Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Stereoselective block of hERG channel by (S)-methadone and QT interval prolongation in CYP2B6 slow metabolizers.

Methadone inhibits the cardiac potassium channel hERG and can cause a prolonged QT interval. Methadone is chiral but its therapeutic activity is mainly due to (R)-methadone. Whole-cell patch-clamp experiments using cells expressing hERG showed that (S)-methadone blocked the hERG current 3.5-fold more potently than (R)-methadone (IC50s (half-maximal inhibitory concentrations) at 37 degrees C: 2 and 7 microM). As CYP2B6 slow metabolizer (SM) status results in a reduced ability to metabolize (S)-methadone, electrocardiograms, CYP2B6 genotypes, and (R)- and (S)-methadone plasma concentrations were obtained for 179 patients receiving (R,S)-methadone. The mean heart-rate-corrected QT (QTc) was higher in CYP2B6 SMs (*6/*6 genotype; 439+/-25 ms; n=11) than in extensive metabolizers (non *6/*6; 421+/-25 ms; n=168; P=0.017). CYP2B6 SM status was associated with an increased risk of prolonged QTc (odds ratio=4.5, 95% confidence interval=1.2-17.7; P=0.03). This study reports the first genetic factor implicated in methadone metabolism that may increase the risk of cardiac arrhythmias and sudden death. This risk could be reduced by the administration of (R)-methadone.

CARDIAC AND MUSCLE CHANNELOPATHIES: ROLES AND REGULATION OF VOLTAGE-GATED SODIUM CHANNELS

Abstract :The contraction of the heart or skeletal muscles is mainly due to the propagation, through excitable cells, of an electrical influx called action potential (AP). The AP results from the sequential opening of ion channels that generate inward or outward currents through the cell membrane. Among all the channels involved, the voltage-gated sodium channel is responsible for the rising phase of the action potential. Ten genes encode the different isoforms of these channels (from Nav1.1 to Nav1.9 and an atypical channel named NavX). Nav1.4 and Nav1.5 are the main skeletal muscle and cardiac sodium channels respectively. Their importance for muscle and heart function has been highlighted by the description of mutations in their encoding genes SCN4A and SCNSA. They lead respectively to neuromuscular disorders such as myotonia or paralysis (for Nav1.4), and to cardiac arrhythmias that can deteriorate into sudden cardiac death (for Nav1.5).The general aim of my PhD work has been to study diseases linked with channels dysfunction, also called channelopathies. In that purpose, I investigated the function and the regulation of the muscle and cardiac voltage-gated sodium channels. During the two first studies, I characterized the effects of two mutations affecting Nav1.4 and Nav1.5 function. I used the HEK293 model cells to express wild-type or mutant channels and then studied their biophysical properties with the patch-clamp technique, in whole cell configuration. We found that the SCN4A mutation produced complex alterations of the muscle sodium channel function, that could explain the myotonic phenotype described in patients carrying the mutation. In the second study, the index case was an heterozygous carrier of a SCNSA mutation that leads to a "loss of function" of the channel. The decreased sodium current measured with mutated Nay 1.5 channels, at physiological temperature, was a one of the factors that could explain the observed Brugada syndrome. The last project aimed at identifying a new potential protein interacting with the cardiac sodium channel. We found that the protein SAP97 binds the three last amino-acids of the C-terminus of Na,, 1.5. Our results also indicated that silencing the expression of SAP97 in HEK293 cells decreased the sodium current. Sodium channels lacking their three last residues also produced a reduced INa. These preliminary results suggest that SAP97 is implicated in the regulation of sodium channel. Whether this effect is direct or imply the action of an adaptor protein remains to be investigated. Moreover, our group has previously shown that Nav1.5 channels are localized to lateral membranes of cardiomyocytes by the dystrophin multiprotein complex (DMC). This suggests that sodium channels are distributed in, at least, two different pools: one targeted at lateral membranes by DMC and the other at intercalated discs by another protein such as SAP97.These studies reveal that cardiac and muscle diseases may result from ion channel mutations but also from regulatory proteins affecting their regulation.Résumé :La contraction des muscles et du coeur est principalement due à la propagation, à travers les cellules excitables, d'un stimulus électrique appelé potentiel d'action (PA). C'est l'ouverture séquentielle de plusieurs canaux ioniques transmembranaires, permettant l'entrée ou la sortie d'ions dans la cellule, qui est à l'origine de ce PA. Parmi tous les canaux ioniques impliqués dans ce processus, les canaux sodiques dépendant du voltage sont responsables de la première phase du potentiel d'action. Les différentes isoformes de ces canaux (de Nav1.1 à Nav1.9 et NavX) sont codées par dix gènes distincts. Nav1.4 et Nav1.5 sont les principaux variants exprimés respectivement dans le muscle et le coeur. Plusieurs mutations ont été décrites dans les gènes qui codent pour ces deux canaux: SCN4A (pour Nav1.4) et SCNSA (pour Nav1.5). Elles sont impliquées dans des pathologies neuromusculaires telles que des paralysies ou myotonies (SCN4A) ou des arythmies cardiaques pouvant conduire à la mort subite cardiaque (SCNSA).Mon travail de thèse a consisté à étudier les maladies liées aux dysfonctionnements de ces canaux, aussi appelées canalopathies. J'ai ainsi analysé la fonction et la régulation des canaux sodiques dépendant du voltage dans le muscle squelettique et le coeur. A travers les deux premières études, j'ai ainsi pu examiner les conséquences de deux mutations affectant respectivement les canaux Nav1.4 et Nav1.5. Les canaux sauvages ou mutants ont été exprimés dans des cellules HEK293 afin de caractériser leurs propriétés biophysiques par la technique du patch clamp en configuration cellule entière. Nous avons pu déterminer que la mutation trouvée dans le gène SCN4A engendrait des modifications importantes de la fonction du canal musculaire. Ces altérations fournissent des indications nous permettant d'expliquer certains aspects de la myotonie observée chez les membres de la famille étudiée. Le patient présenté dans la deuxième étude était hétérozygote pour la mutation identifiée dans le gène SCNSA. La perte de fonction des canaux Nav1.5 ainsi engendrée, a été observée lors d'analyses à températures physiologiques. Elle représente l'un des éléments pouvant potentiellement expliquer le syndrome de Brugada du patient. La dernière étude a consisté à identifier une nouvelle protéine impliquée dans la régulation du canal sodique cardiaque. Nos expériences ont démontré que les trois derniers acides aminés de la partie C-terminale de Nav1.5 pouvaient interagir avec la protéine SAP97. Lorsque que l'expression de la SAP97 est réduite dans les cellules HEK293, cela induit une baisse importante du courant sodique. De même, les canaux tronqués de leurs trois derniers acides aminés génèrent un flux ionique réduit. Ces résultats préliminaires suggèrent que SAP97 est peut-être impliquée dans la régulation du canal Na,,1.5. Des expériences complémentaires permettront de déterminer si ces deux protéines interagissent directement ou si une protéine adaptatrice est nécessaire. De plus, nous avons préalablement montré que les canaux Nav1.5 étaient localisés au niveau de la membrane latérale des cardiomyocytes par le complexe multiprotéique de la dystrophine (DMC). Ceci suggère que les canaux sodiques peuvent être distribués dans un minimum de deux pools, l'un ciblé aux membranes latérales pax le DMC et l'autre dirigé vers les disques intercalaires par des protéines telles que SAP97.L'ensemble de ces études met en évidence que certaines maladies musculaires et cardiaques peuvent être la conséquence directe de mutations de canaux ioniques, mais que l'action de protéines auxiliaires peut aussi affecter leur fonction.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Regulation of sodium transport in the cortical collecting duct : physiological role of GILZ in vitro and in vivo : characterisation of Na+ transport in the mCCDcl1 cell line

SUMMARY Regulation of sodium excretion by the kidney is a key mechanism in the long term regulation of blood pressure, and when altered it constitutes a risk factor for the appearance of arterial hypertension. Aldosterone, which secretion depends upon salt intake in the diet, is a steroid hormone that regulates sodium reabsorption in the distal part of the nephron (functional unit of the kidney) by modulating gene transcription. It has been shown that it can act synergistically with the peptidic hormone insulin through the interaction of their signalisation pathways. Our work consisted of two distinct parts: 1) the in vitro and in vivo characterisation of Glucocorticoid-Induced Leucine Zipper (GILZ) (an aldosterone-induced gene) mechanism of action; 2) the in vitro characterisation of insulin mechanism of action and its interaction with aldosterone. GILZ mRNA, coded by the TSC22D3 gene, is strongly induced by aldosterone in the cell line of principal cells of the cortical collecting duct (CCD) mpkCCDc14, suggesting that GILZ is a mediator of aldosterone response. Co-expression of GILZ and the amiloride-sensitive epithelial sodium channel ENaC in vitro in the Xenopus oocyte expression system showed that GILZ has no direct effect on the ENaC-mediated Na+ current in basal conditions. To define the role of GILZ in the kidney and in other organs (colon, heart, skin, etc.), a conditional knock-out mouse is being produced and will allow the in vivo study of its role. Previous data showed that insulin induced a transepithelial sodium transport at supraphysiological concentrations. Insulin and the insulin-like growth factor 1 (IGF-1) are able to bind to each other receptor with an affinity 50 to 100 times lower than to their cognate receptor. Our starting hypothesis was that the insulin effect observed at these supraphysiological concentrations is actually mediated by the IGF receptor type 1 (IGF-1R). In a new cell line that presents all the characteristics of the principal cells of the CCD (mCCDc11) we have shown that both insulin and IGF-1 induce a physiologically significant increase of Na+ transport through the activation of IGF-1R. Aldosterone and insulin/IGF-1 have an additive effect on Na+ transport, through the activation of the PI3-kinase (PI3-K) pathway and the phosphorylation of the serum- and glucocorticoid-induced kinase 1 (Sgk1) by the IGF-1R, and the induction of Sgk1 expression by aldosterone. Thus, Sgk1 integrates IGF-1/insulin and aldosterone effects. We suggest that IGF-1 is physiologically relevant in the modulation of sodium balance, while insulin can only regulate Na+ transport at supraphysiological conditions. Both hormones would bind to the IGF-1R and induce Na+ transport by activating the PI3-K PDK1/2 - Sgk1 pathway. We have shown for the first time that Sgk1 is expressed and phosphorylated in principal cells of the CCD in basal conditions, although the mechanism that maintains Sgk1 phosphorylation is not known. This new role for IGF-1 suggests that it could be a salt susceptibility gene. In effect, IGF-1 stimulates Na+ and water transport in the kidney in vivo. Moreover, 35 % of the acromegalic patients (overproduction of growth hormone and IGF-1) are hypertensives (higher proportion than in normal population), and genetic analysis suggest a link between the IGF-1 gene locus and blood pressure. RÉSUMÉ La régulation de l'excrétion rénale de sodium (Na+) joue un rôle principal dans le contrôle à long terme de la pression sanguine, et ses altérations constituent un facteur de risque de l'apparition d'une hypertension artérielle. L'aldosterone, dont la sécrétion dépend de l'apport en sel dans la diète, est une hormone stéroïdienne qui régule la réabsorption de Na+ dans la partie distale du nephron (unité fonctionnelle du rein) en contrôlant la transcription de gènes. Elle peut agir de façon synergistique avec l'hormone peptidique insuline, probablement via l'interaction de leurs voies de signalisation cellulaire. Le but de notre travail comportait deux volets: 1) caractériser in vitro et in vivo le mécanisme d'action du Glucocorticoid Induced Leucine Zipper (GILZ) (un gène induit par l'aldosterone); 2) caractériser in vitro le mécanisme d'action de l'insuline et son interaction avec l'aldosterone. L'ARNm de GILZ, codé par le gène TSC22D3, est induit par l'aldosterone dans la lignée cellulaire de cellules principales du tubule collecteur cortical (CCD) mpkCCDc14, suggérant que GILZ est un médiateur potentiel de la réponse à l'aldosterone. La co-expression in vitro de GILZ et du canal à Na+ sensible à l'amiloride ENaC dans le système d'expression de l'oocyte de Xénope a montré que GILZ n'a pas d'effet sur les courants sodiques véhiculées par ENaC en conditions basales. Une souris knock-out conditionnelle de GILZ est en train d'être produite et permettra l'étude in vivo de son rôle dans le rein et d'autres organes. Des expériences préliminaires ont montré que l'insuline induit un transport transépithelial de Na+ à des concentrations supraphysiologiques. L'insuline et l'insulin-like growth factor 1 (IGF-1) peuvent se lier à leurs récepteurs réciproques avec une affinité 50 à 100 fois moindre qu'à leur propre récepteur. Nous avons donc proposé que l'effet de l'insuline soit médié par le récepteur à l'IGF type 1 (IGF-1R). Dans une nouvelle lignée cellulaire qui présente toutes les caractéristiques des cellules principales du CCD (mCCDc11) nous avons montré que les deux hormones induisent une augmentation physiologiquement significative du transport du Na+ par l'activation des IGF-1 R. Aldosterone et insuline/IGF-1 ont un effet additif sur le transport de Na+, via l'activation de la voie de la PI3-kinase et la phosphorylation de la serum- and glucocorticoid-induced kinase 1 (Sgk1) par l'IGF-1R, dont l'expression est induite par l'aldosterone. Sgk1 intègre les effets de l'insuline et l'aldosterone. Nous proposons que l'IGF-1 joue un rôle dans la modulation physiologique de la balance sodique, tandis que l'insuline régule le transport de Na+ à des concentrations supraphysiologiques. Les deux hormones agissent en se liant à l'IGF-1R et induisent le transport de Na+ en activant la cascade de signalisation PI3-K - PDK1/2 - Sgk1. Nous avons montré pour la première fois que Sgk1 est exprimée et phosphorylée dans des conditions basales dans les cellules principales du CCD, mais le mécanisme qui maintient sa phosphorylation n'est pas connu. Ce nouveau rôle pour l'IGF-1 suggère qu'il pourrait être un gène impliqué de susceptibilité au sel. Aussi, l'IGF-1 stimule le transport rénal de Na+ in vivo. De plus, 35 % des patients atteints d'acromégalie (surproduction d'hormone de croissance et d'IGF-1) sont hypertensifs (prévalence plus élevée que la population normale), et des analyses génétiques suggèrent un lien entre le locus du gène de l'IGF-1 et la pression sanguine. RÉSUMÉ GRAND PUBLIC Nos ancêtres se sont génétiquement adaptés pendant des centaines de millénaires à un environnement pauvre en sel (chlorure de sodium) dans la savane équatoriale, où ils consommaient moins de 0,1 gramme de sel par jour. On a commencé à ajouter du sel aux aliments avec l'apparition de l'agriculture (il y a 5000 à 10000 années), et aujourd'hui une diète omnivore, qui inclut des plats préparés, contient plusieurs fois la quantité de sodium nécessaire pour notre fonction physiologique normale (environ 10 grammes par jour). Le corps garde sa concentration constante dans le sang en s'adaptant à une consommation très variable de sel. Pour ceci, il module son excrétion soit directement, soit en sécrétant des hormones régulatrices. Le rein joue un rôle principal dans cette régulation puisque l'excrétion urinaire de sel change selon la diète et peut aller d'une quantité dérisoire à plus de 36 grammes par jour. L'attention qu'on prête au sel est liée à sa relation avec l'hypertension essentielle. Ainsi, le contrôle rénal de l'excrétion de sodium et d'eau est le principal mécanisme dans la régulation de la pression sanguine, et une ingestion excessive de sel pourrait être l'un des facteurs-clé déclenchant l'apparition d'un phénotype hypertensif. L'hormone aldosterone diminue l'excrétion de sodium par le rein en modulant l'expression de gènes qui pourraient être impliqués dans la sensibilité au sel. Dans une lignée cellulaire de rein l'expression du gène TSC22D3, qui se traduit en la protéine Glucocorticoid Induced Leucine Zipper (GILZ), est fortement induite par l'aldosterone. Ceci suggère que GILZ est un médiateur potentiel de l'effet de l'aldosterone, et pourrait être impliqué dans la sensibilité au sel. Pour analyser la fonction de GILZ dans le rein plusieurs approches ont été utilisées. Par exemple, une souris dans laquelle GILZ est spécifiquement inactivé dans le rein est en train d'être produite et permettra l'étude du rôle de GILZ dans l'organisme. De plus, on a montré que GILZ, en conditions basales, n'a pas d'effet direct sur la protéine transportant le sodium à travers la membrane des cellules, le canal sodique épithélial ENaC. On a aussi essayé de trouver des protéines qui interagissent directement avec GILZ utilisant une technique appelée du « double-hybride dans la levure », mais aucun candidat n'a émergé. Des études ont montré que, à de hautes concentrations, l'insuline peut aussi diminuer l'excrétion de sodium. A ces concentrations, elle peut activer son récepteur spécifique, mais aussi le récepteur d'une autre hormone, l'Insulin-Like Growth Factor 1 (IGF-1). En plus, l'infusion d'IGF-1 augmente la rétention rénale de sodium et d'eau, et des mutations du gène codant pour l'IGF-1 sont liées aux différents niveaux de pression sanguine. On a utilisé une nouvelle lignée cellulaire de rein développée dans notre laboratoire, appelée mCCDc11, pour analyser l'importance relative des deux hormones dans l'induction du transport de sodium. On a montré que les deux hormones induisent une augmentation significative du transport de sodium par l'activation de récepteurs à l'IGF-1 et non du récepteur à l'insuline. On a montré qu'à l'intérieur de la cellule leur activation induit une augmentation du transport sodique par le biais du canal ENaC en modifiant la quantité de phosphates fixés sur la protéine Serumand Glucocorticoid-induced Kinase 1 (Sgk1). On a finalement montré que l'IGF-1 et l'aldosterone ont un effet additif sur le transport de sodium en agissant toutes les deux sur Sgk1, qui intègre leurs effets dans le contrôle du transport de sodium dans le rein.

The metabolic syndrome in hypertension: European society of hypertension position statement.

The metabolic syndrome considerably increases the risk of cardiovascular and renal events in hypertension. It has been associated with a wide range of classical and new cardiovascular risk factors as well as with early signs of subclinical cardiovascular and renal damage. Obesity and insulin resistance, beside a constellation of independent factors, which include molecules of hepatic, vascular, and immunologic origin with proinflammatory properties, have been implicated in the pathogenesis. The close relationships among the different components of the syndrome and their associated disturbances make it difficult to understand what the underlying causes and consequences are. At each of these key points, insulin resistance and obesity/proinflammatory molecules, interaction of demographics, lifestyle, genetic factors, and environmental fetal programming results in the final phenotype. High prevalence of end-organ damage and poor prognosis has been demonstrated in a large number of cross-sectional and a few number of prospective studies. The objective of treatment is both to reduce the high risk of a cardiovascular or a renal event and to prevent the much greater chance that metabolic syndrome patients have to develop type 2 diabetes or hypertension. Treatment consists in the opposition to the underlying mechanisms of the metabolic syndrome, adopting lifestyle interventions that effectively reduce visceral obesity with or without the use of drugs that oppose the development of insulin resistance or body weight gain. Treatment of the individual components of the syndrome is also necessary. Concerning blood pressure control, it should be based on lifestyle changes, diet, and physical exercise, which allows for weight reduction and improves muscular blood flow. When antihypertensive drugs are necessary, angiotensin-converting enzyme inhibitors, angiotensin II-AT1 receptor blockers, or even calcium channel blockers are preferable over diuretics and classical beta-blockers in monotherapy, if no compelling indications are present for its use. If a combination of drugs is required, low-dose diuretics can be used. A combination of thiazide diuretics and beta-blockers should be avoided.

ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

Sodium and potassium balance depends on αENaC expression in connecting tubule.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.

Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited.

Transcription and translation require a high concentration of potassium across the entire tree of life. The conservation of a high intracellular potassium was an absolute requirement for the evolution of life on Earth. This was achieved by the interplay of P- and V-ATPases that can set up electrochemical gradients across the cell membrane, an energetically costly process requiring the synthesis of ATP by F-ATPases. In animals, the control of an extracellular compartment was achieved by the emergence of multicellular organisms able to produce tight epithelial barriers creating a stable extracellular milieu. Finally, the adaptation to a terrestrian environment was achieved by the evolution of distinct regulatory pathways allowing salt and water conservation. In this review we emphasize the critical and dual role of Na(+)-K(+)-ATPase in the control of the ionic composition of the extracellular fluid and the renin-angiotensin-aldosterone system (RAAS) in salt and water conservation in vertebrates. The action of aldosterone on transepithelial sodium transport by activation of the epithelial sodium channel (ENaC) at the apical membrane and that of Na(+)-K(+)-ATPase at the basolateral membrane may have evolved in lungfish before the emergence of tetrapods. Finally, we discuss the implication of RAAS in the origin of the present pandemia of hypertension and its associated cardiovascular diseases.

Phenotypical analysis of mice deficient for the channel activating protease CAP2

Channel activating proteases (CAP) are membrane-bound serine proteases that have been identified as in vitro activators of the epithelial sodium channel (ENaC). Two of them are mainly studied in the laboratory. CAP1/Prss8 was previously shown implicated in colonic sodium homeostasis in vivo. In the first part of this thesis, we generated and characterized mice deficient for CAP2/Tmprss4. The mice are healthy and viable, and they do not show any obvious phenotype. We investigated ENaC activity and expression under regular and sodium- deficient diet, and we could demonstrate that CAP2 is not a major regulator of sodium homeostasis in vivo. We next studied whether CAP2 is implicated in potassium homeostasis. We detected a strong gender-dependency when CAP2 knock-out mice were put under a potassium-deficient diet. We showed in male mice an implication of CAP2 in the regulation of the colonic H+, K+- ATPase, and we propose an implication of membrane-associated progesterone receptors and their binding partners, as well as a possible cleavage-mediated glucocorticoid receptor signalling. We studied the possible interaction between CAPI and CAP2 by generating and characterizing two different mouse study groups, displaying different hypomorphic mutations in the CAPI gene, and deficient for CAP2. We demonstrate that balanced expression of CAPI and CAP2 is required for maintainance of skin integrity and for normal placental development. As CAPI knock-out embryos die due to a placental failure, the additional combined deletion of CAP2 resulted in survival until birth. We could evidence that CAPI and CAP2 are implicated in the same signalling pathway as proposed in cancer studies at the level of the placenta, implicating integrin a5, ERK, AKT, E- and N-cadherin. Furthermore, we investigated whether CAPI is implicated in the pathogenesis and susceptibility to experimental chronic colitis in a mutant rat model. By giving CAPI mutant rats Dextran sodium sulfate, we induced chronic inflammation of the colon, and we highlighted the protective role of CAPI at the histopathological and clinical levels. In conclusion, we showed that CAP2 is not a major regulator of ENaC-mediated sodium homeostasis in vivo, but rather a regulator of potassium homeostasis in a gender-dependent manner implicating the colonic H+, K+-ATPase, membrane progesterone receptors, and the glucocorticoid receptor. We have investigated whether CAPI and CAP2 interact at the functional level, and we show that a balanced expression of CAPI and CAP2 is required in the skin, but also in the placenta. Imbalanced expression of CAPI and CAP2 leads to impaired EMT-associated signalling. We have studied whether CAPI is implicated in the pathogenesis and susceptibility to chronic colitis, and we demonstrated the protective role of CAPI in distal colon. -- Les protéases activatrices de canal (CAP) sont des protéases à serine attachées à la membrane qui ont été identifiées comme activateurs in vitro du canal sodique épithélial (ENaC). Deux de ces protéases sont principalement étudiées dans le laboratoire. CAP1/Prss8 a été identifié préalablement comme impliqué dans l'homéostasie du sodium in vivo au niveau du côlon. Dans la première partie de cette thèse, nous avons généré et caractérisé des souris déficientes pour CAP2/Tmprss4. Les souris sont en bonne santé et viables, et elles ne présentent pas de phénotype visible. Nous avons étudié l'activité et l'expression d'ENaC sous diète normale et déficiente en sodium, et nous avons démontré que CAP2 n'est pas un régulateur essentiel de l'homéostasie sodique in vivo. Nous avons ensuite étudié si CAP2 est impliqué dans l'homéostasie du potassium. Nous avons détecté une forte dépendance du sexe lorsque les souris knock-out pour CAP2 étaient placées sous diète déficiente en potassium. Nous avons démontré dans les souris mâles une implication de CAP2 dans la régulation de la H+, K+- ATPase colonique, des récepteurs membranaires à la progestérone et de leur partenaires de liaison, ainsi que dans la possible signalisation médiée par le clivage du récepteur aux glucocorticoïdes. Nous avons étudié l'interaction possible entre CAPI et CAP2 en générant et en caractérisant deux groupes d'étude de souris différents, porteurs de différentes mutations hypomorphiques dans le gène de CAPI, et déficients pour CAP2. Nous avons pu montrer qu'une expression équilibrée de CAPI et CAP2 est requise pour le maintien de l'intégrité de la peau et pour le développement normal du placenta. Les embryons knock-out pour CAPI meurent suite à une défaillance placentaire, et la délétion additionnelle et combinée de CAP2 permet la survie jusqu'à la naissance. Nous supposons que CAPI et CAP2 sont impliqués dans la même voie de signalisation au niveau du placenta que celle proposée dans les études de cancer, impliquant l'intégrine a5, ERK, AKT, E- et N-cadhérine. De plus, nous avons étudié si CAPI est impliqué dans la pathogenèse et la susceptibilité de colite chronique expérimentale dans un modèle de rat mutant. En administrant aux rats mutants pour CAPI du Dextran sodium sulfate, nous avons induit une inflammation chronique du côlon, et nous avons pu mettre en évidence le rôle protecteur de CAPI au niveau histopathologique et au niveau clinique. En conclusion, nous avons démontré que CAP2 n'est pas un régulateur essentiel de l'homéostasie sodique médiée par ENaC in vivo, mais plutôt de l'homéostasie potassique d'une manière dépendante du sexe et impliquant la H+, K+-ATPase colonique, les récepteurs membranaires à la progestérone et le récepteur aux glucocorticoïdes. Nous avons étudié si CAPI et CAP2 interagissent au niveau fonctionnel, et nous avons montré qu'une expression équilibrée entre CAPI et CAP2 est requise dans la peau et le placenta. L'expression déséquilibrée de CAPI et CAP2 mène à une altération de la signalisation associée à l'EMT. Nous avons étudié si CAPI est impliqué dans la pathogenèse et la susceptibilité de colite chronique expérimentale, et nous avons démontré le rôle protecteur de CAPI dans le côlon distal.

Effect of felodipine on myocardial and renal injury induced by aldosterone-high salt hypertension in uninephrectomized rats

It has been recently shown that calcium channel blockers might have a protective effect on cardiac fibrogenesis induced by aldosterone. The objective of this study was to evaluate the protective effect of felodipine, a dihydropyridine calcium channel blocker, against heart and kidney damage caused by aldosterone-high sodium intake in uninephrectomized rats. Wistar rats were divided into three groups: CNEP (uninephrectomized + 1% NaCl in the drinking water, N = 9); ALDO (same as CNEP group plus continuous infusion of 0.75 µg/h aldosterone, N = 12); ALDOF (same as ALDO group plus 30 mg·kg-1·day-1 felodipine in the drinking water, N = 10). All results were compared with those of age-matched, untreated rats (CTL group, N = 10). After 6 weeks, tail cuff blood pressure was recorded and the rats were killed for histological analysis. Blood pressure (mmHg) was significantly elevated (P < 0.05) in ALDO (180 ± 20) and ALDOF (168 ± 13) compared to CTL (123 ± 12) and CNEP (134 ± 13). Heart damage (lesion scores - median and interquartile range) was 7.0 (5.5-8.0) in ALDO and was fully prevented in ALDOF (1.5; 1.0-2.0). Also, left ventricular collagen volume fraction (%) in ALDOF (2.9 ± 0.5) was similar to CTL (2.9 ± 0.5) and CNEP (3.4 ± 0.4) and decreased compared to ALDO (5.1 ± 1.6). Felodipine partially prevented kidney injury since the damage score for ALDOF (2.0; 2.0-3.0) was significantly decreased compared to ALDO (7.5; 4.0-10.5), although higher than CTL (null score). Felodipine has a protective effect on the myocardium and kidney as evidenced by decreased perivascular inflammation, myocardial necrosis and fibrosis.

Immunopharmacology: utilizing antibodies as ion channel modulators

Development of the patch clamp technique by the Nobel Prize winners Bert Sakmann and Erwin Neher led to huge advances in ion channel research. Their work laid the foundations and revolutionized electrophysiological studies of cells and ion channels. These ion channels underlie many basic cellular physiological processes and, therefore, are key therapeutic targets for pharmaceutical companies. However, current pharmacological strategies are hampered by the lack of specific ion channel blockers. Intense research and development programs are now actively employing antibodies to target ion channels in various formats. This review discusses the use of ion channel antibodies and their associated small molecules as pharmacological tools, termed immunopharmacology. In addition, we will review some recent studies looking into clinical applications of immunopharmacology and intrabodies.

Effect of felodipine on myocardial and renal injury induced by aldosterone-high salt hypertension in uninephrectomized rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Investigation of the Effects of a1-Adrenoceptor Antagonism and L-Type Calcium Channel Blockade on Ejaculation and Vas Deferens and Seminal Vesicle Contractility In Vitro

Introduction. Premature ejaculation is one of the most common male sexual dysfunctions. Current pharmacological treatments involve reduction in penile sensitivity by local anesthetics or increase of ejaculatory threshold by selective serotonin reuptake inhibitors. a1-Adrenoceptors (a1-ARs) and L-type calcium channels are expressed in the smooth muscles of the male reproductive tract, and their activations play an important role in the physiological events involved in the seminal emission phase of ejaculation.Aim. To evaluate if the inhibition of the contractility of the vas deferens and seminal vesicle by alpha(1)-AR antagonism or the L-type calcium channel blockade can delay ejaculation.Methods. The effects of the alpha(1)-AR antagonist tamsulosin and of the L-type calcium channel blockers, nifedipine and (S)-(+)-niguldipine, on contractions induced by norepinephrine in the rat vas deferens and seminal vesicles in vitro and on the ejaculation latency of male rats in behavioral mating tests were evaluated.Main Outcome Measure. Tension development of vas deferens and seminal vesicles in response to norepinephrine in vitro and behavioral mating parameters were quantified.Results. Tension development of vas deferens and seminal vesicle to alpha(1)-AR activation was significantly inhibited by tamsulosin, nifedipine, and (S)-(+)-niguldipine. Tamsulosin displayed insurmountable antagonism of contractions induced by norepinephrine in the rat vas deferens and seminal vesicle. Ejaculation latency of male rats was not modified by tamsulosin, nifedipine, or (S)-(+)-niguldipine; however, both the number and weight of the seminal plugs recovered from female rats mated with male rats treated with tamsulosin were significantly reduced.Conclusion. Seminal emission impairment by inhibition of vas deferens or seminal vesicle contractility by L-type calcium channel blockade or alpha(1)-AR antagonism is not able to delay the ejaculation. de Almeida Kiguti LR and Pupo AS. Investigation of the effects of alpha(1)-adrenoceptor antagonism and L-type calcium channel blockade on ejaculation and vas deferens and seminal vesicle contractility in vitro. J Sex Med 2012; 9: 159-168.

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.