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.

肾近端小管的主要功能是将酸分泌到小管腔中，从而重吸收大部分过滤的HCO-3，并输出由身体代谢产生的酸负荷。虽然近端小管（PT）和更远端的肾单位节段可以调节其酸分泌速率以响应血液酸碱状态的变化，但在短时间内发生这种情况的机制尚不清楚。我们研究了HCO-3重吸收率（JHCO 3）在离体灌注兔PT，使用 非平衡（OOE）CO2/HCO-3溶液，以每次改变一个“浴”或基底外侧（BL）酸碱参数。我们发现，虽然JHCO 3是不敏感的pHBL孤立的变化，孤立的增加[CO2]BL导致JHCO 3上升，而孤立的增加[HCO-3]BL导致JHCO 3下降。对CO2的反应表明基底外侧膜附近存在CO2传感器。低剂量血管紧张素II（ANG II）在管腔或浴加重CO2反应，而高剂量ANG II（管腔或浴）或阻断剂， 酪氨酸激酶或蛋白激酶C都显著地减弱CO2反应。这项工作有三个目标：第一，JHCO 3对CO2塑料的反应（是否因慢性酸碱失衡而改变？）它是否延伸到其他转运过程，是否延伸到更多的小管片段？第二，在何种程度上JHCO 3对CO2的反应取决于近端小管中HCO-3重吸收的“硬件”？使用特异性抑制剂和基因敲除/转基因小鼠，我们将询问是否干扰顶端Na-H交换器（NHE 3）或H+泵或 携带这些转运蛋白的顶端囊泡的插入/回收降低了应答。使用免疫细胞化学，我们将询问CO2反应是否引起NHE 3或H+泵9蛋白分布的变化。第三，什么样的信号转导过程参与了对CO2的反应？ 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JHCO 3的作用。我们将研究CO2对潜在第二信使的影响（使用JHCO 3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。拟议的工作将提供重要的新的见解，肾脏如何适当地响应酸碱的挑战，也将阐明机制，血管紧张素II调节PT运输。