Uromodulin and kidney disease: a new entry for an old molecule

1. Uromodulin and kidney disease: a new entry for an old molecule MCKD/FJHN Italian Consortium Gian Marco Ghiggeri Lyon ESPN 2008

2. Uromodulin • Initially described by Tamm and Horsfall in 1950 • Most abundant protein in human urine (50-100 mg/day) Exclusively expressed in TAL and DCT Glomerulus proximal tubule Collecting duct distal tubule Henle’s loop descending limb ascending limb Dahan et al., J Am Soc Nephrol 2003

3. Non linear pH 4 9 150 kDa 5

4. J Exp Med. 1952 Jan;95(1):71-97 J Exp Med. 1952 Jan;95(1):99-104 Tamm Horsfall Glycoprotein Igor TAMM and Frank L. Horsfall. Characterization and separation of an inhibitor of viral hemagglutination present in urine. Proc Soc Exp Biol Med. 1950 May;74(1):106-8

5. Uromodulin structure Y Y Y Y Y Y Y ZP domain I II III D8C 640 22 31 281 334 585 614 • ZP is a large domain, containing around 260 aa, including 8(10) conserved Cys residues, which are involved in disulphide bond formation. • Found in a variety of receptor-like eukaryotic glycoproteins (mouse sperm receptors ZP1, ZP2, ZP3; alpha-tectorin) • ZP domain proteins almost invariably contain single transmembrane domains or GPI anchors that are missing from the secreted mature proteins • Responsible for the ability of ZP domain proteins to assemble into filaments Jovine et al.,Nat Cell Biol 2002

6. Uromodulin structure Y Y Y Y Y Y Y ZP domain I II III D8C 640 22 31 281 334 585 614 cf 568 EVYLCDIINEKCKPTCSGTRFRSGGIIDQSRVLNLGPITRKNVQAVVSRAASSSLGFLKVCLPLLLSATLTLMFQ 642 bt 569 EVYLCDTVNEKCRPTCPETRFRSGSIIDQTRVLNLGPITRKGGQAAMSRAAPSSLGLLQVWLPLLLSATLTLMSP 643 hs 567 EVYLCDTMNEKCKPTCSGTRFRSGSVIDQSRVLNLGPITRKGVQATVSRAFSS-LGLLKVWLPLLLSATLTLTFQ 640 mm 568 EVYLCDSTSEQCKPTCSGTRFRSGNFIDQTRVLNLGPITRQGVQASVSKAASSNLRLLSIWLLLFPSATLIFMVQ 642 rn 570 EVYLCDTMSEQCKPTCSGTRYRSGNFIDQTRVLNLGPITRQGVQASVSKAASSNLGFLSIWLLLFLSATLTLMVH 644 ****** .*:*:***. **:***..***:**********:. ** :*:* .* * :*.: * *: **** : GPI anchor Santambrogio et al.,Biochem Biophys Res Comm 2008

7. Uromodulin function • Urothelial defence against infections • Urothelial defence against calcium oxalate crystals-induced damage • Urothelial defence against ischemic damage • Water / salt balance in TAL and DCT Mo et al, Am J Physiol Renal Physiol, 2003 Mo et al, Kidney Int, 2004 El-Achkar et al, JASN, 2008 Wiggins et al, Clin Chim Acta, 1987

8. Uromodulin Related Diseases

10. ALLELISM of MCKD, FJHN and GCKD • MCKD - Dominant tubulo-interstitial nephritis - Hypostenuria, ESRD, Medullary cysts - Hyperuricemia and gout • FJHN - Phenotypic similarity with MCKD (no medullary cysts) - Tubulo Interstitial Nephropathy, - Hyperuricemia and gout • GCKD - Glomerular cysts: dilatation of Bowman’s space - Sporadic or dominant disorder - Also part of metabolic syndrome (MODY5; HNF-1betamut.)

11. W202S C195P C148W C217G T225M P236R C300Y K307T E188Vdel33 C282R N128S C135S C248W M229R G103C R204G C300G C347G C112R C126R C217R C315R Q316P C148Y C150S C170Y R185S R222P C223Y C255Y C317Y P236L T225L C52W D59A C77Y Y Y Y Y Y Y Y ZP domain D8C I II III IV Aggiornare la lista mutazioni 640 22 31 281 336 585 V93_G97del4ins H177_R185del 614 GPI Summary of UMOD published mutations Exon 4 Exon 5 98% mutations (42/43) 43 mutations 26 Cys-affecting 1 Gly to Cys 13 other missense 3 in frame deletions

12. MCKD/FJHN GCKD

13. Normal MCKD

14. GCKD

15. MCKD/FJHN kidney biopsy Dr. Vivette D’Agati, Dept of Pathology, Columbia Univ

16. Urinary uromodulin is reduced in MCKD2/FJHN patients MCKD#1 1 2 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 6 5 Uromodulin urinary excretion in affected individuals: • Amount of daily excreted uromodulin is 15 to 30 fold decreased (Bleyer et al., Kidney Int 2004) • Excreted uromodulin is wild type only (Dahan et al., J Am Soc Nephrol, 2003)

17. MOLECULAR PATHOGENESIS “In vitro” studies

18. WT C148W Unpermeabilized HEK293 after transfection (6 hrs)

19. WT C150S Mutant uromodulin is retained in the ER

20. ANIMAL MODELS THP-/- • Are more prone to develop • urinary tract infection. • Do no develop MCKD • Knock-in mice ? Raffi et al Kidney In, 2006

21. Mechanisms • Urine concentration defect interaction with ROMK • Hyperuricemia idem • TI fibrosis ? • Cyst formation ?

22. UAKD Uromodulin Associated Kidney Diseases - Chr 17q12 (HNF1-beta) - Chr 1q21 ? - Chr 1q41 ?

23. Atypical familial juvenile hyperuricemic nephropathy associated with a HNF1B mutation Bingham el al, Kidney Int 2003

24. Renal-specific inactivation of HNF1B UMOD can be considered as direct transcriptional targets of HNF1B Gresh et al, EMBO J, 2004

25. UROMODULIN ASSOCIATED KIDNEY DISEASE Control Umod Mutation ? Linkage 1q41 Hodanova et al Kidney Int 2005

26. CONCLUSIONS • The re-discovery of an old actor such as Uromodilun offers the opportunity to open a new area of research on renal fibrosis that may lead to important advancements

27. Thank You!

28. MCKD/FJHN Italian Consortium G. Gaslini Institute, Genoa Gianluca Caridi Gian Marco Ghiggeri Clinical analysis Molecular analysis University of Turin Mario De Marchi Antonio Amoroso Molecular analysis University of Brescia Francesco Scolari Clinical analysis Kidney pathology University of Padua Luisa Murer Kidney pathology DTI, Dibit-HSR, Milan Luca Rampoldi Cellular and animal models Linkage analysis