Apelin-13 Decreases Epithelial Sodium Channel (ENaC) Expression and Activity in Kidney Collecting Duct Cells.

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. METHODS: mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. RESULTS: Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. CONCLUSION: Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination.

Apelin-13 Regulates Vasopressin-Induced Aquaporin-2 Expression and Trafficking in Kidney Collecting Duct Cells.

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APJ (gene symbol Aplnr) are strongly expressed in magnocellular vasopressinergic neurons suggesting that the apelin/APJ system plays a key role at the central level in regulating salt and water balance by counteracting the antiduretic action of vasopressin (AVP). Likewise, recent studies revealed that apelin exerts opposite effects to those of vasopressin induced on water reabsorption via a direct action on the kidney collecting duct. However, the underlying mechanisms of the peripheral action of apelin are not clearly understood. Here, we thus investigated the role of the apelin/APJ system in the regulation of water balance in the kidney, and more specifically its involvement in modulating the function of aquaporin-2 (AQP2) in the collecting duct. METHODS: Mouse cortical collecting duct cells (mpkCCD) were incubated in the presence of dDAVP and treated with or without apelin-13. Changes in AQP2 expression and localization were determined by immunoblotting and confocal immunofluorescence staining. RESULTS: Herein, we showed that the APJ was present in mpkCCD cells. Treatment of mpkCCD with apelin-13 reduced the cAMP production and antagonized the AVP-induced increase in AQP2 mRNA and protein expressions. Immunofluorescent experiments also revealed that the AVP-induced apical cell surface expression of AQP2, and notably its phosphorylated isoform AQP2-pS269, was considerably reduced following apelin-13 application to mpkCCD cells. CONCLUSION: Our data reinforce the aquaretic role of the apelin/APJ system in the fine regulation of body fluid homeostasis at the kidney level and its physiological opposite action to the antiduretic activity of AVP.

PPARγ-induced stimulation of amiloride-sensitive sodium current in renal collecting duct principal cells is serum and insulin dependent.

BACKGROUND/AIMS: Thiazolidinediones (TZDs), such as rosiglitazone or pioglitazone, are peroxisome proliferator-activated receptor gamma (PPARγ) agonists currently used in the treatment of type 2 diabetes. However, their clinical applicability is limited by common and severe side effects including strong water retention, edema and cardiac stroke. The precise mechanisms leading to these disorders are not clearly understood and remain controversial. While the nature of the disorders due to TZDs points to an increase in ENaC-mediated sodium reabsorption in the aldosterone-sensitive distal nephron, some studies suggested that this channel was not targeted by PPARγ agonists. METHODS: Mouse cortical collecting duct cells were incubated in different types of culture medium and treated with or without rosiglitazone. Transepithelial Na(+) current was measured and the changes in SGK and Nedd4 expression were determined by immunoblotting. RESULTS: Herein we demonstrate that rosiglitazone stimulates the amiloride-sensitive transepithelial sodium current in Collecting Duct Principal Cells after 3h and 24h treatment. This activation was dependent of both serum and insulin in culture medium and was mediated by SGK1/Nedd4-2 pathway stimulation. In these conditions, rosiglitazone induced SGK1 expression, Nedd4-2 phosphorylation and thus abolished ubiquitylation and internalization of ENaC channels. This mechanism explains most of the side effects of thiazolidinediones previously observed in humans and animals. CONCLUSION: Our data show an increase in transepithelial sodium amiloride-sensitive current induced by a PPARγ agonist in presence of serum and insulin, thus confirming some in-vitro and in-vivo experiments while providing explanations for previous conflicting findings.

Functional up-regulation of Nav1.8 sodium channel in Aß afferent fibers subjected to chronic peripheral inflammation.

BACKGROUND: Functional alterations in the properties of Aß afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling Aß-fiber excitability following persistent inflammation. METHODS: Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats. RESULTS: Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated Aß-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aß-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in Aß-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in Aß-fibers contributes to inflammatory pain. CONCLUSIONS: Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of Aß-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.

The chemokine CCL2 increases Nav1.8 sodium channel activity in primary sensory neurons through a Gßγ-dependent mechanism.

Changes in function of voltage-gated sodium channels in nociceptive primary sensory neurons participate in the development of peripheral hyperexcitability that occurs in neuropathic and inflammatory chronic pain conditions. Among them, the tetrodotoxin-resistant (TTX-R) sodium channel Na(v)1.8, primarily expressed by small- and medium-sized dorsal root ganglion (DRG) neurons, substantially contributes to the upstroke of action potential in these neurons. Compelling evidence also revealed that the chemokine CCL2 plays a critical role in chronic pain facilitation via its binding to CCR2 receptors. In this study, we therefore investigated the effects of CCL2 on the density and kinetic properties of TTX-R Na(v)1.8 currents in acutely small/medium dissociated lumbar DRG neurons from naive adult rats. Whole-cell patch-clamp recordings demonstrated that CCL2 concentration-dependently increased TTX-resistant Na(v)1.8 current densities in both small- and medium-diameter sensory neurons. Incubation with CCL2 also shifted the activation and steady-state inactivation curves of Na(v)1.8 in a hyperpolarizing direction in small sensory neurons. No change in the activation and inactivation kinetics was, however, observed in medium-sized nociceptive neurons. Our electrophysiological recordings also demonstrated that the selective CCR2 antagonist INCB3344 [N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide] blocks the potentiation of Na(v)1.8 currents by CCL2 in a concentration-dependent manner. Furthermore, the enhancement in Na(v)1.8 currents was prevented by pretreatment with pertussis toxin (PTX) or gallein (a Gßγ inhibitor), indicating the involvement of Gßγ released from PTX-sensitive G(i/o)-proteins in the cross talk between CCR2 and Na(v)1.8. Together, our data clearly demonstrate that CCL2 may excite primary sensory neurons by acting on the biophysical properties of Na(v)1.8 currents via a CCR2/Gßγ-dependent mechanism.

Modulation of canine cardiac sodium current by Apelin.

Apelin, a ligand of the G protein-coupled putative angiotensin II-like receptor (APJ-R), exerts strong vasodilating, cardiac inotropic and chronotropic actions. Its expression is highly up-regulated during heart failure. Apelin also increases cardiac conduction speed and excitability. While our knowledge of apelin cardiovascular actions is growing, our understanding of the physiological mechanisms behind the cardiac effects remains limited. We tested the effects of apelin on the cardiac sodium current (I(Na)) using patch clamp technique on cardiac myocytes acutely dissociated from dog ventricle. We found that apelin-13 and apelin-17 increased peak I(Na) by 39% and 61% and shifted its mid-activation potential by -6.8+/-0.6 mV and -17+/-1 mV respectively thus increasing channel opening at negative voltage. Apelin also slowed I(Na) recovery from inactivation. The effects of apelin on I(Na) amplitude were linked to activation of protein kinase C. Apelin also increased I(Na) "window" current by up to 600% suggesting that changes in intracellular sodium may contribute to the apelin inotropic effects. Our results reveal for the first time the effects of apelin on I(Na). These effects are likely to modulate cardiac conduction and excitability and may have beneficial antiarrhythmic action in sodium chanelopathies such as Brugada Syndrome where I(Na) amplitude is reduced.

Serotonin decreases alveolar epithelial fluid transport via a direct inhibition of the epithelial sodium channel.

Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin (5-hydroxytryptamine, 5-HT) in vitro. However, whether 5-HT contributes to the decrease of alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of 5-HT on ion and fluid transport across the alveolar epithelium. 5-HT caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial type II cell monolayers, but did not affect Na(+)/K(+) ATPase function. Furthermore, we found that the 5-HT induced inhibition of ion transport across the lung epithelium was receptor independent, as it was not prevented by the blockade of 5-HT2R (5-HT receptor 2), 5-HT3R (5-HT receptor 3), or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester). In addition, the stimulation of 5-HT1R (5-HT receptor 1), 5-HT2R (5-HT receptor 2), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) failed to reproduce the 5-HT effect on amiloride-sensitive sodium transport. We ascertained that 5-HT directly inhibited the function of rat alphabetagamma epithelial sodium channel (ENaC), as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor 5-HT receptors (5-HTR). Exposure of mice to hypoxia for 1 hour induced a 30% increase of 5-HT secretion into the distal airways of mice. Finally, the intratracheal instillation of 5-HT inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Together, these results indicate that 5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with ENaC, and thus can be an endogenous inhibitor of this ion channel.

Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.

Proven infection-related sepsis induces a differential stress response early after ICU admission.

INTRODUCTION: Neuropeptides arginine-vasopressin (AVP), apelin (APL), and stromal-derived factor-1α (SDF-1α) are involved in the dysfunction of the corticotropic axis observed in septic ICU patients. Study aims were: (i) to portray a distinctive stress-related neuro-corticotropic systemic profile of early sepsis, (ii) to propose a combination data score, for aiding ICU physicians in diagnosing sepsis on admission. METHODS: This prospective one-center observational study was carried out in a medical intensive care unit (MICU), tertiary teaching hospital. Seventy-four out of 112 critically ill patients exhibiting systemic inflammatory response syndrome (SIRS) were divided into two groups: proven sepsis and non sepsis, based on post hoc analysis of microbiological criteria and final diagnosis, and compared to healthy volunteers (n = 14). A single blood sampling was performed on admission for measurements of AVP, copeptin, APL, SDF-1α, adrenocorticotropic hormone (ACTH), cortisol baseline and post-stimulation, and procalcitonin (PCT). RESULTS: Blood baseline ACTH/cortisol ratio was lower and copeptin higher in septic vs. nonseptic patients. SDF-1α was further increased in septic patients vs. normal patients. Cortisol baseline, ACTH, PCT, APACHE II and sepsis scores, and shock on admission, were independent predictors of sepsis diagnosis upon admission. Using the three first aforementioned categorical bio-parameters, a probability score for predicting sepsis yielded an area under the Receiver Operating Curve (ROC) curves better than sepsis score or PCT alone (0.903 vs 0.727 and 0.726: P = 0.005 and P < 0.04, respectively). CONCLUSIONS: The stress response of early admitted ICU patients is different in septic vs. non-septic conditions. A proposed combination of variable score analyses will tentatively help in refining bedside diagnostic tools to efficiently diagnose sepsis after further validation.

Role of the C-terminal part of the extracellular domain of the alpha-ENaC in activation by sulfonylurea glibenclamide.

The epithelial sodium channel (ENaC) is regulated by hormones and by other intracellular or extracellular factors. It is activated by the sulfonylurea drug glibenclamide. The activator effect of glibenclamide is fast and reversible and was observed in Xenopus oocytes coexpressing the alpha subunit from human, Xenopus, or guinea pig (but not rat) with betagamma-rat ENaC subunits. The mechanism of this effect is not yet well understood. We hypothesize that the extracellular loop of ENaC plays a major role in this activation. Mutants and chimeras of alpha subunits harboring different parts of the rat and guinea pig alpha-subunit extracellular loops were generated and coexpressed with betagamma-rat subunits in Xenopus oocytes. The effect of glibenclamide on ENaC activity was measured using two-electrode voltage-clamp technique. The alpha-rat ENaC chimera containing the C-terminal part of the extracellular loop of the alpha-guinea pig ENaC was significantly stimulated by glibenclamide (1.26-fold), whereas the rat-alpha combination was not activated by this sulfonylurea. Mutagenesis of specific residues on the rat alpha subunit did not generate channels sensitive to glibenclamide, suggesting that the overall structure of the extracellular loop is required for activation of the channel by this drug. These results support the hypothesis of the existence of a role played by the last 100 amino acids of the extracellular loop and confirm that the ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases.

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

Epithelial sodium channel: a ligand-gated channel?

The epithelial sodium channel (ENaC) is a key component of the transepithelial Na+ transport. In epithelia, it is responsible for the maintenance of Na+ balance (which in turn controls extracellular fluid volume and arterial blood pressure) and the regulation of airway surface fluid. While the regulation of channel synthesis and surface density have been well described, the control of channel opening is still poorly understood. The channel has a large extracellular domain of as yet unknown function; a number of extracellular factors have been shown to modulate ENaC activity, including extracellular Na+ itself (through a phenomenon called 'self-inhibition'), several other organic or inorganic cations, which seem to interfere with self-inhibition, and serine proteases. Although a direct interaction with the extracellular domain of ENaC has not yet been demonstrated for each of these modulators, the available data strongly suggest that ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.

Regulatory interdependence of cloned epithelial Na+ channels and P2X receptors.

Epithelial Na+ channels (ENaC) coexist with a family of ATP-gated ion channels known as P2X receptors in the renal collecting duct. Although ENaC is itself insensitive to extracellular ATP, tubular perfusion of ATP can modify the activity of ENaC. To investigate a possible regulatory relationship between P2X receptors and ENaC, coexpression studies were performed in Xenopus oocytes. ENaC generated a persistent inward Na+ current that was sensitive to the channel blocker amiloride (I(am-s)). Exogenous ATP transiently activated all cloned isoforms of P2X receptors, which in some cases irreversibly inhibited I(am-s). The degree of inhibition depended on the P2X receptor subtype present. Activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptor subtypes inhibited I(am-s), whereas activation of P2X1, P2X3, and P2X5 receptors had no significant effect. The degree of inhibition of I(am-s) correlated positively with the amount of ionic charge conducted by P2X receptor subtypes. ENaC inhibition required Na+ influx through I(am-s)-inhibiting P2X ion channels but also Ca2+ influx through P2X4 and P2X(4/6) ion channels. P2X-mediated inhibition of I(am-s) was found to be due to retrieval of ENaC from the plasma membrane. Maximum amplitudes of ATP-evoked P2X-mediated currents (I(ATP)) were significantly increased for P2X2, P2X(2/6), and P2X5 receptor subtypes after coexpression of ENaC. The increase in I(ATP) was due to increased levels of plasma membrane-bound P2X receptor protein, suggesting that ENaC modulates protein trafficking. In summary, ENaC was downregulated by the activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptors. Conversely, ENaC increased the plasma membrane expression of P2X2, P2X(2/6), and P2X5 receptors.

Dual effect of temperature on the human epithelial Na+ channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na+], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (Po, estimated from the number of open channels N.Po, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from approximately 30 to approximately 15 degrees C. The effects of temperature on the single-channel conductance and Po explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.