Oxalate Handling in PAT1 and DRA Knockout Mice

描述（由申请人提供）：高尿酸被认为是草酸钙结石病发生的主要危险因素，约12%的美国人发生草酸钙结石。S.每年估计要花费20亿美元。草酸盐体内平衡由肠道吸收的饮食草酸盐量和肝脏产生的草酸盐量控制，并由肾脏和肠道排泄抵消。因此，肠道和肾脏的上皮膜是生物体与其环境之间交换草酸盐的主要界面，并且平衡和协调以使草酸盐穿过上皮的转运系统在草酸盐稳态中具有重要作用。阴离子交换蛋白长期以来被认为参与肠和肾中的草酸盐运动，其中由SLC 26和可能的SLC 4基因家族成员编码的蛋白质吸引了最多的关注。一个基因（SLC 26 A6），被称为PAT 1是丰富的表达在小肠和近端小管的顶膜，并被认为是参与交换草酸之间的腔和胞质溶胶，但PAT 1的相对重要性一直难以解决的存在下，其他交换。我们使用PAT 1敲除（KO）小鼠的初步研究表明，与野生型（WT）同窝小鼠相比，这些动物显著高尿酸，并支持远端回肠对草酸盐吸收的显著增加。因此，我们已经证明，一个基因的缺失会导致草酸盐处理的巨大变化。基于我们的初步数据，我们提出，PAT 1介导顶端草酸外流，我们假设，SLC 26 A3是主要的顶端转运草酸吸收负责。在目标1中，我们将通过比较WT和3 KO模型（即PAT 1、PAT 2和联合PAT 1/PAT 2缺失小鼠）中肾脏和肠道草酸盐处理，直接讨论PAT 1和PAT 2转运蛋白对草酸盐稳态的生理意义。在目标2中，将通过所有KO小鼠的实时PCR和定量免疫组织化学来确定SLC 26和SLC 4家族其他成员表达模式中可能的补偿性适应。在目标3中，我们将在肾和肠细胞培养模型中使用敲低和功能表达方法来评估单个阴离子交换剂的相对重要性。了解草酸盐在健康和疾病中的处理对于开发潜在的药理学疗法至关重要。

DESCRIPTION (provided by applicant): Hyperoxaluria is considered a major risk factor in the genesis of calcium oxalate stone disease which occurs in about 12% of the U. S. population costing an estimated $2 billion annually. Oxalate homeostasis is governed by the amount of dietary oxalate absorbed in the intestine and that produced by the liver and is offset by both renal and enteric excretion. Thus, the epithelial membranes of the intestinal tract and the kidneys are the principal interfaces for the exchange of oxalate between the organism and its environment and the transport systems that are poised and coordinated to move oxalate across epithelia have significant roles in oxalate homeostasis. Anion exchange proteins have long been considered to be involved in oxalate movements in both intestine and kidney with proteins encoded by members of the SLC26 and possibly the SLC4 gene families attracting the most attention. One gene (SLC26A6), termed PAT1 is abundantly expressed in the apical membrane of small intestine and proximal tubule and is presumed to be involved in exchanging oxalate between the lumen and the cytosol, yet the relative importance of PAT1 has been difficult to resolve given the presence of other exchangers. Our initial studies, using PAT1 knockout (KO) mice show that these animals are significantly hyperoxaluric and support a dramatic increase in oxalate absorption by the distal ileum compared to their wild type (WT) littermates. Thus, we have already demonstrated that a one-gene deletion results in dramatic changes in oxalate handling. Based upon our preliminary data, we have proposed that PAT1 mediates apical oxalate efflux and we hypothesize that DRA (SLC26A3) is the major apical transporter responsible for oxalate uptake. In Aim 1, we will directly address the physiological significance of the PAT1 and DRA transporters to oxalate homeostasis by a comparison of renal and intestinal oxalate handling in WT and 3 KO models, namely PAT1, DRA, and combined PAT1/DRA null mice. In Aim 2, possible compensatory adaptations in the expression patterns of other members of the SLC26 and SLC4 families will be determined by real-time PCR and quantitative immunohistochemistry in all KO mice. In Aim 3, we will use knockdown and functional expression approaches in a renal and intestinal cell culture model to evaluate the relative importance of individual anion exchangers. Understanding oxalate handling in health and disease is essential for the development of potential pharmacological therapies.