Podocyte Loss and Glomerulosclerosis in Inducible Mouse Model of Podocin Mutation-Related Nephrotic Syndrome

Ivana Simic, Mansoureh Tabatabaeifar, Geraldine Mollet, Barbara Bruehl, Corinne Antignac, Franz Schaefer

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

24th European Congress of Pathology, Prague, Czech Republic, 8 - 12 September 2012

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

N-term

C-term

R138Q

• The R138Q mutation causes retention of podocin in the ER, leading to impaired SD formation and foot process effacement

• Knock-in mice carrying the R140Q mutation, the mouse analogue of human R138Q, show developmental arrest of podocytes and lethal renal failure at neonatal age (A. Philippe et al, 2008)

The NPHS2 Gene and Hereditary Nephrotic Syndrome

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

Proteinuria Develops within 1 Week

and Peaks 3-4 Weeks after Induction

0,000

0,200

0,400

0,600

0,800

1,000

1,200

w 0 w1

w2

w3

w4

w5

w6

w7

w8

w9

w10

w11

w12

w13

w14

w15

w16

Pro

tein

uri

a (m

g/g

)

injected

control

Reduced Weight Gain from Week 4

90

100

110

120

130

140

150

160

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Week

% o

f in

itial

wei

ght

+/+

R139Q / -

control

R140Q / -

Moderate Blood Pressure Increase in Sick Animals

Renal Failure, Hypoalbuminemia and Hypercholesterolemia

at 12 to 16 Weeks

+ / + R140Q / -

GF

R (

ml/m

in)

0

100

200

300

400

500

600

+ / + R140Q / -

Ser

um a

lbum

in (

g/L)

10

15

20

25

30

35

40

+ / + R140Q / - C

hole

ster

ol (

mg/

dl)

0

200

400

600

800

control control controlR140Q / - R140Q / - R140Q / -

Progressive Decrease of Podocin Level in the Kidney of Sick Animals

37GAPDH

37

kDa 1 WK 2 WKS 12 WKS 16 WKS KO 16 WKS

After Cre Induction Control

Podocin

Foot Process Effacement upon Podocin Loss

week 2

FP En

L

week 16

FPEn

L

week 2

FP En

L

week 2

FP En

L

week 2

FP En

L

controlcontrol

FP

En

L

control

FP

En

L

PAS Staining - Determination of Glomerular Sclerosis Index (GSI)

• combining the semiquantitative scoring system (el Nahas et al 1991) and Image ProPlus Software to quantify the sclerotic area

control

100 µM

PAS staining

week 4

Glomerular Sclerosis Index (GSI)

Percentage of glomeruli affected with sclerosis

0

20

40

60

80

100

120

w1 w2 w4 w6 w8 w12-16

%

control

sickp<0.01p<0.01p<0.01p<0.01

Glomerular sclerosis index GSI

0,00

0,50

1,00

1,50

2,00

2,50

3,00

w1 w2 w4 w6 w8 w12-16

control

sickp<0.05

Sirius Red Staining - Determination of TIF

• The percentage of Sirius Red – stained fibrotic tubulointerstitial area was measured using Image-Pro Plus software on 30 randomly sampled fields of kidney (magnification 300x) (Zhang G et al, 2003)

control

week 4

Tubulointerstitial Fibrosis

Percentage of total kidney area under fibrosis

02468

101214161820

w1 w2 w4 w6 w8 w12-16

%

control

sick

p<0.01

Podocyte Counting and Density Analysis

• Protocol recommended by Animal Models of Diabetic Complications Consortium (AMDCC) (WT1 Ab and immunoperoxidase)

control

week 4

Average Podocyte Number at Different Time-Points after the Induction

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

week 1 week 2 week 4 week 6 week 8 wek 12-16po

docy

te n

umbe

r (pe

r 4 µ

m c

ut)

sick

control

p<0.01

0

2

4

6

8

10

12

14

16

week 1 week 2 week 4 week 6 week 8 wek 12-16po

docy

te n

umbe

r (pe

r 4 µ

m c

ut)

sick

control

p<0.01

0

20

40

60

80

100

120

week 1 week 2 week 4 week 6 week 8 wek 12-16

podo

cyte

num

ber (

per g

lom

erul

us)

sick

control

p<0.01

Summary

• Postnatal induction of R140Q hemizygosity rapidly leads to massive proteinuria, which is maximal at 4 weeks, hypoalbuminemia, hypercholesterolemia and moderate hypertension

• At the later stage, kidneys show global glomerulosclerosis, tubular atrophy and severe interstitial fibrosis

• Podocyte loss occurred within the second week after the induction

• The inducible NPHS2-R140Q mouse is an ideal model for pharmacological studies

Dr. Ivana Simic

Dr. Mansoureh Tabatabaeifar

Prof. Franz Schaefer

Barbara Brühl

Hopital Necker, France

Dr. Geraldine Mollet

Prof. Corinne Antignac