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Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones.

Urinary crystals in normal and kidney stone patients often have varying sizes; the interaction between renal epithelial cells and COD crystals generated in the tubular fluid could play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. This study aims to compare the cytotoxicity of micro/nano-calcium oxalate dihydrate (COD) crystals (50 nm, 100 nm, 600 nm, 3 mm, and 10 mm) toward African green monkey renal epithelial (Vero) cells to reveal the mechanism of kidney stone formation at the molecular and cellular levels. Methods: Vero cells were exposed to COD crystals of varying sizes at a concentration of 200 mg mL À1 for 6 h. The effects of COD crystals on Vero cell viability, apoptosis rate, and cellular biochemical parameters [lactate dehydrogenase (LDH), superoxide dismutase (SOD), reactive oxygen species (ROS), hyaluronic acid (HA), osteopontin (OPN), and mitochondrial membrane potential (Dj m )] were determined using biochemical and morphological analyses. Results: Vero cell viability and apoptotic rate were closely associated with the size of COD crystals; lower cell viability and higher apoptosis rate were observed in cells exposed to smaller COD crystal size. The expression of SOD, ROS, HA and OPN also changed in a size-dependent manner after exposure to the five different sizes of COD crystals. The area ratio of the (100) face with a high density of Ca 2+ ions to the total surface area was also found to influence the severity of cell injury. Cell injury induced by COD crystals was mainly caused by excessive expression of intracellular ROS and reduction of free-radical scavenger SOD. Moreover, binding of large crystals on the cell membrane surface takes more time to cause cell injury than internalized small-sized crystals. The cell death rate was found to be positively correlated with the amount of internalized COD crystals. Conclusions: although the COD toxicity is often disregarded, the size-dependent cytotoxicity of COD crystals toward Vero cells is demonstrated in this study.

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