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

描述（由申请人提供）：上皮Na通道（ENaC）在远端肾单位中表达，作为Na重吸收的最终位点，从而在细胞外液量和血压（BP）的调节中发挥关键作用。ENaC功能获得性突变与高血压相关，而功能丧失性突变与低血压相关。ENaC由三个结构相关的亚单位组成，称为？，?然后呢？这些亚基在内质网（ER）内组装成三聚体，并且在ER退出后，亚基进行翻译后加工。特别是，在细胞外结构域的特定位点进行切割？蛋白酶弗林蛋白酶和前列腺素对ENaC亚基的抑制作用在异源系统中激活ENaC中具有关键作用，推测是通过在亚基的胞外域内释放抑制结构域。实验建议首先定义的功能作用？ENaC在体内通过产生小鼠远端肾单位特异性？ENaC中断。随后将进行评估功能作用的研究？ENaC亚基蛋白水解在体内产生的小鼠表达？ENaC亚单位在远端肾单位，缺乏关键定义的蛋白水解切割位点弗林蛋白酶或前列腺素。具体目标是：1.缺乏小鼠的生成和表征？ENaC选择性地在肾脏中表达ENaC的细胞中。拟议的研究是针对产生一个小鼠模型，已中断？ENaC在肾单位中的表达。为了实现这一点，将使用两种小鼠：1）在集合管的主细胞或整个肾单位中表达Cre重组酶的小鼠;和2）含有loxP侧翼外显子的小鼠，该外显子对？ENaC函数。这些动物的繁殖将导致远端肾单位敲除？ENaC。这些吗？ENaC基因敲除动物将允许我们定义的作用？ENaC在调节血压、肾电解质排泄和集合管离子转运中的作用。2.野生型或突变型肾脏特异性重建小鼠的产生和表征？ENaC。这些研究旨在确定的作用？ENaC活性的体内调节中的ENaC蛋白水解。前列腺素依赖的分裂？与弗林蛋白酶切割一致，亚基完全激活通道。拟议的研究是针对产生一个小鼠模型，表达？选择性地在远端肾单位内的弗林蛋白酶或前列腺素切割位点中具有突变的亚基。为了实现这一点，天然肾？ENaC会被破坏，然后呢？ENaC表达重建使用; 1）a？在弗林蛋白酶切割位点突变的亚基; 2）a？在前列腺素切割位点内具有突变的亚基;或3）野生型？子单元（作为对照）。介绍的？亚基将带有表位标签以便于检测和蛋白水解加工。这些动物将使我们能够（i）定义蛋白水解加工的程度？亚基在前列腺素或弗林蛋白酶裂解位点的基础条件下，并在设置的体积消耗;及（ii）确定的作用？亚基蛋白水解加工在前列腺素或弗林蛋白酶切割位点的功能通道的表达。公共卫生相关性：拟定的研究旨在确定肾脏上皮钠通道裂解的生理作用。这些研究将确定钠通道内两个不同裂解位点在调节肾脏钠和钾排泄以及维持正常血压中的重要性。这项工作有可能揭示参与高血压和钾排泄障碍的发展和/或维持的机制。

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.