The Role Of P53-CypD In Acute Renal Injury Mediated By Ischemia Reperfusion

Acute kidney injury(AKI)is a common lethal syndrome.The occurrence and mechanism of AKI are complex,and there is no effective intervention means at present.Renal ischemia-reperfusion injury(IRI)is a common cause of AKI.IRI can cause damage and even death of renal tubular epithelial cells,leading to a sharp decline in renal function.Previous studies have shown that Cyclophilin D(CypD)opened mitochondrial membrane permeability transition pore(MPTP),which could cause cell damage and thus mediate renal IRI.However,the mechanism for the open MPTP by CypD has yet to be explained in renal IRI.Moreover,the other study also found that p53 can be combined with cypd to open MPTP in myocardial cell,neurons and so on.Objective: In this study,in vivo experiments were conducted to observe the effect of p53 and CypD on the damage of renal tubular epithelial cells in rats following renal IRI.The mechanism of p53-cypd in renal IRI was further investigated by using in vitro cell model.Methods: 1.In vivo experiments: SD rats were treated by 35 minutes renal ischemia and24 hours reperfusion to establish animal model of IRI.Serum creatinine and urea nitrogen levels were detected,PAS staining was used to observe renal pathological changes,Western blot and TUNEL staining were used to detect apoptosis of renal tubular epithelial cells,mitochondrial morphological changes of renal tubular epithelial cells were observed by transmission electron microscopy,and CypD and p53 changes in renal tubules were observed by Western blot and immunofluorescence staining.2.In vitro experiments: HK-2 cells were treated by ATP depletion model and pretreated with cyclosporine A(CsA)or si-p53;Western blot and Annexin v-FITC /PI staining were used to detect the apoptosis of cells under different treatments;JC-1 staining was used to detect mitochondrial membrane potential transition(MPT)in cells under different treatments;immunofluorescence staining and co-immunoprecipitation assay were used to explore the interaction between CypD and p53.Result: 1.In vivo experiments: a)compared with the control group and the sham operation group,serum blood urea nitrogen and creatinine significantly increased,with tubular epithelial cell vacuolization,sloughing and swelling,and extensive tubular cast formation and obstruction occurred primarily in the cortex and the outer stripe of the outer medulla in rats in the IRI group;b)compared with the control group and the sham operation group,the expression level of cleaved caspase-3 and the percentage of apoptosis in tubular epithelial cells(TUNEL-positive cells)were significantly increased in IRI group;c)Therewas a marked increase in mitochondrial damage in the tubular epithelial cells of the IRI group relative to the control and sham group,characterized by damaged or absent cristae,disruption of the mitochondrial membrane,and mitochondrial swelling and deformation;d)There were no significant changes in CypD expression.Moreover,p53 was significantly increased and colocalized with CypD in the IRI group compared to the control and sham group.2.In vitro experiments: a)compared with the control group,the expression level of cleaved caspase-3,the proportion of apoptosis in HK-2 cells(Annexin v-positive cells),the proportion of cells with MPT,p53 expression were increased,but CypD expression was not significantly changed in ATP-depleted HK-2 cells;b)compared with the ATP depletion group and the solvent control group,cleaved caspase-3 expression level,the proportion of apoptosis in HK-2 cells,the proportion of cells with MPT were significantly decreased in CsA pretreat group,but p53 and CypD expression were not significantly changed;c)compared with the ATP depletion group and the siRNA control group,cleaved caspase-3 expression level,the proportion of Annexin v-positive cells,the proportion of cells with MPT were significantly decreased in si-p53 pretreat group,but CypD expression was not significantly changed.;d)compared with the control group,p53 binds to CypD and forms a complex with it in ATP-depleted HK-2 cells.Conclusion: In the renal IRI,p53 and cypd could regulate the mitochondrial permeability transformation and apoptosis of the renal tubular epithelial cells,and the effect may be achieved through the formation of complex.