Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney
Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney
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DOI：
10.1152/ajprenal.00316.2006
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发表时间：
2007-09-01
影响因子：
4.2
通讯作者：
Praetorius, Jeppe
中科院分区：
文献类型：
--
作者：
Fenton, Robert A.;Brond, Lone;Praetorius, Jeppe
精氨酸加压素 (AVP) 对于维持体液稳态至关重要。 AVP 的抗利尿作用是通过 AVP 与肾集合管 (CD) 中的 2 型加压素受体 (V2R) 结合而启动，导致水通道蛋白 2 (AQP-2) 水通道胞吐插入顶端质膜。在本研究中，我们描述了针对大鼠 V2R NH2 末端的多克隆抗体的生成和表征。过表达大鼠、小鼠或人 V2R 的 HEK-293 细胞显示出强烈的细胞内免疫标记。此外，表达 V2R-绿色荧光蛋白 (GFP) 融合构建体的 M-1 肾细胞的免疫染色显示 GFP 和抗体特异性 V2R 标记之间存在共定位。大鼠肾脏的免疫印迹显示所有区域中的 43-和 47-kDa 蛋白质均被 N-糖苷酶 F 还原为 34-kDa。用非离子去垢剂溶解蛋白质或使用同型双功能交联剂证明大鼠 V2R 在天然肾脏中以蛋白质复合物形式存在。大鼠和小鼠肾脏的免疫组织化学显示有丰富的 CD 标记。双标记共聚焦免疫荧光显微镜[使用远曲小管/连接小管 (CNT) 特异性标记物钙结合蛋白和 CNT/CD 特异性标记物 AQP-2] 显示 CD 和 CNT 中都有 V2R 标记。尽管显微解剖肾小管的 RT-PCR 显示 TAL 中存在 V2R mRNA 表达，但血管结构和其他肾小管（包括粗升肢 (TAL)）中完全没有标记。共聚焦显微镜表明，在亚细胞水平上，V2R 标记主要位于正常肾脏的细胞内，尽管在基底外侧膜域中存在一些明显的染色。分离的内髓集合管的共聚焦显微镜显示 V2R 在细胞内和基底外侧膜域中均表达。
Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct ( CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb ( TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.