Supplementary methods
Identification of early and late EPC
A colony of EPC was defined as a central core of round cells with elongated
sprouting cells at the periphery. The mononuclear cells were isolated and
re-suspended in growth medium (EndoCult liquid medium; StemCell Technologies),
and 5 × 106 mononuclear cells were plated in a fibronectin-coated 6-well plateas
previous described.1, 2After 4 days, the non-adherent cells were collected and plated
onto a fibronectin- coated 24-well plate (1 × 106 cells per well) in triplicate. In the
adherent cells, defined as early and late EPC, the expressed VEGF receptor 2 was
detected by RT-PCR. Moreover, RT-PCR analysis confirmed the expression of
progenitor cell lineage markers, including CD31 and endothelial NO synthase.
Measurement of Plasma Indoxyl Sulfate
Plasma IS (i.e., combined free and protein-bound IS) were analyzed with ultra
performance liquid chromatography (UPLC) system. In brief, samples were
deproteinized by the addition of three parts methanol to one part plasma for
determination of IS. The free concentrations of IS were measured in
plasmaultrafiltrates obtained using Microcon YM-30 separators (Millipore), followed
by the same sample preparation and analysis that was done for plasma IS. A UPLC
assay using detection at the 280 nm setting of the PDA detector was performed at
room temperature on an AglientZorbax XDB-C18 column of 4.6×150mm. Buffer
flow was 0.8 mL/min using 10 mMammonia acetate (pH = 4.0) (A) and 100%
acetonitrile (B) with a gradient from 82.5%A/17.5%B to 55%A/45%B, over 9 min.
Under these conditions, IS appeared at 1.4 min. Standard curves from IS at 0.5, 1, 2.5,
5, and 10 mg/L, processed in the same manner as the serum samples, correlated to the
1
serum samples with average r2 values of 0.999 ± 0.001. Quantitative results were
obtained and calculated as concentrations (mg/L). The sensitivity of this assay was
0.225 mg/L for IS.
Measurement of Nitric Oxide Production
Monolayer cells were grown in six-well plate dishes until 90% confluence and then
starved in a serum-free medium for 4 hours. The cells were treated with various
concentrations of IS (10-5 to 10-3M), and the supernatants were collected at different
time intervals. NO release was determined by measurement of nitrite (NO2−) and
nitrate (NO3−) levels using a commercially available Total NO/Nitrite/Nitrate Assay
Kit (R&D Systems) according to the manufacturer's instructions. 3
In vitro tube formation assay
EPC tube formation assay was performed with an In Vitro Angiogenesis Assay Kit
(BD BioCoat angiogenesis System). After cell harvest, 1×104 late EPCs were placed
in a matrix solution with EGM-2 MV medium and incubated at 37°C for 6 h. Tubule
formation was inspected under an inverted light microscope (×100) with a microscope
video system. Tube formation was measured using the BD AttoVision Software
System with a number of parameters such as tube length.4
DNA cell cycle
For the cell cycle analysis, the cells were trypsinizedand were fixed with 40C
methanol for at least 30 min and then stained with 50 ug/ml propidium iodide and
40ug/ml RNase A for 30 min in the dark. Cell cycle distribution was determined by
flow cytometric analysis (Becton Dickinson FACScan) and Cell Quest software
(Becton Dickinson). All cell cycle distributions represent the mean standard deviation
of at nine independent experiments of flow cytometric analysis.
2
3
Supplementary Tables
Supplementary Table 1.
Primer sequences used in Real time-PCR
Gene
LC3B
LC3A
ITGB7
ITGA4
ITGB1
ICAM1
VCAM1
eNOs(mice)
NGAL(mice)
LC3b (mice)
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Forward
Reward
Sequences
5’-GCCGCACCTTCGAACAAA-3’
5’-TCGTTCTATTATCACCGGGATTTT-3’
5’-CCGACCGCTGTAAGGAGGTA-3’
5’-ACCCTTGTAGCGCTCGATGAT-3’
5’-TGCTTGGACGGCTACTATGGT-3’
5’-AGTCCTGAAGGCCCCACACT-3’
5’-CGAACCGATGGCTCCTAGTG-3’
5’-CACGTCTGGCCGGGATT-3’
5’-CTTCAGAATTGGATTTGGCTCAT-3’
5’-TGGTGCAGTTCTGTTCACTTGTG-3’
5’-GCAGACAGTGACCATCTACAGCTT-3’
5’-CACCTCGGTCCCTTCTGAGA-3’
5’-TGGTCAGCCCTTCCTCCAT-3’
5’-GCTGCCTGCTCCACAGGAT-3’
5’-CACCAGGAAGAAGACCTTTAAGGA-3’
5’-CACACGCTTCGCCATCAC-3’
5’-GCCACTCCATCTTTCCTGTTG-3’
5’-GAAGAGGCTCCAGATGCTCCTT-3’
5’-CGTCCTGGACAATACCAAGT-3’
5’-ATTGCTGTCCCGAATGTCTC-3’
Size (bp)
84
80
102
116
107
80
99
85
84
233
Abbreviations:
LC3B=microtubule-associated protein 1 light chain 3 beta
LC3A=microtubule-associated protein 1 light chain 3 alpha
ITGb7 = integrin, beta 7
ITGa4 = integrin, alpha 7
ITGb1 = integrin, beta 1
ICAM1 = intercellular adhesion molecule 1
VCAM1 = vascular cell adhesion molecule 1
eNOS(NOS3) = nitric oxide synthase 3 (endothelial cell)
NGAL= neutrophil gelatinase-associated lipocalin
4
Supplementary Figures
Supplementary Fig 1.
(A) Flow cytometry analysis of circulating human endothelial progenitor cells (EPCs)
(B) Identification of EPC with immunofluorescence microscopy . KDR, kinase
domain insert receptor; acLDL, acetylated low-density lipoprotein.
(C) Morphology of our early and late human EPCs. Most of the EPC expressed
endothelial and hematopoietic stem cell markers, CD31, CD34, KDR,
VE-cadherin and eNOs, which are considered critical markers of EPCs. Cells
were counterstained with DAPI for the nuclei (blue).
Supplementary Fig 2.
Organic anion transporter (OAT)1 and OAT3 were identified in neither early nor late
EPC.RT-PCR analysisof VEGF receptor 2 (VEGFR2), CD31, endothelial NO
synthase (eNOS), mRNA expression.
5
Supplementary Fig 3.
Cell cycle progression determined by flow cytometry. The distribution and
percentage of cells in the G1and G2/M phases of the cell cycle are indicated. A
histogram plot of flow cytometry analysis of control cells shows that after 24 h of
10-3 M IS treatment, cells in the G0/G1 population increased from 66.9% to 75.7%
compared to the cells in the control group, whereas the increase in cell population
was blunted by anti-oxidative NAC (70.6%). The data shown here are from a
representative experiment repeated 9 times with similar results. (*, P < 0.05; # P =
not significant, vs. untreated control).
6
Supplementary Fig 4
IS stimulated late EPC showed a up-regulation of LC3b messenger RNA and
Relative Quantification
blunted by ATO quantified by real -time PCR.
2.5
*
2.0
Control
1.5
IS 10-3M
1.0
IS+ATO 0.1M
0.5
0.0
LC3A
LC3B
Supplementary Fig 5 a
IS significantly decreased expression of p-eNOs in late EPCs at a concentration of
10-3M. (vs. untreated control)
7
Supplementary Fig 5 b
Effects of IS and ATO on eNOS, Akt, ERK,, VEGF and HO-1 production in cultured
EPCs. Administration of IS on cultured EPCs followed by treatment of ATO
significantly upregulatedeNOS activity, but not ERK, AKT, and VEGF.
8
Supplementary Fig 6.
An in vitro angiogenesis assay was performed with late EPCs to investigate the
effect of IS on EPC neovascularization by ECMatrix gel. The averages of the total
area of complete tubes formed were compared using the BD AttoVision Software
System. The functional capacity for tube formation of late EPCs was blunted by IS
and restored by adding SNP and ATO. However, the recovery capacity of ATO was
downregulated by the nitric oxide synthase inhibitor L-NAME.
Supplementary Fig 7
Is exacerbated the kidney injury at 3days after ischemic injury. However the
kidney function indicator, creatinine was restored after ATO pretreatment. In this
study, removing the right kidney allowed assessment of renal functional recovery in
the left post-ischemic kidney.5
9
Supplementary Fig 8
I/R AKI animals without IS infusion to evaluate the effect of AST120 (capable
of absorbing IS via the intestine) on renal outcome after removal of contralateral
kidney. The results showed that mice pretreated with AST 120 exhibited less kidney
injury, in terms of NGAL and more number of blood EPCs
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