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

MCKD/FJHN Italian Consortium

Gian Marco GhiggeriLyon ESPN 2008

Uromodulin

Most abundant protein in human urine (50-100 mg/day)

Glomerulus

Henle’s loop

Collecting duct

descending limbascending limb

proximal tubule

distal tubule

Exclusively expressed in TAL and DCT

Dahan et al., J Am Soc Nephrol 2003

Initially described by Tamm and Horsfall in 1950

150

5

kDa

4 9Non linear pH

Tamm Horsfall Glycoprotein

J Exp Med. 1952 Jan;95(1):99-104

J Exp Med. 1952 Jan;95(1):71-97

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

Uromodulin structure

Jovine et al.,Nat Cell Biol 2002

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

ZP domain31 334 585 614

640

I III

22 281

YY Y YY

Y Y

D8CII

cf 568 EVYLCDIINEKCKPTCSGTRFRSGGIIDQSRVLNLGPITRKNVQAVVSRAASSSLGFLKVCLPLLLSATLTLMFQ 642 bt 569 EVYLCDTVNEKCRPTCPETRFRSGSIIDQTRVLNLGPITRKGGQAAMSRAAPSSLGLLQVWLPLLLSATLTLMSP 643 hs 567 EVYLCDTMNEKCKPTCSGTRFRSGSVIDQSRVLNLGPITRKGVQATVSRAFSS-LGLLKVWLPLLLSATLTLTFQ 640 mm 568 EVYLCDSTSEQCKPTCSGTRFRSGNFIDQTRVLNLGPITRQGVQASVSKAASSNLRLLSIWLLLFPSATLIFMVQ 642 rn 570 EVYLCDTMSEQCKPTCSGTRYRSGNFIDQTRVLNLGPITRQGVQASVSKAASSNLGFLSIWLLLFLSATLTLMVH 644 ****** .*:*:***. **:***..***:**********:. ** :*:* .* * :*.: * *: **** :

ZP domain31 334 585 614

640

I III

22 281

YY Y YY

Y Y

D8CII

Santambrogio et al.,Biochem Biophys Res Comm 2008

Uromodulin structure

GPI anchor

Uromodulin function

Mo et al, Am J Physiol Renal Physiol, 2003

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, Kidney Int, 2004

Wiggins et al, Clin Chim Acta, 1987

El-Achkar et al, JASN, 2008

Uromodulin Related DiseasesUromodulin Related Diseases

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-1beta mut.)

Summary of UMOD published mutations

43 mutations

26 Cys-affecting

1 Gly to Cys

13 other missense

3 in frame deletions

Aggiornare la lista mutazioni

GPI

640

H177_R185del

ZP domain31 336 585

614

I II III IV22 281

YY Y Y Y Y Y

V93_G97del4ins

D8C

W20

2S

C14

8Y

C12

6RN

128S

G10

3C

C13

5S

C21

7R

C24

8W

C30

0G

C15

0S

C31

5R

C31

7Y

C14

8W

C22

3YT

225L

C25

5Y

D59

A

R18

5S

R20

4G

R22

2P

T22

5M

C11

2R

C17

0Y

C21

7G

C28

2R

E18

8Vd

el33

M22

9R

C52

W

C19

5P

P23

6LP

236R

C34

7G

Q31

6PK

307T

C30

0Y

C77

Y

Exon 4 Exon 598% mutations (42/43)

GCKD MCKD/FJHN

Normal MCKD

GCKD

MCKD/FJHN kidney biopsy

Dr. Vivette D’Agati, Dept of Pathology, Columbia Univ

Urinary uromodulin is reduced in MCKD2/FJHN patients

1 2

1 2 3 4 5

1 2 3 4

6 7

5 6 7 8 9 10 11 12 13

1 2 3 4 65

MCKD#1

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)

MOLECULAR PATHOGENESIS

“In vitro” studies

WT

C148W

Unpermeabilized HEK293 after transfection (6 hrs)

0,0%

10,0%

20,0%

30,0%

40,0%

50,0%

60,0%

70,0%

80,0%

90,0%

100,0%

WT C150S T225K C347G

% o

f gold

part

icle

s

ER

Golgi

PM

WT C150S

Mutant uromodulin is retained in the ER

ANIMAL MODELS THP -/-

Raffi et al Kidney In, 2006

-Are more prone to develop urinary tract infection.

- Do no develop MCKD

-Knock-in mice ?

Mechanisms

• Urine concentration defect interaction with

ROMK

• Hyperuricemia idem

• TI fibrosis ?

• Cyst formation ?

UAKDUromodulin Associated Kidney Diseases

- Chr 17q12 (HNF1-beta)

- Chr 1q21 ?

- Chr 1q41 ?

Atypical familial juvenile hyperuricemic nephropathy associated with a HNF1B

mutation

Bingham el al, Kidney Int 2003

UMOD can be considered as direct transcriptional targets of HNF1B

Gresh et al, EMBO J, 2004

Renal-specific inactivation of HNF1B

Hodanova et al Kidney Int 2005

UROMODULIN ASSOCIATED KIDNEY DISEASE

Control Umod Mutation

Linkage1q41

?

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

Thank You!

MCKD/FJHN Italian Consortium

University of TurinMario De MarchiAntonio AmorosoMolecular analysis

G. Gaslini Institute, GenoaGianluca Caridi

Gian Marco GhiggeriClinical analysis

Molecular analysis

University of BresciaFrancesco Scolari

Clinical analysisKidney pathology

DTI, Dibit-HSR, MilanLuca Rampoldi

Cellular and animal modelsLinkage analysis

University of PaduaLuisa Murer

Kidney pathology