Abstract
Calcium crystal internalization into proximal tubular (PT) cells results in acute kidney injury, nephrocalcinosis, chronic kidney disease (CKD), and kidney-stone formation. Ca2+ 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 Ca2+ homeostasis and ER stress, remain largely unknown. We have recently described a transepithelial Ca2+ transport pathway regulated by receptor-operated Ca2+ 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 Ca2+ signaling by measuring dynamic changes in Ca2+ 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 Ca2+ concentration ([Ca2+]i) via store-operated Ca2+ entry (SOCE); suggesting that the mode of Ca2+ 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 [Ca2+]i rise and ER stress. Our data provide insight into the molecular mechanism of crystal-induced Ca2+ 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 Ca2+ signaling can be used as a tool to reverse the pathological consequence of crystal-induced conditions including cardiovascular calcification.
Highlights
IntroductionCrystals are often present in the renal tubular fluid of both stone-forming and nonstone-forming individuals[1], inhibitors of crystallization rapidly cover such crystals
Crystals are often present in the renal tubular fluid of both stone-forming and nonstone-forming individuals[1], inhibitors of crystallization rapidly cover such crystalssuch elimination processes could be hindered, when the crystals adhere to the tubular lining[3]
We found that crystal internalization elicited an increase in Ca2+ release and produced a greater response in the Ca2+ rise due to Ca2+ entry compared with the noninternalized cells (Fig. 1a–d)
Summary
Crystals are often present in the renal tubular fluid of both stone-forming and nonstone-forming individuals[1], inhibitors of crystallization rapidly cover such crystals. Such elimination processes could be hindered, when the crystals adhere to the tubular lining[3]. Several studies have proposed that the onset of nephrolithiasis lies within the nephron, moreso, renal biopsies of some stone-formers presented with crystals inside renal tubular cells[4,5]. Thereafter, many in vitro studies have shown that calcium crystals, such as CaOx monohydrate, are taken up by renal tubular cells and subsequently influence the process of kidney-stone formation[6]. Crystal size and shape determine the extent of internalization in various tubular cell types[9]
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