Volume 85, Issue 2, Pages 276-288 (January 2014) Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production  Kang He, Xiaosong Chen, Conghui Han, Longmei Xu, Jianjun Zhang, Ming Zhang, Qiang Xia  Kidney International  Volume 85, Issue 2, Pages 276-288 (January 2014) DOI: 10.1038/ki.2013.342 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Lipopolysaccharide (LPS) activated hypoxia-inducible factor (HIF)-2α via nuclear factor (NF)-κB. (a) HIF-2α expression in renal medulla. HIF-2α-/- mice and their wild-type (WT) littermates were subjected to intraperitoneal LPS (3mg/kg) or normal saline (NS; 0.9%, vehicle) injection. At 24h after the injection, the right kidneys were harvested (preischemic kidney) and the left kidney was subjected to 25min of ischemia followed by 3h of reperfusion (IR) and then harvested (postischemic kidney). Nuclear HIF-2α expression was evaluated by western blot analysis and co-detection of TATA-box binding protein (TBP) was performed to assess equal loading (n=4 for each group). HIF-2α protein bands were then quantified and normalized to TBP. Data were expressed as mean±s.d. R, the right kidney (preischemic); L, the left kidney (postischemic). *P<0.05 vs. NS-treated right kidney from WT mice. (b) Effects of LPS and NF-κB inhibition on HIF-2α expression in human umbilical vein endothelial cells (HUVECs). Cells were treated with LPS (1μg/ml) or NS for 24h, followed by exposure to 90min of hypoxia (1% O2) and 1h of reoxygenation (HR). Then, total RNA was isolated and analyzed for HIF-2α expression. For NF-κB inhibition, JSH-23 was added to the medium at a final concentration of 30μmol/l, immediately after the administration of LPS. Dimethyl sulfoxide (DMSO; vehicle) treated cells served as controls. All data were normalized to β-actin and expressed as mean±s.d. from 4 to 6 samples; *P<0.05 vs. DMSO groups treated with the same procedures. (c) Nuclear extracts were isolated from HUVECs and subjected to immunoblotting analysis to evaluate HIF-2α protein level. A representative figure of four experiments is shown. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Serum creatinine and blood urea nitrogen (BUN) levels in conditional hypoxia-inducible factor (HIF)-α knockout mice and their Cre- (wild type) littermates. At 24 h after exposure to either normal saline (NS) or lipopolysaccharide (LPS; 3mg/kg, intraperitoneal), mice were subjected to right nephrectomy, followed by ischemia–reperfusion (IR; 25min of left renal ischemia and 24h of reperfusion) or non-IR sham operation. Concentrations of (a) serum creatinine and (b) BUN were measured and data were expressed as mean±s.d. from 8 to 14 animals per group. *P<0.05 vs. isogenic mice that were treated with NS/IR procedures. NS, not significant. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Effects of nitric oxide synthase (NOS) inhibition or NO elimination on lipopolysaccharide (LPS)-mediated renoprotection in wild-type and hypoxia-inducible factor (HIF)-2α-/- mice. At 24h after exposure to normal saline (NS) or LPS, mice were subjected to right nephrectomy, and then intraperitoneal administration of AG (a selective iNOS inhibitor), or intravenous injection of L-nitroarginine methyl ester (L-NAME; a nonselective NOS inhibitor) or carboxy-PTIO potassium salt (C-PTIO; an NO scavenger), followed by ischemic insult. At 24h after the initiation of reperfusion, blood was harvested and concentrations of (a) serum creatinine and (b) blood urea nitrogen (BUN) were measured and presented. Data were expressed as mean±s.d. from 6 to 8 animals per group. *P<0.05 vs. wild-type mice that were treated with NS+IR (ischemia–reperfusion) procedures. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Histological findings. (a) Representative periodic acid–Schiff (PAS)-stained renal sections from hypoxia-inducible factor (HIF)-2α-/- mice or their wild-type littermates treated with either vehicle+IR (ischemia–reperfusion) or lipopolysaccharide (LPS)+IR procedures (original magnification × 200). (b) Abnormalities based on PAS-stained sections were graded by a semiquantitative histomorphological scoring system from 0 to 4. (c) Representative renal sections immunostained for myeloperoxidase to show polymorphonuclear (PMN) leukocyte infiltration (original magnification × 100). (d) PMN infiltration was scored on a scale of 1–4. (e) Representative renal sections showing positive nuclear staining by fluorescent antibodies for DNA fragmentation in apoptotic cells (original magnification × 400). (f) A summary of the quantitative analysis of apoptotic cells per field. Data were expressed as mean±s.d. from 6 to 8 animals per group. *P<0.05 vs. wild-type mice that were treated with normal saline (NS)+IR procedures. C-PTIO, carboxy-PTIO potassium salt; L-NAME, L-nitroarginine methyl ester. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Effects of the nitric oxide (NO) donor (spermine NONOate) or erythropoietin (EPO) supplementation in hypoxia-inducible factor (HIF)-2α-/- mice. (a, b) At 24h after exposure to either normal saline (NS) or lipopolysaccharide (LPS) treatment, mice were subjected to right nephrectomy, followed by intravenous injection of spermine NONOate (10mg/kg) or phosphate-buffered saline (PBS; vehicle). After 5min, the left kidney was subjected to ischemia–reperfusion (IR) procedures. At 24h after the initiation of reperfusion, mice were killed and serum creatinine and blood urea nitrogen (BUN) levels were measured. Data were expressed as mean±s.d. from 6 to 8 animals per group. *P<0.05 vs. isogenic mice that were treated with NS+PBS+IR. (c, d) LPS/IR procedures were as described above. EPO was given subcutaneously 5min before reperfusion at a dose of 1000IU/kg. Data were expressed as mean±s.d. from four mice per group. *P<0.05 as compared with vehicle-treated wild-type mice. NS, not significant. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Postischemic renal microcirculatory blood flow recovery in wild-type and hypoxia-inducible factor (HIF)-2α-/- mice. (a, b) Baseline renal blood flow was obtained by laser doppler flowmetry monitoring in the right renal medulla before lipopolysaccharide (LPS) or normal saline (NS) treatment. The blood flow was measured again in the left kidney at different time points after the initiation of reperfusion, and the value vs. the baseline result was defined as postischemic vs. preischemic renal perfusion. Some mice were treated with L-nitroarginine methyl ester (L-NAME) or carboxy-PTIO potassium salt (C-PTIO) before ischemia. The data described at different time points were based on the results from separate groups of mice. Data were expressed as mean±s.d. from 4 to 6 mice at each time point per group. *P<0.05 for LPS+IR (ischemia–reperfusion) group vs. all the other groups of wild-type mice at the same time point. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Postischemic vs. preischemic nitric oxide (NO) production in renal medulla. (a) Detection of active NO production in vivo was achieved by electrochemical measurement. (b) NO level was also estimated indirectly with an NO colorimetric assay kit, which determined total concentrations of nitrite plus nitrate present in the renal medulla. Baseline NO release was obtained from the right kidney before the ischemic insult by both methods. At the indicated time points after the initiation of reperfusion, the left kidney was subjected to the measurements to obtain postischemic values. Relative NO production and relative nitrite/nitrate levels were defined as the ratio of postischemic value vs. the baseline. Data were expressed as mean±s.d. from 6–8 animals per group. *P<0.05 for wild-type lipopolysaccharide+ischemia–reperfusion (WT LPS+IR) group vs. all the other groups at the same time point. #P<0.05 for hypoxia-inducible factor (HIF)-2α-/- LPS+IR group vs. all the other groups at the same time point. NS, normal saline. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Expression and activity of nitric oxide synthase (NOS)/arginase in renal medulla. At 24h after the lipopolysaccharide (LPS) or normal saline (NS) treatment, the right kidneys were harvested (preischemic kidney) and the left kidney was subjected to 25min of ischemia followed by (a) 1h or (b) 3h of reperfusion and then harvested (postischemic kidney). The expression levels of inducible NOS (iNOS), endothelial NOS (eNOS), neuronal NOS (nNOS), arginase1, and arginase2 in the renal medulla were determined by real-time reverse transcriptase–PCR and were normalized to β-actin. For every mouse, the value of the left kidney vs. the right one was defined as postischemic vs. preischemic ratio, which reflected the inducibility of the genes in renal medulla by the ischemia–reperfusion (IR) insult. (c) eNOS and iNOS protein levels in the 3-h group were evaluated by western blot analysis and the value of the left kidney vs. the right kidney was defined as postischemic vs. preischemic ratio. (d) Activities of iNOS and constitutive NOS (cNOS) in the 3-h group were evaluated using a NOS detection kit. All data were expressed as mean±s.d. from 6 to 8 animals per group; *P<0.05 vs. isogenic mice that were treated with NS/IR. R, the right kidney (preischemic); L, the left kidney (postischemic). HIF, hypoxia-inducible factor; NS, not significant; WT, wild type. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 Efficacy of RNAi targeting hypoxia-inducible factor (HIF)-2α in human umbilical vein endothelial cells (HUVECs). (a) HUVECs were transfected with an siRNA duplex targeting HIF-2α at a final concentration of 50nmol/l by using Lipofectamine 2000. A scrambled siRNA duplex was used as control. After 28h, mRNA levels of HIF-2α were measured by quantitative realtime reverse transcriptase–PCR. (b) At 28h after siRNA transfection, HUVECs were subjected to 90min of hypoxia (1% O2), followed by 1h of reoxygenation (HR). Nuclear HIF-2α was detected by immunoblotting. Cells without HR or siRNA treatment were used as controls. Results were expressed as mean±s.d. from six experiments; *P<0.05 vs. scrambled siRNA group, #P<0.05 vs. the other three groups. TBP, TATA-box binding protein. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 10 Effect of hypoxia-inducible factor (HIF)-2α knockdown (KD) on nitric oxide synthase (NOS) and arginase expression levels in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with an siRNA duplex targeting HIF-2α (HIF-2α KD) or a scrambled siRNA duplex (HIF-2α wild type (WT)) at a final concentration of 50nmol/l for 4h, followed by lipopolysaccharide (LPS; 1μg/ml) or normal saline (NS) administration. After 24h, cells were exposed to 90min of hypoxia, followed by 1, 3, or 6h of reoxygenation (HR). Then, cells were collected and subjected to analysis for mRNA levels of (a) HIF-2α, (b) neuronal NOS (nNOS), (c) inducible NOS (iNOS), (d) endothelial NOS (eNOS), (e) arginase 1, and (f) arginase 2. All data were normalized to β-actin expression and expressed as mean±s.d. from six experiments; *P<0.05 vs. HIF-2α WT cells treated with the same procedures. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 11 Contribution of hypoxia-inducible factor (HIF)-2α in inflammatory cells or hepatocytes to the renoprotective effects of lipopolysaccharide (LPS). (a, b) Bone marrow transplantation (wild-type→wild-type and HIF-2α-/-→wild-type) was performed 30 days before renal ischemia–reperfusion (IR) procedures. At 24h after the initiation of reperfusion, blood was harvested and concentrations of serum creatinine and blood urea nitrogen (BUN) were measured and presented. Data were expressed as mean±s.d. from 6 to 8 animals per group. (c, d) Serum creatinine and BUN levels in mice with hepatocyte-specific HIF-2α knockout and their Cre- littermates (wild type) were measured, and data were expressed as mean±s.d. from 8–10 animals per group. *P<0.05 vs. isogenic mice that were treated with normal saline (NS)/IR procedures. Alb, albumin; NS, not significant. Kidney International 2014 85, 276-288DOI: (10.1038/ki.2013.342) Copyright © 2014 International Society of Nephrology Terms and Conditions