CTRP3 Overexpression Attenuates Cisplatin Induced Acute Kidney Injury Through Nrf2/MAPK Pathway

Background:Cisplatin is a highly effective and widely used anti-tumor drug,its clinical application is limited by various side effects,nephrotoxicity is one of the most common side effects,and the most common cisplatin related nephrotoxicity is acute kidney injury,its associated with high morbidity and mortality worldwide,but the underlying mechanism remains unclear,and effective approaches to ameliorate cisplatin-induced AKI are not available.AKI caused by cisplatin mainly includes oxidative stress,inflammation and apoptosis.CTRP3 is a new adipokine,which has been found to have antioxidant stress,anti-inflammatory and anti-apoptotic effects,is involved in the pathological progression of several diseases,such as type 2 diabetes mellitus,atrial fibrillation,heart failure,renal fibrosis and IgA nephropathy.Recently,CTRP3 has shown to protect against oxygen-glucose deprivation/reperfusion induced cerebral injury,cardiac ischemia/reperfusion injury,uric acid-induced endothelial injury,by inhibiting inflammation,oxidase stress,and apoptosis.However,the role and regulation of CTRP3 in AKI are remain largely unknown.Methods:1.To study the expression of CTRP3 in cisplatin-induced acute kidney injury model in vivo.The animal model of acute kidney injury induced by cisplatin was established.Blood samples were collected from orbit at 24h,48h and 72h after cisplatin treatment for determination of serum creatinine,urea nitrogen and CTRP3.The mice were sacrificed on the third day after cisplatin treatment.PAS staining was used to observe the morphological changes of renal injury,the apoptosis rate was determined by TUNEL,immunohistochemistry was used to detect the expression of CTRP3,RTPCR and Western blot were used to detect the expression of CTRP3 mRNA and protein in renal tissue.2.The expression of CTRP3 was studied by establishing an in vitro model of acute kidney injury induced by cisplatin in HK-2 cells.HK-2 cells were induced with different concentrations of cisplatin,and the optimal concentration of cisplatin was determined by MTT assay to prepare for the next experiment.RT-PCR and Western blot were used to determine the expression of CTRP3 mRNA and protein in HK-2 cells induced by cisplatin.3.To study the protective effect of CTRP3 on cisplatin-induced acute kidney injury by establishing an in vitro model of cisplatin-induced kidney injury in HK-2 cells.HK-2 cells were transfected with pcDNA-CTRP3 and siRNA-CTRP3,respectively,to up-regulate and down-regulate the expression of CTRP3 in HK-2 cells,and to study the oxidative stress,inflammation,apoptosis and proliferation of HK-2 cells treated with cisplatin.The oxidative stress indexes of SOD,CAT and MDA were measured.Meanwhile,the mRNA expression levels of SOD1 and SOD2 were determined by RTPCR.The inflammatory indexes of TNF-α and MCP-1 were measured by ELISA.The cell activity was measured by MTT,apoptosis-related proteins Bcl-2 and Bax were determined by RT-PCR and Western blot,cell apoptosis was detected by flow cytometry,and cell proliferation was measured by EdU.4.HK-2 cells were transfected with pcDNA-CTRP3,and then treated with cisplatin for RNA transcriptome sequencing.GSEA enrichment analysis of signal pathway showed that MAPK pathway was enriched in HK-2 cells after cisplatin induction.The protein expressions of p-ERK,p-JNK and p-P38 in HK-2 cells of control group,cisplatin treatment group and CTRP3 overexpression+cisplatin treatment group were determined by Western blot.5.HK-2 cells were transfected with siRNA-CTRP3,and the expression of apoptosis protein cleaved caspase-3 in HK-2 cells after cisplatin treatment with p-ERK inhibitor U0126,p-JNK inhibitor SP600125 and p-P38 inhibitor SB203580 was determined by Western blot.The inflammatory indexes of TNF-α and MCP-1 were determined by ELISA,and the apoptosis rate was determined by TUNEL.6.HK-2 cells were transfected with pcDNA-CTRP3,and the protein expressions of Nrf2,p-ERK,p-JNK and p-P38 after si-Nrf2 treatment were determined by Western blot,and the oxidative stress indexes of SOD,CAT and MDA were determined,the mRNA expression levels of SOD1 and SOD2 were determined by RT-PCR.Results:1.In the cisplatin induced AKI model,immunohistochemistry,RT-PCR and Western blot results showed that CTRP3 expression was down-regulated in mouse kidney and human proximal tubular epithelial cells(HK-2).ELISA results showed that compared with the control group,the expression level of CTRP3 began to decrease after 24h of cisplatin treatment.2.In cisplatin induced in vitro experiments,overexpressing CTRP3 inhibited oxidative stress through decreasing MDA levels and increasing the activity of SOD and CAT.The mRNA levels of SOD1 and SOD2 were increased in response to CTRP3 overexpression.CTRP3 also decreased the levels of inflammatory cytokines TNF-α and MCP-1.In addition,CTRP3 overexpression increased cisplatin induced cell activity,decreased cell apoptosis and increased cell proliferation.Consistent with these results,overexpression of CTRP3 effectively elevated the mRNA and protein levels of Bcl-2 and reduced the mRNA and protein levels of Bax.In contrast,inhibition of CTRP3 expression by siRNA-CTRP3 reversed these indices induced by cisplatin.3.CTRP3 overexpression increased cisplatin induced Nrf2 expression and inhibited p-ERK,p-JNK,p-P38 phosphorylation activation.p-ERK inhibitor U0126,pJNK inhibitor SP600125 and p-P38 inhibitor SB203580 attenuated cisplatin-induced inflammation and apoptosis induced by CTRP3 knockdown.The inhibitory effects of CTRP3 overexpression on oxidative stress as well as the expression of p-P38,p-JNK,and p-ERK were significantly reversed by Nrf2 knockdown.Conclusion:CTRP3 attenuates cisplatin-induced acute kidney injury by inhibiting oxidative stress,inflammation and apoptosis through Nrf2/MAPK pathway.