The Role And Mechanism Of Gut Microbial Metabolite Ursodeoxycholic Acid Protects Against Renal Ischemia-reperfusion Injury

Background and ObjectivesRenal ischemia-reperfusion injury(IRI)injury is a pathophysiological phenomenon that refers to kidney tissue damage caused when blood and oxygen supply are restored after a period of renal ischemia.Renal IRI is an inevitable consequence of multiple clinical conditions,including partial nephrectomies,kidney transplantation and cardiac surgery with cardiopulmonary bypass support.Renal IRI significantly affects the survival and quality of patients’ life.Unfortunately,the clinic has no effective prevention and therapeutic strategies for renal IRI.Gut microbes and their metabolites play essential roles in multiple kidney diseases.The mechanisms responsible for the beneficial effect of the microbiome in renal IRI are not fully elucidated.In the present preliminary study,we aimed to investigate the functional role and regulatory mechanisms of gut microbial metabolite ursodeoxycholic acid(UDCA)during renal IRI pathogenesis.The findings of this study would be helpful to provide a theoretical basis for the prevention and treatment of renal IRI,and gutkidney axis theory.MethodsThe murine bilateral kidney IRI(ischemia for 35 min and reperfusion for 24 h)model and human renal tubular epithelial cells(HK-2 cells)hypoxia 24 h and reoxygenation 12 h(H/R)models were performed.The gut microbiota composition and metabolites were analysed by 16S rDNA sequencing,targeted metabolomics and fecal microbiota transplantation.In vivo and in vitro assays validated the phenotype of UDCA protecting renal tubular epithelial cells from injury.RNA-Seq was used to analyse the differential gene expression in kidney tissues after UDCA treatment.Differentially expressed hub genes related to chromatin regulators from GEO and FACER datasets were identified.Bioinformatics data was validated by murine renal IRI model and HK-2 H/R model.The molecular mechanism of UDCA protecting against renal IRI and H/R injury was explored by PPARy antagonist(T0070907)in a murine model and HK-2 cells.The kidney injury was estimated by serum creatinine and blood urea nitrogen in the renal IRI model.The mRNA expression level of kidney injury biomarkers(NGAL and KIM-1)and inflammatory factors(IL-6,TNF-αand MCP-1)in kidney tissues also could be used to evaluate the renal injury.The kidney tissues were stained by PAS and observed under microscopy to estimate the pathologic damage of the kidney tissues.Lipid accumulation in kidney tissues was evaluated by Oil Red O staining.Cell viability and cell death ratio were assessed by CCK8 and LDH assays respectively.The mRNA expression levels were detected by qRT-PCR.The protein expression was detected by immunohistochemistry,immunofluorescence and Western Blot assays.Results1.Renal IRI induced dysbiosis of gut microbiota.The abundance of Eubacterium_xylanophilum_group had significantly decreased after renal IRI and was associated to the content of UDCA.The function of differential gut microbiota was related to lipid metabolism,energy metabolism and biosynthesis of other secondary metabolites.2.Renal IRI induced changes in gut microbiota metabolites.In particular,the content of UDCA decreased after renal IRI.The intervention of UDCA provided protective effects against renal IRI.3.Renal IRI inhibited fatty acid oxidation.Transplanting fecal microbiota from renal IRI to mice further exacerbated renal tissue injury and suppressed fatty acid oxidation.4.In vitro and in vivo experiments demonstrated that the gut microbial metabolite UDCA activated PPARy and promoted fatty acid oxidation,thereby improving renal IRI.5.Construction of GADD45A and GADD45B-associated renal IRI risk predictive model.GADD45A has been implicated in renal IRI.UDCA might improve renal IRI by activating PPARy and inhibiting GADD45A.Conclusion1.Renal IRI induced the dysregulation of gut microbiota and metabolites.2.Gut microbial metabolite ursodeoxycholic acid may protect against renal IRI by activating PPARy and improving fatty acid oxidation.3.Gut microbial metabolite ursodeoxycholic acid may protect against renal IRI by activating PPARy and inhibiting GADD45A.