Cellular and Subcellular Immunolocalization of CIC-5 Chloride Channel in the mouse Kidney : Colocalization with H^+-ATPase

小鼠肾脏中 CIC-5 氯离子通道的细胞和亚细胞免疫定位：与 H+ -ATP 酶共定位

• 批准号: 09671153
• 负责人: SAKAMOTO Hisato
• 金额: $ 1.92万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671153/
• 关键词: CIC-5 chloride channel; cytoplasmic vesicle; endocytosis; H^+-ATPase; low molecular V proteinuria weight; monoclonal antibody; Dent's disease; クロライドチャネル; ClC-5; CLCN5; 腎臓; ゲノムDNA; Dent病

CIC-S chloride channel has attracted special interest in relation to three different inherited renal disease associated with nephrolithiasis, but cellular and subcellular localization of this protein has yet to be detennined. To analyze localization of CIC-5 in the kidney, a CIC-5-specific- rat monoclonal antibody (MAb) was developed to an authentic peptide containing amino acids near the. Carboxyl terminal of CIG-5 using rat lymph node method. Specificity of MAb was established by Western blotting and immunohistochemistry of the stably-CIC-5 transfected mammalian cultured cells. Furthermore the epitope recognizing 5- ammino acid residues was determined by multipin-peptide scanning. With the use of high-resolution immunohistochemical technique using confocal microscope, CIC-5 was found in the apical cytoplasm of proximal tubule cell (PT), as well as in the apical plasma membrane of PT, and in the apical plasma membrane of partial collecting duct cells in mouse kidney. These subcellular localizations were confirmed by transmission electron microscopy. In addition, the cellular localization of CIC-5 in the collecting duct was determined by double staining of the same section with antibodies against CIC-5 and aquaporin-2 (AQP-2), by which an exclusive staining of CIC-5 was found only in the cells without staining of AQP-2.These findings suggest that CIC-5 might be involved in the endocytosis and/or the H^+-secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney. The establishment of CIC-5-specific MAb would provide insight into the pathogenesis of inherited kidney diseases associated with mutations of the CIC-5 gene.

CIC-S氯离子通道与三种不同的肾结石相关的遗传性肾脏疾病有关，但该蛋白的细胞和亚细胞定位尚未确定。为了分析CIC-5在肾脏中的定位，开发了CIC-5特异性大鼠单克隆抗体（MAb），其为含有在肾脏中CIC-5附近的氨基酸的真实肽。CIG-5羧基端的大鼠淋巴结法。通过Western blotting和免疫组织化学法检测稳定转染CIC-5的哺乳动物培养细胞的特异性。用多针肽扫描法确定了识别5-氨基酸残基的表位。应用免疫组化共聚焦显微镜观察，发现小鼠肾脏近曲小管上皮细胞（PT）的顶端胞质、PT的顶端质膜和部分集合管细胞的顶端质膜中均有CIC-5的表达。这些亚细胞定位证实了透射电子显微镜。此外，通过用抗CIC-5和水通道蛋白-2（AQP-2）的抗体对同一切片进行双重染色来确定CIC-5在集合管中的细胞定位，结果表明，CIC-5可能参与了细胞内吞和/或H^+-ATPase的合成。小鼠肾脏近曲小管细胞和皮质集合管A型闰细胞分泌。CIC-5特异性单克隆抗体的建立将为深入了解与CIC-5基因突变相关的遗传性肾脏疾病的发病机制提供依据。

项目成果

SAKAMOTO,H.et al.: "Cellular and Subcellular Immunolocalization of CIC-5 Chloride Channel in the Kidney" J Amer Soc Nephrol. 9. 43A (1998)

SAKAMOTO,H.et al.：“肾脏中 CIC-5 氯离子通道的细胞和亚细胞免疫定位”J Amer Soc Nephrol。

坂本尚登、佐渡義一 他: "クロライドチャネルCLCN5の腎臓内における局在部位" 日本腎臓学会誌. 40・3. 112 (1998)

Naoto Sakamoto、Yoshikazu Sado 等：“肾脏中氯离子通道 CLCN5 的定位位点”日本肾病学会杂志 40・3.112（1998 年）。

MORIMOTO,T.et al.: "Mutations in CLCN5 chloride channel in Japanese patients with low molecular weight" J Am Soc Nephrol. 9. 811-818 (1998)

MORIMOTO,T.et al.：“日本低分子量患者的 CLCN5 氯离子通道突变”J Am Soc Nephrol。

Sakamoto H,Sado Y,Naito I,Endo K,Kawasaki M,Fushimi K,Uchida S,Sasaki S and Marumo F.: "Cellular and Subcellular immunolocalization of CLC-5 Chloride Channel in the Kidney" J Amer Soc Nephrol. 9. 43A (1998)

Sakamoto H、Sado Y、Naito I、Endo K、Kawasaki M、Fushimi K、Uchida S、Sasaki S 和 Marumo F.：“肾脏中 CLC-5 氯离子通道的细胞和亚细胞免疫定位”J Amer Soc Nephrol。

坂本尚登、佐藤義一 他: "クロライドチャネルCLCN5の腎臓内における局在部位" 日本腎臓学会誌. 40・3. 112 (1998)

Naoto Sakamoto、Yoshikazu Sato 等：“肾脏中氯通道 CLCN5 的定位位点”日本肾病学会杂志 40・3.112（1998 年）。