The Role And Mechanism Of TOPK In Cisplatin-induced Acute Kidney Injury

Objective:Acute kidney injury(AKI)is a common clinical syndrome characterized by loss of renal function within a short period of time.Cisplatin(CP)is one of the most widely used antitumor drugs.However,it is nephrotoxic and can cause AKI,therefore its clinical application is limited.T-lymphokine-activated killer cell-derived protein kinase(TOPK)is a serine/threonine protein kinase that is highly expressed in proliferating active tissues and is involved in the pathophysiological processes of several diseases.TOPK activation has been reported to ameliorate ischemia-reperfusion-AKI,but its role in CP-AKI is unclear.Therefore,this study aimed to investigate the role and mechanism of TOPK in CP-AKI.Methods:(1)Establishment of CP-AKI in vivo model: 18 male C57BL/6 mice at 8 weeks of age were randomly divided into three groups: saline injection group,CP injection group for 48 h and CP injection group for 72 h,Serum creatinine and urea nitrogen were measured,q RTPCR was performed to detect kidney injury molecule-1(KIM-1)and monocyte chemoattractant protein-1(MCP-1)m RNA in the renal cortex,Western blots(WB)were used to detect Neutrophil gelatinase-associated lipocalin(NGAL),TOPK,p-TOPK(Thr-9),Aurora kinase B(Aurora B),the cell apoptosis indicator Bcl2 and Bax,antioxidant molecule Heme Oxygenase-1(HO-1)and autophagy marker Microtubule-associated protein light chain 3(LC3)protein expression;(2)Construction of CP-AKI in vitro model: Human kidney tubular epithelial cells(HK-2)were cultured in vitro and stimulated with 20 μM CP for 0 h,6 h,12 h and 24 h.q RTPCR detected the m RNA of KIM-1 and MCP-1,WB measured the expression of TOPK,pTOPK,Aurora B,Bcl2,Bax,HO-1 and LC3 protein expression;(3)Retrieved the GEO database to obtain the RNA sequencing dataset of cisplatinAKI mouse kidney samples for functional analysis of TOPK;(4)HK-2 cells were stimulated with 20 μM CP for 24 h,and combined with TOPKspecific inhibitor OTS514 to inhibit or TOPK-activated plasmid(TOPK-T9E)to activate TOPK,and WB was performed to detect the expression of Aurora B,Bcl2,Bax,HO-1,nuclear factor E2-related factor(Nrf2),LC3,and protein kinase B(PKB/AKT),p-AKT(Ser-473)expression,and flow cytometric analyses were conducted to determine the levels of cell cycle,apoptosis and reactive oxygen species;(5)The 20 μM CP and 10 n M OTS514 combined with 10 μM AKT specific inhibitor VIII were used to stimulate HK-2 cells for 24 h.The expression of p-AKT,AKT,Aurora B,Bcl2 and Bax were detected by WB,and the cell cycle and apoptosis were detected by flow cytometry.Results:(1)CP treatment elevated KIM-1 and MCP-1 m RNA levels,increased NGAL protein levels in renal cortex and HK-2 cells,and decreased p-TOPK,Aurora B,Bcl2/Bax and HO-1 protein expression;(2)In CP-AKI mice sequencing samples,the function of TOPK was focused on the regulation of cell cycle as well as apoptosis;(3)TOPK inhibition exacerbated CP-induced downregulation of Aurora B,Bcl2/Bax,HO-1,Nrf2 and p-AKT proteins in HK-2 cells;(4)Inhibition of AKT exacerbated the decrease in Aurora B expression and Bcl2/Bax ratio in HK-2 cells induced by CP and OTS514,and increased G2/M cycle distribution and apoptosis levels.Conclusion:TOPK activity was significantly inhibited in the CP-AKI disease model,and further studies revealed that TOPK inhibition aggravated CP-induced G2/M phase arrest,apoptosis,and oxidative stress in HK-2 cells,and that TOPK inactivation facilitated G2/M phase arrest and apoptosis via inhibition of AKT signaling pathway,and negatively regulated Nrf2/HO-1 signaling pathway to induce oxidative stress,ultimately aggravating the development of CP-AKI.