NADPH Oxidase 4 Promotes Cisplatin-induced Acute Kidney Injury By Initiating ROS-mediated Necroptosis,Apoptosis And Inflammation

Acute kidney injury(AKI)is characterized by sudden decline of renal function and tends to induce high morbidity and mortality rate of patients.Emerging evidence shows that the pathological feature of AKI includes excessive inflammation and programmed cell death of tubular epithelial cells.In recent years,a large number of literatures showed that various diseases caused by cisplatin,such as ototoxicity,were closely correlated with increased level of reactive oxygen species(ROS).It is well known that ROS is crucial for maintaining the kidney function and structure under physiological and pathological conditions.The major enzymatic sources of ROS production in cisplatin nephrotoxicity is proved to be nicotinamide adenine dinucleotide phosphate oxidases(NADPH oxidases,also known as Noxs).Of note,it has been reported that Nox4 was mainly expressed in the kidney.Moreover,recent studies demonstrated that Nox4 was involved in the pathological process of many kidney diseases.Considering the roles of Nox4 in AKI have not been fully elucidated,here we studied the expression of Nox4 and their roles in cisplatin-induced AKI both in vitro in HK2 cells and in vivo in cisplatin nephropathy treated with apocynin(as Noxs inhibitor)and lentivirus-packaged Nox4 sh RNA plasmid respectively.The main contents are divided into six sections,as follows:1.Inhibition of ROS reduced cisplatin-induced acute kidney injury through RIP-mediated necroptosis,apoptosis and inflammatory in HK2 cell.HK2 cells were treated with or without NAC(50u M)for 12 h and then incubated with20 u M cisplatin for 24 h.Cells were harvested in order to detect the level of the following indicators: reactive oxygen species(ROS),kidney injury molecule-1(KIM-1),necroptosis indexes such as RIP1,RIP3 and phosphor-MLKL protein,apoptosis indexes such as cleaved-caspase3 and inflammatory response such as TNF-?,IL-1?,IL-6 and MCP-1 mRNA by western blot analysis,real-time PCR and other methods.It was found that cisplatin promoted the level of ROS expression,while inhibition of ROS reduced cisplatin-induced renal injury.The results showed that ROS was involved in cisplatin-induced acute renal injury.2.Protective effect of NADPH oxidases inhibitor on cisplatin-induced acute kidney injury in vitro and in vivo.1)The pharmacological effect of apocynin in cisplatin-treated HK2 cells was then detected.Results of western blot analysis showed that overnight preincubation of apocynin largely decreased the protein and mRNA level of KIM-1 in cisplatin-treated HK2 cells.In addition,treatment of apocynin reduced the percentage of cisplatin-induced apoptotic and necrotic cells.Consistently,apocynin significantly reduced cisplatin-activated programmed cell death-related signaling pathways and expression of proinflammatory factors mRNA.These results indicated that blockade of NADPH oxidases by apocynin may prevent cisplatin-induced nephrotoxicity through inhibiting Nox-derived ROS production.2)Results of PAS staining showed that apocynin attenuated the degree of renal injury,consistent with the findings that apocynin downregulated the creatinine and BUN at 3days after intraperitoneal injection of cisplatin(20mg/kg).In addition,intraperitoneally injection of apocynin attenuated cisplatin-induced KIM-1 protein and mRNA level,which was further confirmed by the IHC of KIM-1.Moreover,we also discovered the effect of apocynin on inflammatory response triggered by cisplatin in mice.Results indicated that apocynin largely suppressed the level of TNF-? protein in cisplatin-induced AKI mouse model.Real-time PCR results also showed that apocynin significantly downregulated the mRNA levels of TNF-?,IL-1?,IL-6 and MCP-1compared with cisplatin-induced AKI group.Taken together,apocynin prevented from renal damage and inflammatory in response to cisplatin possibly by affecting ROS level in vivo.3.The role of Nox4 in cisplatin-induced acute renal injury and its possible mechanism.1)20?M cisplatin significantly induced Nox4 in both mRNA and protein level in HK2 cells.Additionally,result of immunohistochemistry and quantitative data indicated that Nox4 was induced in cisplatin nephropathy,which was further confirmed by real-time PCR and western blot in vivo.Results showed cisplatin promoted the level of Nox4 expression both in vitro and in vivo.2)Western blot analysis showed that knockdown of Nox4 significantly inhibited the level of KIM-1,apoptosis and RIP-mediated protein expression in response to cisplatin.The result of real-time PCR also showed that silence of Nox4 successfully decreased cisplatin-upregulated mRNA level of KIM-1.Moreover,disturbance of Nox4 also obviously reduced cisplatin-induced inflammatory cytokines expression such as TNF-?,IL-1? and IL-8.These results suggested that knockdown of Nox4 prevented cisplatin-induced acute kidney injury by apoptosis,RIP-mediated necroptosis and inflammatory.3)Overexpression of Nox4 promoted cisplatin-induced KIM-1,apoptosis and RIP-mediated necroptosis,which was further confirmed by immunofluorescence analysis.In the current study,we found that overexpression of Nox4 further increased cisplatin-induced inflammatory cytokines expression including TNF-?,IL-1? and IL-6 mRNA level compared with control group.Taken together,we found overexpression of Nox4 accelerated cisplatin-induced acute kidney injury.4.Treatment of NAC,a ROS inhibitor,suppressed Nox4 overexpression-promoted kidney injury in cisplatin-treated HK2 cells.Treatment of NAC suppressed the level of KIM-1,apoptosis and RIP-mediated protein promoted by Nox4 overexpression in cisplatin-treated group,which was confirmed by real-time PCR detecting the mRNA level of KIM-1.Moreover,Nox4-induced upregulation of inflammatory cytokines,such as TNF-?,IL-1? and IL-6 mRNA,were suppressed by NAC.These results indicated that Nox4 promoted cisplatin-induced acute kidney injury by generating ROS mediated apoptosis,necroptosis and inflammatory.5.Lentivirus-mediated knockdown of Nox4 protected against cisplatin-induced AKI in vivo.Mice were slowly injected with 100 ul lentivirus-packaged Nox4 sh RNA plasmid with a concentration of 1*107 TU/ml through tail vein using a 0.5ml insulin syringe at the speed of approximately 0.2ul/min.One week later,mice were intraperitoneally injected with either 20mg/kg cisplatin or the equal volume of saline for further analysis.Disruption of Nox4 relieved cisplatin-induced kidney damage.Moreover,results of serum creatinine and BUN assays showed that deficiency of Nox4 protected against the loss of renal function in response to cisplatin(20mg/kg)which was further confirmed by the results of real-time PCR,western blot and immunohistochemistry detecting the mRNA and protein level of KIM-1.In addition,loss of Nox4 prevented the activation of RIP1/RIP3/MLKL and cleavage of caspase-3,and accordingly attenuated cisplatin-induced renal inflammation as shown by downregulation of TNF-? in both protein and mRNA levels.Moreover,mRNA levels IL-1?,IL-6 and MCP-1 were also inhibited by Nox4 knockdown in cisplatin-treated kidney.These results further indicated knockdown of Nox4 protected cisplatin-induced AKI by preventing programmed cell death and inflammation.In this setting,we presumed that Nox4 may be a potential therapeutic target for cisplatin-induced AKI.6.Knockdown of Nox4 didn't reduce the sensitivity of tumor cells to cisplatin in BEL-7401,SGC-7901 and C6 tumor cells.To identify whether Nox4-targeted therapy is applicable to prevent AKI in cancer patients treated with cisplatin,the critical issue is to identify that whether it compromises the kill effects of cisplatin on tumor cells.In this regard,we then investigated the effect of Nox4 on tumor cells.Results of MTT showed that knockdown of Nox4 gently increased,but not decreased,the sensitivity of tumor cells to cisplatin in BEL-7401 liver tumor cell lines.Additionally,loss of Nox4 didn't alter the killing effects of cisplatin on SGC-7901 and C6 tumor cells.