Khaskhali Muhammad H, Byer Karen J, Khan Saeed R
Department of Chemistry, Shah Abdul Latif University, Khairpur, Sindh, Pakistan.
Urol Res. 2009 Feb;37(1):1-6. doi: 10.1007/s00240-008-0160-6. Epub 2008 Nov 13.
Since hypercalciuria is a common feature of idiopathic calcium oxalate (CaOx) nephrolithiasis, renal epithelial cells of stone patients are exposed to various crystals in the presence of high calcium. This study was performed to determine the effect of high calcium levels on CaOx crystal-induced cell injury. We exposed human renal epithelial cell line, HK2 in vitro to CaOx monohydrate crystals at a concentration of 133 microg/cm(2) for 1, 3, 6 or 12 h in the presence or absence of 5 or 10 mM/L calcium Ca(++). We determined the release of lactate dehydrogenase as marker of injury and hydrogen peroxide (H(2)O(2)) and 8-isoprostane (8-IP) as sign of oxidative stress. Cells were also examined after trypan blue and nuclear DNA staining with 4',6-diamidino-2-phenylindole to determine their membrane integrity and apoptosis respectively. Exposure of cells to 5 or 10 mM/L of Ca(++,) for up-to 6 h, resulted in increased trypan blue and DAPI staining and production of H(2)O(2). Similarly an exposure to CaOx crystals also resulted in increased trypan blue and DAPI staining and H(2)O(2) production. An exposure to 5 mM/L Ca or CaOx crystals also resulted in increased production of 8-IP. A combination of the two treatments, Ca and CaOx crystals, did not show anymore changes than exposure to high Ca or CaOx crystals alone, except in the case of a longer exposure of 12 h. Longer exposures of 12 h resulted in cells sloughing from the substrate. These results indicate that exposure to high levels of Ca or CaOx crystals is injurious to renal epithelial cells but the two do not appear to work synergistically. On the other hand, results of our earlier studies suggest that oxalate and CaOx crystals work in synergy, i.e., CaOx crystals are more injurious in the presence of high oxalate. Perhaps Ox and CaOx crystals activate different biochemical pathways while Ca and CaOx crystals affect the identical pathways.
由于高钙尿症是特发性草酸钙（CaOx）肾结石的常见特征，结石患者的肾上皮细胞在高钙环境中会接触到各种晶体。本研究旨在确定高钙水平对CaOx晶体诱导的细胞损伤的影响。我们将人肾上皮细胞系HK2在体外分别暴露于浓度为133μg/cm²的一水合CaOx晶体中1、3、6或12小时，同时存在或不存在5或10 mM/L的钙离子（Ca²⁺）。我们测定了乳酸脱氢酶的释放作为损伤标志物，以及过氧化氢（H₂O₂）和8-异前列腺素（8-IP）作为氧化应激指标。还用台盼蓝和4',6-二脒基-2-苯基吲哚对细胞核DNA进行染色，分别检测细胞的膜完整性和凋亡情况。将细胞暴露于5或10 mM/L的Ca²⁺中长达6小时，会导致台盼蓝和DAPI染色增加以及H₂O₂的产生。同样，暴露于CaOx晶体也会导致台盼蓝和DAPI染色增加以及H₂O₂的产生。暴露于5 mM/L的Ca或CaOx晶体也会导致8-IP的产生增加。除了12小时的较长暴露情况外，Ca和CaOx晶体这两种处理方式的联合作用与单独暴露于高钙或CaOx晶体相比，没有显示出更多变化。12小时的较长暴露导致细胞从底物上脱落。这些结果表明，暴露于高水平的Ca或CaOx晶体对肾上皮细胞有损伤作用，但两者似乎没有协同作用。另一方面，我们早期研究的结果表明，草酸盐和CaOx晶体具有协同作用，即CaOx晶体在高草酸盐存在时更具损伤性。也许草酸盐和CaOx晶体激活不同的生化途径，而Ca和CaOx晶体影响相同的途径。
