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Research LBL - Slide 1

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Research LBL - Slide 1

  1. 1. Tuesday: Andrea Chambers, Darcy Cooke Thrombospondin-1 Is an Endogenous Activator of TGF-β in Experimental Diabetic Neuropathy Tuesday: Steven Hager, Zachary Tong
  2. 2. 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
  3. 3. About the Authors… Christoph Daniels: Kathrin Schaub: Kerstin Amann:
  4. 4. About the Authors… Jack Lawler: Boston College Ph.D. , Biophysics , 1971 — 1976 Professor of Pathology Beth Israel of Deconess Medical Center Christian Hugo:
  5. 5. 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
  6. 6. Just to Recap Get into groups of 5 and complete the exercise
  7. 7. Tackling the Title “Thrombospondin-1 Is an Endogenous Activator of TGF-β in Experimental Diabetic Nephropathy In Vivo” What does this mean to you?
  8. 8. 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?
  9. 9. What was their experimental model? The Experiment C57B16 & 129SVJ
  10. 10. Inducing Diabetes  Induced Type I diabetes, how? STZ? Streptozotocin
  11. 11. Renal Corpuscle
  12. 12. Figure 1: Experimental Design Potential Problems?
  13. 13. Figure 2: Conclusion •Showed TSP-1 Expression in a diabetic mouse model •Show comparable kidney health
  14. 14. Levels of Diabetes in Humans
  15. 15. Figure 2: Diabetic Assessment
  16. 16. Figure 2: Continued…
  17. 17. Any problems?
  18. 18. TGF- β Pathway - Don’t Be Scared!
  19. 19. 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
  20. 20. Figure 3: Active TGF-β
  21. 21. Figure 4: Total TGF-β
  22. 22. 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
  23. 23. 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
  24. 24. Figure 3
  25. 25. 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?
  26. 26. Figure 5: Conclusion •Show decreased matrix accumulation •Look at hallmarks of Diabetic Neuropathy •Collagen IV, Fibronectin, Podocytes
  27. 27. Figure 5:
  28. 28. Points of Interest
  29. 29. Figure 5 PAS Staining= Periodic Acid Schiff, Stains dark
  30. 30. Figure 5
  31. 31. Any Problems? •Podocyte Damage assessed in 20 week only •Not comparable to human kidneys
  32. 32. Figure 6: Conclusion •Decreased inflammatory response in TSP-1 deficient mice •TSP-1 deficiency is the protective effect
  33. 33. Figure 6
  34. 34. Any problems? •Claim of being a protective mechanism is a stretch •20 week results are not significantly significant •Chronic immune response
  35. 35. Figure 7: Conclusion •Statistical significance of decreased glomerular cells in TSP- 1 knockout •For 20 week
  36. 36. Figure 7
  37. 37. 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
  38. 38. Figure 8: Conclusions •TSP-1 knockout mice had healthier kidneys •Based off of Proteinuria, Albuminuria, Serum Urea
  39. 39. Figure 8
  40. 40. 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
  41. 41. 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
  42. 42. 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
  43. 43. HAVE A WONDERFUL SPRING BREAK!!!!!!!!

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