EPITHELIAL OXALATE AND CITRATE TRANSPORT

草酸盐通过肾小球滤过和近端肾小管分泌的组合排泄到尿液中。 草酸盐的肠吸收反过来可能是其在尿中排泄的主要决定因素， 结石病的自然史我们和其他人已经表明顶端阴离子交换剂Slc 26 a6， 已知在鼠近端小管和肠中表达，能够介导Cl ′-草酸盐，SO 42 ″。 - 草酸盐、Cl '-甲酸盐、Cl'-HCCy和Cl '-OH-交换。爪蟾草酸盐转运的直接测定 卵母细胞揭示了几种其它的SLC 26和SLC 4交换剂能够转运草酸盐， 表明上皮草酸盐交换存在显著的分子异质性。我们建议在 目的1系统评价SLC 26交换体和SLC 4交换体在肠道草酸代谢中的作用 转运，从而确定负责通过这种转运的经上皮草酸盐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， 其特征在于单个cDNA在爪蟾卵母细胞中的异源表达。我们还将 描述Caco-2肠上皮细胞系中草酸盐转运机制，我们将评估 使用RNA干扰的特定交换剂的定量作用。在肾脏内，顶膜 近端小管的结构中含有Cl '-草酸盐、草酸盐基和SO 42”-草酸盐 Slc 26 a6显然能够进行所有这些活性，并且可能是主要的顶端草酸盐交换。 交换器在这些细胞中。因此，认为由Slc 26 a6介导的顶端草酸盐交换在细胞凋亡中起作用。 近端肾小管分泌草酸盐，与Slc 26 a1介导的基底外侧草酸盐交换一致。到 为了阐明Slc 26 a6在近端小管中的作用，我们将在Aim 2中产生具有loxP位点侧翼的小鼠。 Slc 26 a6基因的外显子5-7（“floxed小鼠”），用于这些外显子的细胞类型特异性缺失。这些小鼠 和/或具有外显子5-7的种系缺失的小鼠最初将用于检查 Slc 26 a6蛋白质在近端小管顶端的草酸盐交换，使用各种技术。最后 很大一部分草酸钙结石患者表现出摄入的草酸盐吸收过度， 目标1中确定的主要肠道草酸盐转运蛋白的遗传变异可能易患 肾结石。利用遗传核心的资源和专业知识，我们将定义 特定SLC 26和SLC 4基因的遗传变异，从顶端交换器SLC 26 A6开始。的 因此，SLC 26 A6基因中的非保守编码序列变异的功能后果将是 研究使用异源表达在非洲爪蟾卵母细胞。我们还将确定函数 WNK 4编码序列变异的后果，WNK 4是一种对两种蛋白都具有强效抑制作用的新型激酶， SLC 26 A2和SLC 26 A6，小肠中的顶端草酸盐交换体。这种表型特征 将与这些多态性对结石病遗传易感性的影响相关， 高尿酸，如项目3所确定。

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.