White Peter, Gu Luo, Chen Jing
Department of Natural Sciences, Colby-Sawyer College, New London, NH, USA.
Clin Physiol Funct Imaging. 2002 Sep;22(5):312-9. doi: 10.1046/j.1475-097x.2002.00437.x.
The microvilli of the apical membrane of proximal tubule (PT) cells are supported by the underlying actin cytoskeleton. Ischaemic or anoxic ATP-depletion leads to the disruption of the actin cytoskeleton, resulting in microvillar retraction and loss of membrane polarity. Using isolated PT cells, we have previously demonstrated that actin filaments (F-actin) are likely severed during ATP-depletion. A sequential extraction protocol revealed a decrease in actin solubility, resulting in the sequestration of a distinct F-actin pool with the insoluble cellular complex in ATP-depleted PT cells. We demonstrate here that decreased actin solubility is not only a reliable end-marker of ATP-depletion induced injury in freshly isolated PT cells, but also serves as a biochemical marker in the cultured proximal tubular cell line LLC-PK1. In the present studies, we also investigated specific actin-binding drugs to determine if they mimic the effects observed during energy depletion. Jasplakinolide (JP), a compound which binds F-actin and prevents depolymerization, did not effect actin solubility during ATP-depletion. Furthermore, swinholide A (SA), an F-actin severing agent, resulted in decreased actin solubility, mimicking the effects of ATP-depletion. Interestingly, latrunculin A (LA), an agent which depolymerizes F-actin, did not reduce actin solubility, but rather resulted in an increase in digitonin-soluble actin. Taken collectively, our results support previous work and suggest that disruption of the actin cytoskeleton during ATP-depletion is mediated by F-actin severing/fragmentation and not depolymerization. The differential effects of F-actin disrupting agents and the consistencies observed in both models of ischaemic injury will provide a basis for a more detailed understanding of the pathological events of PT-cell dysfunction.
近端小管（PT）细胞顶端膜的微绒毛由其下方的肌动蛋白细胞骨架支撑。缺血或缺氧导致的ATP耗竭会致使肌动蛋白细胞骨架遭到破坏，进而引起微绒毛回缩以及膜极性丧失。我们之前利用分离出的PT细胞证实，在ATP耗竭过程中肌动蛋白丝（F-肌动蛋白）可能会发生切断。一种顺序提取方案显示，ATP耗竭的PT细胞中肌动蛋白溶解度降低，导致一个独特的F-肌动蛋白池与不溶性细胞复合物结合。我们在此证明，肌动蛋白溶解度降低不仅是新鲜分离的PT细胞中ATP耗竭诱导损伤的可靠终末标志物，也是培养的近端肾小管细胞系LLC-PK1中的生化标志物。在本研究中，我们还研究了特定的肌动蛋白结合药物，以确定它们是否能模拟能量耗竭期间观察到的效应。茉莉素内酯（JP）是一种能结合F-肌动蛋白并阻止其解聚的化合物，在ATP耗竭期间对肌动蛋白溶解度没有影响。此外，F-肌动蛋白切断剂斯氏藻素A（SA）导致肌动蛋白溶解度降低，模拟了ATP耗竭的效应。有趣的是，F-肌动蛋白解聚剂拉特罗毒素A（LA）并没有降低肌动蛋白溶解度，反而导致洋地黄皂苷可溶性肌动蛋白增加。综合来看，我们的结果支持了之前的研究工作，并表明ATP耗竭期间肌动蛋白细胞骨架的破坏是由F-肌动蛋白切断/断裂介导的，而非解聚。F-肌动蛋白破坏剂的不同效应以及在两种缺血性损伤模型中观察到的一致性，将为更详细地理解PT细胞功能障碍的病理事件提供基础。
