Epithelial Sodium Channel Subunits Research Articles

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC β- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

The diverse functions of the DEG/ENaC family: linking genetic and physiological insights.

The DEG/ENaC family of ion channels was defined based on the sequence similarity between degenerins (DEG) from the nematode Caenorhabditis elegans and subunits of the mammalian epithelial sodium channel (ENaC), and also includes a diverse array of non-voltage-gated cation channels from across animal phyla, including the mammalian acid-sensing ion channels (ASICs) and Drosophila pickpockets. ENaCs and ASICs have wide ranging medical importance; for example, ENaCs play an important role in respiratory and renal function, and ASICs in ischaemia and inflammatory pain, as well as being implicated in memory and learning. Electrophysiological approaches, both in vitro and in vivo, have played an essential role in establishing the physiological properties of this diverse family, identifying an array of modulators and implicating them in an extensive range of cellular functions, including mechanosensation, acid sensation and synaptic modulation. Likewise, genetic studies in both invertebrates and vertebrates have played an important role in linking our understanding of channel properties to function at the cellular and whole animal/behavioural level. Drawing together genetic and physiological evidence is essential to furthering our understanding of the precise cellular roles of DEG/ENaC channels, with the diversity among family members allowing comparative physiological studies to dissect the molecular basis of these diverse functions.

Type 1 Angiotensin Receptors on CD11c-Expressing Cells Protect Against Hypertension by Regulating Dendritic Cell-Mediated T Cell Activation.

Type 1 angiotensin (AT1) receptors are expressed on immune cells, and we previously found that bone marrow-derived AT1 receptors protect against Ang (angiotensin) II-induced hypertension. CD11c is expressed on myeloid cells derived from the bone marrow, including dendritic cells (DCs) that activate T lymphocytes. Here, we examined the role of AT1 receptors on CD11c+ cells in hypertension pathogenesis. Mice lacking the dominant murine AT1 receptor isoform, AT1a, on CD11c+ cells (dendritic cell [DC] AT1aR knockout [KO]) and wild-type (WT) littermates were subjected to Ang II-induced hypertension. Blood pressures were measured by radiotelemetry. DC AT1aR KO mice had exaggerated hypertensive responses to chronic Ang II infusion with enhanced renal accumulation of effector memory T cells and CD40+ DCs. CCL5 (C-C motif chemokine ligand 5) recruits T cells into injured tissues, and CCR7 (C-C motif chemokine receptor 7) facilitates DC and T cell interactions in the kidney lymph node to allow T cell activation. DCs from the hypertensive DC AT1aR KO kidneys expressed higher levels of CCL5 and CCR7. mRNA expressions for CCR7 and tumor necrosis factor-α were increased in CD4+ T cells from the renal lymph nodes of DC AT1aR KO mice. During the second week of Ang II infusion when blood pressures between groups diverged, DC AT1aR KO mice excreted less sodium than WTs. Expressions for epithelial sodium channel subunits were increased in DC AT1aR KO kidneys. Following activation of the renin angiotensin system, AT1aR stimulation on DCs suppresses renal DC maturation and T cell activation with consequent protection from sodium retention and blood pressure elevation.

Medullary overexpression of sphingosine‐1‐phosphate receptor 1 transgene attenuates sodium retention and hypertension in deoxycorticosterone acetate (DOCA) salt‐treated mice

Our previous studies have showed that sphingosine‐1‐phosphate (S1P) agonist stimulates urinary sodium excretion and the S1P type 1 receptor (S1PR1) knockout in the collecting duct led to increased sodium retention and enhanced salt‐sensitive hypertension under the treatment of deoxycorticosterone (DOCA) and high salt. We hypothesized that the increase in medullary S1PR1 would attenuate sodium retention by promoting sodium excretion in such model. Male C57BL/6J mice around 25g bodyweight received intramedullary transfection of S1PR1 plasmids to achieve renal medullary S1PR1 overexpression (OE). The mice were then implanted with the silastic DOCA sheet subcuntaneously and 1% NaCl drinking water to induce salt‐sensitive hypertension (SS‐HTN). Thereafter, the mice underwent either acute or chronic assessments on blood pressure and urinary sodium excretion. Under DOCA‐salt challenge, mice with S1PR1‐OE showed increased urine volume (UV) and urinary sodium excretion (UNAV) in responses to the acute sodium loading (IV of normal saline at 5% body weight in 30 min); the UV and UNAV were 12.51±2.31 vs. 28.50±4.23mL/min/gKW and 0.93±0.27 vs. 4.72±1.12mmole/min/gKW (p<0.05) in control and S1PR1‐OE mice, respectively. The mice with S1PR1‐OE also showed enhanced pressure natriuretic responses (UV, 18.73±7.57 vs. 40.61±6.43mL/min/gKW, p<0.05; UNAV, 3.58±1.77 vs. 9.52±1.38nmole/min/gKW, p<0.05). In the long‐term DOCA‐salt treatment, S1PR1‐OE mice showed less daily sodium balance with the greater sodium excretion (2.72±0.56 vs. 5.60±0.72mmole/day, p<0.05) and ameliorated sodium retention (3.05±0.39 vs. 1.65±0.39mmole/72hr, p<0.05), which was accompanied with the attenuation of SS‐HTN (SBP 134.2±6.79 vs. 109.8±3.54mmHg, p<0.05). The level of alpha subunit of epithelial sodium channel (αENaC) was not changed. However, the beta subunit (βENaC) was significantly decreased (55%, p<0.01). Although ENaCγ was significantly increased (350%) in the S1PR1‐OE group, immunostaining showed that the DOCA‐stimulated translocation of γENaC onto the apical membrane was inhibited. These results suggested that the overexpression of S1PR1 in the renal medulla attenuates sodium retention by the inhibition of ENaC function in the DOCA‐induced SS‐HTN.

Na+-Retaining Action of COX-2 (Cyclooxygenase-2)/EP1 Pathway in the Collecting Duct via Activation of Intrarenal Renin-Angiotensin-Aldosterone System and Epithelial Sodium Channel.

The collecting duct (CD) is a major site of both biosynthesis and action of prostaglandin E2 as highlighted by the predominant expression of COX-2 (cyclooxygenase-2) and some E-prostanoid (EP) subtypes at this nephron site. The purpose of this study was to determine the relevance and mechanism of CD COX-2/prostaglandin E2/EP1 signaling for the regulation of Na+ hemostasis during Na+ depletion. Mice with Aqp2Cre-driven deletion of COX-2 (COX-2fl/flAqp2Cre+) or the EP1 subtype (EP1fl/flAqp2Cre+) were generated and the Na+-wasting phenotype of these mice during low-salt (LS) intake was examined. EP subtypes responsible for prostaglandin E2-induced local renin response were analyzed in primary cultured mouse inner medullary CD cells. Following 28-day LS intake, COX-2fl/flAqp2Cre+ mice exhibited a higher urinary Na+ excretion and lower cumulative Na+ balance, accompanied with suppressed intrarenal renin, AngII (angiotensin II), and aldosterone, expression of CYP11B2 (cytochrome P450 family 11 subfamily B member 2), and blunted expression of epithelial sodium channel subunits compared to floxed controls (COX-2fl/flAqp2Cre-), whereas no differences were observed for indices of systemic renin-angiotensin-aldosterone system. In cultured CD cells, exposure to prostaglandin E2 stimulated release of soluble (pro)renin receptor, prorenin/renin and aldosterone and the stimulation was more sensitive to antagonism of EP1 as compared other EP subtypes. Subsequently, EP1fl/flAqp2Cre+ mice largely recapitulated Na+-wasting phenotype seen in COX-2fl/flAqp2Cre+ mice. The study for the first time reports that CD COX-2/EP1 pathway might play a key role in maintenance of Na+ homeostasis in the face of Na+ depletion, at least in part, through activation of intrarenal renin-angiotensin-aldosterone-system and epithelial sodium channel.

Inflammatory alveolar macrophage-derived microvesicles damage lung epithelial cells and induce lung injury

Emerging evidence has demonstrated that several microvesicles (MVs) are secreted in bronchoalveolar lavage fluid (BALF) during the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the impact of alveolar macrophage (AM)-derived MVs on epithelial cells and their in vivo effects on ALI/ARDS require further exploration. In this study, MVs were isolated from BALF of mice or mouse alveolar macrophage (MHS) cells by sequential centrifugation and then delivered to epithelial cells or mice. Enzyme-linked immunosorbent assay revealed that BALF-derived MVs (BALF-MVs) and MHS-derived MVs (AM-MVs) were rich in tumor necrosis factor-α (TNF-α) at the early stage of lung injury. In vitro, both inflammatory BALF-MVs and AM-MVs decreased the expression of α subunit of epithelial sodium channel (α-ENaC), γ-ENaC, and Na+,K+-ATPase α1 and β1 in lung epithelial cells. However, antibodies against TNF-α inhibited the effects of inflammatory AM-MVs in epithelial cells. In vivo, the inflammatory AM-MVs, delivered intratracheally to mice, impaired lung tissues and increased the injury score. They also resulted in decreased alveolar fluid clearance and increased lung wet weight/dry weight ratio. Furthermore, inflammatory AM-MVs downregulated the α-ENaC, γ-ENaC, and Na+,K+-ATPase α1 and β1 levels in lung tissues. According to our results, inflammatory AM-derived MVs may potentially contribute to lung injury and pulmonary edema, thereby indicating a potential novel therapeutic approach against ALI/ARDS based on AM-MVs.

Association of cystic fibrosis transmembrane conductance regulator with epithelial sodium channel subunits carrying Liddle's syndrome mutations.

The association of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) in the pathophysiology of cystic fibrosis (CF) is controversial. Previously, we demonstrated a close physical association between wild-type (WT) CFTR and WT ENaC. We have also shown that the F508del CFTR fails to associate with ENaC unless the mutant protein is rescued pharmacologically or by low temperature. In this study, we present the evidence for a direct physical association between WT CFTR and ENaC subunits carrying Liddle's syndrome mutations. We show that all three ENaC subunits bearing Liddle's syndrome mutations (both point mutations and the complete truncation of the carboxy terminus), could be coimmunoprecipitated with WT CFTR. The biochemical studies were complemented by fluorescence lifetime imaging microscopy (FLIM), a distance-dependent approach that monitors protein-protein interactions between fluorescently labeled molecules. Our measurements revealed significantly increased fluorescence resonance energy transfer between CFTR and all tested ENaC combinations as compared with controls (ECFP and EYFP cotransfected cells). Our findings are consistent with the notion that CFTR and ENaC are within reach of each other even in the setting of Liddle's syndrome mutations, suggestive of a direct intermolecular interaction between these two proteins.

LIDDLE SYNDROME – DETECTION OF NOVEL MUTATION IN A FAMILY WITH HYPERTENSION

Abstract Objective: Liddle syndrome is a rare monogenic form of arterial hypertension with autosomal dominant inheritance. It is caused by mutation of genes coding one of three subunits of epithelial sodium channel (ENaC), which is located in distal nephron. Under physiological conditions, its expression and activity is regulated to maintain fluid and electrolytes homeostasis by reabsorbing Na+. Mutated channel cannot be degraded and therefore, it is accumulated on cell membrane; this results in enhanced sodium reabsorption. The most common clinical presentation of Liddle syndrome is early onset of hypertension, hypokalemia, suppressed plasma renin activity and low plasma aldosterone level. Up to now, 36 mutations causing Liddle syndrome were described. Design and method: Genetic assessment was performed in a Czech family of three generations (n = 14, see Figure) where hypertension and tendency to hypokalemia was present in several members. We used PCR amplification of specific alleles of SCNN1B and SCNN1G genes which was followed by Sanger sequencing. Results: We present a novel mutation causing Liddle syndrome: Tyr604∗ in beta-subunit of epithelial sodium channel. This mutation is supposed to generate a premature stop codon at position 604, resulting in truncated beta-subunit of EnaC. It was found in 7 members of the family: I-1, II-1 and 2, and III-1 to 4. Phenotypes of these subjects were different: from severe hypertension and hypokalemia (subjects I-1 and III-3, manifestation in 10 and 5 years, respectively) to normotension with normal potassium levels (III-1 and 2, now aged 8 and 10 years, respectively); however, all the seven subjects with mutation had hypoaldosteronism (serum level < 0.13 ng/ml). Conclusions: A new mutation causing Liddle syndrome is described. It is manifested with different severity and mild forms that are not manifested in early childhood exist; thus, Liddle syndrome may be more frequent among adult hypertensive subjects than currently supposed. Proper diagnosis is important as the patients respond well to treatment with ENaC blocker amiloride.

Clinical characteristics and treatment requirements of children with autosomal recessive pseudohypoaldosteronism.

Autosomal recessive forms of pseudohypoaldosteronism are caused by genetic defects in the epithelial sodium channel. Little is known about the long-term outcome and medication needs during childhood and adolescence. This study reports a single-centre experience of children affected with this ultra-rare condition over a 37-year period. We report the clinical presentation, growth, neuro-development, associated conditions, mortality and medication dosing and administration for 12 affected children from eight families. All children were presented within the first 2 weeks of life with life-threatening, severe hyperkalaemia and hyponatraemia. All parents were consanguineous and of South Asian, Middle Eastern or African ethnic origin. Eight children had homozygous mutations in the SCNN1A and SCNN1G genes, encoding the epithelial sodium channel subunits alpha and gamma, respectively, including one novel mutation. Three children died (25%) and two (16%) had severe neurological impairment post-cardiac arrest secondary to hyperkalaemia. One affected female had a successful pregnancy at the age of 28 years. Despite high mortality and morbidity in this condition, survival with normal physical and neurological outcome is possible, justifying intensive management to prevent electrolyte imbalance.

The δ subunit of epithelial sodium channel in humans-a potential player in vascular physiology.

Vascular epithelial sodium channels (ENaCs) made up of canonical α, β, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, δ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, δ-ENaC is the least understood subunit. However, the recent discovery of δ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the δ subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of δ-ENaC in the vasculature to identify whether δ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of δ-ENaC and implications for vascular physiology and pathophysiology in humans.

SUN-LB93 Mineralocorticoid Receptor Mediates Sex-Dependent Anticontractile Effect of Perivascular Adipose Tissue in Obese Mice

Obesity, a condition of excessive fat mass and subclinical inflammation, reached epidemic proportions with higher prevalence in women compared to men worldwide. Expansion of the perivascular adipose tissue (PVAT) is observed in obesity and clinical studies indicate a positive correlation between PVAT amount and body mass index. PVAT, a fat depot surrounding most of the vessels, modulates vascular function by releasing PVAT-derived factors such as adipokines that exert anticontractile effect in health individuals. Despite sexual dimorphism on PVAT morphology, it is still unknown whether or not there is sex differences in the PVAT modulating vascular function in the setting of obesity. Aldosterone-mineralocorticoid receptor (MR) signaling pathway has been demonstrated to be adipogenic and proinflammatory in classical fat depots and treatment with MR antagonists (A) might reverse vascular dysfunction and remodeling in obese models, especially in female sex. Therefore, we aimed to evaluate the anticontractile effect of PVAT in male and female obese mice and hypothesized that MR signaling would be involved in possible sex differences in PVAT dysfunction in obesity. Male and female C57Bl6/J mice were fed a chow or a high-fat diet (HFD, 60% energy from fat) for 20 weeks. At the last 4 weeks of HFD, female and male mice were treated with the MRA spironolactone (Spi, 100 mg/kg/day). HFD feeding significantly increased body weight and visceral adipose tissue, which was not modified by Spi treatment in both sexes. Resistance mesenteric arteries were isolated with or without PVAT and mounted in a wire myograph to evaluate vascular contractile responses. Lean male and female mice PVAT had an anticontractile effect in the response to phenylephrine that was greater in females than males. The anticontractile effect of PVAT was significantly impaired in obese females but not modified in males. HFD-induced dysfunctional PVAT was prevented by Spi treatment in females. Next, we evaluated the protein expression of aldosterone-synthase CYP11B2, serum and glucocorticoid-regulated kinase 1 (SGK1), and epithelial sodium channel subunits (ENaCs) in isolated mesenteric PVAT of lean and obese male and female mice. There was an increased expression of CYP11B2, SGK1 and ENaCs only in obese female PVAT. Protein expression of adiponectin, a major PVAT-released adipokine was also increased in female mesenteric PVAT. In conclusion, the findings suggest sexual dimorphism in PVAT function in health and in obesity. Although anticontractile role of PVAT was exacerbated in lean female mice, female sex was more susceptible to develop PVAT dysfunction in the setting of obesity which was prevented by MR blockade. HFD-induced PVAT dysfunction in females was associated with increased expression of SGK1 and ENaCs. Therefore, data suggest MR activation as a mechanism mediating sex differences in PVAT dysfunction.FAPESP, CAPES.

Interaction between Epithelial Sodium Channel γ-Subunit and Claudin-8 Modulates Paracellular Sodium Permeability in Renal Collecting Duct.

Water and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient. To investigate whether transcellular sodium transport controls tight-junction composition and paracellular permeability via modulating expression of the transmembrane protein claudin-8, we used cultured mouse cortical collecting duct cells to see how overexpression or silencing of epithelial sodium channel (ENaC) subunits and claudin-8 affect paracellular permeability. We also used conditional kidney tubule-specific knockout mice lacking ENaC subunits to assess the ENaC's effect on claudin-8 expression. Overexpression or silencing of the ENaC γ-subunit was associated with parallel and specific changes in claudin-8 abundance. Increased claudin-8 abundance was associated with a reduction in paracellular permeability to sodium, whereas decreased claudin-8 abundance was associated with the opposite effect. Claudin-8 overexpression and silencing reproduced these functional effects on paracellular ion permeability. Conditional kidney tubule-specific ENaC γ-subunit knockout mice displayed decreased claudin-8 expression, confirming the cell culture experiments' findings. Importantly, ENaC β-subunit or α-subunit silencing or kidney tubule-specific β-ENaC or α-ENaC knockout mice did not alter claudin-8 abundance. Our data reveal the specific coupling between ENaC γ-subunit and claudin-8 expression. This coupling may play an important role in preventing the backflow of reabsorbed solutes and water to the tubular lumen, as well as in coupling paracellular and transcellular sodium permeability.

Target-wide Induction and Synapse Type-Specific Robustness of Presynaptic Homeostasis.

Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of adaptive neuromodulation and is observed at both central and peripheral synapses. In this work, we make several fundamental advances by interrogating the synapse specificity of PHP. We define how PHP remains robust to acute versus long-term neurotransmitter receptor perturbation. We describe a general PHP property that includes global induction and synapse-specific expression mechanisms. Finally, we detail a novel synapse-specific PHP expression mechanism that enables the conversion from short- to long-term PHP expression. If our data can be extended to other systems, including the mammalian central nervous system, they suggest that PHP can be broadly induced and expressed to sustain the function of complex neural circuitry.

A novel SCNN1G mutation in a PHA I infant patient correlates with nephropathy

Systematic form of pseudohypoaldosteronism Type I (PHA I) is a rare recessive homozygous inherited syndrome characterized by severe salt loss, hyperkalemia, hyponatremia, metabolic acidosis, hyperaldosteronism and hyperreninemia. It is caused by mutations in one of the genes encoding the α, β and γ subunits of epithelial sodium channels (ENaC). In this study, we performed whole exome sequencing on an infant patient with PHA I as well as nephropathy. The result presented a novel homozygous six-base deletion in the γ subunit encoding gene SCNN1G. Then we correlated the mutant to kidney damage, along with transcriptional alterations of genes involved in inflammation, oxidative stress and apoptosis, via in vitro and in vivo tests. In addition, it was demonstrated that the SCNN1G defects triggered programmed cell death via inhibiting miR-21 and upregulating PTEN, which then orchestrated the key downstream regulators, including Bcl2, Bax2, and cleaved Caspse-3 in a way that favors cell apoptosis. The study enhances our understanding of the pathophysiology of the disorder of PHA I and the mechanisms of renal damage induced by the novel defect.

Cholinergic Mediated Renal Sodium Retention in Young Spontaneously Hypertensive Rats

Cholinergic activation with systemic nicotine infusion induces renal inflammation and the premature development of hypertension in young pre-hypertensive spontaneously hypertensive rats (SHR), a genetic model of essential hypertension. We hypothesized that the nicotine induced inflammatory response is associated with renal sodium retention in the young SHR. Young pre-hypertensive SHR and age matched Wistar Kyoto (WKY) controls received subcutaneous infusion of either saline (n=10 for each strain) or nicotine (15mg/kg/day) (n=10 for each strain) for 14 days. Animals received normal chow for the first 7 days of infusion and were changed to either a “low-salt” (LSD, 0.01% NaCl) (n=10) or “high-salt” (HSD, 4% NaCl) (n=10) diet. Animals were maintained in metabolic cages during the LSD and HSD periods. Nicotine did not alter sodium excretion significantly in WKY fed LSD or HSD, nor SHR fed LSD. In contrast, nicotine induced a 24% increase in renal sodium retention in SHR fed HSD, compared to controls (2.0±0.1 mM/gm vs 2.6±0.7mM/gm) (p<0.01). Moreover, nicotine-induced sodium retention in young SHR fed HSD was associated with an increase in renal gene expression of serum & glucocorticoid kinase (SGK1) by 34% (p<0.033) and renal Na-K-Cl cotransporter (NKCC2) by 58% (p<0.01), compared to controls. Similarly, there was a 20% increase in the full length form (85kDa) of the alpha subunit of epithelial sodium channel (αENaC) 20% (p<0.05); however, no increase in the cleaved form (25kDa) of αENaC was observed, when compared to controls. No significant change in renal expression of the thiazide-sensitive sodium-chloride co-transporter (NCC) was noted in response to nicotine when young SHR were fed HSD. We conclude that cholinergic mediated hypertension in the young SHR may be a form of salt-sensitive hypertension, possibly related to renal inflammation and supported by renal sodium retention, and increased expression of renal sodium transporters. Support or Funding Information NIH 5K08HL119588-02; Foundation de Leduq Nicotine Induces Renal Sodium Retention in Young Pre-hypertensive Spontaneously Hypertensive Rats (SHR) on a High-Salt Diet (HSD). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased sodium transporters in distal nephron segments are associated with clozapineinduced hypertension

Dopamine receptor subtype 4 (D4R) antagonists, such as clozapine, are used in the treatment of schizophrenia. A common side effect of clozapine is hypertension but the mechanism of the clozapine‐induced hypertension is unknown. D4R plays an important role in blood pressure (BP) regulation; deletion of the D4R gene in mice causes hypertension. To determine if renal mechanisms are involved in clozapine‐induced hypertension, we characterized the distribution of D4R in mouse kidney and measured BP, water and sodium balance, and renal protein expression of sodium transporters in clozapine‐ and vehicle‐treated mice. D4R (immunocytochemistry) was mainly located in thick ascending limb, distal convoluted tubule and cortical and medullary collecting ducts; there is slight staining of D4R in the proximal convoluted but not straight tubule. Clozapine (20mg/kg/day) or vehicle was delivered subcutaneously to adult male C57BL/6J mice for seven days via osmotic mini‐pump (n=5/group). In clozapine‐treated mice, systolic (110±0.5, mm Hg, under anesthesia) and diastolic BPs (76.6±1),) were elevated, relative to vehicle‐treated mice (SBP=97.6±1.6, DBP=69.5±2.3). Food and water intake, body weight, serum electrolytes and creatinine, urine volume, and creatinine clearance were similar in the two groups. The sodium excretion tended to decrease while sodium excretion and blood pressure plot in clozapine‐treated mice was shifted to the right of the vehicle‐treated mice. Renal protein expressions (immunoblotting) of sodium hydrogen exchanger 3 and sodium phosphate cotransporter 2 were similar in clozapine‐ and vehicle‐treated mice. However, the renal protein expressions of sodium potassium 2 chloride cotransporter (180±21, % of control) and sodium chloride cotransporter (171±22% ), and the three subunits of epithelial sodium channel, α (140±7%), β (166±21%) and γ (55 kDa=130±7%, 85kDa=114±10% ), were greater in clozapine‐than vehicle‐treated mice. Renal protein expression of a‐sodium potassium ATPase was also slightly increased (118±4%) in clozapine‐treated mice. These findings suggest that inhibition of D4R by clozapine increases sodium transporters in distal nephron segments. Furthermore, a similar increase in the protein expression of sodium chloride cotransporter was also found in the immortalized mouse renal distal convoluted tubule cells in vitro with inhibition of D4R with its siRNA and antagonists. Thus, inhibition of renal D4R may cause the impaired sodium excretion that may be responsible for the increased blood pressure in clozap‐inetreated mice.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits, ENaC β and γ

The epithelial sodium channel (ENaC) plays a pivotal role in sodium homeostasis, and the development of drugs that modulate ENaC activity is of great potential therapeutic relevance. We screened 6100 chemicals for their ability to activate sodium permeability of ENaC. We used a two-step strategy: a high throughput cell-based assay and an electrophysiological assay. Five compounds were identified showing common structural features including an indole or benzothiophene ring. ENaC consists of three subunits: α, β, and γ. Changing the heteromeric combination of human and mouse ENaC αβγ subunits, we found that all five compounds activated the human β subunit but not the mouse subunit. However, four of them exhibited lower activity when the human γ subunit was substituted by the mouse γ subunit. Our findings provide a structural basis for designing human ENaC activity modulators. Abbreviations: ENaC: Epithelial sodium channel; ΔRFU: delta relative fluorescence units; EC50: Half-maximal effective concentration; Emax: maximum effect value.

Truncated Epithelial Sodium Channel β Subunit Responsible for Liddle Syndrome in a Chinese Family

Background/Aims: Liddle syndrome (LS) is a rare autosomal dominant disease caused by mutations in genes coding for epithelial sodium channel (ENaC) subunits. The aim of this study was to identify the mutation responsible for the LS in an extended Chinese family. Methods: DNA samples from the proband with early-onset, treatment-resistant hypertension, and hypokalemia and 19 additional relatives were all sequenced for mutations in exon 13 of the β-ENaC and γ-ENaC genes, using amplification by polymerase chain reaction and direct DNA sequencing. Results: Genetic testing of exon 13 of SCNN1B revealed duplication of guanine into a string of 3 guanines located at codon 602. This frameshift mutation is predicted to generate a premature stop codon at position 607, resulting in truncated β-ENaC lacking the remaining 34 amino acids, including the crucial PY motif. Among a total of 9 participants with the identical mutation, different phenotypes were identified. Tailored treatment with amiloride was safe and effective in alleviating disease symptoms in LS. No mutation of SCNN1G was identified in any of the examined participants. Conclusions: We report here a family affected by LS harboring a frameshift mutation (c.1806dupG) with a premature stop codon deleting the PY motif of β-ENaC. Our study demonstrates that the earlier LS patients are diagnosed by genetic testing and treated with tailored medication, the greater the likelihood of preventing or minimizing complications in the vasculature and target organs.

Aldosterone Mediated Regulation of Epithelial Sodium Channel (ENaC) Subunits in the Rat Hypothalamus

Current evidence suggests that the epithelial Na+ channel (ENaC) in the brain plays a significant role in the development of hypertension. ENaC is present in vasopressin (VP) neurons in the hypothalamus, suggesting that ENaC in VP neurons is involved in the regulation of blood pressure. Our recent study demonstrated that high dietary salt intake caused an increase in the expression and activity of ENaC that were responsible for the more depolarized basal membrane potential in VP neurons. A known regulator of ENaC expression, the mineralocorticoid receptor (MR), is present in VP neurons, suggesting that ENaC expression in VP neurons is regulated by aldosterone. In this study, the effects of aldosterone and corticosterone on ENaC were examined in acute hypothalamic slices. Real-time PCR and Western blot analysis showed that aldosterone and corticosterone treatment resulted in a significant increase in the expression of γENaC, but not α- or βENaC, and that this expression was attenuated by MR and glucocorticoid receptor (GR) antagonists. Moreover, chromatin immunoprecipitation demonstrated that the aldosterone-MR complex directly interacts with the promoter region of the γENaC gene. However, the treatment with aldosterone did not cause subcellular translocation of ENaC toward the plasma membrane nor an increase in ENaC Na+-leak current. These results indicate that expression of γENaC in VP neurons is induced by aldosterone and corticosterone through their MR and GR, respectively; however, aldosterone or corticosterone alone is not sufficient enough to increase ENaC current when they are applied to hypothalamic slices in vitro.

Expression of the epithelial sodium channel (ENaC) in the endometrium – Implications for fertility in a patient with pseudohypoaldosteronism

Pseudohypoaldosteronism type 1 (PHA) is a syndrome of unresponsiveness to aldosterone. The severe form of this disease results from mutations in the genes that encode for the epithelial sodium channel subunits, SCNN1A, SCNN1B, and SCNN1G. A PHA patient under our care failed to conceive after many years and IVF trials. Our earlier studies had shown that ENaC is expressed in the female reproductive tract. We hypothesized that a defective ENaC expression may be responsible for the infertility of the patient. To test this hypothesis we examined ENaC expression in endometrial Pipelle biopsy samples from three healthy women and the PHA patient with an Arg508X mutation in the SCNN1A gene. The formalin fixed samples were reacted with anti-ENaCA (alpha subunit) antisera, followed by secondary antibodies to visualize ENaC expression by immunofluorescence. Confocal microscopy imaging of the samples showed strong ENaC immunofluorescence along the luminal border (apical membrane) of the epithelial cells in Pipelle samples from healthy women. In contrast, none of the samples from the PHA patient showed ENaC immunofluorescence. The Arg508X mutation interrupts the transport of ENaC subunits to the cell surface, yet it would not be expected to disrupt ENaC localization in the cytoplasm. In contrast to endometrium where ENaC is localized in the apical membrane of the epithelial cells, in keratinocytes ENaC is expressed in cytoplasmic pools. Therefore, we examined ENaC immunofluorescence in plucked hair follicles. As expected, ENaC immunofluorescence was detected in the cytoplasm of keratinocytes of both normal and PHA samples. Our results support the hypothesis that lack of expression of ENaC on the endometrial surface may be responsible for the infertility of the PHA patient.