The Function of Claudin-7 in Renal Epithelial Cells

描述(申请人提供)：肾上皮氯离子转运功能障碍与人类疾病如巴特综合征和吉特尔曼综合征以及盐敏感性高血压有关。WNK4激酶的突变与假性醛固酮增多症II型(PHAII)的高血压有关。引起PHAII的突变体WNK4增加了细胞旁的氯离子通透性，并使紧密连接(TJ)蛋白Claudins磷酸化。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WNK4激酶的一个特异性TJ靶点。Claudin-7基因敲除小鼠(Cln7-/-)表现出盐耗和失水的表型，提示肾小管离子重吸收受损。我们的长期目标是了解TJ蛋白在肾脏中的功能及其在高血压等人类疾病离子失衡中的作用。本项目将验证这一假说，即claudin-7对肾上皮细胞的TJ功能是必不可少的，并与WNK4相互作用，调节细胞旁的氯离子渗透。这项应用有三个具体的目标：(1)表征我们最近产生的Cln7-/-小鼠，并确定claudin-7是否在允许氯离子渗透的细胞旁毛孔的形成中是必不可少的。我们将使用从Cln7/和Cln7-/-小鼠的集合管(CD)分离的原代上皮细胞来确定它们的细胞旁离子选择性。我们将野生型claudin-7基因导入Cln7-/-CD细胞，以确定是否可以恢复claudin-7的功能。我们还将Claudin-7的胞外区(ED)突变体通过RNAi技术导入Cln7-/-CD细胞和LLC-PK1细胞，以确定Claudin-7的胞外区(ED)在细胞外离子选择性中的作用；(2)研究WNK4对Claudin-7介导的细胞外Cl-通透性的调节作用。我们将确定WNK4在Cln7-/-小鼠远端肾单位上皮细胞和Cln7-/-CD细胞中的表达和定位是否发生改变。我们将通过WNK4及其导致PHAII的突变体在CD细胞中的表达来确定细胞旁氯通透性的变化。将WNK4位点的Claudin-7磷酸化缺失和模拟突变体分别导入Cln7-/-CD细胞和LLC-PK1细胞，以确定其在Cln7-/-CD细胞和LLC-PK1细胞中的作用；(3)探讨Claudin-7缺失在Cln7-/-小鼠肾脏盐耗和急性肾小管坏死(ATN)发生中的作用。TJ超微结构、屏障功能、血和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天进行比较，以确定Cln7-/-表型在出生后如何发展。新生Cln7-/-小鼠将接受氯化钠补充，以确定其是否延迟ATN表型并延长Cln7-/-小鼠的寿命。 公共卫生相关性本项目将验证Claudin-7对肾上皮细胞紧密连接功能至关重要的假设，并与WNK4相互作用，调节细胞旁的氯离子渗透。本项目有三个具体目标：(1)鉴定我们最近获得的claudin-7基因敲除(Cln7-/-)小鼠系的特征，并确定Claudin-7是否在形成允许氯离子渗透的细胞旁毛孔中起关键作用；(2)根据我们的发现，研究Claudin-7与WNK4相互作用并被WNK4磷酸化；(3)研究claudin-7缺失在Cln7-/小鼠肾脏盐耗和急性肾小管坏死发生发展中的作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.