Role Of MTOR Activation In Cadmium-induced Autophagy Inhibition In Rat Proximal Tubular Cells

Cadmium(Cd)is a common heavy metal in the ecological environment,with stable,accumulation and difficult to remove the characteristics of the environment,mainly through the respiratory tract and digestive tract into the body.Cadmium has multiple organism and multi-tissue toxicity in mammals,which have been classified as human carcinogens by international cancer research institutions.Kidney is the main site of cadmium accumulation,is the main target organ of acute or chronic cadmium exposure,and proximal tubule is the main injury site of cadmium nephrotoxicity.Autophagy is a highly dynamic multi-step biological process responsible for the degradation and recycling of damaged organelles,misfolded proteins,and long-lived macromolecules in lysosomes,which acts as a cytoprotective mechanism in maintaining cellular homeostasis and adaptation to adverse stress conditions.Autophagy is a cellular defense process in which cytosolic components,organelles,and invading bacteria are transported by autophagosomes to lysosomes for degradation.Dysregulation of autophagy has been implicated in the pathogenesis of kidney diseases such as acute kidney injury and chronic kidney disease.mTOR is a class of evolutionally very conserved serine / threonine protein kinases that are members of the phosphatidylinositol kinase-related kinase family.mTOR can be affected by growth factors,nutrients and other factors.It affect autophagy,apoptosis and a series of biological activities by downstream of the target protein phosphorylation,involved in gene transcription and protein expression.In mammalian cells,mTOR functions at least as two complexes,mTORC1 and mTORC2,with distinct substrate specificities.mTORC1 regulates phosphorylation of p70 S6 kinase 1(S6K1)and eukaryotic initiation factor 4E(eIF4E)binding protein 1(4E-BP1).mTORC2 regulates phosphorylation or activity of Akt,and small GTPases.In addition,mTOR is the main regulator of initiation of autophagy.Experimental studies have shown that Cd can inhibit autophagy,but whether the activation of mTOR on Cd induced autophagy inhibitory effect inrPT cells is not clear.In this study,primary cultured rPT cells as a model,the cadmium exposure and mTOR inhibitor co-incubation treatment for 12 h.The protein levels of mTOR and its substrate were analyzed by Western blotting.The effect of mTOR activation on autophagy in rPT cells was detected by laser confocal microscopy and Western blotting.At the same time,the effects of mTOR activation on lysosomal function and V-ATPase were also examined.The results showed that Cd induced activation of mTOR in rPT cells,both mTORC1 and mTORC2 pathways play an important role in this.Phosphorylation of mTOR plays a major role,mTOR total protein has no effect.At the same time,mTOR activation on rPT cells autophagy has an inhibitory effect,blocking the fusion of autophagosomes and lysosomes.In addition,mTOR activation affects the function of lysosomes in rPT cells,which is manifested in the inhibition of lysosomal V-ATPase expression and the lysosomal acidic environment.In conclusion,the activation of mTOR in rat renal tubular epithelial cells induced by Cd exposure can be mediated by translocation to the lysosomal membrane to inhibit the activity of V-ATPase in lysosomes,thereby attenuating the lysosomal acidic environment,And thus damage the function of lysosomes,and finally inhibit the fusion of lysosomes and autophagosomes,in Cd-induced rPT cells play an inhibitory effect of autophagy.