Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron.
Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron.
复制标题
布美他尼和噻嗪类敏感的 Na-K-Cl 协同转运蛋白沿大鼠肾单位的定位。
DOI：
10.1152/ajprenal.1996.271.4.f931
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Briggs,JP
中科院分区：
文献类型：
--
作者：
Yang,T;Huang,YG;Singh,I;Schnermann,J;Briggs,JP
本研究旨在研究两种主要的肾脏特异性钠-钾-氯转运蛋白，布美他尼敏感性协同转运蛋白（兔NKCC 2和大鼠BSC 1）和噻嗪敏感性协同转运蛋白（TSC）的mRNA定位。来自兔和小鼠的NKCC 2已被证明以三种亚型（指定为A、B和F）存在，其仅在96 bp区域不同。通过基于聚合酶链反应（PCR）的策略从大鼠肾脏中克隆了三种NKCC 2亚型中每一种亚型的趋异区，并选择亚型特异性引物。RNA和cDNA制备肾皮质和髓质和显微解剖的肾单位段。使用逆转录（RT）-PCR，B亚型仅在皮质和F亚型仅在髓质中检测到，而A从被发现在这两个。在肾单位解剖段中，B型仅见于皮质粗升支（CTAL）和含致密斑段（MDCS），F型仅见于髓质粗升支（MTAL）和外髓集合管，A型见于CTAL、MDCS和MTAL。一个额外的亚型，包括A和F外显子被确定为直接测序的一个592 bp的产物从髓质。AF产物仅在髓质中发现，并且仅局限于MTAL中。TSC mRNA仅在远曲小管中检测到。NKCC 2亚型的肾单位差异定位表明，钠-钾-氯协同转运蛋白可能在其转运特性上不同，以解释盐转运沿着肾单位的调节。
The present study was undertaken to investigate the mRNA localization of the two major kidney-specific Na-K-Cl transport proteins, the bumetanide-sensitive cotransporter (NKCC2 in rabbit and BSC1 in rat) and the thiazide-sensitive cotransporter (TSC). NKCC2 from rabbit and mouse has been shown to exist in three isoforms (designated A, B, and F) that differ only in a 96-bp region. The divergent region of each of the three NKCC2 isoforms was cloned from rat kidney by a polymerase chain reaction (PCR)-based strategy, and isoform-specific primers were chosen. RNA and cDNA were prepared from renal cortex and medulla and from microdissected nephron segments. Using reverse transcription (RT)-PCR, the B isoform was detected only in cortex and the F isoform only in medulla, whereas the A from was found in both. In dissected nephron segments, the B form was found exclusively in cortical thick ascending limb (CTAL) and macula densa-containing segment (MDCS), the F form only in medullary thick ascending limb (MTAL) and outer medullary collecting duct, and the A form in CTAL, MDCS, and MTAL. An additional isoform including both A and F exons was identified by direct sequencing of a 592-bp product from medulla. The AF product was found only in the medulla and was localized exclusively in MTAL. TSC mRNA was detected exclusively in the distal convoluted tubule. Differential nephron localization of NKCC2 isoforms suggests that Na-K-Cl cotransporters may differ in their transport characteristics to explain regulation of salt transport along the nephron.