Epithelial Sodium Channel Subunits Research Articles

Renal Denervation Attenuates Nicotine‐Induced Increase in Thiazide‐Sensitive Na+/Cl− Cotransporter in the Young Pre‐Hypertensive Spontaneously Hypertensive Rat

Systemic infusion of nicotine induces renal inflammation and the development of premature hypertension in young spontaneously hypertensive rats (SHR). Renal denervation (RDN) prevents this nicotine-mediated hypertension and attenuates renal inflammation. We hypothesized that these findings were related to alterations in renal sodium handling. Following bilateral RDN or sham surgery in young (3–5 week old SHR), we continuously infused saline or nicotine bitartrate (15mg/kg/day) subcutaneously for 2 weeks. Urinary sodium was assessed via spectrophotometric analysis on spot urine samples obtained at the end of the infusion. Renal homogenates were assessed by Western blot for the presence of the epithelial sodium channel subunits (ENaC-alpha, -beta, and -gamma), sodium-potassium-chloride (NKCC2), and thiazide-sensitive sodium-chloride co-transporter (NCC). Systemic infusion of nicotine induced significant sodium retention, compared to saline, in Sham treated animals, as measured by a significant decrease in urinary sodium (from 252±55 mM to 137±26, p<0.05). During nicotine infusion in animals with RDN, there was a trend towards lesser sodium retention and a higher urinary sodium (192±46 mM, p=0.09). Western blot demonstrated that nicotine infusion in Sham treated animals induced a 1.7 fold increase in renal NCC expression, compared to saline (p<0.05). However, there was no nicotine effect on the expression of the three ENaC subunits or NKCC2 (p>0.05). RDN prevented the increase in renal NCC expression in response to nicotine infusion (p<0.05). We conclude that reduced renal sodium retention and decreased expression of NCC may contribute to the prevention of nicotine-mediated hypertension and renal inflammation by RDN. Support or Funding Information National Institutes of Health (NIH K08 HL119588, PPG HL14388) Spot urinary sodium following Sham surgery or RDN followed by 2 week infusion of subcutaneous saline or nicotine. Urine samples were collected at the end of infusion. Renal Thiazide-Sensitive Na+/Cl− Cotransporter (NCC) Expression Following Renal Denervation and 2-week Subcutaneous Infusion of Saline or Nicotine. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DOCA/salt hypertension alters Period1 and orexin-related gene expression in the medulla and hypothalamus of male rats: Diurnal influences.

In peripheral tissues, aldosterone alters expression of multiple genes, including the clock gene Period 1 (Per1), 11 beta-hydroxysteroid dehydrogenase-2 (11-HSD2), and α-ENAC, the epithelial sodium channel subunit. We evaluated the impact of chronic aldosterone exposure (DOCA) and salt intake on nocturnal changes in gene expression in the male Sprague Dawley rat brain. Additionally, genes associated with the orexin (ORX) system were also evaluated based on the role of this neuropeptide in arousal, feeding and hypertension and an interconnection with Per1 expression. DOCA/salt treatment increased saline intake primarily at night, elevated arterial pressure and lowered heart rate. In the medulla oblongata, DOCA/salt upregulated Per1, 11-HSD2, and α-ENAC expression independent of time of day, but did not change ORX receptor type 1 (ORX-R1) or type 2 (ORX-R2) expression. ORX-R1, and ORX-R2 expression in the medulla did however correlate with Per1 expression following DOCA/salt treatment but not in controls. In the hypothalamus, DOCA/salt treatment upregulated Per1, ORX-A, and ORX-R2 expression, in general, and Per1 and ORX-A expression at night. ORX-A, ORX-R1 and ORX-R2 expression in the hypothalamus correlated with Per1 expression following DOCA/salt but not in controls. These findings demonstrate for the first time that DOCA/salt hypertension modulates ORX gene expression in the brain and suggest that changes in expression in the ORX system may occur directly or indirectly via aldosterone-induced changes in Per1 expression. Our findings also build on emerging evidence that monitoring gene expression during both the day and night is critical to understanding the role of specific genes in hypertension.

Loss of β Epithelial Sodium Channel Function in Meibomian Glands Produces Pseudohypoaldosteronism 1–Like Ocular Disease in Mice

Human subjects with pseudohypoaldosteronism-1 because of loss-of-function mutations in epithelial sodium channel (ENaC) subunits exhibit meibomian gland (MG) dysfunction. A conditional βENaC MG knockout (KO) mouse model was generated to elucidate the pathogenesis of absent ENaC function in the MG and associated ocular surface disease. βENaC MG KO mice exhibited a striking age-dependent, female-predominant MG dysfunction phenotype, with white toothpaste-like secretions observed obstructing MG orifices at 7 weeks of age. There were compensatory increases in tear production but higher tear sodium and indexes of mucin concentration in βENaC MG KO mice. Histologically, MG acinar atrophy was observed with ductal enlargement and ductal epithelial hyperstratification. Inflammatory cell infiltration was observed in both MG and conjunctiva of βENaC MG KO mice. In older βENaC MG KO mice (5 to 11 months), significant ocular surface pathologies were noted, including corneal opacification, ulceration, neovascularization, and ectasia. Inflammation in MG and conjunctiva was confirmed by increased cytokine gene and protein expression and positive Ly-6B.2 immunostaining. Cell proliferation assays revealed lower proliferation rates of MG cells derived from βENaC MG KO than control mice, suggesting that βENaC plays a role in cell renewal of mouse MG. Loss of βENaC function resulted in MG disease and severe ocular surface damage that phenocopied aspects of human pseudohypoaldosteronism-1 MG disease and was sex dependent.

The muscular dystrophic chicken is hypernatremic

ABSTRACT1. The E3 ubiquitin protein ligase 1 (WWP1) gene, the mutation of which causes muscular dystrophy in chickens, is expressed not only in the pectoral muscle, but also in a number of tissues such as the kidney. Therefore, this study examined some parameters related to kidney function in muscular dystrophic (MD) chickens.2. Plasma osmolality, Na+ and K+ concentrations, aldosterone levels, and the expression of aquaporin (AQP) 2, AQP3, and α subunits of the amiloride-sensitive epithelial sodium channel (αENaC) were analysed in the kidneys of 5-week-old MD chickens and White Leghorn (WL) chickens under physiological conditions or after one day of water deprivation.3. Plasma osmolality, Na+ concentrations, and plasma aldosterone levels were significantly higher in MD chickens than in WL chickens. αENaC mRNA expression levels were lower in MD chickens than in WL chickens. AQP2 and AQP3 mRNA expression levels were similar in the two strains of chickens.4. Plasma osmolality correlated with aldosterone levels and AQP2 and αENaC mRNA levels in WL chickens. In MD chickens, plasma osmolality correlated with AQP2 mRNA levels, but not with plasma aldosterone or αENaC mRNA levels.5. These results suggest that neither water reabsorption nor the expression of AQP2 and AQP3 is impaired in MD chickens and that a WWP1 gene mutation may or may not directly induce an abnormality in Na+-reabsorption in the kidneys of MD chickens, potentially through αENaC.

MiR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinalstem cell (ISC) homeostasis. We found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Finally, we provide evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Middle ear inflammation of rat induced by urban particles

ObjectiveThe aim of this study was to evaluate the histologic change of middle ear mucosa and the expression levels of epithelial sodium channel (ENaC) subunits and mucin production genes, after the injection of urban particulate matter (UPM) into the middle ear cavity of rats. MethodsFifty pathogen-free, male Sprague Dawley rats were assigned to the study. Transtympanic injection of UPM solution (300μg/ml, 50μl) was made into the middle ear cavity of rats. Rats were sacrificed at day 1 (group1); day 3 (group2); day 5 (group3); and day 14 (group4) after the procedure. The expression levels of ENaC subunits (α, β and γ) and mucin producing genes (MUC5AC and MUC5B) were analyzed using semi-quantitative real-time reverse transcriptase–polymerase chain reaction. Thickness of middle ear mucosa was measured and analyzed. ResultAfter transtympanic injection, the thickness of middle ear mucosa increased significantly on day 1, 3 and 5 (p<0.05) and was normalized on day 14, compared to the control group. Inflammatory changes observed in the middle ear mucosa were subepithelial widening, inflammatory cell infiltration and vascular space widening on day 1, 3 and 5. These changes had reverted to normal on day 14. The level of ENaC-α expression decreased 0.60 fold on day 1 (p<0.05), but was normalized thereafter. The level of ENaC-β and γ decreased 0.39 and 0.27 fold, respectively, on day 1, was normalized on days 3 and 5, and increased 2.30 and 2.47 fold on day 14, respectively (p<0.05). The level of MUC5AC expression increased 1.97-fold on day 1 (p<0.05) and 2.58-fold on day 5 (p<0.05), but was normalized on day 14. The level of MUC5B expression increased 5.4-fold on day 1, 3.14-fold on day 3, 3.85-fold on day 5, and 2.46-fold on day 14, respectively (p<0.05). ConclusionTranstympanic injection of UPM solution into the middle ear cavity of rat induced a characteristic inflammatory response and altered gene expression related with inflammation and mucin production. These findings provide a useful clue for the understanding of how air pollutants, particularly UPM, contribute to the development of otitis media.

Mineralocorticoid effects in the late gestation ovine fetal lung.

This study was designed to determine the effects of corticosteroids at MR in the late‐gestation fetal lung. Since both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) are expressed at relatively high levels in the fetal lung, endogenous corticosteroids may act at MR as well as GR in the preterm fetal lung. The GR agonist, betamethasone, the MR agonist, aldosterone, or both were infused intravenously for 48 h in ovine fetuses of approximately 130 days gestation. Effects on airway pressures during stepwise inflation of the in situ lung, expression of ENaC alpha (SCNN1A), ENaC beta (SCNN1B), and Na,K ATPase (ATP1A1), and elastin and collagen content were determined after the infusions. We found that aldosterone significantly reduced the airway pressure measured during the initial step in inflation of the lung, although aldosterone had no overall effect on lung compliance, nor did aldosterone induce expression of ENaCα, ENaCβ or Na,K ATPaseα1. Betamethasone significantly increased expression of the epithelial sodium channel (ENaC) subunit mRNAs, and collagen and elastin content in the lungs, although this dose of betamethasone also had no effect on lung compliance. There was no synergy between effects of the MR and GR agonists. Transcriptomic analysis suggested that although aldosterone did not alter genes in pathways related to epithelial sodium transport, aldosterone did alter genes in pathways involved in cell proliferation in the lungs. The results are consistent with corticosteroid‐induced fluid reabsorption at birth through GR rather than MR, but suggest that MR facilitates lung maturation, and may contribute to inflation with the first breaths via mechanisms distinct from known aldosterone effects in other epithelia.

Following-up genome-wide association study signals: lessons learned from Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study.

Family studies have for decades documented a critical role of both common and rare inherited variations contributing to interindividual differences in the risks of common heart, lung and blood diseases and their risk factors. Examples include Mendelian forms of disease such as familial hypercholesterolemia caused by low-density lipoprotein receptor mutations and hypertension resulting from epithelial sodium channel subunit mutations. For most conditions, however, the identity of the contributing loci and allelic variants has remained elusive. In the late 1990s, 2 unlikely scientific tools were combined to seed a revolution in gene and locus discovery for many common disease traits. First, the HapMap Project (www.hapmap.org) produced a collection of haplotype-tagging variants that could capture information across the entire genome in common variants potentially influencing risk to disease. Second, large deeply phenotyped cohort studies in both the United States and Europe formed collaborations that took advantage of available DNA, appropriate informed consent, and high-quality longitudinal information about multiple phenotypes.1 These tools formed the basis of many genome-wide association studies (GWAS) which have identified hundreds of loci underlying common chronic conditions, infectious diseases, and cancer susceptibilities (www.genome.gov/gwastudies/). Because of the small effect sizes expected for most loci of interest, the large number of marker loci measured, and thus the corresponding number of statistical tests, large sample sizes are the norm for most contemporary GWAS. To obtain such sample sizes, multiple groups and studies have combined to form collaborative consortia. For instance, Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE)1 Consortium, which focuses on the genetics of cardiovascular disease and aging, has produced more than 300 publications reporting the results of GWAS for many cardio-, renal-, and cerebrovascular disease-related traits, including metabolic and inflammation traits. Several commentators have raised questions about the utility of GWAS, which have been celebrated …

Binding affinity of epithelial sodium channel subunits and peptides measured through surface plasmon resonance (997.5)

Salt levels play a crucial role in regulating blood pressure, and cells are able to maintain salt levels via ion channels embedded within the plasma membrane. The Epithelial Sodium Channel (ENaC) is a heterotrimeric membrane protein that allows the passage of sodium, and this study’s aim is to further understand what structural aspects of ENaC are responsible for maintaining proper function of the channel. Potential residues critical for interaction between ENaC alpha, beta, and gamma subunits were identified and peptide segments containing the targeted residues were PCR amplified, ligated onto a pGEX‐4t‐2 vector, and transformed into Top10 E. coli. This vector contains a GST‐tag that was used to purify the peptides and immobilize them for further analysis by surface plasmon resonance (SPR). Binding between sets of peptides predicted to be important for stabilizing interactions in ENaC was investigated using SPR. One peptide from a potential interacting set of peptide regions was immobilized through the GST‐tag and binding with the corresponding partner (with GST‐tag removed) was quantified. Two interactions ‐one between the alpha‐palm and beta‐thumb, and the other between the beta‐palm and gamma‐thumb regions of ENaC ‐ were analyzed. The alpha‐palm and beta‐thumb interaction had a measured KD of 2.9 x 10‐8 M, whereas the interaction from the beta‐palm, gamma‐thumb region was in the micromolar range for KD. Additional sets of peptides have been expressed and will be analyzed for binding interactions. Results obtained from this initial study will be compared to those obtained from a mutation screen of ENaC function to confirm whether the predicted residues play a crucial structural role related to function.Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth &amp; W. David) and Welch Foundation Departmental Grant AI‐0045.

Conserved charged residues at the surface and interface of epithelial sodium channel subunits – roles in cell surface expression and the sodium self‐inhibition response

The epithelial sodium channel (ENaC) is composed of three homologous subunits that form a triangular pyramid-shaped funnel, anchored in the membrane with a stem of six transmembrane domains. We examined the structure-function relationships of 17 conserved charged residues on the surface of the ectodomain of human γ-ENaC subunit by alanine mutagenesis and co-expression with α- and β-ENaC subunits in Xenopus oocytes. The results showed that Na(+) conductance of cells expressing these mutants can be accounted for by two parameters: (a) the ENaC density on the cell surface as measured by the fluorescence of an α-EnaC-yellow fluorescent protein hybrid and (b) the sodium self-inhibition (SSI) response that reflects the open probability of the channel (Po). Overall, the activity of all 17 mutants was correlated with surface levels of ENaC. There was no significant correlation between these parameters measured for α- and γ-ENaC subunit mutants at nine homologous positions. Thus, the functions of most of the homologous surface residues examined differ between the two subunits. Only four mutants (K328, D510, R514 and E518) significantly reduced the SSI response. The α-ENaC homologs of three of these (R350, E530 and E538) also severely affected the SSI response. The cASIC1 homologs of these (K247, E417, Q421) are located at the interface between subunits, on or about the ion pathway at the rotational symmetry axis in the center of the trimer. Thus, it is likely that these residues are involved in conformational changes that lead to channel constriction and the SSI response upon Na(+) ion flooding.

Increased Renal Epithelial Na Channel Expression and Activity Correlate With Elevation of Blood Pressure in Spontaneously Hypertensive Rats

Elevation of blood pressure with age is one of the hallmarks of hypertension in both males and females. This study examined transcriptomic profiles in the kidney of 12-, 40-, and 80-week-old spontaneously hypertensive rats and 4 recombinant inbred strains in search for functional genetic elements supporting temporal dynamics of blood pressure elevation. We found that both in males and females of spontaneously hypertensive rats and hypertensive recombinant inbred strains age-dependent blood pressure increment was accompanied by 50% heightened expression of epithelial sodium channel β- and γ-subunits. Epithelial sodium channel subunit expression correlated positively with blood pressure but correlated negatively with renin expression. Increased epithelial sodium channel activity was observed in cultured epithelial cells isolated from the kidney medulla of 80-week-old spontaneously hypertensive rats but not in age-matched normotensive Wistar Kyoto. This difference remained evident after 24-hour treatment with aldosterone. 22Na uptake in the perfused kidney medulla was increased whereas the urinary Na/K ratio was decreased in old spontaneously hypertensive rats compared with normotensive controls. The difference was eliminated by the administration of epithelial sodium channel inhibitor benzamil. Observations in recombinant inbred strains representing various mixtures of parental hypertensive and normotensive genomes suggest that Scnn1g and Scnn1b genes themselves are not implicated in heightened expression and that the increased expression is neither secondary nor required for a partial elevation of blood pressure in contrast to spontaneously hypertensive rats. We suggest that spontaneously hypertensive rats display an intact negative feed-back between renin-angiotensin-system and epithelial Na channel activity whose upregulated expression is supported by a yet unknown mechanism.

Reduced Expression of Enac in Placenta Tissues of Patients with Severe Preeclampsia Is Related to Compromised Trophoblastic Cell Migration and Invasion during Pregnancy

The purpose of the study is to investigate the expression of epithelial sodium channel (ENaC) in normal pregnancy and severe preeclampsia placenta and to explore the underlying mechanism of the relationship between the altered ENaC expression and onset of preeclampsia. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to check epithelial sodium channel subunits expression in mRNA and protein level in first term and full term placental tissue. ENaCα specific RNAi were used to knockdown ENaC expression and cell invasion and migration assay were used to check whether reduced expression of ENaC can compromise trophoblast cell function. The result showed that ENaCα was highly expressed in first term placental trophoblast cells; while EnaCβ was highly expressed in full term placenta. Knockdown ENaCα expression by using small interfering RNA reduced the invasive and migration abilities of HTR-8/SVneo cell. Real time-PCR and Western blot analysis showed that the expression levels of ENaCβ were also significantly lower in severe preeclampsia compared with normal pregnancy. It is concluded that the ENaC played an important role in trophoblast cell invasion and migration. Reduced expression and activity of epithelial sodium channel in trophoblast cells may be involved in the pathogenesis of preeclampsia.

Regulation of plasma aldosterone levels and renal sodium handling by the circadian clock protein Per1

Regulation of sodium balance by the kidney is an important determinant of long term blood pressure (BP) control. We have previously shown that the circadian clock protein Per1 regulates expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Together with our recent finding that Per1 knockout (KO) mice exhibit dramatically lower BP than wild type (WT) mice, these data led to the hypothesis that the BP phenotype involves a renal Na handling mechanism. Consistent with our hypothesis, metabolic balance studies showed that Per1 KO mice excreted more urinary Na compared to WT mice. We measured plasma aldosterone (Aldo) levels in WT and Per1 KO mice at noon and midnight. WT mice experienced the expected circadian surge in Aldo. Importantly, the circadian Aldo surge did not occur and Aldo levels were significantly lower in Per1 KO mice. Thus we examined the expression of the circadian target gene Hsd3b6, an adrenal glomerulosa cell‐specific enzyme in the Aldo synthesis pathway. Whereas Hsd3b6 was expressed in a circadian manner in WT mice, that pattern was lost in Per1 KO mice. Hsd3b6 expression was significantly less in Per1 KO mice, providing one possible explanation for the lower plasma Aldo levels observed in Per1 KO mice. Taken together, these data support the hypothesis that the BP phenotype observed in mice lacking Per1 may involve a primary adrenal defect in addition to a renal defect in Na conservation.

Mechanism of Per1 Action in Blood Pressure Control: Role of the Circadian Repressor Cry 2

The circadian clock plays an integral role in blood pressure (BP) control. In the canonical model of the circadian clock, the transcription factors Clock and Bmal1 activate circadian target genes, while Period 1 (Per1) and Cryptochrome (Cry 1 or 2) repress this action. Others have shown that mice lacking Clock or Bmal1 are hypotensive, while Cry 1/2 double knockout (KO) mice exhibit salt‐sensitive hypertension. Interestingly, our data show that Per1 KO mice exhibit lower BP than wild type (WT) mice and that Per1 positively regulates expression of the alpha subunit of the renal epithelial sodium channel (αENaC). These results suggest a non‐canonical role for Per1 in the kidney and in the regulation of BP. To address this finding, we first evaluated expression of Bmal1, Clock and Cry2 in renal cortical collecting duct (CCD) cells stably transfected with Per1 shRNA and in nuclear extracts from the renal cortex of WT or Per1 KO mice. Whereas loss of Per1 had no effect on Clock or Bmal1 protein expression, Cry2 protein levels were consistently upregulated in the absence of Per1 in vitro and in vivo. Knockdown of Clock using siRNA resulted in decreased αENaC expression in CCD cells in vitro, mimicking the effect seen with Per1 knockdown. Conversely, Cry2 knockdown appeared to cause increased αENaC expression. Taken together, these data suggest that Per1 action on αENaC regulation and BP may involve the circadian repressor Cry 2.

Sphingosine‐1‐phosphate Modulates Aldosterone‐induced Epithelial Sodium Channel Subunit Trafficking in Renal Cortical Collecting Duct Cells

We reported previously that sphingosine‐1‐phosphate (S1P) inhibited amiloride‐sensitive epithelial Na+ channels (ENaC) in the renal medulla. The present study aimed to investigate the underlying molecular mechanisms using cultured M1‐cortical collecting duct cell line. Transepithelial voltage and resistance were measured with a volt ohm meter. Equivalent current (Ieq) was calculated as the ratio of voltage to resistance. Results showed that S1P significantly inhibited aldosterone (ALDO)‐induced Ieq increase. In the presence of S1P1 receptor antagonist W146, however, the inhibitory effect of S1P was blocked (Control, 1.79 ± 0.11; ALDO, 3.57 ± 0.32; ALDO + S1P, 2.42 ± 0.05; ALDO + S1P + W146, 3.22 ± 0.30). In contrast, S1P2 or S1P3 antagonists had no affect. Furthermore, protein kinase A inhibitor 8‐ bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) attenuated ALDO‐induced Ieq increase, but it had no additive inhibitory effect with S1P (Control, 1.82 ± 0.10; ALDO, 3.69 ± 0.30; ALDO + 8‐Br‐cAMP, 2.41 ± 0.39; ALDO + 8‐Br‐cAMP + S1P, 2.35 ± 0.34). In addition, S1P blocked ALDO‐induced cAMP production. Finally, S1P prevented ALDO‐induced α‐ENaC subunit translocation to apical membrane. It is suggested that S1P reduces epithelial Na+ transport via S1P1 receptor‐mediated inhibition of cAMP production and ENaC translocation. (NIH grants HL89563, HL106042 and DK54927).

Role of Per1 and the mineralocorticoid receptor in the coordinate regulation of αENaC in renal cortical collecting duct cells

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.

Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγK189AENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock-regulatory kinases CK1δ/ε

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function and blood pressure. We previously showed that the circadian clock protein Period (Per)1, positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC), which contributes to blood pressure regulation. Casein kinases 1δ and 1ε (CK1δ/ε) are critical regulators of clock proteins. CK1δ/ε must phosphorylate the circadian clock protein Per1 in order for the latter to enter the nucleus. We used a commercially available CK1δ/ε inhibitor, PF670462, to test the effect of CK1δ/ε blockade and inhibited Per1 nuclear entry on αENaC in a model of the renal cortical collecting duct (mpkCCD(c14) cells). CK1δ/ε blockade prevented Per1 and Clock from interacting with an E-box from the αENaC promoter. CK1δ/ε inhibition reduced αENaC mRNA levels by <60%. A similar decrease in αENaC mRNA was observed following siRNA-mediated CK1δ/ε knock-down. Inhibition of CK1δ/ε effectively prevented the transcriptional response of αENaC to aldosterone, suggesting an interaction between the circadian clock and aldosterone-mediated regulation of αENaC. CK1δ/ε inhibition significantly reduced αENaC but increased Caveolin-1 membrane protein levels; transepithelial current, a measure of ENaC activity, was decreased. Importantly, single channel analysis in amphibian renal cells demonstrated a dramatic decrease in the number of patches with observable ENaC current following CK1δ/ε inhibition. The present study shows for the first time that CK1δ/ε inhibition and impaired Per1 nuclear entry results in decreased αENaC expression and ENaC activity, providing further support for direct control of ENaC by the circadian clock.

Antihypertensive Role of Tissue Kallikrein in Hyperaldosteronism in the Mouse

Tissue kallikrein (TK) is synthesized in arteries and distal renal tubule, the main target of aldosterone. Urinary kallikrein excretion increases in hyperaldosteronism. We tested the hypothesis that TK is involved in the cardiovascular and renal effects of high aldosterone. Kallikrein-deficient mice (TK-/-), and wild-type (WT) littermates, studied on two different genetic backgrounds, were treated with aldosterone and high-NaCl diet for 1 month. Control mice received vehicle and standard NaCl diet. Treatment induced 5- to 7-fold increase in plasma aldosterone, suppressed renin secretion, and increased urinary TK activity. In 129SvJ-C57BL/6J mice, blood pressure monitored by radiotelemetry was not different between control TK-/- and WT mice. In TK-/- mice, aldosterone induced larger increases in blood pressure than in WT mice (+47 vs. +27 mm Hg; genotype-treatment interaction, P < 0.05). Night-day difference was also exacerbated in treated TK-/- mice (P < 0.01). Moderate cardiac septal hypertrophy was observed in hypertensive animals without major change in heart function. Aldosterone-salt increased kidney weight similarly in both genotypes but induced a 2-fold increase in renal mRNA abundance of epithelial sodium channel subunits only in TK-/- mice. The hypertensive effect of TK deficiency was also documented in treated C57BL/6J mice. In this strain, aldosterone-induced hypertension was only observed in TK-/- mice (+16 mm Hg, P < 0.01). These findings show that TK deficiency exacerbates aldosterone-salt-induced hypertension. This effect may be due at least in part to enhanced sodium reabsorption in the distal nephron aggravating sodium retention. The study suggests that kallikrein plays an antihypertensive role in hyperaldosteronism.

The Circadian Protein Period 1 Contributes to Blood Pressure Control and Coordinately Regulates Renal Sodium Transport Genes

The circadian clock protein period 1 (Per1) contributes to the regulation of expression of the α subunit of the renal epithelial sodium channel at the basal level and in response to the mineralocorticoid hormone aldosterone. The goals of the present study were to define the role of Per1 in the regulation of additional renal sodium handling genes in cortical collecting duct cells and to evaluate blood pressure (BP) in mice lacking functional Per1. To determine whether Per1 regulates additional genes important in renal sodium handling, a candidate gene approach was used. Immortalized collecting duct cells were transfected with a nontarget small interfering RNA or a Per1-specific small interfering RNA. Expression of the genes for α-epithelial sodium channel and Fxyd5, a positive regulator of Na, K-ATPase activity, decreased in response to Per1 knockdown. Conversely, mRNA expression of caveolin 1, Ube2e3, and ET-1, all negative effectors of epithelial sodium channel, was induced after Per1 knockdown. These results led us to evaluate BP in Per1 KO mice. Mice lacking Per1 exhibit significantly reduced BP and elevated renal ET-1 levels compared with wild-type animals. Given the established role of renal ET-1 in epithelial sodium channel inhibition and BP control, elevated renal ET-1 is one possible explanation for the lower BP observed in Per1 KO mice. These data support a role for the circadian clock protein Per1 in the coordinate regulation of genes involved in renal sodium reabsorption. Importantly, the lower BP observed in Per1 KO mice compared with wild-type mice suggests a role for Per1 in BP control as well.