Mechanism of cytotoxicity of micron/nano calcium oxalate monohydrate and dihydrate crystals on renal epithelial cells

Abstract

Urinary crystals in normal and kidney stone patients often contain a larger proportion of micron/nano calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals. However, the effect of their varying sizes and crystal phases in inducing the formation of kidney stones remains unclear. This study aims to comparatively investigate the cytotoxicity and aggregation capabilities of micron/nano COM and COD in vitro to reveal the mechanism of kidney stone formation. The effects of the exposure of African green monkey renal epithelial (Vero) cells to 50 nm (COM-50 nm and COD-50 nm) and 10 μm (COM-10 μm and COD-10 μm) calcium oxalate crystals were investigated by determining cell viability, cell membrane integrity, cell morphology change, adhesion and internalization, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψ_m), cell cycle progression, and cell death rate by apoptosis and/or necrosis. The cell viability and cytomembrane integrity of Vero cells were significantly decreased, in size- and concentration-dependent manners, after the treatment with micron/nano COM and COD crystals. Cell injury increased with the reduction in crystal size and increase in crystal concentration; COM caused a more serious injury in Vero than COD of the same size. COM-10 μm and COD-10 μm caused mild injury in Vero cells because they could only adhere on the cell surface and could not be completely internalized into cells. Meanwhile, the adhered COM-50 nm and COD-50 nm were internalized into cells, caused severe injury, and the effects were more concentration-dependent. Excessive expression of ROS led to a decrease in Δψ_m and cell cycle dysregulation; a series of cell responses ultimately caused a significant number of necrotic cell deaths and few apoptotic cell deaths. Micron-sized COM and COD crystals induced cell injury by damaging the membrane integrity. Nano-COM and COD crystals could damage membrane integrity by adhering to the cell surface, as well as directly damage the mitochondria via internalized crystals. Thus, nano-sized crystals possess greater toxicity than micron-sized crystals.