Cadmium Exposure Induces Autophagy Via PLC/IP₃/IP₃R Pathway In Duck Renal Tubular Epithelial Cells

In order to investigate the effect of cadmium（Cd）exposure on autophagy of duck renal tubular epithelial cells through PLC/IP₃/IP₃R pathway,primary duck renal tubular epithelial cells were exposed to 2.5μM and 5.0μM Cd,or co-exposed to 5.0μM Cd and 10.0μM 2-APB or 0.125μM U-73122（pretreatment for 1 h）for 12 h,and then the cell samples were collected and analyzed by flow cytometry,ELISA,transmission microscope,laser confocal microscope,MDC staining,RT-q PCR and Western blotting.The levels of cytoplasmic calcium([Ca²⁺]_c),mitochondrial calcium([Ca²⁺]_mit)and endoplasmic reticulum calcium([Ca²⁺]_ER),PLC activity,IP₃ content and IP₃R protein expression level,the acidic autophagy vesicles,the ultrastructure of autophagy,the LC3 immunofluorescence,and the mRNA and protein expression levels of calcium homeostasis-and autophagy-related genes were measured.The results were as follows:1.Under normal culture(with Ca²⁺medium)conditions,[Ca²⁺]_c of 2.5μM Cd group and 5.0μM Cd group was 1.22 and 1.73 times higher than that of control group,respectively.Under Ca²⁺-free medium conditions,their values were 1.21 and 1.71 times higher than that of control group,respectively.Under the condition of Ca²⁺-free medium,their values were1.21 and 1.71 times higher than that of control group,respectively.Compared with control group,[Ca²⁺]_c and[Ca²⁺]_mit in 2.5μM Cd group and 5.0μM Cd group were significantly increased,and[Ca²⁺]_ER was obviously decreased in a dose-dependent manner.After adding 2-APB to inhibit Ca²⁺outflow from endoplasmic reticulum,compared with control group,[Ca²⁺]_c and[Ca²⁺]_mit in Cd group and 2-APB+Cd group were significantly increased,and[Ca²⁺]_ER were evidently decreased.Compared with Cd group,[Ca²⁺]_c and[Ca²⁺]_mit in 2-APB+Cd group were obviously decreased,and[Ca²⁺]_ER were significantly increased.These results indicated that Cd could cause subcellular calcium redistribution,that is,[Ca²⁺]_ERdecreased,[Ca²⁺]_c and[Ca²⁺]_mit overloaded,and the[Ca²⁺]_c overload was mainly caused by the Ca²⁺outflow from endoplasmic reticulum.2.Compared with control group,the PLC activities,IP₃ contents,IP₃R protein expression levels and the mRNA and protein expression levels of calcium homeostasis related genes（GRP78,GRP94,CRT,CaN,CaMKⅡand CaMKKβ）in 2.5μM group and 5.0μM Cd group were obviously increased.Compared with 2.5μM Cd group,the mRNA and protein expression levels of CRT,CaN and CaMKⅡin 5.0μM Cd group were obviously increased,and the protein expression trends were consistent with their gene expression.After adding U-73122,compared with Cd group,[Ca²⁺]_ER in U-73122+Cd group was significantly increased,[Ca²⁺]_c,[Ca²⁺]_mit,PLC activity,IP₃ content,IP₃R protein expression level,as well as the mRNA expression levels of GRP78,GRP94,CaN,CaMKⅡ,CRT and CaMKKβ,and the protein expression levels of CRT,CaN,and CaMKⅡwere obviously decreased.These results indicated that Cd could cause activation of PLC/IP₃/IP₃R pathway and calcium homeostasis imbalance,and inhibition of this pathway could alleviate Cd-induced calcium homeostasis imbalance.3.Compared with control group,the numbers of autophagosomes,acidic autophagic vacuoles,and LC3 fluorescent spots in Cd group and U-73122+Cd group were increased.Compared with Cd group,the numbers of U-73122+Cd group were reduced.Compared with control group,the mRNA expression levels of Atg5 and Beclin-1 in U-73122 group were significantly decreased,and the mRNA expression level of p62 were significantly increased.The changes in Cd group and U-73122+Cd group were opposite.Compared with Cd group,the mRNA expression levels of LC3A,LC3B,Atg5 and Beclin-1,and the protein expression levels of LC3Ⅱ/LC3Ⅰand Beclin-1 in U-73122+Cd group were significantly decreased,while the mRNA and protein expression levels of p62 were significantly increased.These results indicated that Cd could induce autophagy,and inhibition of PLC/IP₃/IP₃R pathway could alleviate Cd-induced autophagy.Conclusion:Cd exposure led to subcellular calcium redistribution,that is,[Ca²⁺]_ERdecrease,[Ca²⁺]_c and[Ca²⁺]_mit overload,and led to changes in the expression levels of calcium homeostasis related factors.[Ca²⁺]_c overload was mainly derived from the release Ca²⁺of endoplasmic reticulum mediated by PLC/IP₃/IP₃R pathway.[Ca²⁺]_c overload further induced autophagy in duck renal tubular epithelial cells.