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分泌酸 进入肾小管腔，从而重新吸收大部分过滤后的 HCO-3，并且 将新陈代谢产生的酸负荷输出到全身。 尽管近端小管可以调节其 酸分泌率随酸碱状态变化而变化 血液，在短时间内发生这种情况的机制 根本不清楚。该项目的长期目标是了解 血液 [C02]、[HCO-3] 和 pH 的变化如何敏锐地控制 HCO-3 近曲小管的重吸收。拟议的一般方法 实验将在体外分离并灌注单个近端组织 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 (JHCO3) 和体积重吸收 (Jv)。在其他方面 实验中，荧光信号的数字成像将用于 监测肾小管细胞内的 pH (Ph/I) 或钙 ([Ca/++]i)。一个 所提出的实验的新颖之处在于切换的可能性 不平衡 (OOE) CO2/HCO-3 的基底外侧（“浴”）溶液 溶液几乎具有 [CO2]、[HCO-3] 和 pH 值的任意组合。 关于灌注 CO2/HCO-3 的肾小管的初步数据表明，转换 浴液从不含 CO2/HCO-3 的 HEPES 变为 OOE“纯 HCO-3” 溶液（含有生理 [HCO-3] 和 pH，但 CO2 可以忽略不计） 显着降低 JHCO3 和 JV。相反，“纯CO2”（生理[CO2] 和 pH，但可忽略不计的 NCO-3) 浴液中的 JHCO/3 和 合资企业。将浴槽切换为 CO2/HCO-3 也会导致显着的 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，[HCO- 3]BL 或 pH/BL 影响 JHCO/3 和 Jv？该方法将是切换 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 CO2-HCO-3 溶液到 OOE 仅更改 [HCO-3]BL 的解决方案。在其他实验中， pH/BL 会发生变化，而不是 [HCO-3]BL。二、如何隔离 [HCO-3]BL 或 pH/BL 的变化会影响由 生电 Na/HCO3 协同转运蛋白 (NBC)？该方法将被表达 爪蟾卵母细胞中的 NBC，并研究 NBC 对分离的敏感性 [HCO-3]0 PR Ph/0 发生变化。目标是确定程度 目标 1 中获得的数据可以解释对 NBC 的影响。 第三，[CO2]/BL 的孤立变化如何影响 JHCO3 和 JV？这 方法与目标 1 相同，除了 [CO2]BL（而不是 [HCO-3]BL 或 pH/BL) 将发生变化。四、PT细胞如何 [C02]BL 的增加转导 H/+ 分泌的增加？这 方法是确定 [Ca/++]i、一氧化氮是否发生变化 产生、蛋白质磷酸化或膜囊泡的插入 促进目标 3 中观察到的 JHC03 的变化。拟议的工作 应该提供关于血液酸碱参数如何急剧变化的新见解 调节近端小管对 HCO-3 的重吸收。