1. Glucocorticoid Action in Podocytes, or Models and Their Caveats in Cultured Podocytes Richard F. Ransom, Ph.D. Research Assistant Professor Department of Pediatrics Nephrology Division University of Michigan Medical School

3. Podocyte Proteome

4. Confessions of a Culturian n Worked with MPC ("Mundelocytes") for 10 yrs n Limited studies using MPC #2 ("Endlichocytes") and RPC ("Kuriharocytes") n Are they podocytes? How could you tell? (i.e., what would a podocyte look like in culture?) How much does it matter? (i.e., what model is appropriate to the question at hand?) Perhaps the better question is: Are they enough like podocytes for my purposes?

5. Example of Cultured Podocytes in a Model "Steroid-Responsive NS"

6. Nephrotic Syndrome (NS) Common kidney disease in children and adults Various etiologies: genetic, environmental, hemo-dynamic, idiopathic Clinical Findings:  Massive proteinuria / Hypoalbuminemia  Edema / Hypercholesterolemia Histologic Findings:  Effacement of podocyte foot processes  Disruption and disorganization of actin filaments in foot processes Primary therapy for NS is oral glucocorticoids

7. Soluble Mediator Hypothesis n Shalhoub proposed soluble mediator hypothesis in 1974 (Shalhoub (1974) Lancet 2:556-560) NS is an immunologic disorder Circulating factor from T-lymphocytes leads to NS n In more than 30 years, no single soluble mediator identified that is both: Present and increased in serum or urine in NS Able to induce experimental NS in animals n However, Shalhoub hypothesis remains the dominant paradigm for pathogenesis of NS Glucocorticoid therapy presumed to act via inhibition of soluble mediator release by T-lymphocytes

8. Glucocorticoids (GC) n Can act via genomic or non-genomic mechanisms n Genomic effects Mediated by GC receptor (GR) GR-hormone complex moves to nucleus to affect transcription Resulting effects mediated by alterations in protein expression n GR present in podocytes, moves to nucleus after hormone binding (Kidney Int 56, 65-73) n GC treatment of cultured murine podocytes can: (Kidney Int 68, 2473-83) Protect and enhance recovery from puromycin aminonucleoside injury Reduce actin filament disruption by cytochalasin D or latrunculin A

9.  B-Crystallin and Hsp27 Both  B-crystallin and hsp27 can confer resistance to heat shock and other cellular stresses n Hsp27 expression increases in glomerular podocytes during experimental nephrotic syndrome n Both proteins are known to be upregulated by glucocorticoids in certain cells and tissues n Both are known to interact with actin and intermediate filament cytoskeletal structures n Hsp27 can act as a barbed-end actin filament capping protein in vitro n No known direct interaction between either protein and actin in vivo

10. Hypothesis The beneficial effects of glucocorticoids in nephrotic syndrome result, at least in part, from a direct action on glomerular podocytes through an increase in the expression of small heat shock proteins

11. DEX Protects From and Ameliorates PAN Injury by a GR-Dependent Mechanism Control DEX - 30 min Medium - 7 d Medium - 30 min PAN - 7 d DEX - 30 min PAN - 7 d PAN - 5 d Medium - 7 d PAN - 5 d DEX - 7 d PAN - 5 d DEX + RU486 - 7 d DEX + RU486 - 30 min PAN - 7 d Treatments with 1 µM DEX, 5 µg/mL PAN, and 20 µM RU486, phalloidin labeled

12. DEX Protects Against Direct Actin Filament Disruption Podocytes treated with 20 µM RU486, 1 µM DEX, or 1 µM DEX + 20 µM RU486 for 30 min, cultured for 3 d, then treated with 0.2 µM latrunculin B or 2 µM cytochalasin D for 10 min, and then fixed, stained with Texas Red phalloidin to label actin filaments RU486DEXDEX + RU Pretreatment Cyto D LAT B

13. Podocyte Proteins Altered by Glucocorticoids Podocyte proteins with expression altered by 1 µM DEX 3 d after treatment (n = 7, 2- tailed unpaired T-test) 2D-PAGE separation of proteins from sham-treated podocytes 2D-PAGE separation of proteins from DEX-treated podocytes

14. CNTF and Small Heat Shock Proteins Induced by Glucocorticoids Representative Western blots of protein extracts from podocytes made at various times after 30 min treatment with 1 µM DEX and probed for CNTF,  B-crystallin, and hsp25 5

15. Hsp25 and  B-Crystallin Expression in Glomeruli Fluorescence micrographs of rat kidney cryosections probed with antibodies against  B-crystallin (aBC) or hsp25 (Hsp25) and also labeled with DAPI

16. Alterations of Small Heat Shock Protein Expression Modulate Podocyte Response to Glucocorticoids Fluorescence micrographs of podocytes transfected with hsp25 (murine), hsp27 antisense (human, but inhibits murine hsp25 expression),  B-crystallin, and  B-crystallin antisense adenoviral constructs and 3 d later treated for 30 min with vehicle alone or 1 µM DEX followed by transfer to medium containing 5 µg/mL PAN for a further 7 d Medium - 30 min PAN - 7 d DEX - 30 min PAN - 7 d

17. Summary n Glucocorticoids (DEX) can protect cultured podocytes from PAN injury and direct actin cytoskeletal disruption n Protection from PAN injury and actin disruption induced by DEX are blocked by RU486 n A proteomic analysis of cultured podocytes identified 92 proteins, 88 unique and 4 isoforms, of 106 spots yielding MALDI-TOF results n The expression of 7 identified proteins was altered 3 d after treatment with 1 µM DEX Western blot analysis confirmed over-expression of  B-crystallin and identified hsp27 as a DEX-induced protein in podocytes n Altering the expression of small heat shock proteins by adenoviral infection with specific sense and antisense constructs modulated the podocyte response to glucocorticoids

18. Conclusions n The therapeutic effects of glucocorticoids in nephrotic syndrome may result, at least in part, from direct effects on glomerular podocytes Increases in the expression of the small heat shock proteins,  B-crystallin and/or hsp27, may be responsible for the protective effects of glucocorticoids in podocytes

19. Podocyte Models n What are you modeling? n Is it possible to model in vivo behavior using an in vitro system? n How can you tell if your model is working? n How can you tell if your model isn’t working? n A tale oft repeated - microscopy as output

20. PAN Model of “NS” n PAN Treatment Typically 50 - 100 µg/ml, <48 h PAN action in animals >5 d Chose concentration with effects >3 d 1.25 µg/ml = "minimal change" 5 µg/ml = "FSGS"

21. Taking it Back In Vivo: Steroid-Responsive PAN Model of NS

22. Alternative Model - Protamine Sulfate

23. A Step Further - Protamine + Heparin

24. A Step Too Far - 1.0 mg/ml Protamine

25. Alternative II - Latrunculin A

26. Outcome Measures n Are you measuring what you think you’re measuring? n Are you sure you want to use a microscope?

27. Be Careful What You Wish (Look) For... Hsp27 Phalloidin Hsp27 + Phalloidin

28. False color images of MPC infected with adenoviruses expressing citrine-ArpC1a and CFP-Hsp27 Co-Localization of Hsp27 and ArpC1a

29. Taking it Live - CFP-Hsp27 in Podocytes

30. Frozen rat kidney sections probed with anti-ArpC1a antibody and DAPI Taking it Back In Vivo: ArpC1a is Expressed in Podocytes

31. And Now - Movie Time! n MPC MPC 1280x1024 10 fps.mov n EPC EPC 1280x1024 10 fps.mov n KPC Y-Cell #2 1280x1024 10 fps.mov n More KPC Y-Cell #2 Detail 5 fps.mov

32. Contributors n William E. Smoyer, M.D. n Tad E. Eichler, B.S. n Virginia Vega-Warner, Ph.D. n David Geller, M.D., Ph.D. Yale University n Daniel Streetman, Pharm.D. n Lynda Welage, Pharm.D. n Jody Martin, Ph.D. Loyola University

33. Altered Hsp27 Expression by Stable Transfection (Selection) ( ++ P < 0.001 vs. Vector; n = 4)

34. Effect of Hsp27 on Morphology (Cell Area in PAN Model) ( ++ P < 0.01 vs. Vector; *P < 0.05, **P < 0.01 vs. Untreated; n = 60 cells/group)

35. Effect of Hsp27 on Physiology (Total F-Actin in PAN Model) ( + P < 0.05, ++ P < 0.01 vs. Vector; *P < 0.05; **P < 0.01 vs. Untreated; n = 3)

36. Average FRET efficiency measurements in transfected COS cells (left) or adenovirus infected MPC cells (right). FRET was also measured in specific subcellular regions of MPC cells. FRET in Podocytes

37. Effect of ArpC1a & Hsp27 on Physiology (Cell Survival After Heat Shock) MTT Assay; x ± SE; n = 8; *P < 0.05 vs. pcDNA control * * * *