CITRATE TRANSPORT IN THE PROXIMAL TUBULE

近端小管中的柠檬酸盐转运

• 批准号: 8167896
• 负责人: Kathleen S Hering-Smith
• 金额: $ 17.13万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167896
• 关键词: Acids; Acute; Address; Apical; Calcium; Cell Line; Cells; Chronic; Citrates; Computer Retrieval of Information on Scientific Projects Database; Diagnosis; Excretory function; Funding; Grant; Human; Hypokalemia; In Vitro; Individual; Institution; Kidney Calculi; Lead; Metabolic acidosis; Modeling; Nephrolithiasis; Nephrons; Physiological; Production; Proteins; Regulation; Research; Research Personnel; Resources; Source; Structure of retinal pigment epithelium; System; Transport Process; United States National Institutes of Health; Vesicle; apical membrane; base; citrate carrier; dicarboxylate; dicarboxylate-binding protein; extracellular; improved; in vitro Model; inhibitor/antagonist; mRNA Stability; novel; trafficking; uptake; urinary

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 各种研究估计，19-63% 的含钙肾结石患者患有低柠檬酸尿症。 了解柠檬酸盐转运的调节机制有望改善对低柠檬酸尿原因的诊断。尿柠檬酸盐是钙肾结石的重要抑制剂，主要由近曲小管的重吸收分数决定。二羧酸转运蛋白 (NaDC1) 可能是沿肾单位顶端摄取过滤柠檬酸盐的主要机制。尿柠檬酸盐排泄最重要的生理调节剂是酸碱状态。随着尿钙的增加，尿柠檬酸盐也会增加。拟议的研究将解决钙对柠檬酸盐转运的急性调节，以及酸碱扰动和低钾血症对柠檬酸盐转运的慢性调节。使用新表征的柠檬酸盐转运体外模型，在特定条件下研究 OK 细胞，柠檬酸盐和二羧酸盐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型柠檬酸转运蛋白。 最近开发了另一种二羧酸运输细胞系。 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。将检验两个假设： 1. 钙强烈抑制哺乳动物近曲小管细胞中一种新的柠檬酸盐转运过程。 2.近端小管柠檬酸盐转运的慢性调节是通过冗余机制完成的，包括NaDC1蛋白产生的变化以及将预先存在的NaDC1蛋白从根尖下囊泡插入根尖膜。 具体目标是： 1. 通过以下方式描述钙敏感性柠檬酸盐转运过程：证明钙敏感性柠檬酸盐转运过程是一种新型转运蛋白，而不是NaDC1，并确定细胞外钙改变该柠檬酸盐转运过程的细胞机制。 2. 描述酸碱扰动和低钾血症对柠檬酸盐转运的慢性调节机制。 为了实现这一目标，将检查三种调节模式：转录（或 mRNA 稳定性）调节、蛋白质水平调节以及代谢性酸中毒和低钾血症条件下 NaDC1 从根尖下囊泡进出顶膜的调节。