Regulation Of ENaC Research Articles

ENaC regulation by [Ca 2+ ] i is determined by mitochondrial Ca 2+ uptake

Apical ATP inhibits ENaC through P2Y2 receptor-mediated Ca2+ release but basal ATP stimulates ENaC via P2X4 Ca2+ channels. We have shown that mitochondria are located in CCD cells so as to prevent free diffusion of Ca2+. We tested if mitochondrial Ca2+ uptake contributes to the polarized effect of [Ca2+]i on ENaC. When the Ca2+ ionophore, ionomycin, was applied apically, single channel measurements showed ENaC open probability (Po) decreased from 0.53 ± 0.08 to 0.18 ± 0.02 (N 蠅 6, P<0.01). In contrast, basal ionomycin increased ENaC Po (0.09 ± 0.02 to 0.18 ± 0.04, N 蠅 6, P<0.05). To determine if the polarized effect of ionomycin was due to mitochondrial Ca2+ uptake, we used the inhibitor Ru360 to inhibit VDAC-mediated Ca2+ uptake across the mitochondrial outer membrane. In cells pretreated with Ru360, apical or basal ionomycin produced identical effects, a small stimulation followed by a small inhibition. These data show that mitochondrial Ca2+ uptake allows polarized [Ca2+]i signaling and ENaC regulation in CCD cells. To confirm the effect of mitochondrial Ca2+ uptake on Na+ uptake in vivo, we used mice lacking VDAC1 or VDAC3. Increased ENaC activity would lead to Na+ and water uptake and increased blood pressure. VDAC1 -/- mice had increased systolic blood pressure compared to littermate controls (109 ± 5.5 vs 137 ± 3.9 mmHg, N蠅5, P<0.05). VDAC3-/- mice had normal systolic blood pressure at baseline, but did not tolerate a high salt (8% NaCl) diet. After 7 days of high salt, VDAC3-/- mice had a systolic BP of 128mmHg ±6.89 vs 109mmHg ±3.77 for littermate controls (N蠅6, P<0.05). These data show that mitochondrial Ca2+ uptake contributes to ENaC regulation both in vitro and in vivo. Support: NIH R37-DK037963 and T32-DK07656 and AHA 13POST16820072.

Compromised Salt Dependence of ENaC Activity Upon Deletion of AT1 Receptors

ENaC-mediated sodium reabsorption in the collecting duct (CD) is an essential determinant of blood pressure in humans. Cumulative evidence suggests direct action of Angiotensin II (Ang II) on ENaC activity in the CD, independently of the classical aldosterone signaling. The significance of regulation by Ang II becomes particularly important during Ang II dependent hypertension, when overactive ENaC is poorly regulated via aldosterone – mineralocorticoid receptor (MR) axis. Stimulatory Ang II actions on ENaC are mediated by AT1 receptors (AT1R). AT1R knockouts have decreased blood pressure despite elevated aldosterone levels. This indicates that aldosterone-MR cascade alone cannot provide sufficient input to adequately regulate ENaC-dependent sodium re-uptake. Using freshly isolated split-opened CDs we found that genetic deletion of AT1R markedly diminishes ENaC activity on low and regular sodium intake, significantly blunting salt-dependent regulation of ENaC. The observed decrease of ENaC activity was mainly attributed to a reduced channel open probability (Po). Pharmacological inhibition of AT1R with losartan in wild-type animals reproduces the effect of genetic ablation of AT1R on ENaC activity. Interestingly, saturation of aldosterone signaling with DOCA has minimal stimulatory effect on ENaC upon AT1R deletion, which can be explained by a markedly diminished MR abundance in the CD cells of the knockouts. Overall, genetic ablation of AR1R elicits a complex inhibitory effect on ENaC activity, directly attenuating ENaC Po and precluding adequate compensation by aldosterone cascade due to decreased MR expression.

Nox4‐mediated and Hydrogen Peroxide Dependent Regulation of ENaC In Salt‐Sensitive Hypertension

Nox4‐mediated and Hydrogen Peroxide Dependent Regulation of ENaC In Salt‐Sensitive Hypertension

FoxO1 inhibits transcription and membrane trafficking of epithelial Na+ channel.

The epithelial Na(+) channel (ENaC), regulated by insulin, is of fundamental importance in the control of Na(+) reabsorption in the distal nephron. The potential role of Forkhead box O1 (FoxO1), downstream of insulin signaling, in the regulation of ENaC remains to be investigated. Here, we found that the overexpression of a constitutively active form of FoxO1 (ADA-FoxO1) suppressed the mRNA level of the ENaC α subunit (α-ENaC; also known as SCCN1A) and the apical density of ENaC in mouse cortical collecting duct (mCCD) cells. Conversely, knockdown of FoxO1 increased the apical membrane levels of α-ENaC and Na(+) transport under basal conditions. Insulin elevated α-ENaC expression and induced FoxO1 phosphorylation; however, the increase in α-ENaC and phosphorylated FoxO1 expression observed with insulin treatment was blunted ∼ 60% in cells expressing ADA-FoxO1. Moreover, insulin induced the interaction between phosphorylated FoxO1 and 14-3-3ε, indicating that FoxO1 phosphorylation promotes ENaC membrane trafficking by binding to 14-3-3ε. FoxO1 also suppressed activity of the α-ENaC promoter, and the putative FoxO1 target site is located in the -500 to -200 nt region of the α-ENaC promoter. These findings indicate that FoxO1 is a key negative regulatory factor in the insulin-dependent control of ENaC expression and forward trafficking in mCCD epithelia.

Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency.

Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation, etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na(+) flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane-permeabilized cells. A decrease in the Na(+)-K(+)-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid reabsorption by MTEs. ERK1/2, but not Akt, phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs. those of wild-type controls. Expression of caspase 8, however, did not differ between wild-type and uPA(-/-) mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na(+)-K(+)-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function.

Expression of ENaC, SGK1 and Nedd4 isoforms in the cochlea of guinea pig.

It has been demonstrated that the epithelial sodium channel (ENaC) may play critical roles in (re)absorbing Na+ from apical plasma membrane in various tissues and cells. Moreover, the serum glucocorticoid-inducible kinase 1 (SGK1) and the ubiquitin-protein ligase neural precursor cell-expressed, developmentally downregulated isoforms Nedd4 are involved in ENaC regulation in response to hormones such as aldosterone, vasopressin and insulin. The aim of the study was to investigate the cellular localizations of ENaC subunits, SGK1, and Nedd4 isoforms in the cochlea of guinea pig. Ten adult guinea pigs were sacrificed and their cochleas were collected. The expression patterns of ENaC subunits, SGK1 and Nedd4-2 isoforms in the guinea pig cochlea were studied by immunohistochemistry with the specific polyclonal rabbit antibodies against rat α-, β- and γ-ENaC, SGK1, Nedd4-1/2 and Nedd4-2. The results showed that all these proteins were extensively expressed in various regions of the cochlea. They were found in the spiral ligament, organ of Corti, spiral limbus, spiral ganglion and Reissner's membrane with different staining patterns. The results indicated that a Na+ transport system may exist in the cochlea of guinea pig consisting of ENaC, SGK1 and Nedd4, which may work in concert to transport Na+ and to maintain homeostasis in inner ear as it does in other epithelia.

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.

Novel roles of raft domain in the molecular mechanism of activation of epithelial Na+ channel at the plasma membrane (892.26)

ENaC expressed in the apical membrane of epithelial cells plays a critical role in homeostasis of fluid content and blood pressure. Na+ transport via ENaC depends on not only the open probability of the channel, but also the number of channel expressed on the apical membrane. ENaC is cleaved by aldosterone‐induced proteases, resulting in an increase in open probability. The lifetime of ENaC at the apical membrane is regulated by ubiquitin ligase (Nedd4‐2). To elucidate roles of raft domain in the regulation of ENaC activation, we quantified the abundance and localization of ENaC at the plasma membrane with/without aldosterone stimulation in renal epithelial cells. Aldosterone stimulated expression of both full‐length and cleaved ENaCs; i.e., the full‐length ENaC was expressed in both cytoplasm and plasma membrane (raft domain), whereas a large portion of the cleaved ENaC was observed at the non‐raft plasma membrane. Depletion of cholesterol from the raft domain diminished expression of cleaved ENaC not only in raft but also non‐raft domain by aldosterone stimulation. Aldosterone also increased expression of Nedd4‐2 and SGK1 in raft domain. Together, these results suggest that the ubiquitination‐mediated regulation of ENaC would occur in raft domain. Our findings demonstrate novel roles of the lipid raft domain on the regulation of ENaC activity/trafficking.

Regulation of ENaC, AE1 and H+ ATPase in renal intercalated cell specific NBCe2 knockout mice (1098.5)

Regulation of ENaC, AE1 and H+ ATPase in renal intercalated cell specific NBCe2 knockout mice Fredrik D Pedersen, Jeppe Praetorius and Helle H Damkier, Department of Biomedicine, Health, Aarhus University, Denmark NBCe2 is an electrogenic Na+ HCO3‐ cotransporter expressed in the brain and kidney. In mouse, NBCe2 mRNA is localized in the collecting duct principal cells; however, attempts to locate the protein in mice have been unsuccessful. In humans, immunohistochemical studies localized NBCe2 to the apical membrane of apparent intercalated cells. Mice lacking NBCe2 are hypertensive and have lower urinary pH, but the mechanism behind and cellular origin of this phenotype is unknown.The aim of the study is to define the cell type responsible for development of hypertension in NBCe2 knockout mice.Intercalated cell specific NBCe2 knockout mice (IC‐KO) were generated using the loxP/cre system. Mice expressing cre recombinase driven by the promotor for the intercalated cell H+ ATPase, B1 were crossed with floxed NBCe2 mice.Tail cuff measurements and urine electrolyte analyses revealed no differences in blood pressure or urine composition between the IC‐KO mice and wildtype littermates. However, immunoblot analysis revealed decreased abundance of the membrane transporters AE1, αENaC and H+ ATPase in the IC‐KO mice. Furthermore, immunohistochemical analysis showed increased γENaC abundance in the apical plasma membrane of the IC‐KO mice.In conclusion, the IC‐KO mouse model does not display the hypertensive phenotype of the full NBCe2 knockout mouse. Nevertheless, the changes in abundance and subcellular localization of key renal membrane transporters indicate a significance of NBCe2 in intercalated cells in mice. However, the changes could also reflect the activity of cre recombinase in a subset of cortical principal cells that has been described for the cre mouse.

EGF/RhoGDIα/Rac1 pathway modulates ENaC activity and contributes to the development of salt‐sensitive hypertension (892.27)

Epithelial sodium channel (ENaC) performs sodium reabsorption in the aldosterone sensitive distal nephron. Amongst other hormonal agents, ENaC‐mediated sodium reabsorption is regulated by the members of epidermal growth factor (EGF) family; specifically, chronic treatment with the EGF‐family members downregulates ENaC activity in vitro. We have recently shown that EGF deficiency is involved in improper ENaC activity and contributes to the development of salt‐induced hypertension in Dahl salt‐sensitive (SS) rats. Investigation of signaling pathways potentially involved in regulation of ENaC by EGF, revealed that high salt diet in SS rats decreases abundance of Rho GDP‐dissociation inhibitor 1(RhoGDIα), a well characterized modulator of Rho‐family small GTPases. RhoGDIα deficiency in cultured mCCDcl1 principal cell line increases ENaC‐mediated current formation and causes enhanced response of ENaC activity to EGF treatment. Further experiments revealed that these effects are mediated via Rac1 small GTPase. We conclude that renal EGF deficiency in the SS rats on a high salt diet provokes excessive ENaC activity via RhoGDIα/Rac1pathway and contributes to the development of salt‐induced hypertension.Grant Funding Source: Supported by AHA, NHLBI

Regulation of epithelial sodium channel activity in A6 cells via the peptide hormone, prolactin (1181.9)

Prolactin (PRL) is a peptide hormone well‐known for its role in lactation. PRL also has osmoregulatory actions via alteration of ion and water transport, actions characterized mostly in aquatic life. In 2003, researchers reported that PRL stimulates epithelial sodium channel (ENaC) activity in adult frog skin. Since that time, PRL‐mediated regulation of ENaC has been largely ignored. Given that ENaC present in the kidney plays an essential part in blood pressure control, we hypothesized that PRL may control blood volume and blood pressure via regulation of ENaC activity. To investigate this hypothesis, we used a well‐known frog kidney cell model (A6‐2F3) possessing amiloride‐sensitive transepithelial current and highly selective sodium channels with a conductance of ~5pS. Amiloride‐sensitive (ENaC‐mediated) current dramatically increased within 30 minutes after application of 1µg/mL PRL to the basal side of the cells ‐ an effect sustained even at 24 hours after treatment. We confirmed this finding using cell attached patch clamp. We found an almost 6 fold increase in ENaC NPo in PRL‐treated versus vehicle‐treated cells. The mechanism, although still not clear, at least partially involves JAK2 signaling since AG‐490, a JAK2 inhibitor, reduced the effect of PRL on amiloride‐sensitive current in A6 cells. Inhibitors of PI3K and SRC had no effect on PRL’s induction of amiloride‐sensitive current. In conclusion, we observed a significant induction of ENaC activity in frog kidney cells exposed to PRL ‐ an effect partially mediated through JAK signaling pathways. Future studies will finalize the signaling mechanisms involved in PRL’s activation of ENaC and the role of these pathways in blood pressure control.Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118

Regulation of ion channels in cystic fibrosis (869.8)

Cystic Fibrosis (CF) is an autosomal recessive disease, characterized by mutations in the CFTR gene, incorrect protein folding, and impaired chloride secretion. In addition to the maintenance of chloride secretion, CFTR is also suggested to be an important regulator of epithelial sodium channel (ENaC) activity. We previously identified the chloride channel ClCN2 as coexpressed with the mutant CFTR and hypothesized that in addition to restoring chloride secretion, ClCN2 can act as a negative regulator of ENaC. Coimmunoprecipitation analysis shows a novel physical interaction between ClCN2 and ENaC in pulmonary epithelium, which is accompanied by changes in ClCN2 and ENaC protein localization, as well as transepithelial resistance. Furthermore, ClCN2 has been shown to play an important role in the regulation of epithelial cell junctions and to this end, we show that changes in ClCN2 localization and activity may also affect cell junctions in CF cells. In conclusion, our data is the first to identify a novel interaction between ClCN2 and ENaC subunits. We also show potential changes in junction formation which may be regulated by changes in ClCN2 activity and localization. Therefore, when CFTR is nonfunctional, as in Cystic Fibrosis, ClCN2 may regulate ENaC activity and tight junction formation, which suggests that modulation of ClCN2 activity might be an advantageous target for treatment of the disease.Grant Funding Source: Supported by 5T32HL72748‐11: Ruth L Kirschstein National Research Service Award

Cytoskeletal regulation of ENaC: the role of ankyrin G (892.28)

The epithelial sodium channel (ENaC) is a major determinant of sodium (Na+) and water balance in the kidney. The hormone aldosterone increases ENaC subunit translation and modifies expression of key regulators that alter ENaC function and membrane density. Using a microarray screen we identified the scaffolding protein, ankyrin G (Ank3), as an aldosterone‐induced protein. We hypothesize that Ank3 regulates Na+ transport by increasing ENaC density at the apical membrane. Ank3 overexpression in a mouse kidney collecting duct epithelial cell line (mCCD‐cl1) increased ENaC‐mediated Na+ transport by 67%±11% (n=12), whereas knockdown reduced ENaC activity by 69%±2% (n=15). Ank3 depletion blunted the ENaC response to aldosterone stimulation in this cell line, from a 70.4±7% increase in control cells to 19.8±5% (n=5) increase in Ank3 knock‐down cells. Membrane permeabilization experiments confirmed the reduction in Na+ transport was due to reduced ENaC, and not Na/K‐ATPase activity. Future studies will identify critical domains governing Ank3’s association with ENaC and the regulation of this interaction. These findings introduce Ank3 as a key regulator in kidney Na+ transport.Grant Funding Source: Supported by DK078917, DK079307

CaMKII‐dependent filamin phosphorylation attenuates the MARCKS regulation of ENaC (892.24)

Filamin A is an actin crosslinking protein regulated by phosphorylation. Several kinases including protein kinase A (PKA), protein kinase B (Akt), protein kinase C (PKC), and Ca(2+)/calmodulin‐dependent protein kinase II (CaMKII) phosphorylate filamin A. Xenopus laevis distal nephron epithelial (2F3) cells pretreated with the specific CaMKII inhibitor, KN‐93, showed an increase in amiloride‐sensitive transepithelial current calculated from transepithelial voltage and resistance measurements obtained with a volt‐ohmmeter, and an increase in ENaC open probability measured by single‐channel patch clamp studies. Whole cell lysates from 2F3 cells pretreated with KN‐93 or vehicle control were used to immunoprecipitate filamin, phosphofilamin, MARCKS, and ENaC before immunoblotting for each protein using specific antibodies. Immunoblot and densitometric analysis showed the level of filamin phosphorylation decreased with KN‐93 treatment compared to vehicle control. The association between ENaC and MARCKS was augmented in cells treated with KN‐93 compared to cells treated with vehicle only. ENaC‐MARCKS interaction is necessary for normal ENaC activity. Sucrose density gradient fractions from KN‐93 or vehicle treated cells were analyzed by immunoblotting. Similar to various actin cytoskeleton proteins, filamin protein expression was observed in non‐lipid raft cytoplasmic fractions. The expression of phosphofilamin between these fractions was different between the KN‐93 treated group compared to the vehicle treated control group. Taken together, these studies indicate filamin is an integral component of the actin cytoskeleton and phosphorylation of filamin by CaMKII disrupts the interaction between MARCKS and ENaC. Grant Funding Source: DK093255‐01

Proteolytic activation of the human epithelial sodium channel by trypsin and trypsin IV involves distinct cleavage sites (1181.2)

Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC) but is not yet fully understood. The serine protease trypsin is known to activate ENaC in vitro but may not be relevant in vivo. In this study, we wanted to investigate whether the trypsin‐like serine protease trypsin IV, known to be expressed in several epithelial tissues, can activate human ENaC. Moreover, using site‐directed mutagenesis we wanted to identify functionally relevant cleavage sites in the γ‐subunit of the channel. In the Xenopus laevis oocyte expression system, we monitored the proteolytic activation of ENaC currents and the appearance of γ‐ENaC cleavage products at the cell surface. We demonstrated that trypsin IV can stimulate ENaC and that this activation involves a critical cleavage site (K189) in the extracellular domain of the γ‐subunit. Moreover, we showed that channel activation by trypsin was prevented by mutating three putative trypsin cleavage sites (K168; K170; R172) in addition to mutating previously described prostasin (RKRK178), plasmin (K189), and neutrophil elastase (V182;V193) sites. In conclusion, we demonstrated for the first time that trypsin IV can activate ENaC and that trypsin and trypsin IV use specific cleavage sites to achieve channel activation. Preferential cleavage sites may provide a mechanism for differential ENaC regulation by tissue‐specific proteases.Grant Funding Source: Supported by grants of the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) (S.H., M.K.)

A microRNA cluster miR‐23/24/27 is regulated by aldosterone to alter Na+ transport in the kidney distal nephron (711.5)

The epithelial sodium channel (ENaC) is a major determinant of sodium (Na+) and water balance in the kidney. We previously reported that aldosterone (aldo) modifies expression of microRNAs (miRs) to alter target gene expression and regulate Na+ transport in principal cortical collecting duct (CCD) epithelial cells. These studies focus on 2 clusters of miRs that are significantly upregulated by aldo, and are members of the miR‐23/24/27 family. In cultured mCCD‐cl1 cells, aldo stimulation (50nM, 24hr) resulted in a ~1.7 fold increase in all family members as determined by microarray and qPCR. In vivo, these miRs were significantly increased (1.7‐3.5 fold) in isolated CCD cells from mice on low Na+ vs standard salt diets. Exogenous overexpression on the miRs, using oligonucleotide mimics, increased ENaC‐mediated Na+ transport in the mCCD cell line (44±5.9%, n=8, p&lt;0.002), in the absence of aldo stimulation. Using our newly developed algorithm, ComiR, we predicted and verified regulation of intersectin 1&amp;amp;2 as targets of these miR clusters, and possible mediators of ENaC regulation. These data confirm a central role for aldo‐regulated miRs in the maintenance of Na+ transport in the distal kidney nephron.Grant Funding Source: Supported by DK078917

ENaC activity is decreased in alveolar epithelial cells from protein kinase C‐α knockout mice (716.2)

ENaC open probability (Po) requires the association of ENaC with anionic phospholipids like PIP2. Protein kinase C is also alters ENaC activity, but the mechanism is unclear. Recently we showed that PKC can affect ENaC through phosphorylation of myristolyated‐alanine‐rich‐C‐kinase‐substrate protein (MARCKS). MARCKS acts to sequester PIP2 in close proximity to ENaC in the membrane and phosphorylation of MARCKS by protein kinase C (PKC) causes MARCKS to dissociate from the membrane. Without MARCKS in the membrane, PIP2 no longer associates with ENaC and ENaC Po decreases. To test whether PKC‐α influences ENaC activity in the lung, we examined ENaC regulation in a PKCα‐/‐ murine model. Primary cultures of alveolar type II cells were prepared from wild‐type and knockout animals, and were subjected to cell‐attached patch clamp. In PKC‐α KO, ENaC Po was significantly less (0.100 ± 0.0241, n = 18) compared to wildtype SV129 mice (0.200 ± 0.0310, n = 28, p = 0.025). The number of channels per patch (N) also decreased. Evan’s blue assays showed a two‐fold decrease in ENaC‐mediated fluid uptake in the KO mice, while wet‐dry assays also showed a decrease in ENaC activity in the KO animals. To confirm this decrease, a lung from each animal was perfused with biotin and membrane protein pulled down with streptavidin. While total ENaC protein did not change in PKCα‐/‐ mice compared with wildtype, membrane localization of all three ENaC subunits decreased. Using confocal microscopy, we observed an increase in reactive oxygen species (ROS) in the alveolar type II cells of PKCα‐/‐ mice. The increased ROS stimulated PKCδ which phosphorylatyed MARCKS in lieu of PKCα and decreased ENaC in Po in the knockout animals.Grant Funding Source: Supported by NIH

Down-Regulation of the Epithelial Na+ Channel ENaC by Janus kinase 2

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na(+) transport, effects at least partially involving regulation of the epithelial Na(+) channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na(+) channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function (V617F)JAK2 or inactive (K882E)JAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, (V617F)JAK2 or (K882E)JAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I(amil)) in dual electrode voltage clamp. Moreover, I(amil) was determined in colonic tissue utilizing Ussing chambers. As a result, the I(amil) in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Coexpression of JAK2 and Nedd4-2 decreased I(amil) in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I(amil). In colonic epithelium, I(amil) was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.

The Cyp2c44 Epoxygenase Regulates Epithelial Sodium Channel Activity and the Blood Pressure Responses to Increased Dietary Salt

Hypertension is a major risk factor for cerebral, cardiovascular, and renal disease, and its prevalence and devastating consequences raises a need for new strategies for its early diagnosis and treatment. We show here that lack of a Cyp2c44 epoxygenase causes dietary salt-sensitive hypertension, a common form of the human disease. Cyp2c44(-/-) mice on normal salt diets are normotensive but become hypertensive when fed high salt. Hypertensive Cyp2c44(-/-) mice show a hyperactive kidney epithelial sodium channel (ENaC) and reductions in ERK1/2 and ENaC subunit phosphorylation. The demonstration that amiloride, an ENaC inhibitor, lowers the blood pressure of hypertensive Cyp2c44(-/-) mice identifies a role for the channel in the hypertensive phenotype of the animals. These studies: (a) identify an antihypertensive role for the kidney Cyp2c44 epoxygenase and for its epoxyeicosatrienoic acid (EET) metabolites in the in vivo control of ENaC activity and the activation of mitogenic kinase pathways; (b) provide evidence for a Cyp2c44 epoxygenase, EET-mediated mechanism of ENaC regulation involving an ERK1/2-catalyzed threonine phosphorylation of the channel γ subunit: and (c) characterize a common scientific platform that could explain the seemingly unrelated biological activities attributed to the epoxygenase metabolites in cell proliferation, angiogenesis, channel activity, and blood pressure control. It is expected that these results will serve as a basis for the development of novel strategies for the early diagnosis and treatment of hypertension and of pathophysiologies associated with dysfunctional mitogenic signaling.

Colon-Specific Deletion of Epithelial Sodium Channel Causes Sodium Loss and Aldosterone Resistance

Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.