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