Yang T, Huang Y G, Singh I, Schnermann J, Briggs J P
Department of Physiology, University of Michigan, Ann Arbor 48104, USA.
Am J Physiol. 1996 Oct;271(4 Pt 2):F931-9. doi: 10.1152/ajprenal.1996.271.4.F931.
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.
本研究旨在调查两种主要的肾脏特异性钠-钾-氯转运蛋白的mRNA定位，即布美他尼敏感共转运体（兔中的NKCC2和大鼠中的BSC1）和噻嗪类敏感共转运体（TSC）。已证明来自兔和小鼠的NKCC2存在三种同工型（分别命名为A、B和F），它们仅在一个96bp的区域有所不同。通过基于聚合酶链反应（PCR）的策略从大鼠肾脏中克隆出三种NKCC2同工型各自的差异区域，并选择了同工型特异性引物。从肾皮质、髓质以及显微解剖的肾单位节段制备RNA和cDNA。使用逆转录（RT）-PCR，仅在皮质中检测到B同工型，仅在髓质中检测到F同工型，而A同工型在两者中均有发现。在解剖的肾单位节段中，B型仅存在于皮质厚升支（CTAL）和含致密斑节段（MDCS），F型仅存在于髓质厚升支（MTAL）和外髓集合管，A型存在于CTAL、MDCS和MTAL中。通过对来自髓质的一个592bp产物进行直接测序，鉴定出一种包含A和F外显子的额外同工型。AF产物仅在髓质中发现，并且仅定位在MTAL中。TSC mRNA仅在远曲小管中检测到。NKCC2同工型在肾单位中的差异定位表明，钠-钾-氯共转运体的转运特性可能不同，以解释沿肾单位的盐转运调节。
