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 NPHS2 Gene and Hereditary Nephrotic Syndrome
• 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)
N-term
C-term
R138Q
Creation of a Conditional Knock-in Mouse Model of
R140Q Mutation
Bl6
X
Cre
Bl6
Cre
Nphs2R140Q/+
Nphs2lox2/lox2
Cre+/+
Cre
Nphs2lox2/R140Q
Cre+
Tamoxifen induction
Nphs2R140Q/Cre
Proteinuria Develops within 1 Week
and Peaks 3-4 Weeks after Induction
1,200
0,800
injected
0,600
control
0,400
0,200
w16
w15
w14
w13
w12
w11
w10
w9
w8
w7
w6
w5
w4
w3
w2
w1
0,000
w0
Proteinuria (mg/g)
1,000
Reduced Weight Gain from Week 4
160
% of initial weight
150
140
control
+/+
130
120
R140Q
R139Q/ -/ -
110
100
90
0
1
2
3
4
5
6
7
8
Week
9
10 11 12 13 14 15 16
Moderate Blood Pressure Increase in Sick Animals
120
i njected
control s
115
Mean BP (mm Hg)
110
105
100
95
90
85
80
75
w1 w2 w3 w4 w5 w6 w7 w8 w9 w10 w11 w12 w13 w14 w15 w16
Renal Failure, Hypoalbuminemia and
Hypercholesterolemia
at 12 to 16 Weeks
600
40
800
400
300
200
Cholesterol (mg/dl)
35
Serum albumin (g/L)
GFR (ml/min)
500
30
25
20
600
400
200
100
15
0
10
control
+/+
R140Q//-R140Q
0
control
+/+
R140Q//-R140Q
control
+/+
R140Q/ /- R140Q
Progressive Decrease of Podocin Level in the Kidney of
Sick Animals
After Cre Induction
kDa
37
1 WK
2 WKS
12 WKS
Control
16 WKS
KO
16 WKS
Podocin
37
GAPDH
Foot Process Effacement upon Podocin Loss
control
week 2
L
FP
En
En
L
FP
week 16
FP
En
L
En
PAS Staining - Determination of Glomerular Sclerosis
Index (GSI)
control
• combining the
semiquantitative scoring
system (el Nahas et al 1991)
and Image ProPlus Software
to quantify the sclerotic
area
PAS staining
100 µM
week 4
Glomerular Sclerosis Index (GSI)
Glomerular sclerosis index GSI
Percentage of glomeruli affected with sclerosis
120
2,50
100
2,00
80
control
p<0.01
p<0.05
1,50
sick
%
3,00
1,00
40
0,50
20
0,00
control
60
sick
0
w1
w2
w4
w6
w8
w12-16
w1
w2
w4
w6
w8
w12-16
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
20
18
16
14
12
10
8
6
4
2
0
p<0.01
control
sick
w1
w2
w4
w6
w8
w12-16
Podocyte Counting and Density Analysis
• Protocol recommended by
Animal Models of Diabetic
Complications Consortium
(AMDCC) (WT1 Ab and
immunoperoxidase)
control
week 4
16,00
16
120
100
p<0.01
80
sick
60
control
40
20
0
podocyte number (per 4 µm cut)
podocyte number (per glomerulus)
Average Podocyte Number at Different Time-Points
after the Induction
p<0.01
14,00
14
p<0.01
12,00
12
10,00
10
sick
8,00
8
control
6,00
6
4,00
4
2,00
2
0,00
0
week 1
week 2
week 4
week 6
week 8
wek 12-16
week
week
1 1
week
week
22
week
week4 4
week
week66
week
week88
wek 12-16
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