Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney.
Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney.
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DOI：
10.1152/ajprenal.2000.278.2.f327
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发表时间：
2000-02
期刊：
影响因子：
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通讯作者：
T. Kwon;A. Pushkin;N. Abuladze;S. Nielsen;I. Kurtz
中科院分区：
文献类型：
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作者：
T. Kwon;A. Pushkin;N. Abuladze;S. Nielsen;I. Kurtz
在本研究中，我们制备了一种兔肽源性cooh末端多克隆抗体，该抗体选择性识别NBC3，利用免疫细胞化学和免疫电镜技术确定NBC3在大鼠肾脏中的细胞和亚细胞定位。免疫细胞化学低温切片和半薄低温切片显示连接小管和皮质、外髓和初始内髓收集管中的嵌入细胞（ic）特异性染色。在连接小管以及皮质和髓质收集管中，标记与A型和B型ic相关。在A型ic中，标记仅限于根尖和亚根尖结构域，而在B型ic中，基底结构域被完全标记。相比之下，收集管主细胞一直未被标记，这是用抗水通道蛋白-2抗体证实的，该抗体在平行半薄冷冻切片中标记主细胞。肾小球、近端小管、降细肢、升细肢、厚升肢、远端曲小管和血管结构未标记。在免疫电镜下，组织样品被冷冻替代，并在超薄的Lowicryl HM20切片上进行免疫标记。免疫电镜显示，NBC3标记在A型ic的根尖质膜、细胞内囊泡和根尖下结构域的管状池内非常丰富。在B型ic中，NBC3主要存在于基底外侧质膜。使用肽吸收抗体的免疫标记对照一致为阴性。综上所述，NBC3在A型碳化硅的顶端质膜和B型碳化硅的基底外侧质膜中含量丰富。这表明NBC3在调节连接管和收集管中的碳酸氢盐运输中起重要作用。
In the present study, we produced a rabbit peptide-derived polyclonal COOH-terminal antibody that selectively recognizes NBC3, to determine the cellular and subcellular localization of NBC3 in rat kidney, using immunocytochemistry and immunoelectron microscopy. Immunocytochemistry with cryostat sections and semithin cryosections revealed specific staining of intercalated cells (ICs) in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting ducts. In the connecting tubule and in the cortical and medullary collecting duct, the labeling was associated with both type A and type B ICs. In type A ICs, labeling was confined to the apical and subapical domains, whereas in type B ICs, basal domains were exclusively labeled. In contrast, collecting duct principal cells were consistently unlabeled, and this was confirmed using anti-aquaporin-2 antibodies, which labeled principal cells in parallel semithin cryosections. Glomeruli, proximal tubules, descending thin limbs, ascending thin limbs, thick ascending limbs, distal convoluted tubules, and vascular structures were unlabeled. For immunoelectron microscopy, tissue samples were freeze-substituted, and immunolabeling was performed on ultrathin Lowicryl HM20 sections. Immunoelectron microscopy demonstrated that NBC3 labeling was very abundant in the apical plasma membrane, in intracellular vesicles, and in tubulocisternal profiles in the subapical domains of type A ICs. In type B ICs, NBC3 was mainly present in the basolateral plasma membrane. Immunolabeling controls using peptide-absorbed antibody were consistently negative. In conclusion, NBC3 is highly abundant in the apical plasma membrane of type A ICs and in the basolateral plasma membrane of type B ICs. This suggests that NBC3 plays an important role in modulating bicarbonate transport in the connecting tubule and collecting duct.