Epithelial Sodium Channel Subunits Research Articles

Coordinate Action of the Circadian Clock Protein Per1 and the Mineralocorticoid Hormone Aldosterone

Many metabolic and physiological processes, including renal function and blood pressure, exhibit a circadian pattern of variation. The molecular mechanism underlying the regulation of these processes by the circadian clock is not understood. We have previously shown that the circadian clock protein Per1 positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC); and negatively regulates the expression of Claudin 1 (Cldn1), a component of epithelial tight junctions; and endothelin‐1 (ET‐1), a blood pressure‐regulating peptide hormone. In the present study we found that Per1 also positively regulates Rh family C glycoprotein (RhCG), an ammonia transporter in the kidney. The sodium‐regulating hormone aldosterone has been well established to regulate the transcription of αENaC, and was also found to upregulate the levels of Cldn1, ET‐1, and RhCG mRNA. Co‐immunoprecipitation of Per1 from nuclear extracts obtained from mouse renal cortex showed an interaction between Per1 and the glucocorticoid receptor (GR), a nuclear receptor that can bind aldosterone. Taken together, these results suggest a coordinate regulation of several genes by Per1 and aldosterone.

Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock‐regulatory kinases CKIδ/ε

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function. We previously showed that the circadian clock protein Per1 positively regulates the expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Casein kinase I is a critical regulator of clock proteins. In order to enter the nucleus, Per1 must be phosphorylated by casein kinase Iε (CKIε). Renal cortical collecting duct (mpkCCDc14) cells were treated with the CKIδ/ε inhibitor PF670462. DNA affinity purification assays showed that CKIδ/ε inhibition prevented Per1 from interacting with a target E‐box from the αENaC promoter. Steady state αENaC mRNA levels were reduced more than 60% following CKIδ/ε inhibition. CKIδ/ε inhibition significantly reduced αENaC protein levels. ENaC activity is tightly controlled by aldosterone and αENaC is a well‐known transcriptional target of aldosterone. Inhibition of CKIδ/ε effectively prevented the aldosterone response of αENaC, suggesting an interaction between Per1 and aldosterone‐mediated regulation of this gene. Importantly, single channel analysis in the amphibian renal cell line 2F3 demonstrated that CKIδ/ε inhibition resulted in decreased ENaC NPo. The present study shows for the first time that CKIδ/ε inhibition represses αENaC expression and ENaC activity, providing further evidence for direct control of ENaC by the circadian clock.

A novel splice site mutation of the beta subunit gene of epithelial sodium channel (ENaC) in one Turkish patient with a systemic form of pseudohypoaldosteronism type 1

Pseudohypoaldosteronism Type 1 (PHA1) is a rare heterogeneous syndrome characterized by severe salt loss, hyperkalemia, hyponatremia, metabolic acidosis, hyperaldosteronism and hyperreninemia. Multi-system form of PHA1 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 presented a novel splice site mutation in the beta-gene of ENaC in a patient with multi-system PHA. We performed DNA sequencing analysis of SCNN1A, SCNN1B, SCNN1G and NR3C2 genes. We found a novel c.1266-1G>C homozygous splice site mutation in intron 8 of the SCNN1B gene. Initially elevated plasma renin activity (PRA) and aldosterone levels of the patient returned to normal with large amounts of dietary salt and serum sodium (Na+) and potassium (K+) levels were within normal range at the end of the first year of life. This improvement may be due to partial activity of mutated ENaC subunits, reduced dependence on aldosterone in salt homeostasis with increasing age, and alternative regulating mechanisms in sodium homeostasis. The results enhance our understanding of the pathophysiology of this disorder and the mechanisms of renal salt conservation.

Pioglitazone‐induced sodium retention in rat is independent of alpha subunit of epithelial sodium channel (ENaCá) expression in distal nephron

We studied the role of ENaC-alpha in renal sodium retention induced by pioglitazone (PGZ), a thiazolidinedione hypoglycemic agent. Male Sprague-Dawley rats in metabolic balance cages were fed on a 1% NaCl diet (control group) and the experimental group received the same diet plus PGZ (40 mg/kg bw/day for 5 days). To suppress aldosterone production, rats were fed on a 3% NaCl diet with or without PGZ. To further suppress aldosterone production, enalapril (25 mg/kg bw/day) was added to olmesartan (15 mg/kg bw/day). Western blotting of the membrane-enriched fractions from the renal cortex and medulla was performed and ENaCα density normalized to beta-actin measured. PGZ treatment led to sodium retention. In 4 control rats on a 1% NaCl diet, ENaCα expression was 0.54±0.02 in the medulla, similar to 0.60±0.04 in 4 PGZ-treated rats, and was 0.68±0.11 in the cortex in PGZ-treated rats, similar to 0.75±0.16 in the control rats. In the rats fed on a 3% NaCl diet, the ENaCα expression was similar in PGZ-treated and control rats both in the medulla and cortex. In PGZ-treated rats receiving a 3% NaCl diet, enalapril and olmesartan, ENaCα expression in the medulla and cortex was also similar in PGZ-treated and control rats. These results show that sodium retention induced by PGZ is independent of ENaCα expression in the apical membrane of the distal nephron, a finding also extended to the situations where aldosterone production is suppressed. Research support: institutional research fund

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na(+) conductance (assayed by the two-electrode voltage-clamp method), Na(+)-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na(+) conductance (to 40-80% of WT). Mutation of only six residues showed a significant effect on the Na(+) self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression (r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ~30% of WT, and Asp393 and Glu530 with τ = ~170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na(+) self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na(+) self-inhibition, most are essential for transport to the oocyte cell surface.

Altered ion transport by thyroid epithelia from CFTR−/− pigs suggests mechanisms for hypothyroidism in cystic fibrosis

Subclinical hypothyroidism has been linked to cystic fibrosis, and the cystic fibrosis transmembrane conductance regulator (CFTR) shown to be expressed in the thyroid. The thyroid epithelium secretes Cl⁻ and absorbs Na(+) in response to cAMP. Chloride secretion may provide a counter-ion for the SLC26A4 (pendrin)-mediated I⁻ secretion which is required for the first step of thyroid hormonogenesis, thyroglobulin iodination. In contrast, few models exist to explain a role for Na(+) absorption. Whether CFTR mediates the secretory Cl⁻ current in thyroid epithelium has not been directly addressed. We used thyroids from a novel pig CFTR(-/-) model, generated primary pig thyroid epithelial cell cultures (pThECs), analysed these cultures for preservation of thyroid-specific transcripts and proteins, and monitored the following parameters: (1) the Cl⁻ secretory response to the cAMP agonist, isoprenaline; and (2) the amiloride-sensitive Na(+) current. Baseline short-circuit current (I(sc)) did not differ between CFTR(+/+) and CFTR(-/-) cultures. Serosal isoprenaline increased I(sc) in CFTR(+/+), but not CFTR(-/-), monolayers. Compared with CFTR(+/+) thyroid cultures, amiloride-sensitive Na(+) absorption measured in CFTR(-/-) pThECs represented a greater fraction of the resting I(sc). However, levels of transcripts encoding epithelial sodium channel (ENaC) subunits did not differ between CFTR(+/+) and CFTR(-/-) pThECs. Immunoblot analysis verified ENaC subunit protein expression, but quantification indicated no difference in expression levels. Our studies definitively demonstrate that CFTR mediates cAMP-stimulated Cl⁻ secretion in a well-differentiated thyroid culture model and that knockout of CFTR promotes increased Na(+) absorption by a mechanism other than increased ENaC expression. These findings suggest several models for the mechanism of cystic fibrosis-associated hypothyroidism.

Clinical improvement in patients with autosomal recessive pseudohypoaldosteronism and the necessity for salt supplementation

Keywords Pseudohypoaldosteronism Aldosterone Epithelial sodium channel ENaC Salt supplementation Electrolyte regulation Na–Cl cotransporterTo the Editor,In a recent article Adachi et al. [1] reported clinicalimprovement with age in one patient with multi-systempseudohypoaldosteronism (PHA) carrying two heterozy-gous mutations in the c subunit of epithelial sodiumchannel (ENaC), noting that this patient ‘‘may be the firstexample of clinical improvement in a molecularly provenAR-PHA1.’’ We would like to draw attention to four majorissues of age-dependent amelioration in multi-system PHAraised by this study.1. Locus of mutation in ENaC genes: The authorscomment that the amelioration observed could be due tothe presence of an intact a-ENaC subunit. About 2 yearsago, we reported clinical improvement in four patients withmulti-system PHA starting at 6–10 years of age, althoughthey continue to exhibit salt-losing episodes even in olderages [2]. One of these patients carries a mutation in thea-ENaC gene and another in the b-ENaC gene. Thus, theclinical amelioration is not due to the locus of the mutationin the c-ENaC gene and can be observed in patients withmutations in any of the three subunits. Moreover, wereported that the degree of the amelioration depends on thegenotype, as one patient with a missense mutation showedamelioration after infancy, necessitating relatively smalleramounts of NaCl with only rare salt-wasting episodes afterthe age of 9 years [2].2. Activity of truncated ENaC subunit: Adachi et al. [1]note that a mutation that leads to the truncation of an ENaCsubunit (without the second trans-membrane domain)should ‘‘lead to the complete loss of its function’’. Recentlywe showed that a truncated human b-ENaC subunit maysupport low partial activity of ENaC expressed in Xenopusoocytes [3]. We further noted that this partial activity maybe partly responsible for long-term clinical ameliorationthat we observed.3. Physiological mechanism of the age-dependent ame-lioration: The study of Adachi et al. [1] provides the firstevidence for a compensatory increased expression of theNa

Increased Na reabsorption via the Na–Cl cotransporter in autosomal recessive pseudohypoaldosteronism

The autosomal recessive form of pseudohypoaldosteronism type 1 (AR-PHA1) is caused by loss-of-function mutations in the epithelial sodium channel subunit genes and is characterized by a multisystemic and lifelong severe salt-wasting tendency. However, we observed a male AR-PHA1 patient who exhibited less frequent salt wasting with advancing age, despite the cessation of daily salt supplementation. To elucidate the mechanism for the above phenomenon. We evaluated the sodium-reabsorption ability of his distal nephrons (from the distal convoluted tubules to the collecting ducts) and compared it to that of a patient with the dominant form of PHA1 (AD-PHA1) carrying a heterozygous NR3C2 (mineralocorticoid receptor) gene mutation. In addition, immunoblotting of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) protein was conducted using urine samples from the AR- and AD-PHA1 patients. The levels of sodium reabsorption that occurred via the distal nephrons were almost identical in the two PHA1 patients, despite their different molecular pathogeneses. Immunoblotting showed an increased urinary NCC protein level in the AR-PHA1 patient. Taken together, increased sodium reabsorption via the upregulation of the expression of NCC might have been responsible, at least in part, for the clinical improvement seen in an AR-PHA1 patient.

An ASIC1 and ENaC hybrid channel is the amiloride sensitive channel in human glioma.

High grade gliomas express multiple members of the Epithelial Sodium Channel (ENaC)/Degenerin (Deg) family, displaying a basally active amiloride sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. Using immunohistochemisty we show a higher expression level of ASIC1, αENaC, and γENaC in glioblastoma biopsy tissue compared with non‐neoplastic brain tissue. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, and γENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed reduced protein expression and a significant reduction in the amiloride sensitive whole cell current as compared with untransfected D54‐MG cells. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC at the plasma membrane of D54‐MG cells, consistent with these subunits interacting with each other to form an ion channel. Data using Total Internal Resolution Fluorescent Microscopy (TIRFM) also suggest that ASIC1 and ASIC2 interact with γENaC in CHO‐K1 cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grants CA101952 and DK037206.

Truncated beta epithelial sodium channel (ENaC) subunits responsible for multi-system pseudohypoaldosteronism support partial activity of ENaC

Aldosterone regulated epithelial sodium channels (ENaC) are constructed of three homologous subunits. Mutations in the α-, β- and γ-ENaC subunit genes (SCNN1A, SCNN1B and SCNN1G) are associated with multi-system pseudohypoaldosteronism (PHA), and mutations in the PY motif of carboxy-terminal region of β and γ subunits are associated with Liddle syndrome of hereditary hypertension. In this study we identified two frameshift mutations in the SCNN1B alleles of a female infant diagnosed with multi-system PHA inherited from her parents. This is the first case of PHA in an Ashkenazi family in Israel. The p.Glu217fs (c.648dupA in exon 4) and p.Tyr306fs (c.915delC in exon 6) mutations produce shortened β-ENaC subunits with 253 and 317 residues respectively instead of the 640 residues present in β-ENaC subunit. Expression of cRNAs carrying these mutations in Xenopus oocytes showed that the mutations drastically reduce but do not eliminate ENaC activity. The findings reveal that truncated β-ENaC subunits are capable of partially supporting intracellular transport of the other two subunits to the membrane and the final assembly of a weakly active channel together with normal α- and γ-ENaC subunits. Moreover, these results enhance our understanding of the long-term consequences of these types of mutations in PHA patients.

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, alphaENaC, and gammaENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, alphaENaC, gammaENaC, and deltaENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, alphaENaC, or gammaENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down alphaENaC or gammaENaC also abolished the high P(K)(+)/P(Na)(+) of D54-MG cells. Knocking down deltaENaC in D54-MG cells reduced deltaENaC protein expression but had no effect on either the whole cell current or K(+) permeability. Using co-immunoprecipitation we show interactions between ASIC1, alphaENaC, and gammaENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, alphaENaC, or gammaENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

Candesartan Differentially Regulates Epithelial Sodium Channel in Cortex Versus Medulla of Streptozotocin-Induced Diabetic Rats

Diabetes is associated with an activated renal renin-angiotensin-aldosterone system (RAAS) and it was shown that streptozotocin (STZ)-induced diabetic rats had increased whole kidney protein levels of the epithelial sodium channel subunits (α-, β- and γ-ENaC). However, the role of the RAAS on the regional, i.e., cortical versus medullary, regulation of ENaC is unclear. Male Sprague-Dawley rats were injected with STZ (intravenous, 65 mg/kg·bw, n=12/group). After 14 days, half of them received drinking water with candesartan (2 mg/kg·bw/day), an angiotensin-II type-1 receptor (AT1R) antagonist, for one week. In the medulla, i.e., inner stripe of the outer medulla (ISOM), base and/or tip of the inner medulla, immunoblotting revealed increased protein abundances of α1 Na-K-ATPase and ENaC subunits with diabetes (200-600% of controls), which were not reversed by candesartan. In fact, candesartan increased all ENaC subunits and α1 Na-K-ATPase in the ISOM and/or base in control rats. In contrast, in the cortex, diabetes did not increase these proteins. However, candesartan reduced cortical β- and γ-ENaC regardless of diabetic state. In summary, diabetes-induced increases in ENaC were seen preferentially in the medulla. These changes appeared to be due to a mechanism clearly distinct from AT1R activation, because they were not abolished by candesartan. In fact, candesartan treatment tended to increase some of these medullary proteins, perhaps in compensation for increased NaCl load. In contrast, cortical β- and γ-ENaC were reduced by candesartan regardless of diabetic state suggesting their regulation by AT1R at this site; however this did not appear to be a site of diabetes-induced ENaC up-regulation.

The (F)low Down on the Endothelial Epithelial Sodium Channel

The epithelial sodium channel (ENaC) contributes to blood pressure homeostasis through renal salt and water transport; increases in ENaC activity increase salt and water reabsorption along the distal nephron, increasing extracellular fluid volume and blood pressure. The importance of tubular ENaC in cardiovascular homeostasis is manifested in Liddle’s disease and type I pseudohypoaldosteronism, diseases associated with the gain and loss of ENaC function, respectively. However, several recent studies suggest that ENaC proteins may play a more ubiquitous role in blood pressure control as mechanosensors. Because ENaC proteins are closely related to a family of mechanosensitive proteins in the nematode termed “degenerins,” several laboratories have addressed the potential role of ENaC proteins as mechanosensors.1–3 In addition to their role in salt and water homeostasis, ENaC proteins and their relatives, acid-sensing ion channels (ASICs), may contribute to control of blood pressure through reflex regulation of the autonomic nervous system (baroreflex, chemoreflex, and metaboreflex) and local control of vascular tone.1 Recent literature reviews suggest that ENaC proteins may function as sensors of pressure-induced vascular stretch and laminar flow.1–3 The role of ENaC as a mediator of pressure-induced constriction in certain vessels is supported by several investigations; however, the role of ENaC as a flow sensor remains unclear.1 Because of their evolutionary relationship to mechanotransduction in the nematode, several laboratories have suggested that ENaC channels may act as flow …

Epithelial Sodium Channel and Acid Sensing Ion Channel Subunits Interact With Each Other To Play A Role In Glioblastoma Whole Cell Current

High grade gliomas like glioblastoma (GBM) characteristically display a basally active amiloride sensitive cation current not seen in normal human astrocytes. We hypothesize this current to be mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC)/ Degenerins (Deg) subunits. Using semi‐quantitative RT‐PCR we have shown higher expression of ASIC1, αENaC, and γENaC in primary GBM cells compared to primary human astrocytes. We have also shown the expression of these subunits in GBM biopsies and on the plasma membrane of GBM cells using immunocytochemistry. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed 50‐ 70% reduction in protein expression and significant inhibition of whole cell patch clamp current compared with untransfected D54‐MG. Knocking down δENaC in D54‐MG cells reduced δENaC protein expression by 50%, but had no effect on the whole cell patch clamp current. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grant CA101952.

Mutations in the amiloride-sensitive epithelial sodium channel in patients with cystic fibrosis-like disease

We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.

Characterization of the epithelial sodium channel α subunit coding and non-coding transcripts and their corresponding mRNA expression levels in Dahl R versus S rat kidney cortex on normal and high salt diet

Aims/hypothesisThe α subunit of the amiloride-sensitive epithelial sodium channel (α ENaC) is critical for the expression of functional channels. In humans and rats, non functional alternatively spliced forms of α ENaC have been proposed to act as negative regulatory components for ENaC. The purpose of this study was to examine the presence and consequently investigate the mRNA expression levels of alternatively spliced forms of α ENaC in kidney cortex of Dahl salt-resistant rats (R) versus Dahl salt-sensitive rats (S) on high salt and normal diets.MethodsUsing quantitative RT-PCR strategy, we examined the mRNA expression levels of previously reported α ENaC-a and -b alternatively spliced forms in kidney cortex of Dahl S and R rats on normal and four-week high salt diet and compared their corresponding abundance to wildtype α ENaC mRNA levels. We identified 2 novel non-coding C-terminus spliced forms and examined their mRNA expression in Dahl R versus S rat kidney cortex. We also tested the presence of five previously reported lung-specific α ENaC spliced forms in Dahl rat kidney cortex (CK479583, CK475461, CK364785, CK475819, and CB690980).ResultsPreviously reported α ENaC-a and -b alternatively spliced forms are present in Dahl rat kidney cortex and are significantly higher in Dahl R versus S rats (P < 0.05). Four-week high salt diet significantly increases α ENaC-b (P < 0.05), but not α ENaC-a transcript abundance in Dahl R, but not S rats. Two non-coding α ENaC spliced forms -c and -d are newly identified in the present study, whose levels are comparable in Dahl R and S rats. Compared to α ENaC-wt, α ENaC-a, -c and -d are low abundance transcripts (4 +/- 2, 110 +/- 20, and 10 +/- 2 fold less respectively), in contrast to α ENaC-b abundance that exceeds α ENaC-wt by 32 +/- 3 fold. We could not identify any of the five previously reported lung-specific α ENaC spliced forms (CK479583, CK475461, CK364785, CK475819, and CB690980) in Dahl rat kidney cortex.Conclusion/interpretationα ENaC alternative splicing might regulate α ENaC by the formation of coding RNA species (α ENaC-a and -b) and non-coding RNA species (α ENaC-c and -d). α ENaC-a and -b mRNA levels are significantly higher in Dahl R versus S rats. Additionally, α ENaC-b is a salt-sensitive transcript whose levels are significantly higher 4-weeks post high salt diet compared to normal salt diet in Dahl R rats. Among the four α ENaC transcripts (-a, -b, -c and -d), α ENaC-b is a predominant transcript that exceeds α ENaC-wt abundance by ~32 fold. α ENaC-a and -b spliced forms, particularly, α ENaC-b, might potentially act as dominant negative proteins for ENaC activity, thereby rescuing Dahl R rats from developing salt-sensitive hypertension on high salt diet. On the other hand, non-coding α ENaC-c and -d might assist alternative splicing, facilitate RNA processing, or regulate α ENaC as well as each other.

A Novel Nonsense Mutation of the Mineralocorticoid Receptor Gene in the Renal Form of Pseudohypoaldosteronism Type 1

Pseudohypoaldosteronism type 1 (PHA1) is a rare congenital disease characterized by salt loss resistant to mineralocorticoids. Most patients are identified by failure to thrive or poor weight gain in early infancy. Plasma renin activity and aldosterone are markedly elevated. PHA1 is caused by mutations in genes encoding either subunits of the amiloride-sensitive epithelial sodium channel (ENaC) or mineralocorticoid receptor (MR) inherited in an autosomal recessive or dominant form, respectively. Patients with the autosomal dominant form of PHA1 show gradual clinical improvement with advancing age; however, the reason for this remains unclear. We report the renal form of PHA1 in a Japanese family. Polymerase chain reaction and direct sequencing revealed a heterozygous nonsense mutation changing codon 861 Arg (CGA) to stop (TGA) in the index patient. Segregation analysis revealed an identical mutation in the patient's father and older sister. Inheritance in this case is assumed to be of the autosomal dominant type.

Plasmin Activates Epithelial Na+ Channels by Cleaving the γ Subunit

Proteolytic processing of epithelial sodium channel (ENaC) subunits occurs as channels mature within the biosynthetic pathway. The proteolytic processing events of the alpha and gamma subunits are associated with channel activation. Furin cleaves the alpha subunit ectodomain at two sites, releasing an inhibitory tract and activating the channel. However, furin cleaves the gamma subunit ectodomain only once. A second distal cleavage in the gamma subunit induced by other proteases, such as prostasin and elastase, is required to release a second inhibitory tract and further activate the channel. We found that the serine protease plasmin activates ENaC in association with inducing cleavage of the gamma subunit at gammaLys194, a site distal to the furin site. A gammaK194A mutant prevented both plasmin-dependent activation of ENaC and plasmin-dependent production of a unique 70-kDa carboxyl-terminal gamma subunit cleavage fragment. Plasmin-dependent cleavage and activation of ENaC may have a role in extracellular volume expansion in human disorders associated with proteinuria, as filtered plasminogen may be processed by urokinase, released from renal tubular epithelium, to generate active plasmin.

Renin–aldosterone response, urinary Na/K ratio and growth in pseudohypoaldosteronism patients with mutations in epithelial sodium channel (ENaC) subunit genes

Multi-system pseudohypoaldosteronism (PHA) is a rare syndrome of aldosterone unresponsiveness characterized by symptoms of severe salt-losing caused by mutations in one of the genes that encode α, β or γ subunit of epithelial sodium channels (ENaC). We examined long-term changes in the renin–aldosterone response in patients with different mutations. Four PHA patients were followed-up for 7–22 years. Patient A with a heterozygous Gly327Cys missense mutation in αENaC is a mild case and patients B, C and D are severe cases. Two additional patients with renal PHA served as controls. In patient A, serum aldosterone and plasma renin activity (PRA) decreased with age, PRA reaching near normal values at age 11. In contrast, patients B–D showed a positive correlation between age and aldosterone ( r > 0.86 for all). In patient B with Arg508 stop mutation, aldosterone reached 166 nmol/L at age 19 (>300-fold higher than normal). Urinary Na/K ratios normalized gradually with age in all patients. Growth curves of the patients were reflective of the severity of PHA and compliance with salt therapy. Functional expression studies in oocytes showed that ENaC with αGly327Cys mutation, as observed in patient A, showed nearly 40% activity of the wild type ENaC. In contrast, stop mutation as in patient B reduces ENaC activity to less than 5% of the normal. Our results demonstrate distinct genotype–phenotype relationships in multi-system PHA patients. The degree of ENaC function impairment affects differently the renin–aldosterone system and urinary Na/K ratios. The differences observed are age-dependent and PHA form specific.

Epithelial Sodium Channel Is a Key Mediator of Growth Hormone-Induced Sodium Retention in Acromegaly

Acromegalic patients present with volume expansion and arterial hypertension, but the renal sites and molecular mechanisms of direct antinatriuretic action of GH remain unclear. Here, we show that acromegalic GC rats, which are chronically exposed to very high levels of GH, exhibited a decrease of furosemide-induced natriuresis and an increase of amiloride-stimulated natriuresis compared with controls. Enhanced Na(+),K(+)-ATPase activity and altered proteolytic maturation of epithelial sodium channel (ENaC) subunits in the cortical collecting ducts (CCDs) of GC rats provided additional evidence for an increased sodium reabsorption in the late distal nephron under chronic GH excess. In vitro experiments on KC3AC1 cells, a murine CCD cell model, revealed the expression of functional GH receptors and IGF-I receptors coupled to activation of Janus kinase 2/signal transducer and activator of transcription 5, ERK, and AKT signaling pathways. That GH directly controls sodium reabsorption in CCD cells is supported by: 1) stimulation of transepithelial sodium transport inhibited by GH receptor antagonist pegvisomant; 2) induction of alpha-ENaC mRNA expression; and 3) identification of signal transducer and activator of transcription 5 binding to a response element located in the alpha-ENaC promoter, indicative of the transcriptional regulation of alpha-ENaC by GH. Our findings provide the first evidence that GH, in concert with IGF-I, stimulates ENaC-mediated sodium transport in the late distal nephron, accounting for the pathogenesis of sodium retention in acromegaly.