The Role And Mechanism Of MTORC1/mTORC2 On Cadmium-induced Kidney Injury In Mice

Cadmium pollution is a serious environmental problem that threatens animal and human health.Cadmium produced in agricultural and industrial activities accumulates in the body through the food chain and shows toxic effect,and the kidney is the main target organ of acute and chronic cadmium exposure.Previous studies have confirmed that lysosome dysfunction mediated autophagy inhibition and caspase dependent apoptosis are important causes of cadmium-induced kidney injury.mTOR(mammalian target of rapamycin)is a serine/threonine kinase,which is an important regulator of cell growth and proliferation.The mTOR kinases exist in two multiprotein complexes:mTORC1 and mTORC2.mTORC1 is very sensitive to rapamycin,sensing amino acid,pressure,energy and other signals,then regulates cell growth state by changing anabolic and catabolic processes.In contrast,mTORC2 is insensitive to rapamycin,and has only been reported to respond to growth factors and regulate cell survival and cytoskeleton.The roles and regulation mechanisms of the two mTOR complexes in cadmium-induced kidney injury remain unclear.In this study,Cdh16Cre tool mice and mice proximal tubular epithelial cells(KTPTS cells)were used as models to study whether mTORC1/mTORC2 involved in cadmium-induced mice kidney injury in vitro and in vivo,and to clarify the regulatory mechanisms.This study will provides important theoretical support for the prevention and control of cadmium pollution to animals and humans.The main research contents are described as follows:1.The role of mTORC1/mTORC2 in cadmium-induced kidney injury in miceIn order to explore the effect of cadmium on mTOR signaling pathway in mice kidney,cadmium(5 μ M,12 h),mTOR activator MHY1485(5 μ M,12 h)and mTOR inhibitor Torin1(100 nM,12 h)were used to set up the control test,KTPTS cells were treated with 0,1.25,2.5,and 5 μM CdCl2 to establish cell damage models in vitro,and 1.5 mg/kg/day intraperitoneal injection for 5 consecutive days to establish kidney injury models in vivo.Then the effect of cadmium on the activity of mTORC1 and mTORC2 were verified by protein immunoblotting,immunofluorescence and immunohistochemistry.The results showed that the protein expression levels of p-mTOR(Ser2448)and p-mTOR(Ser2481)were increased with the increase of cadmium concentration(P<0.05 or P<0.01),the protein expression levels of mTORCl downstream marker p-4EBP1 and mTORC2 downstream marker p-AKT(Thr308)and p-AKT(Ser473)increased significantly(P<0.01),In order to detect the role of mTOR in cadmium-induced kidney injury in mice,small interfering RNA(in vivo)and RNAi adenoassociated virus(in vitro)of the core component Raptor and Rictor were used to inhibit the activity of mTORCl and mTORC2,respectively.Changes in cytotoxicity and kidney injury were detected by CCK-8,RTCA,bright field observation and serum biochemical analysis.The results showed that inhibition of mTORC1 or mTORC2 could further reduce the survival rate of KTPTS cells after cadmium exposure(P<0.0 1),aggravate the damage of KTPTS cells and kidney tissues,and increased the levels of renal index,kidney injury markers(Kim-1,NGAL)and serum renal enzymes(Scr,BUN)(P<0.05 or P<0.01).The results showed that cadmium exposure activates mTORC1 and mTORC2 signaling pathways in mice kidney in vivo and in vitro,and the inhibition of mTORC1 or mTORC2 resulted in increased sensitivity of TKPTS cells and mice kidney tissue to cadmium toxicity damage,the activation of two mTOR complexes may be the survival mechanism of renal cells after cadmium exposure.2.The role of mTORCl/mTORC2 in cadmium-induced autophagy inhibition of mice kidneyIn order to explore the effect of mTORC1 and mTORC2 on cadmium-induced autophagy inhibition in mice kidney,small interfering RNA(in vivo)and RNAi adenoassociated virus(in vitro)of the core component Raptor and Rictor were used to inhibit the activity of mTORC1 and mTORC2,respectively.The autophagy level of KTPTS cells and kidney tissues were detected by western blotting,plasmid transfection and immunofluorescence.The results showed that inhibition of mTORCl significantly decreased the protein expression levels of LC3 and p62(P<0.01),significantly reduced the number of autophagosomes(P<0.01),relieve cadmium-blocked autophagy flow in cadmium-exposed KTPTS cells and kidney tissues.Inhibition of mTORC1 significantly increased the expression of TFEB in the nucleus of cadmium-exposed KTPTS cells(P<0.01),and then restore the expression of lysosome membrane proteins(LAMP1,LAMP2),lysosome hydrolases(CTSB,CTSD,CTSL,GNS,SGSH,TPP1),V-ATPase(ATP6V1A,ATP6V1D,ATP6V1B1,ATP6V1B2),and repair the lysosomal function of cadmium damage.Consistent with mTORC1 inhibition,inhibiting mTORC2 also alleviates cadmium-blocked autophagy flow in vivo and in vitro.mTORC1 inhibition model was established to further test the effect of mTORC2 on cadmium-induced KTPTS cells autophagy inhibition,and the results showed that mTORC2 mediated cadmiuminduced renal autophagy inhibition independently of mTORC1.Further studies showed that inhibition of mTORC2 significantly increases the expression of FOXO1 in the nucleus of cadmium-exposed KTPTS cells(P<0.01),and then significantly restore the expression of lysosome fusion functional protein Rab7(P<0.01).The results indicate that mTORC1 regulates cadmium-induced lysosome degradation by TFEB,and mTORC2 regulates cadmium-blocked autophagosomes and autophagy fusion by FOXO1,by which two mTOR complexes co-mediates cadmium-induced autophagy inhibition in mice renal cells.3.The role of mTORC1/mTORC2 in cadmium-induced apoptosis of mice kidneyIn order to explore the effect of mTORC1 and mTORC2 on cadmium-induced apoptosis in mice kidney,small interfering RNA(in vivo)and RNAi adenoassociated virus(in vitro)of the core component Raptor and Rictor were used to inhibit the activity of mTORC1 and mTORC2,respectively.Changes in the level of apoptosis in the kidney cells in vitro and in vivo were determined by protein immunoblotting,flow cytometry,immunohistochemistry,and TUNEL staining.The results showed that the inhibition of both mTORC1 and mTORC2 further increased the expression level of Cleaved-PARP and Cleaved-Caspase 3,increased the ratio of Bax/Bcl-2,and increased the apoptosis rate(P<0.05 or P<0.01).Further study on the mechanism of mTORC1 and mTORC2 regulating apoptosis of mice renal cells.The results showed that inhibition of mTORC1 and mTORC2 could significantly inhibit the phosphorylation activity of AKT.By adding AKT activator SC79(5 μM,12 h)and inhibitor MK2206(0.5μM,12 h),it was found that AKT played an anti-apoptotic role in cadmiuminduced apoptosis of mice renal cells.By adding mTOR activator MHY1485(5 μM,12 h)and inhibitor Torin1(100 nM,12 h)to explore the relationship between autophagy and apoptosis under the regulation of mTOR,it was found that mTORC1 and mTORC2 are the important proteins that balance the level of autophagy and apoptosis in cadmium-exposed mice renal cells,and the imbalance of two mTOR complexes can lead to the abnormal transformation of autophagy and apoptosis.In conclusion,①mTORC1 and mTORC2 were activated after cadmium exposure in mice kidney in vivo and in vitro,and their activation is the survival mechanism of kidney cells after cadmium exposure.②Both mTORC1and mTORC2 mediates cadmium-induced renal autophagy inhibition.mTORC1 mediates cadmium-induced lysosome dysfunction by inhibiting the activity of transcription factor TFEB.mTORC2 mediates cadmium-blocked autophagosome lysosome fusion by inhibiting the activity of transcription factor FOXO1.③Both mTORCl and mTORC2 could antagonize cadmium-induced apoptosis of kidney cells by regulating the activity of AKT.mTORCl and mTORC2 are important balance factors between renal autophagy and apoptosis after cadmium exposure,which protect the kidney from cadmium-induced toxic damage by limiting the autophagy and antagonizing apoptosis.