EPITHELIAL OXALATE AND CITRATE TRANSPORT

上皮草酸盐和柠檬酸盐运输

• 批准号: 7727081
• 负责人: David Bruce Mount
• 金额: $ 41.08万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7727081
• 关键词: Acidosis; Acids; Affect; Anions; Apical; Bicarbonates; Brush Border; Caco-2 Cells; Calcium; Calcium Oxalate; Calcium-Sensing Receptors; Calculi; Cell Line; Cells; Characteristics; Citrate; Citrates; Code; Collaborations; Complementary DNA; Condition; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dependency; Dietary Factors; Disease; Electrophysiology (science); Epithelial; Epithelial Cells; Excretory function; Exhibits; Exons; Formates; Gene Family; Genes; Genetic; Genetic Polymorphism; Genetic Predisposition to Disease; Genetic Risk; Genetic Variation; Heterogeneity; Homeostasis; Human; Hyperoxaluria; Hypokalemia; Individual; Intake; Intestinal Absorption; Intestines; Kidney; Kinetics; Measurement; Measures; Mediating; Metabolic; Metabolism; Molecular; Mus; Natural History; Nephrolithiasis; Nuclear; Oxalates; Pathway interactions; Patients; Pharmacology; Phenotype; Phosphotransferases; Physiological; Physiology; Play; Protein Kinase; Proteins; RNA Interference; Regulation; Research Personnel; Residual state; Resources; Risk; Role; SLC26A3 gene; Site; Small Intestines; Sodium; Substrate Specificity; Techniques; Thinking; Tissues; Tubular formation; Urate; Urine; Variant; Vitamin D; Vitamin D3 Receptor; Xenopus oocyte; absorption; apical membrane; base; basolateral membrane; cell type; citrate carrier; expression cloning; gain of function mutation; glomerular filtration; member; mutant; novel; programs; urinary

Oxalate is excreted in the urine via a combination of both glomerular filtration and proximal tubular secretion. The intestinal absorption of oxalate may in turn be a major determinant of its excretion in the urine and the natural history of stone disease. We and others have shown that the apical anion exchanger Slc26a6, known to be expressed in the murine proximal tubule and intestine, is capable of mediating Cl'-oxalate, SO42" -oxalate, Cl'-formate, CI'-HCCy, and CI'-OH" exchange. Direct measurement of oxalate transport in Xenopus oocytes has revealed that several other SLC26 and SLC4 exchangers are capable of oxalate transport, indicating that there is significant molecular heterogeneity of epithelial oxalate exchange. We propose in Aim 1 to systematically evaluate the role of SLC26 exchangers and SLC4 exchangers in intestinal oxalate transport, so as to define the major transporters responsible for transepithelial oxalate absorption by this tissue. The kinetics, pharmacology, and electrophysiology of oxalate transport will thus be evaluated and characterized by heterologous expression of individual cDNAs in Xenopus oocytes. We will also characterize oxalate transport mechanisms in the Caco-2 intestinal epithelial cell line, in which we will assess the quantitative role of specific exchangers using RNA interference. Within the kidney, the apical membrane of the proximal tubule contains mechanisms capable of Cl'-oxalate, oxalate-base, and SO42"-oxalate exchange; Slc26a6 is clearly capable of all these activities and is likely the dominant apical oxalate exchanger in these cells. Apical oxalate exchange mediated by Slc26a6 is thus thought to function in proximal tubular secretion of oxalate, in concert with basolateral oxalate exchange mediated by Slc26a1. To clarify the role of Slc26a6 in the proximal tubule we will generate mice in Aim 2 with loxP sites flanking exons 5-7 of the Slc26a6 gene ("floxed mice"), for cell type-specific deletion of these exons. These mice and/or mice with a germline deletion of exons 5-7 will initially be utilized to examine the contribution of the Slc26a6 protein to apical oxalate exchange in the proximal tubule, using a variety of techniques. Finally, a significant fraction of patients with calcium-oxalate stones exhibit hyperabsorption of ingested oxalate, such that genetic variation in the major intestinal oxalate transporters identified in Aim 1 may predispose to nephrolithiasis. Using the resources and expertise of the Genetics Core we will define the spectrum of genetic variability in specific SLC26 and SLC4 genes, beginning with the apical exchanger SLC26A6. The functional consequence of non-conservative coding sequence variation in the SLC26A6 gene will thus be studied using heterologous expression in Xenopus oocytes. We.will also determine the functional consequences of coding sequence variation in WNK4, a novel kinase with potent inhibitory effects on both SLC26A2 and SLC26A6, apical oxalate exchangers in the small intestine. This phenotypic characterization will be correlated with the impact of these polymorphisms on the genetic predisposition to stone disease and hyperoxaluria, as determined in Project 3.

草酸盐通过肾小球滤过和近端管状分泌的结合在尿液中排泄。 草酸盐的肠道吸收反过来又可能是其在尿液中排泄的主要决定因素 石材疾病的自然历史。我们和其他人表明，顶的阴离子交换器SLC26A6， 已知在鼠近端小管和肠中表达，能够介导Cl'-Oxalate，SO42” - 氧化，cl'-formate，ci'-hccy和ci'-oh“交换。 卵母细胞表明，其他几个SLC26和SLC4交换器具有草酸盐的运输， 表明上皮草酸盐交换存在明显的分子异质性。我们建议 目标1可以系统地评估SLC26交换器和SLC4交换器在肠道中的作用 运输，以定义由此负责使草酸酯吸收的主要转运蛋白 组织。因此，将评估草酸盐转运的动力学，药理学和电生理学 以异武卵母细胞中各个cDNA的异源表达为特征。我们也会 表征CACO-2肠上皮细胞系中的草酸盐转运机制，我们将评估 使用RNA干扰的特定交换器的定量作用。在肾脏中，顶膜 近端小管中包含能够Cl'-Oxalate，Oxalate碱和SO42“ - 氧化的机制 交换; SLC26A6显然具有所有这些活动，可能是主要的草酸盐 这些细胞中的交换器。因此，由Slc26a6介导的草酸根交换被认为起作用 草酸盐的近端管状分泌，与SLC26A1介导的基底外侧草酸盐交换。到 阐明SLC26A6在近端小管中的作用，我们将在loxp站点侧面的AIM 2中生成小鼠 SLC26A6基因（“ Floxed小鼠”）的外显子5-7，用于这些外显子的细胞类型特异性缺失。这些老鼠 和/或具有外显子5-7种系缺失的小鼠最初将用于检查该贡献 SLC26A6蛋白使用多种技术在近端小管中的草酸根蛋白交换。最后，一个 钙 - 氧化石的大部分患者表现出摄入的草酸盐的超吸收，例如 AIM 1中确定的主要草酸肠转运蛋白主要肠道转运蛋白的遗传变异可能倾向于 肾结石病。使用遗传学核心的资源和专业知识，我们将定义 特定SLC26和SLC4基因的遗传变异性，从顶端交换器SLC26A6开始。这 因此，SLC26A6基因中非保守编码序列变化的功能后果将为 使用异爪蟾卵母细胞中的异源表达进行了研究。我们将确定功能 WNK4中编码序列变化的后果，一种新型激酶，对这两者都有有效的抑制作用 SLC26A2和SLC26A6，小肠中的根尖草酸盐交换器。这种表型表征 这些多态性将与这些多态性对石材疾病和遗传易感性的影响相关 高黄油尿症，如项目3中确定。