ENaC regulation and its role in blood pressure homeostasis

Epithelial Na+ channels (ENaCs) are expressed in the aldosterone-sensitive distal nephron (ASDN) and participate in the regulated reabsorption of filtered Na+. ENaCs are also expressed in non-epithelial cells, including dendritic cells (DCs) and monocytes where they affect blood pressure in a salt-sensitive manner. We recently described links between variants within the genes encoding the four ENaC subunits in humans (a, b, g and d) and blood pressure, including significant associations between variants within SCNN1D encoding the d subunit and blood pressure. This was surprising as the d subunit is not expressed in human ASDN, but is expressed in human DCs and monocytes. We have also identified human SCNN1D variants result in either a gain- or loss-of-function of human dbg channels. Studies proposed in Aim 1 will address the contribution of the d subunit of ENaC in immune cells to salt-sensitive hypertension, using immune deficient mice that will be adoptively transferred with DCs and monocytes derived from human induced pluripotent stem cells that lack or have mutations introduced into the d subunit. Human immune cells will be used as the d subunit is not expressed in mice. There are a number of extracellular and cytoplasmic factors that have important roles in regulating ENaC activity. These factors include extracellular Na+ that inhibits the channel, a process referred to as Na self-inhibition, channel subunit cleavage by proteases and channel subunit modification by Cys- palmitoylation that both activate ENaC. We have generated and characterize mice with mutations introduced into ENaC subunits that lead to gain-of-function due to a loss of Na+ self-inhibition, or a loss-of-function due to enhanced Na+ self-inhibition. Other mouse models include mutations that prevent protease cleavage or Cys palmitoylation of ENaC subunits. The question of whether these factors affect ENaC activity in immune cells and the immune cell response to dietary salt has not been addressed. Proposed studies in Aim 2 will examine the response of immune deficient mice that have been adoptively transferred with DCs and monocytes from mice bearing ENaC mutants that alter the Na+ self-inhibition response or channel modifications by proteases or by palmitoylation. Studies will use patch clamp electrophysiology to define the functions properties of wild type and mutant ENaCs in dendritic cells.