Oxalate Handling in PAT1 and DRA Knockout Mice

PAT1 和 DRA 敲除小鼠中的草酸盐处理

基本信息

批准号：
7577538
负责人：
Marguerite Hatch
金额：
$ 29.16万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7577538
关键词：
APPBP2 gene Address Adherent Culture Animals Anion Exchangers (Proteins)Anions Apical Attention Bicarbonates Biological Models Caco-2 Cells Calcium Oxalate Calculi Cell Culture Techniques Cell model Cells Chloride Ion Chlorides Clinical Complement Creatinine Cytosol Data Deposition Development Disease Distal Enteral Environment Epithelial Epithelium Excretory function Family Gene Deletion Gene Family Genes Goals Health Homeostasis Hyperoxaluria Immunofluorescence Immunologic Immunohistochemistry Individual Inherited Intestines Kidney Kidney Failure Knock-out Knockout Mice Large Intestine Liver Measures Mediating Membrane Metabolic Diseases Modeling Molecular Movement Mus Organism Oxalates Pattern Physiological Physiology Play Population Primary Hyperoxaluria Proteins Regulation Relative (related person)Renal clearance function Research Personnel Residual state Risk Factors Role Route SLC26A3 gene Small Interfering RNA Small Intestines Source System Techniques Testing Time Tissues Vesicle Western Blotting absorption apical membrane base brush border membrane cost effective therapy expectation hatching ileum in vivo kidney cell liver transplantation mRNA Expression member monolayer novel oxalosis programs solute sulfate transporter treatment strategy uptake

项目摘要

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.

描述（由申请人提供）：高草酸尿症被认为是草酸钙结石病发生的主要危险因素，大约 12% 的美国人口患有草酸钙结石病，每年造成的损失估计为 20 亿美元。草酸盐稳态由肠道吸收的膳食草酸盐量和肝脏产生的草酸盐量控制，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盐交换的主要界面，而准备和协调跨上皮移动草酸盐的运输系统在草酸盐稳态中具有重要作用。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盐运动有关，其中由 SLC26 成员编码的蛋白以及可能由 SLC4 基因家族编码的蛋白最受关注。一种称为 PAT1 的基因 (SLC26A6) 在小肠和近端小管的顶膜中大量表达，据推测参与管腔和细胞质之间的草酸盐交换，但鉴于以下因素，PAT1 的相对重要性一直难以确定：其他交换器的存在。我们使用 PAT1 敲除 (KO) 小鼠进行的初步研究表明，与野生型 (WT) 同窝小鼠相比，这些动物具有显着的高草酸尿症，并且支持远端回肠的草酸盐吸收显着增加。因此，我们已经证明单基因缺失会导致草酸盐处理发生巨大变化。根据我们的初步数据，我们提出 PAT1 介导顶端草酸盐流出，并且我们假设 DRA (SLC26A3) 是负责草酸盐吸收的主要顶端转运蛋白。在目标 1 中，我们将通过比较 WT 和 3 KO 模型（即 PAT1、DRA 和组合 PAT1/DRA 缺失小鼠）中肾脏和肠道草酸盐处理，直接探讨 PAT1 和 DRA 转运蛋白对草酸盐稳态的生理意义。在目标 2 中，将通过实时 PCR 和定量免疫组织化学在所有 KO 小鼠中确定 SLC26 和 SLC4 家族其他成员的表达模式可能的补偿性适应。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各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物疗法至关重要。