Salt sensitivity of volume and blood pressure in a mouse with globally reduced ENaC γ-subunit expression.

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γmt) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γmt/mt mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γmt/mt mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γmt/mt mice and controls. γmt/mt mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na+ diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γmt/mt mice relative to the controls. Lower blood pressures were variably observed in γmt/mt mice on a high-salt diet compared with the controls. γmt/mt also exhibited reduced diurnal blood pressure variation, a "nondipping" phenotype, on a high-Na+ diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure.NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na+ channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na+ channel in regulating body fluid and blood pressure beyond classical transepithelial Na+ transport mechanisms.

Western diet induces renal artery endothelial stiffening that is dependent on the epithelial Na+ channel.

Consumption of a Western diet (WD) induces central aortic stiffening that contributes to the transmittance of pulsatile blood flow to end organs, including the kidney. Our recent work supports that endothelial epithelial Na+ channel (EnNaC) expression and activation enhances aortic endothelial cell stiffening through reductions in endothelial nitric oxide (NO) synthase (eNOS) and bioavailable NO that result in inflammatory and oxidant responses and perivascular fibrosis. However, the role that EnNaC activation has on endothelial responses in the renal circulation remains unknown. We hypothesized that cell-specific deletion of the α-subunit of EnNaC would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Twenty-eight-week-old female αEnNaC knockout and wild-type mice were fed either mouse chow or WD containing excess fat (46%), sucrose, and fructose (17.5% each). WD feeding increased fat mass, indexes of vascular stiffening in the aorta and renal artery (in vivo pulse wave velocity and ultrasound), and renal endothelial cell stiffening (ex vivo atomic force microscopy). WD further impaired aortic endothelium-dependent relaxation and renal artery compliance (pressure myography) without changes in blood pressure. WD-induced renal arterial stiffening occurred in parallel to attenuated eNOS activation, increased oxidative stress, and aortic and renal perivascular fibrosis. αEnNaC deletion prevented these abnormalities and support a novel mechanism by which WD contributes to renal arterial stiffening that is endothelium and Na+ channel dependent. These results demonstrate that cell-specific EnNaC is important in propagating pulsatility into the renal circulation, generating oxidant stress, reduced bioavailable NO, and renal vessel wall fibrosis and stiffening.

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

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

The Absence of Endothelial Sodium Channel α (αENaC) Reduces Renal Ischemia/Reperfusion Injury.

The epithelial sodium channel (ENaC) has a key role in modulating endothelial cell stiffness and this in turn regulates nitric oxide (NO) synthesis. The physiological relevance of endothelial ENaC in pathological conditions where reduced NO bioavailability plays an essential role remains largely unexplored. Renal ischemia/reperfusion (IR) injury is characterized by vasoconstriction and sustained decrease in renal perfusion that is partially explained by a reduction in NO bioavailability. Therefore, we aimed to explore if an endothelial ENaC deficiency has an impact on the severity of renal injury induced by IR. Male mice with a specific endothelial sodium channel α (αENaC) subunit gene inactivation in the endothelium (endo-αENaCKO) and control littermates were subjected to bilateral renal ischemia of 22 min and were studied after 24 h of reperfusion. In control littermates, renal ischemia induced an increase in plasma creatinine and urea, augmented the kidney injury molecule-1 (Kim-1) and neutrophil gelatinase associated lipocalin-2 (NGAL) mRNA levels, and produced severe tubular injury. The absence of endothelial αENaC expression prevented renal tubular injury and renal dysfunction. Moreover, endo-αENaCKO mice recovered faster from renal hypoxia after the ischemia episode as compared to littermates. In human endothelial cells, pharmacological ENaC inhibition promoted endothelial nitric oxide synthase (eNOS) coupling and activation. Altogether, these data suggest an important role for endothelial αENaC in kidney IR injury through improving eNOS activation and kidney perfusion, thus, preventing ischemic injury.

Early Postnatal Secondhand Smoke Exposure Disrupts Bacterial Clearance and Abolishes Immune Responses in Muco-Obstructive Lung Disease.

Secondhand smoke (SHS) exposure has been linked to the worsening of ongoing lung diseases. However, whether SHS exposure affects the manifestation and natural history of imminent pediatric muco-obstructive airway diseases such as cystic fibrosis remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice to SHS from postnatal day (PND) 3-21 and lung phenotypes were examined at PND22. Although a majority of filtered air (FA)-exposed Scnn1b-Tg+ (FA-Tg+) mice successfully cleared spontaneous bacterial infections by PND22, the SHS-exposed Scnn1b-Tg+ (SHS-Tg+) mice failed to resolve these infections. This defect was associated with suppressed antibacterial defenses, i.e., phagocyte recruitment, IgA secretion, and Muc5b expression. Whereas the FA-Tg+ mice exhibited marked mucus obstruction and Th2 responses, SHS-Tg+ mice displayed a dramatic suppression of these responses. Mechanistically, downregulated expression of IL-33, a stimulator of type II innate lymphoid cells, in lung epithelial cells was associated with suppression of neutrophil recruitment, IgA secretions, Th2 responses, and delayed bacterial clearance in SHS-Tg+ mice. Cessation of SHS exposure for 21 d restored previously suppressed responses, including phagocyte recruitment, IgA secretion, and mucous cell metaplasia. However, in contrast with FA-Tg+ mice, the SHS-Tg+ mice had pronounced epithelial necrosis, alveolar space consolidation, and lymphoid hyperplasia; indicating lagged unfavorable effects of early postnatal SHS exposure in later life. Collectively, our data show that early postnatal SHS exposure reversibly suppresses IL-33 levels in airspaces which, in turn, results in reduced neutrophil recruitment and diminished Th2 response. Our data indicate that household smoking may predispose neonates with muco-obstructive lung disease to bacterial exacerbations.

PPARγ agonist-induced fluid retention depends on αENaC expression in connecting tubules.

BACKGROUND/AIMS: Thiazolidinediones (TZDs, like rosiglitazone (RGZ)) are peroxisome proliferator-activated receptor γ (PPARγ) agonists used to treat type 2 diabetes. Clinical limitations include TZD-induced fluid retention and body weight (BW) increase, which are inhibited by amiloride, an epithelial-sodium channel (ENaC) blocker. RGZ-induced fluid retention is maintained in mice with αENaC knockdown in the collecting duct (CD). Since ENaC in the connecting tubule (CNT) rather than in CD appears to be critical for normal NaCl retention, we aimed to further explore the role of ENaC in CNT in RGZ-induced fluid retention. METHODS: Mice with conditional inactivation of αENaC in both CNT and CD were used (αENaC lox/lox AQP2-Cre; 'αENaC-CNT/CD-KO') and compared with littermate controls (αENaC lox/lox mice; 'WT'). BW was monitored and total body water (TBW) and extracellular fluid volume (ECF) were determined by bioelectrical impedance spectroscopy (BIS) before and after RGZ (320 mg/kg diet for 10 days). RESULTS: On regular NaCl diet, αENaC-CNT/CD-KO had normal BW, TBW, ECF, hematocrit, and plasma Na(+), K(+), and creatinine, associated with an increase in plasma aldosterone compared with WT. Challenging αENaC-CNT/CD-KO with a low NaCl diet unmasked impaired NaCl and K homeostasis, consistent with effective knockdown of αENaC. In WT, RGZ increased BW (+6.1%), TBW (+8.4%) and ECF (+10%), consistent with fluid retention. These changes were significantly attenuated in αENaC-CNT/CD-KO (+3.4, 1.3, and 4.3%). CONCLUSION: Together with the previous studies, the current results are consistent with a role of αENaC in CNT in RGZ-induced fluid retention, which dovetails with the physiological relevance of ENaC in this segment.

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.

Hypoxia-induced inhibition of epithelial Na(+) channels in the lung. Role of Nedd4-2 and the ubiquitin-proteasome pathway.

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the ß-Liddle mouse strain mice carrying a truncation of ß-ENaC C-terminus abolishing the interaction between ß-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated ß-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.

[Extravascular lung water: from genes to telemedicine]. / Acqua extravascolare polmonare: dalla genetica alla telemedicina.

Extravascular lung water is a crucial parameter for the management of many different pathological conditions, especially heart failure. Many modalities can be employed for its evaluation, each with different advantages and limitations. In a near future, we hope that extravascular lung water assessment could be performed by non-invasive wearable devices, allowing remote continuous monitoring of pulmonary congestion. Recently, it has been proposed that some specific genetic variations may be responsible for the heterogeneous individual response to the development of extravascular lung water. These data could be relevant to titrate and personalize both treatments and preventive interventions.

Epithelial sodium channel stiffens the vascular endothelium in vitro and in Liddle mice.

Liddle syndrome, an inherited form of hypertension, is caused by gain-of-function mutations in the epithelial Na(+) channel (ENaC), the principal mediator of Na(+) reabsorption in the kidney. Accordingly, the disease pathology was ascribed to a primary renal mechanism. Whether this is the sole responsible mechanism, however, remains uncertain as dysregulation of ENaC in other tissues may also be involved. Previous work indicates that ENaC in the vascular endothelium is crucial for the regulation of cellular mechanics and thus vascular function. The hormone aldosterone has been shown to concomitantly increase ENaC surface expression and stiffness of the cell cortex in vascular endothelial cells. The latter entails a reduced release of the vasodilator nitric oxide, which eventually leads to an increase in vascular tone and blood pressure. Using atomic force microscopy, we have found a direct correlation between ENaC surface expression and the formation of cortical stiffness in endothelial cells. Stable knockdown of αENaC in endothelial cells evoked a reduced channel surface density and a lower cortical stiffness compared with the mock control. In turn, an increased αENaC expression induced an elevated cortical stiffness. More importantly, using ex vivo preparations from a mouse model for Liddle syndrome, we show that this disorder evokes enhanced ENaC expression and increased cortical stiffness in vascular endothelial cells in situ. We conclude that ENaC in the vascular endothelium determines cellular mechanics and hence might participate in the control of vascular function.

Reduced ENaC activity and blood pressure in mice with genetic knockout of the insulin receptor in the renal collecting duct.

To elucidate the role of the insulin receptor (IR) in collecting duct (CD), we bred mice with IR selectively deleted from CD principal cells using an aquaporin-2 promoter to drive Cre-recombinase expression. Young, adult male knockout (KO) mice had altered plasma and electrolyte homeostasis under high- (HS) and low-sodium (LS) diets, relative to wild-type (WT) littermates. One week of LS feeding led to a significant reduction in urine potassium (K(+)) and sodium (Na(+)) excretion in KO, and a reduction in the ratio of Na(+) to chloride (Cl(-)) in plasma, relative to WT. HS diet (1 wk) increased plasma K(+) and reduced urine Na(+) to Cl(-) ratio in the KO. Furthermore, KO mice had a significantly (P = 0.025) blunted natriuretic response to benzamil, an epithelial sodium channel (ENaC) antagonist. Western blotting of cortex homogenates revealed modestly, but significantly (∼15%), lower band density for the ß-subunit of ENaC in the KO vs. WT mice, with no differences for the α- or γ-subunits. Moreover, blood pressure (BP), measured by radiotelemetry, was significantly lower in KO vs. WT mice under basal conditions (mmHg): 112 ± 5 (WT), 104 ± 2 (KO), P = 0.023. Chronic insulin infusion reduced heart rate in the WT, but not in the KO, and modestly reduced BP in the WT only. Overall, these results support a fundamental role for insulin through its classic receptor in the modulation of electrolyte homeostasis and BP.

Postnatal requirement of the epithelial sodium channel for maintenance of epidermal barrier function.

In skin, the physiological consequence of an epithelial sodium channel (ENaC) deficiency is not obvious directly at birth. Nevertheless, within hours after birth, mice deficient for the alpha-subunit of the highly amiloride-sensitive epithelial sodium channel (alphaENaC/Scnn1a) suffer from a significant increased dehydration. This is characterized by a loss of body weight (by 6% in 6 h) and an increased transepidermal water loss, which is accompanied by a higher skin surface pH in 1-day-old pups. Although early and late differentiation markers, as well as tight junction protein distribution and function, seem unaffected, deficiency of alphaENaC severely disturbs the stratum corneum lipid composition with decreased ceramide and cholesterol levels, and increased pro-barrier lipids, whereas covalently bound lipids are drastically reduced. Ultrastructural analysis revealed morphological changes in the formation of intercellular lamellar lipids and the lamellar body secretion. Extracellular formation of the lamellar lipids proved to be abnormal in the knockouts. In conclusion, ENaC deficiency results in progressive dehydration and, consequently, weight loss due to severe impairment of lipid formation and secretion. Our data demonstrate that ENaC expression is required for the postnatal maintenance of the epidermal barrier function but not for its generation.