Article
Reference
Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution
RAJARAM, Renuga Devi, et al.
Abstract
NADPH oxidase 4 (NOX4) catalyzes the formation of hydrogen peroxide (H2O2). NOX4 is highly expressed in the kidney, but its role in renal injury is unclear and may depend on its specific tissue localization.
RAJARAM, Renuga Devi,
et al. Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution.
Redox Biology, 2019, vol. 26, p. 101234
DOI : 10.1016/j.redox.2019.101234 PMID : 31247506
Available at:
http://archive-ouverte.unige.ch/unige:122903
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Supplementary Figure 2: Scheme illustrating the strategy employed for the generation of tubular cell specific Nox4 knock-in mice.
Supplementary Figure 3: Gene expression analysis of Renin and Epo in baseline (A).
Hematoxylin and Eosin staining on the sections of Wt and Nox4KI mice (B). Representative images of active caspase-3 immunostaining on the kidney sections of Wt and Nox4KI mice at baseline (C).
Supplementary Figure 4: Expression analysis of cellular stress (cMyc), angiogenesis (Hif1a, Vegf), pro-inflammatory (Il1b, Il18 and Ccl5) and fibrosis (Tgfb1) genes in the kidney cortical extracts of Wt and Nox4KI mice collected after 3 days (A) and 10 days (B) of UUO. The non- obstructed kidneys were used as controls.
Supplementary Figure 5: Representative images of proliferation marker Ki-67 immunostaining on the kidney sections of Wt and Nox4KI mice collected after 3 days of UUO (A). The non- obstructed kidneys were used as controls. Quantification of Ki-67 immunostaining (B) and apoptosis marker active caspase-3 (C) on the kidney sections of Wt and Nox4KI mice collected after 3 days of UUO.
Supplementary Figure 6: Representative images of macrophage marker F4/80 immunostaining and quantification on the kidney sections of Wt and Nox4KI mice collected after 3 days (A) and 10 days (B) of UUO. The non-obstructed kidneys were used as control.
Suppl. Figure 1 Rajaram et al
50μm
20μm 20μm
50μm 20μm
50μm 100μm
A
B C
D E
F G
FSGS
DN HTN
Targeted allele
mNOX4 cDNA
CAG STOP Neor
mNOX4 cDNA
CAG STOP Neor DTA
Conditional knock-in allele after flp mediated recombination
mNOX4 cDNA CAG STOP
Constitutive knock-in allele expression in the tubular cells after Pax8Cre mediated recombination
mNOX4 cDNA CAG
mRosa26 exon Homology arm LoxP site
Frt site PolyA tail
F1
R1
Suppl.Figure 3 Rajaram et al
Wt Nox4KI 0.0
0.5 1.0 1.5 2.0
Normalized mRNA level
Wt Nox4KI 0.0
1.0 2.0 3.0 4.0
Normalized mRNA level
Wt Nox4KI -2.0
0.0 2.0 4.0 6.0
Normalized mRNA level
Wt Nox4KI 0.0
1.0 2.0 3.0
Normalized mRNA level
ReninEpo
A
Cortex Medulla
50μm
Wt Nox4KI
B Wt Nox4KI
C
50μm
Wt con Nox4KI
con Wt UUO
Nox4KI UUO 0.0
2.0 4.0 6.0 8.0
NormalizedIl1β Expression
ns **
ns
Wt con Nox4KI
con Wt UUO
Nox4KI UUO 0.0
0.5 1.0 1.5 2.0
NormalizedIl18 Expression
nsns ns
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO 0.0
5.0 10.0 15.0
NormalizedCcl5 Expression
ns*** ***ns
10d
Wt con Nox4KI
con Wt UUO
Nox4KI UUO 0.0
5.0 10.0 15.0 20.0
NormalizedcMyc Expression
ns** ***ns
Wt con Nox4KI
con Wt UUO
Nox4KI UUO 0.0
0.5 1.0 1.5
NormalizedHif1α Expression
nsns nsns
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO 0.0
5.0 10.0 15.0
NormalizedIl1β Expression
ns* ** ns
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO 0.0
0.5 1.0 1.5 2.0 2.5
NormalizedIl18 Expression
nsns nsns
Wt con Nox4
KIcon Wt UUO
Nox4KI UUO 0.0
10.0 20.0 30.0 40.0
NormalizedCcl5 Expression
ns** ***ns Wt con
Nox4KI con
Wt UUO Nox4KI
UUO 0.0
0.5 1.0 1.5 2.0
NormalizedVegf Expression
ns**** ****ns
B
Wt con Nox4
KIcon Wt UUO
Nox4KI UUO 0.0
2.0 4.0 6.0 8.0
NormalizedTgfβ1 Expression
ns**** ***ns
Suppl. Figure 5 Rajaram et al
A
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO 0.0
10.0 20.0 30.0
% of Ki-67 +ve cells
ns**** ****ns
B C
100μm
Nox4
KI- Con Wt- UUO Nox4
KI- UUO Wt- Con
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO 0.0
0.2 0.4 0.6 0.8 1.0
% of caspase- 3 +ve cells ns ns** **
50μm
Nox4KI
50μm
B Con UUO
WtNox4KI
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO -2.0
0.0 2.0
% of F4/8
Wt con Nox4
KIcon Wt UUO
Nox4
KIUUO 0.0
10.0 20.0 30.0
% of F4/80 +ve area
ns**** ** * 10d
10d
