Pharmacological Nephroprotection in a Novel Mouse Model of Hereditary Nephrotic Syndrome

Ivana Simic, Mansoureh Tabatabaeifar, Helga Denc, Tanja Wlodkowski, Geraldine Mollet, Corinne Antignac, Franz Schaefer

Division of Pediatric Nephrology, University of Heidelberg Department of Human Genetics, Hopital Necker, Paris

25th European Congress of Pathology, Lisbon, 31 August – 4 September 2013

The NPHS2 Gene and Hereditary Nephrotic Syndrome • Mutations in the NPHS2 gene, encoding podocin, cause autosomal recessive steroid-

resistant nephrotic syndrome• R138Q, the most common podocin mutation in Europeans, causes early disease onset

and rapid progression to end-stage renal disease

• Recently, we generated and characterized an inducible knock-in mouse carrying the R140Q podocin mutation, the murine analogue of the most common human mutation R138Q

N-term

C-term

R138Q

Creation of a Conditional Knock-in Mouse Model of R140Q Mutation

Cre

Cre

Cre

Cre

Nphs2R140Q/+ Nphs2lox2/lox2

Cre+/+

Nphs2lox2/R140Q

Cre+

Nphs2R140Q/-

X

Tamoxifen induction

Bl6 Bl6

Prophylactic RAS Blockade in a Conditional Knock-in Mouse Model of R140Q Mutation

Tamoxifen-induced mice received 10 mg/kg/day of:

•ACE inhibitor Ramipril (R)

•AT1 receptor blocker Candesartan (C)

•the combination of Ramipril and Candesartan (R+C)

•non-RAS antihypertensive amlodipine (A)

Control groups:

•Tamoxifen induction (sick controls) •Vehicle injections (healthy controls)

Blood Pressure Effect of RAS Antagonists vs. Amlodipine

0

20

40

60

80

100

120

1 2 3 4Week of observation

MA

BP

(m

mH

g)

healthy untreated Ramipril Candesartan C+R Amlodipine

***

***

*** ******

***

*** ***

***

***

****

***

* p<0.05; ** p<0.01; *** p<0.001

Markedly Attenuated Proteinuria in Podocin R140Q Knock-In Mice Treated with RAS Antagonists

Normoalbuminemia in Podocin R140Q Knock-In Mice Treated with RAS Antagonists

* p<0.05; ** p<0.01; *** p<0.0001

Reduced Plasma Creatinine Levels in All Treated Animals

Subtotal Loss of Podocin Protein in All Induced Animals Irrespective of Pharmacological Treatment

Podocin

GAPDH

37

37

kDa 4 WKS Control R C R+C A

The Loss of Podocin Signal at Immunofluorescence Analysis

healthy untreated

R+C Amlodipine

Preserved Podocin mRNA Expression in All Induced Animals

0

500

1000

1500

2000

2500

3000

healthy sick Ramipril Candesartan R+C Amlodipine

Type of treatment

Po

do

cin

exp

ress

ion

(%

18S

)

*

* p>0.05 comparing to both sick untreated and healthy animals

Attenuated Glomerulosclerosis in Animals Treated with RAS Antagonists

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

1,80

healthy untreated Ramipril Candesartan R+C Amlodipine

Type of treatment

Glo

mer

ula

r sc

lero

sis

ind

ex

****** ***

*

* p>0.05; ** p<0.05; *** p<0.01; **** p<0.001

A.

B.

PAS staining of renal tissue in induced animals: A. Untreated (notable GS); B. Mice treated with R+C.

Insignificantly Attenuated Tubulointerstitial Fibrosis in Animals Treated with RAS Antagonists

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

9,00

10,00

healthy untreated Ramipril Candesartan R+C Amlodipine

Type of treatment

Are

a af

fect

ed b

y fi

bro

sis

(% o

f to

tal

kid

ney

are

a)

p>0.05

Sirius-Red-staining: A. Untreated animals; B. Mice treated with R+C.

A.

B.

Improved Podocyte Survival in Animals Treated with RAS Antagonists

0

20

40

60

80

100

120

healthy untreated Ramipril Candesartan R+C Amlodipine

Type of treatment

Po

do

cyte

nu

mb

er (

per

glo

mer

ulu

s)

***

**

***

* p<0.05; ** p<0.01; *** p<0.001

A.

B.

WT1 immunostaining of glomeruli of induced mice: A. Untreated (reduced podocyte number); B. Treated with R+C.

Summary

• RAS antagonists markedly attenuate proteinuria in mice hemizygous for podocin R140Q mutation

• After 4 weeks, mice receiving R+C were normo-albuminemic and serum creatinine was increased less than in untreated or animals treated with Amlodipine

• Reduced glomerulosclerosis and better podocyte survival in animals treated with RAS antagonists

• RAS blockade provides effective pharmacological nephroprotection in this hereditary podocytopathy model

Dr. Ivana Simic

Dr. Mansoureh

Tabatabaeifar

Tanja Wlodkowski

Dr. Helga Denc

Prof. Dr. Franz Schaefer

Hopital Necker, France

Dr. Geraldine Mollet

Prof. Dr. Corinne Antignac