Zager R A, Burkhart K M, Conrad D S, Gmur D J, Iwata M
Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.
J Am Soc Nephrol. 1996 Jan;7(1):64-72. doi: 10.1681/ASN.V7164.
Addition of phospholipase A2 (PLA2) to isolated proximal tubular segments (PTS) has previously been shown to decrease hypoxic cell death without altering ATP concentrations. The study presented here was undertaken to identify determinant(s) of this protection, and to define the spectrum of injuries against which it can operate. PTS were extracted from mouse kidneys and subjected to diverse forms of injury (hypoxia/reoxygenation, antimycin A, Ca2+ ionophore, amphotericin B, FeSO4, and myohemoglobin). In subtoxic doses, addition of PLA2 significantly reduced hypoxic- and antimycin A-induced injury (percentage of lactate dehydrogenase release); however, a dose-dependent exacerbation of all other forms of injury resulted. The ability of PLA2 to mitigate hypoxic injury remained intact despite the inhibition of Na,K-ATPase (ouabain) or the inducement of cytoskeletal disruption (cytochalasin D). However, it was negated by minimally toxic amphotericin B or Ca2+ ionophore doses, indicating its dependence on preserved ionic gradients. Nevertheless, neither lowering/removing buffer Ca2+ or NaCl concentrations, nor hypertonic mannitol addition reproduced the cytoprotective effect of PLA2. PLA2 induced synergistic deacylation in hypoxic tubules, suggesting that unsaturated fatty-acid accumulation might mediate its cytoprotective effect. The fact that the addition of exogenous arachidonate, but not palmitate, to tubules protected against hypoxia, but worsened nonhypoxic forms of injury, supported this hypothesis. Since arachidonate might induce "feedback" inhibition of intracellular PLA2, the ability of an intracellular phospholipase inhibitor (ONO-RS-082; Biomol, Plymouth, PA) to blunt hypoxic damage was tested. This agent fully reproduced the cytoprotective effect of PLA2. It was concluded that: (1) PLA2-induced cytoprotection is relatively specific for ATP depletion injury; (2) it is dependent on, but not explained by, maintenance of NaCl and Ca2+ gradients; (3) it does not require Na,K-ATPase activity or cytoskeletal integrity for its expression; and (4) extracellular PLA2, via arachidonate release, may cause feedback inhibition of intracellular PLA2, thereby protecting critical intracellular targets from attack.
先前的研究表明，向分离的近端肾小管节段（PTS）中添加磷脂酶A2（PLA2）可减少缺氧细胞死亡，且不会改变ATP浓度。本文开展的研究旨在确定这种保护作用的决定因素，并明确其可发挥作用的损伤范围。从小鼠肾脏中提取PTS，并使其遭受多种形式的损伤（缺氧/复氧、抗霉素A、钙离子载体、两性霉素B、硫酸亚铁和肌红蛋白）。在亚毒性剂量下，添加PLA2可显著降低缺氧和抗霉素A诱导的损伤（乳酸脱氢酶释放百分比）；然而，却导致所有其他形式的损伤呈剂量依赖性加重。尽管抑制了钠钾ATP酶（哇巴因）或诱导了细胞骨架破坏（细胞松弛素D），PLA2减轻缺氧损伤的能力依然存在。然而，最低毒性剂量的两性霉素B或钙离子载体可使其作用消失，表明其依赖于维持离子梯度。尽管如此，降低/去除缓冲液中的钙离子或氯化钠浓度，以及添加高渗甘露醇均无法重现PLA2的细胞保护作用。PLA2可诱导缺氧肾小管中的协同脱酰作用，提示不饱和脂肪酸积累可能介导其细胞保护作用。向肾小管中添加外源性花生四烯酸而非棕榈酸可保护其免受缺氧损伤，但会加重非缺氧形式的损伤，这一事实支持了该假说。由于花生四烯酸可能诱导细胞内PLA2的“反馈”抑制，因此测试了一种细胞内磷脂酶抑制剂（ONO-RS-082；Biomol公司，宾夕法尼亚州普利茅斯）减轻缺氧损伤的能力。该药物完全重现了PLA2的细胞保护作用。得出的结论是：（1）PLA2诱导的细胞保护作用对ATP耗竭损伤具有相对特异性；（2）它依赖于氯化钠和钙离子梯度的维持，但无法用其来解释；（3）其表达不需要钠钾ATP酶活性或细胞骨架完整性；（4）细胞外PLA2可通过花生四烯酸释放，引起细胞内PLA2的反馈抑制，从而保护关键的细胞内靶点免受攻击。
