Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations
Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations
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DOI：
10.1152/ajprenal.1999.276.4.f544
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发表时间：
1999-04-01
影响因子：
4.2
通讯作者：
Molitoris, BA
中科院分区：
文献类型：
--
作者：
Schwartz, N;Hosford, M;Molitoris, BA
肾近端小管细胞的顶膜对缺血极为敏感，结构改变发生在5分钟内。这些变化被认为是肌动蛋白细胞骨架破坏的次要原因，但其机制尚不清楚。肌动蛋白解聚因子(ADF)是一种19 kDa的肌动蛋白结合蛋白，最近被证明在肌动蛋白细丝动力学的调节中发挥重要作用。由于ADF是已知的介导依赖于pH的F-肌动蛋白结合、解聚和断裂的媒介，而且ADF的激活是通过去磷酸化发生的，我们质疑ADF是否在缺血时微绒毛微丝破坏中起作用。为了验证我们的假设，我们用钳夹模型诱导了大鼠肾脏缺血。对皮质组织的初步免疫荧光和免疫印迹研究证实ADF存在于近端小管细胞中。在生理条件下，ADF均匀分布于细胞质中，主要分布在TritonX-100可溶组分中，存在磷酸化和非磷酸化两种形式。在缺血期间，发生了显著的变化。腔内小泡/滤泡结构含有极高浓度的ADF和G-肌动蛋白，但不含F-肌动蛋白。Western印迹显示ADF迅速发生时间依赖性的去磷酸化。在缺血0~30min时，总ADF水平无明显变化，而PADF分别在5min和15min显著下降至对照水平的72%和19%。生理条件下采集的尿液中不含ADF或肌动蛋白，而缺血30分钟后采集的尿液中既含有ADF又含有肌动蛋白。再灌流与细胞PADF水平的正常化、PADF在细胞内的分布以及顶端微绒毛的修复有关。这些数据表明，ADF在缺血时通过去磷酸化激活，部分是导致顶端肌动蛋白断裂导致微绒毛破坏和管腔内小泡形成的部分原因。
Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the TritonX-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.