Volume 86, Issue 3, Pages 525-537 (September 2014) Carbon monoxide potently prevents ischemia-induced high-mobility group box 1 translocation and release and protects against lethal renal ischemia–reperfusion injury  Yongle Ruan, Lu Wang, Yue Zhao, Ying Yao, Song Chen, Junhua Li, Hui Guo, Changsheng Ming, Shi Chen, Feili Gong, Gang Chen  Kidney International  Volume 86, Issue 3, Pages 525-537 (September 2014) DOI: 10.1038/ki.2014.80 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Establishment of a lethal renal ischemia–reperfusion injury (IRI) model in mice. (a) Survival of mice that had undergone 40 or 50min of renal ischemia and were observed for 14 days. (b) Serum creatinine (Cr) levels of mice that had undergone 40min of renal warm ischemia, on days 1, 3, or 7 after reperfusion. (c) Serum Cr levels of mice that had undergone 50min of renal ischemia at 24h after reperfusion were similar to those of mice at 24h after bilateral nephrectomy (P>0.05). NS, nonsignificant. Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 CO–releasing molecule-2 (CORM-2) pretreatment almost completely protects against lethal renal ischemia–reperfusion injury (IRI). Following 50min of renal warm ischemia and reperfusion, the survival (a) of CORM-2–, phosphate-buffered saline (PBS)– , and inactive CORM-2 (iCORM-2)–treated mice was observed for 14 days (**P<0.001 vs. the iCORM-2– and PBS–treated groups; n=8 per group). (b) The serum creatinine (Cr) and (c) blood urea nitrogen (BUN) levels of sham-operated, PBS–, and iCORM-2–treated mice on day 1 after reperfusion and of CORM-2–treated mice on days 1, 3, and 7 after reperfusion were measured to assess the degree of renal dysfunction (**P<0.0001 vs. the iCORM-2– and PBS–treated groups; n=6–8 per group). The serum Cr and BUN levels in CORM-2–treated mice remained normal when measured at days 1, 3, and 7 after ischemia/reperfusion (I/R) (P>0.05 vs. the sham-operated group). Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 CO–releasing molecule-2 (CORM-2) pretreatment significantly attenuates lethal ischemia–reperfusion injury (IRI)–induced pathological damage. Mouse kidneys were subjected to 50min of ischemia, followed by 24h of reperfusion. The kidneys were harvested from the unclamped mice (sham) or mice clamped and pretreated with phosphate-buffered saline (PBS), inactive CORM-2 (iCORM-2), or CORM-2. (a) Hematoxylin and eosin (H&E) staining was performed to evaluate renal tubular damage (bar=60μm; original magnification, × 400); immunofluorescence staining was performed to detect the expression of CD31 in renal glomeruli and peritubular capillaries (bar=74μm; original magnification, × 200; green, CD31); myeloperoxidase (MPO) assays were performed to evaluate renal neutrophil infiltration; and in situ terminal deoxynucleotidyl transferase–mediated uridine triphosphate nick-end labeling (TUNEL) assays were performed to assess renal cell apoptosis (bar=60μm; magnification, × 400). Images are representative kidney sections from eight mice per group. (b) Quantitative assessment of tubular damage (n=8 per group), CD31 expression (n=8 per group), MPO–positive cells (n=8 per group), and apoptotic cells (n=8 per group), performed as described in Materials and Methods (**P<0.01 vs. the PBS– and iCORM-2–treated groups). The data shown are the means±s.d. HPF, high-power field. Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 CO–releasing molecule-2 (CORM-2) pretreatment potently inhibits high-mobility group box 1 (HMGB1) nucleocytoplasmic shuttling during renal ischemia. Mouse kidneys were subjected to 50min of ischemia without reperfusion. (a) Immunohistochemical staining of HMGB1 from sections of kidneys harvested from the (A) unclamped sham-operated mice or (B) mice clamped and pretreated with phosphate-buffered saline (PBS), (C) inactive CORM-2 (iCORM-2), or (D) CORM-2 (bar=20μm; original magnification, × 1000). Images are representative renal sections from eight mice per group. (b, c) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from sham-operated kidneys (24h after sham surgery) or the ischemic kidneys from PBS–, iCORM-2–, or CORM-2–pretreated mice (n=3, randomly selected from eight animals per group), with each lane representing a (b) separate animal. (c) The corresponding densitometric analyses are shown as bar graphs. The mean±s.d. is reported (**P<0.01 vs. the iCORM-2 and PBS control groups). (d) Western blot analysis for heme oxygenase-1 (HO-1) was performed using total protein samples from kidneys collected before (0) or immediately after ischemia (50min) in the CORM-2–treated group, with each lane representing a separate animal. The unclamped sham-operated kidneys were used as negative controls. The unclamped kidneys from mice that had been treated with Co(III)Protoporphyrin IX chloride (CoPP) for 24h served as positive controls. The blots shown are representative of three experiments with similar results. Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 CO–releasing molecule-2 (CORM-2) pretreatment decreases nuclear histone acetyltransferase (HAT) activity and increases the ratio of nuclear histone deacetylase (HDAC)/HAT activity during renal ischemia. (a, b) Mouse kidneys were subjected to 50min of ischemia without reperfusion. Colorimetric assays were performed to assess nuclear (a) HDAC and (b) HAT activity in renal tissues from the unclamped sham-operated mice or mice clamped and pretreated with phosphate-buffered saline (PBS), iCORM-2, or CORM-2 (*P<0.05, **P<0.01, vs. the sham-operated group; ##P<0.01, vs. the sham-operated, iCORM-2, and PBS control groups; n=7 per group). (c) The ratio of nuclear HDAC/HAT activity was significantly increased in the CORM-2–treated group (**P<0.01, vs. the sham-operated, iCORM-2, and PBS control groups). Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 CO–releasing molecule-2 (CORM-2) pretreatment inhibits the nuclear-cytoplasmic translocation of high-mobility group box 1 (HMGB1) after renal ischemia/reperfusion (I/R). Mice were treated with either inactive CORM-2 (iCORM-2) or CORM-2 1h before renal I/R. (a) Immunohistochemical staining of HMGB1 from sections of kidneys subjected to 50min of ischemia and 0, 1, or 3h of reperfusion (bar=60μm; original magnification, × 1000). Images are representative renal sections from six mice per group. (b, c) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from kidneys subjected to 50min of ischemia and 0, 1, or 3h of reperfusion, with each lane representing a separate animal. (b) The blots shown are representative of three experiments with similar results. (c) The corresponding densitometric analyses are shown as bar graphs. The mean±s.d. is reported (**P<0.01, n=6 per group). OD, optical density. Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 CO–releasing molecule-2 (CORM-2) pretreatment inhibits high-mobility group box 1 (HMGB1) release into the circulation after renal ischemia/reperfusion (I/R). Mice were treated with either inactive CORM-2 (iCORM-2) or CORM-2 1h before renal I/R. A time course of plasma HMGB1 levels was established by enzyme-linked immunosorbent assay (ELISA) in mice after 1, 3, 6, 12, and 24h of renal reperfusion. The data shown are means±s.d. (*P<0.05, **P<0.01; n=5 per group). Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 CO–releasing molecule-2 (CORM-2) pretreatment inhibits mRNA upregulation of high-mobility group box 1 (HMGB1) receptors and downstream proinflammatory cytokines and chemokines after renal ischemia/reperfusion (I/R). Mouse kidneys were subjected to 50min of ischemia and 24h of reperfusion. Real-time PCR was performed to measure mRNA expression of (a) Toll-like receptor 4 (TLR4), (b) receptor for advanced glycation end products (RAGE), (c) tumor necrosis factor-α (TNF-α), (d) interleukin-1β (IL-1β), (e) IL-6, and (f) monocyte chemoattractant protein-1 (MCP1) in the kidneys from unclamped sham-operated mice or mice clamped and pretreated with phosphate-buffered saline (PBS), inactive CORM-2 (iCORM-2), or CORM-2 (*P<0.05, **P<0.01 vs. the CORM-2–treated group; n=6–8 per group). Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 CO–releasing molecule-2 (CORM-2) pretreatment inhibits the acetylation and release of high-mobility group box 1 (HMGB1) during in vitro hypoxic culture of tubular epithelial cells (TECs). In vitro primary cultured renal TECs were pretreated with medium, inactive CORM-2 (iCORM-2), or CORM-2 1h before hypoxia. (a) Immunofluorescence staining of HMGB1 was performed on TECs subjected to hypoxia (1% O2) for 4h. TECs cultured under normal conditions served as normal controls. Confocal images shown are representative of three experiments with similar results (original magnification, × 600 and × 1000; red, HMGB1; green, F-actin; blue, nuclei). (b, c) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from TECs subjected to hypoxia for 4h. (b) Representative western blot of three experiments. (c) Quantification by densitometry (*P<0.05, **P<0.01 vs. the CORM-2–treated group; n=3 per group). (d, e) Coimmunoprecipitation analysis was performed using whole-cell lysates of TECs exposed to hypoxia for 4h. Lysates were immunoprecipitated with an anti-acetyl-lysine antibody and immunoblotted for HMGB1. (d) The blot shown is representative of three experiments with similar results. (e) The corresponding densitometric analyses are shown as bar graphs. Data shown are means±s.d. (**P<0.01 vs. the CORM-2–treated group; n=3 per group). OD, optical density. Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 10 Administration of recombinant high-mobility group box 1 (rHMGB1) largely eliminates the protective effect of CO–releasing molecule-2 (CORM-2) on kidney ischemia–reperfusion injury (IRI). Mice were treated with either inactive CORM-2 (iCORM-2) or CORM-2 (20mg/kg) for 1h before renal ischemia/reperfusion (I/R). rHMGB1 (20μg per mouse) was given intraperitoneally to some CORM-2–pretreated mice immediately after renal reperfusion (CORM-2+rHMGB1–treated group). Following 50min of renal warm ischemia and 24h of reperfusion, the (a) serum creatinine (Cr) and (b) blood urea nitrogen (BUN) levels of sham-operated, iCORM-2–, CORM-2–, and CORM-2+rHMGB1–treated mice were measured to assess the degree of renal dysfunction (**P<0.0001, n=6 per group). Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 11 Neutralizing antibody against high-mobility group box 1 (HMGB1) protects against renal ischemia–reperfusion injury (IRI). Following 50min of renal warm ischemia and 24h of reperfusion, the (a) serum creatinine (Cr) and (b) blood urea nitrogen (BUN) levels of sham-operated, control immunoglobulin G (IgG)–, and anti-HMGB1–neutralizing antibody (α-HMGB1)–treated mice were measured to assess the degree of renal dysfunction (**P<0.0001, n=6 per group). Kidney International 2014 86, 525-537DOI: (10.1038/ki.2014.80) Copyright © 2014 International Society of Nephrology Terms and Conditions