ION TRANSPORT IN ADPKD EPITHELIA

ADPKD 上皮细胞中的离子传输

• 批准号: 2152178
• 负责人: Bruce A. Stanton
• 金额: $ 14.27万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2152178
• 关键词: adenosine; antiport; apical membrane; arginine vasopressin; autosomal dominant trait; basolateral membrane; biological signal transduction; cellular pathology; chloride channels; confocal scanning microscopy; cyclic AMP; cytoskeleton; epithelium; human tissue; intracellular transport; ion transport; membrane permeability; membrane transport proteins; microtubules; polycystic kidney; prostaglandin E; sodium potassium exchanging ATPase; time resolved data; video microscopy; voltage /patch clamp

The long-term objective of this proposal is to understand the cellular mechanisms responsible for abnormal transepithelial NaCl and fluid secretion by cysts in human autosomal dominant polycystic kidney (ADPKD) kidneys. The ultimate goal is to identify an approach to slow or arrest the progression of ADPKD. To this end studies will be conducted to test the primary hypothesis that NaCl and fluid secretion by human ADPKD cysts is driven by electrogenic Cl secretion and that secretion is regulated by arginine vasopressin (AVP), adenosine and prostaglandins (PG) via an increase in cAMP. It is proposed that electrogenic Cl secretion involves uptake of Cl across the basolateral membrane by Na/K/2Cl cotransport and Cl/HCO exchange and diffusion out of the cells across the apical membrane by CFTR Cl channels. The Na-K-ATPase, by maintaining a low intracellular Na concentration, provides the driving force for Cl uptake across the basolateral membrane via the Na/K/2Cl cotransporter. Studies will also be conducted to test the hypothesis that intracellular trafficking of CFTR Cl channels in ADPKD cells is regulated by cAMP and is dependent on microtubules and the actin-based cytoskeleton. We propose that the observed changes in the cyoskeleton in ADPKD cells may alter the trafficking and insertion of CFTR Cl channels into the apical membrane. There are three specific aims. Specific Aim #1: To elucidate the cellular mechanisms of electrogenic Cl secretion across ADPKD epithelia. Transport mechanisms will be identified and characterized in human ADPKD cells using a number of experimental approaches including measurements of short circuit current, confocal-immunofluorescent microscopy and the patch clamp technique. Specific Aim #2: To elucidate the signal transduction pathways regulating Cl secretion across ADPKD epithelia. The regulation of Cl secretion by AVP, adenosine and PG and the signal transduction pathways mediating Cl secretion by these hormones will be evaluated using the patch clamp technique and measurements of short circuit current. Specific Aim #3: To elucidate the intracellular trafficking of CFTR Cl channels in ADPKD epithelia. Intracellular trafficking of CFTR and the regulation of trafficking by microtubules and the actin-based cytoskeleton will be evaluated by time-lapse, video-confocal microscopy of a CFTR-green fluorescent protein (GFP) fusion protein transfected into human ADPKD cells. An important aspect of this application is that the proposed studies will be performed on human ADPKD cells. Thus, the information derived from-these studies will provide a comprehensive and integrative understanding of the cellular mechanisms mediating fluid secretion into the lumen of cysts in human ADPKD kidneys and may ultimately lead to the identification of an approach to slow or arrest the progression of ADPKD.

该提案的长期目标是了解细胞 跨上皮 NaCl 和液体异常的机制 人常染色体显性多囊肾 (ADPKD) 中囊肿的分泌 肾脏。最终目标是找到一种减缓或阻止的方法 ADPKD 的进展。为此，将进行研究以测试 主要假设是人类 ADPKD 囊肿分泌 NaCl 和液体 由生电 Cl 分泌驱动，并且该分泌受以下因素调节 精氨酸加压素 (AVP)、腺苷和前列腺素 (PG) cAMP 增加。据推测，生电 Cl 分泌涉及 通过 Na/K/2Cl 协同转运跨基底外侧膜摄取 Cl Cl/HCO 交换并穿过顶膜扩散出细胞 通过 CFTR Cl 通道。 Na-K-ATP酶，通过维持低细胞内 Na 浓度为整个细胞吸收 Cl 提供驱动力 基底外侧膜通过 Na/K/2Cl 协同转运蛋白。研究也将 进行该假设是为了检验 CFTR Cl 的细胞内运输 ADPKD 细胞中的通道受 cAMP 调节，并且依赖于 微管和基于肌动蛋白的细胞骨架。我们建议 ADPKD 细胞中观察到的细胞骨架变化可能会改变 CFTR Cl 通道运输并插入顶膜。 具体目标有三个。具体目标#1：阐明细胞 ADPKD 上皮细胞产生电 Cl 分泌的机制。运输 将使用以下方法在人类 ADPKD 细胞中鉴定和表征机制 许多实验方法，包括短的测量 电路电流、共焦免疫荧光显微镜和膜片钳 技术。具体目标#2：阐明信号转导途径 调节 ADPKD 上皮细胞的 Cl 分泌。 Cl的调节 AVP、腺苷、PG的分泌及信号转导途径 将使用贴片评估这些激素介导的 Cl 分泌 钳位技术和短路电流测量。具体目标 #3：阐明 CFTR Cl 通道的细胞内运输 ADPKD 上皮细胞。 CFTR 的细胞内运输及其调控 微管和基于肌动蛋白的细胞骨架的运输将 通过 CFTR-green 的延时视频共聚焦显微镜进行评估 荧光蛋白 (GFP) 融合蛋白转染人 ADPKD 细胞。该应用的一个重要方面是所提议的 研究将在人类 ADPKD 细胞上进行。因此，信息 源自这些研究将提供全面且综合的 了解介导液体分泌的细胞机制 人类 ADPKD 肾脏囊肿的管腔，可能最终导致 确定减缓或阻止 ADPKD 进展的方法。