The Role And Mechanism Research Of TWEAK In Cisplatin-Induced Acute Kidney Injury

BackgroundCisplatin is one of the most widely used anti-tumor drugs in clinical practice,which has significant efficacy for solid tumors such as breast cancer and cervical cancer.At the same time,cisplatin has serious side effects.Nephrotoxicity is the dose-limiting toxicity of cisplatin,which greatly limits its clinical application.TWEAK is a member of the tumor necrosis factor ligand superfamily involved in inflammatory response,tissue repair,and other processes.The expression levels of TWEAK and Fn14 are low in normal kidney tissue and significantly upregulated when exposed to stress or injury.Up-regulated TWEAK has been observed in kidney diseases such as renal interstitial injury and fibrosis.However,the expression and function of TWEAK in cisplatin-induced acute kidney injury remains unclear.ObjectiveThis project aims to explore the expression and mechanism of TWEAK in cisplatininduced acute kidney injury by establishing cisplatin-induced acute kidney injury animal and cell models.So as to provide new drug target for clinical prevention and treatment of cisplatin nephrotoxicity.MethodsIn vivo experiments:Male Balb/C mice were used to establish cisplatin-induced acute kidney injury mice model.Plasma creatinine and urea nitrogen values were measured using kits.HE staining was used to observe kidney tissue structure and evaluate the degree of acute kidney injury.The change of gene expression in the kidneys of mice in the control group and cisplatin administration group was detected by RNA sequencing technology.We Screened differentially expressed genes.The protein-protein interaction network was constructed to determine the key genes.The expression of TWEAK,Fn14,GSDMD,IL-1β in mice kidneys were detected by Western blot.The distribution and expression changes of TWEAK and Fn14 in kidney tissue were detected by immunohistochemistry.The value of soluble TWEAK in plasma was determined by ELISA.The expression of Fn 14,GSDMD,GSDMD-N,NLRP3,caspase-1,ASC in kidneys after TWEAK administration were detected by Western blot.TWEAK monoclonal antibody was used to block TWEAK expression in cisplatin-induced acute kidney injury mice.The kidney damage of mice was evaluated by body weight change,plasma creatinine,plasma urea nitrogen,HE staining results and tubular injury scores.The expression of TWEAK,Fn14,GSDMD,GSDMD-N,NLRP3,and IL-18 were detected by Western blot,ELISA kit was used to detect the change of plasma IL-18.qRT-PCR was used to detect the gene expression of Gsdmd,Nlrp3,Asc,Casp1,1118.In vitro experiments:HEK293 cells were used in vitro experiments,and CCK-8 method was used to determine the optimal concentration of cisplatin induced cell damage.The expression of TWEAK,Fn14,GSDMD,GSDMD-N were detected by Western blot.Hoechst 33342/PI staining was used to assess the level of pyroptosis.Western blot was used to detect the expression of GSDMD and GSDMD-N in cells after TWEAK treatment.The expression of TWEAK was silenced by siRNA technique.The expression levels of GSDMD-N and NLRP3 were detected by Western blot,The pyroptosis level was assessed by Hoechst 33342/PI staining.Research effect of blocking NF-κB pathway on TWEAK-induced pyroptosis:The expression of GSDMD,GSDMD-N,NF-κB p65,and p-NF-κB p65 were detected by Western blot.ResultsPlasma creatinine and urea nitrogen levels in cisplatin-induced acute kidney injury mice were significantly increased.A large number of tubular necrosis and a small amount of protein casts could be seen on HE staining.The expression of TWEAK,Fn14,GSDMD,IL-1β in kidney significantly increased.sTWEAK concentration in plasma increased with the increase of cisplatin dose.The expression of Fn14,GSDMD,GSDMD-N,NLRP3,caspase-1,and ASC in kidneys were upregulated after TWEAK treatment.After TWEAK expression was blocked,the weight decline trend induced by cisplatin was significantly reduced.The plasma creatinine and urea nitrogen were significantly reduced.Renal tubular injury was improved.The expression of TWEAK,Fn14,GSDMD,GSDMD,NLRP3,and IL-18 in the kidneys were reduced.The content of plasma IL-18 was decreased.Gsdmd,Nlrp3,Asc,Casp1,Il18 gene expression were significantly reduced.TWEAK,Fn14,GSDMD,GSDMD-N expression were upregulated in HEK293 cells after cisplatin stimulation.Hoechst 33342/PI staining results showed a significant increase in the incidence of pyroptosis.The expression of GSDMD and GSDMD-N in HEK293 cells after TWEAK stimulation were also significantly upregulated.Compared with cisplatin treating group,GSDMD-N,NLRP3 expression in TWEAK-silencing HEK293 cells were reduced.Staining results showed a significant reduction in the incidence of pyroptosis.Western blot results showed that the expression of GSDMD,GSDMD-N,NF-κB p65,and p-NF-κB p65 in cells with inhibition of the NF-κB pathway were reduced.ConclusionsTWEAK and Fn14 were significantly upregulated in cisplatin-induced acute kidney injury mice and cells.Blocking TWEAK could significantly reduce cell pyroptosis caused by cisplatin,and significantly alleviate acute kidney injury.Therefore,TWEAK is a potential target for clinical prevention and treatment of cisplatin-induced nephrotoxicity.