Apical localization of the Na-K-Cl cotransporter, rBSC1, on rat thick ascending limbs
Apical localization of the Na-K-Cl cotransporter, rBSC1, on rat thick ascending limbs
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DOI：
10.1038/ki.1996.6
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发表时间：
1996-01-01
影响因子：
19.6
通讯作者：
Hebert, SC
中科院分区：
文献类型：
--
作者：
Kaplan, MR;Plotkin, MD;Hebert, SC
一个布美他尼敏感的Na-K-Cl共转运蛋白（rBSC1）最近从大鼠肾外髓质（OM） cDNA文库中克隆出来，并显示其主要在肾[1]中表达。本研究的目的是研究大鼠肾脏中共转运蛋白转录本和蛋白的分布。原位杂交显示，rBSC1转录本仅在外髓质中表达，并沿髓皮质射线延伸，这与髓质（MTAL）和皮质（CTAL）厚升肢中rBSC1转录本的表达一致。兔中针对rBSC1羧基末端独特的67个氨基酸片段的多克隆抗体在大鼠肾皮质(C)和外髓质（OM）（但不包括内髓质（IM））匀浆的Western blotting上鉴定出130至160（中点150）kDa的宽主带和至少两个50至70 kD的小带。因此，rBSC1多克隆抗体在大鼠肾脏中检测到的Na-K-CI共转运蛋白大小与其他抗体在小鼠和狗肾脏中检测到的主要相似的150kd布美他胺结合蛋白相似。利用抗rbsc1多克隆抗体在大鼠肾切片上进行免疫荧光研究，发现MTAL和CTAL节段的顶端表面有强烈的信号。抗tamm - horsfall抗体存在于除黄斑致密细胞外的所有TAL细胞中，共定位证实黄斑致密细胞中不存在抗rbsc1荧光。这些结果与rBSC1编码粗升肢顶端Na-K-CI共转运蛋白的主要亚型一致。在黄斑致密细胞中功能性检测到的Na-K-CI共转运蛋白可能由不同的BSC亚型编码。
A bumetanide-sensitive Na-K-Cl cotransporter (rBSC1) was recently cloned from a rat renal outer medulla (OM) cDNA library and shown to be expressed predominantly in the kidney [1]. The purpose of the present study was to examine the nephron distribution of cotransporter transcripts and protein in rat kidney. In situ hybridization showed an intense signal only in the outer medulla and extending along cortical medullary rays consistent with expression of rBSC1 transcripts in medullary (MTAL) and cortical (CTAL) thick ascending limbs. Polyclonal antibodies raised in rabbits against a unique 67 amino acid segment from the carboxyl terminus of rBSC1 identified a broad major band of 130 to 160 (midpoint of 150) kDa and at least two minor bands of 50 to 70 kD on Western blotting of homogenates from cortex (C) and outer medulla (OM), but not inner medulla (IM), of rat kidney. Thus the Na-K-CI cotransporter protein detected by the polyclonal rBSC1 antibody in rat kidney was similar in size to the major similar to 150 kD bumetanide binding protein detected by others in mouse and dog kidneys. Immunofluorescence studies using the anti-rBSC1 polyclonal antibody on rat kidney sections showed an intense signal limited to apical surfaces of MTAL and CTAL segments. Colocalization with anti-Tamm-Horsfall antibody which is present in all TAL cells except macula densa cells confirmed the absence of anti-rBSC1 fluorescence in the macula densa cells. These results are consistent with rBSC1 encoding the, or the major isoform of the, apical Na-K-CI cotransporter in the thick ascending limb. The Na-K-CI cotransporter functionally detected in macula densa cells may be encoded by a different BSC isoform.