Effects Of Autophagy On Cadmium Induced Apoptosis Of Rat Renal Tubular Epithelial Cells And Its Regulatory Mechanism

Cadmium(Cd)is recognized as one of the most toxic heavy metal of environmental and industrial pollutants,and it causes adverse effects in multi-organ fellowing either acute or chronic exposure.With the increased industrial production and consumption,cadmium pollution is getting worse in recent years.Unlike complex organic pollutants,cadmium cannot be degraded by microorganisms.It has an extremely long biological half-life and can be accumulated in human body through cascading effect of the food chain.Since the adverse health effects caused by cadmium,which provoked a significant public health concern.The kidney is the target organ and the primary accumulation site of chronic cadmium exposure.The research of cadmium induced nephrotoxicity mechanism in aspect of environmental pollution,occupational exposure and animal experiments have been carried out extensively by domestic and foreign scholars.Most of the studies focus on the mechanism of apoptosis,however autophagy and its relationship with apoptosis in the process of cadmium induced nephrotoxicity are relatively few.1.Cadmium induces autophagy in rat renal cortex and proximal tubular cellsThe primary cultures of rat proximal tubular(rPT)cells were cultured by mechanical grinding,filtering and chemical digestive methods.The first passage was used to perform the experimental design when it was in its highest cell viability,NRK-52E cell line was applied with the same treatment.Effects of different concentrations of cadmium exposure for different times on the activity of rPT cells and NRK-52E cells were measured by CCK-8.Effects of different concentrations of cadmium exposed for different times on the autophagy level of rPT cells and NRK-52E cells were measured by MDC staining;After 2.5 ?M and 5 ?M cadmium treated rPT cells,5 ?M and 10 ?M cadmium treated NRK-52E cells,LC31I?p62 and Beclin-1 expression levels were quantified by immunoblotting.The results showed that rPT cells exposed to 2.5?M and 5 ?M cadmium,NRK-52E cells exposed to 5 ?M and 10 ?M cadmium for 6 h were with the highest activity and the highest autophagy level compared with control group(P<0.01).While,cadmium treated for 6 h,the cells were with the lowest autophagy level compared with control group(P<0.01).In vivo studies were carried out on 100±5 g male SD rats.Thirty-two rats were allocated randomly to four groups of eight animals each.The experimental period was one week,(1)Control:rats intraperitoneal injected with saline.(2)Cd treated group:rats intraperitoneal injected with CdAc2(50 mg/kg·bw).The experimental period was eight weeks.(3)Control:rats intraperitoneal injected with saline.(4)Cd treated group:rats intraperitoneal injected with CdAc2(50 mg/kg·bw).Changes of gene expression levels of LC3,p62 and Beclin-1 in the rat renal cortex were quantified by Real Time-PCR(RT-PCR).Changes of protein expression levels of LC3?,p62 and Beclin-1 in the renal cortex were quantified by immunoblotting.The results showed that after one week of treatment,compared with the control group,the autophagy levels was significantly increased(P<0.01)and Beclin-1 expression level was significantly increased compared with control group(P<0.01).While,after eight weeks of treatment,compared with the control group,the autophagy levels was significantly decreased(P<0.01).These results illustrated that low dose of cadmium exposure for a short time enhanced autophagy,while for a long time inhibited autophagy.2.Cadmium induces apoptosis in rat renal cortex and proximal tubular cellsThe primary cultures of rat proximal tubular(rPT)cells were cultured by mechanical grinding,filtering and chemical digestive methods.The first passage was used to perform the experimental design when it was in its highest cell viability,NRK-52E cell line was applied with the same treatment.Effects of different concentrations of cadmium exposed for different time on apoptosis of rPT cells and NRK-52E cells were measured by DAPI staining.After 2.5 ?M and 5 ?M cadmium treated rPT cells,5?M and 10 ?M cadmium treated NRK-52E cells for 12 h,changes of apoptosis rat was measured by flow cytometry and changes of protein expression levels of Cyt C in cytoplasm,Cleaved caspase-9,Cleaved caspase-3,ATF-6,GRP78,IREla,Cleaved caspase-12,Fas,FasL,FADD and Cleaved caspase-8 were quantified by immunob lotting.The results showed that rPT cells exposed to 2.5 ?M and 5?M cadmium,NRK-52E cells exposed to 5 ?M and 10 ?M cadmium for 6 h,the cells were with the lowest apoptosis rate(P<0.01).For 12 h,the cells were with the highest apoptosis rate(P<0.01)and proteins of mitochondrial apoptosis pathway,endoplasmic reticulum apoptosis pathway and Fas/FasL apoptosis pathway were increased significantly,compared with control group(P<0.05 or P<0.01).In vivo studies were carried out on 100±5 g male SD rats.Thirty-two rats were allocated randomly to four groups of eight animals each.The experimental period was one week,(1)Control:rats intraperitoneal injected with saline.(2)Cd treated group:rats intraperitoneal injected with CdAc2(50 mg/kg·bw).The experimental period was eight weeks.(3)Control:rats intraperitoneal injected with saline.(4)Cd treated group:rats intraperitoneal injected with CdAc2(50 mg/kg·bw).One week later,changes of gene expression levels of Caspase-3 in the rat renal cortex were quantified by Real Time-PCR(RT-PCR).Changes of protein expression levels of Cleaved caspase-3 in the renal cortex were quantified by immunoblotting.Eight weeks later,changes of protein expression levels of Cyt C in cytoplasm,Cleaved caspase-9,Cleaved caspase-3,ATF-6,GRP78,IREla,Cleaved caspase-12,Fas,FasL,FADD and Cleaved caspase-8 were quantified by immunoblotting.The results showed that after one week of treatment,there was no significant difference on the gene and protein expression levels of Caspase-3 compared with control group(P>0.05).After eight weeks of treatment,genes and proteins of mitochondrial apoptosis pathway,endoplasmic reticulum apoptosis pathway and Fas/FasL apoptosis pathway were increased significantly(P<0.05 or P<0.01).These results illustrated that low dose of cadmium exposure for a short time activate protective processing and reduced apoptosis,while exposure for a long time induced apoptosis through mitochondrial apoptosis pathway,endoplasmic reticulum apoptosis pathway and Fas/FasL apoptosis pathway simultaneously.3.The relationship between autophagy and apoptosis during cadmium damage in rat renal cortex and proximal tubular cellsThe primary cultures of rat proximal tubular(rPT)cells were cultured by mechanical grinding,filtering and chemical digestive methods.The first passage was used to perform the experimental design when it was in its highest cell viability.Effects of 3-MA and RAPA on apoptosis were detected by flow cytometry.Interaction between Beclin-1 and Cyt C,Caspase-3,Cleaved caspase-3,Caspase-9,Cleaved caspase-9,Bcl-2,Bax,ATF-6,GRP78,IREla,Caspase-12,Cleaved caspase-12,Fas,FasL,FADD,Caspase-8,Cleaved caspase-8 were detected by co-immunoprecipitation.Change of ROS level was measured by DCFH-DA fluorescent probe.Change of number of lysosomes devour mitochondria was measured by transmission electron microscopy.The results showed that 3-MA promoted cadmium induced apoptosis significantly(P<0.01)and RAPA inhibited cadmium induced apoptosis(P<0.01)compared with control group.rPT cells exposed to 2.5 ?M and 5 ?M cadmium for 6 h,Beclin-1 interacted with Cyt Ccyto,Caspase-9,Caspase-3,Baxmito,Caspase-12,and Cleaved caspase-8 in a dose-dependent manner,while interacted with Bcl-2total and Bcl-2mito in negative correlation.Beclin-1 silence significantly inhibited autophagy and promoted apoptosis induced by cadmium compared with cadmium group(P<0.01).rPT cells exposed to 2.5 ?M and 5 ?M cadmium for 6 h,compared with the control,the ROS level was increased significantly(P<0.05 or P<0.01),while compared with 2.5 ?M cadmium group,the ROS level in 5?M cadmium group was decreased significantly(P<0.05)and the number of lysosomes devour mitochondria was increased significantly(P<0.01)in a dose-dependent manner,respectively.These results illustrated that autophagy via both regulating effect of Beclin-1 and elimination of damaged mitochondria to protect cells.