| BACKGROUND Renal ischemia/reperfusion injury(IRI)is a injury process of renal tissue caused by the restoration of blood flow(reperfusion)after the compromised blood flow(ischemia)under the influence of surgery and other factors.Renal IRI is one of the important cause of perioperative acute renal injury,and is also one of the more common pathological events in renal transplantation and one of the main harmful factors that influence the kidney transplant.The mechanism involved in the renal IRI is very complicated,and the causes and molecular pathological mechanism are not fully clear.It is known that the attack of excessive reactive oxygen free radicals to capillaries and tissue cells after reperfusion is the direct cause of tissue IRI.At the same time,the ischemia in tissues can cause apoptosis and necrosis of the renal tubular cells and endothelial cells,while the reperfusion can cause the secretion of chemokines and cytokines from renal proximal tubule cells and dendritic cells,macrophages,neutrophils,lymphocytes,and other leukocytes to promote the inflammation and aggravate the injury of renal tissues. Stromal cell derived factor 1(SDF-1)is one of the members of the family of CXC chemokines,also known as CXCL12,and an important participator of inflammation.SDF-1 can combine with CXCR4 to form the SDF-1/CXCR4 axis,which is the crucial regulator of bone marrow cells homing and stem cells migration and differentiation,and participate in the regulatory processes of normal development of central nervous system,immune system and others.Besides,the roles of SDF-1/CXCR4 axis have been reported in various pathological conditions,including tumorigenesis and growth,tumor angiogenesis,inflammation,hypoxic ischemic brain injury,myocardial ischemia-reperfusion injury,bone repairing,liver stem cell transplant,and other important pathological events.However,whether SDF-1/CXCR4 play roles in renal IRI is not yet clear.In conclusion,as an important regulator of bone marrow mesenchymal stem cells transport and inflammation,SDF-1/CXCR4 axis plays important roles in the injury and repair of tissues and other pathological processes.Then,is there abnormal expression of SDF-1/CXCR4 in renal IRI? Dose it has roles in the progression of renal IRI? Whether is the molecular mechanism associated with the change of renal microvascular endothelial cells activity? Answers to these questions is of great significance to further demonstrate the molecular pathology of renal IRI.Based on these issues,we built the cell model of hypoxia/reoxygenation injury in renal microvascular endothelial cells and animal model of renal ischemia reperfusion injury in rats,to investigate the expression and function of SDF-1/CXCR4 in renal IRI in vivo and in vitro.OBJECTIVES To analyze the expression of SDF-1 and CXCR4 after ischemia reperfusion injury in vivo and in vitro,based on the cell model of hypoxia/reoxygenation injury in renal microvascular endothelial cellsand animal model of renal ischemia reperfusion injury in rats.And elucidate the effects of changes in SDF-1 and CXCR4 expressionon theinjury induced by ischemia reperfusion in renal tissues of rats,and whether SDF-1/CXCR4 play roles in the apoptosis and immunocompetence of renal microvascular endothelial cells after hypoxia/reoxygenation injury.Based upon above,to analyze the relationships and mechanisms between changes in SDF-1 and CXCR4 expression as well as renal ischemia reperfusion injury,to provide new evidences for understanding of pathological mechanism of renal ischemia reperfusion injury and searching strategies for prevention and treatment.METHODS1.Establishing the cell model of hypoxia/reoxygenation injury in renal microvascular endothelial cells and animal model of renal ischemia reperfusion injury in rats The cell model was established bytreatingprimary renal microvascular endothelial cells with hypoxia/reoxygenation.Establishing the animal model of renal ischemia reperfusion injury in rats by transabdominal blocking bilateral renal artery using no damaged vessel clamp.And the serum urea nitrogen,serum creatinine value and NGAL concentration were detected in kidney tissues of rat models.HE staining was used to measure the pathological changes of renal tissues,and the renal tubular necrosis score was performed.2.Detecting the expression of SDF-1 and CXCR4 in animal model and cell model of injury q RT-PCR and ELISA assay were performed to detect the expression of SDF-1 and CXCR4 inthe animal model of renal ischemia reperfusion injury in rats and cell model of hypoxia/reoxygenation injury in renal microvascular endothelial cells.3.Analyzing the association of SDF-1 expression and renal tubular necrosis scorein rat model The renal tubular necrosis scorewas performedin the rat model of renal ischemia reperfusion injury.And the correlation between changes of SDF-1 expressionand renal tubular necrosis scorewas analyzed.4.Analyzing theeffect of SDF-1/CXCR4 on the apoptosis of renal microvascular endothelial cells after hypoxia/reoxygenation injury Renal microvascular endothelial cells of hypoxia/reoxygenation injury were treated by recombinant SDF-1(0?25?50?100 ?g/L)or CXCR4 inhibitor AMD3100(0?5?10?20 ?M)for 24 h.Then the Caspase-3 levels were detected by Western Blot,and the apoptosis was anylyzed using flow cytometry.5.Analyzing theeffect of SDF-1/CXCR4 on theimmunocompetenceof renal microvascular endothelial cells after hypoxia/reoxygenation injury The model cells were treated with SDF-1or CXCR4 inhibitor AMD3100,and co-cultured with lymphocytes from peripheral blood.The proliferative activity of lymphocytes were analyzed after different treatment,and the expression of ICAM-1 and IL-2 were measured by ELISA assay.6.Analyzing theprotective effect of SDF-1/CXCR4 on the renal ischemia reperfusion injury in rat models The rat models of renal ischemia reperfusion injury were treated with SDF-1or CXCR4 inhibitor AMD3100 by tail intravenous injection,then the injury was analyzed in renal tissuesof rat models after different treatment.RESULTS1.The cell model of hypoxia/reoxygenation injury in renal microvascular endothelial cells andanimal model of renal ischemia reperfusion injury in rats were established The cell model was established bytreatingprimary renal microvascular endothelial cells with hypoxia/reoxygenation.The ratsmodel of renal ischemia reperfusion injurywere establishedby transabdominal blocking bilateral renal artery using no damaged vessel clamp.And the serum urea nitrogen,serum creatinine value and NGAL concentration were increased in kidney tissues of rat models.Thepathological changes of renal tissues were aggravated.2.SDF-1 and CXCR4 were abnormally expressedin animal model and cell model of injury The expression of SDF-1 and CXCR4 were significantly up-regulated inthe rats model of renal ischemia reperfusion injury and renal microvascular endothelial cells model of hypoxia/reoxygenation injury.And the expression of SDF-1 and CXCR4 were increased gradually with the time of surger.3.Changes of SDF-1 expression were positive association with renal tubular necrosis scorein rat model The renal tubular necrosis scorewas enhanced in the rat model of renal ischemia reperfusion injury.And the changes of SDF-1 expression were positive correlated with renal tubular necrosis scorein rat model4.The inhibitory role of SDF-1/CXCR4 in the apoptosis of renal microvascular endothelial cells after hypoxia/reoxygenation injury In vitro,the apoptosis of renal microvascular endothelial cells after hypoxia/reoxygenation injury was suppressed byrecombinant SDF-1,but induced by CXCR4 inhibitor AMD3100.5.The inhibitory role of SDF-1/CXCR4 in theimmunocompetenceof renal microvascular endothelial cells after hypoxia/reoxygenation injury In vitro,theimmunocompetenceof renal microvascular endothelial cells after hypoxia/reoxygenation injury was suppressed byrecombinant SDF-1,but induced by CXCR4 inhibitor AMD3100.6.Theprotective effect of SDF-1/CXCR4 on the renal ischemia reperfusion injury in rat models The injury of renal tissuesin rat models with ischemia reperfusion was suppressed byrecombinant SDF-1,but aggravated by CXCR4 inhibitor AMD3100.CONCLUSION This study found that the expression of SDF-1/CXCR4 were significantly increased in renal tissues of rats after renal ischemia reperfusion.And the high expressed SDF-1/CXCR4 play a protective role in the renal ischemia-reperfusion injury in rats by inhibiting the apoptosis and immune activity of renal microvascular endothelial cells induced by ischemia-reperfusion injury,to delay the progression of tissue injury. |
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