Role Of CaMK?-Mediated Intracellular Calcium Redistribution In Lead-Induced Apoptosis Of Renal Tubular Epithelial Cells

The toxic heavy metal lead?Pb?was widely distributed in the ecological environment,and will leading damage of the multi-system and multi-organ in the body.The renal is an important target organ for the toxic effects of lead,and the proximal tubule is the target site for Pb-induced renal injury.Previous studies confirmed that calcium homeostasis-induced apoptosis plays an important role in primary cultures of rat proximal tubular?rPT?cells exposed of Pb.The endoplasmic reticulum?ER?serves as an important calcium store in cells and regulate intracellular calcium homeostasis;subcellular Ca²⁺redistribution mediates ER stress and cell apoptosis.Ca²⁺/calmodulin-dependent protein kinase II?CaMK??is a critical regulator of intracellular calcium signaling and has a role in regulating apoptosis pathway of ER stress.However,the underlying mechanism of CaMK? on intracellular calcium in rPT cells remains to be elucidated,and the detailed mechanism of CaMK? mediate ER stress and apoptosis remains to be studied.Therefore,this study was conducted focusing on the role of CaMK? in the subcellular Ca²⁺redistribution,and the intrinsic link between calcium homeostasis and ER stress in apoptosis in the Pb-exposed rPT cell were explored.First,in order to investigate the effect of lead on ER stress in rPT cells.Western bloting used to detect the levels of the ER stress marker proteins?GRP78,GRP94,CRT,CHOP,caspase-12?by lead exposure.The results showed that Pb induced ER stress,and simultaneously activate ER stress-mediated apoptosis in rPT cell.Secondly,three Ca²⁺modulating agents?2-APB,TG,BAPTA-AM?used to treat rPT cells and examined the levels of[Ca²⁺]c and[Ca²⁺]ER by flow cytometry.As a result,it was found that three Ca²⁺modulators can effectively regulate[Ca²⁺]c and[Ca²⁺]_ERR levels.We analyzed the expression of ER stress marker proteins under the treatment of[Ca²⁺]c and[Ca²⁺]_ERR by western bloting.The results showed that subcellular Ca²⁺redistribution induces ER stress in Pb-exposed rPT cells.Thirdly,western bloting used to detected the protein levels of CaMK??t-CaMK?,p-CaMK??in Pb-exposured rPT cell;As a result,it was found that lead causes activation of CaMK? in rPT cells.Finally,rPT cells treated with two inhibitors of CaMK??KN-93 and KN62?.ER stress-induced apoptosis was detected by western bloting and apoptotic was detected by flow cytometry and nuclear staining.The changes of[Ca²⁺]c and[Ca²⁺]_ERR were detected by laser confocal microscopy.The results showed that CaMK? inhibitors?KN-93 and KN-62?inhibited the phosphorylation of CaMK? and also slowed the ER stress-mediated apoptosis in Pb-exposed rPT cells.Similarly,apoptotic assays also showed that CaMK? inhibitor KN93and KN62 can effectively inhibit lead-induced apoptosis.In addition,treatment with KN93and KN62 significantly ameliorated Pb-induced ER calcium release and cytosolic calcium overload in rPT cells.In summary,in low-concentration Pb-treated rPT cells,activated CaMK? regulates intracellular Ca²⁺homeostasis distribution,thereby inducing a detailed mechanism of ER stress-mediated apoptosis.This study provides the basis for basic research on nephrotoxicity caused by Pb accumulation.