The Mechanism Of Twist1 Regulating Fatty Acid Metabolism In Renal Tubular Epithelial Cells Injury Induced By Chronic Hypoxia

Background Chronic kidney disease(CKD)has been regarded as one of the most threatening diseases for its 10.8% incidence rate in our country,which seriously endangers the health of the people in China and the world.The the cellular and molecular mechanism of the development of CKD has not yet been elucidated.The sparse microvessels around the renal tubules were the histopathological characteristics of renal tubular epithelial cells that were often damaged due to stress such as hypoxia.CKD has become an important public health problem in chronic diseases to the world Renal interstitial fibrosis is a common pathological manifestation of various CKDs progressing to end-stage renal disease.Therefore,it is quite urgent to investigate the cellular and molecular mechanism of the hypoxia induced kidney injury and seek the potent interventional target position,by which will be essential for the alleviation of the development and progression of CKD.Proximal tubular epithelial cell(PTC)is the main epithelial cell type in the cortex.More and more evidences showed that renal tubular epithelial cells played an irreplaceable role in the process of repair of CKD.PTCs have the highest energy demand of all cells in the body,these cells utilize fatty acids as a major energy source.Thus,the hypoxia injury level was extremely sensitive for TECs and the fatty acid metabolism homeostasis also played the key role.In the process of sustained injury,the surviving of PTC was restricted by fatty acid metabolism and showed a phenotypic change,from epithelial cell phenotype to mesenchymal cell phenotype(EMT)with lower energy requirements,so the accidental blocking of fatty acid oxidation in renal tubular epithelial cells during hypoxia was the main cause of AKI.Renal tubular injury induced by chronic hypoxia is an important cause of renal interstitial fibrosis.However,the mechanism of renal interstitial fibrosis induced by hypoxia has not been fully elucidated.In the early study,we found that Twist1 regulated renal fibrosis induced by hypoxia via Epithelial-Mesenchymal Transition(EMT).The overexpression of Twist1 promoted the lipid droplets deposition in tubular cells,which revealed that Twist1 might play other important role in the renal interstitial fibrosis through any other mechanisms.We further found that some metabolism related transcription factors including PGC-1??PGC-1??PPAR? and PPAR? significantly decreased in the hypoxia induced tubular epithelial cells accompanied by the increasing expression of Twist1 and accumulation of lipid droplets.The reporter gene assay proved that Twist1 can directly regulate the expression of PGC-1? and PPAR?.Therefore,we assume that Twsit may play an important role in the tubular fatty acids metabolism handicap by inhibiting the expression of PGC-1? and PPAR?,CPT1 and ACOX1 and consequently lead to renal interstitial fibrosis.We will prove this hypothesis through the in vivo and in vitro assays and provide the theoretical basis for clarifying the mechanism of the hypoxia induced renal damage and seeking the new prevention methods.Objectives 1)To investigate the effect of chronic hypoxia on fatty acid metabolism in renal tubules.2)To screen and identify key molecules that regulated hypoxia-induced fatty acid metabolism and promoted renal interstitial fibrosis.3)To study the role of Twist1 in hypoxia-induced fat metabolism homeostasis in renal tubules by inhibiting PGC-1?.4)To investigate the protective effect and mechanism of Twist1 on fat metabolism in renal fibrosis.Methods 1)To identify the triglyceride accumulation caused by fatty acid oxidation disorders via Oil Red O(Oil Red)staining and electron microscopy.2)To measure the oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)of renal tubular epithelium under normoxic and hypoxic conditions by Seahorse XFe Extracellular Flux Analyzer.3)To investigate the mechanism of Twist1 regulating fatty acid metabolism disorders in renal tubular by Genetic sequencing,Histopathology,Western Blot,and Immunohistochemistry in Twist1(-/-)transgenic mice.4)To investigate the regulation of Twist1/PGC-1?/PPAR? on fatty acid metabolism disorders in renal fibrosis by kidney function indicators,renal histopathology,Luciferase reporter gene,Ch IP and other methodsResults 1)Fatty acid oxidative metabolism of renal tubular epithelial cells was unbalanced under hypoxic conditions.2)Hypoxia promotes the expression of Twist1 and significantly decreased the fatty acid oxidation metabolism in renal tubular epithelial cells.Silencing Twist1 in vitro under hypoxia can relieve hypoxia-induced fatty acid metabolism disorders 3)Over-expressing Twist1 under hypoxia inhibited PGC-1? and PPAR? to promote fatty acid metabolism disorders and induced renal interstitial fibrosis.4)Twist1 KO in renal tubules mice can alleviate fatty acid metabolism disorder caused by hypoxia,thereby protecting renal function and renal interstitial fibrosis.Conclusion In summary,our study showed that Twsit1 may play an important role in the tubular fatty acids metabolism handicap by inhibiting the expression of PGC-1?/PPAR?,CPT1,ACOX1 and consequently lead to renal interstitial fibrosis.We will prove this hypothesis through the in vivo and in vitro assays and provide the theoretical basis for clarifying the mechanism of the hypoxia induced renal damage and seeking the new prevention methods.