Renal-Electrolyte Division, Department of Medicine, and.
Renal-Electrolyte Division, Department of Medicine, andDepartment of Cell Biology, University of Pittsburgh, Pittsburgh, PA 15213.
Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8506-11. doi: 10.1073/pnas.1402195111. Epub 2014 May 27.
The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We found that FSS also affects apical endocytosis in PT cells. Exposure of immortalized PT cell lines to physiologically relevant levels of FSS led to dramatically increased internalization of the megalin-cubilin ligand albumin as well as the fluid phase marker dextran. FSS-stimulated apical endocytosis was initiated between 15 and 30 min postinduction of FSS, occurred via a clathrin- and dynamin-dependent pathway, and was rapidly reversed upon removing the FSS. Exposure to FSS also caused a rapid elevation in intracellular Ca(2+) [Ca(2+)]i, which was not observed in deciliated cells, upon treatment with BAPTA-AM, or upon inclusion of apyrase in the perfusion medium. Strikingly, deciliation, BAPTA-AM, and apyrase also blocked the flow-dependent increase in endocytosis. Moreover, addition of ATP bypassed the need for FSS in enhancing endocytic capacity. Our studies suggest that increased [Ca(2+)]i and purinergic signaling in response to FSS-dependent ciliary bending triggers a rapid and reversible increase in apical endocytosis that contributes to the efficient retrieval of filtered proteins in the PT.
肾脏具有在广泛的肾小球滤过率 (GFR) 范围内维持稳定的溶质和液体重吸收分数的非凡能力。过滤的低分子量蛋白质、维生素、激素和其他小分子的内化是由近端小管 (PT) 多配体受体 megalin 和 cubilin 介导的。GFR 的变化和伴随的流体切应力 (FSS) 调节 PT 离子转运的急性变化，据认为这些变化是由微绒毛弯曲介导的。我们发现 FSS 还会影响 PT 细胞的顶端内吞作用。将永生化的 PT 细胞系暴露于生理相关水平的 FSS 会导致 megalin-cubilin 配体白蛋白以及液相标记物右旋糖酐的内化显著增加。FSS 刺激的顶端内吞作用在诱导 FSS 后 15 至 30 分钟开始，通过网格蛋白和动力蛋白依赖性途径发生，并在去除 FSS 后迅速逆转。暴露于 FSS 还会导致细胞内 Ca(2+) [Ca(2+)]i 的快速升高，在用 BAPTA-AM 处理或在灌注培养基中包含 apyrase 时，未观察到纤毛去极化的细胞中观察到这种情况。引人注目的是，纤毛去极化、BAPTA-AM 和 apyrase 也阻断了依赖流动的内吞作用增加。我们的研究表明，对 FSS 依赖性纤毛弯曲的增加 [Ca(2+)]i 和嘌呤能信号转导会触发顶端内吞作用的快速和可逆增加，这有助于在 PT 中有效回收过滤的蛋白质。
