Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, 630 West 168th St., New York, NY 10032-3784, USA.
Am J Physiol Renal Physiol. 2010 Apr;298(4):F1041-50. doi: 10.1152/ajprenal.00115.2009. Epub 2010 Jan 6.
We previously showed that the inhalational anesthetic isoflurane protects against renal proximal tubule necrosis via isoflurane-mediated stimulation and translocation of sphingosine kinase-1 (SK1) with subsequent synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells (Kim M, Kim M, Kim N, D'Agati VD, Emala CW Sr, Lee HT. Am J Physiol Renal Physiol 293: F1827-F1835, 2007). We also demonstrated that the anti-necrotic and anti-inflammatory effect of isoflurane is due in part to phosphatidylserine (PS) externalization and subsequent release of transforming growth factor-beta1 (TGF-beta1) (Lee HT, Kim M, Kim J, Kim N, Emala CW. Am J Nephrol 27: 416-424, 2007). In this study, we tested the hypothesis that isoflurane, via TGF-beta1 release, increases caveolae formation in the buoyant fraction of the cell membrane of human renal proximal tubule (HK-2) cells to organize SK1 and S1P signaling. To detect SK1 protein in the caveolae/caveolin fractions, we overexpressed human SK1 in HK-2 cells (SK1-HK-2). SK1-HK-2 cells exposed to isoflurane increased caveolae/caveolin formation in the buoyant membrane fractions which contained key signaling intermediates involved in isoflurane-mediated renal tubule protection, including S1P, SK1, ERK MAPK, and TGF-beta1 receptors. Furthermore, treating SK1-HK-2 cells with recombinant TGF-beta1 or PS liposome mixture increased caveolae formation, mimicking the effects of isoflurane. Conversely, TGF-beta1-neutralizing antibody blocked the increase in caveolae formation induced by isoflurane in SK1-HK-2 cells. The increase in SK1 activity in the caveolae-enriched fractions from isoflurane-treated nonlentivirus-infected HK-2 cells, while smaller in magnitude, was qualitatively similar to that found in the SK1-HK-2 cell line. Finally, isoflurane also increased caveolae formation in the kidneys of TGF-beta1 +/+ mice but not in TGF-beta1 +/- mice (mice with reduced levels of TGF-beta1). Our study demonstrates that isoflurane organizes several key cytoprotective signaling intermediates including TGF-beta1 receptors, SK1 and ERK, within the caveolae fraction of the plasma membrane. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.
我们之前的研究表明，吸入麻醉剂异氟烷通过异氟烷介导的鞘氨醇激酶-1（SK1）的刺激和易位以及随后在肾近端小管细胞中合成鞘氨醇-1-磷酸（S1P）来保护肾近端小管坏死（Kim M，Kim M，Kim N，D'Agati VD，Emala CW Sr，Lee HT。Am J Physiol Renal Physiol 293：F1827-F1835，2007）。我们还证明，异氟烷的抗坏死和抗炎作用部分归因于磷脂酰丝氨酸（PS）的外翻和随后转化生长因子-β1（TGF-β1）的释放（Lee HT，Kim M，Kim J，Kim N，Emala CW。Am J Nephrol 27：416-424，2007）。在这项研究中，我们测试了这样一个假设，即异氟烷通过 TGF-β1 的释放，增加人肾近端小管（HK-2）细胞质膜的浮力部分中的小窝形成，以组织 SK1 和 S1P 信号。为了检测小窝/窖蛋白中的 SK1 蛋白，我们在 HK-2 细胞中过表达了人 SK1（SK1-HK-2）。暴露于异氟烷的 SK1-HK-2 细胞增加了浮力膜部分中小窝/窖蛋白的形成，其中包含参与异氟烷介导的肾小管保护的关键信号中间物，包括 S1P、SK1、ERK MAPK 和 TGF-β1 受体。此外，用重组 TGF-β1 或 PS 脂质体混合物处理 SK1-HK-2 细胞可增加小窝形成，模拟异氟烷的作用。相反，TGF-β1 中和抗体阻断了异氟烷诱导的 SK1-HK-2 细胞中小窝形成的增加。与 SK1-HK-2 细胞系相比，异氟烷处理的非慢病毒感染的 HK-2 细胞中小窝富含部分中 SK1 活性的增加虽然幅度较小，但在性质上是相似的。最后，异氟烷还增加了 TGF-β1+/+ 小鼠肾脏中小窝的形成，但在 TGF-β1+/− 小鼠（TGF-β1 水平降低的小鼠）中则没有增加。我们的研究表明，异氟烷将包括 TGF-β1 受体、SK1 和 ERK 在内的几种关键细胞保护信号中间物组织在质膜的小窝部分中。我们的发现可能有助于揭示挥发性麻醉剂介导的肾保护的细胞信号通路，并为围手术期吸入麻醉剂的新治疗应用提供帮助。
