Tsc1/mTORC1 Signaling Regulates Energy Metabolism And Participates In Renal Interstitial Fibrosis And Its Mechanism

Part ? The relationship between mTORC1 in tubular epithelial cells and renal interstitial fibrosisRenal fibrosis,particularly tubulointerstitial fibrosis,is the inevitable common outcome of all progressive chronic kidney diseases.Although the mechanism of tubulointerstitial fibrosis has been studied,there is still a lack of effective treatment to relieve the progress of CKD.As a cytoplasmic serine / threonine protein kinase,mTOR plays an important role in the growth,proliferation,differentiation and survival of mammals.Previous studies have shown that abnormal activation of mTORC1 was involved in many pathophysiological processes such as PKD,DN and renal fibrosis.Here,we applied the NRK-52 E cell and animal models of renal fibrosis to research the relationship between mTORC1 signal of renal tubular epithelial cells and renal interstitial fibrosis.The results showed that TGF-?1 stimulated NRK-52 E cells to activate mTORC1 signal in vitro,while rapamycin could alleviate the effect of TGF-?1.At the same time,mTORC1 signal was activated in renal fibrosis models,including unilateral ureteral ligation(UUO),ischemia-reperfusion injury(IR)and folic acid(FA)renal injury model,while rapamycin could alleviate renal fibrosis.Therefore,we speculated that activation of mTORC1 signal was involved in the process of renal interstitial fibrosis.Part ? Role of Tsc1 dependent mTORC1 signal in the process of renal interstitial fibrosisAs an important negative regulatory factor of mTORC1,Tsc includes Tsc1(also known as hamartin)and Tsc2(also known as tuberin).The down-regulation or absence of Tsc1 can cause the continuous activation of mTORC1 and its downstream signals.To explore the direct role of mTORC1 in the process of renal interstitial fibrosis,Tsc1 was knocked down by transfection of small interfering RNA(siRNA)to induce mTORC1 activation in vitro.In addition,Tsc1 knockdown alone could induce the phenotype changes of NRK-52 E cells,which can be inhibited by rapamycin.To further examine the role of Tsc1-dependent mTORC1 signaling in proximal tubules,we created conditional knockout mice in which the Tsc1 gene was specifically deleted in proximal tubule cells by the Cre-Loxp system.The absence of Tsc1 in the proximal tubule led to the activation of mTORC1 signal.The deletion of Tsc1 in proximal tubules resulted in enlarged kidneys characterized by high proportion of proliferative tubular epithelial cells,dilated tubules with cyst formation,and vast area of interstitial fibrosis.In this part of the study,we directly confirmed that Tsc1-dependent mTORC1 signaling was involved in the process of renal interstitial fibrosis in vitro and in vivo.Part ? Tsc1 mediated mTORC1 activation promotes glycolysis in tubular epithelial cells in renal fibrosisMetabolism reprogramming to glycolysis is related to the development of CKD.As an important negative regulatory factor of mTORC1,Tsc1 is also a key regulator of glycolysis.We investigated whether Tsc1/mTORC1 could mediate the progress of renal interstitial fibrosis by regulating glycolysis in tubular epithelial cells.mTORC1 signaling was activated in the tubular epithelial cells in UUO model,accompanied by increased proliferation of tubular epithelial cells and up-regulation of the glycolytic enzymes.Rapamycin pre-treated to inhibit the activation of mTORC1 abolished the enhanced glycolysis and tubular epithelial cell proliferation.Furthermore,knockdown of Tsc1 promoted glycolysis in NRK-52 E cells.The deletion of Tsc1 in proximal tubules resulted in interstitial fibrosis in conjunction with elevated glycolysis.These findings demonstrated that Tsc1-associated mTORC1 signaling mediated the progress of renal interstitial fibrosis by regulating glycolysis in tubular epithelial cells.Part ? Regulating glycolysis and reversing Tsc1/mTORC1 signaling mediated renal interstitial fibrosisCKD is a worldwide problem threatening human health.Although a large number of studies on CKD have been carried out,there is still a lack of effective measures to inhibit or alleviate the progress of CKD.We have previously confirmed that Tsc1/mTORC1 was involved in the process of renal interstitial fibrosis by regulating glycolysis.In this part of the study,2-deoxyglucose(2-DG),a glycolysis inhibitor,was used to inhibit the glycolysis of renal tubular epithelial cells.We found that 2-DG can improve the proliferation of renal tubular epithelial cells and renal interstitial fibrosis.In addition,we also found that HIF-1? siRNA and HK2 siRNA can improve the phenotype of renal tubular epithelial cells induced by Tsc1 siRNA.Inhibition of glycolysis can reduce the proliferation of renal tubular epithelial cells and renal interstitial fibrosis induced by mTORC1 activation,which was expected to provide new ideas for the treatment of CKD.