Zager R A, Burkhart K
Fred Hutchinson Cancer Research Center, Scattle, Washington, USA.
Kidney Int. 1997 Mar;51(3):728-38. doi: 10.1038/ki.1997.104.
The purpose of this study was to gain direct insights into mechanisms by which myoglobin induces proximal tubular cell death. To avoid confounding systemic and hemodynamic influences, an in vitro model of myoglobin cytotoxicity was employed. Human proximal tubular (HK-2) cells were incubated with 10 mg/ml myoglobin, and after 24 hours the lethal cell injury was assessed (vital dye uptake; LDH release). The roles played by heme oxygenase (HO), cytochrome p450, free iron, intracellular Ca2+, nitric oxide, H2O2, hydroxyl radical (-OH), and mitochondrial electron transport were assessed. HO inhibition (Sn protoporphyrin) conferred almost complete protection against myoglobin cytotoxicity (92% vs. 22% cell viability). This benefit was fully reproduced by iron chelation therapy (deferoxamine). Conversely, divergent cytochrome p450 inhibitors (cimetidine, aminobenzotriazole, troleandomycin) were without effect Catalase induced dose dependent cytoprotection, virtually complete, at a 5000 U/ml dose. Conversely, -OH scavengers (benzoate, DMTU, mannitol), xanthine oxidase inhibition (oxypurinol), superoxide dismutase, and manipulators of nitric oxide expression (L-NAME, L-arginine) were without effect. Intracellular (but not extracellular) calcium chelation (BAPTA-AM) caused approximately 50% reductions in myoglobin-induced cell death. The ability of Ca2+ (plus iron) to drive H2O2 production (phenol red assay) suggests one potential mechanism. Blockade of site 2 (antimycin) and site 3 (azide), but not site 1 (rotenone), mitochondrial electron transport significantly reduced myoglobin cytotoxicity. Inhibition of Na, K-ATPase driven respiration (ouabain) produced a similar protective effect. We conclude that: (1) HO-generated iron release initiates myoglobin toxicity in HK-2 cells; (2) myoglobin, rather than cytochrome p450, appears to be the more likely source of toxic iron release; (3) H2O2 generation, perhaps facilitated by intracellular Ca2+/iron, appears to play a critical role; and (4) cellular respiration/terminal mitochondrial electron transport ultimately helps mediate myoglobin's cytotoxic effect. Formation of poorly characterized toxic iron/H2O2-based reactive intermediates at this site seems likely to be involved.
本研究的目的是直接深入了解肌红蛋白诱导近端肾小管细胞死亡的机制。为避免混淆全身和血流动力学影响，采用了肌红蛋白细胞毒性的体外模型。将人近端肾小管（HK-2）细胞与10mg/ml肌红蛋白孵育，24小时后评估致死性细胞损伤（活体染料摄取；乳酸脱氢酶释放）。评估了血红素加氧酶（HO）、细胞色素p450、游离铁、细胞内Ca2+、一氧化氮、H2O2、羟基自由基（-OH）和线粒体电子传递所起的作用。HO抑制（锡原卟啉）几乎完全保护细胞免受肌红蛋白细胞毒性（细胞活力92%对22%）。铁螯合疗法（去铁胺）完全再现了这种益处。相反，不同的细胞色素p450抑制剂（西咪替丁、氨基苯并三唑、醋竹桃霉素）无效。过氧化氢酶在5000U/ml剂量时诱导剂量依赖性细胞保护，几乎完全保护。相反，-OH清除剂（苯甲酸盐、二甲基硫脲、甘露醇）、黄嘌呤氧化酶抑制（奥昔嘌醇）、超氧化物歧化酶以及一氧化氮表达调节剂（L-NAME、L-精氨酸）无效。细胞内（而非细胞外）钙螯合（BAPTA-AM）使肌红蛋白诱导的细胞死亡减少约50%。Ca2+（加铁）驱动H2O2产生的能力（酚红测定）提示了一种潜在机制。阻断线粒体电子传递的位点2（抗霉素）和位点3（叠氮化物），而非位点1（鱼藤酮），显著降低了肌红蛋白细胞毒性。抑制Na，K-ATP酶驱动的呼吸（哇巴因）产生了类似的保护作用。我们得出结论：（1）HO产生的铁释放引发HK-2细胞中的肌红蛋白毒性；（2）肌红蛋白而非细胞色素p450似乎是有毒铁释放的更可能来源；（3）H2O2的产生，可能由细胞内Ca2+/铁促进，似乎起关键作用；（4）细胞呼吸/线粒体末端电子传递最终有助于介导肌红蛋白的细胞毒性作用。在此部位形成特征不明的基于有毒铁/H2O2的反应性中间体似乎参与其中。
