The Protective Effects Of Carbon Monoxide On Renal Ischemia-reperfusion Injury And Its Novel Mechanisms

Part Ⅰ Carbon monoxide-releasing molecule CORM-2protects against renal ischemia-reperfusion injury in mice[Objective] To investigate if the administration of CORM-2can provide protection against reversible renal injury from ischemia-reperfusion.[Methods] Murine renal ischemia was induced by clamping left renal pedicles for40min with vascular micro clamps at32℃, then the contralateral kidney was removed. CORM-2(20mg/kg) or vehicle was administered via intravenous infusion1hour before the onset of ischemia. The blood plasma and renal samples were obtained at24hours after reperfusion to assess renal function and cellular injury. Plasma CREA and UREA levels, HE was performed to estimate the magnitude of renal damage. Kidneys were retrieved from indicated animals at various time points after renal IRI, and the sections were prepared for histological evaluation. MPO immunochemistry, CD3, CD4and CD8fluorescent staining procedures were performed to assess the neutrophils, lymphocytes infiltration in the IRI renal. Besides, Immunofluorescent stain of TNF-α was performed on the kidneys were retrieved from indicated animals to determine the production of inflammatory mediators in renal I/R.[Results]The plasma CREA and UREA rise to high levels after24hours after reperfusion in control mice, CORM-2treatment markedly limited the increase of plasma CREA and UREA in mice subjected to I/R. In parallel, histological analysis demonstrate that CORM-2treatment markedly reduced necrosis of the renal tubular epithelium cells and haemorrhage. IRI caused marked infiltration and accumulation of the MPO-positive neutrophils in renal interstitium. Neutrophils infiltration peaked on day3and reduced on day7in renal IRI. Administration of CORM-2before ischemia dramatically inhibited neutrophils infiltration compared with IRI control or iCORM-2group (P<0.05). The interstitial infiltration of lymphocytes usually appear lately after I/R. CD3-positive T cells infiltration increased on day7after reperfusion, and were mainly CD8-positive cells but CD4-positive cells. CORM-2treatment decreases the infiltration of lymphocytes significantly. Furthermore, we confirmed that CORM-2markedly decreased production of TNF-a using immunofluorescent stain.[Conclusion] Carbon monoxide-releasing molecule CORM-2could ameliorate inflammation to protect against the renal IRI in mice. Part Ⅱ Carbon monoxide-releasing molecule CORM-2protects against lethal renal ischemia-reperfusion injury in mice[Objective] To investigate if the administration of CORM-2can provide protection against lethal renal injury from ischemia-reperfusion inmice.[Methods] Murine renal ischemia was induced by clamping left renal pedicles for50min at32℃, then the contralateral kidney was removed. CORM-2(20mg/kg) or vehicle was administered via intravenous infusion1hour before the onset of ischemia. The blood plasma and renal samples were obtained at24hours after reperfusion to assess renal function and cellular injury. Plasma CREA and UREA levels, HE, TUNEL, CD31immunofluorescence were performed to estimate the magnitude of renal damage.MPO immunochemistry was performed to assess the neutrophils infiltration in the IRI renal. Besides, we also examined the mRNA levels of expression of tumor necrosis factors-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6(IL-6) by real-time PCR. Immunofluorescent stain of TNF-α was performed on the kidneys were retrieved from indicated animals to determine the production of inflammatory mediators in renal I/R. [Results] Murine renal undergo50minutes warm ischemia treated without or with iCORM-2result in irreversible renal damage as nearly all mice dead within3days of reperfusio.n. In contrast, the survival time of the mice pretreated with CORM-2was significantly prolonged (>14days). In parallel, histological analysis demonstrated that CORM-2treatment markedly reduced necrosis of the renal tubular epithelium cells (TUNEL) and capillary endothelial cells (CD31). IRI caused marked infiltration and accumulation of the MPO-positive neutrophils in renal interstitium. Administration of CORM-21hour before ischemia dramatically inhibited neutrophils infiltration compared with IRI control or iCORM-2group (P<0.05). In addition, CORM-2also suppressed proinflammatory gene TNF-α, IL-1β and IL-6expression in I/R. Furthermore, we confirmed that CORM-2markedly decreased production of TNF-α using immunofluorescent stain.[Conclusion] Our study demonstrated that CORM-2had a significant protection on ischemia-reperfusion induced lethal acute renal failure (ARF) through meliorate inflammation in mice Part Ⅲ Carbon monoxide-releasing molecule CORM-2protects against lethal renal ischemia-reperfusion injury in mice by decrease HMGB1nucleocytoplasmic shuttling[Objective] To examine the relationship betweenthe the protective effect of CORM-2and translocation of HMGB1in murine lethal renal ischemia-reperfusion injury.[Methods] In the lethal renal ischemia-reperfusion injury of mice, immunohistochemistry and western blot were performed to evaluate the translocation of HMGB1at0hour,1hour and3hour reperfusion after50minutes renal ischemia. CORM-2was administered1h before ischemia. In addition, primary mice renal proximal tubular epithelial cells (MRPTEpiC) were subjected to hypoxia (1%O2) for4hours or cultured in epithelial cell medium containing500μM H2O24h in vitro, and CORM-2(100p.mol/L) was administered1h before. We use the immunofluorescence to examine the HMGB1nucleocytoplasmic shuttling in the TEpiC. Furthmore, post-translational modifications of HMGB1was detected by using co-immunoprecipitation (IP).[Results] We found that the amount of HMGB1released from the renal epithelium nucleic into cytoplasma was significantly increased at0h,1h and3h reperfusion after renal ischemia, peaking at0h reperfusion. In contrast, CORM-2pretreated significant decreased the HMGB1level in renal tubular epitheliums cytoplasm. Immunofluorescent stain of HMGB1from TEpiC showed that amount of HMGB1translocated from nucleus to cytoplasm suffering hypoxia or oxidative stress (H2O2), whereas, pretreatment of CORM-2significantly inhibited HMGB1nuclecytoplasmic shuttling. Furthermore, co-IP analysis demonstrated hypoxia and oxidative stress induced hyperacetylation of HMGB1, and then released from nucleus, the treatment of CORM-2can inhibits this pathway.[Conclusion] CORM-2inhibits renal epithelium releasing HMGB1, an early mediator of inflammation, to attenuate the renal I/R injury in mice. CORM-2might be possible to block the acetylation process itself to block harmful secretion of HMGB1. Part Ⅳ The relationship between the protective effect of CORM-2and the expression of HO-1[Objective] To investigate the relationship between the protection of CORM-2and the overexpression of HO-1.[Methods] To independently examine the effects of CORM-2on the expression of HO-1, mice received CORM-2at a final dose20mg/kg of body weight intravenously without underwent surgical procedure, the expression of HO-1was evaluated in kidney after1hour and24hours of CORM-2, iCORM-2or CoPP administration. Immunohistochemistry and western blot analysis were performed to evaluate the level of HO-1in kidney. Mice renal ischemia was induced by clamping left renal pedicles for50min warm ischemia and CORM-2/iCORM-2/CoPP was administered via intravenous infusion1hour or24hours before the onset of ischemia. Plasma CREA and UREA levels, HE, TUNEL, and CD31immunofluorescence were performed to estimate the magnitude of renal damage. MPO immunochemistry, TNF-a immunofluorescent stain were performed to to determine the inflammatory reaction in renal I/R.[Results] Interesting, HO-1expression was markedly induced in the CORM-2, iCORM-2or CoPP infusion kidney after24h without I/R, and HO-1protein was localized predominantly in renal tubular epithelium. Whereas, overexpression of HO-1in CORM-2-24h, iCORM-2-24h or CoPP groups mice kidney didn’t show obvious protection against renal damage in ischemia induced acute renal failure compared with CORM-1-1h group (P <0.05). Whereas, CORM-21hour pretreatment markedly reduced apoptosis of the renal tubular epithelium cells (TUNEL) and capillary endothelial cells (CD31), ameliorate interstitial infiltration of inflammatory cell (MPO) and production of proinflammatory mediator(TNF-a).[Conclusion] Our results suggested that the mainly protective effects of CORM-2were not through the HO-1overexpression in mice renal IRI.