Redistribution Of Subcellular Calcium And Its Effect On Apoptosis In Primary Cultures Of Rat Proximal Tubular Cells Exposed To Lead

Lead(Pb) is an important environmental and occupational pollutant which can enter into body through many ways and cause serious damage to human health. As a multi-organ toxicant, lead exerts potent toxic effects on different tissues. Kidney is a sensitive target organ for lead exposure, of which proximal tubular epithelium is the main site of Pb-induced nephrotoxicity. Generally, the toxic mechanism of lead is ascribed to the action of oxidative stress and apoptosis. And our research group found that the apoptotic death induced by oxidative stress played a key role in Pb-induced nephroxicity in vitro. Meanwhile, our previous studies have shown that cytoplasm calcium overload was accompanied by apoptosis in primary cultures of rat proximal tubular(rPT) cells during the Pb exposure. The pro-apoptotic effects of Ca2+ are mediated by a diverse range of Ca2+-sensitive factors that are compartmentalized in various intracellular organelles including the ER, and mitochondria, which are the important intracellular Ca2+ storage compartments. However, the source of elevated Ca2+ and effect of potential subcellular Ca2+ redistribution on apoptosis are still unknown.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 Pb-exposed rPT cells to monitor the imaging of concentrations of Ca2+ in the cytoplasm([Ca2+]c), mitochondria([Ca2+]mit) and ER([Ca2+]ER), respectively, under the confocal microscope. The rPT cells were cultured in Ca2+-containing medium and Ca2+-free medium, changes of [Ca2+]c in Pb-exposed were measured by flow cytometry. Cells were pretreated with 10 μM BAPTA-AM for 30 min, and then exposed to Pb for 12 h to measure changes of [Ca2+]c and [Ca2+]mit, repectively. 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 Pb exposure. Effects of Pb on protein expression levels of two IP3 R isoforms were analyzed by immunoblot; mRNA transcription levels of their encoding genes were assessed by Q-PCR method; the amount of IP3 was determined by specific IP3 [3H] radio receptor assay. Finally, cells were treated with 2-APB or BAPTA-AM in the presence of Pb to measure the apoptotic rates.Data indicate that evated [Ca2+]c induced by Pb exposure was primarily generated intracellularly; elevations of [Ca2+]c and [Ca2+]mit with depletion of [Ca2+]ER were revealed in Pb-treated rPT cells, but this subcellular Ca2+ redistribution could be significantly suppressed by 2-APB. Simultaneously, Pb-mediated mitochondrial Ca2+ overload can be partially suppressed by the cytosolic Ca2+ chelator BAPTA-AM, suggesting that Ca2+ uptake into mitochondria occurs via diverse pathways and ER Ca2+ storage was the chief source. Furthermore, elevated IP3 levels with up-regulated IP3R-1 and IP3R-2(mRNA and protein) levels were revealed in Pb-exposed rPT cells. Additionally, Pb-induced apoptosis was markedly inhibited by 2-APB and BAPTA-AM, respectively. In summary, IP3R-mediated ER Ca2+ release promoted the elevations of [Ca2+]c and [Ca2+]mit in Pb-exposed rPT cells, which played a chief role in apoptosis induced by impaired calcium homeostasis.