The Effect Of Vitamin D Receptor On The Glucose Metabolism Reprogramming Of Sepsis-Associated Acute Kidney Injury

Background and objective: Sepsis associated acute kidney injury(SAKI)is a critical clinical syndrome with a rapid onset and high mortality rate.Recently,the role of glucose metabolism reprogramming in S-AKI progression has become a hot topic.Pyruvate Dehydrogenase E1 alpha Subunit(PDHA1)is the key active site of pyruvate dehydrogenase.In glucose metabolism reprogramming,elevated levels of p-PDHA1/PDHA1 is a critical step that inhibits aerobic respiration and promotes glycolysis.Vitamin D receptor(VDR)is a nuclear transcription factor and it regulates glucose metabolism reprogramming in various diseases,such as tumors.However,its impact on glucose metabolism reprogramming in S-AKI has not been explored.Our study aims to investigate the effect of VDR on pPDHA1/PDHA1 in renal tubular epithelial cells of S-AKI and its underlying mechanism,providing novel insights for the strategies to prevent acute kidney injury.Methods:1.A mouse model of sepsis-associated acute kidney injury(S-AKI)was established in wild-type C57BL/6 mice(pre-treating with a VDR agonist,paricalcitol),VDR knockout(VDR-KO)mice,and renal proximal tubular-specific VDR-overexpressing(VDR-OE)mice,aiming to investigate the impact of VDR on glucose metabolism reprogramming and its renal protective effects.Blood samples were collected from the mice to evaluate kidney function,and kidney tissues were obtained for pathological analysis.Mitochondrial morphology of proximal tubular epithelial cells was observed using electron microscopy.In kidney tissue,immunofluorescence was performed to detect F4/80,p-PDHA1,PDHA1,and TUNEL;RT-q PCR was used to determine IL-6 and MCP-1 mRNA levels,western blot was performed to measure VDR,p-PDHA1,PDHA1,cleaved caspase3,and bcl2 protein expression;and biochemical assays was used to measure hexokinase activity and lactate levels.2.VDR knockout(VDR-KO)human proximal tubular epithelial cells(HK-2)and HK-2 cells transfected with VDR-overexpressing plasmids(VDR-OE)were used to explore the impact of VDR on glucose metabolism reprogramming and its protective effects on cells after lipopolysaccharide(LPS)intervention at the cellular level.RT-q PCR was performed to determine IL-6 and MCP-1 mRNA expression levels,and western blot was used to measure VDR,p-PDHA1,PDHA1,cleaved caspase3,and bcl2 protein expression levels.Seahorse metabolic analyzer was used to determine the oxygen consumption rate(OCR)of cells.Biochemical assays were performed to measure lactate levels.3.The protective effect of VDR on LPS-induced HK-2 cells and its relationship with p-PDHA1 inhibition was investigated at the cellular level.HK-2 cells were treated with dichloroacetate(DCA,a p-PDHA1 inhibitor)and VDR agonist.The detection indices were roughly the same as those in method 2.4.The JASPAR platform was used to predict the VDR binding site(VDRE)in the PDHA1 promoter region,chromatin immunoprecipitation(ChIP)and RT-q PCR were used for validation.Fluorescent luciferase plasmids containing wild-type or mutant PDHA1 promoter sequences were constructed,and a dual-luciferase reporter gene was used to further verify whether PDHA1 is a target of VDR transcriptional regulation.5.This section explored whether VDR can exert renal protective effects in S-AKI by upregulating p-AMPK to impact p-PDHA1/PDHA1 of glucose metabolism reprogramming.Western blot was used to detect the expression of p-AMPK and AMPK in S-AKI mice and HK-2 cells treated with LPS.Compound C(an AMPK pathway activator inhibitor)was used to treat HK-2 cells,followed by VDR agonist treatment.The detection indices were roughly the same as those in method 2.Results:1.In S-AKI mice,the levels of blood urea nitrogen and creatinine were significantly increased,pathological analysis showed a pathological characteristics of interstitial inflammation cell infiltration,tubular lumen expansion,and tubular epithelial cell shedding;electron microscopy indicated significant mitochondrial damage;and there was an increased levels in inflammatory cytokine,cell apoptosis,hexokinase activity,lactate concentration and p-PDHA1/PDHA1 ratio.All above changes were more severe in VDR-KO mice,while alleviated by VDR agonists and VDR-OE treatment.2.Following LPS intervention,HK-2 cells showed an increased levels in inflammatory cytokine,cell apoptosis,lactate accumulation,pPDHA1/PDHA1 ratio,and a decreased level in cellular basal respiration OCR and maximal respiration OCR.The aforementioned changes were more severe in VDR-KO HK-2 cells,but it was relieved in VDR-OE HK-2 cells.3.After DCA treatment,HK-2 cells had a higher levels of basal respiration OCR and maximal respiration OCR,a lower level of lactate concentration,p-PDHA1/PDHA1 ratio,inflammatory cytokine and cell apoptosis compared with the cells treated with LPS.However,intervention with a VDR agonist in follow of DCA treatment did not enhance the aforementioned effects of DCA.4.ChIP-q PCR showed that the PDHA1 promoter contained three functional VDREs with an enrichment ratio of about 1.2-fold.Dual luciferase reporter gene experiments showed that the fluorescence intensity increased by about 30% in cells containing the VDR-overexpressing plasmid compared with the wild-type PDHA1 plasmid.However,the fluorescence activity was significantly reduced after mutate VDREs of PDHA1.5.The expression of p-AMPK increased in S-AKI mice and LPStreated HK-2 cells,and VDR-KO decreased the protein expression of pAMPK,while VDR-OE and VDR agonists increased p-AMPK expression.After Compound C treatment of HK-2 cells,basal respiration OCR levels and maximal respiration OCR levels were decreased,the levels of lactate concentration,p-PDHA1/PDHA1 ratio,inflammatory cytokine and cell apoptosis were increased compared with the cells treated with LPS.However,intervention with a VDR agonist in follow of compound C treatment did not fully reverse the aforementioned effects of Compound C.Conclusion:1.Vitamin D receptor alleviated acute kidney injury of S-AKI by regulating glucose metabolism reprogramming.2.Vitamin D receptor regulates glucose metabolism reprogramming in S-AKI,it mainly attributed to the inactivation of PDHA1 phosphorylation via the AMPK pathway.There were 35 figures,7 diagram and 172 references in the paper.