The Role And Mechanism Of Renal Tubular Epithelial Cells RIPK3 In The Progression From Acute Kidney Injury To Chronic Kidney Disease

BackgroundAcute kidney injury(AKI)is a clinical syndrome with rapid decline of renal function,which could increase the risk of long-term chronic kidney diseases(CKD).Receptor interacting protein kinase-3(RIPK3)is a protein mediating necroptosis and previous studies confirm that RIPK3 is up-regulated in a variety of renal cells in CKD[1,2,3].Due to the lack of specific intervention on renal tubular epithelial cells RIPK3,it is still unclear whether renal tubular epithelial cell RIPK3 mediates the AKI to CKD progression.G2/M cell cycle arrest in renal tubular epithelial cells is one of the important mechanisms of the AKI to CKD progression[4],and previous studies found that proteins phosphorylated by RIPK3 are highly enriched in the cell cycle regulation pathway[5].Therefore,RIPK3 may participate in the regulation of G2/M cell cycle arrest in renal tubular epithelial cells and the AKI to CKD transition.objectivesThe purpose of this study was to clarify the role of renal tubular epithelial cells RIPK3 in the AKI to CKD progression,and to explore the mechanism that RIPK3 regulate G2/M phase cell cycle arrest and promote the AKI to CKD progression.Methods(1)The expression of RIPK3 in renal puncture tissue from CKD patients after cardiac surgery-associated AKI was detected using immunofluorescence and RNA in situ hybridization.The expression of RIPK3 was detected in the AKI to CKD progression animal model constructed by renal ischemia-reperfusion injury using Western blot,fluorescence quantitative PCR and immunofluorescence.Renal fibrosis was evaluated after RIPK3 kinase inhibitor,GSK’872 intervention after IRI or construction of AKI to CKD animal model using transgenic mice with specific knockout of proximal renal tubular epithelial cells RIPK3.Inhibition of RIPK3 or overexpression of phosphorylated RIPK3 were used to explore the fibrosis of human kidney-2(HK-2)cells induced by Transforming growth factor β(TGFβ).(2)The effects of RIPK3 on G2/M phase arrest of renal tubular epithelial cells were studied in animal experiments and in vitro experiments.(3)The effects of RIPK3 on renal tubular epithelial cells phosphorylated cyclin-dependent kinases 1(p-CDK1)were studied in animal experiments and in vitro experiments.Co-Immunoprecipitation and pull-down experiments were used to explore the interaction between the phosphorylated RIPK3 and CDK1.The mechanism that RIPK3 could phosphorylate CDK1 and inhibit its activity,mediate G2/M phase cell cycle arrest and finally promote the progression of AKI to CKD were explored in vitro experiments.Results(1)Renal tubular epithelial cells RIPK3 was up-regulated in renal puncture tissues of CKD patients after cardiac surgery-associated AKI.In AKI to CKD progression animal models,renal tubular epithelial cells RIPK3 and p-RIPK3 were up-regulated.GSK’872 intervention or specific knockout ofp roximal renal tubular epithelial cells RIPK3 could reduce the area of renal interstitial fibrosis,the level of serum creatinine and blood urea nitrogen,and the expression of fibrotic protein including α-SMA and fibronectin after AKI.Inhibiting RIPK3 kinase activity or silencing RIPK3 after TGFβ stimulation could reduce the expression of α-SMA and fibronectin,and overexpression of phosphorylated RIPK3 could increase the expression of α-SMA and fibronectin.(2)GSK’872 intervention after AKI or specific knockout of proximal renal tubular epithelial cells RIPK3 reduce the expression of renal tubular epithelial cells phosphorylated histone H3(p-H3).After TGFβ treatment in HK-2 cells,GSK’872 intervention or silencing RIPK3 decrease the expression of p-H3 and the proportion of G2/M phase,while overexpression of phosphorylated RIPK3 increased the expression of p-H3 and the proportion of G2/M phase.(3)GSK’872 intervention after AKI or specific knockout of proximal renal tubular epithelial cells RIPK3 could reduce the expression of renal tubular epithelial cells phosphorylated CDK1(Thr 14).After TGFβ treatment in HK-2 cells,GSK’872 intervention or silencing RIPK3 decreased the expression ofp-CDK1,while overexpression ofphosphorylated RIPK3 increased its expression.Co-Immunoprecipitation and pull-down experiments confirmed that phosphorylated RIPK3 and CDK1 could bind directly.GSK’872 intervention or silencing RIPK3 decreased the expression of fibronectin after TGFβ intervention.CDK1 kinase inhibitor—Ro-3306 intervention could increase the expression of fibronectin after inhibiting RIPK3.ConclusionsRenal tubular epithelial cells RIPK3 is up-regulated and activated during the AKI to CKD progression and drive the AKI progressing to CKD.The possible mechanism may be that RIPK3 can phosphorylate and inhibit the activity of CDK1,and mediate the G2/M cell cycle arrest of renal tubular epithelial cell,which could provide a new theoretical and therapeutic target for delaying the AKI to CKD progression.