CITRATE TRANSPORT IN THE PROXIMAL TUBULE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 各种研究估计，1963%的含钙肾结石患者有低柠檬酸尿症是原因之一。了解柠檬酸转运的调节机制将有望改善对低柠檬酸尿症原因的诊断。尿柠檬酸盐是钙性肾结石的重要抑制物，主要由近端肾小管的部分重吸收决定。二羧酸转运体(NaDC1)可能是肾单位顶端摄取滤过性柠檬酸的主要机制。尿中柠檬酸排泄最重要的生理调节因素是酸碱状态。随着尿钙的增加，尿液中的柠檬酸也随之增加。拟议的研究将解决钙对柠檬酸盐运输的急性调节，以及酸碱扰动和低钾血症对柠檬酸盐运输的慢性调节。利用一种新的柠檬酸转运体外模型，OK细胞在特定条件下被研究，柠檬酸盐和二元酸的摄取对细胞外钙敏感。这些研究表明OK细胞柠檬酸转运系统可能是一种新的柠檬酸转运系统。最近，又培育出了一种二元酸转运细胞系。稳定转染人NaDC1的人视网膜色素上皮细胞(Cubs细胞)在体外对酸碱条件有反应，因此将代表一种强大的新模型。我们将检验两个假设：1.钙能强烈地抑制哺乳动物近端小管细胞中一种新的柠檬酸转运过程。2.柠檬酸盐近端小管运输的慢性调节是通过冗余机制完成的，包括改变NaDC1蛋白的产生和将预先存在的NaDC1蛋白从根尖下小泡插入到顶膜中。具体目的是：1.阐明钙敏感的柠檬酸转运过程是一种新的转运蛋白，而不是NaDC1，并确定细胞外钙改变这一柠檬酸转运过程的细胞机制。2.阐明酸碱紊乱和低钾血症对柠檬酸转运的慢性调节机制。为了达到这一目的，我们将研究三种调节模式：转录调节(或信使核糖核酸稳定性)调节，蛋白质水平调节，以及在代谢性酸中毒和低血钾条件下，NaDC1从根尖下小泡转运进入和流出根尖膜的调节。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Various studies estimate that 1963% of individuals with calcium containing kidney stones have hypocitraturia as a contributing cause. Understanding the mechanisms of the regulation of citrate transport will hopefully lead to improved diagnosis of causes of hypocitraturia. Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by fractional reabsorption in the proximal tubule. The dicarboxylate transporter (NaDC1) is presumably the main mechanism of apical uptake of filtered citrate along the nephron. The most important physiologic regulator of urinary citrate excretion is acid-base status. Also urinary citrate increases as urinary calcium increases. The proposed studies will address the acute regulation of citrate transport by calcium, and chronic regulation of citrate transport by acid-base perturbations and hypokalemia. Using a newly characterized in vitro model of citrate transport, OK cells studied under particular conditions, citrate and dicarboxylate uptake are sensitive to extracellular calcium. These studies indicate that the OK cell citrate transport system is likely a novel citrate transporter. Recently another cell line of dicarboxylate transport was developed. Human retinal pigmented epithelial cells stably transfected with human NaDC1 (CUBS cells) are responsive to acid-base conditions in vitro and will therefore represent a powerful new model. Two hypotheses will be examined: 1. Calcium acutely inhibits a novel citrate transport process in mammalian proximal tubule cells. 2. Chronic regulation of proximal tubule transport of citrate is accomplished by redundant mechanisms including changes in NaDC1 protein production and insertion of pre-existing NaDC1 protein into the apical membrane from sub-apical vesicles. The specific aims are: 1. To delineate the calcium sensitive citrate transport process by: demonstrating that the calcium sensitive citrate transport process is a novel transporter, not NaDC1, and determining the cellular mechanisms whereby extracellular calcium alters this citrate transport process. 2. To delineate the mechanisms of chronic regulation of citrate transport by acid-base perturbations and hypokalemia. To achieve this aim three modes of regulation will be examined: transcriptional (or mRNA stability) regulation, regulation at the protein level, and regulation by trafficking of NaDC1 into and out of the apical membrane from sub-apical vesicles under conditions of metabolic acidosis and hypokalemia.