| Backgroud: Cisplatin is one of the medicine for tumor treatment and also is the main causes of drug-induced acute kidneyinjury,and its mechanism of inducing renal tubular epithelial cell injury is complex.Besides the DNA injury,whether respiration oxygen species(ROS)and mitochondrial,the main organ for the production of ROS,damage are involved in cisplatin induced acute renal injury is not clear.Objective: In the cisplatin-induced acute renal tubule injury,we explore whether there is a change in mitochondrial related protein expression and ROS formation.Method: Balb/c mice were divided into two groups In vivo experiment,the one was control group,the other was experimental group(intraperitoneal injection of cisplatin).HK-2 cells were incubated with different concentrations of cisplatin in vitro.Serum creatinine,urea nitrogen were deteced by biochemical method.Use PAS and PASM to observe the renal pathological changes.TUNEL and immunofluorescence staining were used to observe the apoptosis of renal cells and the expression of cleaved caspase3.Immunohistochemical staining was used to observe the expression of F4/80 positive cells.We used western block to detect the proteins change of cleaved caspase3/caspase3,SOD,catalase,GPX4,DRP1,P-DRP1,FIS1,MFN1,ND1,ND2,ND3,ND4,ND4 L.Real-ti me PCR was used to detect the transcription of BAX,Bcl-2,Bcl-x and mt DNAl.ROS and cell apoptosis were observed by immunofluorescence.ROS was detected by DCFH-DA,and the change of mitochondrial membrane potential was detected by JC-1.Results: Cisplatin induced acute renal injury in Balb/c mice.The serum creatinine in the control group and the experimental group was 8.79±1.21 mol/L and 68.03±1.16 mol/L(P <0.05)respectively,and the two groups of urea nitrogen were16.00±5.29mmol/L and 42.33±5.51mmol/L(P < 0.05),respectively.The kidney of the mice in the experimental group was increased and the color was deeper than that of the control group.PAS staining showed that the tubule cells in the kidney of the experimental group were swollen obviously,and the vacuolated denaturation was significant.F4/80 positive cells in the experimental group were significantly higher than those in the control group,suggesting that cisplatin induced inflammatory cell infiltration in acute renal injury.TUNEL staining showed that the cell apoptosisc of experimental group compared with control group increased,the experimental group cleaved caspase 3/caspase 3 expression increased 206.04±26.94%(P < 0.05),Bax increased compared with the control group of 825.35±60.69%(P < 0.05),Bcl-2 and Bcl-xl respectively compared with the control group by 99.20±11.55%(P < 0.05),86.26±28.35%(P < 0.05)indicates the presence of apoptosis.The co-staining of cleaved caspase 3 and 3 nitrotyrosine(3-NT)in the experimental group was significantly higher than that in the control group.The transcription of SOD,catalase and GPX4 genes in the experimental group decreased by 99.45±6.65%(P<0.05),89.27±1.01%(P <0.05)and 76.59±14.73%(P < 0.05),respectively.The protein expression was decreased by 59.50±16.26%(P < 0.05)、60.00±2.83%(P < 0.05)and57.00±14.14%(P < 0.05),respectively.In vitro HK-2 cells,JC-1 staining showed increased following the cisplatin concentration,HK-2 cells by red fluorescence gradually to green fluorescence.Cleaved caspase 3/caspase 3 in 200 μM treatment group was 1.55±0.18(P < 0.05),20μM,100μM,200μM treatment group Bax transcripts were increased by 3.63±0.28(P < 0.05),4.33±0.19(P < 0.05),5.67±0.40(P < 0.05),Bcl-2 transcription decreased 64.69 ± 1.15%(P < 0.05),84.74 ± 9.22%(P < 0.05),73.00 ± 20.93%(P < 0.05).ROS fluorescence showed that the fluorescence of the200μM treatment group was significantly enhanced,suggesting that ROS was produced.20μM,100μM and 200μM treated group SOD expression wasdecreaded by16.85±4.88%(P < 0.05),20.02 ± 8.92%(P < 0.05),41.29 ± 8.62%(P < 0.05),respectively;the expression of catalase was 1.11±0.15(P > 0.05),0.61±0.04(P <0.05), 0.15±0.02(P < 0.05),GPX4 expression decreased 43.94 ± 11.44%(P < 0.05),47.37±7.11%(P < 0.05),49.08±8.09%(P < 0.05),respectively.N-acetyl cysteine(NAC)inhibits ROS and improves cell apoptosis.Cisplatin induced protein expression of Balb/c mouse in FIS1 was 1.71±0.43(P < 0.05)higher than those in control group,the expression of P-DRP1/DRP1 was 0.68 ± 0.35(P < 0.05),MFN1 declined49.53±25.45%(P<0.05),mitochondrial DNA was decreased 62.74±3.26%,expression of ND1,ND3,ND4 and ND4 L protein decreased 45.89±15.79%(P < 0.05),48.66±6.29%(P < 0.05),45.12±11.66%(P < 0.05)and 47.41±8.18%(P <0.05).20 μM,100μM and 200μM cisplatin treated group as the control group the expression of P-DRP1/DRP1 was 0.32±0.24(P < 0.05),0.45±0.23(P < 0.05),0.12±0.47%(P < 0.05),200 M treatment group FIS1 protein expression increased compared with the control group,it is 2.17±0.29(P < 0.05),the expression of MFN1 protein was 0.41±0.09(P <0.05).The expression of ND1,ND2 and ND3 in the mitochondrial complex I of the200 M treatment group was0.29±0.22(P < 0.05),0.05±0.07(P < 0.05)and 0.05 ±0.02(P < 0.05).Conclusion: Cisplatin can induce acute renal injury and increase the apoptosis of renal tubular epithelial cells.The changes in the mitochondrial complex I protein and the production of ROS may be involved in the acute renal injury induced by cisplatin... |
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