Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells
Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells
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DOI：
10.1038/ki.1996.378
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发表时间：
1996-09-01
影响因子：
19.6
通讯作者：
Zager, RA
中科院分区：
文献类型：
--
作者：
Iwata, M;Zager, RA
肾毒性损伤后，肾脏修复依赖于肾小管再生。在肌红蛋白尿性急性肾功能衰竭(ARF)的情况下，肌红蛋白在肾小管细胞内持续存在，或在暴露于肌红蛋白的高度时遭受亚致死性损伤，可能会延缓这一过程。为了验证这一假设，在没有或存在临床相关肌红蛋白浓度(0.5、1、2、4 mg/ml)的情况下，对人近端肾小管细胞系(HK-2)进行了24小时的培养。肌红蛋白去除后立即导致致命细胞损伤(重要染料摄取)、脂质过氧化。检测DNA损伤(碱性解旋试验)。通过四甲基偶氮唑盐(四甲基偶氮唑蓝)试验和细胞内总乳酸脱氢酶(LDH)的测定，估计在接下来的恶劣天气中细胞增殖的程度。肌红蛋白对蛋白质和脱氧核糖核酸合成的影响也被评估(S-35-蛋氨酸和溴脱氧尿苷掺入分别)。肌红蛋白诱导剂量依赖性的脂质过氧化(丙二醛的产生)和细胞死亡(在4 mg/ml的浓度下，高达80%的活性染料摄取)。虽然1 mg/ml的肌红蛋白没有引起细胞死亡，但却导致了几乎完全的生长停滞。在肌红蛋白从培养液中去除后，这种情况持续了大约三天。两种对照蛋白(白蛋白：溶菌酶)和第二种肾毒素(庆大霉素；1 mg！ml)都不能复制这种效应。肌红蛋白浓度为1 mg/ml时，蛋白质和柔红霉素的合成受到80%~90%的抑制。通过碱性解旋试验(P<；0.01)，它还引起了显著的DNA损伤。铁络合疗法(脱氧乙胺)可减轻肌红蛋白诱导的细胞杀伤。然而，随着肌红蛋白的负载，它的加入通过发挥直接的抗增殖作用而恶化了HK-2的生长。这些结果表明：(1)亚致死性肌红蛋白毒性可诱导近端肾小管细胞生长停滞，潜在地减缓ARF的恢复；(2)这种作用与血红素诱导的DNA损伤和DNA/蛋白质合成受阻有关；(3)去铁胺可抑制近端肾小管细胞的增殖。在设计临床试验VIIth DFO治疗肌红蛋白尿性ARF时需要考虑这种可能性。
Following nephrotoxic injury, renal repair is dependent on tubular regeneration. In the case of myoglobinuric acute renal failure (ARF), persistence of myoglobin within tubular cells, or sublethal injury sustained at the height of exposure to it, might retard this process. To test this hypothesis, a human proximal tubular cell line (HK-2) was cultured for 24 hours in the absence or presence of clinically relevant myoglobin concentrations (0.5, 1, 2, 4 mg/ml). Immediately following myoglobin removal, lethal cell injury (vital dye uptake), lipid peroxidation. and DNA damage (alkaline unwinding assay) were assessed. The extent of cell proliferation was estimated over the next foul days by a tetrazolium based (MTT) assay and by determining total intracellular LDH. Myoglobin's effects on protein and DNA synthesis were also assessed (S-35-methionine and bromodeoxyuridine incorporation, respectively). Myoglobin induced dose-dependent lipid peroxidation (malondialdehyde generation) and cell death (up to 80% vital dye uptake with the 4 mg/ml challenge). Although 1 mg/ml myoglobin caused no cell death, it induced nearly complete growth arrest. This lasted for approximately three days following myoglobin removal from the media. Neither of two control proteins (albumin: lysozyme) nor a second nephrotoxin (gentamicin; 1 mg!ml) reproduced this effect. The 1 mg/ml myoglobin challenge caused an 80 to 90% depression in protein and DNR synthesis. It also induced significant DNA damage, as assessed by the alkaline unwinding assay (P < 0.01). Iron chelation therapy (defetoxamine) mitigated myoglobin-induced cell killing. However, its addition following myoglobin loading worsened HK-2 outgrowth by exerting a direct anti-proliferative effect. These results indicate that: (i) sublethal myoglobin toxicity can induce transient proximal tubular cell growth arrest, potentially slowing recovery from ARF: (2) this effect correlates with, and could result from, heme-induced DNA damage and a blockade in DNA/protein synthesis, and (3) deferoxamine can inhibit proximal tubular cell proliferation. This possibility needs to be considered in designing clinical trials viith DFO for myohemoglobinuric ARF.