Volume 91, Issue 3, Pages 616-627 (March 2017) Hypoxia inducible factor stabilization improves defective ischemia-induced angiogenesis in a rodent model of chronic kidney disease  Isabel N. Schellinger, Nada Cordasic, Julian Panesar, Björn Buchholz, Johannes Jacobi, Andrea Hartner, Bernd Klanke, Joanna Jakubiczka-Smorag, Nicolai Burzlaff, Eva Heinze, Christina Warnecke, Uwe Raaz, Carsten Willam, Philip S. Tsao, Kai-Uwe Eckardt, Kerstin Amann, Karl F. Hilgers  Kidney International  Volume 91, Issue 3, Pages 616-627 (March 2017) DOI: 10.1016/j.kint.2016.09.028 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 Immunohistochemical analysis of skeletal muscle capillary density in sham-operated (SHAM) controls and in subtotally nephrectomized (SNX) rats following limb ischemia. (a) Frozen sections were obtained from gastrocnemius muscles and stained for Laminin (green) and CD31 (red). (b) Capillary density in nonischemic and ischemic limbs of sham-operated (SHAM) and subtotally nephrectomized (SNX) rats. Capillary density was measured as CD31-positive percentage/Laminin-positive percentage 14 days after induction of ischemia. Values are means ± SEM. N = 18 for SHAM and n = 17 for SNX. One-way Anova with Bonferroni correction *P < 0.05 versus all other groups. Bar = 100 μm. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 Immunohistochemistry for HIF-1a in skeletal muscles 24 hours after induction of ischemia. (a) Representative images of sham-operated (SHAM) and subtotally nephrectomized (SNX) animals. Frozen sections were obtained from gastrocnemius muscles and stained for HIF-1a. Bar = 100 μm. (b) Analysis of HIF-1a immunohistochemistry in nonischemic and ischemic limbs of SHAM and SNX rats. Values are means ± SEM. N = 5 for SHAM and SNX. Unpaired t-test. ∗Significant (P < 0.05) differences versus nonischemic limb of the same group. n.s., no significant difference. Bar = 100μm. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Influence of different stimuli on HIF-1α and HIF-2α expression in endothelial cells after 6 hours (a) or 24 hours (b) of incubation. Protein expression was measured via Western-Blot in SK-HEP1 cells. Ctrl = control; Hypoxia (Hyp) = 1% O2; 2,2-Dipyridyl (DP, as positive control) and ICA at a concentration of 100 μM. β-Actin served as a loading control. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 Expression of Vegf (a), Vegf-r1 (b), Vegf-r2 (c), and Ho-1 (d) in ischemic skeletal muscles. mRNA expression of HIF target genes was measured by qRT-PCR 24 h after induction of ischemia. Sham-operated group (SHAM), subtotally nephrectomized group (SNX), carbon monoxide–treated group (CO), and pharmaceutically treated group (ICA). Values are means ± SEM. n = 7. One-way ANOVA. ∗Significant (P < 0.05) differences versus SHAM. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 Capillary density in skeletal muscles of SHAM or subtotally nephrectomized rats (SNX) without any treatment and after pre-ischemic treatment with carbon monoxide (CO) or the PHD-inhibitor ICA (ICA). Capillary density was measured as CD31-positive percentage/Laminin-positive percentage 14 days after induction of ischemia. Values are means ± SEM. n = 18 for SHAM; n = 17 for SNX; n = 6 for CO; n = 7 for ICA-pre; *significant (P < 0.05) differences between ischemic and the respective nonischemic limb; #significant (P < 0.05) difference between ischemic limbs of SNX versus all other ischemic limb groups. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 6 Expression of Vegf (a), Vegf-r1 (b), Vegf-r2 (c), and Ho-1 (d) in ischemic skeletal muscles treated with ICA after onset of ischemia. mRNA expression of HIF target genes was measured by qRT-PCR 24 h after induction of ischemia. Subtotally nephrectomized group (SNX) and pharmaceutically treated group (ICA). Values are means ± SEM. n = 7. Unpaired t-test. *P < 0.05. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 7 Capillary density of skeletal muscles after post-ischemic treatment with the pharmaceutical PHD inhibitor ICA (ICA-post). Capillary density was measured as CD31-positive percentage/Laminin-positive percentage in sham-operated animals (SHAM), subtotally nephrectomized animals (SNX), subtotally nephrectomized animals treated with the pharmaceutical ICA 2 h and 6 h after induction of hind-limb ischemia, as well as sham-operated animals treated with the pharmaceutical ICA 2 h and 6 h after induction of hind-limb ischemia (SHAM-ICA). Values are means ± SEM. n = 18 for SHAM; n = 17 for SNX; n = 7 for SHAM-ICA-post; n = 7 for SNX-ICA-post. *Significant (P < 0.05) differences between ischemic and the respective nonischemic limb. #Significant (P < 0.05) difference between ischemic limbs of SNX versus all other ischemic limb groups. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 8 Immunohistochemistry for HIF-1a in skeletal muscles 24 h after induction of ischemia. Frozen sections were obtained from gastrocnemius muscles of sham-operated (SHAM), subtotally nephrectomized (SNX) rats and subtotally nephrectomized animals treated with the pharmaceutical ICA 2 h and 6 h after induction of hind-limb ischemia (SNX+ICA post) and stained for HIF-1a. Analysis of HIF-1a immunohistochemistry in nonischemic and ischemic limbs of SHAM, SNX, and SNX+ICA post rats. Values are means ± SEM. n = 5 for SHAM and SNX, and n = 4 for SNX+ICA-post. *Significant (P < 0.05) differences versus nonischemic limb of the same group. n.s, no significant difference. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 9 Tube formation assay using the HUVEC cell line. (a) Representative images of tube forming HUVECs with or without PHD inhibition and with control antibody or VEGF-neutralizing antibody. Bar = 50 μm. (b) Analysis of tube formation. Tubule branches were counted and analyzed using ImageJ and are presented as percentage from ICA untreated conditions *P < 0.05 indicating significant differences between ICA-treated and untreated cells. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 10 Expression of chemokines and cytokines in skeletal muscles. mRNA expression inflammatory genes was measured by qRT-PCR 24 h after induction of ischemia. Sham-operated group (SHAM), subtotally nephrectomized group (SNX), and pharmaceutically treated group (ICA). Values are means ± SEM. n = 6. Panels (a–d) indicate expression changes relative to mean levels in nonischemic skeletal muscle for Mcp-1 (a), Cxcr4 (b), Il6 (c), and Il1B (d). *Significant (P < 0.05) differences versus the respective nonischemic limb. Panel (e) indicates expression changes relative to the expression level of ischemic muscle in untreated SNX animals. #Significant differences versus ischemic-limb of SNX animals. Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S1 Flow diagram of the experimental protocol. CKD was induced in 2 steps. First, 1 kidney was removed (uninephrectomy), and two-thirds of the remaining kidney were removed (5/6 nephrectomy) 1 week later. After 8 weeks, animals were either treated with CO or ICA before induction of hind-limb ischemia (CO or ICA-pre group) or after induction hind-limb ischemia (ICA-post group). Kidney International 2017 91, 616-627DOI: (10.1016/j.kint.2016.09.028) Copyright © 2016 International Society of Nephrology Terms and Conditions