Barac-Nieto M, Constantinescu A, Pina-Benabou M H, Rozental R
Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA.
Exp Biol Med (Maywood). 2004 Dec;229(11):1162-8. doi: 10.1177/153537020422901111.
In the kidney, cell injury resulting from ischemia and hypoxia is thought to be due, in part, to increased cytosolic Ca(2+) levels, [Ca(2+)]i, leading to activation of lytic enzymes, cell dysfunction, and necrosis. We report evidence of a progressive and exponential increase in [Ca(2+)]i (from 245 +/- 10 to 975 +/- 100 nM at 45 mins), cell permeabilization and propidium iodide (PI) staining of the nucleus, and partial loss of cell transport functions such as Na(+)-gradient-dependent uptakes of (14)C-alpha-methylglucopyranoside and inorganic phosphate ((32)Pi) in proximal convoluted tubules of adult rabbits subjected to hypoxia. The rise in [Ca(2+)]i depended on the presence of extracellular [Ca(2+)] and could be blocked by 50 microM Ni(2+)but not by verapamil (100 microM). Presence of 50 microM Ni(2+) also reduced the hypoxia-induced morphological and functional injuries. We also used HEK 293 cells, a kidney cell line, incubated in media without glucose and exposed for 3.5 hrs to 1% O(2)-5% CO(2) and then returned to glucose-containing media for another 3.5 hrs in an air-5% CO(2) atmosphere and finally exposed for 1 min to media containing 1 microM PI. NiCl(2) (50 microM) or pentobarbital (300 microM) more than phenobarbital (1.5 mM), when present in the incubation medium during both the hypoxic and the reoxygenation periods, induced significant (P < 0.001) reductions in the number of cell nuclei stained with PI, similar to their relative potency as inhibitors of T channels. Our findings indicate that hypoxia-induced alterations in calcium level and subsequent cell injury in the proximal convoluted tubule and in HEK cells involve a nickel-sensitive and dihydropyridine insensitive pathway or channel.
在肾脏中，缺血和缺氧导致的细胞损伤被认为部分归因于胞质钙离子（Ca²⁺）水平升高，即[Ca²⁺]i升高，进而导致溶解酶激活、细胞功能障碍和坏死。我们报告了成年兔近端小管在缺氧时[Ca²⁺]i呈进行性指数升高（45分钟时从245±10 nM升至975±100 nM）、细胞通透性增加、细胞核碘化丙啶（PI）染色以及细胞转运功能部分丧失的证据，如（¹⁴）C-α-甲基葡萄糖苷和无机磷酸盐（³²Pi）的Na⁺梯度依赖性摄取减少。[Ca²⁺]i的升高依赖于细胞外[Ca²⁺]的存在，可被50 μM Ni²⁺阻断，但不能被维拉帕米（100 μM）阻断。50 μM Ni²⁺的存在也减轻了缺氧诱导的形态和功能损伤。我们还使用了肾细胞系HEK 293细胞，将其在无糖培养基中孵育，暴露于1% O₂ - 5% CO₂环境3.5小时，然后在空气 - 5% CO₂环境中回到含葡萄糖的培养基中再孵育3.5小时，最后暴露于含1 μM PI的培养基中1分钟。在缺氧和复氧期，孵育培养基中存在NiCl₂（50 μM）或戊巴比妥（300 μM）时比苯巴比妥（1.5 mM）更能显著（P < 0.001）减少PI染色的细胞核数量，这与它们作为T通道抑制剂的相对效力相似。我们的研究结果表明，缺氧诱导的近端小管和HEK细胞中钙水平改变及随后的细胞损伤涉及镍敏感且对二氢吡啶不敏感的途径或通道。
