REGULATION OF PROXIMAL TUBULE BICARBONATE TRANSPORT

近端小管碳酸氢根运输的调节

• 批准号: 6725893
• 负责人: Walter F. Boron
• 金额: $ 18.54万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6725893
• 关键词: acid base balance; angiotensin II; basolateral membrane; bicarbonates; biological signal transduction; carbon dioxide; genetically modified animals; hydrogen channel; immunocytochemistry; ion transport; laboratory mouse; laboratory rabbit; mass spectrometry; membrane permeability; membrane transport proteins; nitric oxide synthase; protein kinase C; protein tyrosine kinase; renal tubular transport; sodium hydrogen exchanger

A major function 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 body metabolism. Although it is well established that the proximal tubule (PT) and more distal nephron segments 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 a short time frame are not clear. We have studied rates of HCO-3 reabsorption (JHCO3) in isolated perfused rabbit PTs, using out-of-equilibrium (OOE) CO2/HCO-3 solutions to vary, one at a time, the 'bath' or basolateral (BL) acid-base parameters. We found that, although JHCO3 is insensitive to isolated changes in pHBL, isolated increases in [CO2]BL cause JHCO3 to rise whereas isolated increases in [HCO-3]BL cause JHCO3 to fall. The response to CO2 suggests the presence of a CO2 sensor near the basolateral membrane. Low-dose angiotensin II (ANG II) in either the lumen or bath accentuates the CO2 response, whereas high-dose ANG II (lumen or bath) or blockers of either tyrosine kinases or protein kinase C all markedly blunt the CO2 response. The proposed work has three aims: First, is the JHCO3 response to CO2 plastic (is it altered by chronic acid-base disturbances?), does it extend to other transporter processes, and does it extend to more tubule segments? Second, to what extent does the JHCO3 response to CO2 depend on the 'hardware' of HCO-3 reabsorption in the proximal tubule? Using specific inhibitors and knockout/transgenic mice, we will ask whether interfering with the apical Na-H exchanger (NHE3) or H+ pump or the insertion/retrieval of apical vesicles carrying these transporters reduces the response. Using mmunocytochemistry, we will ask if the CO2 response entails a change in the distribution of NHE3 or H+-pump 9roteins. Third, what signal-transduction processes are involved in the response to CO2? We will use ANG II and blockers or agonists of specific signal-transduction steps to block or mimic the effects of CO2 on JHCO3. We will examine the effects of CO2 on potential second messengers (using JHCO3 or pHi changes as indices of transport), and use 2-D gels and mass-spectroscopy to identify candidate proteins involved in signal transduction. The proposed work will provide important new insight into how the kidney responds appropriately to acid-base challenges, and will also elucidate the mechanism by which ANG II modulates PT transport.

肾近曲小管的主要功能是将酸分泌到肾小管腔内，从而重新吸收大部分过滤后的HCO-3，并输出体内代谢产生的酸负荷。尽管众所周知，近端肾小管（PT）和更远端的肾单位段可以响应血液酸碱状态的变化来调节其酸分泌速率，但在短时间内发生这种情况的机制是不清楚。我们研究了离体灌注兔 PT 中的 HCO-3 重吸收率 (JHCO3)，使用 不平衡 (OOE) CO2/HCO-3 溶液每次改变一个“浴”或基底外侧 (BL) 酸碱参数。我们发现，虽然 JHCO3 对 pHBL 的孤立变化不敏感，但 [CO2]BL 的孤立增加会导致 JHCO3 上升，而 [HCO-3]BL 的孤立增加会导致 JHCO3 下降。对 CO2 的反应表明基底外侧膜附近存在 CO2 传感器。管腔或浴中的低剂量血管紧张素 II (ANG II) 会增强 CO2 反应，而高剂量 ANG II（管腔或浴）或二者之一的阻滞剂 酪氨酸激酶或蛋白激酶 C 都会显着减弱 CO2 反应。拟议的工作有三个目标：首先，JHCO3 是否对 CO2 塑料有反应（是否会因慢性酸碱紊乱而改变？），是否会扩展到其他转运过程，是否会扩展到更多的小管段？其次，JHCO3 对 CO2 的反应在多大程度上取决于近曲小管中 HCO-3 重吸收的“硬件”？使用特定的抑制剂和基因敲除/转基因小鼠，我们将询问是否干扰顶端 Na-H 交换器 (NHE3) 或 H+ 泵或 携带这些转运蛋白的顶端囊泡的插入/回收会降低反应。使用免疫细胞化学，我们将询问 CO2 反应是否会导致 NHE3 或 H+-泵 9 蛋白的分布发生变化。第三，对二氧化碳的反应涉及哪些信号转导过程？ 我们将使用 ANG II 和特定信号转导步骤的阻断剂或激动剂来阻断或模拟 CO2 对 JHCO3 的影响。我们将研究 CO2 对潜在第二信使的影响（使用 JHCO3 或 pHi 变化作为运输指标）， 并使用二维凝胶和质谱来鉴定参与信号转导的候选蛋白质。拟议的工作将为肾脏如何适当应对酸碱挑战提供重要的新见解，并将阐明 ANG II 调节 PT 转运的机制。