High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.     

The epithelial sodium channel (ENaC): Mediator of the aldosterone response in the vascular endothelium?

In the kidney the epithelial sodium channel (ENaC) is regulated by the mineralocorticoid hormone aldosterone, which is essential for long-term blood pressure control. Evidence has accumulated showing that ENaC is expressed in endothelial cells. Moreover, its activity modifies the biomechanical properties of the endothelium. Therefore, the vascular system is also an important target for aldosterone and responds to the hormone with an increase in cell volume, surface area, and mechanical stiffness. These changes occur in a concerted fashion from minutes to hours and can be prevented by the specific sodium channel blocker amiloride and the mineralocorticoid receptor (MR) blocker spironolactone. Aldosterone acts on cells of the vascular system via genomic and non-genomic pathways. There is evidence that the classical cytosolic MR could mediate both types of response. Using a nanosensor covalently linked to aldosterone, binding sites at the plasma membrane were identified by atomic force microscopy. The interaction of aldosterone and this newly identified surface receptor could precede the slow classic genomic aldosterone response resulting in fast activation of endothelial ENaC. Recent data suggest that aldosterone-induced ENaC activation initiates a sequence of cellular events leading to a reduced release of vasodilating nitric oxide. We propose a model in which ENaC is the key mediator of aldosterone-dependent blood pressure control in the vascular endothelium.     

Aldosterone regulates rapid trafficking of epithelial sodium channel subunits in renal cortical collecting duct cells via protein kinase D activation

Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKCdelta. The activation of PKCdelta and PKCepsilon coincided with PKD1 activation and while a complex was formed between PKD1 and PKCepsilon after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKCdelta. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, alpha, beta, and gamma ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKCdelta /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.     

Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.     

Mechanisms of Aldosterone's Action on Epithelial Na + Transport

Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.     

Implication of ENaC in salt-sensitive hypertension

Arterial blood pressure is critically dependent on sodium balance. The kidney is the key player in maintaining sodium homeostasis. Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the highly selective, amiloride-sensitive epithelial sodium channel (ENaC). Direct evidence that dysfunction of ENaC participates in blood pressure regulation has come from the molecular analysis of two human genetic diseases, Liddle’s syndrome and pseudohypoaldosteronism type 1 (PHA-1). Both, increased sodium reabsorption despite low aldosterone levels in Liddle’s patients and decreased sodium reabsorption despite high aldosterone levels in PHA-1 patients, demonstrated that ENaC is an effector for aldosterone action. Gene-targeting and classical transgenic technology enable the generation of mouse models for these diseases and the analysis of the involvement of the epithelial sodium channel (ENaC) in the progress of these diseases. A first mouse model using ENaC transgenic knockout mice [ENaC(−/−)Tg] mimicked several clinical features of PHA-1, like salt-wasting, metabolic acidosis, high aldosterone levels, growth retardation and increased early mortality. Such mouse models will be necessary in testing the involvement of genetic and/or environmental factors like salt-intake in hypertension.     

The epithelial sodium channel: from molecule to disease

Genetic analysis has demonstrated that Na absorption in the aldosterone-sensitive distal nephron (ASDN) critically determines extracellular blood volume and blood pressure variations. The epithelial sodium channel (ENaC) represents the main transport pathway for Na⁺ absorption in the ASDN, in particular in the connecting tubule (CNT), which shows the highest capacity for ENaC-mediated Na⁺ absorption. Gain-of-function mutations of ENaC causing hypertension target an intracellular proline-rich sequence involved in the control of ENaC activity at the cell surface. In animal models, these ENaC mutations exacerbate Na⁺ transport in response to aldosterone, an effect that likely plays an important role in the development of volume expansion and hypertension. Recent studies of the functional consequences of mutations in genes controlling Na⁺ absorption in the ASDN provide a new understanding of the molecular and cellular mechanisms underlying the pathogenesis of salt-sensitive hypertension.     

Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.     

Inducible renal principal cell-specific mineralocorticoid receptor gene inactivation in mice

To investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MR ablation (MR(AQP2Cre) mice). To restrict Cre expression to principal cells, we have used the regulatory elements of the mouse aquaporin-2 (AQP2) gene to drive Cre expression. Since AQP2 is already expressed during renal development, MR ablation took place long before the analysis performed at the adult stage. To investigate whether the early onset of MR ablation affected the adult renal sodium handling, we developed a transgene expressing the CreER(T2) fusion protein under control of the regulatory elements of the AQP2 gene (AQP2CreER(T2)). Immunofluorescence revealed MR loss in the collecting duct (CD) and late connecting tubule after induction of MR ablation by tamoxifen in MR(AQP2CreERT2) mice that equals the MR loss in MR(AQP2Cre) mice. Surprisingly, tamoxifen-independent MR loss is observed in CDs of noninduced mutants without affecting circulating aldosterone levels. Under a low-salt diet, the induced ablation of MR at the adult stage recapitulates the renal sodium wasting observed in mice with constitutive early-onset MR ablation. The AQP2CreER(T2) transgene is a new tool for investigating in vivo the function of genes downstream of MR in renal ENaC-mediated sodium reabsorption by inducible somatic gene inactivation.     

Cystic fibrosis transmembrane conductance regulator-dependent up-regulation of Kir1.1 (ROMK) renal K+ channels by the epithelial sodium channel

The epithelial sodium channel (ENaC) and the secretory potassium channel (Kir1.1/ROMK) are expressed in the apical membrane of renal collecting duct principal cells where they provide the rate-limiting steps for Na(+) absorption and K(+) secretion. The cystic fibrosis transmembrane conductance regulator (CFTR) is thought to regulate the function of both ENaC and Kir1.1. We hypothesized that CFTR may provide a regulatory link between ENaC and Kir1.1. In Xenopus laevis oocytes co-expressing both ENaC and CFTR, the CFTR currents were 3-fold larger than those in oocytes expressing CFTR alone due to an increased expression of CFTR in the plasma membrane. ENaC was also able to increase Kir1.1 currents through an increase in surface expression, but only in the presence of CFTR. In the absence of CFTR, co-expression of ENaC was without effect on Kir1.1. ENaC-mediated CFTR-dependent up-regulation of Kir1.1 was reduced with a Liddle's syndrome mutant of ENaC. Furthermore, ENaC co-expressed with CFTR was without effect on the closely related K(+) channel, Kir4.1. We conclude that ENaC up-regulates Kir1.1 in a CFTR-dependent manner. CFTR may therefore provide the mechanistic link that mediates the coordinated up-regulation of Kir1.1 during the stimulation of ENaC by hormones such as aldosterone or antidiuretic hormone.     

Distribution of epithelial sodium channels and mineralocorticoid receptors in cardiovascular regulatory centers in rat brain

Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for alpha-, beta-, and gamma-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of gamma-ENaC was undetectable, whereas alpha- and beta-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial Na+ concentration as well as neuronal excitation.     

Dissecting mineralocorticoid receptor structure and function

The molecular mechanisms by which aldosterone regulates epithelial sodium transport in the distal colon and the distal nephron remain to be fully elucidated. Aldosterone acts via the mineralocorticoid receptor (MR) to induce the expression of genes whose products are involved in sodium transport. The structural basis of MR interactions with aldosterone has been examined by creating chimeras of the MR and the closely related glucocorticoid receptor; we have exploited differences in ligand-binding specificity to determine the region(s) of the MR that confer aldosterone-binding specificity. These findings have been related to a three-dimensional model of the MR based on the crystal structure of the progesterone receptor. These studies have been extended to include the characterisation of interactions between the N- and C-termini of the MR. We have characterised six genes that are regulated in vivo in the distal colon and/or kidney of the rat that are directly and acutely regulated by aldosterone administration: the three subunits of the epithelial sodium channel, serum and glucocorticoid-induced kinase, channel-inducing factor and K-ras2A. These studies provide insights into the molecular pathways that mediate aldosterone-induced amiloride-sensitive epithelial sodium transport.     

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.     

The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na⁺ transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,β,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.