Epithelial Na⁺ Channels, Immune Cells, and Salt

Abstract

Epithelial Na⁺ channels (ENaCs) are known to affect blood pressure through their role in transporting Na⁺ in the distal nephron of the kidney. While expressed in other epithelial tissues, there is growing evidence that ENaCs are also expressed in nonepithelial tissues where their activity influences blood pressure. This review provides an overview of ENaCs and key mechanisms that regulate channel activity. The role of ENaCs in antigen-presenting dendritic cells is discussed, where ENaC-dependent sensing of increases in the extracellular Na⁺ concentration leads to activation of a signaling cascade, T cell activation with the release of proinflammatory cytokines, and an increase in blood pressure. The potential contribution of this pathway to human hypertension is discussed.

INTRODUCTION

Sodium (Na⁺) transporters in the aldosterone-sensitive distal nephron (ASDN) play key roles in regulating the reabsorption of filtered Na⁺, extracellular fluid volume, and blood pressure. Individuals with gene variants that result in activation of specific Na⁺ transporters in this segment have hypertension that is responsive to diuretics that target these transporters. The epithelial Na⁺ channel (ENaC) is expressed in the late distal convoluted tubule, the connecting tubule, and throughout the collecting duct and is one of the key distal nephron Na⁺ transporters that regulates blood pressure. The channel also facilitates K⁺ secretion through defined K⁺ secretory pathways mediated by ROMK (renal outer medullary K⁺) and BK (large-conductance Ca²⁺-activated K⁺) channels. Liddle syndrome, a well-described but rare cause of hypertension, occurs when ENaCs have mutations that interfere with their regulatory interactions with the ubiquitin ligase Nedd4–2 (neural precursor cell expressed developmentally downregulated 4-like) (1, 2). Liddle syndrome is responsive to ENaC blockers such as amiloride and triamterene (1, 2).

ENAC SUBUNIT COMPOSITION, STRUCTURE, AND DISTRIBUTION

In the ASDN, ENaCs are formed by three structurally related subunits, termed α, β, and γ, which have two membrane-spanning domains linked by large, highly folded extracellular regions that function as sensors of factors in the extracellular environment that regulate channel activity. These subunits assemble with a 1:1:1 subunit stoichiometry, with the transmembrane domains (primarily the second transmembrane domains) from each subunit forming the channel pore, which contains a selectivity filter that allows for Na⁺ or Li⁺ permeation while restricting K⁺ permeation, a site that has a key role in regulating channel gating (referred to as the degenerin or deg site), and a site that interacts with specific channel inhibitors, such as amiloride (1). A fourth ENaC subunit, referred to as δ, may substitute for the α subunit in channels at specific sites outside of the ASDN. Although the δ subunit is generally expressed in species where the other ENaC subunits are found, it is a pseudogene in rats and mice, limiting the ability to study its functional role in whole animals (1).

The ENaC/degenerin family of ion channels have large, highly folded extracellular domains formed primarily by β strands (referred to as the β ball and palm domains) and α helices (referred to as the thumb, finger, and knuckle domains) (Figure 1). The structure of the extracellular region of an acid-sensing ion channel (ASIC1), a member ENaC/degenerin family, was first resolved in 2007 (3). Human ENaC structures have been resolved with cryo-electron microscopy and are remarkedly similar to the structure of ASIC1, with an addition of a peripheral domain formed by β strands that have key protease cleavage sites (discussed below), referred to as the GRIP (gating relief of inhibition by proteolysis) domain (1, 4, 5). The subunit arrangement is α, β, and γ, when viewed from above in a counterclockwise fashion (4).

Figure 1.

Epithelial sodium channel (ENaC) structure. (a) Structure of a human ENaC αβγ heterotrimer. A side view ribbon is illustrated, and the gating relief of inhibition by proteolysis (GRIP) containing key protease cleavage sites and embedded inhibitory tracts in the α and γ subunits are illustrated with surface rendering. The α subunit is in green, the β subunit in blue, and the γ subunit in red. Side (left) and top (right) views are shown. ENaC structures are based on Protein Data Bank codes 6BQN (4). (b) Arrangement of extracellular and transmembrane domains in an ENaC subunit. Panel b adapted from Reference 4.

ENaC Regulation

Aldosterone is one of the key hormones that regulates ENaC in the ASDN. It is released from adrenal glands in response to a decrease in extracellular fluid volume, states of volume expansion associated with decreases in effective atrial volume as seen in the settings of heart or liver failure, and in response to an elevated serum K⁺ concentration (6). In the ASDN, aldosterone binds to and activates mineralocorticoid receptors, increasing the expression of specific genes including those encoding the α subunit of ENaC and a serum and glucocorticoid regulated kinase 1 (SGK1) (6, 7). One of the key targets of SGK1 is Nedd4–2, and phosphorylation of specific sites in Nedd4–2 has been shown to attract a 14–4-3 protein that blocks the binding of Nedd4–2 to ENaC, preventing channel ubiquitination and internalization from the cell surface (7–9). In the late distal convoluted tubule (or DCT2) and early connecting tubule, ENaC is activated by circulating glucocorticoids via the mineralocorticoid receptor, reflecting low levels of a cytoplasmic enzyme hydroxysteroid 11-beta dehydrogenase 2 (HSD-11B2) that degrades cortisol (10, 11).

Several factors in the external environment affect ENaC activity. Proteases activate ENaC by cleaving the α and γ subunits at defined sites within the peripheral GRIP domains that contain imbedded inhibitory tracts (1, 12). Cleavage at sites both preceding and following the α or γ subunit inhibitory tract leads to release of the tracts and transitions channels to higher open probability states (1, 12–14). The hormone aldosterone, secreted in response to volume depletion or hyperkalemia, activates ENaCs in the ASDN in association with an increase in proteolytic processing of the α and γ subunits (15–17). The serine protease furin, a member of the proprotein convertase family, resides primarily in the trans-Golgi and cleaves proteins transiting through the biosynthetic pathway. Furin cleaves the α subunit twice, releases its inhibitory tract, and partially activates the channel, transitioning channels from a low to a moderate activity state. It also cleaves the γ subunit once at a site preceding the inhibitory tract. Subsequent cleavage by a surface protease distal to the γ subunit inhibitory tract releases the tract, transitioning channels to a high activity state (13, 14, 18). Many serine and metalloproteases have been shown to activate ENaC by cleaving the γ subunit at a site distal to its inhibitory tract (1). Cleavage sites flanking the inhibitory tracts in the α and γ subunits coevolved and first appeared as vertebrates transitioned from an aquatic to a terrestrial environment, supporting the notion that protease activation of ENaC reflects release of inhibitory tracts (19).

A majority of the work demonstrating the role of proteases in activating ENaC has been performed using heterologous expression systems. While cleavage of ENaC subunits occurs in vivo, the role of this process in activating ENaC is still unclear. In support of proteases activating ENaC in vivo, mice with nephrotic syndrome responded to aprotinin, a nonselective serine protease inhibitor, with enhanced natriuresis (20). However, another report noted that ENaC subunit cleavage in kidneys occurred early following the initiation of a low-Na⁺ diet, while ENaC activity accessed in principal cells ex vivo was a relatively late response (21). Furthermore, a recent report of mice lacking the γ subunit furin cleavage site found that ENaC open probability in isolated cortical collecting ducts (CCDs) and net Na⁺ transport in microperfused CCDs were similar to those seen in CCDs from littermate control mice when the mice were placed on a low-Na⁺ diet to activate ENaC (22). To date, genetic deletion of specific proteases in the ASDN has not suppressed ENaC activity in vivo (1, 23, 24), although apparent reductions in ENaC activity have been reported with site-specific genetic deletions of PRSS8 encoding the serine protease prostasin in alveolae and distal colon (25, 26). These findings suggest that (a) there are other factors that activate ENaC in the ASDN and compensate for reduced protease activation, and (b) there are likely redundant proteases that cleave the γ subunit and activate ENaC in the ASDN.

Aside from transporting Na⁺, the binding of Na⁺ to a defined site in the extracellular domain of the α subunits results in allosteric changes that are transmitted to the pore region, leading to a reduction in channel open probability (1, 12). The process, referred to as Na⁺ self-inhibition, is modified by ENaC subunit proteolysis. Noncleaved channels are inhibited by low concentrations of Na⁺ (<10 mM), whereas channels that have released the α subunit inhibitory tract require higher concentrations of Na⁺ to inhibit the channel, and channels that have released both the α and γ subunit inhibitory tracts lack Na⁺ self-inhibition (1, 14, 27). An Na⁺ binding site that is required for the Na⁺ self-inhibition response, including a key Asp residue, was identified within the extracellular domain of the α subunit (Figure 2). Mutations introduced within the vicinity of this site alter Na⁺ self-inhibition (5, 28). Furthermore, mutations at additional multiple sites throughout the extracellular domains of ENaC subunits, primarily the α and γ subunits, have also been shown to affect Na⁺ self-inhibition, presumably alerting allosteric transitions that occur subsequent to Na⁺ binding (1, 29–31). This Asp residue in the Na⁺ binding site has been conserved through evolution. Surprisingly, this residue is not conserved as the δ subunit evolved. It appears that species lacking this δ subunit Asp residue (e.g., human δ) have lost the Na⁺ self-inhibition response (32). While Na⁺ self-inhibition is difficult to assess in vivo, a report of siblings with a mild Liddle syndrome phenotype that was responsive to ENaC inhibition and were noted to have a gain-of-function mutation at an extracellular site in the α subunit that is associated with a reduced Na⁺ self-inhibition response, suggests that human ENaC variants with a loss of Na⁺ self-inhibition contribute to hypertensive phenotype (31, 33).

Figure 2.

Na⁺ binding site. A peripheral region of the human α subunit (Protein Data Bank code 6WTH) illustrating a bound cation adjacent to a loop connecting the β6–β7 strands. Key residues that coordinate cation binding, including Asp338, are shown (4, 28).

ENaC Distribution

In addition to their expression in the ASDN, ENaCs are expressed in other epithelial tissues where they impact total body Na⁺ content, extracellular fluid volume, and blood pressure (1, 2). These sites include distal colon and lingulae epithelia (1). ENaCs are expressed in airway and alveolar epithelia, where they have roles in regulating airway surface liquid volume that impacts rates of mucociliary clearance, as well as the volume of surface liquids in alveolae (34, 35). ENaCs are expressed in nonepithelial tissues where they may have a role in influencing blood pressure. They are expressed in vascular endothelium where they have a role in modulating the activity of nitric oxide synthase (2, 36, 37). However, studies of genetic deletion of ENaC subunits in endothelia have not shown a role of endothelial ENaC in regulating blood pressure (38). ENaC subunits and related family members, including acid-sensing ion channel (ASIC) subunits, are expressed in vascular smooth muscle where they have a role in functioning as mechanical sensors and mediating pressure induced vasoconstriction (39). There is evidence that these channels in vascular smooth muscle influence blood pressure, based on knockout or knockdown studies (39). While the ENaC δ subunit is poorly expressed in the human ASDN (40), the δ subunit is expressed in human monocytes and vasculature (41, 42).

IMMUNE ACTIVATION BY Na⁺

Activation of the immune system by elevated Na⁺ has been increasingly implicated in blood pressure regulation, hypertension, renal damage, and cardiovascular disease by mechanisms that are starting to be elucidated. Tissue Na⁺ accumulation has been observed in muscle and skin and occurs at other sites including the kidney and possibly in lymphoid organs (43). High dietary salt is associated with increases in interstitial Na⁺ in rodent skin without changes in plasma Na⁺ concentrations (44). Na⁺ accumulation in human skin and skeletal muscle has also been observed using ²³Na magnetic resonance imaging in the settings of hypertension and aging (45). However, the detection of a hypertonic compartment in the skin remains a controversial issue, as one study suggested that Na⁺ accumulation in the skin and other tissues is primarily in isotonic fluids, where tissue Na⁺ excess may simply reflect extracellular volume expansion (46).

Interstitial tissue Na⁺ accumulation has been implicated in volume regulation and development of hypertension, heart failure, and chronic kidney disease. Recent studies have found that interstitial Na⁺ storage in skin and muscle is electrostatically associated with glycosaminoglycans (43) that can accumulate in tissues without changing plasma concentrations in hypertension (44) and in aging (45). As mentioned above, other studies have found that Na⁺ accumulation in tissues is not hypertonic, and that the accumulation of salt and water is systemic, leading to cellular rarefaction and edema (46). However, our research shows that regardless of osmolarity, kidney injury leads to intrarenal accumulation of Na⁺ (47), and that elevated Na⁺, not osmolality, activates immune cells, as equimolar mannitol solutions had no effect (48).

We found that increased tissue Na⁺ accumulation is associated with the accumulation of isolevuglandins (IsoLGs), products of lipid oxidation, in monocytes. These cells are activated in subjects with higher tissue salt, as indicated by increased expression of CD83 (48). The renal medulla has a high concentration of interstitial Na⁺ compared to the cortex (49, 50). We found increased accumulation of antigen-presenting cells, including dendritic cells in the renal cortico-medullary junction of hypertensive people when compared to normotensive people (48), in keeping with the concept that elevated Na⁺ activates immune cells and can contribute to hypertension (51–53). Na⁺ accumulation in specific tissues has relevance to circulating monocytes, which can transit sites with minimal to no differentiation (54). It is conceivable that the increased intrarenal tissue accumulation of Na⁺ activates immune cells via IsoLG production in antigen-presenting cells. We previously reported that IsoLG adducts accumulate on the surface of antigen-presenting cells and act as neoantigens to promote both T cell activation and hypertension (55). IsoLGs are highly reactive products of lipid oxidation that posttranslationally modify proteins by forming covalent bonds with lysine residues (56). Elevated Na⁺ is a potent stimulus for IsoLG adduct formation in dendritic cells (57). A hypertensive response to angiotensin II at subpressor doses has been reported following adoptive transfer of salt-exposed dendritic cells (57). Pharmacological scavenging of IsoLGs prevents dendritic cell activation, along with hypertension and end-organ damage. Furthermore, mice lacking NADPH oxidase have an absence of IsoLG protein adduct formation in dendritic cells (55, 57). These studies suggest that therapeutic strategies to reduce tissue Na⁺ levels may reduce immune cell activation with associated inflammation and hypertension.

Mouse dendritic cells express the α and γ subunits of ENaC (57). These cells respond to increases in extracellular concentration of Na⁺ in an ENaC-dependent manner. Na⁺ entry via ENaC leads to Ca²⁺ influx via the Na⁺/Ca²⁺ exchanger, activation of protein kinase C (PKC) that phosphorylates the p47 subunit of the NADPH oxidase, leading to superoxide production, activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, and formation of immunogenic IsoLG adducts. These events result in T cell activation, release of inflammatory cytokines, and an increase in blood pressure (57, 58) (see Figure 3).

Figure 3.

(a) ENaC-dependent pathway of dendritic cell activation and an (b) Na⁺-dependent T cell activation pathway leading to inflammation and salt-sensitive hypertension. (a) Na⁺ is transported into dendritic cells via ENaC in an SGK1-dependent manner. A high extracellular Na⁺ concentration enhances Na⁺ entry, leading to an increase in intracellular Ca²⁺ (via the Na⁺/Ca²⁺ exchanger) that activates protein kinase C. Subsequent downstream events include activation of NADPH oxidase and generation of ROS. This leads to generation of IsoLGs that adduct to intracellular proteins creating an immunogenic complex that is processed on MHC molecules, leading to T cell activation. Activation of NLRP3 inflammasomes and increases in NF-κB expression promote innate and T cell activation, infiltration of the kidney by activated T cells, secretion of proinflammatory cytokines, and inflammation that contributes to salt-sensitive hypertension. (b) Th17 cells are activated directly in the presence of a high Na⁺ environment by activating NFAT5, leading to immune cell activation and infiltration of the kidney and causing vascular damage and hypertension. Abbreviations: AF9, ALL1-fused gene from chromosome 9; ENaC, epithelial sodium channel; FOXO1, forkhead box protein O1; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN-γ, interferon gamma; IL, interleukin; IL-23R, interleukin 23 receptor; IsoLGs, isolevuglandins; MHC II, major histocompatibility molecule II; NEDD4–2, neural precursor cell expressed developmentally downregulated 4-like; NF-κB, nuclear factor kappa B; NFAT5, nuclear factor of activated T cells 5; NLRP3, NLR family pyrin domain-containing 3; p38/MAPK, p38 mitogen-activated protein kinase; PDK1, 3-phosphoinositide-dependent protein kinase 1; RORγt, RAR-related orphan receptor gamma; ROS, reactive oxygen species; SGK1, serum and glucocorticoid-induced protein kinase 1; TCR, T cell receptor; Th17, T helper cell 17; TNF-α, tumor necrosis factor alpha; Ub, ubiquitination; WNK4, no lysine kinase 4.

In dendritic cells as well as other ENaC-expressing nonepithelial cells including endothelial cells, ENaC appears to be functioning as a sensor of the extracellular [Na⁺], modulating signaling pathways associated with an increase in intracellular [Na⁺] when the [Na⁺] increases above 140 mM (1, 2). Although the role of ENaC proteolysis in modulating channel activity and dendritic cell activation has not been determined, our unpublished work suggests that Na⁺ self-inhibition has a role in regulating dendritic cell activation in vivo (J.A. Ishimwe, E.C. Ray, A.J. Nickerson, S. Sheng, A. Marciszyn, M. Saleem, A. Kirabo and T.R. Kleyman, unpublished data).

The induction of hypertension resulting from inflammation is due to oxidative stress injury leading to vascular damage, endothelial dysfunction, and kidney injury. Vascular remodeling and fibrosis that narrow arterial lumens in the kidney from the effect of inflammatory mediators are what primarily lead to hypertension (59–61). For example, tumor necrosis factor alpha (TNF-α) has been shown to activate NF-κB and NADPH oxidase, increase the expression of adhesion molecules in the vessels, and increase Na⁺ retention while reducing nitric oxide production (62–66). Interleukin-17 (IL-17) in mice has been reported to increase T cell infiltration in the kidney and promote oxidative stress whereas interferon gamma (IFN-γ) promotes renal fibrosis and has been reported to decrease glomerular filtration (67–70). Our studies indicate that antigen-presenting cells accumulate in the hypertensive kidney at the cortico-medullary junction and that elevated Na⁺ increases immune chemoattractants including VCAM (vascular cell adhesion molecule) and ICAM (intercellular adhesion molecule) (48). This results in infiltration of the activated immune cells into the kidney, which release cytokines known to induce vascular dysfunction and modulation of renal Na⁺ transporters. For example, previous studies have found that activation of the IL-1β receptor on renal-myeloid cells during hypertension prevents nitric oxide production and activation of NKCC2, leading to blood pressure elevation through enhanced Na⁺ reabsorption (71). In addition, IL-17A increased expression of NHE3(Na⁺/H⁺ exchanger isoform 3) and NCC (Na⁺-Cl⁻ cotransporter) in the proximal and distal convoluted tubule cells through phosphorylation of SGK1 (72).

SGK1 Is Required for Na⁺-Induced ENaC Activation

SGK1 is a well-characterized activator of ENaC in epithelial cells. In the ASDN, aldosterone increases SGK1 transcription and protein expression. The kinase requires serial phosphorylation events to be activated. These include phosphorylation within a hydrophobic motif (at Ser422) by the mammalian target of rapamycin 2 (mTORC2) to induce a conformational change and phosphorylation within its kinase domain (Thr256) by 3-phosphoinositide-dependent protein kinase 1 (PDK1), a kinase that is downstream of the insulin receptor (73, 74).

SGK1 has a key role in the aldosterone-dependent activation of ENaC in the ASDN by several distinct mechanisms. As discussed above, SGK1-dependent phosphorylation of Nedd4–2 recruits a 14–3-3 protein, preventing Nedd4–2-dependent ubiquitination of ENaC at the plasma membrane and subsequent channel internalization and degradation (7, 75). SGK1 can directly phosphorylate the α subunit of ENaC at Ser621, activating the channel in vitro (76). Interestingly, DYRK2 (dual-specificity tyrosine phosphorylated and regulated kinase 2) also activates the channel by phosphorylating this residue (77). SGK1 enhances transcription of SCNN1A (encoding the α subunit) through inhibition of a repressor of SCNN1A transcription mediated by Dot1a (histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a) and Af9 (ALL1-fused gene from chromosome 9) (78, 79).

We previously found that SGK1 has a role in the Na⁺-activated signaling cascade in dendritic cells, leading to renal inflammation and hypertension. Using a model of salt-sensitive hypertension, mice with a genetic deletion of Sgk1 in dendritic cells had reduced expression of the α and γ subunits in these cells, reduced systolic blood pressure, and kidney injury in response to a high-salt diet (80). Although mechanisms by which SGK1 activates ENaCs in dendritic cells are still being defined, we believe that it is likely that similar mechanisms are used by SGK1 in the ASDN and dendritic cells to activate ENaCs. It remains unclear whether the above mechanisms reflect, in part, aldosterone-dependent signaling through the mineralocorticoid receptor or glucocorticoid signaling through either the glucocorticoid or mineralocorticoid receptor, or whether they are independent of these signaling pathways. These questions are clinically relevant, particularly in the setting of primary aldosterone-associated hypertension and the growing clinical use of mineralocorticoid receptor antagonists.

When CD4⁺ T cells are activated, they transition to IL-17-producing Th17 T cells that infiltrate and increase vascular and renal damage through inflammatory responses (57, 58, 81). It is interesting to note that CD4⁺ T cells can also be activated directly by high salt in an SGK1-dependent manner to promote differentiation to Th17 cells (53, 82). Na⁺ entry via an undefined pathway into CD4⁺ T cells promotes the activation of NFAT5 (nuclear factor of activated T cells 5) via p38/MAPK (p38 mitogen-activated protein kinases) and subsequent activation of SGK1. Subsequent SGK1 signaling inhibits FOXO1 (O of forkhead box transcription factors) to relieve RORγt (RAR-related orphan receptor gamma). This facilitates IL-23 receptor and SGK1-RORγt signaling, promoting transcription and translation of IL-17A, TNF-α, and GM-CSF (granulocyte-macrophage colony-stimulating factor) (Figure 3). Furthermore, activated dendritic cells secrete IL-23 that is important in promoting the SGK1-RORγt signaling pathway in Th17 cells. In summary, elevated levels of extracellular Na⁺ activates T cells to promote hypertension either via an ENaC-SGK1-dependent pathway in dendritic cells or by directly activating T cells via the NFAT5 pathway. SGK1 has a role in the differentiation of naïve CD4⁺ T cells into Th17 cells and regulatory T (Treg) cells and by repressing the function of Treg cells and reciprocally promoting the development of Th17 cells (83).

The NF-κB-Inflammasome Contributes to Salt-Induced Hypertension

The activation of inflammasomes and signaling through NF-κB is an important pathway contributing to salt-sensitive hypertension. Inflammasomes are cytosolic protein complexes found in innate immune cells and other cells that function as PRRs (pattern recognition receptors) and sensors for recognition of PAMPs (pathogen-associated molecular patterns) and DAMPS (danger- or damage-associated molecular patterns) (84). There are several types of inflammasomes. The most studied in association with cardiovascular disease and hypertension is the NLRP3 inflammasome [nucleotide-binding domain, leucine-rich repeat containing proteins (NLR) family pyrin domain containing 3]. The NLRP3 inflammasome is activated by many stimuli associated with hypertension, including ROS (reactive oxygen species), cellular K⁺ efflux, cellular Ca²⁺ influx, and increases in extracellular Cl⁻ (85–87).

The NLRP3 inflammasome is a complex of multiple proteins, including the adaptor protein ASC (apoptosis-associated speck-like adaptor protein), NLRP3, and procaspase-1 (88). The NLRP3 protein has a C-terminal-containing LRR (leucine-rich repeat), a PYD (N-terminal pyrin domain), and a central ATPase-containing NACHT (84). The assembly and activation of the NLRP3 inflammasome occur in two stages. The first stage or priming stage is initiated by recognition of PAMPs or DAMPs by PRRs and signaling through myeloid differentiation primary response protein (MyD88) and IL-1 receptor-associated kinase (IRAK), leading to activation and nuclear translocation of NF-κB, where it induces the transcription of NLRP3 and proinflammatory cytokines pro–IL-1β and pro-IL-18 (89). Priming can also be induced through signaling via Toll-like receptors (TLRs), tumor necrosis factor receptor (TNFR), or IL-1 receptor ligand-mediated signaling. The second stage is assembly of the active NLRP3 inflammasome with subsequent activation of caspase-1 and cleavage of pro-IL-1β and pro-IL-18 into the active cytokines IL-1β and IL-18 that are released inducing an inflammatory response (90). These cytokines are elevated in hypertension and are associated with vascular dysfunction (91, 92).

Increased salt intake and associated ENaC-dependent Na⁺ entry in dendritic cells lead to NADPH oxidase activation, as discussed above, and production of ROS that activates the NLRP3 inflammasome and leads to the secretion of inflammatory cytokines that contribute to hypertension (Figure 4). Activation of the NLRP3 inflammasome induced by Na⁺ is ENaC and IsoLG dependent (58). We found that an increase in salt intake also influences expression of NLRP3 inflammasome components and the activation marker IL-1β, using cellular indexing of transcriptomes and epitopes (58). Interestingly, pharmacological inhibition of the NLRP3 inflammasomes protects mice from salt-sensitive hypertension development and renal damage (93, 94). Similarly, when the NF-κB is inhibited, blood pressure is reduced, resulting in protective effects against hypertension-induced end-organ damage (95, 96).

Figure 4.

NF-κB-inflammasome pathway. The NF-κB pathway is activated by several pathways, mainly via MyD88 signaling involving TLRs, the IL-1R, and the TNFR. NF-κB activation leads to transcription of pro-IL-1β and pro-IL-18. NF-κB also activates NLRP3 inflammasomes, leading to activation of caspase-1 that cleaves and converts pro-IL-1β and pro-IL-18 into their active forms, IL-1β and IL-18. These inflammatory cytokines are secreted and contribute to inflammation, renal Na⁺ retention, and hypertension. Abbreviations: IL-1R, interleukin 1 receptor; IRAK1/4, interleukin 1 receptor-associated kinase 4; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NLR family pyrin domain-containing 3; ROS, reactive oxygen species; TLR, Toll-like receptor; TNFR, tumor necrosis factor receptor.

ENaC GENE VARIANTS

We recently examined phenotype and whole-genome sequencing data in the Trans-Omics in Precision Medicine (TOPMed) project to determine whether low-frequency and rare ENaC variants within the genes SCNN1A, SCNN1B, SCNN1D, and SCNN1G, encoding the four ENaC subunits α, β, δ, and γ, respectively, were associated with blood pressure levels using a sequence kernel association test. While we found significant associations of diastolic pressure and mean arterial pressure and variants in SCNN1A and SCNN1B, we were surprised to find significant associations between SCNN1D variants and systolic pressure, diastolic pressure, pulse pressure and mean arterial pressure (41). Furthermore, the significant SCNN1D associations were only seen with low-frequency variants (mean allele frequencies between 0.01 and 0.05), suggesting that humans with SCNN1D variants contributing to a blood pressure phenotype are not rare in the general population (41). Additional studies are needed to address whether specific SCNN1D variants in human monocytes contribute to hypertension.

CONCLUSIONS

In summary, there is a growing body of evidence in mouse models that an increase in the extracellular Na⁺ concentration results in an ENaC-dependent increase in intracellular Na⁺ in dendritic cells, activating a signaling cascade that eventually results in the release of proinflammatory cytokines from T cells and an increase in blood pressure. There is also evidence in humans that ENaC-dependent salt sensing in monocytes contributes to hypertension, providing new therapeutic targets to control blood pressure in humans.