Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells.
Cytoprotective effects of calbindin-D(28k) against antimycin-A induced hypoxic injury in proximal tubular cells.
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DOI：
10.1016/s0024-3205(02)01710-1
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发表时间：
2002-06
期刊：
影响因子：
6.1
通讯作者：
Ming-Ju Wu;L. Lai;Y. Lien
中科院分区：
文献类型：
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作者：
Ming-Ju Wu;L. Lai;Y. Lien
细胞内钙离子在缺氧性细胞损伤的发病机制中起重要作用。钙结合蛋白-D28 k是一种依赖维生素D的钙结合蛋白，可作为一种缓冲剂，抑制细胞内钙离子浓度（[Ca 2 +]i）的升高。为探讨钙结合蛋白-D28 k对近端肾小管上皮细胞缺氧损伤的保护作用，将CMV即刻早期基因启动子调控下的钙结合蛋白-D28 k cDNA质粒转染小鼠近端肾小管上皮细胞（MCT）。转染细胞经北方印迹分析、Western印迹分析和免疫荧光染色证实钙结合蛋白-D28的表达。将未转染和转染的MCT细胞置于抗霉素A（10 μM）诱导的化学缺氧和葡萄糖剥夺30-120 min。在30，60，与未转染的细胞相比，缺氧后90和120分钟（所有p < 0.05）。缺氧损伤后，转染细胞的细胞活力也显着高于非转染细胞。转染无钙结合蛋白-D28 kcDNA的质粒不影响化学缺氧损伤后LDH释放或细胞活力。[Ca+ 2]i在暴露于化学缺氧后用Fura-2比率测量。转染细胞内[Ca ~（2+）]i的初始上升速率和30-120 min的最终[Ca ~（2+）]i显著降低。本研究结果表明，转染Calbindin-D28 k基因的MCT细胞对化学性缺氧损伤具有保护作用，其机制可能与其缓冲细胞内[Ca+2]i有关。
Intracellular calcium plays an important role on the pathogenesis of hypoxia-induced cellular injury. Calbindin-D28k, a cytosolic vitamin D-dependent calcium binding protein, can serve as a buffer to limit a surge in intracellular Ca2+concentration ([Ca2+]i) induced by various stimulations. To evaluate the possible cytoprotective effect of calbindin-D28kagainst hypoxic injury in proximal tubular cells, a plasmid containing calbindin-D28kcDNA under the control of CMV immediate-early gene promoter was transfected into the murine proximal tubular epithelial (MCT) cells. The expression of calbindin-D28kin the transfected cells was verified with Northern blot analysis, Western blot analysis, and immunofluorescent staining. The non-transfected and transfected MCT cells were subjected to chemical hypoxia induced by antimycin A (10 μM) and glucose deprivation for 30–120 min. The transfection of calbindin-D28kreduced lactate dehydrogenase (LDH) release by 41%, 41%, 24%, and 24%, respectively, at 30, 60, 90 and 120 min after hypoxia when compared to the non-transfected cells (all p < 0.05). Cell viability after hypoxic injury was also significantly higher in transfected cells than non-transfected cells. Transfection with the plasmid without calbindin-D28kcDNA did not affect LDH release or cell viability after chemical hypoxic injury. [Ca+2]i was measured ratiometrically with fura-2 after exposure to chemical hypoxia. The rate of initial rise in [Ca2+]i and final [Ca+2]i at 30–120 min were significantly lowered in transfected cells. In conclusion, this study demonstrated that transfection of calbindin-D28kgene into MCT cells provide protective effects against chemical hypoxic injury probably through its buffering effects on [Ca+2]i.