Long Kimberly R, Shipman Katherine E, Rbaibi Youssef, Menshikova Elizabeth V, Ritov Vladimir B, Eshbach Megan L, Jiang Yu, Jackson Edwin K, Baty Catherine J, Weisz Ora A
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Mol Biol Cell. 2017 Sep 15;28(19):2508-2517. doi: 10.1091/mbc.E17-04-0211. Epub 2017 Jul 18.
Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na/K-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.
近端小管（PT）内衬的细胞具有独特的膜特化结构，这对于维持该肾单位节段的高容量离子转运和内吞功能是必需的。体内的PT细胞会根据肾小球滤过率（GFR）的变化急性调节离子转运，以维持球管平衡。培养的PT细胞会根据流体剪切力（FSS）的急性变化上调内吞能力；然而，尚不清楚GFR是否会调节PT内吞作用，以使体内过滤蛋白的摄取效率最大化。在这里，我们表明在持续FSS下培养的细胞会形成扩展的顶端内吞途径、增加内吞能力和溶酶体生物发生。此外，完全分化细胞的内吞能力会因FSS的变化而迅速调节。暴露于持续FSS的PT细胞还获得了广泛的刷状缘和基底外侧膜内陷，类似于在体内观察到的情况。在次优水平的FSS下培养会导致中间表型，表明存在阈值效应。暴露于FSS的细胞表达了更高水平的PT功能所需的关键蛋白，包括离子转运体、受体和膜运输机制，并增加了腺嘌呤核苷酸水平。使用雷帕霉素抑制雷帕霉素机制靶点（mTOR）可防止细胞能量水平、溶酶体生物发生和内吞摄取的增加，表明这些代表了一个协调的分化程序。相比之下，雷帕霉素并不能阻止FSS诱导的Na/K-ATP酶水平的增加。我们的数据表明，FSS变化对内吞反应的快速调节可能有助于体内球管平衡。此外，FSS通过上调内吞作用和离子转运能力的单独途径，为PT细胞的分化提供了必要的刺激。FSS的变化也可能有助于肾脏发育过程中以及肾损伤后修复过程中PT细胞的成熟。
