EPITHELIAL OXALATE AND CITRATE TRANSPORT

草酸通过肾小球滤过和近端肾小管分泌物在尿液中排泄。 草酸的肠道吸收可能反过来是其在尿液中排泄的一个主要决定因素 结石病的自然病史。我们和其他人已经证明了顶端阴离子交换剂Slc26a6， 已知在小鼠近端小管和肠道表达，能够介导Cl‘-草酸，SO42“ -草酸、Cl‘-甲酸盐、CI’-HCCy和CI‘-OH“交换。非洲爪哇草酸转运的直接测量 卵母细胞已经揭示了其他几个SLC26和SLC4交换器能够运输草酸， 说明上皮草酸交换存在显著的分子异质性。我们建议在 目的1系统评价SLC26交换器和SLC4交换器在肠道草酸中的作用 运输，从而确定负责跨上皮草酸吸收的主要转运体 组织。因此，将评估草酸盐运输的动力学、药理学和电生理学 以单个cDNA在非洲爪哇卵母细胞中的异源表达为特征。我们还将 描述Caco-2肠上皮细胞系中草酸的转运机制，我们将在其中评估 使用RNA干扰的特定交换器的定量作用。在肾脏内，顶膜 近端小管的部分含有能够产生氯-草酸、草酸碱和SO42“-草酸的机制 交换；Slc26a6显然能够进行所有这些活动，并且很可能是主要的顶端草酸 这些电池中的交换器。因此，SLC26a6介导的尖端草酸交换被认为在 近端小管分泌草酸，与SLc26a1介导的基侧草酸交换相一致。至 阐明slc26a6在近曲小管中的作用，我们将在Aim 2中建立loxP位点两侧的小鼠 SLc26a6基因的第5-7外显子(“Floted小鼠”)，用于特定细胞类型的这些外显子的缺失。这些老鼠 和/或外显子5-7胚系缺失的小鼠最初将被用来研究 SLC26a6蛋白到顶端草酸在近端小管中的交换，使用各种技术。最后，一个 相当大一部分草酸钙结石患者表现出摄取草酸的高吸收，如 目标1中确定的主要肠道草酸转运体的遗传变异可能易患 肾结石。利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，从顶端交换器SLC26A6开始。这个 因此，SLC26A6基因非保守编码序列变异的功能后果将是 研究了异源基因在非洲爪哇卵母细胞中的表达。我们还将确定功能 WNK4编码序列变异的后果，WNK4是一种对两者都有有效抑制作用的新激酶 SLC26A2和SLC26A6是小肠中的顶端草酸交换器。这种表型特征 将与这些多态对结石病遗传易感性的影响相关 高草酸尿症，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.