The Function of Claudin-7 in Renal Epithelial Cells

描述（由申请人提供）：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相关。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。引起PHAII的突变体WNK 4增加细胞旁Cl-渗透性并磷酸化紧密连接（TJ）蛋白claudin。最近，我们发现claudin-7在调节细胞旁Cl-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-/-）显示盐耗和失水表型，表明肾小管中离子重吸收受损。我们的长期目标是了解TJ蛋白在肾脏中的功能以及它们对人类疾病如高血压中离子失衡的贡献。本项目将检验以下假设：claudin-7对肾上皮细胞中TJ功能至关重要，并与WNK 4相互作用以调节细胞旁Cl-渗透。本申请具有三个具体目的：（1）表征我们最近产生的Cln 7-/-小鼠并确定密蛋白-7是否在允许Cl-渗透的细胞旁孔的形成中是必需的。我们将使用从Cln 7 +/+和Cln 7-/-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。我们将检测野生型紧密连接蛋白-7以确定紧密连接蛋白-7功能是否可以在Cln 7-/- CD细胞中恢复。我们还将claudin-7的胞外结构域（艾德）突变体转染到Cln 7-/- CD细胞和经RNAi敲低claudin-7的LLC-PK 1细胞中，以确定claudin-7艾德在细胞旁离子选择性中的作用;（2）研究WNK 4激酶对claudin-7介导的细胞旁Cl-渗透性的调节。我们将确定在Cln 7-/-小鼠以及Cln 7-/- CD细胞的远端肾单位上皮中WNK 4的表达和定位是否改变。我们将通过在CD细胞中表达WNK 4及其引起PHAII的突变体来确定细胞旁Cl-渗透性的变化。将在WNK 4位点处的封闭蛋白-7磷酸化无效和模拟突变体转染到Cln 7-/- CD细胞以及LLC-PK 1细胞中，以确定封闭蛋白-7的WNK 4磷酸化对细胞旁离子选择性的作用，以及（3）研究封闭蛋白-7缺失在Cln 7-/-小鼠肾脏中盐耗和急性肾小管坏死（ATN）的发展中的作用。将在出生后第1、4和7天的Cln 7-/-小鼠中比较TJ超微结构、屏障功能、血液和尿液离子浓度以及血浆肾素水平，以确定Cln 7-/-表型在出生后如何发育。新生Cln 7-/-小鼠将经受NaCl补充以确定其是否延迟ATN表型并延长Cln 7-/-小鼠的寿命。 公共卫生相关性本项目将检验以下假设：claudin-7对于肾上皮细胞中的紧密连接功能是必需的，并且在调节细胞旁Cl-渗透中与WNK 4相互作用。该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是否在允许Cl-渗透的细胞旁孔的形成中是必需的;（2）基于我们的研究结果，即claudin-7与WNK 4相互作用并被WNK 4磷酸化，研究WNK 4对claudin-7介导的细胞旁Cl-渗透性的调节，以及（3）研究claudin-7缺失在Cln 7-/-小鼠肾脏中盐耗和急性肾小管坏死的发展中的作用。

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.