1. Journal Club 09/16/08 Patricia Weng

2. Autosomal Dominant Polycystic Kidney Disease (ADPKD) Most prevalent, potentially lethal, monogenic disorder Prevalence 1/400-1/1000 Olmsted County, MN Annual incidence rate for ESRD due to ADPKD in men & women 8.7 and 6.0/million, respectively, in USA Clinical: multiple epithelial-lined kidney cysts, results in kidney failure in majority of individuals by 5 th -6 th decade, extrarenal cysts Genetically heterogeneous: PKD1: chr 16p13.3: 85% cases PKD2: chr 4q21: 15% cases Two-hit mechanism (germline and somatic inactivation of two PKD alleles) Mochizuki et al Science 1996, European Polycystic Kidney Disease Consortium Cell 1994, Watnick et al Mol Cell 1998

3. PKD1 and PKD2 PKD1 PKD2 Polycystin-1 Polycystin-2 Torres et al Lancet 2007

4. Copyright ©2007 American Society of Nephrology Hildebrandt et al. J Am Soc Nephrol 2007

5. Tumor necrosis factor alpha (TNF α) First isolated in 1975 by Carswell in association with necrosis of sarcoma Proinflammatory cytokine with multiorgan effects, produced by many cells, especially macrophage All functions transmitted through 55- and 70 kDa polypeptide receptors Possesses growth stimulating & growth inhibitory processes induces neutrophil proliferation during inflammation induces neutrophil apoptosis when binds to TNF-R55 receptor Low levels of TNF α regulate body’s circadian rhythm and promote remodeling or replacement of injured tissue by stimulating fibroblast growth. Plays role in: immune response to bacterial, viral, parasitic, fungal infections CV system with vascular contraction and proliferation Carswell et al PNAS 1975, Murray et al Blood 1997, Beutler et al Science 1985b

6. TNF-α Converting Enzyme (TACE) Moss et al Nat Clinic Pract Rheum 2008 ADAM17 Mediator of TNF-α shedding

7. TACE and TGF-α Shah et al Trends in Pharm Sci 2006

8. TACE and Polycystic Kidney Disease TGF-α abnormally expressed in PKD EGFR in cystic epithelia overexpressed and mislocalized in ARPKD and ADPKD bpk model of ARPKD : kidney TGF-α expression Dell et al Kid Int 2001

9. Effect of TACE inhibitor (WTACE2) on bpk mice Whole kidney micrograph from day 21 WTACE2-treated and untreated cystic bpk mice and noncystic littermates. Micrograph demonstrating relative kidney sizes of WTACE2-treated and untreated cystic bpk mice and noncystic littermates. (A) WTACE2-treated noncystic, (B) untreated noncystic, (C) WTACE2-treated cystic, and (D) untreated cystic. Kidneys were harvested at day 21. Cystic-treated mice have decreased kidney size compared to untreated cystic mice. Untreated and treated noncystic kidneys are not significantly different in size. Dell et al Kid Int 2001

10. FIP-2 Cellular protein identified via yeast two-hybrid system, interacts with Ad anti-TNF-α protein E3-14.7K FIP-2 colocalizes with and causes redistribution of E3-14.7K In vitro and in vivo interaction between E3-14.7K and FIP-2 FIP-2 reverses protective effect of E3-14.7K on TNF receptor-induced cytolysis FIP-2 is component of TNF-α signaling pathway FIP-2 found to be involved in Huntington’s Disease Huntington protein linked to Rab8 protein through FIP-2 (yeast 2 hybrid system) Huntington, FIP-2 and Rab8 regulate membrane trafficking and cellular morphogenesis Li et al Mol Cell Biol 1998, Hattula et al Curr Biol 2000

12. Background Summary ADPKD results from germline inactivation of a single allele (PKD1 or PKD2) and second hit mutation to inactivate second functional copy TNF-α is proinflammatory cytokine, increased after renal injury, stress seen in ADPKD TNF-α released from membrane by TACE, which also releases TGF-α which binds to and activates the EGF receptor TACE inhibitor reduces cyst formation in bpk mouse model of ARPKD FIP-2 is TNF-α induced protein which is involved in vesicular trafficking

13. Effects of TNF-α on FIP2 and Polycystin-2 in IMCD cells

14. Immunofluorescence Staining of Endogenous Polycystin-2

15. TNF-α signaling disrupts PC2 cilia localization in IMCD cells

16. TUNEL staining of the TNF-α treated IMCD cells

17. Transfection of siRNA against FIP2 into IMCD cells inhibited FIP2 expression and rescued the TNF-α effect on PC2 localization

18. TNF-α disrupts PC1 and PC2 complex formation but not PC1 cilia localization

19. Summary I TNF-α elevates expression of FIP-2 PC-2 and FIP-2 coimmunoprecipitate from IMCD cells TNF-α can affect the normal localization of PC-2 through the induction of FIP-2 in IMCD cells TNF-α disrupts the PC-1-PC-2 interaction, but does not affect ciliary localization of PC-1 in IMCD cells Data suggests that TNF-α promotes cyst formation by disrupting the normal localization of PC-2

20. TNF- α triggers cyst formation in cultured embryonic kidneys

21. TNF-α initiates cyst formation from collecting ducts and proximal tubules

22. Immunoblot analysis of FIP2, TNFR-I and TACE protein abundance in WT and Pkd2 +/- embryonic kidneys with or without TNF-α stimulation TNF-α stimulation: 6.25 ng/ml x 5 days

23. Quantification of TNF-α concentration in 10 human ADPKD kidneys

24. Immunoblot analysis of FIP2 and TNFR-I protein abundance comparing cultured NHK cells and PKD cells FIP2TNFR

25. TNF-α stimulated cyst formation in Pkd2+/- kidneys

26. The TNF-α inhibitor Etanercept prevents cyst formation in Pkd2+/- mice

27. A functional network connecting TNF- signaling, polycystin complex and cystogenesis

28. Conclusion Data suggest TNF- α promotes renal cyst development in the genetic background associated with ADPKD Results of this study are consistent with the previous finding that a TACE inhibitor reduced cyst formation in the bpk mouse model of ARPKD TNF- α/FIP-2/PC-2 network may contribute to the transition from normal tubule development to cystic disease onset in the heterozygous genetic background associated with ADPKD Potential for therapeutic intervention for ADPKD with Etanercept