Role And Mechanism Of Mitophagy In Contrast-induced Acute Kidney Injury

Objective:1.To explore the rat model of contrast-induced acute kidney injury(CI-AKI).2.To evalute the changes of autophagy,mitophagy,oxidative stress,mitochondrial injury,renal tubular epithelial cell apoptosis induced by contrast meida.3.To explore the effects of enhancing autophagy and mitophagy by rapamycin on contrast-induced abnormalities in oxidative stress,mitochondrial injury and renal tubular epithelial apoptosis in a CI-AKI rat model.Methods:1.Fifty four healthy Sprague-Dawley rats were randomly divided into nine groups: control group(group A);tail vein injection groups with iohexol at the dose of3.5,5.25,8.75 g I/kg(group B1-3);intraperitoneal injection groups with iohexol at the dose of 5.25,8.75,12.25 g I/kg(group C1-3)and two consecutive tail vein injection groups with iohexol at the dose of 5.25,7.0 g I/kg(group D1-2).Renal function and renal histology were determined on the second day following contrast administration.2.Twenty four healthy Sprague-Dawley rats were divided into control group(group Con),CI-AKI group(group CI-AKI)and rapamycin-pretreated groups at the dose of 2 or 5 mg/kg(group Rap1/2).CI-AKI was induced by intraperitoneal injection of iohexol at the dose of 12.25 g iodine/kg.Rapamycin(diluted in 0.9%saline to a final volume of 0.5 ml)or its vehicle was intraperitoneally administrated for 7 consecutive days before contrast intraperitoneal injection.Renal function and renal histology were evaluated one day following contrast administration.The expression of LC3,Beclin-1,Pink-1 and cytochrome c(Cyt c)was measured by western-blot.Mitochondrial membrane potential(MMP)was determined by JC-1,colocalization of LC3-labeled autophagosomes with LAMP2-labeled lysosomes or TOMM20-labeled mitochodria was observed by fluorescence microscope.Renal tubular epithelial cell apoptosis and malondialdehyde(MDA)levels were examined by the methods of TUNEL and ELISA,respectively.Results:1.Compared with group A,increased serum creatinine(Scr)and severe renal injury were induced by contrast using intraperitoneal injection with the largest dose.However,Scr and renal injury were moderately elevated in other groups.2.Significantly increased Scr and obvious renal injury were induced by contrast administration(Con vs CI-AKI,P<0.05),which were dramaticlly and dose-dependently attenuated by rapamycin administration(Rap1/2 vs CI-AKI,P<0.05;Rap1 vs Rap2,P<0.05).3.Activated autophagy as demonstrated by the overexpression of LC3Ⅱ/Ⅰand Beclin-1(Con vs CI-AKI,P<0.05)and the integration of LC3-labeled autophagosomes with LAMP2-labeled lysosomes in rats in CI-AKI,which was obviously and dose-dependently enhanced by rapamycin pretreatment.4.Activated mitophagy as evidenced by the upregulated expression of pink-1and the integration of LC3-labeled autophagosomes with TOMM20-labeled mitochodria was observed in rats in CI-AKI.And the integration of LC3-labeled autophagosomes with TOMM20-labeled mitochodria was remarkably and dose-dependently enhanced by rapamycin pretreatment.5.Significantly increased MDA was induced by contrast administration(Con vs CI-AKI,P<0.05),which was decreased by rapamycin pretreatment(Rap1/2 vs CI-AKI,P<0.05;Rap1 vs Rap2,P<0.05).6.The contrast-induced obvious mitochondrial injury including increase in cytosolic/mitochondrial Cyt c and decrease in MMP.And the contrast-induced increase in cytosolic/mitochondrial Cyt c and decrease in MMP were obviously and dose-dependently suppressed by rapamycin.7.The increased renal tubular epithelial cell apoptosis induced by contrast was obviously and dose-dependently alleviated by rapamycin administration.Conclusion:1.The animal model of CI-AKI was successfully induced by intraperitoneal injection with largest doses of contrast.2.Contrast exposure causes mitochondrial injury,which activates autophagy and mitophagy.But,the activated autophagy and mitophagy are relatively inadequent for the large quantities of damaged mitochondria.So large quantites of damagedmitochondria accumulated in cytoplasm,which can further induce ROS and Cyt c release from the mitochondria and result in oxidative stress injury and renal tubular epithelial cell apoptosis.3.Enhancing autophagy and mitophagy by rapamycin administration could exert protective effects on CI-AKI by suppressing mitochondrial injury and subsequent oxidative stress injury and renal tubular epithelial cell apoptosis.