Advanced Glycation End-products Regulate Podocyte Autophagy Through MTOR/TFEB Pathway In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is the leading cause of chronic and end-stage renal disease worldwide[1].Podocyte injury play a key role in the pathogenesis and progression of DN and is associated with impaired autophagy.Autophagy is an intracellular adaptation that maintains the homeostasis of cells[2,3].mTOR is a key negative regulator of autophagy[4].Hyperactivation of mTOR in DN results in disease progression[5-7].TFEB is a traIscription factor and a positive regulator of autophagy 8g].Studies have found that mTOR can phosphorylate TFEB to inhibit its activity[9,10].The autophagy activity was inhibited in the glomeruli from patients with DN[11].In streptozotocin(STZ)-induced diabetic mice,podocyte autophagy was induced at early stage but was inhibited at late stage[12].The turnover of autophagy accompanied with diabetes progression prompted us to investigate the pathogenic factor of long-term of diabetes,advanced glycation end products(AGEs).Our previous study observed in vitro that AGEs inhibit podocyte autophagy by activating mTOR.Object and methodThis study aimed to verify the role of AGEs in podocyte injury and autophagy impairment in DN from in vivo.We further investigated whether mTOR and TFEB are involved in the regulation of podocyte autophagy by AGEs in vitro and in vivo.MethodIn this study,we used immunofluorescence,immunoblotting,electron microscopy and other methods to investigate the key molecules of autophagy of LC3,beclinl,p62 and observe autophagic vacuoles.Dual-fluorescence probes and chloroquine were used to intervene podocytes to detect the dynamic changes of autophagy flux.This study observed the changes of autophagy,mTOR and TFEB in podocytes from patients with DN.In vivo experiments,AGEs generation inhibitor pyridoxamine was used to establish DN mice models with different AGEs levels in db/db mice,and Torin1 was used to inhibit mTOR activity.Changes in podocyte and kidney structure,autophagy,mTOR and TFEB were observed.In vitro experiments,with or without the stimulation of AGE-BSA,cultured podocytes were treated with Torin or TFEB siRNA to determine autophagy,mTOR,and TFEB.The chromatin immunoprecipitation was used to verify the regulation of AGEs on TFEB.The interaction of mTOR and TFEB under the stimulation of AGEs was explored by co-immunoprecipitation in vivo(extraction of glomeruli)and in vitro.Conclusion and SignificanceAGEs are the key factors causing podocyte autophagy dysfunction in DN.TFEB can maintain podocyte autophagy and homeostasis.AGEs inhibit autophagy through activating mTOR and inhibiting TFEB nuclear translocation.AGEs inhibit TFEB by activating mTOR and promote their interaction.This study clarified the key pathogenic factors and its molecular mechanism that lead to podocyte autophagy inhibition in DN,and provided new targets for prevention of podocyte injunry and the treatment of DN.