REGULATION OF PROXIMAL TUBULE BICARBONATE TRANSPORT

近端小管碳酸氢根运输的调节

• 批准号: 6354689
• 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: 2000-09-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6354689
• 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.

肾近曲小管的主要功能之一是泌酸 进入小管腔，从而重吸收大部分过滤的HCO-3， 将新陈代谢产生的酸负荷输出到全身。 虽然近端小管可以调节其 酸分泌率响应于酸碱状态的变化， 血液，这在短时间内发生的机制 一点都不清楚这个项目的长期目标是了解 血液中[CO2]、[HCO-3]和pH值的变化如何快速控制HCO-3 近端小管的重吸收。建议的一般方法 实验将在体外分离和灌注单个近端组织 从兔子的肾脏中分离出来。在一些实验中， 微量热法和3 H-菊粉的组合将用于测量 HCO-3重吸收（JHCO 3）和体积重吸收（Jv）。换句 实验中，荧光信号的数字成像将用于 监测肾小管细胞内pH（Ph/I）或钙（[Ca/++]i）。一 新的方面提出的实验是切换的可能性 将基底外侧（“浴”）溶液置于失衡（OOE）CO2/HCO-3 几乎任何[CO2]、[HCO-3]和pH组合的溶液。 用CO2/HCO-3灌注的肾小管的初步数据表明， 从不含CO2/HCO-3的HEPES到OOE“纯HCO-3”的槽液 溶液（含有生理[HCO-3]和pH值，但CO2可忽略） 显著降低JHCO 3和JV。相反，“纯CO2”（生理[CO2]） 和pH值，但可忽略的NCO-3）在浴显着增加JHCO/3和 合资企业。将浴切换到CO2/HCO-3也导致显著的pH/i 增加，由于刺激顶端Na-H交换和H/+泵。 拟议的工作有四个目标：第一，如何孤立的变化[HCO- 3]BL或pH/BL影响JHCO/3和Jv？我们的做法是， 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CO2-HCO-3溶液到OOE 溶液中只有[HCO-3]BL发生了变化。在其他实验中， 将改变pH/BL，而不是[HCO-3]BL。二、如何孤立 [HCO-3]BL或pH/BL的变化会影响由 生电Na/HCO 3共转运蛋白（NBC）？该方法将是明示的 本文研究了非洲爪蟾卵母细胞中的NBC，并研究了NBC对分离的 [HCO-3]0 PR Ph/0的变化。目标是确定 目标1中获得的数据可以用来解释对NBC的影响。 第三，[CO2]/BL的孤立变化如何影响JHCO 3和JV？的 方法与目标1相同，除了[CO2]BL（而不是 [HCO-3]BL或pH/BL）。四、PT细胞如何 [C 02]BL的增加导致H/+分泌的增加？的 方法将是确定[Ca/++]i、一氧化氮 膜囊泡的产生、蛋白磷酸化或插入 有助于目标3中观察到的JHC 03变化。拟议工作 应该提供新的见解，血液酸碱参数如何急性 调节近端小管对HCO-3的重吸收。