REGULATION OF CITRATE TRANSPORT

柠檬酸盐运输的监管

• 批准号: 6517548
• 负责人: L Lee HAMM
• 金额: $ 19.85万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517548
• 关键词: acid base balance; calcium; citrates; divalent cations; estrogens; hypokalemia; laboratory rabbit; magnesium; protein biosynthesis; protein localization; renal tubular transport; starvation; tissue /cell culture; transport proteins

Urinary citrate is one of the most important known endogenous inhibitors of calcium nephrolithiasis. Urinary citrate excretion is primarily determined by its fractional reabsorption in the proximal tubule. Previously, a major impediment to studies of citrate transport has been the lack of a suitable cell culture model. However, we have now clearly identified a method to study citrate reabsorption in a proximal tubule cell line (see preliminary data). In addition, the recent cloning of an apical dicarboxylate transporter from the proximal tubule by Pajor allows new insight into the regulation of citrate transport. This with our new culture model will allow us to examine several critical issues in the molecular regulation of citrate transport. The specific aims are to characterize both acute and chronic regulation of citrate absorption in the proximal tubule. Acute regulation of citrate transport by pH and divalent cations, in particular will be addressed. Although a variety of previous studies have addressed the influence of pH on citrate transport, our proposed studies will be able to directly address simultaneously the role of intracellular pH (to be measured with fluorescent probes) as well as extracellular pH. In regards to divalent cations, our preliminary data demonstrate dramatic effects of extracellular calcium and magnesium on citrate transport. Our findings suggest that changes in luminal calcium and magnesium concentrations within the ranges seen clinically may be important in determining urinary citrate excretion. The proposed studies will also address chronic regulation of citrate reabsorption. Our cell culture method will allow us to directly examine the mechanism of chronic regulation by pH, hypokalemia, and starvation. We hypothesize that changes in citrate reabsorption with each of these result from changes in the abundance and/or cellular distribution of the citrate transporter. In the proposed studies we will be able to measure the direct in vitro effects of these stimuli on citrate transport in polarized cells, and on mRNA and protein levels and cellular distribution of the dicarboxylate transporter.

尿柠檬酸盐是已知的最重要的钙肾结石内源性抑制剂之一。 尿柠檬酸盐排泄主要取决于其在近曲小管中的重吸收分数。 此前，柠檬酸盐转运研究的一个主要障碍是缺乏合适的细胞培养模型。 然而，我们现在已经明确确定了一种研究近曲小管细胞系中柠檬酸盐重吸收的方法（参见初步数据）。 此外，Pajor 最近从近端小管克隆了顶端二羧酸转运蛋白，为了解柠檬酸盐转运的调节提供了新的见解。我们的新培养模型将使我们能够研究柠檬酸盐运输分子调节中的几个关键问题。具体目标是表征近曲小管柠檬酸盐吸收的急性和慢性调节。 将特别讨论 pH 值和二价阳离子对柠檬酸盐转运的急性调节。 尽管之前的各种研究已经解决了 pH 对柠檬酸盐转运的影响，但我们提出的研究将能够直接同时解决细胞内 pH（用荧光探针测量）和细胞外 pH 的作用。 关于二价阳离子，我们的初步数据表明细胞外钙和镁对柠檬酸盐转运的显着影响。 我们的研究结果表明，临床上观察到的范围内管腔钙和镁浓度的变化对于确定尿柠檬酸盐排泄可能很重要。拟议的研究还将解决柠檬酸盐重吸收的长期调节。 我们的细胞培养方法将使我们能够直接检查 pH、低钾血症和饥饿的慢性调节机制。 我们假设柠檬酸盐重吸收的变化是由于柠檬酸盐转运蛋白的丰度和/或细胞分布的变化造成的。 在拟议的研究中，我们将能够测量这些刺激对极化细胞中柠檬酸转运以及二羧酸转运蛋白的 mRNA 和蛋白质水平以及细胞分布的直接影响。