REGULATION OF PROXIMAL TUBULE BICARBONATE TRANSPORT

近端小管碳酸氢根运输的调节

• 批准号: 6574318
• 负责人: Walter F. Boron
• 金额: $ 24.29万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6574318
• 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。 进入管腔，从而重新吸收大部分过滤的HCO-3，以及 输出全身新陈代谢产生的酸负荷。 虽然已经确定近端小管可以调节它的 对酸碱状态变化作出反应的酸分泌速率 血液，在短时间内发生这种情况的机制 完全不清楚。这个项目的长期目标是了解 血中[CO_2]、[HCO-3]和pH的变化对HCO-3的影响 近端小管的重吸收。建议的一般方法 实验将分离并灌流体外单个近端组织。 从兔子的肾脏中分离出来。在一些实验中， 微量热法与~(3/H)-菊糖联用测定 HCO3重吸收(JHCO3)和体积重吸收(JV)。在其他 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)或钙离子([Ca/++]i)。一个 拟议中的实验的新方面是切换的可能性 非平衡(OOE)CO_2/HCO-3的基侧(“浴”)溶液 具有几乎任何[CO2]、[HCO-3]和pH组合的溶液。 关于用CO2/HCO-3灌流的小管的初步数据表明， 从无CO_2/HCO_3的HEPES到OOE“纯HCO_3”的镀液 溶液(含有生理的[HCO-3]和pH，但可忽略二氧化碳) 显著降低JHCO3和JV。相反，“纯二氧化碳”(生理性的[二氧化碳] 和pH，但可以忽略的NCO-3)显著增加了JHCO/3和 JV。将浴液切换到CO2/HCO-3也会导致相当大的pH/i 增加，这是由于顶端Na-H交换和H/+泵的刺激。 拟议的工作有四个目标：第一，如何在[HCO- 3)BL值或pH/BL值影响JHCO3和JV？方法将是要么改变 从HEPES到可变基面的纯HCO-3溶液的浴液 [HCO-3](即[HCO-3]BL)，或从标准的CO2-HCO-3溶液到OOE 仅改变了[HCO-3]BL的解决方案。在其他实验中， PH/BL将是变化的，而不是[HCO-3]BL。第二，孤立的 [HCO3]BL值或pH/BL值的变化影响HCO3的外流。 生电Na/HCO3共转运蛋白(NBC)？这种方法将是明确的 NBC在非洲爪哇卵母细胞中的表达，并研究NBC对分离的 [HCO-3]0 PR Ph/0的变化。目标是确定以下方面的程度 在目标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JHCO3和JV？这个 方法与目标1相同，不同之处在于[CO2]BL(而不是 [HCO-3]BL值或pH/BL值)将改变。第四，PT细胞是如何 将[C02]BL的增加转化为H/+分泌的增加？这个 方法将是确定[Ca/++]i、一氧化氮的变化 膜泡的产生、蛋白磷酸化或插入 对目标3中观察到的JHC03的变化作出贡献。拟议工作 应该为血液酸碱参数如何尖锐地提供新的见解 通过近端小管调节HCO-3的重吸收。