RIPK3 Promotes Sepsis-induced Acute Kidney Injury Via TFEB Mediated Autophagy Flow Dysfunction

BackgroundSepsis is one of the main causes of acute kidney injury(AKI).Proximal renal tubule epithelial cells(RTECs)injury play a central role in the pathogenesis and progression of AKI and is closely related to autophagy dysfunction.Autophagy is an adaptive response to different extracellular pressure.And efficient autophagic process play important role to mainta:in metabolism and homeostasis of cells.The transcription factor EB(TFEB)acts as an important regulator of autophagy.TFEB regulated autophagy is phosphorylated-dependent process.TFEB present in cytoplasm under normal physiological state.TFEB translocated from the cytoplasm to the nucleus when starvation-induced autophagy.RIPK3 is a protein with serine/threonine kinase.C terminus of RIPK3 is RIP homotypic interaction motif(RHIM).N terminus of RIPK3 is the kinase and death domains(KD).Studies have shown that the level of RIPK3 increased in the blood or urine of patient with sepsis induce AKI.Previous studies have demonstrated autophagy activation in mouse model of sepsis induced kidney injury by LPS or cecal ligation and puncture(CLP).In additional,autophagy dysfunction aggravated kidney injury.However,the mechanism of autophagy in regulating RTECs injury is still unclear.Taken together,the current study intends to discuss the role of RIPK3 in LPS induced renal injury,and preliminarily clarify the mechanism how RIPK3 regulates TFEB mediated autophagy flow dysfunction in RTECs.Methods1)In vivo experiment:generated sepsis associated acute kidney injury mouse model by LPS intraperitoneal injection,we examined the expression of autophagy related protein(LC3,p62)and lysosomal function protein(LAMP1,Cathepsin B,VPS 11)by western blot.We further observed autophagic vacuoles by transmission electron microscopy(TEM).The autophagic flow changes of RTECs were dynamically observed by immunofluorescence in LPS induced mRFP-GFP-LC3 fluorescence transgenic mice.To observe the role of GSK'872 in sepsis induced AKI,we tested renal function and autophagy of mice pre-treated with GSK'872.In additional,immunofluorescence staining were performed to observe the interaction and distribution of RIPK3 and TFEB in patient with sepsis induce AKI.2)In vitro experiment:established RTECs autophagy flow dysfunction model by LPS stimulated.With the stimulation of LPS,cultured RTECs were pre-treated with GSK'872 or RIPK3 siRNA or TFEB siRNA to observe autophagy and translocation of TFEB.Co-immunoprecipitation was used to examine the interaction between RIPK3 and TFEB.We finally tested the target gene level of TFEB downstream and expression of lysosomal function protein under LPS stimulation by qPCR and western blot.Results1)In vivo experiment:compared with control group,serum creatinine(SCR),blood urine nitrogen(BUN),the expression of protein RIPK3,LC3,p62 were significantly increased in mice of LPS group.PAS staining observed the swelling of RTECs and vacuolation degeneration,narrowing of the lumen.We observed autophagosome accumulate in RTECs of mice treated with LPS by TEM.Immunofluorescence showed the decreased expression of nucleus TFEB(p<0.001).On the contrary,SCR,BUN,the expression of protein LC3,p62 were obviously reduced in mice of LPS+GSK'872 group compared with the LPS group.In additional,PAS staining observed the damage of RTECs was relieved in mice treated with GSK'872 prior to LPS.Autolysosome in LPS+GSK'872 group were observes by TEM.Immunofluorescence showed the elevated expression of nucleus TFEB(p<0.001).In additional,immunofluorescence staining were performed to observe the interaction of RIPK3 and TFEB in patient with sepsis induce AKI.2)in vitro experiment:(1)The expression of p62,LC3 and RIPK3 were significantly increased in RTECs stimulated with LPS compared with control group(p<0.001).However,the level of nucleus TFEB decreased in RTECs exposed to LPS.On the contrary,knockout of RIPK3(siRNA)or suppressed activity of RIPK3 kinase(GSK'872)reduced LPS induced the expression of LC3 and p62,and raised the content of nucleus TFEB in TRECs exposed to LPS.(2)Compared with LPS group,knockout of TFEB increased the level of LC3 and p62 in control group.However,forced expression of TFEB suppressed up-regulation of LC3 and p62.(3)Co-immunoprecipitation confirmed the interaction between RIPK3 and TFEB.(4)The target gene level of TFEB downstream and the expression of lysosomal function protein(LAMP1,Cathepsin B)decreased in RTECs stimulated with LPS compared with control group,whereas suppressed activity of RIPK3 kinase or knockout of RIPK3 raised the target gene level of TFEB downstream and the expression of lysosomal function protein under LPS stimulated.ConclusionsRIPK3 promotes LPS induced renal injury by autophagy flow dysfunction of RTECs.RIPK3 is involved in autophagy flow dysfunction of RTECs by suppressing nuclear translocation of TFEB.Suppression nuclear translocation of TFEB might damage lysosomal function.