Research On The Role And Mechanism Of Autophagy In NiCl₂-induced Apoptosis In Kidney

Nickel,one of the essential microelements for human,has been widely applied in health,industry and daily life.Nickel has also been recognized as a toxic and carcinogenic environmental pollutant.The kidney is the major target organ for nickel toxicity.Our previous studies showed that nickel chloride（Ni Cl₂）induced renal toxicity,however,the role of autophagy in nickel-induced nephrotoxicity is unclear.Therefore,in our present study,we established a Ni Cl₂ toxicity model in vivo and in vitro,and an oxidative stress,autophagy,mitochondrial autophagy and apoptosis intervention model.Then,we used electron microscopy,immunohistochemistry,immunofluorescence,western blotting,flow cytometry and q RT-PCR to explore the effects and mechanism of nickel on autophagy in the kidney,as well as clarify the role and mechanism of autophagy in nickel-induced apoptosis of renal cell.The findings will provide new ideas for nickel toxicological mechanism.Part 1:The effect and mechanism of nickel on autophagy in the kidney1.The effect and mechanism of nickel chloride on autophagy in the kidney7-weeks-old male Institute for Cancer Research Mouse（ICR）mice and mouse renal tubular epithelial cells（TCMK-1）were used as the research object.Mice were intragastrically administrated with different doses of Ni Cl₂（0 mg/kg,7.5 mg/kg,15 mg/kg,30 mg/kg）for 28 days to establish Ni Cl₂ poisoning model in vivo.TCMK-1 cells were treated with 0μM,200μM,400μM,and 800μM Ni Cl₂ for 24 h to establish Ni Cl₂poisoning model in vitro.For the in vivo study,Ni Cl₂ induced renal injury in mice characterized by reduction in kidney index,obvious pathohistological changes,and an increase in renal function indexes（CRE and BUN）.Immunohistochemical results showed that Ni Cl₂ treatment significantly increased LC3 expression and decreased p62 expression in the kidney.Electron microscope results showed that Ni Cl₂ increased the number of autophagic lysosomes in mouse kidney.In addition,Ni Cl₂ also caused a significant decrease in p62,PI3K,AKT and m TOR protein and m RNA expression levels,and increase in LC3,Beclin1,Atg5/12/7/3,Atg16L1,AMPKα,ULK1 protein and m RNA expression levels.For the in vitro study,MTT assay showed that Ni Cl₂ inhibited TCMK-1 cell activity.TEM observation showed a large number of autophagosomes in TCMK-1 cells induced by Ni Cl₂.Immunofluorescence results showed that Ni Cl₂ increased LC3 expression.Ni Cl₂also caused a significant decrease in p62,PI3K,AKT and m TOR protein and m RNA expression levels,and increase in LC3,Beclin1,Atg5/12/7/3,Atg16L1,AMPKα,ULK1protein and m RNA expression levels.To further verify that Ni Cl₂ induces autophagy through AKT/AMPK-m TOR signaling pathway in renal cell,we used AKT promoter（SC79）and AMPK inhibitor（Com C）to intervene in the Ni Cl₂ poisoning model in vitro,respectively.The results showed that AKT-m TOR pathway was activated and Ni Cl₂-induced autophagy was inhibited after SC79 treatment.On the other hand,AMPK-m TOR pathway was inhibited and Ni Cl₂-induced autophagy was also inhibited after Com C treatment.These results proved once again that AKT/AMPK-m TOR signaling pathway was involved in Ni Cl₂-induced autophagy in TCMK-1 cells.2.Oxidative stress-mediated AKT/AMPK-m TOR pathway was involved in Ni-induced autophagyThe Ni Cl₂ poisoning model in vivo and in vitro were established by referring to part I.In addition,an animal model of oxidative stress intervention was established in mice co-treated with acetylcysteine（NAC）and Ni Cl₂.NAC and mitochondrial targeted antioxidant Mito-TEMPO were used for oxidative stress intervention,to establish an in vitro cell model of oxidative stress intervention.We observed that in the in vivo study,flow cytometry and biochemical analysis showed that Ni Cl₂ treatment significantly increased ROS level and MDA content in kidney,but significantly decreased T-AOC,CAT,SOD and GSH content.However,after NAC intervention,the effects of Ni Cl₂ on these indexes were alleviated.Moreover,NAC significantly alleviated the histopathological damage caused by Ni Cl₂,and the elevation of CRE and BUN caused by Ni Cl₂ was also inhibited.The results of western blot showed that co-treatment with NAC and Ni Cl₂ decreased LC3Ⅱ/Ⅰand p-AMPKα^Thr172/AMPKαprotein levels,and increased p62,p-AKT^Ser473/AKT and p-m TOR^Ser2448/m TOR protein levels.For the in vitro experiments,flow cytometry and fluorescence microscopy showed that Ni Cl₂ increased ROS levels in TCMK-1 cells.However,the co-treatment with NAC and Ni Cl₂ reduced the ROS level.MTT assay also showed that NAC could alleviate the inhibition of Ni Cl₂ on cell activity.The results of western blot showed that co-treatment with NAC and Ni Cl₂ decreased LC3Ⅱ/Ⅰand p-AMPKα^Thr172/AMPKαprotein levels,and increased p62,p-AKT^Ser473/AKT and p-m TOR^Ser2448/m TOR protein levels.In addition,the results of Mito-TEMPO intervention experiment also showed consistent results with the NAC intervention experiment.Part 2:The role and mechanism of autophagy in nickel-induced apoptosis of renal cell1.Effect of Ni Cl2 on apoptosis in the kidneyThe Ni Cl₂ poisoning model in vivo and in vitro was established by referring to part I.The results from the in vivo study showed that for TUNEL staining,Ni Cl₂ increased the number of TUNEL-positive cells in kidney tissue.The results of western blot and q RT-PCR showed that Ni Cl₂ treatment significantly increased protein levels of Bax/Bcl-2,Cleaved-Caspase-3/8/9 and Cleaved-RARP in the kidney,significantly increased the m RNA expression levels of Bax,Caspase-3,Caspase-8,Caspase-9 and RARP,and significantly decreased Bcl-2 m RNA expression level.In vitro study also revealed that the DAPI staining results showed that Ni Cl₂ increased the number of apoptotic cells in TCMK-1 cells.Flow cytometry showed that Ni Cl₂treatment significantly increased the apoptosis rate of TCMK-1 cells.The results of western blot and q RT-PCR showed that the changing trends of Bax,Bcl-2,Caspase-3/8/9,RARP also were consistent with in vivo experiment.2.The role and mechanism of autophagy in Ni Cl2-induced apoptosis of TCMK-1 cellsAutophagy inhibitor 3-MA and autophagy activator Rapa were used to establish Ni Cl₂poisoning model via autophagy intervention in vitro.Caspase inhibitor Z-VAD-FMK was used to establish Ni Cl₂ poisoning model by apoptosis intervention in vitro.In the autophagy intervention experiment,MTT assay showed that 3-MA could enhance the inhibition of Ni Cl₂ on cell activity,whereas Rapa alleviates the inhibition of Ni Cl₂ on cell activity.Immunofluorescence results showed that the fluorescence intensity of LC3 decreased after co-treatment with 3-MA and Ni Cl_2.The fluorescence intensity of LC3 increased after co-treatment with Rapa and Ni Cl₂.Flow cytometry also showed that the apoptosis rate of cells was significantly increased after co-treatment with 3-MA and Ni Cl₂,the apoptosis rate of cells was significantly decreased after co-treatment with Rapa and Ni Cl₂.In addition,after co-treatment with 3-MA and Ni Cl₂,LC3Ⅱ/Ⅰprotein level was significant decreased,and Cleaved-Caspase-3/8/9 and Cleaved-RARP protein levels were significant increased.After co-treatment with Rapa and Ni Cl₂,LC3Ⅱ/Ⅰprotein level was significant increased,and Cleaved-Caspase-3/8/9 and Cleaved-RARP protein levels were significant decreased.These results suggest that 3-MA enhance Ni Cl₂-induced apoptosis in TCMK-1 cells,whereas Rapa inhibits Ni Cl₂-induced apoptosis in TCMK-1 cells.In the apoptosis intervention experiment,the result of MTT assay showed that Z-VAD-FMK could alleviate the inhibition of Ni Cl₂ on cell activity.DAPI staining results showed that the number of apoptotic cells decreased after Z-VAD-FMK intervention.The results from flow cytometry showed that the apoptosis rate was significantly decreased after Z-VAD-FMK intervention.The results of western blot showed that Z-VAD-FMK treatment significantly decreased protein expression levels of Bax/Bcl-2,Cleaved-Caspase-3/8/9,and Cleaved-RARP.The proteins expression of LC3Ⅱ/Ⅰand p62 were not significantly different between the Ni Cl₂ group and Z-VAD-FMK+Ni Cl₂ group.These results suggest that Z-VAD-FMK can inhibit Ni Cl₂-induced apoptosis in TCMK-1 cells,but has no significant effect on Ni Cl₂-induced autophagy in TCMK-1 cells.3.The role and mechanism of mitophagy in Ni Cl2-induced apoptosis of TCMK-1 cellsThe Ni Cl₂ poisoning model in vivo and in vitro was established by referring to part I.Mitophagy inhibitor Mdivi-1 and mitophagy activator CCCP were used to establish Ni Cl₂poisoning model by mitophagy intervention in vitro.From the in vivo and vitro study,the results of western blot showed that the protein levels of LC3Ⅱ/Ⅰand p62 in mitochondria were significantly increased in Ni Cl₂ treatment groups.These results suggest that Ni Cl₂ can increase the level of mitophagy in vivo and vitro.In the mitophagy intervention experiment,MTT assay showed that Mdivi-1 could enhance the inhibition of Ni Cl₂ on cell activity,and CCCP could alleviate the inhibition of Ni Cl₂ on cell activity.Flow cytometry results showed that the apoptosis rate of cells was significantly increased after co-treatment with Mdivi-1 and Ni Cl₂,however,the apoptosis rate of cells was significantly decreased after co-treatment with CCCP and Ni Cl₂.Co-treatment with Mdivi-1 and Ni Cl₂ significantly decreased the protein expression of LC3Ⅱ/Ⅰand p62 in mitochondria,whereas co-treatment with CCCP and Ni Cl₂ significantly increased the expression of these proteins.The results of apoptotic protein showed that Bax/Bcl-2,Cleaved-Caspase-3/8/9,and Cleaved-RARP protein levels were significantly increased after co-treatment with Mdivi-1 and Ni Cl₂.However,the expression levels of these apoptotic proteins were significantly decreased after co-treatment with CCCP and Ni Cl₂.These results suggest that Mdivi-1 can enhance Ni Cl₂-induced apoptosis in TCMK-1 cells,while CCCP inhibit Ni Cl₂-induced apoptosis in TCMK-1 cells.Conclusion:Autophagy was involved in Ni Cl₂-induced nephrotoxicity of mice,and oxidative stress-mediated AKT and AMPK pathways were important inducements of Ni Cl₂-induced autophagy in renal cells.Mitophagy could inhibit the apoptosis of TCMK-1cells induced by Ni Cl₂,and autophagy plays a protective role in Ni Cl₂-induced nephrotoxicity.