Tuesday: Andrea Chambers, Darcy Cooke

Thrombospondin-1 Is an

Endogenous Activator of TGF-β

in Experimental Diabetic

Neuropathy

Tuesday: Steven Hager, Zachary Tong

Objectives:

Understand how TSP-1 affects TGF-β

Explain how TSP-1 affects Diabetic

Nephropathy

Critically evaluate methods and

experiments

Design and propose a new experiment to

improve renal function in Diabetic

Nephropathy

About the Authors…

Christoph Daniels:

Kathrin Schaub:

Kerstin Amann:

About the Authors…

Jack Lawler:Boston College

Ph.D. , Biophysics , 1971 — 1976

Professor of Pathology

Beth Israel of Deconess Medical Center

Christian Hugo:

Diabetes (The Journal)

“Diabetes publishes original research about

the physiology and pathophysiology of

diabetes mellitus. Submitted manuscripts can

report any aspect of laboratory, animal, or

human research.”

Impact Factor: 8.398 (PNAS: 9.380)

We want you to be very critical of this paper

Just to Recap

Get into groups of 5 and complete

the exercise

Tackling the Title

“Thrombospondin-1 Is an Endogenous Activator of TGF-β in Experimental

Diabetic Nephropathy In Vivo”

What does this mean to you?

Motivation

What did they know?

What did they want to know?

Why did they bother to study this?

TGF-β activation may be the causative agent of diabetic nephropathy

TGF-β is activated by TSP-1

TGF-β and TSP-1 are both upregulated in human diabetic patients

Diagnostic analysis of diabetic nephropathy is well characterized

Is TSP-1 deficiency capable of suppressing TGF-β activation in

mouse diabetic models?

What was their experimental model?

The Experiment

C57B16 & 129SVJ

Inducing Diabetes

Induced Type I diabetes, how?

STZ? Streptozotocin

Renal Corpuscle

Figure 1: Experimental Design

Potential

Problems?

Figure 2: Conclusion

•Showed TSP-1 Expression in a

diabetic mouse model

•Show comparable kidney health

Levels of Diabetes in Humans

Figure 2: Diabetic Assessment

Figure 2: Continued…

Any problems?

TGF- β Pathway - Don’t Be

Scared!

Figure 3 & 4: Conclusion

•TGF-β activity is reduced in TSP-1

KO mice•Active TGF-β decreased

•Total TGF-β normal

•Activity of downstream components

decreased

Figure 3: Active TGF-β

Figure 4: Total TGF-β

Any Problems?

•Quantification by immunohistochemistry

•Why not western blot for Total TGF?

•Why not rtPCR for Total TGF mRNA?

•Scoring System

•Semi-quantitative and subjective

Smad2/3 and PAI

•SMAD2/3

•Transcription factors

•Activated by TGF signaling

•Activity mechanism is phosphorylation

dependent

•Plasminogen activator inhibitor-1(PAI-1)

•Function not important

•Target gene of SMAD2/3

Figure 3

Potential Problems?

•Only assessed phosphorylated SMAD2/3

•Why not total SMAD2/3?

•Quantification by immunohistochemistry

•Why not western blot for Total SMAD?

•Why not rtPCR for Total SMAD mRNA?

•Nuclear vs. Cytoplasmic SMAD?

•Assumed PAI-1 expression is only driven by

SMAD

•PAI-1 reporter construct?

Figure 5: Conclusion

•Show decreased matrix accumulation

•Look at hallmarks of Diabetic Neuropathy•Collagen IV, Fibronectin, Podocytes

Figure 5:

Points of Interest

Figure 5

PAS Staining= Periodic Acid Schiff, Stains

dark

Figure 5

Any Problems?

•Podocyte Damage assessed in 20 week

only

•Not comparable to human kidneys

Figure 6: Conclusion

•Decreased inflammatory response in

TSP-1 deficient mice

•TSP-1 deficiency is the protective

effect

Figure 6

Any problems?

•Claim of being a protective mechanism is a

stretch

•20 week results are not significantly

significant

•Chronic immune response

Figure 7: Conclusion

•Statistical significance of

decreased glomerular cells in TSP-

1 knockout

•For 20 week

Figure 7

Any problems?

•Statistical significance is for 20 week

TSP-1 deficient mice only

•Cells proliferated at similar levels

•Not statistically significant

•Cells apotosed at similar levels

Figure 8: Conclusions

•TSP-1 knockout mice had healthier kidneys

•Based off of Proteinuria, Albuminuria,

Serum Urea

Figure 8

Experimental Problems

•TSP-1 isn’t the only activator of

TGF

•STZ injection induces cytotoxicity

•Increases Albuminuria

•Tested in mice, not the same for

humans

•Scoring

Be an editor

•Groups of 5

•Suggest improvements for the paper

•Better designs, new designs

•Write how TSP-1 can be used in

treatment of Diabetic Nephropathy

•How else can you effectively treat

Diabetic Nephropathy?

THIS WILL BE HANDED IN

Objectives:

Understand how TSP-1 affects TGF-β

Explain how TSP-1 affects Diabetic

Nephropathy

Critically evaluate methods and

experiments

Design and propose a new experiment to

improve renal function in Diabetic

Nephropathy

HAVE A WONDERFUL SPRING BREAK!!!!!!!!