Involvement of the nuclear factor-jB pathway in the adhesionof neutrophils to renal tubular cells after injury inducedby neonatal postasphyxial serum

Tao Xiong • Wenbin Dong • Hui Fu •

Qingping Li • Cunliang Deng • Xiaoping Lei •

Lin Guo

Received: 30 July 2013 / Accepted: 15 November 2013 / Published online: 26 November 2013

� Springer Science+Business Media New York 2013

Abstract Nuclear factor jB (NF-jB) plays an important

role in the regulation of inflammatory proteins. However, it

is unclear whether the NF-jB/intercellular adhesion mol-

ecule-1 (ICAM-1) pathway is involved in the adhesion of

neutrophils and renal injury after hypoxia–ischemia (HI) in

neonates. In this report we investigated whether NF-jB and

its downstream molecule ICAM-1 were involved in renal

injury induced by postasphyxial serum (PS) from neonates.

Human renal proximal tubular (HK-2) cells were preincu-

bated with 10 % fetal calf serum (control), 20 % neonatal

PS, or 20 % PS plus pyrolidine dithiocarbamate (PDTC).

The expression of IjBa, NF-jB p65, and ICAM-1 in HK-2

cells was determined by Western blot and/or immunohis-

tochemistry. Nuclear translocation of NF-jB p65 in HK-2

cells was detected by immunofluorescence and Western

blot. The ICAM-1 mRNA was determined by RT-PCR.

Then HK-2 cells were cultured with neutrophils from

neonates with asphyxia. After HK-2 cells had been cultured

with neutrophils, we detected myeloperoxidase (MPO)

activity, the leakage rate of lactate dehydrogenase (LDH),

and cell viability. We found that PS preincubation resulted

in significantly decreased IjBa expression and increased

expression of NF-jB and ICAM-1, and facilitated the

nuclear translocation of NF-jB in HK-2 cells. PS prein-

cubation increased MPO activity, leading to elevated

leakage rates of LDH and decreased cell viability after

neutrophil exposure. Furthermore, the inhibition of NF-jB

activity by PDTC significantly upregulated IjBa expres-

sion, decreased NF-jB and ICAM-1 expression, down-

regulated the nuclear translocation of NF-jB, and

decreased MPO activity. This leads to decreased leakage

rates of LDH and increased cell viability after neutrophil

exposure. Our findings suggest that NF-jB/ICAM-1 path-

way may be involved in neutrophil–endothelial interactions

and neonatal renal injury after HI.

Keywords Postasphyxial serum � Nuclear factor-

jB � Renal injury � Intracellular adhesion molecule-1 �Adhesion � Neonate

Introduction

Asphyxia continues to be a major cause of death or later

neurodevelopmental sequelae in neonates. After asphyxia,

renal injury commonly occurs as a severe complication [1].

Renal cell death in hypoxia–ischemia (HI) is partly due to

inflammation. Following HI, circulating neutrophils selec-

tively infiltrate ischemic kidney tissues. Adherent neutro-

phils could release toxic products, which contribute to

endothelial barrier injury and tissue destruction [2–4].

NF-jB, a ubiquitous transcription factor activated by

various stimuli, is implicated in ischemia–reperfusion

injury. NF-jB regulates the expression of genes associated

with inflammation, which is likely to play a pivotal role in

the pathophysiology of HI renal injury [5, 6]. Prevention of

NF-jB could result in attenuated neutrophil infiltration [7].

T. Xiong � W. Dong (&) � H. Fu � Q. Li � X. Lei � L. Guo

Department of Newborn Medicine, Affiliated Hospital of Luzhou

Medical College, No. 25, Tai Ping Road, Luzhou 646000,

Sichuan, People’s Republic of China

e-mail: dongwenbin2000@163.com

T. Xiong

Department of Pediatrics, West China Second University

Hospital, Sichuan University, No. 20, Section 3, Renmin Nan

Lu, Chengdu, Sichuan 610041, People’s Republic of China

C. Deng

Department of Infection, Affiliated Hospital of Luzhou Medical

College, Luzhou 646000, Sichuan, People’s Republic of China

123

Mol Cell Biochem (2014) 388:85–94

DOI 10.1007/s11010-013-1901-6

The inflammatory process starts with the interaction of

endothelial adhesion molecules and their ligands located on

leukocytes. Intercellular adhesion molecule-1 (ICAM-1),

which belongs to the immunoglobulins, is an important

endothelial adhesion molecule in inflammatory processes

[8]. ICAM-1 is an important downstream effector of NF-

jB. After NF-jB activation, ICAM-1 could be activated on

the surface of endothelial cells during HI, and then mediate

tight neutrophil adhesion [9, 10].

Since kidney proximal tubule cells are susceptible to HI

injury during renal injury [11], we used human renal

proximal tubular (HK-2) cells to establish an in vitro injury

model. We have previously demonstrated that neutrophils

play an important role in renal injury from neonates with

asphyxia [12], and inflammatory factors participate in the

process of HI renal injury [13]. However, the intracellular

signal transduction mechanism mediating renal injury after

HI is poorly understood. Therefore, the present study

focused on whether NF-jB and its downstream effector

ICAM-1 were involved in the injury of HK-2 cells induced

by challenge with postasphyxial serum (PS). We hypoth-

esized that NF-jB/ICAM-1 signaling pathway may be

involved in renal injury after HI. To test this hypothesis, we

tested the expression of IjBa, NF-jBp65, and ICAM-1, as

well as MPO, the leakage of LDH, and cell viability in HK-

2 cells. Our research was aimed at revealing the potential

mechanism of renal injury of neonates after asphyxia.

Materials and methods

Materials

The HK-2 cell line (American Type Culture Collection,

Manassas, VA) was cultured in Dulbecco’s modified eagle

medium (DMEM) (GIBCO, Carlsbad, CA) with 10 % fetal

calf serum (FCS) (Invitrogen Corp., Carlsbad, Calif., USA)

and supplemented with penicillin (100 IU/mL) and strepto-

mycin (100 IU/mL). Pyrrolidine dithiocarbamate (PDTC)

was purchased from Sigma Chemical Co. (St. Louis, MO.,

USA). Anti-IjBa, anti-NF-jB p65, and anti-ICAM-1 poly-

clonal antibodies were purchased from Cell Signaling

Technology (Beverly, Mass., USA); the Streptavidin-Per-

oxidase Kit was supplied by Maixin Biotech (Fuzhou,

China); and the NF-jB Nuclear Translocation Assay Kit

was obtained from Beyotime Biotechnology Co. (Jiangsu,

China).

Preparation of postasphyxial serum and neutrophils

in neonate

Thirty term neonates (gestational age, 37–39 weeks) with

asphyxia (Apgar score lower than 7 points at 1 min)

admitted to our neonatal intensive care unit between March

2008 and September 2010 were enrolled in this study. The

parents of the neonates gave informed consent to partici-

pation in the study. This study was approved by the Ethics

Committee of our institution. None of the patients were

administered immune depressants and none had infectious

disease. Since the climax of inflammatory reaction after

asphyxia reaches a maximum at 24 h, whole blood cells

were obtained from peripheral venous blood within 24 h of

birth (5 mL from each, anticoagulation with heparin. The

total amount of blood driven from asphyxiated newborns

was about 150 mL). Serum was collected by centrifugation

at 2,500 rpm for 20 min followed by deactivation of

complement at 56 �C for 30 min. Serum was sterilized and

adjusted to a final concentration of 20 % in DMEM med-

ium, as described by Dong et al [14]. Neutrophils were

drawn off by pipette following centrifugation, washed by

PBS buffer solution for three times, centrifuged at 800 rpm

for 10 min at 20 �C every time, and adjusted to a final

concentration of 1 9 106/mL in DMEM medium.

Experimental procedure

Incubation conditions generally followed our published

protocols [15]. PDTC was solubilized in DMEM medium

at 40 lM, and the pH was adjusted to 7.2. Cells were

initially plated in 100 mL flasks, grown to 60–80 % con-

fluence, and then plated at a density of 8 9 104 cells per

well in 6-well cell culture plate. Controls were maintained

in the standard medium and the asphyxia group was treated

with 20 % PS. Cells in the PDTC-blocking group were

incubated in PS containing 40 lM PDTC. All cells were

cultured at 37 �C in humidified atmosphere with 95 % air–

5 % CO2 for 24 h.

Immunochemistry

Cells were grown on coverslips before fixation and staining.

Coverslips were incubated in methanol containing 0.3 %

hydrogen peroxide for 20 min and then in 5 % normal

bovine serum for 15 min. The coverslips were then incu-

bated overnight at 4 �C with a rabbit anti-human ICAM-1

antibody (1:100). A parallel set of sections was incubated

with the same dilution of normal rabbit serum as a control

for non-specific binding. The sections were incubated with

secondary antibody (Santa Cruz Biotechnology, 1:3,000)

for 30 min and then processed using the Streptavidin-Per-

oxidase Kit. Coverslips were then dehydrated and mounted.

Confocal microscopy for NF-jB p65 translocation

Cells were plated onto baked glass coverslips in 6-well

plates at a density of 9 9 106 cells per plate. After

86 Mol Cell Biochem (2014) 388:85–94

123

preincubation, the cells were washed twice with PBS and

then fixed with 3 % formaldehyde in PBS for 20 min at

room temperature. The cells were subsequently washed

thrice with PBS, and 1,000 lL of anti-p65 antibody was

added. After 12 h at room temperature, the cells were

washed thrice with PBS. A 1,000 lL volume of Cyanine-

3 (Cy3) anti-rabbit antibody (1:100) (1:500, Jackson

Immunoresearch) was added to the well and incubated for

1 h at room temperature, then washed twice with PBS.

Then 40,60-diamidino-2-phenylindole (DAPI) was used to

counterstain the nuclei. We detected intracellular dye

distribution by confocal microscopy (Leica TCS SP2,

Germany). Fluorescence was measured at 358 nm exci-

tation/461 nm emission and 554 nm excitation/570 nm

emission.

Western blot analysis

4 9 107 Cells were homogenized in ice-cold lysis buffer

containing cytosol extraction buffer that contained HEPES

(pH 7.9; 10 mmol/L), KCL (10 mmol/L), EDTA (ethyle-

nediaminetetraacetic acid; 0.1 mmol/L), EGTA (ethylene

glycol tetraacetic acid; 0.1 mmol/L), DTT (dithiothreitol;

1 mmol/L), PMSF (phenylmethanesulfonyl fluoride;

0.5 mmol/L), protease inhibitor aprotinin (5 mg/mL), and

leupeptine (5 mg/mL). Lysates were centrifuged at

14,000 rpm for 30 min at 4 �C. Cytosol and nuclear pro-

teins were purified as described by Li et al [16]. Protein

concentration was determined by a BCA protein assay kit

(Pierce, Rockford, IL, USA) using bovine serum albumin

as the standard. Protein samples (60 mg per lane) were

separated on an 8 % SDS-polyacrylamide gel. The protein

was then transferred to polyvinylidene fluoride membranes.

The membranes were blocked in 5 % bovine serum albu-

min in Tris-buffered saline containing 0.05 % Tween-20

for 1 h at room temperature with rotation. Then the

membranes were separated for loading control a-tubulin

(Santa Cruz Biotechnology, USA, 1:500) or other specific

proteins. The separated membranes were incubated for 1 h

at room temperature with the following antibodies: Anti-a-

tubulin, anti-IjBa, anti-NF-jB p65, or anti-ICAM-1 anti-

bodies, and incubated overnight at 4 �C. Membranes were

then incubated with peroxidase-conjugated IgG (Santa

Cruz Biotechnology, Santa Cruz, CA, USA, 1:3,000) in

blocking solution for 1 h. Signals of bound antibodies were

developed by enhanced chemiluminescence (Pierce). NIH

image was used to measure the densities of protein signals

on X-ray films after scanning. Protein levels were nor-

malized to a-tubulin as a loading control. Relative optical

density of protein bands was measured after subtracting the

film background.

Semiquantitative reverse transcription PCR analysis

Total cellular RNA was extracted from HK-2 cells by the

Tri-Zol reagent (GIBCO-BRL) and a Superscript kit

(GIBCO-BRL) was used for the RT synthesis of cDNA.

PCR amplification was performed with 35 cycles of 30 s at

94 �C, 30 s at 62 �C, and 2 min at 72 �C. The oligonu-

cleotide primers were as follows: ICAM-1: sense primer 50-dGCAAGCTCCCAGTGAAATGCAAAC-30 and antisense

primer 50-dTGTCTA CTGACCCCAACCCTTGATG-30. A

498-bp product was expected in the reaction. b-actin: sense

primer 50-ACACTGTGCCCATCTAGGAGG-30 and anti-

sense primer 50-AGGGGCCGGACTCGTCATACT-30.PCR products were electrophoresed on 2 % agarose gels

(OXOID). The integrated density value (IDV) of each band

was assessed using a Gelpro image analyzer (Bio-Rad), and

mRNA levels were normalized to b-actin as a loading

control.

Assessment of adhesion of neutrophils to HK-2 cells

Since neutrophils contain myeloperoxidase (MPO), MPO is

used as a marker enzyme for measuring neutrophils accu-

mulation in tissue samples. After 24 h of preincubation,

0.1 mL prepared neutrophil suspension (106/mL) was

placed onto HK-2 cells monolayer, and kept for 60 min

under static conditions to induce cell attachment at 37 �C

in humidified atmosphere with 95 % air–5 % CO2. Each

culture well was gently washed thrice with PBS buffer to

remove non-adherent cells, and then attached neutrophils

were quantified using a MPO assay. Optical density was

measured at 450 nm with an ELISA plate reader. Assays

were performed in duplicate, and the results were nor-

malized for protein content.

Assessment of LDH leakage

LDH leakage was assessed as a marker of loss of cellular

integrity following exposure of HK-2 cells to neutrophils.

LDH activity in cell medium was assessed using a com-

mercially available kit (Sigma, NSW, Australia) adapted to

a 96-well format as Vranyac-Tramoundanas et al. [17]

described. 250 lL of reaction mixture containing

0.139 mM NADH and 4.63 mM pyruvate was added to

each microplate well, and the reaction was initiated by the

addition of 40 lL of cell medium. LDH activity was based

on the conversion of NADH to NAD? and was calculated

as the difference between the natural logarithms of the

absorbance at 340 nm (A340) at 25 �C for three time points;

1, 2, and 3 min. Results were expressed as a percentage of

activity in cell lysed with 1 % triton 9100.

Mol Cell Biochem (2014) 388:85–94 87

123

Cell viability assay

Cell viability was quantified by a short-term microculture

tetrazolium (MTT) assay. In a 96-well microplate, 3 9 103

cells per well were exposed to neutrophils. The media were

replaced with 90 lL of serum-free medium and 10 lL of

MTT solution (5 mg/mL in sterile PBS). After 4-h incu-

bation at 37 �C, the MTT solution in the wells was

replaced with 100 lL dimethylsulfoxide. The absorption at

570 nm (OD570) was measured on a spectrophotometer

(Bio-RAD Benchmark Plus). The cell viability was con-

verted and expressed as the percentage of the control.

Results were expressed as the mean OD for the selected

paradigms performed in duplicate (n = 5). The cells

without any treatment were used as negative control.

Statistical analysis

Results are expressed as mean ± SD. The statistical sig-

nificance between treatments was analyzed using one-way

ANOVA, where p \ 0.05 was considered significant.

Results

PS challenge induced decreased expression of IjBa

NF-jB exists in the cytoplasm in an inactive form while

binding with IjBa. The decreased IjBa expression repre-

sents the activation of NF-jB. To quantify the expression

of IjBa in HK-2 cells, we performed Western blot analysis

on the control, asphyxia group, and PDTC group. We

found that basal level of IjBa was detectable in the cyto-

solic fraction in HK-2 cells. When cells were subjected to

PS challenge for 24 h, the expression of IjBa was signif-

icantly reduced, which represented activation of NF-jB.

Furthermore, NF-jB inhibitor, PDTC, could block the

reduction in IjBa (Fig. 1).

PS challenge induced up-expression and nuclear

translocation of NF-jB

The immunofluorescence approach was used to determine

whether PS was responsible for the upregulation and

nuclear translocation of NF-jB. All samples were posi-

tively immunostained with the anti-NF-jB p65 subunit

antibodies and DAPI. NF-jB immunostaining was

observed in the cytoplasm in normal cells (Fig. 2a). When

HK-2 cells were cultured for up to 24 h with 20 % PS,

there was a conspicuous increase in the level of the NF-jB

p65. Also, intense nuclear NF-jB immunostaining was

typically present in the majority of the asphyxial cells

(Fig. 2b). Moreover, we found that PDTC administration

obviously reduced immunostaining and nuclear transloca-

tion of NF-jB p65 (Fig. 2c).

To further quantify the total NF-jB protein expression

in HK-2 cells, we performed Western blot. We found that a

basal level of NF-jB p65 subunit was detectable in the

control. When cells were subjected to PS challenge for

24 h, the expression of NF-jB was significantly increased

in the asphyxia group. However, PDTC could interrupt the

PS-mediated increase in NF-jB. Each immunoblot is from

a single experiment and is representative of five separate

experiments. Densitometry results are expressed as

mean ± SE of five separate experiments (Fig. 3).

To identify nuclear translocation of NF-jB in HK-2

cells, we extracted nuclear and cytosolic proteins from HK-

2 cells and quantified NF-jB expression separately in the

nucleus and cytoplasm using Western blot analysis

(Fig. 4). We found that the nuclear NF-jB protein was

obviously increased after PS challenge for 24 h in the

asphyxia group. On the contrary, cytoplasmic NF-jB

protein was evidently decreased after PS challenge. How-

ever, PDTC obviously blocked the upregulation of NF-jB

in the nucleus and the downregulation of NF-jB in the

cytoplasm (Fig. 4).

PS challenge induced upregulation of ICAM-1

ICAM-1 is a downstream protein of NF-jB. We speculated

whether activation of NF-jB induced by PS would promote

ICAM-1 transcription in this model. To answer this ques-

tion, we detected the expression of ICAM-1 mRNA after

Fig. 1 Western blot analysis of the expression of IjBa in HK-2 cells.

a The expression of IjBa in HK-2 cells. b Relative optical density of

IjBa. a One band at 40 kDa corresponding to the IjBa protein

significantly decreased after PS challenge compared with that of

controls. However, PDTC could increase IjBa expression after PS

challenge. b *p \ 0.05 versus that of control, #p \ 0.05 versus values

for asphyxia. Results are representative of at least five independently

repeated experiments

88 Mol Cell Biochem (2014) 388:85–94

123

PS challenge using PCR. We found that ICAM-1 mRNA

levels were significantly induced at 24 h after PS challenge

compared with the control group. However, ICAM-1

mRNA levels were significantly reduced in the PDTC

group compared with the asphyxia group (Fig. 5).

We further investigated whether the expression of

ICAM-1 protein was changed after its changed mRNA

level. To answer this question, we quantified ICAM-1

expression using immunochemistry and Western blot ana-

lysis. We found that HK-2 cells were positively

immunostained with the anti-ICAM-1 antibodies, although

ICAM-1 immunoreactivity was weak in control (Fig. 6a-

A). When HK-2 cells were cultured for up to 24 h in 20 %

PS, there was a conspicuous increase in the level of ICAM-

1. Intense immunostaining was specifically present in the

membrane of the asphyxial cells (Fig. 6a-B). In contrast,

addition of PDTC reduced immunostaining for ICAM-1 to

a weak level (Fig. 6a-C). Western blot analysis showed

that ICAM-1 was present in the normal cells. When cells

were subjected to PS challenge for 24 h, the ICAM-1 level

Fig. 2 Immunofluorescence of NF-jB p65 in HK-2 cells. Left panels

show NF-jB p65 (Cyanine-3, red) staining, middle panels show

nuclear staining (DAPI, blue), right panels show both (overlap).

a control. b asphyxia group. c PDTC-blocking group. All samples

were positively immunostained with the anti-NF-jB p65 subunit

antibodies (red) and DAPI (blue). NF-jB immunostaining was

observed in the cytoplasm (arrow) in normal cells. a When HK-2

cells were cultured for up to 24 h with 20 % PS, there was a

conspicuous increase in the level of NF-jB p65. Also, intense nuclear

NF-jB immunostaining was typically present in the majority of the

asphyxial cells, purple immunostaining for nucleus was shown in the

overlay (arrow), indicating a high nuclear NF-jB concentration.

b Moreover, we found that PDTC administration obviously reduced

immunostaining and nuclear translocation of NF-jB p65 (arrow).

c Results are representative of at least five independent repeat

experiments. (Color figure online)

Mol Cell Biochem (2014) 388:85–94 89

123

was significantly upregulated. In addition, PDTC could

block ICAM-1 expression induced by PS to a normal level

(Fig. 6b, c).

PS challenge induced increased MPO activity

after neutrophils cultivation

To determine whether neutrophils were implicated in kid-

ney proximal tubule cell injury after HI, we assessed

adhesion of neutrophils using MPO activity analysis. We

found that PS induced significantly increased MPO activ-

ity, which represented upregulated adhesion of neutrophils

in asphyxia group. Also, we found that PDTC pretreatment

resulted in a significant decrease in MPO activity (Fig. 7a).

PS challenge induced increased LDH leakage

after neutrophils cultivation

To evaluate HK-2 cells injury caused by adhesion of neu-

trophils following PS challenge, LDH leakage was deter-

mined. As shown in Fig. 7b, we found that LDH leakage of

HK-2 cells in the asphyxia group was increased after neu-

trophil cultivation, in comparison with the control group.

Pre-treatment with PDTC inhibited LDH leakage (Fig. 7b).

PS challenge induced decreased cell viability

after neutrophils cultivation

To further understand whether cell viability was regulated

by adhesion of neutrophils following PS challenge, the cell

viability of HK-2 cells was measured by MTT methods. As

shown in Fig. 7c, we found that the cell viability of HK-2

cells in PS group was decreased after neutrophil cultiva-

tion, in comparison with the control group. Pre-treatment

with PDTC resulted in a significant increase in cell via-

bility (Fig. 7c).

Discussion

Despite the progress in understanding the roles of NF-jB in

HI injury, the roles and mechanisms of NF-jB in the

pathologic processes of neonatal renal injury caused by HI

are not clear. In this study, we found that NF-jB/ICAM-1

pathway is involved in renal tubular cell injury after

asphyxia.

Fig. 3 Western blot analysis of the expression of NF-jB p65 in HK-2

cells. a The expression of NF-jB p65 in HK-2 cells. b Relative

optical density of NF-jB p65 a. One band at 65 kDa corresponding to

the NF-jB protein significantly increased after PS challenge

compared with that of controls. However, PDTC could block NF-

jB upregulation induced by PS. b *p \ 0.05 versus values for

control, #p \ 0.05 versus values for asphyxia. Results are represen-

tative of at least five independent repeat experiments

Fig. 4 Western blot analysis of nuclear translocation of NF-jB p65

in HK-2 cells. a The expression of NF-jB p65 in nucleus and

cytoplasm of HK-2 cells. b Relative optical density of NF-jB p65 in

nucleus and cytoplasm a. One band at 65 kDa corresponding to the

NF-jB protein could be detected in nucleus and cytoplasm of HK-2

cells. After PS challenge for 24 h, the nuclear NF-jB protein was

obviously increased in the asphyxia group. On the contrary,

cytoplasmic NF-jB protein was evidently decreased. However,

PDTC obviously blocked the upregulation of NF-jB in the nucleus

and the downregulation of NF-jB in the cytoplasm. b *p \ 0.05

versus values for control, #p \ 0.05 versus values for asphyxia.

Results are representative of at least five independent repeat

experiments

90 Mol Cell Biochem (2014) 388:85–94

123

The evidence in this study supports the involvement of

the NF-jB/ICAM-1 pathway in renal injury after HI. First,

PS induced upregulation and nuclear translocation of the

NF-jB p65 subunit, while decreasing expression of the NF-

jB inhibitor protein IjBa, indicating that NF-jB was

activated in HK-2 cells after HI injury. Second, ICAM-1, a

downstream component of NF-jB, was increased in the

HK-2 cells. This increase led to elevated MPO activity and

injury of HK-2 cells, suggesting that the downstream

pathway of NF-jB was involved in HI renal injury. Third,

the NF-jB–specific inhibitor PDTC inhibited this pathway

by blocking NF-jB–mediated induction of ICAM-1

expression and adhesion of neutrophils, resulting in less

damage to HK-2 cells. Therefore, the NF-jB/ICAM-1

pathway might have a crucial role in regulating adhesion of

neutrophils involved in renal injury in neonates after HI

(Fig. 8).

Ischemia–reperfusion injury is an uncommon kind of

‘‘inflammation disease,’’ with involvement of leukocytes,

adhesion molecules, chemokines, and cytokines [18].

Activation of NF-jB and its target genes has been impli-

cated in acute inflammatory responses. Normally, NF-jB is

maintained in the cytoplasm and bound to an inhibitor

protein, IjBa. After a variety of stimuli, IjBa is phos-

phorylated by the IjB kinase complex, then degraded by

the 26S proteosome. This process releases NF-jB from the

IjBa proteins. Then, NF-jB can translocate to the nucleus

and bind to NF-jB sites to upregulate a panel of genes

including ICAM-1. Since HI could enhance the production

of cytokines, PS contains a large number of proinflamma-

tory cytokines such as TNF-a and interleukin-6. These

cytokines can induce the activation of NF-jB after PS

challenge. In this study, PS challenge induced decreased

expression of IjBa, with up-expression and nuclear

translocation of NF-jB. These results represented the

activation of NF-jB, which was responsible for upregula-

tion of ICAM-1 (Fig. 8).

It is well known that NF-jB plays a key role in the

regulation of expression of ICAM-1, because the ICAM-1

gene is known to contain NF-jB-binding sites. ICAM-1 is

found in abundance throughout the normal kidney, and is

Fig. 5 PCR analysis of ICAM-1 mRNA in HK-2 cells. a The ICAM-

1 mRNA level in HK-2 cells. b Relative optical density of ICAM-1

mRNA in HK-2 cells. a Two bands corresponding to ICAM-1

(498 bp) and b-actin (661 bp) were amplied. ICAM-1 mRNA levels

were significantly induced at 24 h after PS challenge compared with

the control group. However, ICAM-1 mRNA levels were significantly

reduced in the PDTC group compared with the asphyxia group.

b *p \ 0.05 versus values for control, #p \ 0.05 versus values for

asphyxia. Results are representative of at least five independent repeat

experimentsFig. 6 The expression of ICAM-1 protein in HK-2 cells. a Immuno-

chemical analysis of ICAM-1 in HK-2 cells. A control. B asphyxia

group. C PDTC-blocking group. Magnification, 9400 Immunoreac-

tivity of ICAM-1 in HK-2 cells was detected using immunohisto-

chemistry (n = 5). We found that PS significantly induces expression

of ICAM-1 in HK-2 cells. (B) However, PDTC could block PS-

mediated induction of ICAM-1 protein expression (C). b Western blot

analysis of the expression of ICAM-1 in HK-2 cells. c Relative optical

density of ICAM-1. b One band at 90 kDa corresponding to the

ICAM-1 protein significantly increased after PS challenge compared

with that of controls. However, PDTC could block PS-mediated

induction of ICAM-1 protein expression. c *p \ 0.05 Versus values

for control, #p \ 0.05 versus values for asphyxia. Results are

representative of at least five independent repeat experiments

Mol Cell Biochem (2014) 388:85–94 91

123

expressed constitutively on endothelium, epithelial, and

mesangial cells and fibroblasts. Increased expression of

ICAM-1 could be triggered by reperfusion. Numerous

studies have demonstrated an important role for ICAM-1 in

renal IR [19, 20]. ICAM-1 RNA and protein were upreg-

ulated in a mouse model of renal HI [19]. In HK-2 cells or

porcine proximal tubule cells, the ICAM-1 protein was

increased after renal HI induced by hydrogen peroxide

[20]. Inhibition of ICAM-1 has been found to protect

against both the functional impairment and histological

changes associated with ischemic acute renal failure in the

rat [21].

In our study, the upregulation of adhesion of neutrophils

is partly due to increased expression of ICAM-1 after PS

challenge. This is because induction of adhesion molecules

on endothelial surfaces is the initial step for neutrophil

recruitment under ischemia [2, 22]. Neutrophil–endothelial

interactions are the initial event involved in inflammatory

diseases, which is due to the binding of integrins on neu-

trophils with adhesion molecules on the endothelium [7,

23]. CD11a/CD18 and CD11b/CD18, two integrins on the

surface of neutrophils, play the most important role in

neutrophil–endothelial interactions. Since ICAM-1 is the

ligand for both CD11a/CD18 and CD11b/CD18 integrins,

increased ICAM-1 protein by PS challenge facilitated

neutrophil–endothelial binding in our study.

After HI injury, the cytokines in PS also play key roles

in mediating neutrophil function. In response to cytokines

such as TNF-a, neutrophils have the ability to produce IL-8

as well as other cytokines by de novo mRNA synthesis.

This is thought to contribute to para- or auto-crine cell

activation and may serve as a feed-forward signal for

recruitment of additional neutrophils to their target regions

at sites of lesion. In addition, cytokines and oxyradicals in

PS could simulate integrin expression on neutrophils. Thus,

a PS challenge could not only facilitate ICAM-1 expression

on HK-2 cells, but also upregulate integrin expression on

neutrophil, leading to dramatically activated neutrophil–

endothelial interactions and enhanced MPO activity in our

study (Fig. 8).

Fig. 7 a MPO activity after neutrophil cultivation. The MPO activity

of asphyxia group was significantly increased compared with the

control group. PDTC significantly inhibited the MPO activity

compared with asphyxia group. *p \ 0.05 versus values for control,#p \ 0.05 versus values for asphyxia. b The leakage rate of LDH in

HK-2 cells. The leakage rate of LDH in asphyxia group was

significant increased compared with the control group (p \ 0.01).

PDTC significantly decreased the leakage rate of LDH compared with

asphyxia group. *p \ 0.01 versus values for control, #p \ 0.05 versus

values for asphyxia. c The OD value of Cell Viability in HK-2 cells.

The cell viability of asphyxia group was significantly decreased

compared with the control group. PDTC significantly increased cell

viability compared with asphyxia group. *p \ 0.05 Versus values for

control, #p \ 0.05 versus values for asphyxia

Fig. 8 The possible mechanism of nuclear factor-jB pathway-

mediated adhesion of neutrophils. PS contained cytokines. After PS

challenge, cytokines activated NF-jB in HK-2 cells after decreasing

the cytoplasmic IjBa protein (This step could be blocked by PDTC).

Subsequently, NF-jB translocated into the nucleus, regulating the

transcription of ICAM-1 genes and enhancing expression of ICAM-1

protein. For neutrophils, cytokines in PS contributed to para- or auto-

crine cell activation of neutrophils. This activation not only produced

more cytokines, but also led to greater expression of integrins

92 Mol Cell Biochem (2014) 388:85–94

123

Neutrophils mobilized from blood provide a common

mechanism whereby endothelial injuries progress under

ischemic states. Several reports have described how renal

endothelial injuries and dysfunction were caused by

recruitment of neutrophils [24, 25]. Our results are con-

sistent with these studies. We found that the adherence of

neutrophils is associated with severe injury to HK-2 cells,

which was manifested as increased LDH leakage and

decreased cell viability.

Next, we tried to investigate whether inhibiting the NF-

jB/ICAM-1 pathway reduced HK-2 cells damage in this

model. The NF-jB inhibitor, PDTC, was used when PS

was incubated. PDTC, as an antioxidant, is involved in its

ability to inhibit NF-jB via the stabilization of IjBa or via

the inhibition of the ubiquitin–proteasome pathway [26]. In

our study, we found that PDTC increased IjBa protein

levels after PS challenge. Meanwhile, we found that PDTC

attenuated the expression and nuclear translocation of NF-

jB after PS challenge, which indicated that the active form

of NF-jB was suppressed. Since the promotor regions of

the genes of ICAM-1 contain NF-jB-binding sites, the

reduction in NF-jB activation by PDTC inhibition could

inhibit ICAM-1 expression on HK-2 cells. We found that

PDTC significantly reduced ICAM-1 mRNA and protein

expression after PS challenge. We further studied whether

the reduction in ICAM-1 by PDTC could inhibit adhesion

of neutrophils and HK-2 cells injury. We found that MPO

activity, which represents adhesion of neutrophils, was

dramatically downregulated by PDTC. The downregulation

of MPO was also associated with lower LDH leakage rate

and higher cell viability, indicating attenuated cell injury.

Our findings are consistent with recent reports that NF-jB

inhibition may provide renal protection by the downregu-

lation of adhesion of neutrophils after HI injury [27, 28].

Conclusion

In the current study it was found that the NF-jB/ICAM-1

pathway induced adhesion of neutrophils and was involved

in renal injury after HI. This may provide a new insight

into treatment of renal injury after asphyxia in neonates.

Agents targeting NF-jB might help us to study protective

mechanisms in neonatal HI.

Acknowledgments This research was supported by Sichuan Youth

Science and Technology Foundation (No. 04ZQ026-033 to Wenbin

Dong), and the National Natural Science Foundation of China (No.

81300525 to Tao Xiong). The authors are also grateful for support

provided by the confocal microscope facility at the Institute of

Myocardium Electrophysiology of Luzhou Medical College.

Conflict of interest The authors declare that they have no conflicts

of interest concerning this article.

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