Journal Club09/16/08

Patricia Weng

Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Most prevalent, potentially lethal, monogenic disorderPrevalence 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 5th-6th 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

PKD1 and PKD2

PKD1

PKD2

Polycystin-1

Polycystin-2

Torres et al Lancet 2007

Copyright ©2007 American Society of Nephrology

Hildebrandt et al. J Am Soc Nephrol 2007

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 processesinduces neutrophil proliferation during inflammationinduces 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

TNF-α Converting Enzyme (TACE)

Moss et al Nat Clinic Pract Rheum 2008

ADAM17

Mediator of TNF-α shedding

TACE and TGF-α

Shah et al Trends in Pharm Sci 2006

TACE and Polycystic Kidney Disease

TGF-α abnormally expressed in PKDEGFR in cystic epithelia overexpressed and mislocalized in ARPKD and ADPKDbpk model of ARPKD : kidney TGF-α expression

Dell et al Kid Int 2001

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

FIP-2Cellular 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 cytolysisFIP-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

Background SummaryADPKD 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

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

Immunofluorescence Staining of Endogenous Polycystin-2

TNF-α signaling disrupts PC2 cilia localization in IMCD cells

TUNEL staining of the TNF-α treated IMCD cells

Transfection of siRNA against FIP2 into IMCD cells inhibited FIP2 expression and rescued the TNF-α effect

on PC2 localization

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

Summary ITNF-α elevates expression of FIP-2PC-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

TNF- α triggers cyst formation in cultured embryonic kidneys

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

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

Quantification of TNF-α concentration in 10 human ADPKD kidneys

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

FIP2 TNFR

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

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

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

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