The Role And Mechanism Of SIRT3 Deacetylation PDHA1 Disorder-Mediated Metabolic Reprogramming Signaling In Sepsis Associated Acute Kidney Injury

Sepsis associated acute kidney injury(SAKI)is a frequent complication in critically ill patients,which is related to high morbidity and mortality.In recent years,studies have suggested that metabolic reprogramming is involved in the pathogenesis of SAKI,but the mechanism is unclear.Pyruvate dehydrogenase(PDH)inactivation is thought to be involved in cellular metabolic reprogramming under a variety of pathophysiological conditions.It is noteworthy that the activity of PDH is regulated by post-translational modification.Acetylation of PDH decreases the activity of PDH,while deacetylation of PDH enhances the activity of PDH.In this study,the acetylation level of mitochondrial protein in the renal tissue of septic mice was identified by mass spectrometry.It was found that the acetylation level of pyruvate dehydrogenase E1 component subunit alpha(PDHA1)at the 385 lysine residue(K385)site increased 2.734-fold(P=0.010).This result suggested that enhanced PDHA1 acetylation and inactivation may be key molecular mechanisms that mediate the SAKI metabolic reprogramming.Interestingly,mitochondrial deacetylase SIRT3 protein may be involved in the regulation of PDHA1 acetylation modification.Besides,studies have shown that decreased SIRT3 protein expression in kidney aggravated SAKI,whereas the upregulation of SIRT3 can alleviate SAKI,but whether these effects were related to the regulation of metabolic reprogramming has not been reported.In conclusion,we speculate that enhanced PDHA1 acetylation-induced metabolic reprogramming due to SIRT3 downregulation is a key mechanism for SAKI occurrence.The metabolic reprogramming of SAKI is manifested by the inhibition of pyruvate oxidation,followed by enhanced glycolysis and oxidative phosphorylation inhibition.Objective:In this study,by establishing SAKI-related animal and cell models,we explored the increased acetylation and decreased activity of PDHA1 caused by SIRT3 downregulation,which mediated metabolic reprogramming to participate in the development of SAKI.The results will provide ideas and potential strategies for future SAKI treatment.Methods:In vivo,the SAKI animal model was established by CLP operation on C57BL/6 mice(6～8 weeks old).Western blot technique was used to detect the expression of SIRT3 and PDHA1.PDHA1 activity assay kit was used to detect PDHA1 activity.The proteomics was used to identify PDHA1 lysine acetylation sites.Glycolysis inhibitor 2-deoxy-D-glucose(2-DG)and activator UK 5099 were used to determine the role of glycolysis in SAKI.SIRT3 inhibitor 3-TYP was used to investigate whether SIRT3 inhibition enhanced PDHA1 acetylation.Sodium dichloroacetate(DCA),a PDHA1 activator,was used to investigate whether PDHA1 activation improves SAKI.In vitro,human kidney-2(HK-2)was stimulated with lipopolysaccharide(LPS)to establish SAKI cell model.Adenovirus-mediated SIRT3 overexpression and SIRT3 siRNA were used to SIRT3 knockdown to investigate whether the change in PDHA1 acetylation depends on SIRT3.Results:1.SIRT3 protein expression was downregulated in renal tissue(F=8.363,P=0.001)and HK-2 cells(F=17.147,P<0.001)after sepsis.2.Proteomics found that PDHA1 K385 acetylation level in renal tissue was significantly upregulated after sepsis(P=0.010).3.After sepsis,there was no significant change in the expression of PDHA1 protein in renal tissue(F=0.844,P=0.514)and HK-2 cells(F=1.616,P=0.222).However,the PDHA1 acetylation level was increased,and the protein activity was decreased significantly(kidney,F=6.211,P=0.009;HK-2 cells,F=5.874,P=0.005).4.SIRT3 as an upstream deacetylase reduced PDHA1 acetylation level.5.Sepsis caused the enhancement of glycolysis in renal tissue(F=90.515,P<0.001)and HK-2 cells(F=23.191,P<0.001).6.Inhibition of glycolysis aggravated renal dysfunction in CLP-induced sepsis(BUN,F=36.215,P<0.001;Cr,F=31.742,P<0.001)and reduced mice survival rate(χ2=4.220,P=0.0399).7.Activation of glycolysis did not improve renal dysfunction in septic mice(BUN,P=0.302;Cr,P=0.799).8.Activation of PDHA1 partially restored CLP-induced decrease in ATP content of kidney(F=18.720,P<0.001)and relieved renal dysfunction in septic mice(BUN,F=16.101,P<0.001;Cr,F=12.045,P=0.001).Conclusion:1.The expression of SIRT3 protein in the kidney and HK-2 cells was downregulated after sepsis;2.Downregulation of SIRT3 expression in the kidney and HK-2 cells after sepsis led to upregulation of PDHA1 acetylation with decreased PDHA1 activity;3.Deterioration of metabolic reprogramming aggravated SAKI,but activating PDHA1 to reverse metabolic reprogramming can alleviate SAKI.