Post on 12-Jul-2015
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!!!!!!!!