Oxalate Handling in PAT1 and DRA Knockout Mice

描述(申请人提供)：高草酸尿被认为是草酸钙结石病发生的主要风险因素，草酸钙结石病发生在大约12%的美国人口中，估计每年造成20亿美元的损失。草酸的动态平衡由肠道吸收的草酸和肝脏产生的草酸的量控制，并被肾脏和肠道排泄所抵消。因此，肠道和肾脏的上皮膜是生物体与其环境之间草酸交换的主要界面，准备并协调跨上皮运输草酸的运输系统在草酸动态平衡中具有重要作用。长期以来，阴离子交换蛋白一直被认为与肠道和肾脏中的草酸运动有关，其中SLC26和SLC4基因家族成员编码的蛋白最受关注。有一个基因(SLC26A6)，命名为PAT1，在小肠顶膜和近端小管中大量表达，被认为参与了管腔和细胞液之间的草酸交换，但由于存在其他交换器，PAT1的相对重要性一直难以确定。我们使用PAT1基因敲除(KO)小鼠的初步研究表明，这些动物具有显著的高草酸，并支持远端回肠对草酸的吸收比它们的野生型(WT)窝产仔显著增加。因此，我们已经证明了一个基因缺失会导致草酸处理的戏剧性变化。根据我们的初步数据，我们提出了PAT1介导的顶端草酸外流，我们假设DRA(SLC26A3)是负责草酸吸收的主要顶端转运体。在目标1中，我们将通过比较WT和3个KO模型(即PAT1、DRA和PAT1/DRA联合缺失小鼠)对肾脏和肠道草酸的处理，直接讨论PAT1和DRA转运体对草酸稳态的生理意义。在目标2中，SLC26和SLC4家族其他成员的可能的代偿性适应将通过实时聚合酶链式反应和定量免疫组织化学在所有KO小鼠中确定。在目标3中，我们将在肾和肠细胞培养模型中使用基因敲除和功能表达方法来评估单个阴离子交换器的相对重要性。了解草酸在健康和疾病中的处理对于开发潜在的药物疗法至关重要。

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.