Oxalate Handling in PAT1 and DRA Knockout Mice

描述（由申请人提供）：高草酸尿被认为是草酸钙结石病发生的主要危险因素，该疾病发生在美国约12%的人口中，每年耗资约20亿美元。草酸体内平衡是由肠道吸收的草酸和肝脏产生的草酸量控制的，并由肾脏和肠道排泄抵消。因此，肠道和肾脏的上皮膜是机体与其环境之间草酸交换的主要界面，而在上皮间平衡和协调移动草酸的运输系统在草酸稳态中起着重要作用。阴离子交换蛋白长期以来被认为参与了肠道和肾脏的草酸盐运动，其中由SLC26和SLC4基因家族成员编码的蛋白吸引了最多的关注。一个被称为PAT1的基因（SLC26A6）在小肠顶端膜和近端小管中大量表达，被认为参与在管腔和细胞质之间交换草酸盐，但由于存在其他交换基因，很难确定PAT1的相对重要性。我们最初使用PAT1基因敲除（KO）小鼠进行的研究表明，与野生型（WT）小鼠相比，这些动物的草酸盐含量明显高，回肠远端对草酸盐的吸收显著增加。因此，我们已经证明了一个基因的缺失会导致草酸处理的巨大变化。基于我们的初步数据，我们提出PAT1介导草酸盐的根尖外排，我们假设DRA （SLC26A3）是负责草酸盐摄取的主要根尖转运蛋白。在Aim 1中，我们将通过比较WT和3ko模型（即PAT1、DRA和PAT1/DRA联合无效小鼠）肾脏和肠道对草酸盐的处理，直接探讨PAT1和DRA转运体对草酸盐稳态的生理意义。在Aim 2中，将通过实时荧光定量PCR和定量免疫组织化学在所有KO小鼠中确定SLC26和SLC4家族其他成员表达模式中可能的代偿性适应。在Aim 3中，我们将在肾脏和肠道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作用对于开发潜在的药物治疗是必不可少的。

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.