Role of subcellular calcium redistribution in regulating apoptosis and autophagy in cadmium-exposed primary rat proximal tubular cells

Abstract

Ca2+ signaling plays a vital role in regulating apoptosis and autophagy. We previously proved that cytosolic Ca2+ overload is involved in cadmium (Cd)-induced apoptosis in rat proximal tubular (rPT) cells, but the source of elevated cytosolic Ca2+ concentration ([Ca2+]c) and the effect of potential subcellular Ca2+ redistribution on apoptosis and autophagy remain to be elucidated. Firstly, data showed that Cd-induced elevation of [Ca2+]c was primarily generated intracellularly. Moreover, elevations of [Ca2+]c and mitochondrial Ca2+ concentration ([Ca2+]mit) with depletion of endoplasmic reticulum (ER) Ca2+ levels ([Ca2+]ER) were revealed in Cd-treated rPT cells, but this subcellular Ca2+ redistribution was significantly suppressed by 2-Aminoethoxydiphenyl borate (2-APB). Elevated inositol 1,4,5-trisphosphate (IP3) levels with up-regulated IP3 receptor (IP3R) protein levels were shown in Cd-exposed cells, confirming that IP3R-mediated ER Ca2+ release results in the elevation of [Ca2+]c. Up-regulated sequestosome 1 (p62) protein levels and autophagic flux assay demonstrated that Cd impaired autophagic degradation, while N-acetylcysteine (NAC) markedly attenuated Cd-induced p62 and microtubule-associated protein 1 light chain 3-II (LC3-II) accumulation, implying that the inhibition of autophagic flux was due to oxidative stress. Furthermore, pharmacological modulation of [Ca2+]c with 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and 2-APB alleviated Cd-mediated apoptosis, inhibition of autophagic degradation and subsequent cytotoxicity, while thapsigargin (TG) had the opposite regulatory effect on them. In summary, cytosolic calcium overload originated from IP3R-mediated ER Ca2+ release has a negative impact on Cd nephrotoxicity through its promotion of apoptosis and inhibition of autophagic flux.