Physiologic role of the gamma epithelial sodium channel subunit in the kidney

描述(申请人提供)：上皮性钠通道(ENaC)表达于远端肾单位，是钠重吸收的最终部位，因此在调节细胞外液容量和血压(BP)中起关键作用。ENAC功能获得突变与高血压相关，而功能丧失突变与低血压相关。ENaC由三个结构上相关的亚基组成，分别称为？、？然后呢？这些亚基在内质网(ER)内组装成三聚体，在内质网退出后，这些亚基进行翻译后加工。特别是，在确定的位置的胞外区的切割？蛋白水解酶Furin和Prostasin的亚基在激活异源体系中的ENaC中起着关键作用，推测是通过在亚基的胞外结构域中释放一个抑制域来实现的。建议通过实验首先确定？的功能作用。ENAC在体内通过产生具有远端肾单位特异性的小鼠？ENAC中断。随后将进行评估？功能作用的研究？ENAC亚基在体内蛋白降解产生的小鼠中有什么表达？远端肾单位中的ENAC亚单位，缺乏为Furin或Prostasin定义的关键蛋白水解性裂解位点。具体目的是：1.缺乏小鼠的产生和特性？ENAC选择性地在肾脏表达ENaC的细胞中表达。拟议的研究针对的是产生一种扰乱？ENAC在肾单位的表达。为此，将使用两只小鼠：1)在集合管的主细胞或整个肾单位表达Cre重组酶的小鼠；2)含有loxP侧翼外显子的小鼠，这些外显子对？Enac函数。繁殖这些动物会导致远端肾单位被敲除吗？埃纳克。这些？ENAC基因敲除动物会让我们定义角色吗？ENAC在调节血压、肾脏电解质排泄、集合管离子转运等方面具有重要作用。2.肾组织特异性重组野生型或突变型小鼠的产生及特性埃纳克。这些研究旨在确定？ENAC蛋白在体内对ENaC活性的调节。前列腺素依赖的切割？亚基与Furin裂解协同，完全激活该通道。建议的研究针对的是产生一种表达？在远端肾单位内有选择性地突变Furin或Prostasin裂解位点的亚单位。要做到这一点，天然肾脏？ENAC会被打乱吗？使用：1)a？在Furin裂解位点有突变的亚基；2)a？在前列腺素裂解部位有突变的亚基；或3)野生型？子单元(作为对照)。被介绍的那个？亚基将带有表位标签，以便于检测和蛋白质降解处理。这些动物将允许我们(I)定义蛋白质分解加工的程度？基础条件下前列腺素或呋喃裂解部位的亚基以及在体积耗竭的设定中；以及(Ii)确定？蛋白水解亚单位处理在前列腺素或呋喃裂解位点表达的功能通道。公共卫生相关性：拟议的研究旨在确定肾脏上皮钠通道断裂的生理学作用。这些研究将确定钠通道中两个不同的裂解位点在调节肾脏钠钾排泄和维持正常血压方面的重要性。这项工作有可能揭示高血压和钾排泄障碍的发生和/或维持机制。

DESCRIPTION (provided by applicant): Epithelial Na channels (ENaC) are expressed in the distal nephron where they serve as the final sites of Na reabsorption, thereby playing a key role in the regulation of extracellular fluid volume and blood pressure (BP). ENaC gain-of-function mutations are associated with hypertension, whereas loss-of-function mutations are associated with hypotension. ENaCs are comprised of three structurally related subunits termed ?, ? and ?. These subunits are assembled into a trimer within the endoplasmic reticulum (ER) and, following ER exit, the subunits undergo post-translational processing. In particular, cleavage at defined sites of the extracellular domain of the ? subunit by the proteases furin and prostasin has a key role in activating ENaC in heterologous systems, presumably by releasing an inhibitory domain within the subunit's ectodomain. Experiments are proposed to first define the functional role of ? ENaC in vivo by generating mice with distal nephron-specific ? ENaC disruption. These will be followed by studies assessing the functional role of ? ENaC subunit proteolysis in vivo by generating mice that express ? ENaC subunits in the distal nephron that lack key defined proteolytic cleavage sites for furin or prostasin. The specific aims are: 1. Generation and characterization of mice lacking ? ENaC selectively in ENaC-expressing cells in the kidney. The proposed studies are directed at generating a mouse model that has disrupted ? ENaC expression in the nephron. To accomplish this, two mice will be used: 1) a mouse expressing Cre recombinase either in principal cells of the collecting duct or in the entire nephron; and 2) a mouse containing loxP-flanked exons critical to ? ENaC function. Breeding of these animals will result in distal nephron-knockout of ? ENaC. These ? ENaC knockout animals will allow us to define the role of ? ENaC in the regulation of blood pressure, renal electrolyte excretion, and collecting duct ion transport. 2. Generation and characterization of mice with renal-specific reconstitution of wild-type or mutant ? ENaC. These studies are designed to define the role of ? ENaC proteolysis in the in vivo regulation of ENaC activity. Prostasin-dependent cleavage of the ? subunit, in concert with furin cleavage, fully activates the channel. The proposed studies are directed at generating a mouse model that expresses a ? subunit with mutations in the furin or prostasin cleavage sites selectively within the distal nephron. To accomplish this, native renal ? ENaC will be disrupted and ? ENaC expression reconstituted using; 1) a ? subunit with a mutation in the furin cleavage site; 2) a ? subunit with a mutation within the prostasin cleavage site; or 3) a wild type ? subunit (as a control). The introduced ? subunits will bear epitope tags to facilitate detection and proteolytic processing. These animals will allow us to (i) define the extent of proteolytic processing of the ? subunit at the prostasin or furin cleavage sites under basal conditions and in the setting of volume depletion; and (ii) determine the role of ? subunit proteolytic processing at the prostasin or furin cleavage sites in the expression of functional channels. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to determine the physiologic role of cleavage of the epithelial sodium channel in the kidney. The studies will define the importance of two different cleavage sites within the sodium channel in regulation of kidney sodium and potassium excretion and maintenance of normal blood pressure. This work has the potential to uncover mechanisms involved in the development and/or maintenance of hypertension and disorders of potassium excretion.