Human podocytes perform polarized, caveolae-dependent albumin endocytosis
Human podocytes perform polarized, caveolae-dependent albumin endocytosis
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DOI：
10.1152/ajprenal.00532.2013
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发表时间：
2014-05-01
影响因子：
4.2
通讯作者：
Blaine, Judith
中科院分区：
文献类型：
--
作者：
Dobrinskikh, Evgenia;Okamura, Kayo;Blaine, Judith
肾小球形成选择性过滤屏障，允许水、离子和小溶质通过进入泌尿空间，同时限制细胞和大分子通过。肾小球滤过屏障的三层包括血管内皮、肾小球基底膜（GBM）和足细胞上皮。足细胞能够内化白蛋白，并被假设为清除穿过GBM的蛋白质。本研究通过跟踪FITC标记的白蛋白的命运，来建立足细胞对白蛋白的内吞和加工机制。永生化人足细胞的共聚焦成像和全内反射荧光显微镜显示，FITC-白蛋白内吞作用优先发生在基底膜上。网格蛋白介导的内吞作用和小窝介导的内吞作用的抑制表明，大多数FITC-白蛋白通过小窝进入足细胞。一旦内化，FITC-白蛋白与EEA 1和LAMP 1（内吞标记物）以及新生儿Fc受体（转胞吞标记物）共定位。在用FITC-白蛋白预加载足细胞后，在随后的60分钟孵育期间，大部分加载的FITC-白蛋白丢失。一部分白蛋白损失通过溶酶体降解发生，因为用亮抑酶肽（一种溶酶体蛋白酶抑制剂）预处理可部分抑制FITC-白蛋白的损失。与白蛋白的转胞吞作用一致，预装载的足细胞也逐渐将FITC-白蛋白释放到细胞外介质中。这些研究证实了足细胞内吞白蛋白的能力，并提供了对足细胞中白蛋白处理的细胞机制和结局的机理性见解。
The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes.