Abrogation of store-operated Ca(2+) entry protects against crystal-induced ER stress in human proximal tubular cells

Calcium crystal internalization into proximal tubular (PT) cells results in acute kidney injury, nephrocalcinosis, chronic kidney disease (CKD), and kidney-stone formation. Ca(2+) supersaturation in PT luminal fluid induces calcium crystal formation, leading to aberrant crystal internalization into PT cells. While such crystal internalization produces reactive oxygen species (ROS), cell membrane damage, and apoptosis; the upstream signaling events involving dysregulation of intracellular Ca(2+) homeostasis and ER stress, remain largely unknown. We have recently described a transepithelial Ca(2+) transport pathway regulated by receptor-operated Ca(2+) entry (ROCE) in PT cells. Therefore, we examined the pathophysiological consequence of internalization of stone-forming calcium crystals such as calcium phosphate (CaP), calcium oxalate (CaOx), and CaP + CaOx (mixed) crystals on the regulation of intracellular Ca(2+) signaling by measuring dynamic changes in Ca(2+) transients in HK2, human PT cells, using pharmacological and siRNA inhibitors. The subsequent effect on ER stress was measured by changes in ER morphology, ER stress-related gene expression, endogenous ROS production, apoptosis, and necrosis. Interestingly, our data show that crystal internalization induced G-protein-coupled receptor-mediated sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) via store-operated Ca(2+) entry (SOCE); suggesting that the mode of Ca(2+) entry switches from ROCE to SOCE following crystal internalization. We found that SOCE components—stromal interacting molecules 1 and 2 (STIM1, STIM2) and ORAI3 (SOCE) channel were upregulated in these crystal-internalized cells, which induced ER stress, ROS production, and cell death. Finally, silencing those SOCE genes protected crystal-internalized cells from prolonged [Ca(2+)](i) rise and ER stress. Our data provide insight into the molecular mechanism of crystal-induced Ca(2+) dysregulation, ER stress, and PT cell death and thus could have a translational role in treating crystal nephropathies including kidney stones. Taken together, modulation of Ca(2+) signaling can be used as a tool to reverse the pathological consequence of crystal-induced conditions including cardiovascular calcification.