Interplay Between Autophagy And Apoptosis In Lead(?)-Induced Cytotoxicity Of Primary Rat Proximal Tubular Cells

Lead is a heavy metal pollutant widely existing in natural environment,which is not easy to be degraded.In vivo,lead can interfere with normal life activities by combining with various proteins,amino acids and other substances in the body,and it is characterized by multiple systems and multiple organs damage to the body.Kidney is an important target organ for toxic damage of lead and renal tubules are target sites for toxic damage of lead and kidney.Previous studies showed that apoptosis is the main way of renal tubular epithelial cell death induced by low dose lead exposure.Autophagy,a dynamic multistage biological process in animals,is an important defense and protection mechanism.However,recent studies have found that autophagy inhibition plays an important role in lead induced renal tubular epithelial cell toxicity.In recent years,the interaction and association between autophagy and apoptosis is a hot topic in the field of life science.However,the mechanism of the interaction between autophagy and apoptosis is not clear yet.In light of this,a lead induced rat renal tubular epithelial(rPT)cell was used to establish an in vitro model of lead induced renal injury,and the role of cell autophagy and apoptosis in lead induced renal toxicity was discussed.First,we set up an in vitro model of lead induced renal injury with lead exposed rat renal tubular epithelial(rPT)cells and explore the effect of Pb exposure on the formation of autophagosome.In experiment,we added a autophagy regulating agent that acts on the formation stage of autophagic,that is,autophagic inhibitor 3-MA and autophagic promoter RAPA,to detect the changes in the level of LC3-II protein in Pb.Meanwhile,Western blotting technology was used to detect the expression of 2 key proteins,ATG5 and Beclin-1,during the formation of autophagy.The final results showed that Pb could promote the formation of autophagosome in rPT cells.To investigate whether autophagy is involved in lead(II)-induced apoptosis in rPT cells,autophagic modulators acting on the stage of autophagosome formation,such as 3-MA and Rapa,were applied to verify this idea.We used immunoblotting to detect the protein levels of cleaved caspase-3 and cleaved PARP;simultaneously flow cytometry was used to detect the apoptosis rate and DAPI staining technique.Confocal microscopy was used to observe the apoptosis morphology of rPT cells.The results obtained by three techniques showed that inhibition of autophagic formation can aggravate lead induced apoptosis,and the promotion of autophagic can inhibit the apoptosis of Pb induced rPT cells.Then we used gene knockout technique to silence autophagy related gene Atg5 and re examined the above indicators.The results further confirmed that the inhibition of autophagic formation can aggravate lead induced apoptosis.Thirdly,autophagy is a highly conserved catabolic process responsible for degradation and reuse of organelles and long-lived proteins by lysosomes.Bafilomycin A1(BA),a known inhibitor of autophagosomes degradation was expected to increase the accumulation of autophagosomes.We added the BA and detected the protein level of LC3-II and p62,the apoptosis marker protein cleaved caspase-3 and cleaved PARP by Western blot,and detected the apoptosis rate,and observed the apoptosis morphology of rPT cells.It was found that lead has the same mechanism of action as BA.It is confirmed that autophagy inhibition is directly involved in the apoptosis induced by lead exposure in rat renal tubular epithelial cells.Finally to fully verify the role of autophagy in lead(II)-induced cytotoxicity in rPT cells,a series of chemical and genetic modulators that inhibited or promoted autophagy,were used to evaluate their effects on lead(II)-induced cytotoxicity in rPT cells.We have found that treatment with autophagy-inhibiting drugs,such as 3-MA,Atg5-siRNA and BA,significantly accelerated lead(II)-induced cytotoxicity while treatment with autophagy enhancer Rapa obviously inhibited lead(II)-induced cytotoxicity in rPT cells.Data give us a solid evidence that autophagy inhibition contributes to lead(II)-induced cytotoxicity.In summary,lead(II)treatment not only promotes the formation of autophagosomes,but also inhibits the degradation of autophagosomes,providing us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells.The interplay between autophagy and apoptosis is implicated in the toxic mechanism of lead(II)-induced cell death in rPT cells,highlighting the importance of their interaction to gain an insight into clinical therapeutic strategy for heavy metal poisoning.This study provided us rational idea and scientific reference for prevention of kidney disease induced by lead contamination.