 10, 20

and 30mg/ml) FAF-HSA in serum free media for 24, 48 and 72h. Cytoplasmic

leakage was quantified by measuring released LDH concentrations in the

supernatants. (A) HK2-GF and (B) HK2 cells. Un-treated cells were used as the

control. In addition, the LDH was measured in the standard medium alone for

each type of cells to use as a background control. The data are represented as

means of triplicates ± SD (n = 3) (Unpaired t test p < 0.05vs. control), C: control

sample.

HK2-GF (2,500 cells/well)

HK2 (2,500 cells/well)

c 51

02

03

0 c 51

02

03

0 c 51

02

03

00.0

0.1

0.2

0.3

OD

(490nm

)

24h

48h

72h

mg/ml

Med

ium

c 51

02

03

0 c 51

02

03

0 c 51

02

03

00.0

0.1

0.2

0.3

OD

(490

nm)

24h

48h

72h

mg/ml

Med

ium

157



cells/well) in 96 well plates. Following this, the cells were treated with (5, 10,

20 and 30mg/ml) FAF-HSA in serum free media for 24, 48 and 72h.

Cytoplasmic leakage was quantified by measuring released LDH

concentrations in the supernatants. (A) HK2-GF and (B) HK2 cells. Un-

treated cells were used as the control. In addition, the LDH was measured in

the standard medium alone for each type of cells to use as a background

control. The data are represented as means of triplicates ± SD (n = 3)

(Unpaired t test p < 0.05vs. control), C: control sample.



c 51

02

03

0 c 51

02

03

0 c 51

02

03

00.0

0.1

0.2

0.3

OD

(490 n

m)

24h

48h

72h

mg/ml

Med

ium

c 51

02

03

0 c 51

02

03

0 c 51

02

03

00.0

0.1

0.2

0.3

OD

(490 n

m)

24h

48h

72h

mg/ml

Med

ium

158

c 5 10 20 30

c 5 10 20 30

c 5 10 20 30

0.0

0.1

0.2

0.3

OD

(490

nm)

24h

48h

72h

mg/ml

**

Med

ium

**

**



c 5 10 20 30

c 5 10 20 30

c 5 10 20 30

0.0

0.1

0.2

0.3

OD

(490

nm)

24h

48h

72h

mg/ml

Med

ium

**

*

*



cells/well) in 96 well plates. Following this, the cells were treated with (5, 10,

20 and 30mg/ml) FAF-HSA in serum free media for 24, 48 and 72h.

Cytoplasmic leakage was quantified by measuring released LDH

concentrations in the supernatants. (A) HK2-GF and (B) HK2 cells showed

that there was a steady increase in LDH release in relation to ascending FAF-

HSA concentrations. Significant differences are indicated compared to the

control. Un-treated cells were used as the control. In addition, the LDH was

measured in the standard medium alone for each type of cells to use as a

background control. The data are represented as means of triplicates ± SD (n =

3) (Unpaired t test p < 0.05vs. control), C: control sample.

159

Figure (5.7): Impact of 𝜆-LC on HK2 (+/-GF) cells viability, assessed by


cells/well) in 96 well plates. Following this, the cells were treated with (1, 5

and 10mg/ml) 𝜆-LC in serum free media for 24, 48 and 72 h. Cytoplasmic

leakage was quantified by measuring released LDH concentration in the

supernatants. (A) HK2-GF and (B) HK2 cells showed an increase in LDH

release in relation to ascending 𝜆-LC concentration after 48 and 72h of

incubation time compared to the control. Un-treated cells were used as the

control. In addition, the LDH was measured in the standard medium alone

for each type of cells to use as a background control. The data are

represented as means of triplicate ± SD (n = 3) (Unpaired t test p < 0.05vs.

control), C: control sample.



c 1 51

0 c 1 51

0 c 1 51

00.0

0.1

0.2

0.3

OD

(49

2nm

)

Comparing HK2 LC LDH

24

48

72

mg/ml

Med

ium

**

****

c 1 51

0 c 1 51

0 c 1 51

0

0.0

0.1

0.2

0.3

OD

(490nm

)

Comparing GF LC LDH

24h

48h

72h

mg/ml

Med

ium

****

****

160

FA

F-H

SA

GF

HK2

HK2

C S C S

24h 72h

GF

𝝀-L

C

C2

4h

24

h

C7

2h

72

h

C2

4h

24

h

C7

2h

72

h

C2

4h

24

h

C7

2h

72

h

C2

4h

24

h

C7

2h

72

h0.0

0.5

1.0

1.5

OD

(490nm

)

LC

HSA-FFA

Med

ium

***

**

*** ***

*

***

***

GF HK2GF HK2

FAF-HSA

𝜆-LC

Figure (5.8): Impact of 24 and 72h incubation of HK2 (+/-GF) cells with excess

amounts of FAF-HSA or 𝜆-LC on LDH release into the medium. HK2 (+/-GF) cells

were cultured overnight (5 x 105 cells/well) in 6 well plates. Following this, the

cells were treated with (5mg/ml) FAF-HSA or 𝜆 -LC in serum free media.

Cytoplasmic leakage was quantified by measuring released LDH concentration in

the supernatants. (A) FAF-HSA and 𝜆-LC proteins increased LDH release in a

time-dependent manner’ significant differences were indicated compared to the

control. Un-treated cells were used as controls. In addition, the LDH was measured

in the standard medium alone for each type of cell to use as a background control.

(B) 50𝜇𝑙 aliquot from supernatants measured in A are shown in B. The data are

presented as means of triplicates ± SD (n = 4) (Unpaired t test p < 0.05 vs. control).

C: control sample, and S: stimulated sample.

161

5.2.2 Autophagy as a response of HK2 (+/- GF) to cell damage by protein

overload:

Transmission electron microscopy (TEM) was chosen to detect the presence of autophagy

in cells exposed to protein overload. HK2 (+/-GF) cells were sub-cultured in 6 well plates

and stimulated with FAF-HSA or 𝜆 -LC (5mg/ml) for 24 and 72h, or with 0.2μM

tunicamycin for 16h (positive control to induce autophagy by endoplasmic reticulum stress)

(Moon et al., 2014). The samples were detached, spun and fixed, then processed for TEM.

The subsequent procedure was carried out by Natalie Allcock of the Electron Microscopy

Laboratory, University of Leicester.

Time point matched un-treated cells were used as controls. This analysis benefits from

literature describing ultrastructural criteria of autophagic vacuole formation.

Representative images for stimulated cells with (FAF-HSA, 𝜆-LC and tunicamycin) and

un-stimulated were chosen from a total number of about (150 for HK2-GF with FAF-HSA

and 𝜆 -LC), (130 for HK2 with FAF-HSA and 𝜆 -LC) and (50 for HK2-GF with

tunicamycin) images.

In the electron micrographs, different stages of autophagocytosis were seen in HK2-GF

cells exposed to tunicamycin. Figure 5.9 (A and B) show early/initial autophagosomes

containing intact cytosol or organelles in double membranes, whereas (C and D) show

autophagolysosomes (late autophagy) with more or less degraded cytoplasmic material.

Compared to figure 5.10, the control HK2-GF cell has a normal appearing lobulated

nucleus, empty vesicles, organelles and numerous microvilli. Furthermore, the cells in

(figure 5.10 (A2 and B2)) are in early prophase of mitosis because the chromosomes are

visible and the cell nucleus is still intact.

162

Stimulation HK2-GF cells with FAF-HSA for 24h (figure 5.11 (A1 and A2)) produces

multilamellar bodies. Multilamellar bodies are lysosomal organelles expressed under

various physiological and pathological conditions, and several factors, like autophagy,

contribute in lamellae formation within secondary lysosomes and autolysosomes (Lajoie et

al., 2005). Morphologically, these bodies are possibly lipid storage or organelles that could

be surrounded by a membrane and have a core composed of multilamellar membranes

(Schmitz & Muller, 1991). However, (figure 5.11 (A6)) shows multilamellar vesicles that

have many membrane layers, and multivesicular vesicles encapsulate smaller vesicles. In

(figure 5.11 (A3 and A4)) autophagylysosomes are seen, filled with cellular debris that is

formerly called the degradative or late autophagic vacuoles (Liu et al., 2014). Also, after

72h FAF-HSA stimulation, multilamellar body (figure 5.12 (A1 and A2)) and numerous

autophaglysosomes were clearly detected (figure 5.12 (A3 and A4)) compared with the

controls (figure 5.10).

Next, exposure of HK2 cells to FAF-HSA for 24h illustrates autophaglysosome (figure

5.14 (A1)) and a significant number of unilamellar vesicles with single bilayer, varying

considerably in size (100 nm to 2400 nm) ((figure 5.14 (A2)) (van Swaay, 2013).

Moreover, figure 5.14 (A3) demonstrates a large number of vesicles that could be lipid

vesicles. The overload of FAF-HSA on HK2 cells for the 72h (figure 5.15 (A1)) also shows

visibly large autophagosome compared to control cells (figure 5.13).

The overall morphology of un-treated control cells with a serum free medium alone for 24

and 72h appeared normal with a lobulated nucleus. Numerous mitochondria and microvilli,

typical of differentiated proximal tubular cells, were observed (figure 5.13).

However, exposure of HK2-GF to 𝜆 -LC for 24h shows the different stages of

macroautophagy, a type of autophagy. It is a multistep process starting with the phagophor

stage, which is a double membrane that encloses and isolates the cytoplasmic components

during the macroautophagy (figure 5.16 (A3)) and autophagosome. Figure 5.16 (A4) shows

the last stage in the macroautophagy process that is the autophaglysosomes. The HK2-GF

cells show the same results after stimulation with 𝜆-LC for the longer time of 72h, the

different stages of macroautophagy (figure 5.17 (A1)) and autophaglysosomes (figure 5.17

(A2)). However, it showed a huge number of lipid vesicles (figure 5.17 (A3)) compared to

163

empty vesicles in control cells (figure 5.10 (A2)). The HK2 cells after 𝜆-LC treating for the

same time point display the same response of HK2-GF, phagophor with ribosomes (figure

5.18 (A2 and A3)) and autophaglysosomes (figure 5.18 (A1)) at the 24 and 72h (figure

5.19).

In summary, the same concentration of FAF-HSA and 𝜆 -LC produce comparable

morphological changes in both HK2 (+/-GF) cells.

164

5 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎 1 𝝁𝒎

5 𝝁𝒎

Figure (5.9): Representative transmission electron micrographs (TEM) showing different

stages of autophagocytosis in cells exposed to (0.2 μM) tunicamycin for 16h (positive

control for autophagy). (A and B) autophagosome (early autophagy) (vacuoles with double

membrane), (C and D) autophaglysosomes with degraded cytoplasmic material (late

autophagy), (E) mitochondria.

165

Control 72h HK2-GF

1 2 5 𝝁𝒎 5 𝝁𝒎

Control 24h HK2-GF

2 5 𝝁𝒎

1 5 𝝁𝒎

Figure (5.10): Representative transmission electron micrographs (TEM) of HK2-GF cells

incubated in serum free media for 24 and 72h (control cells). The cells have a lobulated

nucleus, microvilli (A1), (A2) empty vesicles, (B1) are rich in mitochondria. The cells in

(A2) and (B2) are in early prophase of mitosis because the chromosomes are visible and the

cell nucleus is still intact.

166

FAF-HSA 24h HK2-GF

1 𝝁𝒎 5 𝝁𝒎

5 𝝁𝒎

5 𝝁𝒎

1

5 6

4 3

2

1 𝝁𝒎

5 𝝁𝒎


treated with (5mg/ml) FAF-HSA for 24h. (A1 and A2) show multilamellar body. (A3 and

A4) show autophaglysosomes with degraded cytoplasmic material. (A5) shows

autophagosome and finally (A6 ) shows multilamellar vesicles.

167

FAF-HSA 72h HK2-GF

3 4

2 1

1 𝝁𝒎

5 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎


treated with (5mg/ml) FAF-HSA for 72h. (A1 and A2) show multilamellar body. (A3 and

A4) show autophaglysosomes with degraded cytoplasmic material.

168

Control 72h HK2

Control 24h HK2

5𝝁𝒎

5𝝁𝒎

1

1 2

2

1 𝝁𝒎

1 𝝁𝒎


incubated in serum free media for 24 and 72h (control cells). The cells have a lobulated

nucleus (A1). The cells in (A2) and (B2) are in the early prophase of mitosis because the

chromosomes are visible and the cell nucleus is still intact.

169

FAF-HSA 24h HK2

1 𝝁𝒎

1 𝝁𝒎

3

2 1 1 𝝁𝒎


treated with (5mg/ml) FAF-HSA for 24h. (A1) show autophaglysosomes with degraded

cytoplasmic material. (A2) show many autophagosomes with double membranes and (A3)

could be lipid vesicles.

170

5 𝝁𝒎 2 1

1 𝝁𝒎

FAF-HSA 72h HK2


treated with (5mg/ml) FAF-HSA for 72h. (A1 and A2) show clear autophagosomes with

double membranes. In addition, (A2) shows Golgi apparatus (arrow).

171

𝝀-LC 24h HK2-GF

1

4

2

3

5 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎


treated with (5mg/ml) 𝜆-LC for 24h. It shows different stages of macroautophagy process

and autophagosome (A3) and autophagolysosome (A4).

172


treated with (5mg/ml) 𝜆-LC for 72h. (A1) show different stages of macroautophagy. (A2)

show autophaglysosomes with degraded cytoplasmic material. (A3) may be numerous

numbers of lipid vesicles and (A4) show multilamellar body.

𝝀-LC 72h HK2-GF

1 𝝁𝒎

1 𝝁𝒎

4

2

3

1 1 𝝁𝒎

1 𝝁𝒎

173

Figure (5.18): Representative transmission electron micrographs (TEM) of HK2 cells treated

with (5mg/ml) 𝜆-LC for 24h. (A1) shows autophaglysosomes containing partially degraded

cytoplasmic material. (A2 and A3) show ribosomes in autophagosomes.

𝝀-LC 24h HK2

5 𝝁𝒎

5 𝝁𝒎

3

2 1 1 𝝁𝒎

174

Figure (5.19): Representative transmission electron micrographs (TEM) of HK2 treated

with (5mg/ml) 𝜆-LC for 72h shows autophaglysosomes containing partially degraded

cytoplasmic material. (A6) show rough endoplasmic reticulum.

𝝀-LC 72h HK2

1 2

3 4

5 6

5 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎

1 𝝁𝒎

175

5.2.3 Effect of overload protein concentration to induce apoptosis:

To examine whether excessive protein doses can lead to apoptosis, cell injury and death,

HK2 (+/- GF) cells were sub-cultured in 6 well plates and incubated for 24 and 72h in a

serum free medium in the presence of FAF-HSA or 𝜆-LC (5mg/ml). Then, the presence

of apoptotic cells was analysed using ApopTag® In Situ Apoptosis Detection Kit.

Among FAF-HSA treated HK2-GF cells, there were few cells that stained positive for

TdT activity, indicative of apoptosis at 24h (figure 5.20 (A3)). More noticeable

apoptotic bodies and fragmented nuclei appeared after 72h (figure 5.20 (A4)). In

contrast, very few or no apoptotic cells were detected in the control cells incubated with

a serum free medium alone for 24 and 72h (figure 5.20 (A1 and A2)). Consistently,

apoptotic cells were shown after 𝜆-LC incubation for 24h (figure 5.20 (A5)). At 72h, 𝜆-

LC significantly induced more apoptosis HK2-GF cells compared to control cells

(figure 5.20 (A6)).

As a result, FAF-HSA induced apoptosis after treating HK2 cells for 24h (figure 5.20

(B3)), and at 72h of incubation time the number of the apoptotic cells was increased

(figure 5.20 (B4)). Similarly, 𝜆-LC induced apoptosis in 24h and the number of the

apoptotic cells was significantly increased at 72h of incubation time (figure 5.20 (B5

and B6)) compared to no apoptotic cells in control cells at 24h (figure 5.20 (B1)) and

few in the control cells at 72h (figure 5.20 (B2)). The controls were incubated in a

serum free medium.

Quantification by cell counting for 3 different fields for each sample at 10X

magnification showed HK2-GF treatment with (5mg/ml) FAF-HSA induced a 1-fold

increase in apoptosis comparing to the control and a two-fold increase with (5mg/ml) 𝜆-

LC at 24h. At 72h, the number of apoptotic cells after 𝜆-LC treatment was increased

more than 2-fold in 24h, however, FAF-HSA had no additional effect (figure 5.20 (C)).

Moreover, FAF-HSA had no significant effect on HK2 cells during the first 24h

stimulation time compared to the control, but a statistically significant increase by two-

fold in the number of the apoptotic cells after 72h of incubation. Whereas, 𝜆-LC raised

the number of apoptotic cells by 2-fold after 24 and 72h compared with controls (figure

5.20 (D)).

176

HK2-GF

(Control 24h)

(FAF-HSA 72h) (FAF-HSA 24h)

(Control 72h)

4 3

2 1

177

(𝝀-LC 24h) 6 5 (𝝀-LC 72h)

Figure (5.20): Specific staining of DNA fragmentation associated with apoptosis (ApopTag®) in stimulated HK2 (+/-GF) cells with

overload proteins. (A) HK2-GF cells exposed to (5mg/ml) FAF-HSA or 𝜆-LC for 24 and 72h. Reaction of TdT enzyme was

visualised by peroxidase substrate (nuclear brown colour for apoptotic cells) after stimulating the cells compared to the control (un-

treated cells) (red arrows indicate an example of apoptotic cells). Methyl green was used as counterstain. Objective x10.

178

HK2

(Control 24h)

(FAF-HSA 72h) (FAF-HSA 24h)

(Control 72h)

4 3

2 1

179

(𝝀-LC 24h) 5 6

(𝝀-LC 72h)

(B) HK2 cells exposed to FAF-HSA or 𝜆-LC (5mg/ml) for 24 and 72h. Reaction of TdT enzyme was visualised by peroxidase

substrate (nuclear brown colour for apoptotic cells) after stimulating the cells compared to the control (untreated cells) (red

arrows indicate an example of apoptotic cells). Methyl green was used as counterstain. Objective x10.

180

HK2 HK2-GF

Contr

ol

HS

A-F

FA

LC

Contr

ol

HS

A-F

FA

LC

0.0

0.5

1.0

1.5

2.0

24 h 72 h

Arb

itra

ry d

ensi

tom

eter

y u

nits

( x10 f

ield

)**

**

**

**

Contr

ol

HS

A-F

FA

LC

Contr

ol

HS

A-F

FA

LC

0.0

0.5

1.0

1.5

2.0

Arb

itra

ry d

ensi

tom

eter

y u

nits

( x10 f

ield

)

**

*

* **

24 h 72 h

(C and D) Quantitative analysis for immunostaining results of apoptotic HK2 (+/-GF) cells after exposure to FAF-HSA or 𝜆-LC

(5mg/ml) for 24 and 72h. Densitometric analysis of data obtained from the mean of three different fields (x 10) for each sample

and compared to un-stimulated controls (measuring the apoptotic cells (brown colour in each field)). The data are represented

as the mean of three fields ± SD (Unpaired t test p < 0.05). There were statistically significant differences between the

conditions. Image J software was used.

181

Caspase-3 is a downstream effector in this cascade that mediates apoptosis when

activated by various upstream signals (Lin et al., 2014). To investigate the effects of

protein overload on caspase-3 expression, HK2 (+/-GF) cells were incubated with FAF-

HSA or 𝜆-LC (5mg/ml) for 24 and 72h. The mRNA and protein expression of caspase-3

were measured by RT-qPCR and Western blot, respectively.

Figure (5.21) shows the expression of procaspase-3 in HK2 (+/-GF) cells from

stimulated and un-stimulated cells at (33kDa) size; however, activated caspase-3

(17kDa, cleaved product) could not be detected in treated cells, perhaps reflecting the

relatively small number of ApopTag® positive cells (figure 5.20). Caspase-3 mRNA

gene expression was increased with time in HK2 cells after FAF-HSA treatment at 24

and 72h compared to the un-treated control, but HK2-GF did not show any rise after the

stimulation (figure 5.22 (A)). Similarly, HK2 cells treated with 𝜆 -LC showed a

significant increase in caspase-3, as compared to un-treated control cells, however,

HK2-GF cells showed elevation in caspase-3 after 72h of stimulation with 𝜆-LC (figure

5.22 (B)).

In summary, although both FAF-HSA and 𝜆-LC induced apoptosis in HK2 (+/-GF)

cells, 𝜆-LC had a more pronounced effect on cells.

182

C S C S C S C S

24h 72h 24h 72h

HK2-GF HK2

FAF-HSA

𝝀-LC

Caspase-3

33kDa

𝜷 -actin

42kDa

𝜷 -actin

42kDa

Caspase-3

33kDa

Figure (5.21): Western Blot analysis of Caspase-3 from stimulated HK2 (+/-GF)

cells with overload proteins. The effect of FAF-HSA or 𝜆 -LC on caspase-3

protein production from HK2 (+/-GF) cells. Cells were cultured (5 x 105) in 6

well plates and stimulated with FAF-HSA or 𝜆-LC (5mg/ml) for 24 and 72h. Cell

lysates were prepared and analysed by Western blot SDS-PAGE. Cell lysates

(20𝜇𝑔 protein) were used. 𝛽-actin was used as loading control. C: control sample

and S: stimulated sample.

183

FAF-HSA

Contr

ol

24

h

72

h

24

h

72

h0.0

0.5

1.0

1.5

2.0

m R

NA

Cas

pas

-3 e

xpre

ssio

n

(Fold

chan

ge

vs.

contr

ol)

***

**

GF HK2

𝝀-LC

Contr

ol

24

h

72

h

24

h

72

h0

1

2

3

4

5

m R

NA

Cas

pas

-3 e

xpre

ssio

n

(Fold

chan

ge

vs.

contr

ol)

***

GF HK2

Figure (5.22): Effects of FAF-HSA or 𝜆 -LC on Caspase-3 mRNA

expression from HK2 (+/- GF). Cells were sub-cultured in 6 well plates (5

x 105) and incubated with 5mg/ml 𝜆-LC or FAF-HSA for 24 and 72h. (A)

FAF-HSA up-regulated caspase-3 expression from HK2 compared to the

control (B) 𝜆 -LC elevates caspase-3 expression (in a time dependent

manner) in HK2 cells (+/-GF) by (Quantitative RT-PCR) (2^-ΔΔCT value

was used, the mRNA expression normalized to β-actin and calibrated to

the average of un-treated controls). The data are represented as means of

duplicate ± SD (n = 2) (Unpaired t test P < 0.05).

184

5.2.4 Effect of FAF-HSA and 𝝀-LC on mediators of inflammation in

kidney damage:

In this study, after exposing HK2 (+/-GF) cells to the FAF-HSA or 𝜆-LC (5mg/ml),

total mRNA was isolated from control and stimulated cells at 24 and 72h. RT-PCR and

qRT-RCR were used to determine gene expression from stimulated HK2 (+/-GF) cells.

Housekeeping genes are constitutive genes, which are essential for basic cellular

function. They are expressed at a constant level in all cells under normal and

pathophysiological conditions (Eisenberg & Levanon, 2003) However, some of the

housekeeping genes might express differently depending on the experimental condition

(Greer et al., 2010). Because of this, three different housekeeping genes were tested

with our proteinuric condition by RT-PCR and RT-qPCR, glyceraldehyde 3-phosphate

dehydrogenase (GAPDH), beta 2 microglobulin (𝛽2M) and beta-actin (𝛽-actin). Our

results showed a variant expression in GAPDH and 𝛽2M from HK2 (+/- GF) cells

before and after stimulation with FAF-HSA (data not shown). So, FAF-HSA overloads

effects on the expression of both genes. In addition, in tubular proteinuria disease the

low molecular proteins such as 𝛽2M, that is completely reabsorbed by proximal tubules

in normal conditions appears in urine (Pacific Biomarker, 2012), which might explain

the differences in this gene expression from our PTECs in our protein overload

condition (the effect of protein overload on 𝛽2M was investigated and discussed in

chapter 7). However, the expression of β-actin was stayed constant upon stimulation.

Therefore, it was used as a reference (housekeeping gene).

Transforming growth factor-𝛽1 (TGF- 𝛽1) is an important mediator of tubular kidney

disease and one of the most important profibrotic cytokines in renal damage (Hsieh et

al., 2012). Treating HK2-GF cells with FAF-HSA for 24 and 72h up-regulated TGF-β1

mRNA expression significantly. On the other hand, HK2 cells showed an important

increase after 72h of stimulation compared with the control (figure 5.23 (A)). After 𝜆-

LC stimulation, the mRNA expression of TGF-β1 was elevated after 72h compared to

control in HK2-GF cells. The expression levels of TGF-β1 at both time points were

increased significantly in HK2 (figure 5.23 (B)).

Tumor necrosis factor (TNF- 𝛼) is one of the pro-inflammatory cytokines and a

mediator of inflammatory tissue damage. The effect of protein overload on HK2 (+/-

https://en.wikipedia.org/wiki/Constitutive_gene

https://en.wikipedia.org/wiki/Gene_expression

185

GF) cells on TNF- 𝛼 gene expression was measured. As shown in (figure 5.24 (A))

TNF-α expression was clearly increased with the long stimulation time of 72h from

HK2 (+/-GF) cells with FAF-HSA stimulation, compared to control cells. However,

stimulating the HK2 (+/-GF) cells with 𝜆 -LC up-regulated the TNF-α mRNA

expression significantly in a time-dependent manner compared to the control (figure

5.24 (B)).

Kidney Injury Molecule-1 (KIM-1) is a type 1 transmembrane protein and a specific

biomarker of human proximal tubular cell injury (van Timmeren et al., 2006). KIM-1 is

implicated in the pathogenesis of proteinuria induced renal damage or repair (Lim et al.,

2014). The HK2 cell line expresses KIM-1 under normal culture conditions but up

regulates in proximal tubules in nephron toxic injury (Han et al., 2002 and van

Timmeren et al., 2006). Gene expression of KIM-1 as a marker of tubular damage was

measured. KIM-1 was detected in HK2 (+/-GF) cells. At 24h of FAF-HSA stimulation,

the HK2 (+/-GF) did not show any significant change in KIM-1 mRNA levels, however,

it was elevated after 72h compared to the control (figure 5.25 (A)). On the other hand,

exposing the HK2 (+/-GF) cells to 𝜆-LC for 24 and 72h showed a significant decrease

in KIM-1 mRNA level compared to the control (figure 5.25 (B)).

To investigate another kidney cell injury biomarker, the clusterin mRNA level was

measured in stimulated HK2 (+/-GF) cells with FAF-HSA or 𝜆 -LC. Clusterin is a

glycoprotein; the mRNA and protein levels increase in tubular cells after injury like

KIM-1. It has been associated with kidney injury to both glomeruli and tubules (Vaidya

et al., 2008).

Figure (5.26) illustrates that FAF-HSA and 𝜆-LC were elevated in the clusterin gene

expression after stimulating HK2-GF cells compared to no or a very faint band from

untreated control cells at 24 and 72h. However, the HK2 cells did not show any band

with both protein treatments at both time points, the 24 and 72h (data not shown). 𝛽-

actin gene expression was used as a housekeeping gene and load control.

In summary, in this chapter it can be conclusively demonstrated that high concentrations

of FAF-HSA and 𝜆-LC have a cytotoxic effect on HK2 (+/-GF) cells with a long time of

incubation. The different readouts (MTT, LDH, and TEM) are likely to capture a

population of cells, which do not react uniformly to the stimulations. While some

186

undergo apoptosis, other involved in autophagic repair. While some proliferate, others

are leaky. mRNA expression studies analyse those cells, which are metabolically intact.

187

FAF-HSA

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5

2.0

Rat

io o

f m

RN

A (

TG

F-β

1 /β-a

ctin

)**

**

***

GF HK2

Contr

ol

24

h

72

h

24

h

72

h0.0

0.5

1.0

1.5

m R

NA

TG

F-b

1 e

xpre

ssio

n

(Fold

chan

ge

vs.

contr

ol)

***

******

GF HK2

𝝀-LC

Figure (5.23): mRNA expression of TGF-𝛽1 from stimulated HK2 (+/-GF) cells

with overload proteins. HK2 (+/-GF) cells were sub-cultured in 6 well plates (5 x

105) after exposure to 5mg/ml (A) FAF-HSA or (B) 𝜆-LC for 24 and 72h. (A)

Shows significant increase in TGF-𝛽1 level after 72h stimulation with FAF-HSA

from HK2 (+/-GF) cells compared with the controls (untreated cells) by semi-

quantitative RT-PCR. The results are presented as a ratio of densitometry analysis

of the gene relative to β- actin mRNA expression (housekeeping gene) using Image

J software. (B) Shows significant rise in TGF-𝛽1 mRNA expression from HK2

cells (+/-GF) after 𝜆-LC stimulation for 72h by RT-qPCR, 2^-ΔΔCT value was used,

the mRNA expression normalized to β-actin and calibrated to the average of

untreated controls. The data are represented as means of duplicate ± SD (n = 2)

(Unpaired t test P < 0.05), C: control sample.

188

Figure (5.24): mRNA expression of TNF-𝛼 from stimulated HK2 cells (+/-GF)

with overload proteins. HK2 (+/-GF) cells were sub-cultured in 6 well plates (5

x 105) after exposure to 5mg/ml (A) FAF-HSA or (B) 𝜆-LC for 24 and 72h. (A)

Shows significant increase in TNF-𝛼 level after 72h of stimulation with FAF-

HSA from HK2 (+/-GF) cells comparing with the controls (un-treated cells).

Similarly, (B) shows a significant rise in TNF-𝛼 mRNA expression from HK2

(+/-GF) cells after 𝜆-LC stimulation for 72h by semi-quantitative RT-PCR. The

results are presented as a ratio of densitometry analysis of the gene relative to β-

actin mRNA expression (housekeeping gene) using Image J software. The data

are represented as a means of duplicate ± SD (n = 2) (Unpaired t test P < 0.05),

C: control sample.

FAF-HSA

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5R

atio

of

mR

NA

(T

NF

-a/β

-act

in)

**

****

GF HK2

𝝀-LC

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5

Rat

io o

f m

RN

A (

TN

F-a

/β-a

ctin

)

GF

*

***

HK2

*

189

FAF-HSA

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.2

0.4

0.6

0.8

Rat

io o

f m

RN

A (

KIM

-1/β

-act

in)

**

GF HK2

Con

trol

24h

72h

24h

72h

0.0

0.5

1.0

1.5

m R

NA

Kim

-1 e

xpre

ssio

n

(Fol

d ch

ange

vs.

cont

rol)

***

******

GF HK2

𝝀-LC

Figure (5.25): mRNA expression of KIM-1 from stimulated HK2 (+/-GF) cells

with overload proteins. HK2 (+/-GF) cells were sub-cultured in 6 well plates (5 x

105) after exposure to 5mg/ml (A) FAF-HSA or (B) 𝜆-LC for 24 and 72h. (A)

Shows increase in KIM-1 level after 72h of stimulation with FAF-HSA from HK2

(+/-GF) cells compared to the controls (untreated cells) by semi-quantitative RT-

PCR. The results are presented as a ratio of densitometry analysis of the gene

relative to β-actin mRNA expression (housekeeping gene) using Image J

software. (B) Shows significant decrease in KIM-1 mRNA expression from HK2

(+/-GF) cells after 𝜆-LC stimulation for 72h by RT-qPCR; 2^-ΔΔCT value was

used, the mRNA expression normalized to β-actin and calibrated to the average of

untreated controls. The data are represented as a means of duplicate ± SD (n = 2)


190

Figure (5.26): Semi-quantitative analysis of clusterin mRNA expression from

HK2-GF cells after stimulating with 5mg/ml FAF-HSA or 𝜆-LC for 24 and

72h. Clusterin gene expression was increased significantly after treating HK2-

GF cells with FAF-HSA and 𝜆-LC compared with absent or low expression

from untreated control cells. 2𝜇g mRNA was used from each sample. 𝛽-actin

was used as a housekeeping gene, C: control sample and S: stimulated sample.

24h 72h

Clusterin

800 bp

FAF-HSA

𝝀-LC

C S C S

C S C S

𝜷-actin

210 bp

191

5.3 Discussion

Studies have pointed to a direct pathogenic effect of protein on PTC to explain the

relation and extent of diseased kidney and proteinuria (Shalamanova et al., 2007 and Li

et al., 2008).

Treating HK2 (+/-GF) cells with FAF-HSA (5, 10, 20 and 30mg/ml) for (2, 4, 6, 24, 48

and 72h) as a time course to monitor the effect of the FAF-HSA on the proliferation of

the cells showed that after early time points (2, 4 and 6h) there was no difference in the

number of HK2 (+/-GF) cells when compared with their controls. Our results agreed

with a previous study, which used HK2 cells grown in a medium with EGF and

stimulated with (5mg/ml) HSA (95% pure and fatty acid free) for 8h. There was no

statistically significant impact on the proliferation of the HK2 cells using MTSTM

proliferation dye (Newman et al., 2000).

Another study investigated the effects of FAF-HSA (30mg/ml) for a longer incubation

time (24h) with primary cultures of human PTCs and determined the cell number by

hemocytometer. They found that the percentage of the proliferation of human PTC was

significantly increased compared with control cells, thus, no toxic effect (inhibition) on

the proliferation of human PTCs with a high concentration of albumin (Arici et al.,

2003). Interestingly, our HK2-GF cells showed no inhibition on growth with all FAF-

HSA concentrations after 24 and 48h of incubation time. These results agreed with

Arici et al. (2003). Whereas, we could not detect a significant increase in cell growth as

they detected after the 24h incubation, this difference might be due to the different cell

types used in both studies. In contrast, after 72h of incubation there was significant

decrease in cells number with the highest FAF-HSA concentration (30mg/ml) compared

to the control. One explanation may be due to the long-time of the incubation.

The fact that HK2 cells behave differently than HK2-GF when treated with FAF-HSA

might be due to the differences in media culture conditions. From the first 24h of

incubation with same concentrations of FAF-HSA, the viability of HK2 cells decreased

and the greatest reduction was observed with the highest concentration of FAF-HSA

(30mg/ml) compared to the control at all three time points.

An unexpected result with LDH release from stimulated HK2 (+/-GF) cells with a range

of concentrations of FAF-HSA for 24, 48 and 72h, the LDH activity was found to

192

increase at 24 and 48h but decrease at 72h. These results are at variance with MTT

results that showed the maximum decrease in cell activity with longer incubation time

with the range of FAF-HSA concentrations for HK2 (+/-GF) cells. This might be due to

the cell number that was used in the LDH assay (2,500 cells/well). The cells may

release the maximum LDH in the supernatant at 48h, and a higher number of cells were

used in LDH assay. For example, Hills et al. (2013) used (5,000 cells/well) in a 96 well

plates to study the cytotoxic effect of visfatin on HK2 cells for 48h by LDH assay. Also,

Wu et al. (2009) utilised HK2 cells plated in 96 well plates at (10,000 cells/well) for

testing the cytotoxicity of different compounds measured for 5-24h by LDH assay. In

both studies they used higher cell numbers than the one used in our study and for a

shorter incubation time up to 48h.

At 24h, the LDH release was increased significantly from HK2-GF cells stimulated with

FAF-HSA concentrations, when higher number of cells was used. By comparing the

LDH (leakage injured cells) and MTT (cell activity) at 24h of FAF-HSA stimulation,

we concluded that FAF-HSA led to injure the HK2-GF cell membrane (LDH release

increased), but did not let them die, so we did not detect decrease in the cell number

with MTT assay. However, when HK2 cells were treated with a range of FAF-HSA for

24h, the MTT showed an inhibition in growth with all concentrations and, compared

with LDH release for this experiment. In, 72h of incubation with FAF-HSA

demonstrates that the maximum LDH release from the HK2 (+/-GF) cells occur with the

highest concentrations (30 mg/ml) FAF-HSA, however, HK2 cells show more LDH

release compared to HK2-GF. The HK2-GF cells being more differentiated as brush

border; this seemed beneficial to respond to FAF-HSA stimulation. In addition, these

results were comparable with MTT results, which showed the maximum cell death

achieved after 72h of incubation with the highest concentrations for HK2 (+/-GF) cells.

In conclusion, exposure PTECs grown in a medium with or without an EGF cocktail to

high (30mg/ml) or low (5mg/ml) concentrations of albumin for short times will have no

effect on cells proliferation. The EGF cocktail in the growth media of HK2 cells may

play a protective role and save the cells from damage for a longer time. Lastly,

incubating HK2 (+/-GF) cells with a high concentration of albumin led to cell toxicity.

193

When exposing HK2 (+/-GF) cells to different concentrations of 𝜆 -LC (1, 5 and

10mg/ml) for 24, 48 and 72h, a slight decrease in mitochondrial activity was noticed by

MTT assay with 1 and 5 mg/ml (40 and 200 𝜇𝑀, respectively) 𝜆-LC at 24h, however,

with the higher dose 10mg/ml (400𝜇𝑀), a significant reduction was detected.

In 2008, Li and his group determined a significant decrease in the number of HK2 cells,

which were grown in a medium supplemented with EGF, after 24h treated with (50 𝜇𝑀)

LC by MTS cell proliferation/cytotoxic assay. While their study used 𝜅-LC, the present

work used 𝜆-LC. On the other hand, others displayed a significant reduction in HK2 cell

viability treated with 𝜅-LC (50 𝜇𝑀) but after 48h of incubation (Li et al., 2008). The

reason for that might be the different source from which the LC was purified (donor

urine sample) that may affect the LC toxicity. Our results from stimulated HK2-GF

cells with 𝜆-LC are in agreement with Li et al. (2008). The viability of our HK2-GF

cells was decreased with the similar 𝜆-LC dose 1mg/ml (40𝜇𝑀) and as well with higher

doses at 48h and also 72h, although, we used different types of LCs. Similarly, HK2

cells illustrated a significant reduction in cell viability after 48 and 72h of incubation

time with all 𝜆-LC concentrations (1, 5 and 10 mg/ml) compared to the control.

To confirm the toxic effects of 𝜆-LC on HK2 (+/-GF) cells by different assay, LDH

release from injured cells was measured at the end of each incubation time with

different 𝜆-LC concentrations. Exposure of HK2 (+/-GF) cells to 𝜆-LC produced no

significant effects on LDH release to the supernatant at 24h. However, Wang & Sanders

in 2007 showed conflicting results. They demonstrated a significant increase in LDH

release from HK2 cells after 24h incubation with (5mg/ml) 𝜆-LC. Two differences

could explain that; first, the source of the LC, and the toxic effect as explained

previously. Also, their cells growing in a different type of media may play a role in the

cell sensitivity. Nonetheless, after 48 and 72h showed the maximum LDH release from

HK2 (+/-GF) cells with (10mg/ml) 𝜆 -LC. These results are comparable with MTT

results.

Our work and results have given evidence that high concentrations of FAF-HSA and 𝜆-

LC exert an inhibitory effect on the proliferation and viability of HK2 (+/-GF) cells by

measuring the cellular metabolic activity and cell membrane injury with MTT and LDH

assays, respectively. In addition, by comparing the effect of (5mg/ml) FAF-HSA and 𝜆-

LC on HK2 (+/-GF) cells, we can conclude that 𝜆-LC had a more toxic effect on the

194

cells; it showed a significant decrease in cell viability at 48 and 72h with 𝜆-LC, but

FAF-HSA had no effect by MTT assay. These finding are important to prove our

protein overload conditions are able to inhibit HK2 (+/-GF) cell proliferation and had a

pathological effect on the cells, and can be used as proteinuria model in this project for

more investigative experiments.

Liu et al. (2014) investigated the activation of autophagic pathways in response to

urinary protein (UP), which is purified from a minimal change nephrotic syndrome

patient (MCNS), or (HSA with fatty acid) in HK2 cells. Exposure HK2 cells to an

arrangement of UP (0.1-8mg/ml) for 8h showed a significant increase in the number of

microtubule-associated protein 1 light chain 3 (LC3-II, a key marker of autophagy) by

immunofluorescence staining and Western blot. In parallel, typical image of early/late

autophagic vacuoles were shown by TEM. However, the range of HSA (0.1-8mg/ml) on

HK2 cells for 8h showed that only the high dose induced an increase in LC3-II. They

explained the different effect of HSA and UP that might be observed from the complex

components in UP, like immunoglobulin. Another study characterised the ultrastructure

of HK2 cells following (10mg/ml) bovine serum albumin with fatty acid (BSA) for 12h

by TEM. They found that BSA induced HK2 cells to generate secondary lysosomes,

cytoplasmic vacuolisation, and damaged rough endoplasmic reticulum and

mitochondria (Liu et al., 2009).

Similarly, our results demonstrated that FAF-HAS and 𝜆 -LC (5mg/ml) produced

different stages of macroautophagy stages in HK2 (+/-GF) cells at 24 and 72h,

autophagosoms, autolysosomes and a lot of multilamellar bodies.

Taken together, previous studies showed autophagy activation in response to albumin

(with fatty acid) overload. Our results proved that FAF-HAS and 𝜆 -LC induced

autophagy in HK2 (+/-GF) cells. However, the meaning of up-regulation of autophagy

in the kidney proximal tubules is not clear. One study suggested a cytoprotective role of

autophagy in kidney injury; in contrast, another study reported that autophagy is

positively involved in cell death in renal tubules (Takabatake et al., 2014). For example,

although Liu et al. (2014) indicated the autophagy activation in HK2 cells in response to

high HSA, they concludes the autophagy activation was a cytoprotective response

because pretreating HK2 cells with an autophagy inhibitor led to increased autophagic

flux, but reduced the apoptosis and different kidney injury markers.

195

The main aim in our study was to investigate the autophagy activation in HK2 (+/-GF)

cells in response to FAF-HSA and 𝜆-LC, but the role of this activation is not clear.

Stimulating HK2 (+/-GF) cells with 𝜆-LC induced morphological alterations in cultured

cells, including detachment of the cells from the culture plate and aggregation (data not

shown). By contrast, with FAF-HSA appeared less harmful, as most of the cells

remained attached to the flask surface. Li and his group in 2008 showed similar results

with stimulating HK2 cells with 𝜅-LC (50𝜇M) for 24h and comparing with control

cells. So, maybe autophagy plays a cytoprotective role with the FAF-HSA experiment,

but it has a role in cell injury with light chain stimulation. However, more investigation

is needed on this issue.

In vivo, the presence of proximal tubule apoptosis was shown in kidney biopsy

specimens of patients with focal segmental glomerulosclerosis (FSGS); in addition,

there was a significant correlation between the extent of proteinuria and the number of

apoptotic cells. In vitro, a model of albumin overload showed an increase in apoptosis

in a concentration dependent manner in proximal tubule cells (HKC-8) after exposure to

(5 and 10mg/ml) HSA (endotoxin free) for 24h by fluorescence microscopy (stained

with Annexin V, an early marker of apoptosis) and Hoechst 33342 (staining for late

apoptosis), but, there was no additional apoptosis with 20mg/ml HSA. In contrast, the

20mg/ml FAF-HSA induced the maximum necrosis with trypan blue staining (Erkan et

al., 2007). Comparably, the cell culture system used in this project, FAF-HSA induced

apoptosis in HK2-GF cells at 24 and 72h by the same level and this could be because

the protective role of the EGF cocktail, however, the apoptotic cells increased in HK2

cells with incubation time. 𝜆-LC induced apoptosis to the same extent in HK2 (+/-GF)

at 24 and 72h, and this may be due to a high number of cells detaching from the culture

plate or becoming necrotic; remaining attached cells were stained for apoptosis by the

ApopTag® kit, which distinguishes apoptosis from necrosis by specifically detecting

DNA cleavage and chromatin condensation associated with apoptosis.

It was reported that up-regulation apoptosis in the kidney is associated with increases in

caspase-3 activity, protein and mRNA levels (Yang et al., 2001). For example, Erkan et

al. (2005) showed significant elevation in caspase-3 activity HKC-8 treated with

(5mg/ml) HSA (endotoxin free) for 24h. Also, caspase-3 activity increased in

196

stimulation of HK2 cells with (1mg/ml) of two monoclonal FLCs (𝜅2 and 𝜆3) that were

purified from the urine of patients who had multiple myeloma and light chain

proteinuria, at 24 and 48h.

In this project the procaspase-3 (33kDa) was detected from HK2 (+/-GF) cells

simulated with (5mg/ml) FAF-HSA or 𝜆-LC for 24 and 72h; however, the cleaved

caspase-3 (17kDa) could not be detected. This is an unexpected result because the cells

showed apoptosis after stimulation as explained previously, but this could be due to the

low protein concentration (20𝜇g) of the lysates analysed by Western blot. One study

detects the cleaved caspase-3 from (100𝜇g) proximal tubule lysates stimulated with 200

𝜇M H2O2 for 8h (Kaushal et al., 2004). Also, the mRNA caspase-3 level was measured

in HK2 (+/-GF) cells treated with (5mg/ml) FAF-HSA or 𝜆-LC. The results showed a

gradual increase in caspase-3 mRNA level from HK2 cells stimulated with FAF-HSA at

all-time points, but no elevation was detected in treated HK2-GF. Nonetheless, 𝜆-LC

led to an increase the expression of caspase-3 mRNA from HK2-GF at 72h

significantly, and from HK2 cells at 24h and persisting through 72h.

Different kidney injury markers were measured in this project. This study examined the

effect of (5mg/ml) FAF-HSA and 𝜆-LC on TGF-𝛽1 cytokine on HK2 (+/-GF) at 24 and

72h. It was reported that a high concentration of albumin up-regulated TGF-β1 mRNA

expression in PTCs (Zoja et al., 2003). Our result showed that treated HK2-GF with

FAF-HSA increased TGF-β1 mRNA expression significantly. A further study

investigated the effect of treating HK2 cells; grown with a medium supplemented with

EGF, with (5mg/ml) FAF-HSA, a mild increase only was described for TGF-β1

production after 8h of incubation. This mild increase could be due to the short time of

incubation (Newman et al., 2000). On the other hand, HK2 cells showed an important

increase just after 72h of stimulation compared to the control. Li et al. (2008) analysed

the level of TGF-β1 mRNA and protein expression in cell culture medium from

stimulated HK2 cells with 𝜅-LC (25𝜇𝑀) for a time course (8, 24, 48 and 72h). They

found that TGF-β1 mRNA and protein levels were significantly elevated at 24h

persisting through 72h. Comparably, the (5mg/ml) (200 𝜇𝑀) 𝜆-LC in our project up-

regulated the mRNA level significantly from HK2 cells (+/-GF) at 24h and persisted

through 72h. In conclusion, stimulating PTCs with albumin or LC led to elevated TGF-

β1 gene expression with time.

197

The effects of FAF-HSA on the mRNA level of TNF-𝛼 from HK2 (+/-GF) cells was

significantly elevated at 72h, but no change occurred at 24h. By contrast, the (5mg/ml)

𝜆-LC leads to an increase of mRNA for TNF-𝛼 slightly at 24h and significantly at 72h.

However, previous study showed different results, where the exposure of human PTCs

(SV40) to a range of LC (1.5 and 3 mg/ml) and HSA with fatty acid (10 and 30mg/ml)

for 24h, the LC and HSA had no effect on the TNF-𝛼 production in the supernatant

(Sengul et al., 2002). This might be because of the shorter incubation time with

albumin, low 𝜆-LC concentrations or toxic effect.

Our data showed that the mRNA level of KIM-1 did not increase after stimulating HK2

(+/-GF) cells with (5mg/ml) FAF-HSA at 24h, but expression was increased at 72h. Lim

and his group in 2014 presented similar results, where stimulating HK2 cells with

5mg/ml HSA (with fatty acid) for a time course (4, 24, 48 and 72h) significantly

enhanced the KIM-1 mRNA expression with time, but the KIM-1 protein level was

increased significantly in the early (4 and 24h) and reduced with the late time points (48

and 72h). KIM-1 produced from injured PTECs has two roles: to be a marker associated

with tubular damage, or to clear the debris from damaged renal tubules. Clearance of

the necrotic and apoptotic cells from injured tubular is a critical process for restoration

of normal tissue (Lim et al., 2014). The early induction of KIM-1 from an injured

kidney might serve an adaptive function to clear apoptotic, necrotic cells and debris

from injured tubular that lead to a decrease in the inflammatory response in the

damaged tubule. In contrast, the continuous expression of KIM-1 during tubular damage

may cause inflammation and leads to further tubular damage and loss of renal function

(Lim et al., 2014). This may explain the late KIM-1 production from HK2 (+/-GF) cells

at 72h after FAF-HSA stimulation. Nonetheless, exposure of HK2 (+/-GF) cells to 𝜆-LC

showed a decrease in the KIM-1 mRNA level and this could be due to KIM-1 produced

as protein in the supernatants. So, in the conditions reported in this work, KIM-1 may

plays a clearance role of apoptotic cells and debris from damaged renal tubules (Lim et

al., 2014) with FAF-HSA stimulation but it was produced as a marker for cell damage

with 𝜆-LC treatment because the cell culture with 𝜆-LC showed lots of detached and

floating cells in the supernatants.

198

Our results showed a significant increase in clusterin mRNA expression from HK2-GF

cells after exposure to FAF-HSA and 𝜆-LC compared to no or very slight expression in

the control sample. Unexpectedly, HK2 cells did not show any clusterin expression after

stimulating with both proteins.

The novelty of this work is the side-by-side comparison of 𝜆-LC and FAF-HSA on HK2

(+/-GF) cells in parallel. Our study in this chapter proved that FAF-HSA and 𝜆-LC

overload in proximal tubules had an inhibitory effect as assessed by low cell

proliferation/viability, detectable autophagy, and cellular apoptosis. The inflammatory

responses coincide with the measure of damage and, possibly, repair. These effects are

included in figures (8.1 and 8.2).

199

Chapter Six-Megalin Phosphorylation in

Renal Proximal Tubular Epithelial Cells

200

6. Introduction:

Megalin, a luminally expressed receptor of the renal proximal tubules in the kidney. It is

glycoprotein and belongs structurally to the low-density-lipoprotein (LDL-R) receptor

family. It has a pivotal function together with cubilin and other receptors such as CD36

to bind filtered proteins and mediate their uptake by the endocytosis process in PTECs.

Megalin consists of a large extracellular domain and a short cytoplasmic domain/tail.

The large extracellular domain made up of four complementary types of ligand binding

repeats, epidermal growth factor repeats (EGF), YWTD spacer domains, a single

transmembran domain of 22 amino acids and NPXY short cytoplasmic domain. The

human megalin cytoplasmic domain/tail consists of 209 amino acids with three NPXY

motifs; this domain regulates the trafficking, function and endocytic activity of the

megalin receptor (figure 6.1 (A)) (Yuseff et al., 2007 and Marzolo & Farfán, 2011).

The cytoplasmic tail of megalin (MegCT) links the PTECs’protein (filtered proteins)

exposure with signalling effects within the cell. Activation signalling of cascades and

protein-protein interactions lead to protein phosphorylation of MegCT. The

phosphorylation of MegCT has the role of decreasing the expression of megalin on the

cell surface by a negative regulation (reduction) of megalin recycling (Marzolo &

Farfán, 2011). Excessive megalin mediated endocytosis might damage PTECs and

proteinuria will develop (Saito et al., 2010). The megalin receptor is constitutively

phosphorylated by a protein kinase at serine/threonine residues that enhance the

endocytosis rate (Yuseff et al., 2007). The MegCT has multiple potential

phosphorylation sites; these sites will phosphorylate when they are activated by protein

kinase C (PKC), casein kinase-l (CK-1) and glycogen synthase kinase-3 (GSK-3)

(Marzolo & Farfán, 2011) as distinct sites (figure 6.1(B)).

Previous work investigated if the phosphorylation of MegCT occurs, and by which

agents relevant to the urine of proteinuria patients. HSA (essentially fatty acid free) was

used because it is a major component in proteinuric urine and one of megalin receptor

ligands. EGF was used as another megalin receptor ligand, and PDBU a stimulator of

PKC (positive control). They used the same technique that was described in materials

and methods (See 2.16.2 and 2.16.3), however, after collecting the beads and being run

201

on 12% SDS-PAGE, the gel was dried and exposed to autoradiograph film to detect the

results. They found that in primary human proximal tubular cells that were pre-

stimulated with HSA (1mg/ml) and PDBU (1𝜇M), the MegCT-GST phosphorylation

was augmented compared to un-stimulated control cells and the GST (the beads) alone.

When cells were pre-stimulated with different concentration of EGF (10,100 and

500pg/ml), maximal phosphorylation was obtained with 500pg/ml. Thus, they

confirmed that MegCT is a phosphor-acceptor protein and in this case, the HSA and

EGF activate intracellular kinase pathways that as capable to phosphorylate MegCT

such as PDBU activate PKC.

Phosphorylation sites in MegCT Mass Spectrometry of tryptic digests of MegCT-GST

revealed the presence of phosphorylated in vitro by analysis of lysates prepared from

HK2 stimulated with HSA (1 mg/ml) or EGF (500 pg/ml). Five phosphorylated sites

identified in the MegCT (figure 6.1 (B)).

Another study also showed different MegCT phosphorylation sites by PKC and PKA

and investigated if MegCT phosphorylate within the PPPSP motif and GSK-3 is

required for phosphorylation (Yuseff et al., 2007). They identified that the PPPSP site

in the MegCT is significantly phosphorylated mainly by GSK-3, which adds a

phosphate molecule onto the serine amino acid residue. In addition, they displayed that

MegCT phosphorylated in vitro by using a minireceptor construct containing the

complete cytoplasmic domain of human megalin with amino terminal HA epitope

(Hemagglutanin Tag), in labelled LLC-PK1 (Procine kidney proximal tubule cells).

Additionally, they transfected the minireceptor in Chinese ovary hamster (CHO) cells

and Madin-Darby canine kidney (MDCK) cells; the results showed phosphorylation in

the megalin tail minireceptor (Yuseff et al., 2007).

Also, to detect megalin phosphorylation in the PPPSP cytoplasmic motif by GSK-3, the

minireceptor with wild type (PPPSP) or mutant peptide sequence (PPPAP or PPPDP)

with cytoplasmic domain were expressed in MDCK cells, which were labelled with 35S-

methionine/cysteine or 32P-orthophosphate for 2 to 4h and cell lysates were analysed by

SDS-PAGE and autoradiography. The results showed the wild type (PPPSP) motif with

serine constitutes the main phosphosite in the megalin cytoplasmic domain, as the

(PPPDP and PPPAP) mutant receptors were not highly phosphorylated as the wild type

(PPPSP). After that, they showed the GSK-3 binds directly to the megalin tail and

http://en.wikipedia.org/wiki/Serine

202

phosphorylates the receptor cytoplasmic domain by transfected HEK293 cells with

GSK3-HA; cell lysates incubated with MegCT-GST or GST alone as a control and anti-

HA was used by Western blot. The results showed the megalin cytoplasmic domain

interacts with GSK-3 (Marzolo & Farfán, 2011).

Based on these knowledge, three amino acid sequences (FHYRRTGSLLPAL),

(SRRDPTPTYSATE) and (TPPPSPSLPAKP) were phosphorylated in vitro and

injected into a rabbit with Freund’s Adjuvant (providing stimulation) (FHYRRTG-S

(PO 3 H 2) -LLPAL), (SRRDP-T (PO 3 H 2) -P-T (PO 3 H 2) –YSATE) and (TPPP-S

(PO 3 H 2) - PSLPAKP) (modified peptides) in an attempt to raise phosphor-specific

antibodies (Prof. Nigel Brunskill). In parallel, the unmodified peptides (not

phosphorylated, TPPPSPSLPAKP) were used as control peptides to obtain antiserum,

which could be used at the affinity purification stage to remove immunoglobulin that

recognises non-phosphorylated parts of the phosphor peptides. After that, the final

bleeds of immunised rabbits were obtained and affinity purified antisera were tested on

opossum kidney (OK) cells that were pre-stimulated with albumin and on the control

un-stimulated cells, which was done by Dr. Ravinder Chana at University of Leicester.

From all antisera tested, anti (TPPPSPSLPAKP) differentiated best between detecting

phosphorylated MegCT-GST and the un-phosphorylated peptidase sequence. Therefore,

this antiserum was chosen to use in this project to detect the phosphorylation signalling

in the cytoplasmic tail of megalin. By utilising these antibodies directed against specific

activation sites of the intracellular portion of megalin, they could be tested for their

effect on phosphorylation of Meg-CT, using FAF-HSA and 𝜆-LC two components in

the urine of MM patients with LC proteinuria and ligands of the megalin receptor.

This chapter will examine the phosphorylation of the megalin receptor by albumin and

light chain in a specific site of the cytoplasmic tail in vitro. Additionally, the expression

of megalin and CD36 receptors will be followed in our protein overload conditions in

vitro due to the fact that they are important receptors in the renal tubular endocytosis

process.

203

6.1 Aim

The goal of these studies was to first elucidate and follow the expression of megalin and

CD36 expression from PTECs in our vitro protein overload conditions, and secondly to

investigate if MegCT is a phosphor-acceptor protein in vitro by using cell lysate from

HK2 (+/-GF) cells pre-stimulated with overload proteins. This was made possible by

expanding work that characterised megalin phosphorylation (Baines, 2010).

204

A

B

HYRRTGSLLPALPKLPSLSSLVKPSENGNGVTFRSGADLNMDIGVSGFGPETAI

DRSMAMSEDFVMEMGKQPIIFENPMYSARDSAVKVVQPIQVTVSENVDNKNY

GSPINPSEIVPETNPTSPAADGTQVTKWNLFKRKSKQTTNFENPIYAQMENEQ

KESVAATPPPSPSLPAKPKPPSRRDPTPTYSATEDTFKDTANLVKEDSEV

EGF-type repeat

Complement type repeat

YWTD-region

NPXY motif

Transmembrane domain

Figure (6.1): The Megalin receptor. (A) Shows megalin receptor structure (Verroust &

Christensen, 2002) and (B) Demonstrates the 209 amino acid sequence of the human MegCT

(NM_004525.2). The blue highlight line shows the specific site of phosphorylation on the

cytoplasmic tail by GSK-3. The red highlight line shows the identified sites by (Baines, 2010).

The green highlight lines shows the sites of phosphorlation on the cytoplasmic tail by PKC

(Baines, 2010). The yellow lines shows the three peptides that were used to immunise rabbits

in the production of antisera.

205

6.2 Results

6.2.1 Expression of mRNA and protein of Megalin in HK2 (+/-GF)

cells:

To investigate the expression of megalin from HK2 (+/-GF) cells, total mRNA from

HK2 (+/-GF) cells was extracted, then cDNA was prepared from (5𝜇𝑔) mRNA and

analysed by RT-PCR (see 2.7). In addition, HEK 293 was grown to use as a positive

control for this analysis. 𝛽 -actin was utilised as a housekeeping gene because it is

expressed in comparable levels in HK2 (+/-GF) and HEK 293 cells.

As shown in (figure (6.2)), while β-actin expression was easily detectable, no megalin

band with the expected size (300bp) was detected from either HK2 or HK2-GF,

contrasting with an abundant expression of megalin mRNA in HEK293 cells.

L HEK293 HK2 HK2-GF

Figure (6.2): Analysis of megalin mRNA expression in HK2, HK2-GF and

HEK 293 cells. RT-PCR products were run on 1% agarose with 1-kb

DNA ladder (L). (A) mRNA expression for megalin in HEK293 cells

(positive control for megalin expression) but both HK2+/-GF showed no

megalin expression. The same amount of RNA (5 𝜇 g) was used for

HEK293 and HK2 (+/-GF) to prepare cDNA. (B) mRNA expression for β-

actin housekeeping gene.

𝛽-actin

170bp

Megalin

300bp

300 bp

200 bp

100 bp

400 bp

300 bp

200 bp

100 bp

206

Next, RT-qPCR was used in the assumption that mRNA megalin in HK2 (+/-GF) cells

were expressed at very low levels, which escaped the sensitivity of standard PCR (35 x

cycles). First, the efficiency and specificity of the primers (Megalin and β-actin) were

tested using HEK293 cDNA as described in materials and methods (see 2.8.2). Figure

(6.3) shows the amplification, standard curve and melting curve of β-actin and megalin

primers.

After that, HK2 (+/-GF) cells were tested. As presented in (figure 6.4 (A1)) the β-actin

amplification started early in cycle number 10; that means the expression in samples is

abundant, however, (figure 6.4 (B1)) shows the amplification with megalin increased

after cycle number 35, which means the expression of this gene is very scarce. Different

annealing temperatures were used (53-60C˚) to get a clear single band for megalin from

HK2 (+/-GF) cells at the correct size, as judged by running the qPCR samples in 1%

agarose gel (figure 6.5).

For megalin protein production, Western blot analysis was used for HK2 (+/-GF) cells

and HEK293 cells (as positive control), and whole cell lysates from the two cell lines

were prepared as described in material and methods (see 2.9.1). The protein samples

were separated using a gradient SDS-PAGE gel (4-15%) electrophoresis, transferred to

a PVDF membrane overnight at 30V and subsequently incubated with human anti-

megalin antibodies. Two human anti-megalin antibodies were used, commercially goat

anti-human megalin (see Table 2.7), and rabbit anti-MegCT antisera; this primary

antibody was generated as part of Richard Baines’ PhD work at the University of

Leicester (Baines, 2010) to detect intact megalin in HK2 cell lysate. The immunoblot

analysis showed no expression of megalin in HK2 (+/-GF) cell lysates, whereas megalin

was clearly detected in the cell lysates from HEK293 cells (figure 6.6 (A)).

As a last attempt to detect protein megalin expression from HK2 (+/-GF) cells, high

concentrations of protein samples (100-150𝜇g) were run through a 4% SDS-PAGE gel,

and then the Western blot steps were followed as described in materials and methods

(see 2.9.3). The same human anti-megalin antibodies were used. However, the

immunoblot analysis also showed no expression of megalin in HK2 (+/-GF) cell lysates,

whereas it was abundantly expressed in HEK293 cells (150𝜇g loaded) (figure 6.6 (B)).

207

Megalin 𝜷-actin

Figure (6.3): RT-qPCR efficiency for megalin and β-actin gene expression

in HEK293 cells. (A1 and B1) Show the amplification of β-actin

(housekeeping gene) and megalin in the serial dilution of cDNA (1:10)

prepared from (5𝜇𝑔 mRNA) HEK293 cells respectively. (A2) The standard

curve of β-actin (efficiency = 0.99) and (B2) the standard curve of megalin

(efficiency = 0.96). (A3 and B3) Show the melting curves analysis for β-

actin and megalin samples to determine specificity of the products,

respectively. Duplicate reactions were run for each sample.

208

Figure (6.4): RT-qPCR for mRNA megalin and β-actin gene expression in (5𝜇𝑔

mRNA) HK2 (+/-GF) cells (A1) the amplification of β-actin (housekeeping gene)

started early in cycle 10 but (B1) the amplification of megalin started very late at

cycle 35 (58C˚ annealing temperature). (A2 and B2) Show the melting curves

analysis for β-actin and megalin samples to determine specificity of the products,

respectively. Duplicate reactions were run for each sample.

A 𝜷-actin B Megalin

1

2

209

Figure (6.5): Gel electrophoresis analysis for RT-qPCR product (megalin

receptor). The RT-qPCR products were run in 1% agarose gel to show the

expression of (A) 𝛽-actin (170 bp) and (B) Megalin receptor (300bp) from

HK2 (+/-GF) cells at the correct size. Analysis of HEK293 cDNA was

included as a positive control for the expression of both genes. The products

from HK2 (+/-GF) cells are visibly as abundant as from HEK293 cells.

Megalin

300 bp

400bp

300bp

200bp

100bp

B

NTC HEK293 HK2 HK2-GF

800 bp

600bp

500bp

400bp

300bp

200bp

100bp

𝛽-actin

170bp NTC HEK293 HK2 HK2-GF

A

210

50KDa

37KDa

𝛽-actin (42KDa)

200KDa

4%

SD

S-P

AG

E

L 20 50 100 150/𝜇𝑔

Megalin (~600KDa) HEK293 cells

𝛽-actin (42KDa)

50KDa

37KDa

4-1

5%

SD

S-P

AG

E g

rad

ient

gel

200 KDa

Megalin (~600KDa)

L HK2 HK2-GF HEK293

211

6.2.2 MegCT-GST fusion protein phosphorylation in HK2 (+/-GF)

cells:

To test the hypothesis that MegCT phosphorylates at the PPPSP site, cell lysates from

HEK293 and HK2 (+/-GF) cells pre-stimulated with PDBU, FAF-HSA and 𝜆-LC at

different concentrations and times were examined using the PPPSP phosphor specific

antibody (See 2.16.2 and 2.16.3).

Cell lysates derived from HEK293 cells pre-stimulated with 10µM PDBU over a time

course (10, 20, 30 and 60min) showed that MegCT-GST phosphorylation was

augmented at 10 and more at the 20min (figure 6.7). Due to the maximum

phosphorylation on MegCT in HEK293 cells being detected at 20min, HK2 (+/-GF)

cells lysates were examined after being pre-stimulated with 10µM PDBU for 20min;

surprisingly, no phosphorylation in MegCT was detected in this condition. Therefore, to

determine whether the phosphorylation observed in HK2 (+/-GF) cells was

physiologically relevant, a time response experiment to 10µM PDBU was performed.

MegCT-GST phosphorylation was detected within 10min in the pre-stimulated lysate

compared to the control (un-stimulated) cells in HK2-GF cells, but no phosphorylation

detected at 20min of incubation, which explained why initially the phosphorylation of

MegCT-GST could not be detected (figure 6.8 (A)). Similarly, the time course of HK2

cells pre-stimulated with 10µM PDBU showed phosphorylation after 10 min compared

to the control and other stimulation times (figure 6.8 (B)). Based on this finding, HK2-

GF cells were pre-simulated with FAF-HSA (5 and 30mg/ml) for this short time of

10min. MegCT-GST phosphorylation was observed with 30mg/ml concentration

compared to un-stimulated control cells and pre-stimulated cells with 5mg/ml FAF-

HSA that showed no phosphorylation band. In addition, HK2-GF pre-stimulated with

10µM PDBU for 10min was used as positive control and the unmodified peptide (not

phosphorylated, TPPPSPSLPAKP) was detected by anti-peptide antibody in MegCT

Figure (6.6): Western blot analyses of megalin. (A) HK2 (+/-GF) and HEK293

cells lysate (20𝜇g) were loaded onto a 4-15% SDS-PAGE gradient gel, primary

antibody goat anti-human (1:1000) was used to detect megalin. (C) HEK293 cell

lysates (20, 50, 100 and 150 𝜇g) were loaded onto a 4% SDS-PAGE gel; primary

antibody rabbit anti-MegCT antisera (1:1000) detect intact megalin. (B and D) 𝛽-

actine was used as the loading control, (20𝜇g) from each sample were loaded onto

a separate 12% SDS-PAGE gel. The arrow indicates megalin.

212

in each sample as loading control (figure 6.9 (A)). Similarly, HK2 cells that pre-

incubated with (5 and 30 mg/ml) FAF-HSA for 10 min has been phosphorylated the

MegCT-GST with the 30mg/ml FAF-HSA compared to the absence of phosphorylated

band appear with 5mg/ml FAF-HSA stimulation and control cells (figure 6.9 (B)). 𝜆-LC

observes phosphorylation in vitro was investigated by a concentration response

experiment. 𝜆-LC stimulated the phosphorylation of MegCT-GST fusion protein at a

concentration (1mg/ml); however, higher phosphorylation was elicited by (5mg/ml) 𝜆-

LC compared to the un-stimulated control sample HK2-GF cells. The HK2-GF cells

were pre-stimulated with FAF-HSA (30/mg/ml) and PDBU (10µM) as positive controls

(figure 6.10 (A)). HK2 cells also showed similar results when the cells were pre-

stimulated with 𝜆-LC; the MegCT-GST activated and showed phosphorylation with

(1mg/ml) and the phosphorylation augmented with (5mg/ml) 𝜆-LC. As expected, no

phosphorylation was detected in un-stimulated HK2 cells compared to phosphorylate

MegCT-GST with lysates derived from HK2 pre-stimulated with FAF-HSA (30mg/ml)

and PDBU (10 and 20 µM) as positive controls (figure 6.10 (B)).

In summary, because the antibodies successfully discriminated between the

phosphorylation states of this peptide motif, this thesis could show that our protein

overload conditions using HK2 (+/-GF) cells led to phosphorylation of megalin

cytoplasmic tail in vitro. Multiple kinases regulate MegCT-GST fusion protein

phosphorylation (Baines, 2010), but this work specifies GSK-3 due to its proposed

phosphorylation of serine in our peptide of interest, PPPSP.

213

Anti-Phospho peptide

50kDa

Anti-Peptide

50kDa

HEK293 cells

(10µM PDBU)

C 10 20 30 60 /min

Figure (6.7): Time course of PDBU stimulated phosphorylation of MegCT-GST

fusion protein. HEK293 cells were pre stimulated with PDBU (10µM) for 10 up

to 60min. Stimulated cell lysates were incubated with MegCT-GST complex for

60min. Samples were run on 12% SDS-PAGE. PVDF membranes were then

probed with anti-phospho peptide antibody. As shown, HEK293 cells were

maximum phosphorylated after 20min incubation with PDBU compared to no

phosphorylation in control (un-stimulated cells) and less phosphorylation after

10min. Anti-peptide antibody detected the un-phosphorylated peptide was used as

loading control. One representative blot of at least two is shown.

214

Figure (6.8): Time course of PDBU stimulated phosphorylation of MegCT-GST

fusion protein by HK2 (+/-GF) cells. HK2 (+/-GF) cells were stimulated with PDBU

(10µM) for 10 up to 60min. Stimulated cell lysates were incubated with MegCT-

GST complex for 60min. Samples were run on 12% SDS-PAGE. PVDF membranes

were then probed with anti-phospho peptide antibody. (A) Shows HK2-GF cells were

phosphorylated after 10min incubation with 10µM PDBU compared to no

phosphorylation in control (un-stimulated cells) and (B) Show less phosphorylation

after 10min stimulation for HK2 cells. Anti-peptide antibody detected the un-

phosphorylated peptide and was used as loading control. One representative blot of at

least two is shown.


50kDa

Anti-Peptide

50kDa

HK2 cells

(10µM PDBU)

C 30 20 10/min


50kDa

HK2-GF cells

(10µM PDBU)

C 10 20 30 60/min

Anti-Peptide

50kDa

215

Figure (6.9): Effect of FAF-HSA on phosphorylation of MegCT-GST fusion

protein. HK2 (+/-GF) cells were pre-stimulated with FAF-HSA (5 and 30 mg/ml)

for 10min. Stimulated cell lysates were incubated with MegCT-GST complex for

60min. Samples were run on 12% SDS-PAGE. PVDF membranes were then probed

with anti-phospho peptide antibody. (A and B) Show HK2 (+/-GF) cells were

phosphorylated after 10 min incubation with 30mg/ml FAF-HSA and with PDBU

(10µM, positive control) comparing to no phosphorylation in control (un-stimulated

cells) and stimulated cells with 5mg/ml FAF-HSA. Anti-peptide antibody detected

the un-phosphorylated peptide and was used as loading control. One representative

blot of at least two is shown.

HK2-GF cells

10 min


50kDa

Anti-Peptide

50kDa

HK2-GF cells

10 min

Co

ntr

ol

10

µM

PD

BU

5 m

g/m

l F

AF

-HS

A

30

mg/m

l F

AF

-HS

A


50kDa

Anti-Peptide

50kDa

Contr

ol

5 m

g/m

l F

AF

-HS

A

30 m

g/m

l F

AF

-HS

A

216

Co

ntr

ol

10

µM

PD

BU

1 m

g/m

l 𝜆

-LC

30 m

g/m

l F

AF

-HS

A

HK2-GF cells

10 min


50kDa

Anti-Peptide

50kDa

Contr

ol

1 m

g/m

l 𝜆

-LC

30 m

g/m

l F

AF

-HS

A

10µ

M P

DB

U

20

µM

PD

BU

HK2 cells

10 min


50kDa

Anti-Peptide

50kDa

5 m

g/m

l 𝜆

-LC

5m

g/m

l 𝜆

-LC

Figure (6.10): Effect of 𝜆 -LC on phosphorylation of MegCT-GST fusion

protein. HK2 (+/-GF) cells were stimulated with 𝜆-LC (1 and 5 mg/ml) for

10min. Stimulated cell lysates were incubated with MegCT-GST complex for

60min. Samples were run on 12% SDS-PAGE. PVDF membranes were then

probed with anti-phospho peptide antibody. (A and B) Show HK2-GF cells

were phosphorylated after 10min incubation with both 𝜆-LC concentrations,

FAF-HSA (30mg/ml) and PDBU (10µM) as positive controls comparing to no

phosphorylation in control (un-stimulated cells). Similarly. Anti-peptide that

detected the un-phosphorylated peptide was used as loading control. One

representative blot of at least two is shown.

217

6.2.3 Effects of protein overload on mRNA expression for Megalin and

CD36 by HK2 (+/-GF) cells:

Megalin is a receptor that plays a role in the uptake, reabsorption and transport of

urinary protein by endocytosis in renal PTECs (Leheste et al., 1999) . Megalin-

mediated endocytic overload leads to damage of PTECs and this might develop

proteinuria (Saito et al., 2010). In addition, several studies suggest that additional

receptors for proteins in PTECs may exist and have a role in renal proximal tubule

fibrosis when up regulated in proteinuric condition such as CD36 (Yang et al., 2007).

To investigate the effects of protein overload on megalin and CD36 mRNA expression

in vitro, HK2 (+/-GF) cells were sub cultured in 6 well plates, then stimulated with

FAF-HSA or 𝜆-LC (5mg/ml) for 24 and 72h. After each time point the mRNA and

cDNA were prepared and gene expressions were analysed by RT-qPCR for both

receptors. HK2-GF cells exposed to FAF-HSA for 72h showed a significant increase in

mRNA CD36 expression; however, the mRNA CD36 expression from HK2 cells was

down regulated significantly after stimulation with FAF-HSA at the same 72h (figure

6.11 (A1)). On the other hand, stimulation with 𝜆-LC showed no change in CD36

mRNA expression and was observed in HK2 (+/-GF) cells at 24h, compared to a clear

and noticeable increase in mRNA CD36 expression at 72 from HK2 (+/-GF) cells

(figure 6.11 (B1)).

Next, HK2-GF cells stimulated with FAF-HSA showed a slight increase in megalin

receptor mRNA expression level at 72h. In contrast, HK2 cells displayed significant

elevation in megalin expression at 24h and persisted through 72h compared to the

control sample (figure 6.11 (A2)). With 𝜆-LC stimulation, HK2-GF cells had no change

in megalin receptor mRNA expression at both time points compared to the control

sample. Likewise, there was no change in megalin receptor mRNA expression level

after 24h of stimulation with 𝜆-LC, but the level was decreased after 72h in HK2 cells

(figure 6.11 (B2)).

218

FAF-HSA

Contr

ol

24

h

72

h

24

h

72

h0

1

2

3

m R

NA

CD

36 e

xpre

ssio

n

(Fold

chan

ge

vs.

contr

ol)

**

***

**

GF HK2 Contr

ol

24

h

72

h

24

h

72

h0

1

2

3

4

5

m R

NA

Meg

alin

expre

ssio

n

(Fold

chan

ge

vs.

contr

ol)

**

**

GF HK2

*

𝝀-LC

Contr

ol

24

h

72

h

24

h

72

h

0

1

2

3

m R

NA

CD

36 e

xpre

ssio

n

(Fold

change v

s.c

ontr

ol) ***

*

GF HK2

Contr

ol

24

h

72

h

24

h

72

h

0

1

2

3

4

5

m R

NA

Megalin e

xpre

ssio

n

(Fold

change v

s.c

ontr

ol)

***

GF HK2

*

Figure (6.11): Effect of FAF-HSA or 𝜆 -LC on megalin and CD36 mRNA

expression from HK2 (+/- GF) cells. Cells were sub cultured in 6 well plates (5 x

105 cells/well) and incubated with 5mg/ml 𝜆-LC or FAF-HSA for 24 and 72h. (A2)

FAF-HSA elevates megalin expression gradually from HK2 (time dependent)

compared to the control, however, (A1) CD36 expression from HK2-GF at 72h, but

significant decrease from HK2 cells at both time points comparing with the control.

(B2) 𝜆-LC decreased megalin mRNA expression at 72h from HK2 cells and no

change in HK2-GF with both time points, but (B1) It increased CD36 mRNA

expression at 72h from HK2 by (Quantitative RT-PCR) (2^-ΔΔCT value was used,

the mRNA expression normalized to β-actin and calibrated to the average of un-

treated controls). The data are represented as means of duplicate ± SD (n = 3)

(Unpaired t test P < 0.05).

219

In summary, from the results in this chapter it can be confirmed that MegCT is

phosphorylated in vitro by using cell lysates derived from HK2 (+/-GF) cells that were

pre-stimulated with FAF-HSA or 𝜆 -LC in a specific site (PPPSP) in the megalin

cytoplasmic domain, which may have a role in proximal tubular injury in proteinuria.

220

6.3 Discussion:

The mechanism of tubular handling of filtered proteins and pathomechanisms of

developing proteinuria remains an issue of interest. Megalin is the main receptor that is

responsible for protein endocytosis process in PTECs. Therefore, a high concentration

of proteins like albumin on proximal tubular cells is a sign of decreased endocytosis and

lead to proteinuria. Several studies reported that albuminuria might cause tubular

disease (Caruso-Neves et al., 2005).

Our in vitro work initially identified the gene and protein megalin expression in the

HK2 (+/-GF) cell line. In 2012, Sawada and his group examined the gene and protein

megalin expression in HK2 cells that were grown in a DMEM/F-12 medium with 10%

BSA. They found that HK2 cells showed a very slight expression of megalin mRNA

and protein. Our study agreed with Sawada et al. (2012) finding; our results showed no

mRNA expression in HK2 (+/-GF) cells by RT-PCR compared to a clear megalin band

from HEK293 (positive control). We explained that might be due to the low megalin

expression from HK2 (+/-GF) cells in our cultured condition; also, it might be the PCR

technique’s sensitivity to detect a very low expression of megalin from the cells. Thus,

the qRT-PCR’s more sensitive technique was used to detect the megalin expression and

we showed a clear but low megalin mRNA expression from HK2 (+/-GF) cells

compared to a clear abundance of meglain mRNA expression from HEK293 (positive

control). Fifty cycles were required to detect megalin mRNA in HK2 (+/-GF) cells

using the same primers for RT-PCR and qRT-PCR, while the housekeeping gene was

readily detectable. Low abundance of expression was the likely explanation of not

different splicing although this was not investigated.

In addition, our study could not detect megalin protein expression, compared to

considerable production that was detected with HEK293 cells (positive control). In

contrast, these observations were inconsistent with other studies that showed clear

megalin protein expression in HK2 cells. For example, Li et al. (2008) showed a clear

megalin protein production by Western blot from HK2 cells that were cultured in a

keratinocyte serum free medium supplemented with 5ng/ml EGF and 0.05% bovine

pituitary extract. Thus, one reason might explain the different levels of megalin mRNA

and protein expression, which is the cultured medium that was used in our conditions,

because the same Western blot method and anti-human megalin antibody (C-19) that

221

were used in Li et al. (2008) to detect megalin protein expression in HK2 cells were

utilised in this project.

The megalin endocytosis process might contribute to develop proximal tubular cell

damage in proteinuric conditions. This contribution might be mediated by (i) as initially

explained, the excessive amount of filtered protein in tubular filtrate leads to a decrease

in the endocytosis process, (ii) accumulation macromolecules ligands on tubular cells

(Baines, 2010), (iii) megalin ligands activate signalling pathways in proximal tubular

cells that lead to phosphorylate the megalin cytoplasmic domain in a specific site, and

this phosphorylation is able to decrease megalin cell surface expression (negative

recycling) (Marzolo & Farfán, 2011).

Part of this project carried on from a previous study (Baines, 2010), which identified

megalin as phosphor-acceptor protein. This study showed augmented phosphorylation

of MegCT-GST when this recombinant tool was incubated with primary human

proximal tubular cell lysates that were pre-stimulated with FAF-HSA (1mg/ml) or

PDBU (10µM) compared to no phosphorylation detected in the control un-stimulated

cells. This phosphorylation was detected by autoradiography (Baines, 2010). There are

multiple potential sites for phosphorylation in MegCT. As described initially, Baines

(2010) identified several phosphorylation sites in the MegCT (figure 6.1), which could

be affected by different kinases.

Based on previous studies, our project investigates if MegCT phosphorylates in PPPSP

site in vitro by protein-protein interaction. In vitro, our proteins overload conditions

(stimulate HK2 (+/-GF) cells with high concentration of FAF-HSA and 𝜆-LC) were

used to investigate if they phosphorylate the MegCT by incubating MegCT-GST with

pre-stimulated HEK293 and HK2 (+/-GF) cell lysates to activate kinases and this

activation causes the MegCT phosphorylation. Different FAF-HSA concentrations were

used to activate kinases that are capable of phosphorylating MegCT, like PDBU when

activated by PKC. Initially, this project showed MegCT phosphorylated in pre-

stimulated HEK293 and HK2 (+/-GF) cells by PDBU (10µM). Likewise, FAF-HSA

was shown to activate kinases and MegCT phosphorylated in HK2 (+/-GF) cells,

however, the phosphorylation in MegCT for HK2 (+/-GF) cells was detected with a

high concentration of FAF-HSA (30mg/ml) but no phosphorylation occurs with

(5mg/ml) at 10min; maybe the (5mg/ml) FAF-HSA needs a longer time to activate

222

kinases to phosphorylate MegCT. By comparing our finding with Baines (2010), in this

study (1mg/ml) FAF-HSA was used to pre-stimulate the primary human proximal

tubular cells and MegCT was phosphorylated; in contrast, in our study a fivefold higher

concentration was used (5mg/ml) FAF-HSA to pre-stimulate HK2 (+/-GF) cells but no

MegCT phosphorylation was detected compared to the augmented amount with HK2

(+/-GF) cells pre-stimulated with (30mg/ml) FAF-HSA. One reason might explain the

different between our results and Baines (2010) is that the type of proximal tubular

cells. Thus, the primary human proximal tubular cells maybe more sensitive, however,

the main idea, which is MegCT phosphorylates by albumin, was confirmed in both

conditions with primary or cell line proximal tubular cells.

Additionally, our work investigated if 𝜆 -LC protein is capable of phosphorylating

MegCT in the PPPSP site. The results showed a clear phosphorylation in MegCT after

pre-stimulating the HK2 (+/-GF) cells with (1mg/ml) 𝜆 -LC. Likewise, higher

phosphorylation occurred with (5mg/ml) 𝜆-LC in both HK2 (+/-GF) cells, compared to

no phosphorylation that was detected from un-stimulated cells and a clear

phosphorylation with FAF-HSA (30mg/ml) and PDBU (10 and 20𝜇M) as positive

controls.

In 2011, Marzolo & Farfán documented that the phosphorylation of MegCT by GSK-3

is able to decrease its cell surface expression (negative megalin recycling), because

when GSK-3 was blocked with two different inhibitors (LiCl or SB216763) the MegCT

phosphorylation was decreased significantly; more megalin was expressed on the cell

surface and was more efficiently recycled (Yuseff et al., 2007).

The interaction of albumin or 𝜆-LC with proximal tubular epithelial cells and MegCT

phosphorylation in the PPPSP site involving GSK-3 will activate several signalling

pathways that regulate this phosphorylation event. In 2007, Yuseff et al. showed that

the PPPSP site in MegCT was phosphorylated mainly by GSK-3 with a small

contribution from PKC. In addition, they reported that the phosphorylation occurring by

PKC and PKA in different sites in the cytoplasmic domain receptor were not the main

phosphorylation target because the deletion of the region with the PPPSP motif

markedly reduced the megalin phosphorylation; that means PPPSP is the major

phosphorylation and/or a priming site to allow phosphorylation in other sites within the

megalin cytoplasmic domain.

223

Among the kinases that regulate the MegCT phosphorylation, there is PI3-Kinase,

which has role in several cellular functions such as cell growth, proliferation,

differentiation, motility, survival and intracellular trafficking. Many of these functions

relate to the ability of PI3K to activate protein kinase B (PKB) and C (PKC) (Peart &

Gross, 2006 and Cabezas et al., 2011). PKB, also known as AKT, and PKC are

serine/threonine-specific protein kinases. The PKC activate the extracellular-signal-

regulated kinases/ Mitogen-activated protein kinase (ERK/MAPK), and the activation

of ERK and PKB will inhibit GSK-3 (figure 6.12).

PI3K is implicated in several PTCs processes and a main regulator of MegCT

phosphorylation and albumin endocytosis in PTCs (Brunskill et al., 1998). In addition,

it regulates apoptosis by phosphorylating PKB, and phosphorylating PKB will lead to

inhibiting Bcl-2-associated death promoter (BAD) and thereby inhibits apoptosis

(Caruso-Neves et al., 2005); also, albumin-induced phosphorylation of MegCT is

dependent on ERK activity (Dixon & Brunskill, 2000). ERK is stimulated by oxidative

stress and has an inhibitory influence on PKB in PTCs (Sinha et al., 2004). Thus,

MegCT phosphorylation may be the result of a complex series of interactions between

ROS, ERK and PI3k/PKB activity. Also, these kinases are involved in GSK-3

activity/inhibition and regulate the MegCT phosphorylation. PI3K activates PKB/AKT,

which activates MAPK/ERK to inhibit GSK-3, and at the same time it activates PKB

also to inhibit GSK-3. However, when MAPK/ERK activates by ROS and this

activation lead to inhibit PKB/AKT, which lead to activate GSK-3 and phosphorylation

event will occur (Baines, 2010) (figure 6.12). To confirm this observation, the

production of ROS from HK2 (+/-GF) cells in our protein overload conditions will be

investigated in the next chapter.

http://en.wikipedia.org/wiki/Protein_kinase_B

http://en.wikipedia.org/wiki/Serine/threonine-specific_protein_kinase

224

FLC (Free Light Chain)

MegCT (Megalin Cytoplasmic Tail)

PI3K (Phosphatidylinositide 3-kinase)

PKB/AKT (Protein Kinase B)

PKC (Protein Kinase C)

ERK (Extracellular-signal-regulated kinases)

MAPK (Mitogen-activated protein kinase)

GSK3 (Glycogen synthase kinase 3)

ROS (Reaction Oxygen Species)

PTECs (Proximal Tubular Epithelial cells)

Activation

Inhibition

Stop

Phosphorylation

MegCT- XXXXXPPPSPXXXXX MegCT- XXXXXPPPSPXXXXX

P

GSK-3 Activation GSK-3 Inhibition

PI3K

PKB/AKT PKC

MAPK/ERK

GSK-3

ROS

Megalin Receptor

PTECs

GSK-3

PI3K

PKB/AKT PKC

MAPK/ERK

Albumin or FLC

P

ROS

Figure (6.12): Interactions of Albumin or 𝝀-LC with PTECs and different signalling kinase pathways that regulate

MegCT phosphorylation.

225

As explained initially, filtered proteins are reabsorbed in PTECs by receptor-mediated

endocytosis process such as megalin and cubilin receptors. Evidence suggests that other

receptors for those proteins might exist like CD36. In 2005, Caruso-Neves and his

group said a high concentration of albumin like in proteinuric conditions down regulates

megalin; in contrast, in proteinuric nephropathies, albumin up-regulates CD36 (Baines

et al., 2012).

In vitro, Baines et al. (2012) showed an increase in CD36 expression from HK2 cells

stimulated with albumin (10mg/ml) for 48h; also, in vivo they found an increase in

CD36 expression in PTC in kidney sections from patients with three different nephrotic

diseases. These results suggested a direct relation between CD36, albumin uptake and

reabsorption in PTECs. Another study also showed an increase in CD36 expression

from stimulated proximal tubular cells (LLC-PK1) with albumin (1 and 10mg/ml) for

36h, and this increase in CD36 may interact with thrombospondin-1 (TSP-1) and lead to

activating TGF-𝛽, which initiates and regulates fibrosis (Yang et al., 2007). Our results

agreed with previous studies in that stimulated HK2-GF cells with FAF-HSA (5mg/ml)

showed an increase in CD36 mRNA expression at 72h, however, HK2 cells showed the

opposite result. The CD36 expression was down regulated at 72h. Also, stimulating

HK2 (+/-GF) cells with 𝜆-LC (5mg/ml) showed a significant increase in CD36 mRNA

expression at 72h. Thus, our data showed increases in CD36 in our proteinuria

conditions with both albumin and 𝜆-LC, which might contribute to and cause further

progression of renal tubular dysfunction.

On the other hand, in in vivo proteinuric animal models of diabetic nephropathy megalin

expressions are down regulated (Baines et al., 2012). In vitro, the effects of protein

overload conditions on PTC megalin expression were studied by stimulating HK2 (+/-

GF) cells with FAF-HSA and 𝜆-LC for 24 and 72h. Our results showed that megalin

mRNA expression increased slightly in HK2-GF cells at 72h; in contrast, HK2 cells

showed a significant increase in megalin expression at 24h and persisted through 72h in

a time-dependent manner with FAF-HSA.

On the other hand, stimulating the cells with FAF-HSA showed an increase in megalin

expression from HK2-GF cells at 72h, however, HK2 cells showed significant elevation

in megalin mRNA levels at 24h and persisted through 72h. A previous study examined

the effect of a pathophysiological concentration of albumin on megalin mRNA

226

expression by incubating LLC-PK1 cells with a range of albumin (with fatty acid)

concentrations for 24h by RT-PCR. The results showed a decrease in megalin mRNA

by 40% with 20mg/ml (Caruso-Neves et al., 2005). By comparing this study with our

results, we can clarify that the megalin mRNA level increase in our protein overload

conditions could be because of the low concentration of albumin; they used a range of

albumin concentration starting with (0.1 up to 20 mg/ml) and the decrease was detected

just with five times higher concentration of albumin than our concentration. Also, they

used albumin with fatty acid and this might affect the results. In spite of this, 𝜆-LC

(5mg/ml) had no effect on mRNA megalin expression at 24h, but at 72h the level was

decreased significantly in HK2 (+/-GF) cells compared to the 24h expression level.

Taken together, the same (5mg/ml) FAF-HSA and 𝜆 -LC had different effects on

megalin mRNA expression from the HK2 (+/-GF) cells. Further studies are needed to

investigate the function relationship between megalin and CD36 expression.

In summary, our results in this chapter provide confirmation of our hypothesis that

MegCT is phosphorylated in vitro by using cell lysates derived from HK2 (+/-GF) cells

in response to FAF-HSA and 𝜆-LC, which might be has a role in PTECs damage in

proteinuric conditions.

227

Chapter Seven- Effects of Protein

Overload on Proximal Tubular Cells in

the Progression of Damage In Vitro

228

7. Introduction:

Renal failure means a decrease in kidney function. This condition can be a cause for

high morbidity and mortality, so early diagnosis is very important because it might help

to save the kidney from additional damage.

Proteinuria is recognised as a marker of renal disease and could be pre-glomerular

injury. Albuminuria causes PTCs injury that leads to kidney injury; this injury occurs

through the albumin uptake endocytosis process that leads to activate many different

intracellular signaling pathways in PTCs.

Inflammatory and dysregulation mechanisms in kidneys lead to nephron loss (Kriz &

LeHir, 2005). This chapter focuses on protein overloading of PTECs and the resultant

production of key cytokines and chemokines as an important cell type specific

mechanism responsible for the progression of renal insufficiency in proteinuric diseases

such as IL-6, IL-8 and MCP-1, which are made at a basal level by epithelial cells

(Stadnyk, 1994). It has been reported that albumin induces IL-8 in PTECs at both

transcriptional and translational levels (Tang et al., 2003). Also, a normal kidney

expresses MCP-1 (Wang et al., 1997) that is up regulated in PTCs challenged with

protein overload (Zoja et al., 2003). The effect of FAF-HSA and 𝜆 -LC (protein

overload) on mRNA expression and protein production for these cytokines from HK2

(+/-GF) cells was measured in this chapter to investigate their role in PTECs damage.

The application of developed complement inhibition to renal diseases with proteinuria

has previously been discussed (Inal et al., 2003) but the development has been

hampered by the need of treating progressive disease over a long period, more data are

needed. Many pathophysiological mechanisms have been proposed to be a reason for

proteinuria induced kidney injury such as the activation of the complement system

(Morita et al., 2000). There is accumulating evidence showed that complement

activation pathways in the PTECs may have a major role that leads to cell damage; this

might occur if complement components are activated in tubular cells then lead to cell

activation, resulting in tubular damage (Gaarkeuken et al., 2008). The alternative

pathway (AP) of the complement system is involved in kidney diseases. In in vitro

studies there was evidence that PTECs activate complement via AP by measuring the

229

C5b-9 membrane attack complex on cells surface; this measurement is in the presence

of AP-favoring Mg-EGTA conditions. In vivo, C3 and C5b-9 were found in PTCs in rat

kidneys induced by protein overload (Zoja et al., 2003).

In 1999, Peake and his group demonstrated that HK2 cells express mRNA for all the

alternative pathway components by PCR. The hypothesis that the protein overload

effects on the expression of AP complement components was examined in this chapter.

Also, one of the effects in response to protein overload is activation the hydrogen

peroxide (H2O2) pathway and generation H2O2 in human proximal tubular cells (Abbate

et al., 2006). The production of H2O2 from the PTCs as a response to protein overload

may activate different cascades, leading to inflammation, fibrosis or apoptosis for cells.

For example, the production of H2O2 will activate the caspase cascades and lead to cell

apoptosis; also, it will stimulate IL-8 and TGF-𝛽1 and lead to cell fibrosis (Imai et al.,

2004). The effect of FAF-HSA and 𝜆-LC on H2O2 generation and their role in PTECs

damage was investigated in the chapter.

Lastly, this chapter scans for possible biomarkers of proteins that might induce kidney

injury. An array was used in order to identify a potential biomarker from in vitro studies

that could be useful in early diagnosis, guiding targeted intervention and monitoring

disease progression and resolution. 38 different types of protein biomarkers were

examined for kidney injury. They divided to four main categories: low molecular

weight proteins, up regulated proteins, cytokines/chemokines and anti-inflammatory

cytokines (figure 7.17).

7.1 Aim:

The aim of this chapter is to study how protein overloading might modulate/influence

production of inflammatory markers, complementary components and ROS, which is

implicated in the progression of cell injury and kidney disease.

230

7.2 Results:

7.2.1 Evaluation the effect of FAF-HSA and 𝝀-LC on cytokine

production in HK2 (+/-GF) cells:

A significant increase in LDH release was detected in the supernatant of the HK2 (+/-

GF) cells exposed to (5mg/ml) FAF-HSA and 𝜆-LC for 24 and 72h. The supernatants

from the experiments were used to measure cytokines and chemokines (see 5.2.1.2.1).

Interleukin-6 (IL-6), interleukin IL-8 (IL-8) and monocyte chemo attractant protein-1

(MCP-1) were measured in the supernatants using commercial human ELISA kits.

Also, total RNA was isolated from the control and stimulated HK2 (+/-GF) cells at the

different time-points to quantify IL-6, IL-8 and MCP-1 gene expression.

Epithelial cells produce IL-6 constitutively (Krueger et al., 1991). Therefore, the effects

of FAF-HSA and 𝜆-LC on IL-6 production from HK2 (+/-GF) cells were evaluated.

There was a marked effect on IL-6 secretion (7.1 (A)). HK2-GF cells showed a quick

response with FAF-HSA stimulation at 24h and the maximum effect was seen at 72h of

exposure, the longest time interval tested in these studies, compared to un-stimulated

control cells and medium alone. Also, HK2-GF showed a similar response with 𝜆-LC

stimulation; IL-6 secretion was increased significantly at 24h and the highest IL-6

secretion was at 72h. However, HK2 cells show strong late IL-6 production at 72h in

response to both proteins (7.1 (A)).

Figure (7.1 (B)) shows a direct comparison between mRNA levels of IL-6 from HK2

(+/-GF) cells after 24 and 72h treating with (5mg/ml) FAF-HSA. Treating HK2 (+/-GF)

cells with FAF-HSA for 24h up-regulated the IL-6 mRNA expression significantly. On

the other hand, they showed an important decrease at 72h of stimulation compared to

the control by RT-PCR. The results are presented as a ratio of densitometric values for

the gene relative to β-actin mRNA expression from each control and stimulated sample

using Image J software. Next, in figure (7.1 (C)), after 𝜆-LC stimulation the expression

of IL-6 increased in a time-dependent manner from HK2 (+/-GF) cells compared to

control by RT-qPCR; 2^- ΔΔ CT value was used, then the mRNA expression was

normalised to β-actin and calibrated to the average of un-treated controls. HK2 cells

stimulated with (5mg/ml) 𝜆-LC for 72h showed the highest IL-6 expression.

231

C 2

4h

24h

C 7

2h

72h

C 2

4h

24h

C 7

2h

72h0.0

0.5

1.0

1.5

2.0

2.5

Rat

io o

f m

RN

A (

IL

6 /β-a

ctin

)

GF HK2

**

**

* **

FAF-HSA

C2

42

4C

72

72

C2

42

4C

72

72

C2

42

4C

72

72

C2

42

4C

72

720

100

200

300

400

500

IL6 (

pg/m

l)

FAF-HSA

l-LC

Med

ium

HK2 HK2GF

***

***

***

***

*** ***

GF

232

𝝀-LC

Con

trol

24h 72

h

24h

72h0

5

10

15

m R

NA

IL

6 ex

pres

sion

(Fol

d ch

ange

vs.

cont

rol)

***

***

***

**

GF HK2

Figure (7.1): The effects of 𝜆-LC and FAF-HSA on IL-6 protein production and

mRNA expression by HK2 (+/-GF) cells. Cells were sub-cultured in 6 well plates (5

x 105 cells/well) and incubated with 𝜆-LC or FAF-HSA (5mg/ml) for 24 and 72h.

The supernatants were collected and IL-6 secretion was measured (ELISA), and

total RNA (2𝜇g) from each sample was prepared to study IL-6 gene expression

(PCR). Un-treated cells were used as the control. (A) 𝜆 -LC and FAF-HSA

significantly stimulated IL-6 protein production at 72h from HK2 cells, however,

HK2-GF produced IL-6 from the first 24h of stimulation time with both proteins.

(B) The expression of IL-6 mRNA was increased at 24h with FAF-HSA and

decreased after 72h in HK2 (+/-GF) cells (semi-quantitative RT-PCR). The results

are presented as a ratio of densitometry analysis of the gene relative to β-actin

mRNA expression (housekeeping gene) using Image J software. (C) 𝜆 -LC

stimulation leads to a time-dependent increase of IL-6 expression in HK2 (+/-GF)

cells (RT-qPCR) (2^-ΔΔCT value was used, the mRNA expression normalized to β-

actin and calibrated to the average of un-treated controls). The data are represented

as means of duplicate ± SD (n = 2) (Unpaired t test P < 0.05), C: control sample.

233

Tubular epithelial cells produce IL-8 and they are regulated by a variety of pro-

inflammatory cytokines like TNF-α (Tang et al., 2003). The effects of FAF-HSA and 𝜆-

LC on IL-8 secretion by HK2 (+/-GF) cells are shown in figures (7.2 (A)). After 24h of

exposure to 𝜆 -LC IL-8 production was increased, but the levels were significantly

decreased by prolonged treatment for 72h for both HK2 (+/-GF) cells. In contrast,

exposure HK2 (+/-GF) cells to FAF-HSA had no effect on IL-8 levels after 24 and 72h,

but there was a significant rise at 72h of stimulation of HK2 cells with FAF-HSA. IL-8

was measured in the supernatants of un-stimulated cells and medium alone as controls.

𝜆-LC incubation leads to a peak response of IL-8 at 24h regardless of the culture media

used, and with or without the EGF cocktail. By contrast, the late production of IL-8

caused at 72h by FAF-HSA is blunted in the presence of EGF. The expression of IL-8

was increased gradually with time by (5mg/ml) FAF-HSA at 24 and 72h from HK2-GF

cells, but HK2 cells showed an increase at 72h compared to its controls (figure 7.2 (B))

HK2 (+/-GF) cells behaved similarly; IL-8 mRNA expression was up regulated by

(5mg/ml) 𝜆-LC at 24h, and the increase in expression peaked at 72h compared to the

control (figure 7.2 (C)). These 72h increases could indicate that the HK2 cells are not

exhausted in their IL-8 production at this time point, but rather up regulate their mRNA

expression.

C2

4

24

C7

2

72

C2

4

24

C7

2

72

C2

4

24

C7

2

72

C2

4

24

C7

2

720

200

400

600

800

1000

1200

IL8 p

g/m

l

**

**

FAF-HSA λ-LC

Med

ium

**

*

****

**

***

HK2 GFGF HK2

234

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5

Rat

io o

f m

RN

A (

IL

8 /β-a

ctin

)

GF HK2

****

**

FAF-HSA

𝝀-LC

Con

trol

24h

72h 24

h

72h0

1

2

3

4

5

m R

NA

IL8

expr

essi

on

(Fol

d ch

ange

vs.

cont

rol)

***

***

**

**

GF HK2

Figure (7.2): The impacts of 𝜆-LC or FAF-HSA on IL-8 protein production and

mRNA expression by HK2 (+/-GF) cells. Cells were sub-cultured in 6 well plates

(5 x 105 cells/well) and incubated with (5mg/ml) 𝜆-LC or FAF-HSA for 24 and

72h. The supernatants were collected and IL-8 protein production was measured

(ELISA), and total RNA (2𝜇g) from each sample was prepared to study IL-8 gene

expression (PCR). Un-treated cells were used as the control. (A) 𝜆 -LC

significantly stimulated the production of IL-8 in 24h from HK2 (+/- GF) cells,

while FAF-HSA had no effect, except for the significant increase of IL-8

production from HK2 cells in 72h. (B) The expression of IL-8 mRNA was

increased with FAF-HSA at 72h from HK2 (+/-GF) cells (semi-quantitative RT-

PCR). The results are presented as a ratio of densitometry analysis of the gene

relative to β-actin mRNA expression (housekeeping gene) using Image J software.

(C) However, with 𝜆 -LC stimulation of the IL-8 expression rose in a time-

dependent manner in HK2 (+/-GF) cells by (RT-qPCR) (2^-ΔΔCT value was used,

and the mRNA expression normalized to β-actin and calibrated to the average of

un-treated controls). The data are represented as means of duplicate ± SD (n = 2)


235

Proximal tubular epithelial cells are one of major sources of MCP-1. Chemokine MCP-

1 production is increased in proteinuric conditions. In addition, it plays a major role in

the proximal tubule to produce inflammatory mediators (Wang & Sanders, 2007).

Overnight incubation of HK2 cells with FAF-HSA and 𝜆-LC had no effect on MCP-1

secretion, but it was enhanced at 72h compared to the control cells. On the other hand,

HK2-GF cells showed a slight increase in the MCP-1 production after FAF-HSA and 𝜆-

LC at 24h, and a further increase at 72h just by 𝜆-LC (figure 7.3 (A)).

Presence of the EGF cocktail seems to dampen MCP-1 release and this extends 𝜆-LC

and FAF-HSA induced mRNA expression; the expression of MCP-1 mRNA was

significantly up regulated by 𝜆-LC steadily at 24 and 72h from HK2-GF cells. Also,

HK2 cells appeared to increase in MCP-1 expression at 24h, but at 72h the expression

highly increased compared to the control. FAF-HSA had no effect on MCP-1 mRNA

expression with HK2 (+/-GF) cells compared to the controls, except the slight increase

from HK2 cells at 72h (figure 7.3 (B and C)).

In summary, these results suggest that FAF-HSA and 𝜆 -LC stimulated several

inflammatory mediators in HK2 (+/-GF) cells, which may have a role in proximal

tubular inflammation.

236

FAF-HSA

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5

Rat

io o

f m

RN

A (

MC

P-1

/β-a

ctin

)

GF HK2

**

*

C2

42

4C

72

72

C2

42

4C

72

72

C2

42

4C

72

72

C2

42

4C

72

720

100

200

300

400

500

MC

P-1

(p

g/m

l)

*

FAF-HSA

l-LC

Med

ium

**

***

*

***

GF HK2GF HK2

237

𝝀-LC

Con

trol

24h

72h

24h 72h0

5

10

15

m R

NA

MC

P-1

exp

ress

ion

(Fol

d ch

ange

vs.

cont

rol)

**

****

****

**

GF HK2

Figure (7.3): The effects of 𝜆-LC or FAF-HSA on MCP-1 protein production and

mRNA expression from HK2 cells (+/-GF). Cells were sub-cultured in 6 well

plates (5 x 105 cells /well) and incubated with 𝜆-LC or FAF-HSA (5mg/ml) for 24

and 72h. The supernatants were collected and MCP-1 protein production was

measured (ELISA), and total RNA (2𝜇g) from each sample was prepared for

MCP-1 gene expression (PCR). Un-treated cells were used as the control. (A) 𝜆-

LC significantly stimulated the production of MCP-1 in 72h from HK2 (+/-GF)

cells while FAF-HSA had no effect, except the significant increase of MCP-1

production from HK2 cells in 72h. (B) The FAF-HSA had no effects on MCP-1

mRNA expression, except the increase at 72h from HK2 cells (semi-quantitative

RT-PCR). The results are presented as a ratio of densitometry analysis of the gene

relative to β-actin mRNA expression (housekeeping gene) using Image J

software. (C) 𝜆 -LC stimulation MCP-1 expression rise with the time (time-

dependent) in HK2 (+/-GF) cells by (RT-qPCR) (2^-ΔΔCT value was used, the

mRNA expression normalized to β-actin and calibrated to the average of un-



238

7.2.2 Complement component production by HK2 (+/-GF) cells:

To study the alternative pathway (AP) complement components in protein overload

conditions, HK2 (+/-GF) cells were sub-cultured in 6 well plates and incubated with

FAF-HSA or 𝜆-LC (5mg/ml) for 24 and 72h; cell protein lysates and total mRNA from

control and stimulated samples were prepared for protein production by Western blot

and gene expression by PCR.

C3 is an essential complement protein to activate AP (Zoja et al., 2003). The effects of

protein overload on C3 complement components are shown in figure (7.4). HK2 (+/-

GF) cells stimulated with FAF-HSA up regulated C3 mRNA expression after 24h. At

72h, the expression from HK2-GF was decreased comparing to the 24h sample but still

higher than control sample at the same time point, however, HK2 cells persisted the

elevation through 72h (figure 7.4 (A)). Comparably, there is a significant time

dependent increase in C3 mRNA after 𝜆-LC treatment of HK2 (+/-GF) cells over 24

and 72h (7.4 (B)).

Complement factor B (FB) mRNA expression was increased in HK2 (+/-GF) cells

stimulated with FAF-HSA at 24h and it returned to the normal level compared to the

control samples at 72h (figure 7.5 (A)). This contrasted with findings obtained for HK2

(+/-GF) cells stimulated with 𝜆-LC for 24h. The greater increase for FB mRNA was

observed for HK2 stimulated with 𝜆-LC for 72h (figure 7.5 (B)).

Complement Factor H (FH) is the important AP regulator to balance between the

complement activation and inhibition, which might be disturbed in the proteinuria

condition. For example, albumin reduces FH binding (Fearn & Sheerin, 2015). FH

mRNA expresses from HK2 cells (Peake et al., 1999) and the protein production was

detected from human proximal tubular epithelial cells (Zhou et al., 2001).

Properdin or FP reportedly has a major role in stabilising the AP convertase in tubular

epithelium cells when binding to glycosaminoglycans on cell surfaces (Fearn &

Sheerin, 2015). HK2 cells express FP mRNA (Peake et al., 1999).

Our result showed that the mRNA was increased after 24h of stimulation with FAF-

HSA (5mg/ml) for HK2 (+/-GF) cells; however, at 72h HK2-GF decreased the

expression of FH, but the expression was still high from the HK2 cells (figure 7.6 (A)),

239

contrasting with 𝜆-LC stimulation, which led to a significant reduction or no change in

FH mRNA expression from HK2 (+/-GF) cells at 24 and 72h (figure 7.6 (A)). Notably,

the HK2 (+/-GF) cells treated with 𝜆-LC did not significantly increase the mRNA

expression for FH in both 24 and 72h incubation, although, the same samples did yield

enhancement of C3 and FB mRNA. The absence of an increase in mRNA coding for the

main regulator of the AP may be functionally important in increasing complement-

mediated damage, if reduced mRNA coincides with reduced protein production. C3b is

formed by the cleavage of C3 complement component to C3a and C3b. C3b binds to FB

to create unstable C3bBb and to be stable should bind to properdin or factor P (FP),

which promotes the association of C3b with FB and inhibits FH. As shown in figure

(7.7 (A)) the mRNA FP expression was increased significantly from HK2-GF cells after

72h of treatment with FAF-HSA, but there was no change in properdin mRNA

expression in HK2 cells. On the other hand, 𝜆-LC stimulation led to an increase in the

mRNA FP level at 24 and 72h from both HK2 (+/-GF) cells compared to their un-

stimulated controls (figure 7.7 (B)).

240

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5

2.0

2.5

Rat

io o

f m

RN

A (

C3 /β-a

ctin

)

GF HK2

*

* *

***

FAF-HSA

Con

trol

24h 72h

24h

72h0

1

2

3

4

5

m R

NA

C3

expr

essi

on

(Fol

d ch

ange

vs.

cont

rol)

****

****

****

****

GF HK2

𝝀-LC

Figure (7.4): Effects of FAF-HSA or 𝜆-LC on C3 mRNA expression from HK2

(+/-GF). Cells were sub-cultured in 6 well plates (5 x 105 cells/well) and

incubated with (5mg/ml) 𝜆-LC or FAF-HSA for 24 and 72h. (A) The FAF-HSA

up-regulate C3 expression from HK2 (+/-GF) compared to the control by (semi-

quantitative RT-PCR). The results are presented as a ratio of densitometry

analysis of the gene relative to β-actin mRNA expression (housekeeping gene)

using Image J software. (B) 𝜆-LC elevates C3 expression in a time dependent

manner) in HK2 cells (+/-GF) by (RT-qPCR) (2^- ΔΔCT value was used, the

mRNA expression normalized to β-actin and calibrated to the average of un-



241

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.2

0.4

0.6

0.8R

atio

of

mR

NA

(F

B /β-a

ctin

)

GF HK2

**

**

FAF-HSA

Contr

ol

24

h

72

h

24

h

72

h0

1

2

3

4

5

m R

NA

FB

exp

ress

ion

(Fol

d c

hang

e vs

.cont

rol)

**

**

GF HK2

𝝀-LC

Figure (7.5): Impacts of FAF-HSA or 𝜆-LC on FB mRNA expression from HK2

(+/-GF). Cells were sub-cultured in 6 well plates (5 x 105 cells/well) and

incubated with (5mg/ml) 𝜆-LC or FAF-HSA for 24 and 72h. (A) FAF-HSA up-

regulates FB expression from HK2 (+/- GF) at 24h comparing with the control

(semi-quantitative RT-PCR). The results are presented as a ratio of densitometry

analysis of the gene relative to β-actin mRNA expression (housekeeping gene)

using Image J software. (B) 𝜆-LC had no effects on FB expression except the

significant increase from HK2 cells at 72h by (RT-qPCR) (2^-ΔΔCT value was

used, the mRNA expression normalized to β-actin and calibrated to the average of

un-treated controls). The data are represented as means of duplicate ± SD (n = 2)


242

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0.0

0.5

1.0

1.5

2.0R

atio

of

mR

NA

(F

H /β-a

ctin

)

GF HK2

**

**

** ***

FAF-HSA

Contr

ol

24

h

72

h

24

h

72

h0

1

2

3

4

5

m R

NA

FH

expre

ssio

n

(Fold

chan

ge

vs.

contr

ol)

**** ****

GF HK2

𝝀-LC

Figure (7.6): Effects of FAF-HSA or 𝜆-LC on FH mRNA expression from HK2 (+/-GF).

Cells were sub-cultured in 6 well plates (5 x 105 cells/well) and incubated with (5mg/ml) 𝜆-

LC or FAF-HSA for 24 and 72h. (A) FAF-HSA elevates FH expression from HK2 (+/- GF)

at 24h comparing with the control (semi-quantitative RT-PCR). The results are presented as

a ratio of densitometry analysis of the gene relative to β-actin mRNA expression

(housekeeping gene) using Image J software. (B) 𝜆-LC decreased FH expression by (RT-

qPCR)(2^-ΔΔCT value was used, the mRNA expression normalized to β-actin and calibrated

to the average of un-treated controls). The data are represented as means of duplicate ± SD

(n = 2) (Unpaired t test P < 0.05).

243

FAF-HSA

Con

trol

24h

72h

24h 72

h0

2

4

6

m R

NA

Pro

perd

in e

xpre

ssio

n

(Fol

d ch

ange

vs.

cont

rol) ***

GF HK2

Con

trol

24

h

72

h

24

h

72

h0

2

4

6

m R

NA

Pro

perd

in e

xpre

ssio

n

(Fol

d ch

ange

vs.

cont

rol)

****

**

****

GF HK2

𝝀-LC

Figure (7.7): Effects of FAF-HSA or 𝜆-LC on Properdin mRNA expression

from HK2 (+/-GF). Cells were sub-cultured in 6 well plates (5 x 105

cells/well) and incubated with (5mg/ml) 𝜆-LC or FAF-HSA for 24 and 72h.

(A) FAF-HSA elevates properdin expression from HK2-GF at 72h, but no

significant change in HK2 cells at both time points compared to the control.

(B) Also, 𝜆-LC increase properdin mRNA expression at 72h from HK2 (+/-

GF) by (RT-qPCR)(2^- ΔΔ CT value was used, the mRNA expression

normalized to β-actin and calibrated to the average of un-treated controls).

The data are represented as means of duplicate ± SD (n = 2) (Unpaired t test P

< 0.05).

244

Because there is not necessarily concordance between mRNA expression and protein

production, complement components protein production in HK2 (+/-GF) cells were

examined. The extracted protein lysates from stimulated HK2 (+/-GF) cell with FAF-

HSA or 𝜆-LC (5mg/ml) at 24, 72h and un-stimulated control samples were analysed by

Western blot.

The C3 protein production was noticeably elevated from stimulated HK2 (+/-GF) cells

with FAF-HSA at 24h and continually up-regulated with the longer time incubation

72h. Compared to the level of C3 from un-treated control cells, HK2 and HK2-GF

behaved similarly (figure 7.8 (B)). Meanwhile, stimulating the HK2 (+/-GF) cells with

𝜆-LC increased the C3 production at 24h. Nerveless, the C3 level was clearly reduced at

72h compared to the C3 production level from the 24h stimulated sample, but the

production was still higher than the control samples (figure 7.8 (B)).

Following, the results figure (7.9 (C)) displays elevated FB levels from HK-GF cells at

24h of FAF-HSA stimulation, but at 72h the FB production was decreased to the basal

level. However, there was no significant effect on FB at 24 and 72h of stimulation of

HK2 cells with FAF-HSA. Moreover, 𝜆-LC had the same effect as FAF-HSA on HK2-

GF cells in increasing FB production with no effect on HK2 cells at 24h. In contrast, 𝜆-

LC stimulation led to an increase in the FB production significantly at 72h from HK2

cells.

The protein bands were quantified by scanning densitometer ImageJ software in figure

(7.8 (B) and 7.9 (C)), respectively. In addition, FH (polyclonal 150kDa) and properdin

(polyclonal 55kDa) have been detected by Western blot; however, the bands were too

faint to be analysed (data not shown).

245

𝜷 -Actin

42 kDa

C S C S

72h 24h

C S C S

72h 24h

FAF-HSA 𝝀-LC

GF

H

K2

C3

100 kDa

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h

0

1

2

3

4

5

Den

sit

om

erty

rati

on

(C

3 /β-a

cti

n)

HSA-FFA

GF

*

** *****

HK2

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h

0

1

2

3

4

5

LC

Den

sit

om

erty

rati

on

(C

3 /β-a

cti

n)

GF

**

***

HK2

𝝀-LC FAF-HSA

Figure (7.8): Effects of exposure to FAF-HSA or 𝜆-LC on C3 protein production

from HK2 (+/-GF) cells. Cells were sub-cultured (5 x 105 cells/well) in 6 well plates

and stimulated with FAF-HSA or 𝜆-LC (5mg/ml) for 24 and 72h. Cell lysates were

prepared and analysed by Western blot. (A) Strongest C3 production from HK2 (+/-

GF) cells was observed at 72h of stimulation with FAF-HSA, while the 𝜆 -LC

increased C3 abundance early (24h) in HK2 (+/-GF) cells. 𝛽-actin was used as the

loading control. (B) Densitometric analysis of C3 protein production in (A). The

results are presented as a ratio of densitometry analysis of C3 to β-actin using Image J

software. The data are represented as means ± SD (n = 2) (Unpaired t test P < 0.05),

C: control sample and S: stimulated sample.

246

FB

95 kDa

C S C S

24h 72h

C S S

24h 72h

GF

H

K2

𝝀-LC FAF-HSA

kDa

130

100

70

50

Human Plasma

(positive control

for FB)

C 2

4h

24

h

72

h

C 2

4h

24

h

72

h0

2

4

6

HSA-FFA

GF

Den

sito

mer

ty r

atio

n (

FB

/β-a

ctin

)

HK2

**

C 2

4h

24

h

C 7

2h

72

h

C 2

4h

24

h

C 7

2h

72

h0

1

2

3

4

LC

Den

sit

om

erty

rati

on

(F

B /β-a

cti

n)

GF

*****

HK2

𝝀-LC FAF-HSA

Figure (7.9): Effects of exposure to FAF-HSA or 𝝀-LC on FB protein production

from HK2 (+/-GF) cells. Cells were sub-cultured (5 x 105 cells/well) in 6 well

plates and stimulated with FAF-HSA or 𝛌-LC (5mg/ml) for 24 and 72h. Cell

lysates were prepared and analysed by Western blot. (A) Positive control for FB in

25𝝁𝒍 of human serum albumin (1:100 dilutions in H2O). (B) FAF-HSA and 𝝀-LC

elevated FB production from HK2- GF cells at 24h. 𝜷 -actin was used as the

loading control. (C) Densitometric analysis of FB protein production in (A). The

results are presented as a ratio of densitometry analysis of FB to β-actin (loading

control) using Image J software. The data are represented as means ± SD (n = 2)

(Unpaired t test P < 0.05), C: control sample and S: stimulated sample.

247

From figure (7.10) it can be concluded that FAF-HSA and 𝜆-LC up-regulated the C3

mRNA expression and protein production at both time points, except the C3 production

at 72h with 𝜆-LC was decreased compared to the 24h C3 production.

Also, FB mRNA was increased at 24h with FAF-HSA and then decreased at 72h to the

normal control level. In contrast, 𝜆-LC had no effect at 24h on FB expression from HK2

(+/-GF) cells but at 72h HK2-GF decreased and HK2 cells increased FB expression

significantly.

About FH, FAF-HSA had the same effect on HK2 (+/-GF) cells FH mRNA elevated at

24h and 72h except HK2-GF was decreased at 72h. On the other hand, 𝜆-LC had no

effect on FH mRNA expression in HK2 (+/-GF) cells except HK2 was increased at 72h.

Interestingly, it was noticed that FH and FP behaved oppositely. From figure (7.10)

with FAF-HSA stimulation when FH mRNA increased, there was no change in the FP

mRNA but when the FP increased at 72h from HK2-GF cells the FH decreased. Also,

when HK2 (+/-GF) cells were stimulated with 𝜆-LC the FP mRNA expression was

increased at both time points, however, no change in FH mRNA levels was detected.

The lacks of analysis of alternative forms of FH do not allow firm conclusions to be

drawn from this seemingly opposing behaviour FH/FP.

248

AP components 24h 72h 24h 72h 24h 72h 24h 72h

C3 mRNA/Protein

FB mRNA/Protein

P mRNA _ _ _

FH mRNA _ _ _

Figure (7.10): Juxtaposition of mRNA and protein for AP components (C3, FB, P and FH) from HK2 (+/-GF) stimulated with FAF-HSA

and 𝜆-LCfor 24 and 72h. The results in this figure are compiled from comparisons with the control samples for each time point.

Increase Decrease No change

FAF-HSA

GF HK2

𝝀-LC

GF HK2

249

7.2.3 Effect of Protein Overload on H2O2 production:

To investigate if protein overload conditions are capable of generating H2O2 that could

be one of cells damage causes in proteinuric condition, HK2 (+/-GF) cells were sub-

cultured in 96 well plates and exposed to FAF-HSA or 𝜆-LC (5mg/ml) over a time

course (5, 10, 20, 30, 60 and 360min) to measure the H2O2 generation. Intracellular

content and release of H2O2 in supernatants were measured by the oxidant-sensitive

dye, carboxy-H2DCFDA. The medium alone was measured as background control.

FAF-HSA and 𝜆-LC induced intracellular and extracellular release of H2O2 in HK2

(+/GF) cells. The FAF-HSA induced intracellular production of H2O2 from HK2-GF

after 5min of incubation; however, the maximum production was at 10min of

stimulation time and after that the production was decreased gradually with increasing

times of incubation. Similarly, the 𝜆-LC induced the H2O2 production from HK2-GF

cells at 5min of incubation and the maximum production was at 10min of stimulation;

after that the production reduced compared to H2O2 production from the control sample.

However, the amount of H2O2 that was produced from stimulated HK2-GF cells with

(5mg/ml) 𝜆 -LC at 10min was significantly higher over HK2-GF stimulated with

(5mg/ml) FAF-HSA at 10min by approximately a two-fold increased (figure 7.11 (A)).

A parallel and significant increase in the extracellular H2O2 release occurred throughout

the observation period. FAF-HSA showed significant elevation in the H2O2 release from

HK2-GF by the same level at 5, 10 and 20min incubation periods, but from 30 up to

360min the secretion was reduced gradually with the time. Comparably, when HK2-GF

cells were exposed to 𝜆-LC the H2O2 release was significantly raised at 5min, remaining

elevated until 60min. Also, by comparing the amount of the H2O2 that was released

from HK2-GF cells stimulated with 𝜆-LC, it was significantly higher over the HK2-GF

stimulated with FAF-HSA (figure 7.11 (A)). In addition, the extracellular production

remained high until 1h with 𝜆-LC incubation; however, with FAF-HSA it started to

decrease after 30min time of incubation.

HK2 cells showed a slight increase in the H2O2 intracellular production after FAF-HSA

stimulation at all-time points. Likewise, 𝜆-LC induced the H2O2 production from HK2

cells at 5min and the maximum production were at 20min, and then the production

decreased but was still higher than the control sample. After that, the H2O2 released

250

from HK2 cells stimulated with FAF-HSA was elevated at 5min and remained up until

30min, but at 60min the cells showed the maximum release, and at 360min the release

was returned to the normal level compared to the control sample. In contrast, when HK2

cells were exposed to 𝜆-LC, the H2O2 extracellular release was increased at 5min and

persisted throughout the 360min (figure 7.11 (B)).

Moreover, in order to detect H2O2 production in the medium collected from HK2 (+/-

GF) cells sub-cultured in 6 well plates with density (5 x 105 cells/well) and stimulated

with FAF-HSA or 𝜆-LC (5mg/ml) for 24 and 72h (prolonged incubation), Amplex red

was used.

Overnight incubation of HK2-GF cells with FAF-HSA decreased the medium level of

H2O2 at 24 and 72h, when compared with the medium of un-stimulated control cells. In

contrast, exposed HK2-GF cells to 𝜆-LC produced a slightly detectable level of H2O2 at

24h, but at 72h the level returned to the normal level compared to the control (figure

7.12 (A)). On the other hand, HK2 cells showed a reduction in the H2O2 release at both

time points with FAF-HSA stimulation compared to the control samples. Also, with 𝜆-

LC stimulation both incubation periods showed a significant decrease in H2O2 released

in the medium compared to H2O2 levels in the control sample medium (figure 7.12 (B)).

In summary, 𝜆-LC led to an increase in the H2O2 intracellular production more than

FAF-HSA at 10min, and at the longest time of incubation 360min with both proteins the

H2O2 production decreased significantly in HK2-GF cells. However, the extracellular

secretion of H2O2 was higher with 𝜆-LC than FAF-HSA incubation, but it reduced to

the normal level with FAF-HSA and remained high with 𝜆-LC at the 360min incubation

period. The HK2 cells increased the H2O2 cell production at 60min with FAF-HSA and

the level decreased with the longer time of incubation, but 𝜆-LC showed a quicker

response and the significant increase at 20min and the level remained high with the

longest incubation period of 360min.

Comparing the H2O2 intracellular production from HK2 (+/-GF) cells after stimulation

with both proteins showed that HK2-GF cells increased the H2O2 cell production

quicker than HK2 cells with both proteins, but the level was decreased from HK2-GF at

360min; however, it remained high in HK2 cells, which means the HK2 still produce

more H2O2.

251

Regarding extracellular release, HK2-GF cells also showed a quicker response with

FAF-HSA, however, HK2 showed extreme production at 60min. Additionally, HK2-GF

elevated the release of H2O2 and this production decreased at 360min, but HK2 cells

increased the production and this elevation remained until the 360min incubation period

with 𝜆-LC. Lastly, at 24 and 72h of incubation time, HK2 (+/-GF) cells showed a

decrease in H2O2 release with both stimulator proteins.

In conclusion, this experiment has shown that in HK2 (+/-GF) cells, FAF-HSA and 𝜆-

LC elicited rapid H2O2 generation in cell supernatant as well as at the intracellular level.

252

C 5 10 20 30 60 360 C 5 10 20 30 60 36

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253

Figure (7.11): Time course of H2O2 generation in HK2 (+/-GF) cells. HK2 (+/-

GF) cells sub cultured in 96 well plates with density (1 x 103 cells /well) and

exposed to FAF-HSA or 𝜆-LC (5mg/ml) for (5, 10, 20, 30 60 and 360 min).

H2O2 production within the cells or cell supernatants was measured by the

oxidant-sensitive dye, carboxy-H2DCFDA. The medium alone was measured as

the background control. C: control samples. (A) HK2-GF and (B) HK2 cells

showed the H2O2 intracellular and extracellular production after exposed to

FAF-HSA and 𝜆-LC. The data are represented as means of duplicate ± SD (n =

2) (Unpaired t test P < 0.05).

HK2-GF

C 2

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254

HK2

Figure (7.12): Effect of FAF-HSA or 𝜆-LC on H2O2 production by

HK2 (+/-GF) cells. Cells were incubated with (A) FAF-HSA or (B)

𝜆-LC (5mg/ml) for 24 and 72h. At the end of incubation, H2O2

production was measured in cell supernatants by Amplex Red

method. The medium alone was measured as the background control.

C: control samples. The data are represented as means of duplicate ±

SD (n = 3) (Unpaired t test P < 0.05).

C 2

4h

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255

7.2.4 Study of potential biomarkers of human kidney injury using an

in vitro protein stimulation model:

Apoptosis and inflammatory mediators can contribute to kidney injury in proteinuric

condition. The proteome profiler array kit was used to detect different biomarker

proteins that may have a role in renal cell damage.

To identify early kidney cell injury biomarkers, HK2-GF cells were sub-cultured in 6

well plates (1x 106 cells/well) and stimulated with FAF-HSA (5mg/ml), 𝜆-LC (1 and 5

mg/ml) for 72h. Un-treated HK2-GF cells were used as the control. HK2-GF was

chosen because they were grown in the same medium that primary cells grow in; 𝜆-LC

concentrations were decided to examine the dose effect on the cells and the FAF-HSA

to examine the mechanism of damage between 𝜆-LC and FAF-HSA by using the same

concentration. 200μg of protein cell lysate from each sample was analysed as described

previously (Materials and Methods, see 2.15). Image J and Graph prism 6 software were

used for data analysis. Negative (PBS) and positive (reference spots) controls were

tested for accuracy.

The expression of 38 different types of protein biomarkers for kidney injury was

examined. Our results divided and presented the quantification expression level of these

proteins depending on several categories (figure 7.13).

Four low molecular weight proteins will be presented. First, 𝛽2-microglobin (𝛽2M) is

11kDa and Cystatin C is 13kDa proteins. 𝛽2M expresses regularly on the cell surface of

all nucleated cells. Both protein are filtered by the glomerulus and reabsorbed by

PTECs without secretion (Pacific Biomarker, 2012). It reported that Cystatin C is a

sensitive marker of renal injury in patients with MM (Chae et al., 2015). The secretion

of both proteins in urine used as a marker for renal function specifically reflects the

tubular dysfunction (Vaidya et al., 2008).

As shown in figure (7.13 (A)) 𝛽2M was increased after stimulation in HK2-GF cells

compared with the control. Though, 𝛽2M is used as a housekeeping protein, in renal

injury 𝛽 2M has been described as a marker of proximal tubular damage (Pacific

Biomarker, 2012).

256

Similarly, stimulated HK2-GF with 𝜆-LC and FAF-HSA elevated 𝛽2M production at

the same level. In contrast, HK2-GF cells showed a higher increase in Cystatin C with

𝜆-LC stimulation compared to FAF-HSA (figure 7.13 (A)). The mechanism of this

increase is unknown, especially as Cystatin C is thought to be independent of

inflammation (Coll et al., 2000) and there is a slight increase after albumin stimulation.

Lipocalin-2 or neutrophil gelatinase-associated lipocalin (NGAL) protein is one of the

earliest markers for PTECs injury (Pacific Biomarker, 2012) and the most robustly

expressed protein in the kidney after nephrotoxic injury (Chae et al., 2015). In a normal

kidney, NGAL expression is found only in distal tubules and collecting ducts, however,

in the case of kidney injury NGAL protein was also stained in proximal tubule cells

(Geus et al., 2012). For example, one study demonstrated that NGAL was up-regulated

in the first few hours in mouse model after ischemic renal injury. In addition, NGAL

was up-regulated in the kidneys of mice 3h after cisplatin (20 mg/kg), which induced

renal injury and has been proposed as an early biomarker for diagnosing acute kidney

injury (Vaidya et al., 2008). NGAL was raised in HK2-GF cells after stimulating with

FAF-HSA and 𝜆 -LC, however, HK2-GF with (1mg/ml), 𝜆 -LC led to a greater

expression (figure 7.13 (A)).

Fatty acid-binding proteins (FABPs) are small proteins with (15kDa) molecular weight.

Two types of FABP were identified in the human kidney: liver-type FABP (L-FABP) or

(FABP1) and heart-type FABP (H-FABP). L-FABP expresses in PTECs and appears to

be a sensitive biomarker for renal injury (Pelsers, 2008). Stimulated HK2-GF cells with

FAF-HSA and 𝜆-LC showed that FABP expression was significantly reduced compared

to the control sample (figure 7.13 (A)).

Then, the up-regulated proteins. Kidney injury molecule-1 (KIM-1) is a promising and

sensitive marker for proximal tubule injury. It is the best-characterised urinary

biomarkers in animals and human models with renal disease (Lock, 2010). In addition,

it is undetectable in a normal kidney but induced in proximal tubules after toxic injury

like proteinuria (van Timmeren et al., 2006).

257

As demonstrated in (figure 7.13 (A)) KIM-1 expression was raised after treating HK2-

GF with FAF-HSA and 𝜆-LC; the higher production appeared with (1mg/ml) 𝜆-LC.

Similarly, clusterin is associated with tubular kidney injury (Khan & Pandey, 2014) and

was increased after treating the HK2-GF cells with both proteins to the same extent.

Cysteine-rich protein (Cyr61) is normally induced in proximal straight tubules of

kidney and raised in kidney injury (Vaidya et al., 2008). It was elevated after treating

HK2-GF with both proteins; also Cyr61 was elevated more with (1mg/ml) 𝜆-LC (figure

7.13 (A)).

Epithelium growth factor (EGF) produced from PTECs with massive proteinuria

(Rodriguez-Iturbe et al., 2005), and HK2-GF increased the EGF expression slightly

after exposure to overload FAF-HSA or 𝜆-LC (figure 7.13 (A)).

Vascular cell adhesion molecule-1 (VCAM-1) plays an important role in the

inflammatory process (Baek et al., 2010). FAF-HSA had no effects on VCAM-1

expression from HK2-GF compared to the control; however, stimulating HK2-GF cells

with (1 and 5 mg/ml) 𝜆-LC showed a significant increase compared to the control

(figure 7.13 (A)).

Matrix metallopeptidase 9 (MMP-9) is an enzyme that has been implicated as an early

predictive kidney biomarker of tubular epithelial cells injury (Dimas et al., 2013). The

FAF-HSA had no effects on the MMP-9 production from HK2-GF cells after

stimulation, compared to stimulated HK2-GF with both concentrations of 𝜆-LC that

showed significant increase in the MMP-9 production (figure 7.13 (A)). Additionally,

the accumulation of MMP-9 in epithelial cells was controlled by the stimulation of

TGF-β and thrombin promotes the expression of thrombospondin-1 (TSP-1) (Diams et

al., 2013). It has been reported that in diabetes, TGF-β is a key mediator of the cellular

processes that induce renal disease, including renal tubular injury leading to proteinuria.

In addition, TSP-1 is a significant regulator of TGF-β activation and important for the

development of tubular damage (Lu et al., 2011). TSP-1 (THB1) is a glycoprotein that

mediates cell-to-cell interactions. TSP-1 was clearly elevated after treating the HK2-GF

with (1 and 5 mg/ml) 𝜆-LC. On the other hand, the FAF-HSA (5mg/ml) had no effect

on TSP-1 (figure 7.13 (A)).

258

Furthermore, kidney injury as explained previously can be the result of inflammatory

pathogenesis. PTECs produce pro-inflammatory mediators as early biomarkers of

kidney injury such as cytokines and chemokines.

IL-6 expression was elevated after stimulating the HK2-GF; cells stimulated with 𝜆-LC

(1 and 5 mg/ml) produced the higher IL-6 level comparing with FAF-HSA stimulated

cells (figure 7.13 (A)).

Additionally, TNF-𝛼 mediator of inflammatory tissue damage and TNF-RI showed an

increase when treating HK2-GF cells with (5mg/ml) FAF-HSA and 𝜆-LC, but showed a

highly significant increase with (1mg/ml) 𝜆-LC. Similarly, tumor necrosis factor-like

weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily. It is a

multifunctional cytokine in kidney injury involved in cell proliferation, cell death, cell

differentiation, tissue regeneration and inflammation. In addition, it is constitutively

expressed in human and murine renal tubular cells in low level, and it can be up

regulated in tissues damaged by acute kidney injury and inflammatory disease and

proteinuria (Sanz et al., 2011). Figure (7.13 (B)) demonstrated the elevation of TWEAK

level after treating HK2-GF with FAF-HSA, but it increased more with 𝜆 -LC

incubation.

Next, FAF-HSA had no effect on MCP-1 chemokine production from HK2-GF cells

after treating. In contrast, the 𝜆-LC had a clear significant effect by increasing the MCP-

1 production compared to the control (figure 7.13 (B)).

CXCL16 is a chemokine that has a role in several different models of renal injury

causing renal inflammation, fibrosis and renal failure when elevated in kidney tubular

epithelial cells (Norlander et al., 2013). Nonetheless, treating HK2-GF cells with both

(5mg/ml) FAF-HSA and 𝜆-LC did not lead to a raise in the CXCL16 level compared to

a slight increase with (1mg/ml) 𝜆-LC (figure 7.13 (B)). In addition, it found that the

chemokine CXCL16 is induced by angiotensin (Norlander et al., 2013), and by

measuring the angiotensin after stimulating the cells with both proteins; they also had

no effect on angiotensin production (figure 7.13 (A)).

Anti-inflammatory cytokines counterbalance the pro-inflammatory cytokine.

Interleukin-10 (IL-10) is an effective anti-inflammatory cytokine that inhibits

inflammatory and cytotoxic pathways to protect the kidney from injury (Akcay et al.,

259

2009). IL-10 effect was tested in ischemia (acute renal injury) mouse model and it was

found that a single injection of IL-10 inhibited renal damage in general (Deng et al.,

2001). As displayed in figure (7.13 (B)) the IL-10 production from HK2-GF cells after

exposure to FAF-HSA and 𝜆-LC was noticeably very weak.

260

control

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FAF-HSA (5mg/ml)

262

Figure (7.13): The human kidney biomarker array detects multiple analyses cell culture lysates samples. HK2-GF cells were sub-cultured

overnight and stimulated with FAF-HSA (5mg/ml) and 𝜆-LC (1 and 5 mg/ml) for 72h. Protein lysates were prepared and (200𝜇g) from

each sample was run on each array. The data was analysed by Image J soft wear. (A and B) Show the different biomarker behavior in this

project module (protein overload) as a cause of kidney injury. Un-treated cells were used as the control. The biomarkers were divided

depending on the type, such as pro-inflammatory cytokines. LMWP: low molecular weight proteins.

263

One of the highly induced proteins in the proteomic profile assay is trefoil factor 3

(TFF3). TFF3 is a small peptide ~7kDa, produced by epithelial cells. It plays a role in

epithelial regeneration and wound healing, inhibiting apoptosis and promoting survival

(Yu et al., 2010). Also, it is important for mucosal protection, cell proliferation and cell

migration in vivo and in vitro.

At the same time, the serum and urinary level of TFF3 is associated with kidney injury

due to its providing a sign for ongoing inflammation processes (Du et al., 2013). In

addition, possible triggers of the release of TFF3 may include damage or inflammation

(Astor et al., 2011). Interestingly, the human kidney biomarker array kit, which was

used to test different kidney injury biomarkers, found that there was an abundance of

TFF3 in lysates from HK2-GF cells exposed to (1 and 5 mg/ml) 𝜆-LC, however, FAF-

HSA had no effect compared to the control un-stimulated sample at 72h incubation

period (figure 7.13 (A)).

For more investigation and to prove the biomarker array results, TFF3 production was

measured in HK2-GF lysates stimulated with 𝜆 -LC (1 and 5mg/ml) or FAF-HSA

(5mg/ml) by Western blot (data not shown) (Done by Dr. Zina Zwaini). The

densitometric analysis of Western blot results showed similar results to kidney

biomarker array results (figure 7.13 (A)). 𝜆-LC (1mg/ml) showed the highest TFF3

production compared to less production with 𝜆-LC (5mg/ml) and no effect with FAF-

HSA (5mg/ml). The level of TFF3 from the stimulated sample with FAF-HSA and un-

stimulated cells (control) are comparable (figure 7.14)

In addition, to detect the effect of both proteins on TFF3 secretion from HK2 (+/-GF)

cells and to examine if the EGF cocktail had any effect on producing TFF3 from HK2-

GF cells with 𝜆-LC stimulation. The supernatants were collected from HK2 (+/-GF)

cells stimulated with FAF-HSA and 𝜆-LC (5mg/ml) for 24 and 72h quantitatively by

ELIZA (Done by Dr. Zina Zwaini). Also, our results correspond to proteomic array

results (figure 7.13 (A)). FAF-HSA had no effect on TFF3 production from HK2 (+/-

GF) cells at both time points. However, 𝜆-LC showed a significant increase in TFF3

secretion from HK2 (+/-GF) cells at both time points, but higher at 24h compared with

264

the 72h (figure 7.15). Taken together, 𝜆-LC initiates more restitution in HK2 (+/-GF)

cells than FAF-HSA.

In summary, several earlier biomarkers for the recognition of cell damage that can be a

sign or cause of kidney injury have been investigated. Using these biomarkers in the

protein overload model showed the difference between the effects of two proteins on the

proximal tubule cells. Interestingly, by comparing the effect of 𝜆-LC and FAF-HSA on

the HK2-GF cells, the (1mg/ml) 𝜆-LC led to the highest increase of 14/38 proteins

compared to (5mg/ml) 𝜆-LC and FAF-HSA. Moreover, FAF-HSA had no change or

decrease in 6/14 markers compared with 𝜆-LC in both concentrations. These results

may support the observation that 𝜆-LC has more damaging effects on PTECs. Figure

(7.16) showed the summary of different effects of each protein with specific

concentrations on several biomarkers.

Figure (7.14): Densitometric intensity for Western blot semi-quantitative analysis

of TFF3 production from stimulated HK2-GF cell lysates; cells stimulated with

FAF-HSA (5mg/ml) or 𝜆 -LC (1 or 5 mg/ml) for 72h. TFF3 was increased

significantly after treating HK2-GF cells with 𝜆-LC compared with no changes

with FAF-HSA stimulation. Normalised density of TTF3 (7kDa) to loading

control (𝛽-actin).

Contr

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Figure (7.15): The effects of 𝜆-LC and FAF-HSA on TFF3 production by HK2 cells

(+/-GF). Cells were sub cultured in 6 well plates (5 x 105 cells/well) overnight, and

incubated with (5mg/ml) 𝜆-LC or FAF-HSA for 24 and 72h. The supernatants were

collected and TFF3 production was measured (ELISA), un-treated cells were used

as the control. Stimulated HK2 (+/-GF) cells with 𝜆-LC was elevated significantly

in the TFF3 secretion, whereas TFF3 was undetected in HK2 (+/-GF) cells

stimulated with FAF-HSA at both time points compared to control samples. The

data are represented as means duplicate ± SD (n = 2).

C2

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Markers 𝝀-LC (1mg/ml) 𝝀-LC (5mg/ml) FAF-HSA (5mg/ml)

Cystatin C

NGAL

KIM-1

Cyr61

VCAM-1

MMP9

TSP-1

TFF3

IL-6

TNF-𝜶

TNF-RI

TWEAK

MCP-1

CXCL16

Figure (7.16): Compilation of proteins detected by proteome profile human kidney array, which are differentially influenced by

𝜆-LC and FAF-HSA.

Highest increase Lower increase Lowest increase No change

267

7.3 Discussion:

Overloading protein on PTCs induces pro-inflammatory and inflammatory cytokines,

which may have a direct or indirect role in kidney injury (Sengul et al., 2002). In our

project, the effect of (5mg/ml) FAF-HSA and 𝜆-LC on HK2 (+/-GF) cells for 24 and

72h on IL-6, IL-8 and MCP-1 was examined by measuring the mRNA gene expression

and protein production levels in supernatants.

Starting with IL-6, one study showed that stimulated HK2 cells, which were grown in a

medium supplemented with EGF, with a range of FAF-HSA (0.5 up to 5mg/ml) for 8h

had no effect on the IL-6 release (Newman et al., 2000). Similarly, another study

stimulated primary human PTCs with a higher range of HSA (with fatty acid) (3.5 up to

35 mg/ml) for 6h and showed no effect on IL-6 secretion with any of these

concentrations (Sengul et al., 2002). In contrast, our results illustrated that IL-6

production from HK2 (+/-GF) cells elevated at 24 and 72h with FAF-HSA. So, the time

of incubation with albumin could be the reason for not detecting IL-6 before 24h. In

addition, our results displayed a significant increase in IL-6 mRNA expression from

HK2 (+/-GF) cells in the initial 24h but the abundance was notably decreased

at 72h with FAF-HSA. The decrease or lack of increase of IL-6 mRNA expression at

72h could be due to IL-6 produced as a protein in the supernatants of treated cells

because the highest IL-6 secretion at 72h.

The HK2 (+/-GF) cells behaved similarly with 𝜆-LC stimulation compared to FAF-

HSA; IL-6 secretion from HK2 (+/-GF) cells was raised at 24h and persisted through

72h. Our results agreed with Sengul et al. (2002) who showed that (1.5, 3 or 10mg/ml)

of 𝜅 -LC, which was purified from the urine of MM patient, promoted the IL-6

production from human PTCs at 24h. Also, 𝜆-LC elevated the mRNA gene expression

of IL-6 in HK2 (+/-GF) at 24h but the maximum increase was at 72h. By comparing the

mRNA and protein levels for HK2 (+/-GF) cells, HK2 cells showed higher IL-6

secretions than HK2-GF. In addition, the IL-6 mRNA expression decreased at 72h with

FAF-HSA from both HK2 (+/-GF) cells, but with 𝜆-LC the expression was still elevated

compared with 24h, this elevation might cause cell inflammation.

The effect of FAF-HSA on IL-8 showed that FAF-HSA had no effect on IL-8 secretion

with HK2 (+/-GF) cells but the level of IL-8 was significantly raised at 72h with HK2

268

cells. This is consistent with findings in Sengul et al. (2002) who showed that the range

of HSA (with fatty acid) (3.5 up to 35mg/ml) had no effect on IL-8 production from

human PTCs at 6 and 24h stimulation time. With regard to IL-8 mRNA expression,

Tang et al. (2003) presented that IL-8 gene expression in human PTECs was increased

significantly at 3h and then reduced progressively to undetectable levels at 12 and 24h

with (5mg/ml) HSA (with fatty acid). Our results showed a similar response, where the

IL-8 mRNA did not change from HK2 cells stimulated with FAF-HSA for 24h but after

72h a significant increase was detected, and this was comparable with the protein IL-8

secretion results that showed high IL-8 secretion at 72h. On the other hand, stimulated

HK2-GF cells with FAF-HSA led to elevated IL-8 mRNA expression at 24 and 72h,

but, surprisingly, no change in the protein level at both time points. The reason for the

lack of a difference between FAF-HSA stimulated HK2-GF and control cells in IL-8

release is unclear, given the increase in mRNA measured for both time points.

Stimulating HK2 (+/-GF) cells with 𝜆-LC showed augmentation in IL-8 secretion at

24h, but at 72h the production was reduced significantly. Likewise, Sengul et al, (2002)

showed significant elevation in IL-8 production from human PTCs after treatment with

(1.5 and 3mg/ml) 𝜅-LC, purified from the urine of the MM patient at 6 and 24h. IL-8

mRNA expression followed a time-dependent manner; the expression was up regulated

at 24h and reached a maximum at 72h in HK2 (+/-GF) cells. HK2 cells expressed IL-8

more than HK2-GF at 72h. Comparing the IL-8 expression and secretion, at 24h the

HK2 (+/-GF) cells increased the expression and the production of IL-8 significantly.

Nonetheless, at 72h the IL-8 transcription was still increasing, but the protein translation

was significantly decreased. The reason for the significant decrease of IL-8 production

at 72h in spite of the elevation of mRNA levels at the same stimulation time could be

that IL-8 production peaked at 24h. Alternatively, 𝜆-LC may increase expression of

peptidase like anti-peptidase N that degrades and inactivates IL-8 (Kanayama et al.,

1995). Then IL-8 would be antigenically undetectable by ELISA.

Lastly, MCP-1, FAF-HSA had no significant effects on MCP-1 secretion from HK2 (+/-

GF) cells at both time points but at 72h the production was elevated markedly with HK2

cells. Our findings agreed with Sengul and his group in 2002, they determined the

MCP-1 production from human PTCs that stimulated with range of HSA (with fatty

acid) (3.5 up to 35 mg/ml) for 6 and 24h. Their results showed no significant effects on

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MCP-1 production with all HSA concentrations at both time points. However, a

different study showed opposite results. They found that the release of MCP-1 from rat

PTCs increased only with relatively high concentrations of (10-30mg/ml) BSA

(delipidated) at 8h (Newman et al., 2000), so, this could explain the reason of no change

in the MCP-1 level in our protein overload conditions. First could be the low

concentration of FAF-HSA (5mg/ml) or the long-time of stimulation; MCP-1 might be

secreted earlier than 24h and then decreased. In addition, the late MCP-1 production at

72h from HK2 cells may be the cause of inflammation and the EGF cocktail protects

HK2-GF cells from that. The MCP-1 mRNA expression showed no change from HK2

(+/-GF) cells, however, HK2 showed elevation at 72h. Previous study demonstrated that

rat PTCs induced the MCP-1 mRNA expression strongly after 8h of exposure to a range

of (delipidated) BSA (1-30mg/ml) by PCR. In addition, they stimulated the PTCs to

(15mg/ml) (delipidated) BSA for (2, 4, 8 and 24h) and found the MCP-1 mRNA

expression increased with time but reached its peak at 4h and persisted for 24h (Wang

et al., 1997). On the one hand, by comparing our results with Wang et al. (1997), we

can conclude that stimulating HK2 (+/-GF) cells with (5mg/ml) FAF-HSA showed no

increase in MCP-1 mRNA expression and this might be due to the long-time of

stimulation with low albumin concentration. On the other hand, may be because the

protective effects of the EGF cocktail in the HK-GF cells medium. Also, the low FAF-

HSA concentration in our study might need a longer time to induce MCP-1 from HK2

as an inflammatory response at 72h.

HK2 (+/-GF) behaved similarly with 𝜆-LC stimulation. There was a slight increase in

MCP-1 secretion at 24h, but at 72h production was elevated strongly, especially from

HK2 cells. Wang et al. (2007) in a study stimulating HK2 cells overnight with six

different LC (three k and three 𝜆), purified from different urines of MM patients with

LC proteinuria, showed a mixed response with (1mg/ml) of LCs on MCP-1 secretion

from HK2 cells. Because of a mixed effect of different LCs purified from distinct

patients, it was concluded that the type of the LC purified from the donor plays a central

role in the toxicity effects that lead to induce MCP-1. On the other hand, the MCP-1

promoted at 72h in our study could be due to our 𝜆-LC with (5gm/ml) concentration

need a longer time to induce MCP-1 production from our HK2 (+/-GF) cells.

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The mRNA expression was increased from HK2 (+/-GF) cells stimulated with 𝜆-LC at

both time points. By comparing the MCP-1 protein production and mRNA expression,

MCP-1 mRNA expression did not increase in HK2-GF cells, maybe because in the

translations of mRNA to protein in the supernatants, a high number of cells were

already detached from flask and became apoptotic or necrotic cells. Or, the effects of

the EGF cocktail, which also could explain the significant increase in MCP-1 mRNA

and production at 72h from HK2 cells. In addition, HK2 cells showed the highest MCP-

1 production and mRNA expression at 72h with 𝜆 -LC may play the role in cell

inflammation that leads to cells damage.

In brief, our observation is that both FAF-HSA and 𝜆 -LC are likely to mediate

cytokines responses and may lead to cell inflammation or to be responsible for

progression of cell injury in proteinuric condition.

Previously, it was shown that HK2 cells express mRNA for all AP major components

(C3, FH, FB and FP) (Peake et al., 1999). Our stimulated HK2 cells with FAF-HSA

showed increase in C3 mRNA and protein levels at 24 and 72h. However, HK2-GF

cells showed different results; the C3 expression elevated significantly at 24h and

reduced at 72h. In contrast, the protein production of C3 was increased strongly at 24

and 72h at the same level. If greater C3 release in detrimental, then cells grown in the

EGF cocktail are in a protective environment. The C3 component might have additional

and independent pro-inflammatory action in the proteinuric condition, which is

independent of the C5b-9 location (Zoja et al., 2003). The in vitro study stimulating

human PTCs with (5mg/ml) serum protein led to an increase in the C3 gene expression

significantly in a time-dependent manner (Tang et al., 1999), so, increasing the C3 in

PTCs may induce pro-inflammatory cytokines and lead to cell inflammation in the

proteinuric condition. In addition, it was reported that the activation of AP on the PTCs

cells’ surface was followed by synthesis of pro-inflammatory cytokines such as IL-6

and TNF-α (Zoja et al., 2003). From that we can perhaps explain the high significant

increase in C3 expression and production from HK2 cells at 72h with FAF-HSA having

a role in inducing the late IL-6, IL-8 and MCP-1 significant secretions; in addition, the

mRNA level for both TNF-α and TGF-β also increased at the same time. All of that

could lead to inflammation and cell damage.

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In contrast, the reduction of C3 expression and no change in protein level at 72h for

HK2-GF cells stimulated with FAF-HSA might explain the lack of increase in IL-6

secretion and on IL-8 and MCP-1 at 72h, and this might be the protective effect of the

EGF cocktail (Thurman & Renner, 2011). A previous study using biopsy material

demonstrated that IgA nephropathy patients with proteinuria showed elevation in C3

mRNA expression in tubular epithelial cells. The local overproduction of C3 by renal

tubules might contribute to tubular injury (Zhou et al., 2001). Our results displayed

significant up-regulation in C3 gene expression from both cells stimulated with 𝜆-LC at

both time points. Correspondingly, the C3 protein production for both HK2 (+/-GF)

cells was elevated significantly at 24h, however, although the C3 gene expression at

72h was still high, the protein production of C3 was reduced. This may be due to the

huge number of cells that were floating and detached from the plate surface and became

apoptotic or necrotic cells; C3 production was measured in the remaining attached cells.

At 72h, stimulated HK2 (+/-GF) cells with 𝜆-LC showed a significant increase in IL-6

and MCP-1 secretion. Additionally, the mRNA level for TNF-α and TGF-β also

increased at the same time. All that may contribute to inflammation and cell damage.

FB mRNA was detected in tubules of normal human kidney (weak detection) (Zhou et

al., 2001). Our results showed that FB expression from HK2 (+/-GF) cells was detected,

and increased from both cells after FAF-HSA stimulation for 24h, but at 72h the

expression was returned to the normal level. In the same way, HK2 (+/-GF) cells

showed elevation in FB protein production at 24h and reduction at 72h. The 𝜆-LC

effects on FB mRNA expression displayed no change in FB at 24h from stimulated

HK2 (+/-GF) cells; however, HK2-GF cells significantly decreased and HK2 increased

FB expression at 72h. The protein production of FB was increased at 24 and 72h from

HK2 (+/-GF) cells.

Gaarkeuken et al. (2008) showed that in three patients with proteinuric kidney disease,

FP was detected on the tubular brush border, whereas it was absent in the tubular of

healthy kidneys. Exposure of HK2 (+/-GF) cells to FAF-HSA had no effect on FP

expression at both time points, except the strong increase at 72h from HK2-GF.

However, the expression from HK2 (+/-GF) cells was significantly elevated after 𝜆-LC

stimulation. On the one hand, FH mRNA expression was increased after stimulating

HK2 (+/-GF) cells with FAF-HSA at 24h, whereas at 72h the expression was decreased

form HK2-GF but still at the same level from HK2 cells compared with the 24h

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samples. On the other hand, the 𝜆-LC stimulation enhanced the FH expression from

HK2 (+/-GF) cells at 24h and persisted through 72h.

In brief, our protein overload conditions showed the effect on complement AP

components in transcription and translation in proximal tubular epithelial cells that

could have a role in cell damage and kidney injury. There are several explanations:

possibly, that the continuous C3 expression from cells after both protein stimulation; the

increase in mRNA level of FP from HK2 (+/-GF) cells at 24 and 72h after 𝜆-LC

stimulation compared to no effect on FP levels after FAF-HSA could support the idea of

a 𝜆-LC toxicity effect is more than FAF-HSA, which may lead to over-activation of AP

complement system that might contribute to cell damage; the lack of FH expression

from cells after 𝜆-LC stimulation at both time points may be lead to an inability to

control activation of the pathway. A standard rabbit red blood cells lysis test was

performed in an attempt to quantify AP activity (appendix V). This absence of

detectable AP activity in cell culture supernatants collected from stimulated HK2 (+/-

GF) cells were stimulated with FAF-HSA or 𝜆 -LC (5mg/ml) for 24 and 72h

demonstrated that no hemolytically active MAC was generated in the cell culture

supernatants.

It was reported that LC protein is capable of generating H2O2 (Wang & Sanders, 2007).

Our data from the series of experiments showed that stimulated HK2 (+/-GF) cells with

FAF-HSA and 𝜆-LC for several time points led to an increase the H2O2 intra and

extracellularly. Morigi et al. (2002) showed that in a time course of stimulation of HK2

cells, which were supplemented with EGF in their growth medium, HSA (with fatty

acid) and IgG (30mg/ml) increased H2O2 production threefold over the control, and this

increase extended to 60min of incubation. However, our HK2-GF showed maximum

H2O2 cell production at 10min with both stimulations and this elevation decreased with

longer times of incubation; also, the H2O2 released in supernatants increased

significantly with both proteins, however, at 360min the production returned to the

normal level compared to the control with FAF-HSA stimulation. In contrast, with 𝜆-

LC the amount of H2O2 remained high. This could be due to the higher concentrations

of HSA (30mg/ml), and the fatty acid in HSA may have an extra effect on H2O2

generation in the Morigi et al. (2002) study. In addition, they used IgG to stimulate the

cells, which means heavy and light chains together, but in our study we utilized purified

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𝜆 -LC, so IgG may have a more toxic effect to keep the cells producing H2O2.

Furthermore, a high concentration of IgG was used (30mg/ml). All these reasons may

affect the H2O2 production. To prove this concept, Morigi et al. (2002) showed the

effect of increasing concentration of both HSA and IgG (1, 10 and 30mg/ml) H2O2

production for 5min of incubation from HK2 cells. They found that the amount of H2O2

released in the supernatants or produced in cells was dose-dependent.

On the other hand, HK2 cells showed the maximum H2O2 cell production at 60min and

20min with FAF-HSA and 𝜆-LC, respectively, but the production remained high up to

360min of incubation. Likewise, H2O2 released in supernatants from stimulated HK2

cells with FAF-HSA and 𝜆-LC displayed that H2O2 increased more with 𝜆-LC and the

elevation remained to 360min, however, with FAF-HSA the production was increased

significantly at 60min and reduced to the same control level at 360min of incubation.

Noticeably, the amount of H2O2 generated from HK2 (+/-GF) stimulated cells with 𝜆-

LC was higher than the amount after FAF-HSA stimulation and the generation

remained high with the longest time of incubation (360min); this is may reflect the more

toxic effect of 𝜆-LC than FAF-HSA. Also, the H2O2 generated from stimulated HK2-GF

quicker than HK2 cells but it decreased with long-term stimulation with HK2-GF and

remained high from HK2 cells. The explanation of that might be that the effect of the

EGF cocktail has a role in preventing higher H2O2 production.

Previous study demonstrated an increase in H2O2 released in culture supernatant of HK2

cells stimulated with (1 mg/ml) (λ and κ) LC for 24h; the cells were grown in a

medium supplemented with EGF (Basnayake et al., 2011). Also, another study showed

that stimulated HK2 cells with six human LC (5mg/ml) for 24h, which was purified

from different urines of MM patients with LC proteinuria, resulted in the generation of

H2O2 in the medium to different extents (Wang & Sanders, 2007). Our data results

partly agree with Basnayake et al. (2011) and Wang & Sanders (2007), in that exposure

HK2-GF cells with (5mg/ml) 𝜆-LC showed increase in H2O2 production in the medium

at 24h; in contrast, HK2 cells showed reduction at 24h. On the other hand, HK2 (+/-GF)

cells stimulated with FAF-HSA showed decreased H2O2 production in the medium at

24h. Wang & Sanders (2007) results support our findings. They found that exposure of

HK2 cells with (15 mg/ml) HSA (with fatty acid) for 24h had no effect on H2O2

production in the medium. Lastly, 𝜆-LC in the medium was decreased when HK2 (+/-

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GF) cells were incubated with both FAF-HSA and 𝜆 -LC for 72h. Thus, it can be

concluded that both proteins with the same concentration are capable of producing

H2O2from HK2 (+/-GF) cells, but to a different extent.

Several accessible and promising markers for kidney cell injury were measured in

lysates of HK2-GF cells, which were sub cultured in 6 well plates and stimulated with

FAF-HSA (5mg/ml) and 𝜆-LC (1 and 5mg/ml) for 72h using a Proteome Profiler Array

kit. The detected biomarkers will be discussed in categories (figure 7.17).

In vivo study, increased urinary 𝛽2M excretion has been reported as an early sign of

tubular injury such as in patients with nephrotoxicant exposure and renal transplantation

(Vaidya et al., 2008). One vivo study showed that the rate of urinary Cystatin C

excretion is augmented in the proteinuria/albuminuria rat model (Nejat et al., 2012).

Also, Kim and his group in 2014 showed that MM patients with kidney injury

complications showed an increase in total excretion of urinary Cystatin C. All previous

vitro and vivo studies were measured by the urinary secretion of 𝛽2M and Cystatin C

and used as markers for tubular injury. Differently, in our vitro study both markers were

measured as protein production from the HK2 (+/-GF) cells after protein overload

stimulation as a cause of cell injury. FAF-HSA and 𝜆-LC showed increase in 𝛽2M

production by the same level, which could be a sign of cell injury. However, Cystatin C

significantly increased with 𝜆-LC fourfold more than FAF-HSA in HK2-GF cells. So,

the increasing of Cystatin C production from HK2-GF cells also can be used as a good

marker for cell injury.

The relationship between Lipocalin-2 (NGAL) level and renal function in patients with

MM was examined in 199 patients and showed a strong correlation between the level of

NGAL in plasma and the degree of renal injury. A correlation was found between the

NGAL and Cystatin C plasma levels (Chae et al., 2015). Additionally, measuring

urinary NGAL levels might require a 24h urine collection sample; because of that the

use of urinary NGAL as a quantitative marker is limited. In another study, NGAL

plasma and urinary levels were evaluated in 48 patients with newly diagnosed MM.

They showed elevation in NGAL plasma and urinary levels and reported to be an

indicator for early renal tubular damage (Chae et al., 2015). By comparing our vitro

results, NGAL showed a similar result to Cystatin C; it highly increased after 𝜆-LC

stimulation compared to slight elevation with FAF-HSA in HK2-GF cells.

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Similarly, FABP1 appears to be an attractive biomarker for a number of renal diseases

like chronic kidney disease and IgA nephropathy, but additional studies are needed to

determine the utility of FABP1 especially in the setting of ischemia/reperfusion injury

and nephrotoxin exposure (Vaidya et al., 2008). Our study in vitro results showed no

effect or decrease in FABP1 levels with both overload proteins. In contrast, in our lab

FABP1 level was measured from HK2 in vitro ischemia/reperfusion model for 6 and

24h. The results showed a great increase in the FABP1 level compared to control cells

(Zwaini, 2016). Zwaini’s (2016) result agreed with de Geus et al. (2012), who reported

that the urinary FABP1 was undetectable in the healthy control urine but in ischaemic

conditions, tubular FABP1 gene expression was induced.

These four LMW proteins are reabsorbed in proximal tubules by megalin-mediated

endocytosis and proximal tubular injury reduces that reabsorption, so production and

secretion of these proteins in urine will increase. Also, filtered albumin reabsorbs by the

same process; the competition for receptor-mediated transport between albumin and

LMW proteins could be the reason for elevation of the proteins in the proteinuria

condition. The same reasoning applies for LC, which is another ligand of megalin

receptor.

van Timmeren et al. (2006) measured mRNA, protein, and urinary KIM-1 levels in a

protein overload rat model with renal damage. They found that the mRNA by RT-PCR

and protein production levels by Western blot were up-regulated in the kidney of a

proteinuric rat compared with the control rat kidney; also, a low level of urinary KIM-1

protein was found compared with significant high secretion in the proteinuric model.

Another study also showed up-regulation in the KIM-1 gene and protein products in

animal models with acute kidney injury chemically by cisplatin (Lock, 2010).

Furthermore, kidney biopsies from 102 patients with a variety of kidney diseases were

collected and KIM-1 protein stained in tissue specimens. They showed a positive KIM-

1 staining in PTCs correlated with tubulointerstitial fibrosis and inflammation. At the

same time, the urinary KIM-1 level was measured and they found a correlation between

the KIM-1urinary level and the tissue expression of KIM-1 (Vaidya et al., 2008). Our

results showed an increase in protein levels from stimulated cells compared with the

control sample. Thus, KIM-1 appears to play a role in the pathogenesis of tubular cell

damage in kidney disease.

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Next, clusterin is expressed in damaged tubular cells (Adiyanti & Loho, 2012). In 2008,

Viadya and his group said that in vivo rat cisplatin induced renal injury showed an

increase in clusterin mRNA and protein levels. However, until now, there has been no

clinical study that uses clusterin as an early marker of kidney injury in humans. Our

results showed an increase in the protein level of clusterin in stimulated HK2-GF cells.

Thus, up-regulation of clusterin suggests the occurrence of renal injury and proves to be

a potential biomarker of nephrotoxicity.

Another promising biomarker for early diagnosis kidney injury is Cyr61. It is induced in

the kidney after toxic and ischemia injury in the animal model (Mussap & Merlini,

2014). In rats, Cyr61 is rapidly increased in proximal tubules of the kidney after renal

ischemia and secreted in urine. In an in vitro study, Xu and his group in 2014

investigated the effect of hypoxia on Cyr61, which is a method to induce damage in

cells by incubating HK2 cells for 1 up to 4h in 1% O2. They found that the protein

expression of Cyr61 was increased significantly from hypoxic HK2 cells compared to

the normoxic control cells by Western blot. Also, in our vitro study, the HK2-GF cells

in the protein overload model of the Cyr61 levels were increased after stimulating the

cells with FAF-HSA or 𝜆 -LC. So, Cyr61 appears to be a marker of cell damage

independent of the type of damage.

VCAM-1, an adhesion molecule, and was tested with proteomic profile array. Plasma

VCAM-1 was measured in healthy and diabetic patients with nephropathy. VCAM-1

was elevated in patients with macroalbuminuria compared to normal patients and

patient with microalbuminuria; that means it increased with overt nephropathy (Hojs et

al., 2015 and Clausen et al., 2000). Also, Seron and his group (1991) showed an

increase in VCAM-1 in PTCs in kidney sections from IgA nephropathy patients

compared to very low levels in a section of proximal tubules from normal kidneys. Our

vitro results showed significant elevation in VCAM-1 after 𝜆-LC stimulation, but the

FAF-HSA had no effect on the VCAM-1 abundance.

One study supported our finding; they showed no effect in VCAM-1 mRNA and protein

levels from HK2 cells after being stimulated with (5mg/ml) FAF-HSA for 4h; in

contrast, the (5mg/ml) albumin with fatty acid increased the expression of both VCAM-

1 mRNA and protein at the 4h incubation period (Baek et al., 2010).

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The fatty acid bound to the albumin may have a role in inducing VCAM-1 from the

cells due to incubating HK2 with albumin free fatty acid for 4h or 72h showed no effect

on VCAM-1 expression.

Afterward, MMP-9 was tested by proteomic profile as one of the kidney injury markers.

The relation between MMP-9 and kidney damage has been the subject of a limited

number of investigations, specifically the role of MMP-9 in PTCs in conditions of renal

injury. MMPs are involved in acute kidney injury and changes in tubular epithelial

cells; this study said that MMP-9 expression was increased in tubules of rat

ischemia/reperfusion model (Dimas et al., 2013). Another study showed an increase in

MMP-9 activity in supernatant in rat proximal tubular cells treated with delipidated

BSA (1mg/ml) for 72h (Liang et al., 2007). In contrast, our results showed that the

FAF-HSA had no effect on MMP-9 production and this might be due to the MMP-9

secreted in the supernatant or the protective effects of the EGF cocktail. Also, Liang et

al. (2007) used BSA as source of albumin with rat proximal tubular cells (different

species). However, the two concentrations of 𝜆-LC showed a significant increase in

MMP-9 production. Thus, 𝜆-LC might have a more injurious effect than albumin in our

conditions that leads to increasing the MMP-9 production.

As TSP-1/THBS 1 controls MMP-9 (Diams et al., 2013) TSP-1 was investigated an

early marker for the development of tubulointerstitial kidney disease (Neuwirt et al.,

2014). Also, it was involved in TGF- 𝛽 pathways that activated in fibrotic kidney

disease in rat. Moreover, it is an important mediator-induced kidney dysfunction in the

obesity mouse model (Neuwirt et al., 2014). Thus, TSP-1 is involved in several kidney

diseases. When studying the effects of our protein overload model on TSP1 production,

the results showed that FAF-HSA had no effect on TSP-1, however, the 𝜆 -LC

stimulation led to a significant increase of TSP-1 abundance. Both proteins had the

same impact on TSP-1 and MMP-9 production after stimulation; the 𝜆 -LC (1 and

5mg/ml) increased TSP-1 and MMP-9 production but FAF-HSA had no effect on both

markers.

After that, pro-inflammatory cytokines and chemokines were examined as markers for

kidney injury. Several experimental and clinical studies propose that IL-6 has a role in

renal injury in different forms of renal disease. For example in vivo, in an acute kidney

injury patient the circulating level of IL-6 highly increased; also, it increased in kidney

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mRNA levels of nephrotoxin in mouse model induced acute kidney injury (Jones et al.,

2015). The effect of our kidney cell damage (protein overload) in vitro showed an

increase in IL-6 production in response to 𝜆-LC stimulation. By contrast, FAF-HSA had

no effect on IL-6. Regarding the impact of both proteins on IL-6 mRNA and production

levels at 72h on HK2-GF cells that was discussed previously, FAF-HSA led to decrease

in the mRNA and protein production of IL-6, but the IL-6 secretion was increased

significantly in HK2-GF cells at the 72h incubation period. However, the 𝜆-LC showed

a significant increase in mRNA expression, protein production and secretion of IL-6 in

the same cells. This led to conclude that IL-6 simultaneously promotes an injurious

inflammatory response to protect the kidney from further injury (with FAF-HSA

stimulation) or becomes responsible for progression of cell injury in protein overload

conditions (with 𝜆-LC stimulation).

MCP-1 is produced by kidney cells and acts as a mediator of toxic kidney injury (Peres

et al., 2013). An in vivo study of MCP-1 deficient mice showed less tubulointerstitial

damages after development of nephrotoxic, implying a major role for MCP-1 in

tubulointerstitial inflammation. In the kidney, PTCs are the major source of MCP-1. In

proteinuric conditions, MCP-1 production is increased and this relates to the

progression of renal disease (Wang & Sanders, 2007). In vivo, the protein overload

proteinuria model in rats showed MCP-1 protein localised in tubular cells (Wang et al.,

1997). Our vitro study showed a significant increase in MCP-1 protein production from

HK2-GF cells with 𝜆-LC stimulations, but FAF-HSA had no effect. The MCP-1 mRNA

expression and secretion results that were discussed previously showed similar results.

Next, CXCL16, increased CXCL16 serum and urine levels in humans were found in

chronic kidney disease (Norlander et al., 2013). Another study showed that the urinary

CXCL16 level was raised in mice and patients with lupus nephritis, correlating well

with urine protein levels (Wu et al., 2007). Hypertension is one of the causes of

albuminuria and renal failure and involves angiotensin II, which increases CXCL16 in

kidney PTECs (albuminuria renal failure) (Norlander et al., 2013). Thus, from the prior

studies CXCL16 may have a role in several models of renal disease.

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In our protein overload model, FAF-HSA and 𝜆 -LC had no significant effect on

CXCL16 production in HK2-GF cells and this might be due to the possibility that

production of CXCL16 depends on the stimulation by angiotensin II, which was also

tested and showed no increase in our vitro protein overload model.

IL-10 is an important anti-inflammatory cytokine. It inhibits IL-6, IL-8, TNF-α and

attenuates the expression of TNF-α receptors (Opal & DePalo, 2000). IL-10 was tested

in different renal injury diseases such as cisplatin and ischemia mouse models that

showed necrosis in PTCs. It found that the injection of IL-10 inhibited renal damage.

These findings were confirmed by histologic levels; IL-10 treated kidneys had less

necrosis in proximal tubules in both models (Deng et al., 2001). The effect of protein

overload on HK2-GF cells and IL-10 production in our study showed no significant

change was detected in IL-10 protein levels after stimulation with both proteins. Thus,

this could be one of the reasons for cell damage. In addition, no increase in IL-10

mRNA expression was detected by PCR from HK2 cells stimulated with (5mg/ml)

FAF-HSA for 24 and 72h compared to the control samples (MSc student Panayiota,

Albumin mediated nephrotoxicity- characterisation of an in vitro model, 2013).

TNF-α is an important inflammatory cytokine involved in kidney injury. For example,

diabetic patients with advanced renal failure showed an increase in TNF-α plasma

levels, and the concentrations were significantly correlated with urinary protein

excretion, which means it may have a role in the pathogenesis of proteinuria and renal

damage in these patients. This concept was examined in animals with nephrotoxic

nephritis; the administration of antibodies against TNF-α led to reduced histological

lesions and albuminuria (Navarro et al., 1999). In another vivo study, mice injected with

cisplatin (20 mg/kg) and severe renal failure showed significant up-regulation in TNF-α

mRNA and urine secretion (Ramesh & Reeves, 2002). Also, Nath in 2010 documented

that rats with a systemic infusion of nephrotoxic light chains over 3 days had increased

TNF-α renal production. In this vitro study TNF-α mRNA and protein production were

also increased from stimulated HK2-GF cells with overload FAF-HSA and 𝜆-LC as a

source of cell damage. So, the elevation would result in cell damage in proteinuria and

use as a marker for cell injury.

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Furthermore, the TNF-RI also showed a significant increase in HK2-GF cells after

stimulation with both proteins. An increase of TNF-RI with a concomitant decrease of

anti-inflammatory IL-10 as we find in this study has been described before (Joyce et al.,

1994).

Then, TWEAK, for example, mice receiving an overdose of folic acid that developed

acute kidney injury showed an increase in TWEAK in kidney tubular cells. In addition,

patients with MM showed an increase in TWEAK levels (Sanz et al., 2011). In our

protein overload conditions on HK2-GF cells, TWEAK production was significantly

increased with FAF-HSA and 𝜆-LC, so it could be a sign of cell injury.

Finally, TFF3, in 2008, the US Food and Drug Administration (FDA) and the European

Medicines Evaluation Agency (EMEA) jointly accepted seven urinary indicators of

acute drug-induced renal tubular toxicity in drug development. TFF3 is one of these

markers. For example, experimental rats with nephrotoxicity showed a high TFF3

urinary level. In the human model, the high level of TFF3 in diabetic and hypertension

patients with risk factors for chronic kidney disease is associated with ongoing kidney

damage (Astor et al., 2011).

As known from previous studies, TFF3 is involved in the repair of injury, presumably to

the tubular epithelium like in chronic kidney disease. The TFF3 higher levels indicated

in urine are indicative of ongoing repair, which in turn means damage or ongoing

inflammatory processes. If this principle is correct, TFF3 can be useful as a marker for

kidney damage. In our vitro work that examined the effect of protein overload as source

of cell damage, TFF3 was one of the most highly induced proteins in proteomic

analyses from HK2-GF cells stimulated with 𝜆-LC; however, FAF-HSA had no effect

on TFF3 protein production. Western blot analysis of cell lysates for the same

condition, which is HK2-GF stimulated with 𝜆-LC (1 and 5 mg/ml) and FAF-HSA

(5mg/ml), validated this observation. 𝜆-LC stimulated cells showed markedly elevated

TFF3 abundance, contrasting with un-treated and human serum albumin stimulated

cells. From these results, we concluded that 𝜆-LC induced TFF3 in proximal tubular

epithelial cells in vitro more than FAF-HSA. In addition, TFF3 in supernatants secretion

showed that a significant increase of TFF3 was detected from 𝜆-LC stimulated cell

supernatant, whereas there was no detection of TFF3 secretion from FAF-HSA

stimulated cells. Taken together, from our cells toxicity conditions by 𝜆-LC that induce

281

TFF3 but FAF-HSA could not we can explain that 𝜆-LC has more toxic effect on cells

than FAF-HSA due to that the high TFF3 level with 𝜆-LC means ongoing repair, which

in turn, the high level of damage or cells injury. Additionally, the high number of

detached debris cells from the surface of experimental plates after stimulation with 𝜆-

LC but not with FAF-HSA at the same stimulation time (72h) with the same

concentration (5mg/ml), can possibly be used to support this idea.

Taken all together, our protein overload conditions and mediated cell damages might

occur by activating many of the several pathways that lead to cell inflammation,

apoptosis/necrosis and fibrosis, depending on the molecules that are involved in the

mechanisms that induced cell injury. From our studies, we conclude that stimulated

HK2-GF cells with FAF-HSA and 𝜆-LC showed different effects on regulating several

biomarkers. 𝜆-LC with (1mg/ml) showed more of an effect on stimulation of the marker

molecules than (5mg/ml) concentration. By comparing the effect of 𝜆-LC and FAF-

HSA with same concentration (5mg/ml) we found that 𝜆-LC led to stimulating some

marker molecules that FAF-HSA had no effect on. Figure (8.1 and 8.2) showed the

interaction of 𝜆-LC and FAF-HSA with HK2-GF cells, explaining and summarising the

signaling pathways, which might explain the way of proximal tubular damage/repair in

our protein overload conditions

282

LMW and Up regulated

proteins

Pro-inflammatory

(Cytokines/Chemokine’s)

Anti-inflammatory

𝛽2M

Cystatin C

KIM-1

Clusterin

Cyr61

EGF

EGF-R

VCAM-1

MMP9

TFF3

TSP-1

FABP1

IL-6

TNF-𝛼

TNF-R

TWEAK

Lipocalin-2 (NGAL)

MCP1

CXCL16

IL10

Figure (7.17): The biomarkers were measured by proteomic profile assay that might have a role in the process of PTECs injury in our protein

overload condition.

Normal PTECs Damage PTECs

Inflammation, Fibrosis,

Apoptosis, Cell death,

kidney injury and

proteinuria

Biomarkers for early

diagnosis kidney injury

Toxicity by protein

overload

283

8. Summary:

In summary, excessive 𝜆 -LC on HK2-GF cells for 72h led to activating several

pathways leading to production of different injury markers (figure 8.1) C3 complement

component production was increased and this elevation might cause AP activation,

which promotes injury by formation of a membrane attack complex (Abbate et al.,

2006), although our rabbit blood cell lysis system did not detect MAC formation. TNF-

𝛼 production was increased from cells, and also it can be stimulated by C3. TNF-𝛼

activated the IL6. Also, KIM-1 production was increased. These activations led to cell

inflammation; fibrosis and can lead to cells damage. TGF-𝛽 production was increased,

which led to cell fibrosis and can activate IL-8 and MCP-1 and lead to cell

inflammation. Also, TGF-𝛽 can activate PKC or AP-1 to activate clusterine, which also

increased after stimulation and leads to cells fibrosis.

𝜆-LC increased the production of TSP-1, MMP-9, NGAL, Cystatin c, Cyr6 and VCAM-

1; the increased production of all these proteins was used as markers for tubular cell

injury in kidney diseases. TFF3, one of the proteins, which increased in tubular cell

injury but for the purpose of healings cells, was increased significantly from stimulated

cells. From these markers it can be said that 𝜆-LC had a toxic effect on the proximal

tubular cells. The H2O2 and megalin cytoplasmic tail phosphorylation might support this

finding. The extra and intra cellular H2O2 production was increased with 𝜆 -LC

stimulation from HK2-GF cells and this production can lead to activate cytokines and

chemokine production, which end up with cell inflammation. Also, it can activate

caspases, which leads to cells apoptosis.

In addition, H2O2 might play an important role in megalin cytoplasmic tail

phosphorylation by GSK-3, and our results detected megalin tail phosphorylation in

HK2-GF cells pre-stimulated with 𝜆-LC (5mg/ml). This phosphorylation may affect

megalin receptor recycling to the cell surface negatively (reduction), which means no

megalin receptor on cell surfaces to up take light chain and the protein accumulation

might lead to cell damage. By measuring the expression of megalin receptor in

stimulated HK2-GF cells, the results showed a significant decrease in megalin receptor

expression.

284

Lastly, our apoptosis and autophagy results may be able to support our findings as well.

Exposure HK2-GF cells to 𝜆-LC showed an increase in the number of apoptotic cells

significantly. At the same time the autophagy was activated, which might be a

cytoprotective cell response, but autophagy also can be activated in cell stress. In this

condition the autophage play a role in cell damage after 𝜆-LC stimulation because at

this time point a high number of cells were detached and floating in the supernatants.

However, excessive FAF-HSA protein on HK2-GF cells for 72h had a different effect

(figure 8.2). At this stimulation time point, the C3 production was increased and this

elevation might cause AP activation and lead to cell damage. TNF-𝛼 production was

increased from cells, and it can be stimulated by C3. Also, TWEAK, NGAL, Cystatin C

and Cyr61 are markers for tubular injury and they were increased. The elevation of all

these proteins leads to cell inflammation and fibrosis. Also, KIM-1 production was

increased from cells, but the expression of KIM-1 in injured proximal tubule cells could

lead to cell inflammation or repair (Ichimura et al., 2012).

Albumin had no effect on IL6, MCP-1, VCAM-1, MMP-9, TSP-1, TFF-3 and CXCL16

production. Thus, albumin had less effect on proximal tubular cells than 𝜆-LC; the

(5mg/ml) FAF-HSA might cause some cell injury but not as much as 𝜆-LC. The H2O2

production and megalin cytoplasmic tail phosphorylation results may support this

observation. H2O2extracellular production was increased and decreased to the normal

levels at 6h, and had no effect on kinase activation. That plays a role in megalin

cytoplasmic tail phosphorylation because no phosphorylation was detected with

(5mg/ml) FAF-HSA. In addition, albumin increased the megalin receptor expression

and this may be because of the high albumin concentration, so more albumin uptake can

occur. Finally, the apoptosis and autophage results also could help to explain and

support our observation. Albumin showed an increase in the number of apoptotic cells

after stimulation at 24h, but this number did increase at 72h, and at the same time the

stimulated cells with albumin showed autophage activation that may play a

cytoprotective response, because most of the cells were intact to the well surface and

just a few number of cells were floating.

285

The cells that were used in the proteomic profile array were HK2 cells with an EGF

cocktail, which may have some protective effect from cell damage. The EGF cocktail

might have played a protective role with albumin protein overload model, however,

with 𝜆 -LC it did not; this is may be because of the high toxic effect of 𝜆 -LC.

286

Damage PTECs

Cell death, kidney injury

and proteinuria

PTECs

Bruch Border

Megalin Receptor

Excessive Endocytosis

C3

FB

FP

Complement

activation

Apoptosis Fibrosis

Inflammation

TNF-𝜶 H2O2

IL6 IL8 MCP-1

TGF-𝜷

PKC or AP-1

Clusterin

MMP9

TSP-1

TNF-RI

IL10

KIM-1

TWEKA

Caspase

activation

NGAL

Cystatin C

Cyr61

VCAM-1

Excess 𝝀-LC

Cell Repair Cells injury/Healing

TFF3

287

Figure (8.1): Hypothetic signal pathways of proximal tubule cells in response to overload 𝜆-LC. HK2-GF cells were sub-cultured

overnight and stimulated with (1 and 5 mg/ml) 𝜆-LC for 72h. Protein lysates were analysed by proteomic profile array kit. Detected

protein markers involved in potential mechanism in the development of protein overload induce cell damage are presented. In

addition, the mechanism of ROS that involved in response of proximal tubule cells in protein overload conditions. Arrows mean

positive stimulation confirmed in tubular cells.

288

Damage PTECs

Cell death, kidney

injury and proteinuria

PTECs

Bruch Border

Megalin Receptor

Excess HSA-FFA

Excessive Endocytosis

C3

FB

FP

Complement

activation

Apoptosis Fibrosis

Inflammation

TNF-𝜶 H2O2 TGF-𝜷

PKC or AP-1

Clusterin

TNF-RI

IL10

KIM-1

TWEKA

Caspase

activation

NGAL

Cystatin C

Cyr61

Cell Repair

289

Figure (8.2): Hypothetic signal pathways of proximal tubule cells in response to overload FAF-HSA. HK2-GF cells were sub-cultured overnight

and stimulated with (5 mg/ml) FAF-HSA for 72h. Protein lysates were analysed by proteomic profile array kit. Detected protein markers involved

in potential mechanism in the development of protein overload induce cell damage are presented. In addition, the mechanism of ROS that

involved in response of proximal tubule cells in protein overload conditions.

290

Conclusion

It is possible that proteinuria can cause progressive renal damage and lead to end-stage

renal failure. This possibility is supported by increasing the number of experiments and

clinical studies in this issue. This renal damage/injury occurs by activating multiple

pathways in kidney cells, leading to tubular cells inflammation, fibrosis and apoptosis.

This thesis investigated the effect of protein overload conditions causing injury/damage

in proximal tubular epithelial cells using albumin-fatty acid free and light chain purified

from urine of MM patient in vitro, by examining the activation of different pathways

that might have a role in encouraging cell damage in proteinuria conditions.

Measurements involved complement production, inflammatory cytokines and ROS

generation.

In addition, megalin is one of the important receptors in proximal tubular cells to

reabsorb filtered protein from glomerular like albumin and light chain by endocytosis

process. This thesis proved that protein overload conditions using albumin and light

chain lead to phosphorylated MegCT in a specific site (PPPSP) by activating GSK-3

kinase, and this phosphorylation affects megalin receptor recycling to the cell’s surface

negatively (reduction).

From the findings of this thesis it can be concluded that phosphorylation of the megalin

cytoplasmic tail might associate with reduced light chain endocytosis in renal proximal

tubular cells and might impact on the progression of proteinuric nephropathy.

291

Findings and Future work

This table summarises the major findings in this thesis and indications for future work:

Finding Future Work

Characterisation of Renal Proximal

Tubular Epithelial Cells:

Culturing the proximal

tubular cells in vitro with

media supplemented with

EGF cocktail had effect on

cells proliferation, and

some protection from

protein overload impact on

cells.

Investigate the effect of

which component in the

EGF cocktail has the

protective effect on cells

(additive/synergistic

effects of components)

Establishing an in vitro model of

protein-induced epithelial cell

damage:

Proliferation of proximal

tuabular cells in vitro was

inhibited by protein

overload using FAF-HSA.

However, no protective

effect of EGF cocktail on

proximal tubular cells with

protein overload condition

using light chain protein.

Autophage was activated

and apoptotic cells were

detected in both protein

overload conditions using

albumin and light chain.

Examine the effect of EGF

cocktail on protection cells

from protein overload

condition using albumin

with fatty acid.

Investigate the role of

autophagy activation by

inhibiting the process and

measuring apoptosis. In

addition, measuring the

percentage of early/late

cell apoptosis and necrosis

by flow cytometer

technique

292

Megalin Phosphorylation in Renal

Proximal Tubular Epithelial Cells:

Phosphorylation of megalin

cytoplasmic tail using

MegCT-GST fusion with

pre-stimulated cell lysates

with albumin or light chain

derived from HK2 (+/-GF)

cells by Western blot using

specific phosphor-antibody

against (PPPSP) site.

Megalin mRNA expression

was decreased in proximal

tubular cells after light

chain treatment but

increased with albumin.

Investigate the effect of

higher FAF-HSA

concentration on megalin

expression, and examine if

albumin with fatty acid has

different effects.

CD36 mRNA expression

was increased in protein

overload condition.

Inhibiting GSK-3 and

detecting the

phosphorylation. Inhibit

PI3K and measure the

phosphorylation using

albumin with fatty acid.

Time course for 5mg/ml

FAF-HSA.

Optimising conditions to

measure the megalin

protein production by

Western blot in proximal

tubular cells after protein

overload stimulation.

Investigate why megalin

decreased and CD36

increased.

293

Appendix

294

Figure (Appendix I): Poster in The 7th Saudi Student conference, Edinburgh,

UK, February 2014.

295

Figure (Appendix II): Poster in Kidney Week 2014 conference, Glasgow, UK,

April 2014.

296

Figure (Appendix III): Poster in University of Leicester Postgraduate Research Festival, selected as one of the 50 most promising

researchers among 1500 postgraduates at the University, University of Leicester, Leicester, UK, June 2014.

297

A TGF-𝜷

24h

72h

157 bp

GF HK2

1 2 3 4

B IL-6

24h

72h 250 bp

GF HK2

1 2 3 4

C IL-8

72h

24h 111 bp

GF HK2

1 2 3 4

298

D MCP-1

24h

72h

177 bp

GF HK2

1 2 3 4

F FH

24h

72h

276 bp

GF HK2

1 2 3 4

24h

E C3

72h

168 bp

GF HK2

1 2 3 4

299

Figure (Appendix IV): Semi-quantitative analysis (RT-PCR) of mRNA expression

from HK2 (+/-GF) cells after stimulating with FAF-HSA for 24 and 72h (5mg/ml).

2𝜇g mRNA was used from each sample. 𝛽-actin was used as housekeeping gene. (1

and 3) Control sample (2 and 4) stimulated sample.

G FB

548 bp

GF HK2

1 2 3 4

24h

72h

24h

72h

H KIM-1

210 bp

GF HK2

1 2 3 4

GF HK2

1 2 3 4

24h

72h

I 𝜷-actin

160 bp

300

Appendix ( V ) Alternative pathway (AP) hemolytic assay:

The alternative pathway (AP) hemolytic assay aims to quantify the activity of

complement using unmodified rabbit erythrocytes. It is cheap, fast, simple and

insensitive to C3 degradation. The rabbit blood cells (RBC) were used a complement

activator because it is the most AP sensitive of erythrocyte species (Spitzer et al.,

2007) and human serum as a C3 complement source.

The HK2 (+/- GF) cells were seeded in (1X 106 cells/well) in 6 well plates for 24h.

They were then treated with 5mg/ml of FAF-HSA and 𝜆-LC in serum free medium

for 24 and 72h in the incubator 37C˚, 5% CO2. After each time point the supernatants

were collected.

0.5ml from the rabbit red blood cells (RBC) (Rabbit Blood in Alsever's, tcs

bioscience, Cat no. RB053) was centrifuged for 3min at 200g to remove the storage

solution then the pellet was washed twice in PBS. 150𝜇𝑙 from the pellet was mixed

with 15ml of veronal buffer saline to be stable (RBC stock). 50𝜇𝑙 from the diluted

cells was mixed with 200𝜇𝑙 dH2O in cuvette to test the complete hemolysis (100% of

the cells lysis); the OD was measured by spectrophotometer at (413nm) (varian 50 bio

uv-visible spectrophotometer) and should give (1.0). If more than 1 the cells should

be diluted more in buffer (too much cells in the stock) or if less than 1 so the stock is

too dilute and needs more cells. The dH2O was used as a blank.

Different controls were used:

50𝜇𝑙 RBC stock + 200𝜇𝑙 dH2O (100 % hemolysis)

50𝜇𝑙 RBC stock+ 200𝜇𝑙 VBS (No hemolysis)

50𝜇𝑙 RBC stock + 50𝜇𝑙 serum + 100𝜇𝑙 VBS (positive control for hemolysis

by AP- C3)

50𝜇𝑙 RBC stock + 50𝜇𝑙 serum heat in activated for 30min at 56C˚ + 100𝜇𝑙

VBS (less or no hemolysis by AP-C3)

For each test sample 100𝜇𝑙 VBS + 100 𝜇𝑙 sample supernatant + 50𝜇𝑙 RBC stock.

301

In 96 well plate all the samples and controls was added and incubated for 45min in

the incubator 37C˚, because the RBC complete lysis in 30min and does not change

subsequently. Then, the plate was centrifuged for 3min at 200g.

Veronal Buffer Saline (VBS)

4mM barbiturate

145mM NaCl

2mM MgCl2

8mM EGTA

pH 7.4

1- H2O + RBCs

2-Buffer+RBCs

3-Serum+RBCs

4-In active Serum+RBCs

302

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