Volume 89, Issue 3, Pages 574-585 (March 2016) Indoxyl sulfate suppresses endothelial progenitor cell–mediated neovascularization  Szu-Chun Hung, Ko-Lin Kuo, Hsin-Lei Huang, Chia-Chun Lin, Tung-Hu Tsai, Chao-Hung Wang, Jaw-Wen Chen, Shing-Jong Lin, Po- Hsun Huang, Der-Cherng Tarng  Kidney International  Volume 89, Issue 3, Pages 574-585 (March 2016) DOI: 10.1016/j.kint.2015.11.020 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 Plasma indoxyl sulfate and worsened renal function in SNx mice. (a) Mice were divided into 3 groups including sham operation (Sham), subtotal nephrectomy with no treatment (SNx), and SNx treated with AST-120 (SNx+AST-120). (b) Plasma indoxyl sulfate, (c) BUN, and (d) serum creatinine levels were markedly increased after SNx. ∗P < 0.05 versus Sham group; #P < 0.05 versus SNx group; n = 9 in each group. BUN, blood urea nitrogen; HI, unilateral hindlimb ischemia surgery; 2/3 Nx, 2/3 nephrectomy; SNx, subtotal nephrectomy; UNx, uninephrectomy. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 Blood perfusion and EPC mobilization in mice after HI surgery. (a) Blood perfusion in ischemic hindlimb was measured before; immediately after; and 1, 2, 3, and 4 weeks after hindlimb ischemia surgery by laser Doppler. Circulating (b) Sca-1+/Flk-1+ cells and (c) CD34+/Flk-1+ cells were determined by flow cytometry at baseline; 2 days; and 1, 2, 3, and 4 weeks after hindlimb ischemia surgery. ∗P < 0.05 versus Sham group; #P < 0.05 versus SNx group; †P < 0.05 versus baseline. n = 9 in each group. EPC, endothelial progenitor cell; HI, hindlimb ischemia surgery; SNx, subtotal nephrectomy. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Bone marrow–derived EPC homing and then differentiation into endothelial cells in ischemic hindlimb. (a) By immunofluorescent staining, more GFP+/CD31+ double-positive cells (yellow color) were identified in the muscles of the ischemic hindlimb of sham-operated mice than those in SNx mice, both of which received enhanced GFP transgenic mouse bone marrow cell transplantation. The numbers of GFP+/CD31+ cells in the muscle of ischemic limbs were significantly restored in SNx mice treated with AST-120. (b) Quantification of GFP+/CD31+ double-positive cells in the muscle of ischemic hindlimbs of sham-operated, SNx mice, and SNx mice treated with AST-120. (c) Plasma indoxyl sulfate was measured at 2 weeks after hindlimb ischemia. Results are means ± SEM. ∗P < 0.05 versus Sham group; #P < 0.05 versus SNx group; n = 5 in each group. BM-EC, bone marrow–derived endothelial cells; EPC, endothelial progenitor cell; GFP, green fluorescence protein; HPF, high-powered field; SNx, subtotal nephrectomy. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 Histology in ischemic muscles. (a) CD31+ capillaries and α-SMA+ small arteries by costaining for CD31+ (brown), α-SMA+ (brown), and hematoxylin (blue) in ischemic muscles of 3 study groups at 3 weeks after hindlimb ischemia surgery. Quantification of (b) CD31+ capillaries and (c) α-SMA+ in the ischemic muscles. Results are means ± SEM. ∗P < 0.05 versus Sham group; #P < 0.05 versus SNx group; n = 5 in each group. α-SMA+, α-smooth muscle actin+; SNx, subtotal nephrectomy. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 Protein expressions in ischemic hindlimbs. The muscle tissues were taken from ischemic hindlimbs 2 weeks after hindlimb ischemia surgery in 3 study groups. (a) Verification of p-eNOS, eNOS, p-STAT3, STAT3, IL-10, and VEGF proteins by Western blotting. (b–e) Quantification of p-eNOS, eNOS, IL-10, p-STAT3, STAT3, and VEGF protein expressions from ischemic hindlimbs. Results are means ± SEM. ∗P < 0.05 versus Sham group; #P < 0.05 versus SNx group; n = 5 in each group. eNOS, endothelial nitric oxide synthase; IL, interleukin; p, phospho; SNx, subtotal nephrectomy; STAT, signal transducer and activator of transcription; VEGF, vascular endothelial growth factor. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 6 Effects of indoxyl sulfate on ischemia-induced neovascularization in SNx mice. SNx mice were divided into 3 groups: vehicle group, indole group, and indole + AST-120 group. (a) Plasma indoxyl sulfate was measured at 4 weeks after hindlimb ischemia. (b) Blood perfusion in ischemic hindlimb was measured before; immediately after; and 1, 2, 3, and 4 weeks after hindlimb ischemia surgery by laser Doppler. (c) Circulating Sca-1+/Flk-1+ cells were determined by flow cytometry at baseline and 2 days after hindlimb ischemia surgery. (d) Quantification of CD31+ capillaries and α-SMA+ in the ischemic muscles. (e) Verification and quantification of p-eNOS, eNOS, p-STAT3, STAT3, IL-10, and VEGF protein expressions from ischemic hindlimbs by Western blotting. Results are means ± SEM. ∗P < 0.05 versus vehicle group; #P < 0.05 versus indole group; n = 5 in each group. α-SMA+, α-smooth muscle actin+; eNOS, endothelial nitric oxide synthase; HI, hindlimb ischemia surgery; IL, interleukin; p, phospho; SNx, subtotal nephrectomy; STAT, signal transducer and activator of transcription; VEGF, vascular endothelial growth factor. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 7 Indoxyl sulfate inhibited hypoxia-induced HIF-1α/IL-10/STAT3/VEGF pathway in human ECFCs. ECFCs were pretreated with IS for 24 hours, and then exposed to normoxia or hypoxia (1% oxygen) for 12 or 24 hours. (a) ECFCs were analyzed for protein content of HIF-1α by Western blotting. (b) The medium from ECFCs were analyzed for IL-10 by enzyme-linked immunosorbent assay. (c) ECFCs were analyzed for protein content of p-STAT3/STAT3 by Western blotting. (d) The medium from ECFCs was analyzed for VEGF by enzyme-linked immunosorbent assay. Results are means ± SEM. ∗P < 0.05 versus normoxia; ∗∗P < 0.001 versus normoxia; n = 5 in each group. CM, culture medium; ECFC, endothelial colony forming cell; GADPH, glyceraldehyde-3-phosphate dehydrogenase; H12, hypoxia for 12 hours; H24, hypoxia for 24 hours; HIF, hypoxia-inducible factor; IL, interleukin; IS, indoxyl sulfate; N, normoxia; p, phospho; STAT, signal transducer and activator of transcription; VEGF, vascular endothelial growth factor. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 8 Indoxyl sulfate inhibited migration and tube formation of human ECFCs. (a) After pretreatment with indoxyl sulfate for 24 hours, ECFCs were cultured in Boyden chamber for 24 hours under hypoxia (1% oxygen). Cells were stained with crystal violet, and the average of the total number of migrated cells was counted using computer software. (b) Tube formation assay for late ECFCs using ECMatrix gel (Chemicon, Temecula, CA). Representative photos for tube formation are shown, and the average of the total number of branch points was compared using computer software. Results are means ± SEM. ∗P < 0.05 versus normoxia; #P < 0.001 versus hypoxia alone; n = 5 in each group. ECFC, endothelial colony forming cell; HPF, high-powered field; IL, interleukin; IS, indoxyl sulfate; VEGF, vascular endothelial growth factor. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S1 Protocol illustration of the effect of enteral indole ingestion on neovascularization in SNx mice. SNx mice were divided into 3 groups: 1 treated with oil only via gavage twice a week (vehicle group), 1 treated with indole (50 mg/kg) in oil via gavage twice a week (indole group), and 1 treated with indole (50 mg/kg) in oil via gavage twice a week and 5% AST-120 mixed in pulverized chow (indole + AST-120 group). HI, unilateral hindlimb ischemia surgery; IS, indoxyl sulfate; 2/3 Nx, two-thirds nephrectomy; SNx, subtotal nephrectomy; UNx, uninephrectomy. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S2 Hypoxia-induced IL-10 and VEGF production via HIF-1α in human ECFCs. (A) Human ECFCs were characterized as adherent cells positive for Dil-AcLDL up-taken by direct fluorescent staining (red), and characterized by immunofluorescence staining (green) for the expression of eNOS, vWF, CD31, CD34, VE-cadherin, and KDR. Cells were counterstained with propidium iodide for nucleus (blue). (B) After treatment by normoxia or hypoxia (1% oxygen) for 6, 12, or 24 hours, ECFCs were analyzed for protein content of HIF-1α by Western blotting. (C and D) The medium from cultured ECFCs were analyzed for IL-10 and VEGF by enzyme-linked immunoadsorbent assay. (E) ECFCs were exposed to normoxia for 24 hours or treated with IL-10, or exposed to hypoxia (1% oxygen) alone or with cotreatment with IL-10 neutralized antibody (IL-10 Ab) and cucurbitacin I (1 μmol/l). The medium from cultured ECFCs were analyzed for VEGF by enzyme-linked immunoadsorbent assay. (F) ECFCs were pretreated with scramble or HIF-1α small, interfering RNA for 48 hours, and then exposed under normoxia or hypoxia (1% oxygen) for 12 or 24 hours. ECFCs were analyzed for protein content of HIF-1α by Western blotting. (G and H) The medium from cultured ECFCs was analyzed for IL-10 and VEGF by enzyme-linked immunoadsorbent assay. ∗P < 0.05 versus normoxia; ∗∗P < 0.001 versus normoxia; n = 5 in each group. Cur, cucurbitacin I; Dil-AcLDL, acetylated low-density lipoprotein; ECFC, endothelial colony forming cell; eNOS, endothelial nitric oxide synthase; GADPH, glyceraldehyde-3-phosphate dehydrogenase;; HIF-1α, hypoxia-inducible factor-1; HIF-1i, HIF-1α small, interfering RNA; IL, interleukin; KDR, kinase insert domain receptor; S, scramble; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S3 Cell viability of endothelial colony forming cells 24 hours after culture with various concentrations of indoxyl sulfate determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The percentage of cell viability in the indoxyl sulfatelfate determined with 3-(4,5-dimethylthiazol-2-yl)-2,5d group (cell viability = 100%). The data are expressed as the mean ± SEM from 3 independent experiments. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S4 The expressions of IL-10 and VEGF in cultured endothelial colony forming cells did not differ between CM and CM + KH2PO4 groups. CM, culture medium; H12, hypoxia for 12 hours; H24, hypoxia for 24 hours; IL, interleukin; N, normoxia; VEGF, vascular endothelial growth factor. Kidney International 2016 89, 574-585DOI: (10.1016/j.kint.2015.11.020) Copyright © 2016 International Society of Nephrology Terms and Conditions