HIF-1α Mediates Renal Fibrosis By Regulating Metabolic Reprogramming Of Renal Tubule Epithelial Cells

Research backgroundThe number of patients with chronic kidney disease(CKD)continues to increase currently.The persistent inflammatory response and increasing fibrosis in the renal interstitium are often presented in the process of CKD progression.Renal fibrosis is a progressive pathophysiological change related to renal injury and function degradation,which is a complex and dynamic process.Most patients present with progressive renal function degradation and develop into the end-stage renal disease(ESRD)eventually,due to lack of the timely and effective prevention and treatment strategies.The kidney is particularly sensitive to the pathophysiology of hypoxia under various stress.Hypoxia is occurring in different stress stimulation and development of diseases,in which hypoxia inducible factors(HIFs)play a central role in the response and regulation of hypoxia.HIF-1α transcription and protein expression are increased to regulate the initial adaptive response of cells to hypoxia in the early stage of hypoxia,as well as reducing the demand of tissue cells for oxygen,to help the cells to quickly adapt the hypoxic microenvironment and achieve a new balance.In the case of mitochondrial respiratory chain damaged,the energy generation is shifted from oxidative mitochondrial phosphorylation to aerobic glycolysis,to ensure that cells adapt to the low-oxygen and nutrient-poor microenvironment.which initiates metabolic reprogramming(metabolic remodeling)due to the altered metabolism pattern.The role of metabolic reprogramming is not only conducted to the generation of energy,but also involved in the production and regulation of various products in the process of metabolic change.The process of metabolic reprogramming is accompanied by the increase of multiple glycolytic metabolites and the decrease of tricarboxylic acid cycle and oxidative phosphorylated metabolites,such as acetyl-CoA,lactic acid,α-ketoglutarate,etc.The changes of these metabolites play important roles in regulation and catalysis of epigenetic alternations in tissue cells,which are directly related to the disease’s occurrence and progression.Reserch objective:In this study,we investigated whether HIF-1α could affect the metabolic reprogramming of renal tubular epithelial cells in the early stage of renal injury,and whether the changes of metabolites would induce the changes of epigenetics,thus promoting renal fibrosis through changing the methylation level of fibrosis-related genes.This study will provide an important experimental basis for the early prevention and treatment of renal fibrosis through exploring the mechanism of this regulatory axis in the early occurrence and progression of renal fibrosis.MethodsIn vivo experiments:The male C57BL/6 mice were managed with unilateral ureteral occlusion(UUO)to establish the animal model of renal fibrosis.Renal samples were collected at 1,3,and 7 days after intervention.Animals in each group were treated with HIF-1α inhibitor KC7F2 and tested subsequently.In vitro experiments:The renal tubular epithelial cells HK2 were managed with AngⅡ.In addition,HK2 cells were treated with AngⅡ treated conditioned medium and lactic acid respectively to compare the effect.Cells were treated with HIF-1αinhibitor KC7F2 and detected subsequently.The differentially expressed genes and pathways related to renal fibrosis were analyzed by biological information data in sham and UUO treated groups.After intervention,the samples were used for histopathological staining and Masson staining for exporing the renal pathological changes and fibrosis status after early injury.Immunohistochemical staining and immunofluorescent staining were used to observe the protein expression and distribution of HIF-1α,α-SMA and fibronectin.The content of GSH,MDA,ATP and lactic acid in kidney tissue were detected by the commercial kits,and the levels of BUN and SCr in serum were detected.The expression of mitochondrial related proteins,oxidative phosphorylation,and glycolytic related proteins,TET2 protein and fibrosis-related proteins were detected by western blot.The changes of cell metabolic flux,OCR and ECAR,was detected by Seahorse.The changes of mitochondria were detected by fluorescent probe staining and SEM scanning analysis.After bisulfite modification,the methylation levels of CpG sites in α-SMA and HIF-1α DNA promoter regions were detected by sequencing.Results1.In the model of ureteral obstruction,the differentially expressed genes in renal tissue increased significantly that mainly enriched in cell metabolism,cell adhesion molecules,extracellular matrix and other signaling pathways.In the early stage of ureteral obstruction,fibrosis occurs in the obstructed kidney,and HIF-1α expression elevated rapidly.Moreover,the OCR was decreased and the ECAR was increased in renal tubular epithelial cells in early fibrosis.In addition,mitochondrial function and quality was abnormal,the energy metabolism shifted from aerobic oxidation to aerobic glycolysis,which symbolized the occurrence of metabolic reprogramming.2.Metabolic reprogramming of renal tubular epithelial cells in the early stage of renal fibrosis leads to enhanced aerobic glycolysis,increased content of glycolytic metabolites.The elevated metabolites induced expression of TET2,which was contributed to reduction of CpG sites methylation in the DNA promoter region ofα-SMA.The metabolic reprogramming resulted in increased expression of α-SMA and fibronectin proteins to promote the occurrence of renal fibrosis.3.Inhibition of HIF-1α with KC7F2 could reduce metabolic reprogramming and TET2 expression,and suppress the DNA demethylation of α-SMA.Therefore,the renal fibrosis development was alleviated through stabilizing the fibrosis related proteins expression.ConclusionThe rapid increase of HIF-1α expression in the early stage of renal fibrosis induced metabolic reprogramming of renal tubular epithelial cells,which leads to changes in metabolite levels during glucose metabolism,up-regulation of TET2 protein,and increased demethylation of CpG site in the DNA promoter region of fibrosis related protein α-SMA.The increased expression of α-SMA promoted the occurrence of renal fibrosis,and inhibition of HIF-1α effectively suppressed TET2 expression and the development of early renal fibrosis.