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.
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.
