Role Of Subcellular Calcium Redistribution In Regulating Apoptosis And Autophagy In Cadmium-Exposed Primary Rat Proximal Tubular Cells

Cadmium?Cd?is a widespread heavy metal pollutant in the environment,it exerts toxic effects on multiple system and organs in mammals.Kidney is the most sensitive target organs of toxicity,where Cd accumulates primarily in the proximal tubule of the nephron.Our research group found that oxidative stress-mediated apoptotic death played a key role in Cd-induced nephrotoxicity in vitro;moreover,it is important to note that intracellular Ca2+overload is involved in this process.Calcium ion?Ca2+?is a ubiquitous secondary messenger involved in various cellular processes.And Ca2+ signaling plays a vital role in regulating apoptosis and autophagy.But it has not been reported the source of Cd-induced [Ca2+]c elevation and the role of subcellular calcium redistribution in regulating apoptosis and autophagy in cadmium-exposed primary rat proximal tubular cells.Based on this,this study will offer further evidences to elucidate the regulatory effect of elevated [Ca2+]c on autophagy and apoptosis in Cd-exposed rPT cells.It will provide theoretical basis for Cd-induced nephrotoxicity.First,the source of Cd-induced [Ca2+]c elevation was studied.In this study,cytosolic Ca2+ dye Fluo-4-AM,mitochondrial Ca2+ sensitive indicator dihydro-Rhod-2-AM and endoplasmic reticulum?ER?Ca2+ probe Mag-Fluo-4-AM was loaded into Cd-exposed rPT cells to monitor the concentrations of Ca2+ in the cytoplasm?[Ca2+]c?,mitochondria?[Ca2+]mit?and ER?[Ca2+]ER?,respectively by flow cytometry.The rPT cells were cultured in Ca2+-containing medium and Ca2+-free medium,changes of [Ca2+]c in Cd-exposed were measured by flow cytometry.Then the changes of [Ca2+]c,[Ca2+]mit and [Ca2+]ER were measured under Cd exposure.Then cells were co-incubated with 2-APB,a specific inhibitor of inositol 1,4,5-trisphosphate receptor?IP₃R?that functions to release Ca2+ from ER stores,to assess the changes of [Ca2+]ER,[Ca2+]c and [Ca2+]mit during the Cd exposure.The amount of IP₃ was determined by specific IP₃ [3H] radio receptor assay;of Cd on protein expressionlevels of two IP₃ R isoforms were analyzed by immunoblot.Taken together,these findings supported that elevated IP₃ levels activated IP₃ R to facilitate ER-Ca2+ release in Cd-exposed rPT cells.Then,effect of Cd-induced [Ca2+]c elevation on apoptosis was investigated.BAPTA-AM,TG and 2-APB were applied to assess its effect on Cd-induced apoptosis,respectively.Apoptosis is characterized morphologically by condensation and fragmentation of nuclei.Therefore,Hoechst 33,258 staining was firstly applied to assess the morphological changes of treated cells.And flow cytometry,a quantitative analysis of apoptosis,was applied too.Then cell viability was performed using the CCK-8.Given the above results,we can confirm that Cd-induced [Ca2+]c elevation leads to cytotoxicity in rPT cells.Next,the relationship between elevation of [Ca2+]c and autophagy was studied.In order to observe the effect of elevation of [Ca2+]c on autophagic flux and aotophagosome.We measured the expression status of p62 and LC3-II in rPT cells,and transfected cells with RFP-GFP-LC3 and GFP-LC3 plasmid,demonstrating that Cd-induced [Ca2+]c elevation inhibited the autophagic flux and caused autophagosome accumulation in rPT cells.To investigate the role of intracellular Ca2+ in the regulation of autophagosome-lysosome fusion during Cd exposure.Firstly,we analyzed the co-localization of LC3 with LAMP-1,and next we assess the co-localization of LC3 with Rab7.These data suggested that Cd-induced [Ca2+]c elevation inhibited the fusion of autopahgosomes with lysosomes in rPT cells via alternation of localization of Rab7.Finally,we investigated the relationship between oxidative stress and autophagy.To verify whether there is a link between ROS production and elevated [Ca2+]c in this process,changes of ROS levels were assessed by using three calcium modulators.Then,the ROS scavenger,NAC,was applied to treat cells.The expression level of p62 and LC3-II was measured by western blotting in rPT cells.Then,we analyzed the co-localization of LC3 with LAMP-1 and LC3 with Rab7.These data suggested that oxidative stress triggered by cytosolic Ca2+ elevation inhibited the autophagosome-lysosome fusion in Cd-exposed rPT cells.In summary,Cd induced an subcellular calcium redistribution in rPT cells,i.e.,elevations of [Ca2+]c,[Ca2+]mit and decreased [Ca2+]ER.Stimulated ER Ca2+ release due to theactivation of IP₃ Rs is responsible for the increase in [Ca2+]c.Furthermore,elevated [Ca2+]c can promote apoptosis and suppress autophagic flux,demonstrating that disturbance in intracellular Ca2+ signaling regulated by ER plays a dual role in Cd-induced cytotoxicity in rPT cells.Elevated [Ca2+]c blocked the recruitment of Rab7 to autophagosomes,leading to the blockage of autophagosome-lysosome fusion,and resultantly promoted the autophagosome accumulation in Cd-exposed rPT cells.Moreover,Cd-induced elevation of [Ca2+]c triggered ROS overproduction in rPT cells,which exhibited a similar inhibitory effect on autophagy as cytosolic Ca2+ overload.