122 research outputs found
Role of serine proteases in Na+ and K+ handling in the kidney
No full textAbstract
Hypertension affects nearly a billion people worldwide, and it is influenced by both genetic and environmental factors. A high Na+ and low K+ diet, high saturated and low unsaturated fats, physical inactivity, heavy alcohol and tobacco use, and obesity are all known risk factors for hypertension. There is evidence that genetic factors play a role in the anti-hypertensive effect of dietary K+ intake. Our goal was to investigate and characterize the genetic factors that influence blood pressure in response to K+ intake. Epithelial Na+ channel (ENaC) plays a vital role in Na+ reabsorption along the distal nephron and drive K+ secretion via an electrochemical gradient, thereby maintaining extracellular·fluid volume and blood pressure. NR3Cl, GRXCRI, HPN, ATP12A, CAPI, CAP2, CAP3, !MPRSS3, and
TMPRSS9 were discovered to be associated with K+ sensitivity in human subjects using unbiased and targeted-gene approaches. GWAS (unbiased approach) revealed two genes, TMPRSS3 and TMPRSS9, to be strongly associated with K+ sensitivity in serum samples from female subjects. We used wild-type mice (C57BL/6N) supplemented with standard or challenged Na+ and K+ diet compositions, which revealed differences in candidate gene mRNA transcript regulation between male and female mice or on a K+ diet. Tmprss3 was further investigated in Xenopus oocytes, where it produced an ENaC-mediated Na+ current in contrast to their catalytically inactive mutants. Similarly, we studied the role of Tmprss3 in ENaC activation in the kidney, using Tmprss3-I- mice housed in a metabolic cage and fed a standard, high K+ or low Na+ diet. Our findings show that under low Na+ diets, cleavage of the a- and yENaC subunits occurs, plasma aldosterone levels remain unchanged, and i.p. administration of benzamil has no effect on fractional Na+ excretion in Tmprss3-1-· mice compared to Tmprss3+/+ mice. Furthermore, Tmprss3 mRNA was only found in a few puncta during an RNAscope analysis of the wild-type mouse kidney subjected to an 8% high Na+ diet. Collectively, our results suggest that Tmprss3 can activate ENaC in vitro and Tmprss3 does not play arole in ENaC regulation in the kidney under physiological conditions.
--
L'hypertension artérielle touche près d'un milliard de personnes dans le monde et est influencée par des facteurs génétiques et environnementaux. Un régime alimentaire riche en Na+ et pauvre en K+, des graisses saturées et peu insaturées, l'inactivité physique, une consommation importante d'alcool et de tabac, et l'obésité sont autant de facteurs de risque connus d'hypertension. Il existe des preuves que les facteurs génétiques jouent un rôle dans l'effet antihypertenseur de l'apport alimentaire en K+. Notre objectif était d'étudier et de caractériser les facteurs génétiques qui influencent la pression artérielle en réponse à l'apport de K+. Le canal Na+ épithélial (ENaC) joue un rôle vital dans la réabsorption du Na+ le long du néphron distal et entraîne la sécrétion de K+ via un gradient électrochimique, maintenant ainsi le volume de liquide extracellulaire et la pression artérielle. On a découvert que NR3Cl, GRXCRl, HPN, ATP12A, CAPl, CAP2, CAP3, TMPRSS3 et TMPRSS9 étaient associés à la sensibilité au K+ chez les sujets humains en utilisant des approches génétiques non biaisées et ciblées. L'étude d'association pangénomique (approche non biaisée) a révélé que deux gènes, TMPRSS3 et TMPRSS9, étaient fortement associés à la sensibilité au K+ dans les échantillons de sérum de sujets féminins. Nous avons utilisé des souris de type sauvage (C57BL/6N) soumises à un régime alimentaire standard ou contesté à base de Na+ et de K+, ce qui a révélé des différences dans la régulation des transcriptions des gènes candidats entre les souris mâles et femelles ou sous régime K+. La protéine Tmprss3 a été étudiée plus avant dans des ovocytes de Xenopus, où elle a produit un courant Na+ médié par ENaC, contrairement à ses mutants catalytiquement inactifs. De même, nous avons étudié le rôle de Tmprss3 dans l'activation des ENaC dans le rein, en utilisant des souris Tmprss3-/- logées dans une cage métabolique et nourries avec un régime standard, à forte teneur en K+ ou à faible teneur en Na+. Nos résultats montrent que sous un régime à faible teneur en Na+, le clivage des sous-unités - et ENaC se produit, les niveaux d'aldostérone plasmatique restent inchangés, et l'administration i.p. de Benzamil n'a aucun effet sur l'excrétion fractionnelle de Na+ chez les souris Tmprss3-/- par rapport aux souris Tmprss3+/+. De plus, l'ARNm Tmprss3 n'a été trouvé que dans quelques ponctuations lors d'une analyse par ARNscope du rein de souris de type sauvage soumises à un régime à 8% de Na+ élevé. Collectivement, nos résultats suggèrent que Tmprss3 peut activer l'ENaC in vitro et que Tmprss3 ne joue pas de rôle dans la régulation de l'ENaC dans le rein dans des conditions physiologiques
Role of channel activating proteases in renal Na+ and K+ handling
No full textThe epithelium sodium channel (ENaC) is responsible for the final Na+ reabsorption in the kidney tubules. This channel can be activated by several mechanisms such as hormones, ion concentration, phospholipids, and proteases. The activation of ENaC by proteases is well characterized in vitro. Using Xenopus oocytes, it has been shown that the channel-activating protease 1 and 3 (CAP1/Prss8 and CAP3/St14) were able to activate ENaC. However, in vivo this activation has not yet been studied in the kidney. Furthermore, CAP1/Prss8 is known to have other targets like the protease receptor 2 (PAR2) in the skin. While this receptor is known to be implicated in renal K+ handling, no studies have been made on its activation in the kidney. We were thus also interested to see if CAP1/Prss8 was an activator of PAR2 in the kidney.
By using RNAscope analysis, we were first able to co-localize CAP1/Prss8 and CAP3/St14 in the same tubules with ENaC. Then, by using genetically engineered mouse models, we investigated the role of CAP1/Prss8 and CAP3/St14 in proteolytic ENaC activation and Na+/K+ homeostasis. Under low salt diet, CAP1/Prss8 tubule-specific knockout mice were able to maintain proteolytic ENaC activation through an aldosterone independent pathway, whereas CAP3/St14 tubule-specific knockout mice maintained Na+ and K+ homeostasis, but showed an altered protein expression of ENaC subunits. Surprisingly, CAP1/Prss8-CAP3/St14 tubule- specific double knockout mice rescued these phenotypes, showed no difference in ENaC protein expression, and were able to increase plasma aldosterone concentrations as in control mice. Finally, PAR2 tubule-specific knockout mice were able to maintain Na+ and K+ homeostasis and were able to increase plasma aldosterone concentration under low salt diet.
Overall, in the present study we have shown that in vivo CAP1/Prss8 and CAP3/St14 were not involved in direct ENaC proteolysis as previously described in Xenopus oocytes. Under low salt diet, CAP1/Prss8 deficiency uncoupled ENaC activation from the classical aldosterone pathway, while CAP3/St14 appears to be involved in ENaC protein synthesis. Finally, PAR2 deletion does not affect Na+ and K+ balance under low salt diet.
From a more clinical point of view, our findings could be relevant for hypertensive patients. Some patients develop hypertension with low levels of aldosterone. It would be interesting to see if some of these patients could have polymorphisms of CAP1/Prss8 and if the uncoupling of ENaC activation from aldosterone could lead to hypertension.
--
Le canal sodique épithélial (ENaC) est responsable de la réabsorption finale du Na+ dans les tubules rénaux. De nombreuse voies telles que des variations de pH, les hormones ou encore les protéases peuvent activer ce canal. L’activation in vitro d’ENaC par les protéases est bien documentée. Dans le modèle d’ovocyte de xénopes, il a été montré qu’ENaC peut être activé par des protéases dites activatrices de canaux (CAP1/Prss8 et CAP3/St14). Cependant, cette activation n’a pas encore été étudiée in vivo dans le rein. De plus, CAP1/Prss8 est connu pour avoir d’autres cibles d’activation dans d’autres tissues, comme le récepteur à protéase 2 (PAR2) dans la peau. Malgré le fait que ce récepteur soit impliqué dans l’homéostasie rénale du K+, aucune étude n’a été faite sur son activation dans le rein. Nous avons donc également cherché à savoir si CAP1/Prss8 était un activateur de PAR2 dans cet organe.
Dans un premier temps, en utilisant l’analyse par RNAscope, nous avons localisé CAP1/Prss8 et CAP3&St14 dans les mêmes tubules que ENaC. Dans un second temps, en utilisant des modèles de souris génétiquement modifiées, nous avons étudié le rôle de CAP1/Prss8 et CAP3/St14 dans l'activation protéolytique d’ENaC ainsi que leur implication dans la réabsorption de Na+ et K+. Dans le cadre d'un régime faiblement salé, les souris déficientes pour CAP1/Prss8 spécifiquement dans les tubules ont été capables de maintenir l'activation protéolytique des sous unités d’ENaC par une voie indépendante de l'aldostérone. A l’inverse, les souris déficientes pour CAP3/St14 spécifiquement dans les tubules ont maintenu l'homéostasie du Na+ et du K+ tout en montrant une expression protéique altérée des sous-unités ENaC. Étonnamment, les souris doublement déficientes pour CAP1/Prss8 et CAP3/St14 ont compensé ces phénotypes, en ne montrant aucune différence dans l'expression d’ENaC au niveau protéique et ont été capables, tout comme les souris contrôles, d'augmenter les concentrations plasmatiques d'aldostérone. Finalement, les souris knockout PAR2 spécifiques des tubules ont été capables de maintenir l'homéostasie du Na+ et du K+ et d'augmenter la concentration plasmatique d'aldostérone sous un régime pauvre en sel.
En conclusion, dans la présente étude, nous avons montré que in vivo CAP1/Prss8 et CAP3/St14 n'étaient pas impliqués dans la protéolyse directe d’ENaC, comme précédemment décrit dans les ovocytes de xénopes. Sous régime faiblement salé, le knockout de CAP1/Prss8 découple l'activation d’ENaC de l’aldostérone, alors que CAP3/St14 semble être impliqué dans la synthèse protéique d’ENaC. Finalement, la délétion de PAR2 dans le rein n’affecte pas l’homéostasie du Na+ et K+ sous une diète faiblement salée. D'un point de vue plus clinique, nos résultats pourraient être pertinents dans le traitement des patients développant des signes d’hypertension avec de faibles niveaux d'aldostérone. Il serait intéressant de voir si certains de ces patients pourraient présenter des polymorphismes de CAP1/Prss8 entrainant l’activation d’ENaC par découplage de l’aldostérone
Role of glucocorticoid receptor mutations in hypertension and adrenal gland hyperplasia.
No full textHypertension is one of the leading causes of premature death in humans and exhibits a complex aetiology including environmental and genetic factors. Mutations within the glucocorticoid receptor (GR) can cause glucocorticoid resistance, which is characterized by several clinical features like hypercortisolism, hypokalaemia, adrenal hyperplasia and hypertension. Altered glucocorticoid receptor signalling further affects sodium and potassium homeostasis as well as blood pressure regulation and cell proliferation and differentiation that influence organ development and function. In salt-sensitive hypertension, excessive renal salt transport and sympathetic nervous system stimulation may occur simultaneously, and, thus, both the mineralocorticoid receptor (MR) and the GR-signalling may be implicated or even act interdependently. This review focuses on identified GR mutations in human primary generalized glucocorticoid resistance (PGGR) patients and their related clinical phenotype with specific emphasis on adrenal gland hyperplasia and hypertension. We compare these findings to mouse and rat mutants harbouring genetically engineered mutations to further dissect the cause and/or the consequence of clinical features which are common or different
The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes
No full textCellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing
Mechano-electrical transduction in mice lacking the α-subunit of the epithelial sodium channel
No full textV. The epithelial sodium channel and its implication in human diseases
No full textThe epithelial Na+channel (ENaC) controls the rate-limiting step in the process of transepithelial Na+reabsorption in the distal nephron, the distal colon, and the airways. Hereditary salt-losing syndromes have been ascribed to loss of function mutations in the α-, β-, or γ-ENaC subunit genes, whereas gain of function mutations (located in the COOH terminus of the β- or γ-subunit) result in hypertension due to Na+retention (Liddle’s syndrome). In mice, gene-targeting experiments have shown that, in addition to the kidney salt-wasting phenotype, ENaC was essential for lung fluid clearance in newborn mice. Disruption of the α-subunit resulted in a complete abolition of ENaC-mediated Na+transport, whereas knockout of the β- or γ-subunit had only minor effects on fluid clearance in lung. Disruption of each of the three subunits resulted in a salt-wasting syndrome similar to that observed in humans.</jats:p
Lessons from Mouse Mutants of Epithelial Sodium Channel and Its Regulatory Proteins
No full textThe use of gene-modified mouse models allows the experimental in vivo analysis of specific gene defects at the level of target cells. With respect to the epithelial sodium channel and some of its regulatory proteins, gene-modified models that control gene defects in a time- and tissue-dependent conditional or constitutive manner have been generated. The combination of molecular and physiologic approaches in these mouse models increases the understanding of the complex regulation and the cell signaling cascades involved in Na(+) transport in target cells and may ultimately provide new insights into the pathophysiology of renal Na(+) retention and BP regulation. This review summarizes and discusses the gene-targeting approaches that have been applied to the epithelial sodium channel and its regulatory proteins
Dysfunction of epithelial sodium transport: From human to mouse
No full textDysfunction of epithelial sodium transport: From human to mouse. The highly amiloride-sensitive epithelial sodium channel (ENaC) is an apical membrane constituent of cells of many salt-absorbing epithelia. In the kidney, the functional relevance of ENaC expression has been well established. ENaC mediates the aldosterone-dependent sodium reabsorption in the distal nephron and is involved in the regulation of blood pressure. Mutations in genes encoding ENaC subunits are causative for two human inherited diseases: Liddle's syndrome, a severe form of hypertension associated with ENaC hyperfunction, and pseudohypoaldosteronism (PHA-1), a salt-wasting syndrome caused by decreased ENaC function. Transgenic mouse technologies provide a useful tool to study the role of ENaC in vivo. Different mouse lines have been established in which each of the ENaC subunits was affected. The phenotypes observed in these mice demonstrated that each subunit is essential for survival and for regulation of sodium transport in kidney and colon. Moreover, the α subunit plays a specific role in the control of fluid absorption in the airways at birth. Such mice can now be used to study the role of ENaC in various organs and can serve as models to understand the pathophysiology of these human diseases
ENaC activation by proteases
No full textProteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane‐bound and soluble proteases that are defined as channel‐activating proteases (CAPs), since they induce Na(+) ion transport through an autocrine mechanism when co‐expressed with the highly amiloride‐sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel‐activating protease 1, prostasin) followed by CAP2 (channel‐activating protease 2, TMPRSS4) and CAP3 (channel‐activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine‐, cysteine‐ and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ‐specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis
- …
