Involvement of ENaC in the development of salt-sensitive hypertension

Abstract

Salt-sensitive hypertension is associated with renal and vascular dysfunctions, which lead to impaired fluid excretion, increased cardiac output, and total peripheral resistance. It is commonly accepted that increased renal sodium handling and plasma volume expansion are necessary factors for the development of salt-induced hypertension. The epithelial sodium channel (ENaC) is a trimeric ion channel expressed in the distal nephron that plays a critical role in the regulation of sodium reabsorption in both normal and pathological conditions. In this mini-review, we summarize recent studies investigating the role of ENaC in the development of salt-sensitive hypertension. On the basis of experimental data obtained from the Dahl salt-sensitive rats, we and others have demonstrated that abnormal ENaC activation in response to a dietary NaCl load contributes to the development of high blood pressure in this model. The role of different humoral factors, such as the components of the renin-angiotensin-aldosterone system, members of the epidermal growth factors family, arginine vasopressin, and oxidative stress mediating the effects of dietary salt on ENaC are discussed in this review to highlight future research directions and to determine potential molecular targets for drug development.

salt-sensitive hypertension is a widespread disorder associated with increased cardiovascular events and reduced survival (94). Clinical tests for salt sensitivity vary in the literature, but most of them generally define this phenomenon as blood pressure alteration in response to a change in NaCl intake (22). The pathogenesis of salt-sensitive (SS) hypertension includes intrarenal and extrarenal factors affecting water-sodium balance leading to cardiorenal dysfunction; high-salt intake causes sodium retention and plasma volume expansion which, together with an increased total peripheral resistance, are responsible for blood pressure elevation in salt-sensitive subjects (21, 43, 49, 72). In this mini-review, we focus on Na⁺ reabsorption in the distal nephron, which is mainly mediated by the epithelium sodium channel (ENaC). Amiloride (trade name: Midamor), a selective ENaC blocker, is used in the treatment of such monogenic forms of hypertension as Liddle’s syndrome and T594M polymorphism of ENaC (both causing overactivation of the channel), apparent mineralocorticoid excess, and familial hyperaldosteronism-1 (6, 28). Therefore, ENaC is an important therapeutic target involved in the body fluid volume expansion which, in combination with other factors, underlies the genesis of hypertension, especially its salt-induced form (26, 28).

ENaC is a classic effector of the renin-angiotensin-aldosterone system (RAAS); in addition, it is regulated by other neurohumoral factors, such as arginine vasopressin (AVP) and atrial natriuretic peptide, which are involved in modulation of water-salt balance and blood pressure (27, 52, 74, 87, 93). Although aldosterone-sensitive distal parts of nephron and collecting ducts (CDs) account for less than 5–10% of Na⁺ reabsorption, the significance of these segments for total sodium handling is very high (79). First, ENaC is expressed downstream from the macula densa. Therefore, reabsorption in these segments occurs after tubuloglomerular feedback interaction and is critical in determining final urinary sodium concentration (12, 29, 58). As it was recently proposed, ENaC in the connecting tubules serves as the central component of connecting tubule glomerular feedback (CTGF) mechanism, sensing Na⁺ delivery from proximal parts of nephron and regulating afferent arteriole resistance (69–71). Second, ENaC-mediated sodium handling can affect the activity of other sodium transporters. For example, it was shown that suppression of ENaC can lead to a low-sodium chloride symporter function, which strengthens the natriuretic effect of amiloride (85, 89).

Dahl SS Rat as a Model for Studies of Salt-Induced Hypertension

Sufficient data about involvement of ENaC in salt-sensitive hypertension came from studies using the Dahl SS rat strain. This model recapitulates many traits of the human form of salt-sensitive hypertension, such as suppressed plasma renin activity, lower circulating aldosterone concentration, increased vascular resistance, impaired pressure-natriuresis, and decreased venous compliance (13, 64, 65, 86). A hallmark of Dahl SS rats is the rapid progression of hypertension. Typical experimental protocols use 4% or 8% NaCl chows that cause a fast rise in blood pressure, becoming statistically significant just 2 days after the change of diet (15, 17, 30). Additionally, hypertension in Dahl SS rats is accompanied by the marked progression of renal injury. Clinically, there is also evidence that salt-sensitive patients are more susceptible to renal injury (11). Furthermore, Dahl SS rats are characterized by an increased CTGF, severe glomerular damage, and progressive proteinuria (13, 73, 83, 91).

High-Salt Diet Causes Abnormal Renal Hyperactivation of ENaC in Dahl SS Rats

In salt-resistant animal models, ENaC-driven reabsorption is diminished in response to salt load and exaggerated during water deprivation (10, 24, 25, 57, 63, 82); however, in salt-sensitive hypertension, the situation changes. For instance, a large set of data obtained from this strain indicates that ENaC in the central nervous system serves as a sensor of salt load and, thus, contributes to the development of hypertension via RAAS activation (2, 88, 92). However, because of the brief nature of this review, we will only focus on the regulation of ENaC in the kidney. Aoi et al. (3) reported that renal α-ENaC and β-ENaC mRNA levels were increased in SS rats fed an 8% diet for 4 wk compared with control animals fed a 0.3% diet. Later, abnormal upregulation of all three ENaC mRNA subunits, as well as mRNA for SGK1 (serum/glucocorticoid-regulated kinase 1, known to play an essential role in stimulation of ENaC activity), was reported in the SS rats fed a high (8% NaCl) salt diet by the same group of authors (4). Furthermore, Western blot analysis revealed that increased β- and γ-subunits (but not the α-subunit) protein abundance accompanies the development of hypertension on an 8% NaCl diet. Expression of cleaved γ-subunit was also found to be elevated (36). Our recent studies have confirmed these observations; we found higher β-ENaC and γ-ENaC protein abundance (including cleaved form of γ-subunit) in the cortex of SS rats fed a high-salt diet (4%) compared with the low-salt diet (0.4%). This effect was not found in a salt-resistant consomic SS.BN¹³ strain generated by introgression of chromosome 13 from Brown Norway rats into the Dahl SS background. Importantly, we also assessed β-ENaC abundance in a servo-controlled model, where the left kidney was protected from high renal perfusion pressure. Servo-controlled studies allowed us to separate effects of changes in blood pressure from system factors (circulating hormones and other blood components, innervation, diet) since a surgically installed cuff automatically occludes the aorta if BP exceeds 120 mmHg; this procedure protects the left kidney from mechanistic effects of increases in blood pressure, but keeps it in the same neurohumoral environment as the nonprotected right kidney (34, 54). Histological evaluation demonstrated that the right kidneys, which were exposed to high systemic blood pressure, had higher β-ENaC abundance than the left kidneys, in which perfusion pressure was controlled at the normal level (61). These data allow us to conclude that high renal perfusion pressure and/or associated tissue damage increase cortical ENaC abundance during high-salt load in the SS rats.

It is known that neither ENaC mRNA nor protein levels reflect electrical activity of the functional channels in the cell membrane. Therefore, we used patch-clamp experiments on split-open cortical collecting duct (CCD)/connecting tubule (CNT) segments to directly study channel activity in the tubules isolated from 11-wk-old SS animals fed a normal 0.4%, or a 4% diet for 3 wk. The studies revealed that a high-salt diet increases activity of ENaC via the elevation channel number in the apical membrane. Importantly, ENaC activity measured with electrophysiology did not change in salt-resistant SS.BN¹³ rats when animals were challenged with a high-salt diet. Additionally, pharmacological blockade of ENaC with amiloride or benzamil significantly precluded development of hypertension in Dahl SS rats (36, 61). Together, these findings indicate that abnormal high cortical ENaC activity and abundance contribute to the development of salt-sensitive hypertension in Dahl SS rats.

Another study tested ENaC expression in inner medullary collecting duct (1). Comparison of the SS rats with a control salt-resistant (SR) strain on a regular (0.3%) salt diet revealed a greater abundance of basal α-ENaC in SS rats. 8% NaCl diet increased the apical membrane staining for β-ENaC and γ-ENaC but did not affect the apical localization of α-ENaC (1). These observations suggest that the persistent high expression of α-ENaC, increased apical localization of β-ENaC and γ-ENaC, and high channel activity in SS rats on a high-salt diet may contribute to retention of sodium and total body fluid expansion and, therefore, result in elevated blood pressure.

Renin-Angiotensin-Aldosterone System in the Regulation of ENaC in SS Rats

The exact mechanism(s) that mediates effects of a high-salt diet on ENaC in SS rats remains unclear. SS rats are considered to be a systemic low-renin model, and high-salt consumption decreases plasma renin activity even further (5). However, hypertension in these animals is accompanied with activation of the local paracrine RAAS system. Kidney transplantation from SS to SR rats dramatically increases salt susceptibility in the latter strain (16) that illustrates the importance of intrarenal factors for the development of hypertension. Although plasma renin activity and concentration of angiotensinogen, ANG II, and aldosterone are suppressed by changing dietary NaCl from 0.3% to 8%, intrarenal levels of these endocrine factors can be significantly elevated (9, 41, 42). A paradoxical activation of renal mineralocorticoid receptors (MR) was also discovered in salt-sensitive hypertension (77). Furthermore, adrenalectomized SS rats do not develop hypertension on a high-salt diet, whereas exogenous aldosterone supplement reverses this phenomenon (77). The critical role of the RAAS components as a key factor of salt sensitivity and positive regulation of ENaC may be illustrated by experiments on the renin (encoded by Ren gene) knockout Dahl SS rats. These rats are unable to synthetize functional renin and are characterized by loss of urine-concentrating ability and low blood pressure (~60 mmHg) (53, 60). Single-channel patch-clamp analysis revealed decreased ENaC activity in Ren^−/− rats, which was mediated via changes in the channel open probability (60).

The paradoxical MR activation mentioned above can occur independent of aldosterone through activation of a small GTPase Rac1 (38). Rac1 cycles between an inactive guanosine diphosphate and active guanosine triphosphate-bound forms. In its active form, Rac1 regulates ubiquitination, cytoskeleton rearrangements, and membrane trafficking and might be involved in control of ion channel activity. In addition, it serves as a structural unit of NADPH oxidases. Shibata et al. (78) investigated activation of MR and Rac1 in Dahl SS and SR rats and found that an 8% salt diet elevated the abundance of active Rac1, SGK1, and MR in SS rats, but suppressed them in SR rats (78). This aberrant Rac1-MR activation in response to sodium load led to severe albuminuria, high blood pressure, and renal damage, which could be prevented by pharmacological Rac1 inhibition. The authors also demonstrated, using a mouse model in which mice lack RhoGDIα, an adaptor protein keeping Rac1 in the inactive form, which causes excessive renal Rac1 activity and makes this knockout strain salt-sensitive. Hyperactivity of Rac1 has been described earlier to upregulate ENaC-mediated current density in cell cultures and in cortical CDs isolated from Sprague-Dawley rats (37, 59, 80). We also investigated the involvement of Rac1 in 4% salt-induced excessive ENaC activity and found that a high-salt diet decreased the abundance of RhoGDIα in CDs of SS rats. Ex vivo studies revealed that low RhoGDIα levels augmented the bioavailability of Rac1 and activated ENaC-mediated Na⁺ reabsorption (37, 62). These findings indicate that a high-salt diet leads to renal MR activation and RhoGDIα downregulation, which increase ENaC-mediated sodium reabsorption via higher Rac1 activity.

Other Hormones Involved in ENaC Control in Salt-Induced Hypertension

Humoral factors involved in regulation of ENaC in salt-sensitive hypertension spread beyond the components of the RAAS. We have observed that 4% salt diet decreases the level of the EGF in the cortex of SS rats. EGF is a peptide involved in cell migration, differentiation, and proliferation and was also shown to regulate the activity of ion channels. Earlier, we and others demonstrated that chronic EGF treatment decreases ENaC activity (45, 46), and EGF family members are involved in the development of hypertension (81). We found that restoration of EGF levels via an intravenous supplementation decreased ENaC activity in SS rats on an 8% salt diet and prevented the increase in blood pressure and kidney injury. Thus, we conclude that EGF deficiency contributes to the development of salt-sensitive hypertension via ENaC overactivation (61). The low RhoGDIα level caused by MR activation may be a permissive factor for activation of Rac1 during EGF deficiency, but further investigation is needed to test this hypothesis.

Another hormone that is critical for regulation of sodium reabsorption is AVP (7, 8, 67, 68, 90). In the kidney, AVP participates in maintaining body fluid homeostasis by regulating water, urea and ion transport, glomerular filtration rate, and renal blood flow. It is well known that AVP exerts its antidiuretic effect by regulating sodium and water transport via the V₂ receptor, which is expressed in the basolateral membrane of the thick ascending limb of Henle's loop, distal tubules, and the CDs (40). In mice, AVP has a prominent stimulatory effect on ENaCʼs open probability, as well as the number of active channels and is involved in the regulation of plasma tonicity (10, 51, 52, 84). Moreover, in adrenalectomized mice, plasma AVP is increased, and this was shown to upregulate ENaC activity and compensate for the aldosterone deficiency. There are no studies on the involvement of AVP in ENaC regulation during development of SS hypertension, but the observations reported above strongly suggest a possible role for AVP in salt sensitivity. It is well characterized in Sprague-Dawley rats that vasopressin infusion (or water deprivation) increases the mRNA (55) and protein (19, 20) abundance of the β- and γ-subunits of ENaC (39, 50). This effect is similar to the ramifications of a high-salt diet challenge in Dahl SS rats.

Reactive Oxygen Species Production as the Pivotal Mechanism of ENaC Regulation in SS Hypertension

Increased levels of oxidative stress have been many times reported in Dahl SS rats (14, 34, 56) and hypertensive patients (44, 66); this appears to be an attractive signaling mechanism linking a high-salt diet and elevated ENaC activity. Major sources of reactive oxygen species (ROS) production in the kidney include tubular NADPH oxidase enzymatic complexes and infiltration of lymphocytes, both of which are increased by a high-salt diet in SS rats (17, 18, 31, 33, 35). A panel of studies indicates that knockout of NADPH oxidase subunits p67 and Nox4, or proteins responsible for T- and B-lymphocyte maturation, have renoprotective effects and may preclude development of high blood pressure in Dahl SS rats (15, 23, 76, 95). Principal cells of the CCDs are capable of producing and secreting ROS (75). We demonstrated that ROS increases ENaC activity in immortalized CCD cells (32), and activation of ENaC by hydrogen peroxide was reported earlier in amphibian renal cells (48). A direct link between Nox4 activity and ENaC-driven sodium reabsorption using immortalized mouse CCD principal cell line was shown as the mechanism mediating stimulation of ENaC by prorenin (47). However, the role NADPH oxidase produced ROS in ENaC hyperactivity in SS hypertension requires further investigation.

Conclusion

It still remains unclear which intracellular mechanisms activate ENaC in the CNT/CCD segment in response to a salt load, and how they affect the number of channels in the apical membrane, the open probability of individual channels, and their cleavage and modifications. The role of ENaC in the increased CTGF interaction shown in Dahl SS rats as a cause of glomerular barotrauma (91) also remains to be studied. Therefore, while we have reviewed the important role of ENaC-mediated sodium reabsorption in the development of salt-sensitive hypertension, there is still much to be learned about the involvement of different regulatory pathways (illustrated in Fig. 1), and the specific mechanism that results in the susceptibility of an individual to salt-induced hypertension.

Fig. 1.

Schematic illustration of epithelial sodium channel (ENaC) involvement in the development of salt-sensitive (SS) hypertension. High salt intake leads to lack of epidermal growth factor (EGF) in cortical tissue, low abundance of Rho GDP-dissociation inhibitor α (RhoGDIα), and abnormal activation of mineralocorticoid receptors (MR) via high activity of Rac1, which also serves as a structural unit of NADPH oxidase. Production of reactive oxygen species (ROS; as well as some other mechanisms not shown here) increases ENaC activity, which, in turn, contributes to the body fluid volume expansion required for the development of SS hypertension.

GRANTS

This article is supported by National Heart, Lung, and Blood Institute Grants HL-116603 (to T. S. Pavlov), HL-108880, and HL-122662 (to A. Staruschenko).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.S.P. prepared figures; T.S.P. drafted manuscript; T.S.P. and A.S. edited and revised manuscript; T.S.P. and A.S. approved final version of manuscript.