Supplemental Fig. 1 B6 + Abx (12 weeks) B6 B6 B6 + Abx Number of colonies in culture media C. B. A. C57BL/6 (B6) n = 9 I/R 40min Sacrifice Day 0 day 10 Bacteroides spp. Staphylococcus (MSSA) Enterococcus fecalis Lactobacillus murinus Lactbacillus spp. Escherichia coli UD UD UD UD UD UD Bacteroides spp. Staphylococcus (MSSA) Enterococcus fecalis Lactobacillus murinus Lactbacillus spp. Escherichia coli B6 + Abx (12 weeks) n = 9 1X10 5 1X10 6 1X10 7 1X10 8 (cfu/g) 1X10 5 1X10 6 1X10 7 1X10 8 (cfu/g) B6 + Abx B6 D E. Necrosis Brush border region Intraluminal debris *P < 0.05 pre day10 Relative gene expression in kidney tissue X100 X400 X100 X400 * * U-Alb / U-Cr 0.08 BUN Cr B6 I/R B6 + Abx I/R Grade Grade Grade Havcr1 μg/mgCr μg/dL μg/dL
Transcript
Supplemental Fig. 1
B6 + Abx (12 weeks)
B6
B6 B6 + Abx
Number of colonies in culture media C.B.
A.C57BL/6 (B6) n = 9
I/R 40min
Sacrifice
Day 0 day 10
Bacteroides spp.Staphylococcus (MSSA)
Enterococcus fecalisLactobacillus murinus
Lactbacillus spp.Escherichia coli
UDUDUDUDUDUDBacteroides spp.
Staphylococcus (MSSA)Enterococcus fecalis
Lactobacillus murinusLactbacillus spp.
Escherichia coli
B6 + Abx (12 weeks) n = 9
1X105 1X106 1X107 1X108
(cfu/g)
1X105 1X106 1X107 1X108
(cfu/g)
B6+
Abx
B6
D
E.
Necrosis Brush border region Intraluminal debris
*P < 0.05
pre day10Re
lativ
e ge
ne e
xpre
ssio
n in
kid
ney
tissu
e
X100 X400 X100 X400
**
U-Alb / U-Cr0.08
BUN
Cr
B6 I/RB6 + Abx I/R
Grad
e
Grad
e
Grad
e
Havcr1
μg/m
gCr
μg/d
Lμg
/dL
Supplemental Figure 1. Gut microbiota againsts from tubular injury in a mouse model of AKI. (A) The protocol of antibiotic treatment and I/R induction in C57BL/6 (B6) mice. Prior to I/R, WT mice were treated with 1 g/L ampicillin sodium salt , 1 g/L neomycin sulfate, 1 g/L metronidazole, 0.5 g/L vancomycin hydrochloride, and 0.5 g/L gentamicin sulfate in drinking water for 12 weeks prior to I/R. (B) The gut microbiota was reduced to below 1�105 colony-forming units (cfu)/g after treatment with antibiotics. (C) The administration of antibiotics led to an enlargement of the cecum in B6 mice. (D, E) The antibiotic-treated mice showed more severe tubular injury, as reflected in the acute tubular necrosis (ATN) score, compared with the untreated mice on day 10 after I/R. Statistical analysis was performed using Student’s t test. *P < 0.05. Abx, antibiotic treatment.
0
2000
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10000
12000
14000
16000
18000
His
Asn Ser
Gln
ArgAsp G
ly
all-Thr
Glu
ThrAla
ProM
etVal
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
0
200
400
600
800
1000
1200
1400
1600
His
Asn Ser
Gln
ArgAsp G
ly
all-Thr
Glu
ThrAla
ProM
etVal
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
A. Feces (Gut)
B. Plasma
Total amino acid
C. Kidney
Supplemental Fig. 2
(nmol/g)
(nmol/ml)
(nmol/g)
(nmol/ml)
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
His
Asn Ser
Gln
ArgAsp G
ly
all-Thr
Glu Thr
AlaPro
Met
Val
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
0
200
400
600
800
1000
1200
1400
1600
His
Asn Ser
Gln
ArgAsp G
ly
all-Thr
Glu
ThrAla
ProM
etVal
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
D. Urine
B6
B6 Sham day 2
B6 I/R day 2
B6 Sham day 10
B6I/R day 10
n = 4-6/group
UD UD
UD UD
UD UD
Supplemental Figure 2. The profile of all amino acids analyzed
with 2D HPLC. We performed metabolomic analysis of the
amino acids in the AKI mice. All amino acids were evaluated in
the feces (A), plasma (B), kidney (C), and urine (D) of the mice
with/without I/R on days 0, 2, and 10. Data are shown as
means � SEM.
UD, undetectable.
02000400060008000
1000012000140001600018000
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro Met
Val
all0-Ilc Ile Le
uPhe Trs Lys Cys Tyr
0
200
400
600
800
1000
1200
1400
1600
His Asn Ser
Gln Arg Asp Glyall-T
hrGlu Thr
Ala Pro MetVal
all0-Ilc Ile Le
uPhe Trs Lys Cys Tyr
0
2000
4000
6000
8000
10000
12000
14000
16000
His Asn Ser
Gln Arg Asp Glyall-T
hrGlu Thr
Ala Pro MetVal
all0-Ilc Ile Le
uPhe Trs Lys Cys Tyr
L-amino acid
(nmol/g)
(nmol/ml)
(nmol/g)
(nmol/ml)
0200400600800
1000120014001600
His Asn Ser
Gln Arg Asp Glyall-T
hrGlu Thr
Ala Pro MetVal
all0-Ilc Ile Le
uPhe Trs Lys Cys Tyr
B. Plasma
C. Kidney
D. Urine
B6 B6 Sham day 2B6 I/R day 2B6 Sham day 10B6I/R day 10n= 4-6/group
A. Faeces (Gut)
UD UD UD
UD UD
UD UD
UD UD
Supplemental Fig. 3
Supplemental Figure 3. The profile of L-amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in the AKI mice. The L-amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days 0, 2, and 10. Data are shown as means � SEM. UD, undetectable.
020406080
100120140160180200
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
Phe Trs Lys
Cys Tyr
0
2
4
6
8
10
12
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
Phe Trs Lys
Cys Tyr
0
200
400
600
800
1000
1200
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
Phe Trs Lys
Cys Tyr
D-amino acid Supplemental Fig. 4
(nmol/g)
(nmol/ml)
(nmol/g)
(nmol/ml)
0
2
4
6
8
10
12
14
16
18
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
Phe Trs Lys
Cys Tyr
B. Plasma
C. Kidney
D. Urine
B6 B6 Sham day 2B6 I/R day 2B6 Sham day 10B6I/R day 10n= 4-6/group
A. Feces (Gut)
UDUD
UD UD UD UD
UD UD UD UD UD
UD UD UD UD UDUD
Supplemental Figure 4. The profile of D-amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in AKI mice. The D-amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days 0, 2, and 10. Data are shown as means � SEM. UD, undetectable.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
0
0.02
0.04
0.06
0.08
0.1
0.12
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
0
0.2
0.4
0.6
0.8
1
1.2
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
D-amino acid/L-amino acid Supplemental Fig. 5
0
0.001
0.002
0.003
0.004
0.005
0.006
HisAsn Se
rGln Arg Asp Gly
all-Thr
Glu ThrAla Pro
Met
Val
all0-Il
c Ile Leu
PheTrs Ly
sCys Tyr
B. Plasma
C. Kidney
D. Urine
B6 B6 Sham day 2B6 I/R day 2B6 Sham day 10B6I/R day 10n= 4-6/group
A. Feces (Gut)
UDUD
UD UD UD UD
UD UD UD UD UD
UD UD UD UDUD UD UD
Supplemental Figure 5. The profile of amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in in germ-free mice with AKI. The ratios of the D-/ L-amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days 0, 2, and 10. Data are shown as meanS � SEM. UD, undetectable.
A. Total amino acid
B. L-amino acid
C. D-amino acid
D. D/L amino acid
(nmol/g)
Feces (Gut) Supplemental Fig. 6
Gf B6 n = 6Gf B6 I/R 10day n = 5
00.020.040.060.08
0.10.120.140.16
His
Asn
Ser
Gln
Arg
Asp
Gly
all-T
hr Glu
Thr
Ala
Pro
Met Va
l
all0
-Ilc Ile Leu
Phe
Trs
Lys
Cys
Tyr
0100020003000400050006000
His
Asn
Ser
Gln
Arg
Asp
Gly
all-T
hr Glu
Thr
Ala
Pro
Met Va
l
all0
-Ilc Ile Leu
Phe
Trs
Lys
Cys
Tyr
010203040506070
His
Asn
Ser
Gln
Arg
Asp
Gly
all-T
hr Glu
Thr
Ala
Pro
Met Va
l
all0
-Ilc Ile Leu
Phe
Trs
Lys
Cys
Tyr
0100020003000400050006000
His
Asn
Ser
Gln
Arg
Asp
Gly
all-T
hr Glu
Thr
Ala
Pro
Met Va
l
all0
-Ilc Ile Leu
Phe
Trs
Lys
Cys
Tyr
(nmol/g)
(nmol/g)
UDUD
UDUD
UD
UDUDUD
Supplemental Figure 6. The profile of amino acids analyzed with 2D HPLC in germ-free mice. We performed metabolomic analysis of the amino acids in the feces of germ-free mice. All amino acids (A), L-amino acids (B), D-amino acids (C) and the ratio of the D-/L-amino acids (D) were evaluated in the germ-free mice pre-I/R and on day 10 after I/R. Data are shown as means � SEM. UD, undetectable.
ml/
mo
use
/6
da
ys
Water n = 6
20 mM D-Ser n = 6
NS
Supplemental Fig. 7
Supplemental Figure 7. The volume of water intake was
similar between the untreated mice and the D-serine-
treated mice. Statistical analysis was performed using
Student’s t test.
NS, not significant.
Water 20 mM D-Ser
Water 20 mM D-Ser
meas 29.50 29.71
SD 8.09 4.39
TTEST 0.96
O.D.
μg/ m
gCr
***
day10 after I/R
Supplemental Fig. 8
A
B
Supplemental Figure 8. Administration of high-dose D-serine augmented kidney injury. (A) Urine albumin excretion increased after high-dose administration of D-serine. (B) Post-hypoxic tubular proliferation was inhibited by D-serine at a dose of 100 mM. Statistical analysis was performed using Student’s t test compare to control. *P < 0.05.
n = 6/group
Water n = 880 mM D-Serine n = 6
**P < 0.01***P < 0.001
Water 80mM D-Serine
0
1μM
10μM
100μ
M
1mM
10m
M
100m
M
D-Ser
****
*****
******
U-ALB/U-Cr
Supplemental Fig. 9
Pre I/R Post I/RMark
er
Positive
contro
l
Negativ
e contro
l
Supplemental Figure 9. Confirmation of the status of the germ-free mice by 16S rDNA PCR before and after I/R. No PCR product was detected in the feces of the germ-free mice before and after I/R.
