| Acute kidney injury(AKI)is a common complication with high morbidity and mortality worldwide,is a destructive clinical condition induced by multiple insults including ischemic reperfusion,nephrotoxic drugs and sepsis.It is characterized by a sudden decline in renal function,in addition to excessive inflammation,oxidative stress and programmed cell death of renal tubular epithelial cells.And currently has no effective treatment beyond sup-portive care.Therefore,it is important to explore effecitive treatment.Necroptosis,not apoptosis is a type of programmed cell death.Recently,RIPK1-mediated necroptosis plays an important role in AKI and triggers a serious inflammatory response.Necrostatin-1(Nec-1),is a classic RIPK1 inhibitor,has a certain therapeutic effect in acute kidney injury.But it has several drawbacks like the narrow structure activity relationship(SAR)profile,moderate potency and non-ideal pharmacokinetic properties,in vivo and in vitro and failed to be applied in clinic.Therefore in this study,we evaluated the treatment effects of Cpd-71,a novel RIPK1 inhibitor,by comparing with Necrostatin-1(Nec-1).This study mainly performs four parts as followed:1.Cpd-71 attenuated cisplatin-induced actue kidney injury.The effect of Cpd-71 in concentrations of 0.825,1.65 and 3.3mg/kg was evaluated in vivo in cisplatin nephrotoxicity.In order to observe the protective effect of Cpd-71 on cisplatin induced AKI,pathological examination of renal tissue was performed by PAS staining,serum levels of BUN and Cre were detected by BUN and Cre Kit;the renal injury factor KIM1 via western blot,real-time PCR and IHC.Results showed that kidney damage induced by cisplatin was alleviated after pretreatment of Cpd-71.Notably,Cpd-71 has better protective effects compared with Nec-1 in the same concentration.We first evaluated the effect of Cpd-71 on HK2 cells viability via MTT assay.Results showed Cpd-71 at concentrations lower than 0.8 μM had limited effect on the cell growth of HK2 cells.Then we further determined the effects of Cpd-71 on cisplatin-induced cell damage.Results showed Cpd-71 at concentrations of 0.1,0.2 and 0.4 μM restored cell viability in a concentration-dependent manner.Western blot and Real-time PCR showed that cisplatin upregulated KIM1,but Cpd-71(0.1,0.2 and 0.4 μM)suppressed KIM1 expression in HK2 cells.Pretreatment with Cpd-71(0.4 μM)had better suppressive effects on KIM1 expression than Nec-1.The suppressive effect of Cpd-71 on KIM1 was further confirmed by IF analyses2.Cpd-71 reduced inflammatory responseImmunohistochemistry showed Cpd-71 reduced macrophage infiltration and TNF-α positive signal in injured kidney.Consistently,pro-inflammatory cytokines and chemokines TNF-α,IL-6 and MCP-1 m RNA level were down-regulated following Cpd-71 treatment in vivo.In cisplatin-induced HK2 cells,inflammatory factors including TNF-α,IL-1β and IL-6 were markedly inhibited by Cpd-71,and the anti-inflammatory effect of Cpd-71(0.4 μM)was comparable with Nec-1(50 μM).3.Cpd-71 protected against cisplatin-induced oxidative stress levelThe level of oxidative stress was detected by GSH and MDA assay.Results showed GSH was significantly decreased in cisplatin-induced mice but restored after Cpd-71 pretreatment,this is in line with the finding that Cpd-71 suppressed MDA level in mice exposed to cisplatin.Western bolt and Real-time PCR assay showed that Cpd-71 treatment reduced cisplatin-induced Nox4 protein expression and m RNA levels.Likewise,DHE and DCF staining showed that ROS generation was increased in cisplatin-induced HK2 cells,however,ROS was significantly reduced when cisplatin-induced HK2 cells following Cpd-71 or Nec-1 treatment.In addition,Western blot analyses revealed that Cpd-71 shows more suppressive effect on Nox4,a main member of NOX family in kidney,than Nec-1 treatment group,this result was further confirmed by real-time PCR.4.Cpd-71 attenuated renal injury by targeting RIPK1-mediated necroptosisImmunohistochemistry staining showed that Cpd-71 reduced RIPK1 expression in renal tubules.Western blot confirmed that Cpd-71 had more inhibitory effects on RIPK1 and RIPK3 activation compared with Nec-1 in cisplatin nephropathy.Furthermore,IF analysis suggested that Cpd-71 suppressed phosphorylation of MLKL in cisplatin-induced mice in vivo.We performed a CETSA that enables us to evaluate target engagement in vivo.Results showed that the detected soluble RIPK1 proteins clearly differ at denaturation temperatures ranging from 47 to 59℃ with and without Cpd-71 treatment,Cpd-71-treated mice showed a significant increase in the thermal stability of RIPK1.Which indicated that Cpd-71 is directly bound to the RIPK1 protein.Electron microscopy showed that cisplatin-induced HK2 cells exhibited plasma membrane rupture,organelle swelling and loss of cell organelle contents,these effects were alleviated in Cpd-71 treatment group.Consistently,IF analyses showed the suppressive effect of Cpd-71 on phosphorylation and membrane translocation of MLKL was better than Nec-1.We then further detected the mechanisms by which Cpd-71 suppressed necroptosis in two cell necrosis models.RIPK1 and RIPK3 are activated when HK2 cells are exposed to TNF-α and z VAD,and we found that Cpd-71 had a suppressive effect on RIPK1/RIPK3 signaling,which was further confirmed in cisplatin-induced cell model.Importantly,the suppressive effect of Cpd-71 was more significant than Nec-1.Moreover,co-immunoprecipitation revealed RIPK1 interacted with RIPK3 to mediate necroptosis in cisplatin-treated group,but Cpd-71 decreased the formation of RIPK1/RIPK3 complex.In addition,a docking experiment was performed to propose a possible binding mode for Cpd-71 in RIPK1.Cpd-71 exhibited more effectively suppressive effect on cisplatin-induced renal tubular cell necroptosis than Nec-1 by physically binding to the allosteric type III ligand binding site of RIPK1,thereby reduced RIPK1 kinase activity. |
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