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; 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分泌酸到小管腔中，从而重新吸收大多数过滤的HCO-3，并导出人体代谢产生的酸负荷。尽管众所周知，近端小管（PT）和更远端的肾单位段可以根据血液的酸碱状态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尚不清楚。我们已经研究了孤立的灌注兔PTS中HCO-3的重吸收率（JHCO3） 平衡（OOE）CO2/HCO-3溶液一次变化，一次是“浴”或基底外侧（BL）酸碱参数。我们发现，尽管JHCO3对PHBL的孤立变化不敏感，但[CO2] BL的孤立增加导致JHCO3上升，而[HCO-3] BL的分离增加导致JHCO3下降。对CO2的反应表明，基底外侧膜附近存在二氧化碳传感器。腔或浴中的低剂量血管紧张素II（ANG II）突出二氧化碳反应，而高剂量ANG II（腔或浴室）或任何一个的阻滞剂 酪氨酸激酶或蛋白激酶C均显着钝化二氧化碳反应。拟议的工作具有三个目的：首先，JHCO3对CO2塑料的反应（是否会因慢性酸碱干扰而改变吗？），是否扩展到其他转运蛋白过程，并且是否延伸至更多的小管片段？其次，JHCO3对CO2的响应在多大程度上取决于近端小管中HCO-3重吸收的“硬件”？使用特定的抑制剂和敲除/转基因小鼠，我们将询问是否干扰Na-H-H交换器（NHE3）或H+泵或 携带这些转运蛋白的顶囊泡的插入/检索减少了反应。使用mmunocytoothemistion，我们将询问CO2响应是否需要变化NHE3或H+-Pump 9roteins的分布。第三，对CO2的响应涉及哪些信号转移过程？ 我们将使用特定信号转导步骤的ANG II和阻滞剂或阻滞剂或激动剂来阻断或模仿CO2对JHCO3的影响。我们将检查二氧化碳对潜在第二使者的影响（使用JHCO3或PHI变化作为运输指数）， 并使用2-D凝胶和质谱镜检查来鉴定参与信号转导的候选蛋白。拟议的工作将为肾脏如何应对酸碱挑战的适当反应提供重要的新见解，并将阐明ANG II调节PT运输的机制。