REGULATION OF PROXIMAL TUBULE BICARBONATE TRANSPORT

近端小管碳酸氢根运输的调节

• 批准号: 6412911
• 负责人: Walter F. Boron
• 金额: $ 14.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6412911
• 关键词: Xenopus oocyte; acid base balance; basolateral membrane; bicarbonates; biological signal transduction; calcium indicator; carbon dioxide; digital imaging; hydrogen channel; inulin; ion transport; laboratory rabbit; membrane permeability; membrane transport proteins; microcalorimetry; nitric oxide; renal tubular transport

One of the major functions of the renal proximal tubule is to secrete acid into the tubule lumen, thereby reabsorbing most of the filtered HCO-3, and exporting the acid load produced by metabolism throughout the body. Although it is well established that the proximal tubule can modulate its rate of acid secretion in response to changes in the acid base status of the blood, the mechanisms by which this occurs over ra short time frame are not at all clear. The long-term goal of this project is to understand how changes in blood [C02], [HCO-3] and pH acutely control HCO-3 reabsorption by the proximal tubule. The general approach for the proposed experiments will be isolated and perfuse in vitro single proximal tissues isolated from the kidney of the rabbit. In some experiments, the combination of microcalorimetry and 3/H-inulin will be used to measure HCO-3 reabsorption (JHCO3) and volume reabsorption (Jv). In other experiments, digital imaging of fluorescence signals will be used to monitor intracellular pH (Ph/I) or calcium ([Ca/++]i) in tubule cells. A novel aspect of the proposed experiments is the possibility of switching the basolateral ("bath) solution to an out-of-equilibrium (OOE) CO2/HCO-3 solution with virtually any combination of [CO2], [HCO-3] and pH. Preliminary data on tubules perfused with CO2/HCO-3 suggest that switching the bath solution from CO2/HCO-3 free HEPES to an OOE "pure HCO-3" solution (containing a physiological [HCO-3] and pH, but negligible CO2) markedly reduces JHCO3 and JV. Conversely, "pure CO2" (physiological [CO2] and pH, but negligible NCO-3) in the bath markedly increases JHCO/3 and JV. Switching the bath to CO2/HCO-3 also causes a substantial pH/i increase, due to stimulation of apical Na-H exchange and H/+ pumping. The proposed work has four aims: First, how do isolated changes in [HCO- 3]BL or pH/BL affect JHCO/3 and Jv? The approach will be either to switch the bath from HEPES to a "pure HCO-3" solution of varying basolateral [HCO-3] (i.e., [HCO-3]BL), or from a standard CO2-HCO-3 solution to an OOE solution in which only [HCO-3]BL has been changed. In other experiments, pH/BL will be varied, rather than [HCO-3]BL. Second, how do isolated changes in [HCO-3]BL or pH/BL affect the HCO-3 efflux mediated by the electrogenic Na/HCO3 co-transporter (NBC)? The approach will be express NBC in Xenopus oocytes, and study the sensitivity of NBC to isolated changes in either [HCO-3]0 PR Ph/0. The goal is to determine the extent to which the data obtained in Aim 1 can be accounted by for effects on NBC. Third, how do isolated changes in [CO2]/BL affect JHCO3 and JV? The approach will be the same as in Aim 1, except that [CO2]BL (rather than [HCO-3]BL or pH/BL) will be changed. Fourth, how does the PT cell transduce an increase in [C02]BL to an increase in H/+ secretion? The approach will be to determine if changes in [Ca/++]i, nitric oxide production, protein phosphorylation or insertion of membrane vesicles contribute to the changes in JHC03 observed in Aim 3. The proposed work should provide fresh insight into how blood acid-base parameters acutely regulate HCO-3 reabsorption by the proximal tubule.

肾近端小管的主要功能之一是分泌酸 进入小管腔，从而重新吸收大多数过滤的HCO-3，并且 导出整个人体代谢产生的酸负荷。 尽管已经很好地确定近端小管可以调节其 响应于酸碱状态的变化的酸分泌速率 血液，在RA短时间内发生的机制 根本不清楚。该项目的长期目标是了解 血液[C02]，[HCO-3]和pH的变化如何急性控制HCO-3 近端小管的重吸收。提议的一般方法 实验将被分离出来，并在体外单个近端组织进行灌注 从兔子的肾脏中分离出来。在某些实验中， 微量钙化和3/H-氨基蛋白的组合将用于测量 HCO-3重吸收（JHCO3）和体积重吸收（JV）。在其他 实验，荧光信号的数字成像将用于 在小管细胞中监测细胞内pH（pH/I）或钙（[CA/++] I）。一个 提出的实验的新颖方面是切换的可能性 非平衡（OOE）CO2/HCO-3的基底外侧（“浴）溶液 使用[CO2]，[HCO-3]和pH的任何组合。 用CO2/HCO-3灌注小管的初步数据表明切换 从CO2/HCO-3 Free Hepes到OOE“ Pure HCO-3”的浴溶液 解决方案（包含生理学[HCO-3]和pH，但可忽略不计的CO2） 明显减少了JHCO3和JV。相反，“纯CO2”（生理[CO2] 和pH，但可以忽略的NCO-3）在浴中显着增加了JHCO/3和 合资将浴室切换到CO2/HCO-3也会引起大量的pH/I 由于刺激顶端Na-H交换和H/+泵送而增加。 拟议的工作有四个目标：首先，如何孤立的变化[hco- 3] BL或pH/BL影响JHCO/3和JV？该方法要么要切换 从HEPES到不同基底外侧的“纯HCO-3”溶液的浴 [HCO-3]（即[HCO-3] BL），或从标准的CO2-HCO-3解决方案到OOE 仅[HCO-3] BL已更改的解决方案。在其他实验中， pH/bl将变化，而不是[HCO-3] BL。第二，如何隔离 [HCO-3] BL或pH/BL的变化会影响由HCO-3外排介导的。 电源NA/HCO3共转运蛋白（NBC）？该方法将是明确的 NBC在爪蟾卵母细胞中，研究NBC对分离的敏感性 [HCO-3] 0 Pr pH/0的更改。目标是确定 AIM 1中获得的数据可以通过对NBC的影响来解释。 第三，[CO2]/BL的孤立变化如何影响JHCO3和JV？这 方法将与AIM 1相同，只是[CO2] BL（而不是 [HCO-3] BL或PH/BL）将更改。第四，PT单元如何 将[C02] BL的增加增加到H/+分泌的增加？这 方法是确定[CA/++] I，一氧化氮的变化是否变化 膜囊泡的生产，蛋白质磷酸化或插入 有助于AIM 3中观察到的JHC03的变化。拟议的工作 应该提供有关血酸基碱参数如何敏锐的洞察力的新见解 通过近端小管调节HCO-3的重吸收。