RENAL NET ACID SECRETION AND THE NA/K/2C1 CONTRANSPORTER

肾净酸分泌和 NA/K/2C1 转运蛋白

• 批准号: 6524225
• 负责人: SUSAN MARIE WALL
• 金额: $ 20.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524225
• 关键词: acid base balance; acidity /alkalinity; ammonia; apical membrane; basolateral membrane; chloride ion; fluorescent dye /probe; fluorimetry; immunocytochemistry; isolation perfusion; laboratory rat; membrane permeability; membrane transport proteins; microspectrophotometry; polymerase chain reaction; protein isoforms; protein localization; renal tubular transport; tissue /cell culture; urine acidity; western blottings

Two distinct genes (BSC-1 and BSC-2) encode the NA/+-K/+-2CI- co- transporter. BSC-1, or the "absorptive isoform", is kidney-specific and localizes to the apical membrane of the thick ascending limb. BSC-2, the "secretory" isoform, is widely distributed BSC-1 is responsible for the "single effect" of the countercurrent multiplier, and thereby generates an axial gradient in the medullary interstitium for ammonium and other solutes, facilitating net acid secretion and urinary concentration. In the thick ascending limb, NH/4 uptake by the Na/+-K/+-2CI-co-transporter (BSC-1) is highly regulated. Since BSC-2 localizes to the basolateral membrane of the rat alpha intercalated cell and since it is both an NH/4 amd a CO/transporter, it likely mediates net acid secretion. However, regulation of BSC-2 in the kidney and its role in net acid secretion are untested. Regulation of net CI- and H/+ secretion by the collecting duct requires transport of these ions to be regulated in parallel across the apical and the basolateral membrane of the collecting duct cell. With perturbations in acid-base balance such as chronic metabolic acidosis an increase in apical proton secretion is observed which results from up- regulation of the apical H/+-ATPase in parallel with the basolateral CI- HCO/3-exchanger. However, in the rat contribution of CI-/HCO/3-exchange to transepithelial net acid and CI-secretion has not been established. Thus, another mechanism for net H/+ and/or CI-uptake across the basolateral membrane may be important in mediating or modulating transepithelial net acid secretion. We will determine the contribution of BSC-2 to transepithelial net acid and net CI-secretion. Moreover, we will determine if activity of the co-transporter is modulated in a fashion appropriate for correction of perturbations in acid-base balance. With the recent cloning of both isoforms of the co-transporter, the published structure has been exploited to raise antibodies against co-transporter peptides. These antibodies can be used to study the long term regulation of the co-transporter. Changes in protein immunoreactivity and mRNA can be correlated with changes in transport activity to determine the role of the co-transporter in acid-base homeostasis. Specific Aims are to determine the following in the rat OMCD: 1. If BSC-2 modulates net acid secretion through direct NH/4+ uptake. 2. If BSC-2 is a major contributor to transepithelial net CI- secretion. 4. To determine the mechanism of BSC-2 transport regulation. To answer these questions, isolated renal tubules perfused in vitro will be studied. Transport will be studied with microfluorimetry as well as pH sensitive fluorescent probes. Transporter immunoreactivity and message abundance will be studied using immunoblots, immunocytochemistry and RT PCR.

两个不同的基因（BSC-1和BSC-2）编码NA/+-K/+-2CI- co-。 传送器BSC-1或“吸收亚型”是肾特异性的， 局限于粗的上升支的顶膜。BSC-2， “分泌”亚型，广泛分布的BSC-1负责 “单一效应”的逆流乘数，从而产生 在髓核中存在铵和其他元素的轴向梯度 溶质，促进净酸分泌和尿浓度。在 粗升支，Na/+-K/+-2CI-共转运蛋白摄取NH/4 （BSC-1）受到高度监管。由于BSC-2定位于基底外侧 大鼠α闰细胞的膜，因为它既是NH/4 和CO/转运蛋白，它可能介导净酸分泌。然而，在这方面， BSC-2在肾脏中的调节及其在净酸分泌中的作用是 未经测试集合管对净Cl-和H/+分泌的调节 需要这些离子的运输被平行地调节穿过 收集管细胞的顶膜和基底侧膜。与 酸碱平衡紊乱，如慢性代谢性酸中毒和 观察到顶端质子分泌增加，这是由于向上- 顶端H/+-ATP酶的调节与基底外侧CI- HCO/3-交换器。然而，在大鼠Cl-/HCO/3-交换的贡献中， 跨上皮净酸和CI分泌尚未建立。 因此，另一种机制的净H/+和/或CI-吸收跨 基底外侧膜可能在介导或调节 经上皮净酸分泌。我们将决定 BSC-2对跨上皮净酸和净CI分泌的影响。而且我们 将确定协同转运蛋白的活性是否在 适用于校正酸碱干扰的方法 平衡随着最近共转运蛋白两种亚型的克隆， 已公开的结构已被用于产生抗 共转运蛋白肽。这些抗体可用于研究长期 共同运输者的长期监管。蛋白质变化 免疫反应性和mRNA可以与转运的变化相关 活性，以确定协同转运蛋白在酸碱平衡中的作用 体内平衡具体目的是确定大鼠中的以下内容 OMCD：1.如果BSC-2通过直接NH/4+调节净酸分泌， 摄取。2.如果BSC-2是跨上皮净CI的主要贡献者， 分泌物4.确定BSC-2的运输调节机制。 为了回答这些问题，离体肾小管灌流在体外将 被研究。运输将研究与显微荧光，以及 pH敏感荧光探针。转运蛋白免疫反应性和 将使用免疫印迹、免疫细胞化学 和RT PCR。