Size-dependent toxicity and interactions of calcium oxalate dihydrate crystals on Vero renal epithelial cells

Abstract

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 μm, and 10 μm) 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 μg mL⁻¹ 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 (Δψ_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 Ca²⁺ 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.