Mechanisms and Correction of Abnormal Bicarbonate Secretion by DRA in Diarrhea

DRA 治疗腹泻时碳酸氢盐异常分泌的机制及纠正

• 批准号: 9981963
• 负责人: MARK DONOWITZ
• 金额: $ 16.03万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-08 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981963
• 关键词: Acute; Affect; Anion Transport Proteins; Anions; Antidiarrheals; Area; Bicarbonates; Binding; Brush Border; Caco-2 Cells; Cell model; Cells; Cessation of life; Child; Cholera Toxin; Colon; Colon Carcinoma; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dehydration; Development; Diarrhea; Dissociation; Drug Targeting; Early Endosome; Electrolytes; Functional disorder; Future; Goals; Grant; Human; Intestines; Ion Transport; Knowledge; Link; Location; Modeling; Molecular Chaperones; Outcome; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Physiology; Population; Process; Proteins; Public Health; Regulation; Reporting; Research; Role; SLC26A3 gene; Small Intestines; Stem cells; Testing; Time; Undifferentiated; Villus; absorption; antiporter; apical membrane; colon cancer cell line; diarrheal disease; drug development; human subject; ileum; innovation; link protein; monolayer; new therapeutic target; novel; trafficking; virtual

SUMMARY There remain major gaps in understanding regulation of human intestinal electrolyte transport under normal physiologic conditions and in diarrheal diseases. This proposal deals with the role of the brush border (BB) Cl/HCO3 antiporter, SLC26A3 (DRA) in intestinal Na, Cl and HCO3 transport and will resolve the gap in understanding of its acute regulation, intestinal cells involved and ways to reverse the abnormal transport in severe, cAMP related diarrheas. The long term goal of this project is to define the coordinated and dynamic changes in Na, Cl and HCO3 transport that occur in diarrhea so that they can be targeted by drugs to reverse the changes and treat most diarrheal diseases. The Aims of this proposal include to: I.Test the hypothesis that necessary steps in cAMP/cholera toxin inhibition of neutral Na and Cl absorption that involves DRA, which occurs at the same time as stimulation of DRA activity and the related HCO3 secretion, includes increased trafficking of DRA to the BB and reduction in the physical association of DRA with NHE3 that occurs under basal conditions and increased physical association with CFTR. II. Test the hypothesis that cAMP related changes in DRA and NHE3 activities and trafficking involve association with SNX27 in early endosomes and these changes can be reversed by retromer stabilizers, which identify a new drug target to treat diarrhea. To accomplish these studies we will use ileal and proximal colonic enteroids made from healthy human subjects grown as 2D monolayers and compare the results with those from the human colon cancer cell line, Caco-2 cells. Caco-2 cells are used for many studies of intestinal physiology and for drug development, including anti- diarrheal drugs. We predict that Caco-2 cells will give similar information regarding intestinal Na, Cl and HCO3 transport under basal conditions and in models of diarrhea; however it will be important to identify any differences that could be relevant to studies of intestinal function and drug development. These studies are innovative being among the first to study electrolyte transport in human enteroids as monolayers, to examine the interaction of DRA with NHE3 and CFTR in the same cell population with emphasis on their dynamic interactions with each other under basal conditions and with elevated cAMP, are the initial study of the role of the early endosomal protein SNX27 and the retromer in acute DRA regulation, and develop studies of SNX27/retromer as a potential drug target for future development to treat diarrhea. The expected outcomes of this proposal will be to a) understand the coordinated regulation of DRA, NHE3, and CFTR in a model of severe diarrhea that includes consideration of mechanism of HCO3 loss, a gap in understanding the pathophysiology of diarrhea, b) establish that drug therapy of diarrhea must consider the coordinated regulation of DRA with NHE3 and CFTR and not just the regulation of each transporter alone, and c) identify a new drug target in a diarrhea model in human intestine that can reverse the diarrhea related transport changes in DRA and NHE3, and that potentially could be developed into a drug to treat diarrhea.

概括 在正常情况下，在理解人肠电解质转运的调节方面仍然存在重大差距 生理状况和腹泻疾病。该提案涉及刷子边框（BB）的角色 Cl/HCO3抗植物，SLC26A3（DRA）在肠内Na，Cl和Hco3传输中，将解决差距 了解其急性调节，所涉及的肠细胞以及扭转异常转运的方法 严重的，与营地有关的腹泻。该项目的长期目标是定义协调和动态的 腹泻中发生的Na，Cl和HCO3转运的变化，以便可以用药物靶向逆转 变化和治疗大多数腹泻疾病。该提议的目的包括：i.检验假设 营地/霍乱毒素抑制中性Na和Cl吸收的必要步骤，涉及DRA， 与刺激DRA活性和相关HCO3分泌的同时发生，包括增加 将DRA贩运到BB并减少DRA与NHE3的物理关联，发生在 基础条件和与CFTR的物理关联增加。 ii。检验与营地相关的假设 DRA和NHE3活动和贩运的变化涉及早期内体中与SNX27的关联 这些变化可以通过逆转录稳定剂逆转，这些变化剂鉴定出一种用于治疗腹泻的新药物靶标。到 完成这些研究，我们将使用由健康的人类受试者制成的回肠和近端结肠 作为2D单层生长，并将结果与​​人类结肠癌细胞系的结果进行比较 细胞。 CACO-2细胞用于许多肠道生理学和药物发育的研究，包括抗 腹泻药。我们预测Caco-2细胞将提供有关肠NA，CL和HCO3的类似信息 在基础条件下和腹泻模型中运输；但是，必须确定任何 可能与肠道功能和药物发育研究有关的差异。这些研究是 创新的是最早研究人类肠动物中电解质运输的人之一，以检查 DRA与NHE3和CFTR在同一细胞种群中的相互作用，重点是它们的动态 在基础条件下彼此相互作用和与升高的营地相互作用，是对的作用的初步研究 早期内体蛋白SNX27和急性DRA调节中的逆转录器，并开发了 SNX27/逆转录剂是治疗腹泻的未来发育的潜在药物靶标。预期的结果 该提议将是a）理解DRA，NHE3和CFTR的协调调节 严重的腹泻，包括考虑HCO3损失机制，这是理解的差距 腹泻的病理生理，b）确定腹泻的药物治疗必须考虑协调 使用NHE3和CFTR调节DRA，而不仅仅是单独调节每个转运蛋白，c）确定a 人类肠道模型中的新药物靶标可以逆转腹泻相关的转运变化 在DRA和NHE3中，可能会发展为治疗腹泻的药物。