A study of oxidant stress induced cellular injury in cultured mesangial cells

为建立大鼠急性肾功能衰竭的体外模型，用1 mM次黄嘌呤(HX)和50U/ml黄嘌呤氧化酶(XO)联合作用于体外培养的大鼠内髓集合管细胞，建立氧化应激模型。氧化应激导致1)细胞内钙离子浓度(Ca)i增加，并通过荧光指示剂碘化丙啶检测细胞存活率下降。2)氧化应激可通过清除细胞外钙、钙拮抗剂维拉帕米和细胞外代谢酸来预防氧化应激所致的细胞损伤。1 mM HX和5u/mlXO联合作用诱导的氧化应激可引起细胞内钙离子浓度升高，细胞损伤由碘化丙啶强度检测。给予钙拮抗剂维拉帕米和细胞外代谢性酸中毒可预防氧化应激引起的细胞损伤。在该协议中，超级…同时应用更多的超氧化物歧化酶(SOD)，但不能提高细胞存活率，在本实验所考察的任何浓度下，100~3000u/ml，加入氢氧化物清除剂过氧化氢酶可显著提高细胞存活率，达到对照水平。采用体外离体管微灌注法灌流PST。1 mM HX对PST的水重吸率(JV)无影响。HX和0.5u/mlXO联合应用可在20~40min内引起刷状缘膜脱落，常见于急性肾小管坏死。在这种情况下，JV明显下降，在40分钟时，JV降至接近零。维拉帕米的应用部分阻止了HX和XO所致的形态和功能改变，而基底外侧代谢性酸中毒完全阻止了这些改变。而SOD对氧化应激所致的体外培养的PST细胞损伤无明显影响。这些结果提示，次黄嘌呤和黄嘌呤氧化酶联合作用可引起体外培养的内髓集合管、系膜细胞和近端直小管的细胞损伤。钙拮抗剂可部分阻止氧化应激所致的细胞损伤，而细胞外代谢性酸中毒则可完全阻止细胞损伤。单独应用超氧化物歧化酶不能预防氧化应激所致的细胞损伤，而与过氧化氢酶联合应用则可完全阻止氧化应激所致的细胞损伤。较少

To aim to produce a in vitro model of acute renal failure, oxidant stress was produced by the combination of 1 mM Hypoxanthine (HX) and 50u/ml Xanthine oxidase (XO) in cultured rat inner medullary collecting duct cells. Oxidant stress induced 1) increased the intracellular calcium ion concentration (Ca) i, and decreased cellular viability examined by a fluorescent indicator, propidium iodide. 2) oxidant stress induced cellular injury was prevented by a removal of extracellular calcium, administration of calcium antagonist, verapamil, and extracellular metabolic acidosis.Secondary, the same maneuvers were applied in cultured mesangial cells. Oxidant stress, induced by the combination of 1 mM HX and 5 u/ml XO,induced the increase of intracellular calcium, and cellular injury, detected by the intensity of propidium iodide. Oxidant stress induced cellular injury was prevented by an administration of calcium antagonist, verapamil, and extracellular metabolic acidosis. In this protocol, supe … More roxide dismutase (SOD) was also applied, however, SOD did not improve cellular viability, at any concentration examined here, 100 u/ml to 3000u/ml. The addition of catalase, an scavenger of hydroxide, to SOD markedly improved cellular viability to the level of control.Thirdly, the same maneuvers were applied in isolated rabbit proximal straight tubules (PST). PST was perfused by in vitro isolated tubular microperfusion. 1 mM HX did not affect the water reabsorption rate (Jv) in PST.The combination of HX and 0.5 u/ml XO induced brush border membrane detachment in 20-40 min, which is usually seen in acute tubular necrosis. In this condition, Jv decreased markedly, and at 40 min.Jv went down to near zero. The application of verapamil partially prevented the morphological changes and functional changes induced by HX and XO.Basolateral metabolic acidosis completely prevented these changes. However, SOD did not afffect the oxidant stress induced cellular damage in isolated PST in vitro.These data suggested that the combination of Hypoxanthine and xanthine oxidase induced cellular damage, in cultured inner medullary collecting duct, cultured mesangial cells and isolated proximal straight tubules in vitro. Calcium antagonist partially prevents oxidant stress induced cellular damage, and extracellular metabolic acidosis prevents cellular damage completely. The administration of SOD alone did not prevent oxidant stress induced cellular injury, however, the combination with catalase prevent it completely. Less