Myoglobin toxicity in proximal human kidney cells: Roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport
Myoglobin toxicity in proximal human kidney cells: Roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport
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DOI：
10.1038/ki.1997.104
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发表时间：
1997-03-01
影响因子：
19.6
通讯作者：
Burkhart, K
中科院分区：
文献类型：
--
作者：
Zager, RA;Burkhart, K
本研究的目的是直接了解肌红蛋白诱导近端肾小管细胞死亡的机制。为了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10 mg/ml肌红蛋白一起孵育，24小时后评估致死细胞损伤（活体染料摄取; LDH释放）。血红素加氧酶（HO），细胞色素p450，游离铁，细胞内Ca 2+，一氧化氮，H2 O2，羟基自由基（.OH）和线粒体电子传递所发挥的作用进行了评估。HO抑制（Sn原卟啉）赋予几乎完全的针对肌红蛋白细胞毒性的保护（92%对22%细胞活力）。铁螯合疗法（去铁胺）完全再现了这种益处。相反，趋异细胞色素p450抑制剂（西咪替丁，氨基苯并三唑，三乙酰竹桃霉素）没有效果。在5000 U/ml剂量下，过氧化氢酶诱导剂量依赖性细胞保护，几乎完全。相反，.OH清除剂（苯甲酸盐、DMTU、甘露醇）、黄嘌呤氧化酶抑制剂（oxypurinol）、超氧化物歧化酶和一氧化氮表达操纵剂（L-NAME、L-精氨酸）则无作用。细胞内（而非细胞外）钙螯合（BAPTA-AM）导致肌红蛋白诱导的细胞死亡减少50%。Ca 2+（加铁）驱动H2 O2产生（酚红测定）的能力表明了一种潜在的机制。阻断位点2（抗霉素）和位点3（叠氮化物），而不是位点1（鱼藤酮），线粒体电子传递显着降低肌红蛋白的细胞毒性。抑制Na，K-ATP酶驱动的呼吸（哇巴因）产生类似的保护作用。我们的结论是：（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，而不是细胞色素p450，似乎是毒性铁释放的更可能来源;（3）H2 O2的产生，可能由细胞内Ca 2 +/铁促进，似乎起着关键作用;（4）细胞呼吸/末端线粒体电子传递最终有助于介导肌红蛋白的细胞毒性作用。在这个网站上形成的不良特征的有毒铁/H2 O2为基础的反应性中间体似乎可能参与。
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, amino-benzotriazole, 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 similar to 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.