A
0
1
2
3
4** **
Tubu
lar d
amag
e sc
ore
(AU
)
B6
Sh
FVB B6
Nx
FVB
B6 FVB
Sh
Nx
C ShB6 ShFVB NxFVBNxB6
Day
2D
ay 2
8D
ay 5
6
KI6
7
B
Supplemental Figure 1. Tubular injury and cell proliferation after nephron reduction. (A) Representative imagesstained with PAS (left panels, original magnification, X400) and quantification (right panel) of tubular lesions in FVB andB6 mice 2 days after sham-operation (Sh) or subtotal nephrectomy (Nx). n = 4-6 and n = 10-12 for Sh and Nx,respectively in each strain. Data are means ± SEM. ANOVA was followed by the Tukey–Kramer test. Nx versus Shmice: ** P <0.01. (B) mRNA expression of Lcn2 in FVB and B6 mice 2 days after Sh or Nx (n = 5-6 and n = 10 for Shand Nx, respectively in each strain). Data are means ± SEM. ANOVA was followed by the Tukey–Kramer test. Nxversus Sh mice: ** P < 0.01, *** P < 0.001. (C) Representative cortical sections from FVB and B6 mice 2, 28, and 56days after Sh or Nx stained with anti- KI-67 antibody (original magnification, X200; n = 4-6 and n = 10-12 for Sh andNx, respectively in each strain at each time point).
0
20
40
60
80
100
**
***
Rel
ativ
e m
RN
A qu
antit
y (fo
ld)
B6
Sh
FVB B6
Nx
FVB
Lcn2
B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB
Day 56Day 28Day 2
10
15
20
25
30
*
**### ###
Body
wei
ght (
g)
10
15
20
25
30
******
### ###
Body
wei
ght (
g)
10
15
20
25
30## ###
Body
wei
ght (
g)
A
B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB
Day 56Day 28Day 2
0
100
200
300
400
*** **
Kidn
ey w
eigh
t (m
g)
0
100
200
300
400
** *#
Kidn
ey w
eigh
t (m
g)
0
100
200
300
400 ###***
Kidn
ey w
eigh
t (m
g)
B
Supplemental Figure 2. Time-course analysis of body and kidney weight. (A) Body weight and (B) kidney weightin B6 and FVB mice 2, 28, and 56 days after sham-operation (Sh) or subtotal nephrectomy (Nx). n = 4-6 and n = 9-12for Sh and Nx, respectively in each strain at each time point. Data are means ± SEM. ANOVA was followed by theTukey–Kramer test. Nx versus Sh mice: * P < 0.05, ** P < 0.01, ***P < 0.001; FVB versus B6 mice: # P < 0.05, ## P <0.01, ### P < 0.001.
A
B
C
Supplemental Figure 3. Time-course quantification of renal lesions. (A) Glomerular lesions score, (B) tubular
dilatation area and (C) fibrosis score of kidneys from FVB and B6 mice 2, 28, and 56 days after sham-operation (Sh) or
subtotal nephrectomy (Nx). n = 4 and 6 for Sh and Nx, respectively in each strain at each time point. Data are means ±SEM. ANOVA was followed by the Tukey–Kramer test. Nx versus Sh mice: * P < 0.05, ** P < 0.01, *** P < 0.001; FVB
versus B6 mice: # P < 0.05, ### P < 0.001.
B6
Sh
FVB B6
Nx
FVB
Day 2
0
10
20
30
40
50###
Tubu
lar d
ilatio
n ar
ea (%
)
B6
Sh
FVB B6
Nx
FVB
Day 28
0
10
20
30
40
50###
Tubu
lar d
ilatio
n ar
ea (%
)
B6
Sh
FVB B6
Nx
FVB
Day 56
0
10
20
30
40
50 ***###
Tubu
lar d
ilatio
n ar
ea (%
)
B6
Sh
FVB B6
Nx
FVB
Day 2
0
5
10
15
20
Fibr
osis
(%)
B6
Sh
FVB B6
Nx
FVB
Day 56
0
5
10
15
20***
###
***
Fibr
osis
(%)
B6
Sh
FVB B6
Nx
FVB
Day 28
0
5
10
15
20
***
***#
Fibr
osis
(%)
B6
Sh
FVB B6
Nx
FVB
Day 2
0
1
2
3
4
** **
Glo
mer
ular
lesi
ons
scor
e (A
U)
B6
Sh
FVB B6
Nx
FVB
Day 28
0
1
2
3
4
***
Glo
mer
ular
lesi
ons
scor
e (A
U)
B6
Sh
FVB B6
Nx
FVB
Day 56
0
1
2
3
4
*
***###
Glo
mer
ular
lesi
ons
scor
e (A
U)
B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB0
10
20
30
40
50***#
Seru
m C
reat
inin
e (µ
mol
.l-1)
Day 56Day 56Day 56
0
5
10
15
20***###
Urin
e Pr
ot/C
reat
(mg/
mm
ol)
0510
1500
3000
4500
6000###***
Urin
e Al
b/Cr
eat (
mg/
mm
ol)
Supplemental Figure 4. Renal function. Serum creatinine, urine protein-to-creatinine ratio and urine albumin-to-creatinine ratio in FVB and B6 mice 56 days after sham-operation (Sh) or subtotal nephrectomy (Nx). n = 6 and n = 9-11 for Sh and Nx, respectively in each strain. Data are means ± SEM. ANOVA was followed by the Tukey–Kramer test.Nx versus Sh mice: *** P < 0.001; FVB versus B6 mice: # P < 0.05, ### P < 0.001.
Sh
B6 FVB
Nx
Supplemental Figure 5. Cell proliferation interests mainly tubular cells. Representative images of co-localizationexperiments using anti-PCNA (blue) and anti-α-SMA antibodies in B6 and FVB mice 56 days after sham-operation (Sh)or subtotal nephrectomy (Nx). Original magnification, X200; n = 6 both Sh and Nx in each strain.
PCNAα-SMA
Technical quality control
Statistical model
Unbiased analysis
Geneset enrichment analysis (GSEA)
2days
56days
28days
mRNA(n=48)
Compensatoryphase
Deteriorationphase
Quiescent phase
B6
FVB
Surgery
n=4/each time
Supplemental Figure 6. Experimental protocol used for transcriptomic analysis. Four mice per group (sham-operation, Sh; or subtotal nephrectomy, Nx) of each strain (B6 and FVB) at each time point (2, 28, and 56 days aftersurgery, corresponding to the renal compensatory, quiescent, and deterioration phases, respectively) were used forwhole-kidney transcriptomic analysis. Overall, microarray analysis was performed on 48 mice. A full statistical modelwas fitted to the expression data in order to consider: (1) the strain (FVB versus B6), (2) the surgery (Nx versus Sh),and (3) the time after surgery (2 versus 28 versus 56 days) effect. Unbiased clustering analysis was performed usingco-inertia analysis. Gene-set enrichment analysis was then performed using a collection of gene signatures from PID,Pathway Commons and Reactome.
Isg15
0
2
4
6
8
10***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 2
ShFVB NxFVB0
2
4
6
8
10
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 56
ShFVB NxFVB0
2
4
6
8
10
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 28
ShFVB NxFVB
Usp18
0
1
2
3
4
5***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 2
ShFVB NxFVB0
1
2
3
4
5
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 56
ShFVB NxFVB0
1
2
3
4
5
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 28
ShFVB NxFVB
Irf7
0
2
4
6***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 2
ShFVB NxFVB0
2
4
6
*
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 56
ShFVB NxFVB0
2
4
6
*
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 28
ShFVB NxFVB
Ifi202b
0
500
1000
1500
2000 ***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 2
ShFVB NxFVB0
500
1000
1500
2000
***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 56
ShFVB NxFVB0
500
1000
1500
2000
**
Rel
ativ
e m
RN
A qu
antit
y (A
U)
ShFVB NxFVB
Day 28
Ifi204
0
2
4
6
8
10***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 2
ShFVB NxFVB0
2
4
6
8
10
**
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Day 56
ShFVB NxFVB0
2
4
6
8
10
**
Rel
ativ
e m
RN
A qu
antit
y (A
U)
ShFVB NxFVB
Day 28
Supplemental Figure 7. Time-course analysis of type I IFN induced genes expression after nephron reduction.mRNA expression of Ifi202b, Ifi204, Isg15, Usp18, and Irf7 in FVB mice 2, 28, and 56 days after sham-operation (Sh)or subtotal nephrectomy (Nx). n = 4-6 and n = 9-11 for Sh and Nx, respectively in each strain at each time point. Dataare means ± SEM. Mann Withney test. Nx versus Sh mice: * P < 0.05, ** P <0.01, *** P <0.001.
0
1
2
3
4
***
***
***
***
***
***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Irf7
Sh
Day 7
CK OK Sh
Day 14
CK OK Sh
Day 21
CK OK
Ifi204
Sh
Day 7
RK LK Sh
Day 14
RK LK Sh
Day 21
RK LK0
1
2
3
4
***
***
***
***
***
***R
elat
ive
mR
NA
quan
tity
(AU
)
Ifi205
Sh
Day 7
RK LK Sh
Day 14
RK LK Sh
Day 21
RK LK0
1
2
3
4
*
*
*
*
*
*
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Isg15
Sh
Day 7
CK OK Sh
Day 14
CK OK Sh
Day 21
CK OK0
1
2
3
4
*
**
*
**
*
**
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Usp18
Sh
Day 7
CK OK Sh
Day 14
CK OK Sh
Day 21
CK OK0
1
2
3
4
***
***
***
***
***
***
Rel
ativ
e m
RN
A qu
antit
y (A
U)
Supplemental Figure 8. Unilateral ureteral obstruction results in early type I IFN response. mRNA expression ofIfi204, Ifi205, Isg15, Usp18 and Irf7 in B6 mice 7, 14, and 21 days after sham-operation (Sh) or unilateral ureteralobstruction (UUO) in non obstructed control kidney (CK) and obstructed kidney (OK). n = 3-4 and n = 5-6 for Sh andUUO, respectively at each time-point. Data are means ± SEM. ANOVA was followed by the Tukey–Kramer test. OKversus Sh or CK: * P < 0.05, ** P < 0.01, *** P < 0.001.
B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB
Day 56Day 28A
B6
Sh
FVB B6
Nx
FVB
Day 2
0.0
0.2
0.4
0.6
***##
CD
3+ c
ells
/ fie
ld (%
)
0.0
0.2
0.4
0.6 **
CD
3+ c
ells
/ fie
ld (%
)
0.0
0.2
0.4
0.6 *
*
CD
3+ c
ells
/ fie
ld (%
)
B6
Sh
FVB B6
Nx
FVB B6
Sh
FVB B6
Nx
FVB
Day 56Day 28B
B6
Sh
FVB B6
Nx
FVB
Day 2
0.0
0.5
1.0
1.5
2.0
***
F4/8
0+ c
ells
/ fie
ld (%
)
0.0
0.5
1.0
1.5
2.0
F4/8
0+ c
ells
/ fie
ld (%
)
0.0
0.5
1.0
1.5
2.0 **
*
F4/8
0+ c
ells
/ fie
ld (%
)
Supplemental Figure 9. Time-course quantification of renal inflammatory cell infiltration. (A) Quantification of T-lymphocytes and (B) monocytes-macrophages in B6 and FVB mice 2, 28, and 56 days after sham-operation (Sh) orsubtotal nephrectomy (Nx) by CD3 and F4/80 immunohistochemistry, respectively. n = 4 for both Sh and Nx, in eachstrain at each time point. Data are means ± SEM. ANOVA was followed by the Tukey–Kramer test. Nx versus Sh mice:* P < 0.05, ** P < 0.01, *** P < 0.001; FVB versus B6 mice: ## P < 0.01.
Gene Primer Sequence (5’- - - - - 3’)
Cdkn1a F GGAACATCTCAGGGCCGAAAR CTGACCCACAGCAGAAGAGG
Ifi202b F ATTCCAGCGTGTCTGAGGTGR ACCACCACTTTCATTGCTCCT
Ifi204 F TAGAAGTGGTGGGGAGTGGAR GCACCATCACTTGTTTGGGAC
Ifi205 F TTGTGAACGTGTACCAACTCTR TGCCTTGATCTTCTTCACGTC
Isg15 F TCTGACTGTGAGAGCAAGCAR ACCAGGAAATCGTTACCCCC
Usp18 F AAGGACGCAAAGCCTCTGAAR ATGGTCAAGGTTTGGGGCAG
Irf7 F GTCACCACACTACACCATCTACR CCATGAGGAAGTGTTCGATGT
Cd3 F AAGCCTGTGACCCGAGGAAR TGCGGATGGGCTCATAGTCT
Mac1 F CTTCCAGGGCAGGAGTCGTATR CAATGGAGCTGCCCACAAT
SiglecH F CAGAACTCCACAGCCCATGTR TGTGTTGCTGGTCTCTCCAC
Tlr7 F GTTCTTGACCTTGGCACTR CCGTGCATATTCATCGTA
Kim1 F GAGAGTGACAGTGGTCTGTATTGR CCTTGTAGTTGTGGGTCTTCTT
Lcn2 F GGACCAGGGCTGTCGCTACTR GGTGGCCACTTGCACATTGT
Ripk3 F GCCTTCCTCTCAGTCCACACR ACGCACCAGTAGGCCATAAC
Mlkl F AGGATGCAGAGGAAGACGGAR TGGGATCTCCTGTGTGGGTT
Rpl13 F CTCATCCTGTTCCCCAGGAAR GGGTGGCCAGCTTAAGTTCTT
Supplemental Table 1: Primers used in the study.
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