RENAL BASIS OF HYPOCITRATURIA

低柠檬酸尿症的肾脏基础

• 批准号: 6238719
• 负责人: ROBERT J ALPERN
• 金额: $ 15.14万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-14 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238719
• 关键词: Krebs' cycle; Xenopus oocyte; apical membrane; biological transport; citrates; excretion; family genetics; gene mutation; genetic transcription; human genetic material tag; human subject; immunocytochemistry; kidney function; laboratory rat; membrane transport proteins; mitochondria; molecular cloning; nephrolithiasis; northern blottings; nuclear runoff assay; protein structure function; protein transport; western blottings

Hypocitraturia is an important cause of kidney stones. Urinary citrate excretion is regulated principally by the reabsorption of citrate in the proximal tubule. Proximal tubular citrate reabsorption is regulated at two steps: i) apical membrane citrate uptake; and 2) mitochondrial citrate uptake and metabolism. The proposed studies will examine four major aims regarding citrate transport by the proximal tubule. In Aim 1, we will clone the renal apical membrane Na/citrate cotransporter by expression cloning using an oocyte expression system. We will then characterize the transporter with regard to structure, function, and tissue distribution, in order to definitively determine that the clone corresponds to the apical membrane transporter. We will also clone other isoforms expressed in kidney cortex or intestine. Lastly, we will clone the human apical membrane Na/citrate transporter cDNA and gene, in order to allow genetic studies and definition of promoter function. In Aim 2, we will define the molecular basis for chronic regulation of the apical membrane Na/citrate cotransporter. Chronic acidosis and chronic K deficiency have both been demonstrated to cause chronic adaptations in the transporter that persist when the transporter is assayed in vitro. The present studies will examine the chronic regulation of apical membrane Na/citrate transporter: a) activity; b) protein abundance by western blot; c) protein distribution by cell fractionation and western blot; d) mRNA abundance by northern blot; and e) transcriptional rate by nuclear runoff. in Aim 3, we will examine whether there is chronic regulation of mitochondrial citrate transport and/or metabolism. Using the same chronic models of regulation, we will examine whether there are changes in mitochondrial function that persist in vitro. Mitochondrial citrate uptake will be measured as well as the activities of a number of key members of the tricarboxylic acid cycle. Once again, we will use western blotting to determine whether the abundance of any of the TCA cycle enzymes has been altered, northern blotting to determine whether the abundance of their respective mRNAs has been altered, and nuclear runoff studies to examine transcriptional rate. Lastly, using quantitative morphometry, we will examine whether these chronic conditions are associated with alterations in inner mitochondrial membrane surface area. In Aim 4, we will examine whether overactivity of the apical membrane Na/citrate transporter is responsible for familial hypocitraturia. In preliminary studies performed during the previous grant period, we have identified a number of families with frequent kidney stones, in which the proband has idiopathic hypocitraturia. We will examine other members of these families, as well as investigating other families. We will then explore whether any mutations are present in the apical membrane Na/citrate transporter gene that cosegregate with idiopathic hypocitraturia.

低柠檬酸尿是肾结石的重要原因。尿柠檬酸盐 排泄主要受体内柠檬酸盐的重吸收调节。 近端小管近端肾小管柠檬酸盐重吸收调节为2 步骤：i）顶膜柠檬酸盐摄取;和2）线粒体柠檬酸盐 吸收和代谢。拟议的研究将探讨四个主要目标 关于近端小管的柠檬酸盐转运。在目标1中，我们 通过表达克隆肾顶膜钠/柠檬酸盐协同转运蛋白 使用卵母细胞表达系统进行克隆。 然后我们将描述 转运蛋白的结构、功能和组织分布， 为了明确地确定克隆对应于 顶端膜转运蛋白 我们还将克隆表达的其他同种型， 在肾皮质或肠中。最后，我们将克隆人类根尖细胞， 膜钠/柠檬酸盐转运蛋白cDNA和基因，以允许遗传 启动子功能的研究和定义。在目标2中，我们将定义 根尖膜Na/citrate慢性调节的分子基础 协同转运蛋白慢性酸中毒和慢性缺钾都是 证明会导致转运蛋白的慢性适应， 当在体外测定转运蛋白时。本研究将探讨 顶端膜Na/柠檬酸盐转运蛋白的慢性调节：a） 活性; B）蛋白质丰度，通过蛋白质印迹法; c）蛋白质分布， 细胞分级分离和蛋白质印迹; d）通过北方印迹的mRNA丰度; 和e）通过核径流的转录速率。在目标3中，我们将研究 是否存在线粒体柠檬酸盐转运的慢性调节 和/或新陈代谢。使用相同的长期监管模式，我们将 检查是否有线粒体功能的变化， 体外将测量线粒体柠檬酸盐摄取以及 三羧酸循环的许多关键成员的活动。 我们将再次使用蛋白质印迹法来确定 任何TCA循环酶的丰度已经改变，北方 印迹以确定它们各自的mRNA的丰度是否 被改变，核径流研究，以检查转录率。 最后，使用定量形态测量学，我们将检查这些 慢性疾病与线粒体内部的改变有关， 膜表面积在目标4中，我们将研究是否过度活跃， 顶端膜钠/柠檬酸转运蛋白负责家族性 低柠檬酸尿症在上一次赠款期间进行的初步研究中， 在此期间，我们已经确定了一些家庭频繁肾 结石，其中先证者患有特发性低柠檬酸尿症。我们将 检查这些家庭的其他成员，以及调查其他 家庭然后我们将探讨是否有任何突变存在于 顶膜钠/柠檬酸盐转运蛋白基因与 特发性低柠檬酸尿症