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The Function of Claudin-7 in Renal Epithelial Cells

Claudin-7在肾上皮细胞中的功能

基本信息

批准号：
8288763
负责人：
YAN-HUA CHEN
金额：
$ 31.96万
依托单位：
EAST CAROLINA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-10 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8288763
关键词：
Acute Kidney Tubular Necrosis Aldosterone Apical Bartter Disease Biotin Birth Blood Cell Polarity Cells Complementary DNA Defect Development Distal Duct (organ) structure Electron Microscopy Epithelial Epithelial Cells Epithelium Event Extracellular Domain Failure Feedback Functional disorder Goals Hematocrit procedure Histopathology Hypertension Impairment Injection of therapeutic agent Ions Kidney Knock-in Mouse Knock-out Knockout Mice LLC-PK1 Cells Laboratory Finding Life Link Maps Measurement Measures Mediating Methods Mouse Strains Mus Mutation Nephrons Newborn Infant Nitrogen Pattern Permeability Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Plasma Play Property Proteins RNA Interference Regulation Renal tubule structure Renin Role Saline Site Sodium Chloride Structure Suggestion Syndrome System Testing Tight Junctions Tracer Tubular formation Type II Pseudohypoaldosteronism Urine Water Western Blotting base body water loss cell injury human disease in vivo in vivo Model intraperitoneal kidney epithelial cell light microscopy molecular marker mutant postnatal protein function pup research study salt sensitive success wasting

项目摘要

Project Summary Dysfunction of renal epithelial Cl- transport is associated with human diseases such as Bartter and Gitelman syndromes as well as in salt-sensitive hypertension. Mutations in WNK4 kinase have been linked to hypertension in pseudohypoaldosteronism type II (PHAII). PHAII-causing mutant WNK4 increases paracellular Cl- permeability and phosphorylates tight junction (TJ) protein claudins. Recently, we have found that claudin-7 plays a crucial role in regulating paracellular Cl- permeation and is a specific TJ target of WNK4 kinase. Claudin-7 knockout mice (Cln7-/-) display salt wasting and water loss phenotypes, suggesting the impairment of ion reabsorption in renal tubules. Our long- term goals are to understand TJ protein functions in kidneys and their contribution to ionic imbalance in human diseases such as hypertension. This project will test the hypothesis that claudin-7 is essential for TJ functions in renal epithelial cells and interacts with WNK4 in modulation of paracellular Cl- permeation. This application has three Specific Aims: (1) to characterize our recently generated Cln7-/- mice and determine whether claudin- 7 is essential in the formation of paracellular pores allowing Cl- permeation. We will use primary epithelial cells isolated from collecting duct (CD) of Cln7+/+ and Cln7-/- mice to determine their paracellular ion selectivity. We will transfect wild-type claudin-7 to determine if claudin-7 functions can be restored in Cln7-/- CD cells. We will also transfect the extracellular domain (ED) mutants of claudin-7 into Cln7-/- CD cells and LLC-PK1 cells with claudin-7 knockdown by RNAi to determine the role of claudin-7 ED in paracellular ion selectivity; (2) to investigate the regulation of claudin-7 mediated paracellular Cl- permeability by WNK4 kinase. We will determine if the expression and localization of WNK4 are altered in epithelia of distal nephron in Cln7-/- mice as well as in Cln7-/- CD cells. We will determine the changes in paracellular Cl- permeability by the expression of WNK4 and its PHAII-causing mutant in CD cells. Claudin-7 phosphorylation-null and -mimic mutants at WNK4 site will be transfected into Cln7-/- CD cells as well as LLC-PK1 cells to determine the role of WNK4 phosphorylation of claudin-7 on paracellular ion selectivity and (3) to investigate the roles of claudin-7 deletion in the development of salt-wasting and acute tubular necrosis (ATN) in Cln7-/- mouse kidney. TJ ultrastructure, barrier function, blood and urine ion concentration as well as plasma renin level will be compared at postnatal day 1, 4, and 7 Cln7-/- mice to determine how Cln7-/- phenotypes develop after birth. Newborn Cln7-/- mice will be subject to NaCl supplement to determine if it delays ATN phenotype and prolong the life of Cln7-/- mice. Project Narrative This project will test the hypothesis that claudin-7 is essential for tight junction functions in kidney epithelial cells and interacts with WNK4 in modulation of paracellular Cl- permeation. This project has three specific aims to (1) characterize our recently generated claudin-7 knockout (Cln7-/-) mouse line and determine whether claudin-7 is essential in the formation of paracellular pores allowing Cl- permeation; (2) investigate the regulation of claudin-7 mediated paracellular Cl- permeability by WNK4 based on our findings that claudin-7 interacts with and is phosphorylated by WNK4 and (3) investigate the roles of claudin-7 deletion in the development of salt-wasting and acute tubular necrosis in Cln7-/- mouse kidney.

项目摘要肾上皮Cl-转运功能障碍与人类疾病如Bartter 和Gitelman综合征以及盐敏感性高血压。WNK 4激酶的突变与假性醛固酮减少症II型（PHAII）的高血压有关。PHAII致突变体 WNK 4增加细胞旁Cl-渗透性并磷酸化紧密连接（TJ）蛋白密蛋白。最近，我们发现claudin-7在调节细胞旁氯离子渗透中起着至关重要的作用，并且是WNK 4激酶的特异性TJ靶标。Claudin-7敲除小鼠（Cln 7-/-）显示盐消耗和失水表型，提示肾小管离子重吸收受损。我们长久以来- 学期目标是了解TJ蛋白在肾脏中的功能及其对离子失衡的作用人类疾病如高血压。本项目将检验claudin-7对肾上皮细胞TJ功能至关重要的假设。细胞，并与WNK 4相互作用，调节细胞旁氯渗透。该应用程序有三个具体目的：（1）表征我们最近产生的Cln 7-/-小鼠并确定claudin-1是否为Cln 7-/-小鼠。 7是必不可少的形成细胞旁孔允许Cl-渗透。我们将使用primary 从Cln 7 +/+和Cln 7-/-小鼠的集合管（CD）分离的上皮细胞，以确定它们的细胞旁离子选择性我们将检测野生型claudin-7，以确定claudin-7功能是否可以在Cln 7-/- CD细胞中恢复。我们还将检测细胞外结构域（艾德）突变体，通过RNAi将密蛋白-7敲低导入Cln 7-/- CD细胞和LLC-PK 1细胞中，以确定Cln 7-/- CD细胞和LLC-PK 1细胞中的密蛋白-7的表达。 claudin-7艾德在细胞旁离子选择性中的作用;（2）研究claudin-7的调节作用介导的细胞旁氯渗透性的WNK 4激酶。我们将确定表达式和在Cln 7-/-小鼠以及Cln 7-/- CD中，WNK 4的定位在远端肾单位的上皮中改变细胞我们将通过WNK 4的表达来确定细胞旁Cl-渗透性的变化，它在CD细胞中的PHAII突变体。在WNK 4处的Claudin-7磷酸化无效和模拟突变体将该位点转染到Cln 7-/- CD细胞以及LLC-PK 1细胞中以确定WNK 4的作用 claudin-7的磷酸化对细胞旁离子选择性的影响;（3）研究claudin-7的作用在Cln 7-/-小鼠肾中盐耗和急性肾小管坏死（ATN）的发展中的缺失。 TJ的超微结构、屏障功能、血、尿离子浓度以及血浆肾素水平将在出生后第1天、第4天和第7天比较Cln 7-/-小鼠以确定Cln 7-/-表型如何发展出生后新生Cln 7-/-小鼠将接受NaCl补充以确定其是否延迟ATN 表型和延长Cln 7-/-小鼠的寿命。项目叙述本项目将检验claudin-7对细胞紧密连接功能至关重要的假设，肾上皮细胞，并与WNK 4相互作用，调节细胞旁氯渗透。这项目有三个具体目标：（1）表征我们最近产生的claudin-7敲除（Cln 7-/-）小鼠系，并确定claudin-7是否在细胞旁孔的形成中是必需的（2）研究claudin-7介导的细胞旁Cl-的调节基于我们的发现，封闭蛋白-7与WNK 4相互作用并被其磷酸化， WNK 4和（3）研究claudin-7缺失在盐耗和急性脑梗死发生中的作用。 Cln 7-/-小鼠肾脏中的肾小管坏死。