The Function of Claudin-7 in Renal Epithelial Cells

描述（由申请人提供）：肾上皮细胞Cl-转运功能障碍与人类疾病，如Bartter综合征和Gitelman综合征以及盐敏感性高血压有关。WNK4激酶突变与II型假性醛固酮减少症（PHAII）患者的高血压有关。引起phaii的突变体WNK4增加细胞旁Cl-通透性并磷酸化紧密连接（TJ）蛋白。最近，我们发现cladin -7在调节细胞旁Cl-渗透中起着至关重要的作用，是WNK4激酶的特异性TJ靶点。Cln7敲除小鼠（Cln7-/-）表现出盐消耗和水损失表型，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中离子失衡的作用。该项目将验证以下假设：claudin-7对肾上皮细胞TJ功能至关重要，并与WNK4相互作用，调节细胞旁Cl-渗透。该应用程序有三个特定目的：(1)表征我们最近生成的Cln7-/-小鼠，并确定claudin- 7在允许Cl-渗透的细胞旁孔的形成中是否必不可少。我们将使用从Cln7+/+和Cln7-/-小鼠收集管（CD）中分离的原代上皮细胞来确定它们的细胞旁离子选择性。我们将转染野生型claudin-7，以确定claudin-7的功能是否可以在Cln7-/- CD细胞中恢复。我们还将通过RNAi将claudin-7的胞外结构域（ED）突变体转染到Cln7-/- CD细胞和lc - pk1细胞中，以确定claudin-7 ED在细胞旁离子选择性中的作用；(2)研究WNK4激酶对claudin-7介导的细胞旁Cl-通透性的调节。我们将确定WNK4在Cln7-/-小鼠和Cln7-/- CD细胞的远端肾元上皮中的表达和定位是否发生改变。我们将通过WNK4及其在CD细胞中引起phaii的突变体的表达来确定细胞旁Cl-通透性的变化。将WNK4位点的Claudin-7磷酸化缺失和模拟突变体转染到Cln7-/- CD细胞和LLC-PK1细胞中，以确定WNK4磷酸化Claudin-7对细胞旁离子选择性的作用；(3)研究Cln7-/-小鼠肾脏中Claudin-7缺失在盐损耗和急性肾小管坏死（ATN）发生中的作用。将在出生后第1、4、7天比较小鼠TJ超微结构、屏障功能、血、尿离子浓度以及血浆肾素水平，以确定出生后Cln7-/-表型的发展情况。新生Cln7-/-小鼠将接受NaCl补充，以确定其是否延迟ATN表型并延长Cln7-/-小鼠的寿命。

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.