Caveolae May Enable Albumin to Enter Human Renal Glomerular Endothelial Cells
Caveolae May Enable Albumin to Enter Human Renal Glomerular Endothelial Cells
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DOI：
10.1002/jcb.25061
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发表时间：
2015-06-01
影响因子：
4
通讯作者：
Nitta, Kosaku
中科院分区：
文献类型：
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作者：
Moriyama, Takahito;Takei, Takashi;Nitta, Kosaku
人肾小球内皮细胞 (HRGEC) 上的小凹在肾小球疾病中增加，并且与蛋白尿的程度相关。为了评估小凹导致白蛋白尿的机制，我们研究了白蛋白是否通过小凹进入 HRGEC。将 HRGEC 与 Alexa Fluor 488 标记的 BSA 或转铁蛋白一起孵育，然后使用小窝的主要结构蛋白 Caveolin-1 (Cav-1) 或网格蛋白包被凹坑的主要结构蛋白网格蛋白的抗体进行免疫荧光定位，以评估 BSA 是否与 Cav-1 共定位。 HRGEC 还与白蛋白和小凹破坏剂（包括甲基 β 环糊精 (MBCD) 和制霉菌素）一起孵育，以确定破坏小凹是否会干扰白蛋白内吞至 HRGEC 中。在用 Cav-1 小干扰 RNA (siRNA) 转染后，HRGEC 还与白蛋白一起孵育。标记的 BSA 与 Cav-1 共定位，但不与网格蛋白共定位。相反，标记的转铁蛋白与网格蛋白共定位，但不与 Cav-1 共定位。蛋白质印迹和免疫荧光显示，与正常 HRGEC 相比，用 MBCD 或制霉菌素孵育 HRGEC，或用 Cav-1 siRNA 转染，细胞内白蛋白和 Cav-1 的量显着减少。这些发现表明白蛋白通过小凹进入 HRGEC，这表明小凹通过提供白蛋白进入肾小球内皮细胞的途径在白蛋白尿的发病机制中发挥重要作用。 (C) 2015 年 Wiley 期刊公司。
Caveolae on human renal glomerular endothelial cells (HRGECs) are increased in glomerular disease and correlate with the degree of albuminuria. To assess the mechanism by which caveolae contribute to albuminuria, we investigated whether albumin enters into HRGECs through caveolae. HRGECs were incubated with Alexa Fluor 488 labeled BSA or transferrin, followed by immunofluorescence localization with antibody to caveolin-1 (Cav-1), the main structural protein of caveolae, or clathrin, the major structural protein of clathrin coated pits, to assess whether BSA colocalized with Cav-1. HRGECs were also incubated with albumin and caveolae disrupting agents, including methyl beta cyclodextrin (MBCD) and nystatin, to determine whether disrupting caveolae interfered with albumin endocytosis into HRGECs. HRGECs were also incubated with albumin after transfection with Cav-1 small interfering RNAs(siRNAs). Labeled BSA colocalized with Cav-1, but not with clathrin. In contrast, labeled transferrin colocalized with clathrin, but not with Cav-1. Incubation of HRGECs with MBCD or nystatin, or transfection with Cav-1 siRNA, significantly reduced the intracellular amounts of albumin and Cav-1, relative to normal HRGECs, as shown by western blotting and immunofluorescence. These findings indicate that albumin enters HRGECs through the caveolae, suggesting that caveolae play an important role in the pathogenesis of albuminuria by providing a pathway through which albumin can enter glomerular endothelial cells. (C) 2015 Wiley Periodicals, Inc.