Size-dependent cellular uptake mechanism and cytotoxicity toward calcium oxalate on Vero cells.

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.

Adhesion and internalization differences of COM nanocrystals on Vero cells before and after cell damage.

The adhesion and internalization between African green monkey kidney epithelial (Vero) cells (before and after oxidative damage by hydrogen peroxide) and calcium oxalate monohydrate (COM) nanocrystals (97±35nm) were investigated so as to discuss the molecular and cellular mechanism of kidney stone formation. Scanning electron microscope (SEM) was used to observe the Vero-COM nanocrystal adhesion; the nanocrystal-cell adhesion was evaluated by measuring the content of malonaldehyde (MDA), the activity of superoxide dismutase (SOD), the expression level of cell surface osteopontin (OPN) and the change of Zeta potential. Confocal microscopy and flow cytometry were used for the observation and quantitative analysis of crystal internalization. In the process of adhesion, the cell viability and the SOD activity declined, the MDA content, Zeta potential, and the OPN expression level increased. The adhesive capacity of injured Vero was obviously stronger than normal cells; in addition the injured cells promoted the aggregation of COM nanocrystals. The capacity of normal cells to internalize crystals was obviously stronger than that of injured cells. Cell injury increased adhesive sites on cell surface, thereby facilitating the aggregation of COM nanocrystals and their attachment, which results in enhanced risk of calcium oxalate stone formation.

Renal Epithelial Cell Injury Induced by Calcium Oxalate Monohydrate Depends on their Structural Features: Size, Surface, and Crystalline Structure.

Urinary crystals in normal and kidney stone patients often differ in crystal sizes and surface structures, but the effects of different crystal properties on renal tubular epithelial cells remain unclear. This study aimed to compare the cytotoxicity of micron/nano-calcium oxalate monohydrate (COM) crystals with sizes of 50 nm, 200 nm, 1 µm, 3 µm, and 10 µm to African green monkey renal epithelial (Vero) cells, to reveal the effect of crystal size and surface structure on cell injury, and to investigate the pathological mechanism of calcium oxalate kidney stones. Cell viability, cellular biochemical parameters, and internalized crystal amount in Vero cells were closely associated with the size of COM crystals. At the same concentration (200 µg/mL), COM-1 µm induced the most serious injury to Vero cells and caused the most significant change to cellular biochemical parameters, which were related to the specific porous structure and highest internalized amount in Vero cells. By contrast, COM-50 nm and COM-200 nm crystals lost their small size effect because of serious aggregation and weakened their toxicity to cells. COM-3 µm and COM-10 µm crystals were too large for cells to completely internalize; these crystals also exhibited a low specific surface area and thus weakened their toxicity. The excessive expression of intracellular ROS and reduction of the free-radical scavenger SOD were the main reasons for cell injury and eventually caused necrotic cell death. Crystal size, surface structure, aggregation, and internalization amount were closely related to the cytotoxicity of COM crystals.

Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation.

BACKGROUND: Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear. METHODS: African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin-eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry. RESULTS: The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased. CONCLUSION: Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone formation.

Preparation, characterization, and in vitro cytotoxicity of COM and COD crystals with various sizes.

Calcium oxalate crystals in urine often differ in size and crystal phase between healthy humans and patients with kidney stones. In this work, calcium oxalate monohydrate (COM) and dihydrate (COD) with sizes of about 50 nm, 100 nm, 1 µm, 3 µm, and 10 µm were prepared by varying reactant concentration, reaction temperature, solvent, mixing manner, and stirring speed. These crystals mainly had a smooth surface and no obvious pore structure, except COM-1 µm. In cell culture medium, the zeta potential of crystals became increasingly negative with increasing size, and the absolute value of zeta potential of COD was greater than the same-sized COM. Results of cell viability and PI staining assays showed that the order of injury degree in African green monkey renal epithelial (Vero) cells caused by different sizes of COD was COD-50 nm>COD-100 nm>COD-1 µm>COD-3 µm>COD-10 µm, and that of different sizes of COM was COM-1 µm>COM-50~COM-100 nm>COM-3 µm>COM-10 µm. COM-1 µm presented the highest cytotoxicity in Vero cells, which was associated with its rougher surface, larger specific surface area (SBET), and larger pore volume. Overall, these findings indicated that the physical properties of crystals play an important role in their cytotoxicity.

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

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 (Δψm)] 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.