Difference of injury mechanisms on renal epithelial cells induced by micron/nano COM and COD crystals
Difference of injury mechanisms on renal epithelial cells induced by micron/nano COM and COD crystals
复制标题
微米/纳米COM与COD晶体对肾上皮细胞损伤机制的差异
作者：
Sun X-Y;Ouyang J-M;Li Y-B;Wen X-L
正常人和肾结石患者的尿液晶体通常含有较大比例的微米/纳米草酸钙一水合物（COM）和草酸钙二水合物（COD）晶体。然而，它们的不同尺寸和晶相在诱导肾结石形成方面的差异仍不清楚。本研究旨在比较研究微米/纳米COM和COD的细胞毒性和体外聚集能力，以揭示肾结石的形成机制。曝光50 nm的效果（COM-50 nm和COD-50 nm）和10 μm通过检测细胞活力、细胞膜完整性、细胞形态、细胞粘附和内化、细胞内活性氧（ROS）、线粒体膜电位（Δ Km）、细胞凋亡率、细胞凋亡率等指标，研究了草酸钙晶体（COM-10μm和COD-10μm）对非洲绿色猴肾上皮（Vero）细胞的作用，细胞周期进程和细胞凋亡和/或坏死导致的细胞死亡率。微米/纳米COM和COD晶体处理后，非洲绿猴肾细胞的细胞活力和细胞膜完整性显着下降，且呈尺寸和浓度依赖性。细胞损伤随晶体尺寸的减小和晶体浓度的增加而增加; COM对Vero细胞的损伤比相同尺寸的COD更严重。COM-10μm和COD-10μm对Vero细胞的损伤较轻，因为它们只能粘附在细胞表面，不能完全内化到细胞内。同时，粘附的COM-50 nm和COD-50 nm进一步内化到细胞中，造成严重损伤，且作用更具浓度依赖性。ROS的过度表达进一步导致Δ λ m降低和细胞周期失调;一系列细胞反应最终导致大量坏死细胞死亡和少数凋亡细胞死亡。10 μ m大小的COM和COD晶体通过破坏膜完整性诱导细胞损伤。纳米COM和COD晶体可以通过粘附晶体破坏膜的完整性，以及通过内化晶体直接损伤线粒体。因此，纳米尺寸的晶体比微米尺寸的晶体具有更大的毒性。
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 difference of their varying sizes and crystal phases in inducing the formation of kidney stone remains unclear. This study aims to investigate comparatively the cytotoxicity and aggregation capabilities of micron/nano COM and COD in vitro to reveal the mechanism of kidney stone formation. The effect of the exposure 50 nm (COM–50nm and COD–50nm) and 10 μm (COM–10μm and COD–10μm) calcium oxalate crystals toward the African green monkey renal epithelial (Vero) cells were investigated by detecting cell viability, cell membrane integrity, cell morphology change, adhesion and internalization, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), 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 of 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 with the same size. COM-10μm and COD-10μm caused mild injury in Vero because they could only adhere on the cell surface and could not be completely internalized into cells. Meanwhile, the adhered COM-50nm and COD-50nm were further internalized into cells, caused severe injury, and the effects were more concentration-dependent. Excessive expression of ROS further led to the decrease of Δψm and cell cycle dysregulation; a series of cell response ultimately caused 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 crystals, as well as directly damage the mitochondria by internalized crystals. Thus, Nano–sized crystals possess greater toxicity than micron–sized crystals.