The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting duct

Etude du rôle de la phosphatidylinositol 3-kinase dans la réabsorption du sodium par un modèle de tubule distal et collecteur du rein

Cette thèse vise à démontrer l’importance de la PI 3-kinase dans la régulation hormonale de la réabsorption rénale du sodium. Ce contrôle extrêmement précis, notamment par l’aldostérone, s’effectue au niveau du néphron distal. Nous avons utilisé comme modèle l’épithélium de cellules A6, dérivées du tubule distal de Xenopus Laevis. Le transport unidirectionnel de sodium s’effectue en deux étapes: depuis, l’entrée à partir du milieu luminal par des canaux sodiques épithéliaux (ENaCs) insérés dans la membrane apicale, jusqu’à la sortie vers le liquide extracellulaire par des pompes Na+/K+-ATPases, situées dans la membrane basolatérale. L’insuline augmente ce transport de sodium et la PI 3-kinase semble assurer un rôle-clef.Nous avons étudié l’importance de chacun des 3-phosphoinositides produits par la PI 3-kinase, sur le transport du sodium en ajoutant au milieu cellulaire des formes «perméantes» de ces phospholipides. Parmi ceux-ci, le PIP3 et dans une moindre intensité le PI(3,4)P2 augmentent ce transport. En revanche, le PI3P, le PI(3,5)P2, ainsi que le PI(4,5)P2 n’ont pas d’effet sur lui. Nous avons démontré par la technique du Western blot que la 3-phosphatase PTEN est exprimée dans les cellules A6. Cette phosphatase déphosphoryle le PIP3 en PI(4,5)P2. Nous avons surexprimé PTEN dans les cellules A6. Ceci réduit l’augmentation du transport du sodium induite par l’insuline, ainsi que celle induite par addition de la forme «perméante» de PIP3. Nous avons ensuite vérifié si d’autres agents qui activent la PI 3-kinase, stimulent également le transport de sodium à travers cet épithélium. A cette fin, nous avons d’abord vérifié que l’EGF et le peroxyde d’hydrogène, connus pour stimuler la PI 3-kinase dans d’autres systèmes, activent également cette enzyme dans les cellules A6. Tous deux augmentent ce transport. L’importance de l’augmentation induite par H2O2 est comparable à celle de l’insuline, tandis que l’effet de l’EGF est plus transitoire. Un dosage d’activité de la PI 3-kinase, nous a permis de démontrer que l’intensité de l’activation de la PI 3-kinase est corrélée avec l’amplitude de l’augmentation du transport du sodium. Par comparaison avec l’effet de l’insuline et de l’H2O2, l’EGF augmente faiblement l’activité de la PI 3-kinase et induit une faible augmentation du transport. Nous avons également examiné si la voie des MAPK influence la stimulation du transport du sodium par ces différents agents. Cette voie ne semble pas impliquée dans l’effet de l’insuline ou du peroxyde d’hydrogène. Par contre, elle diminue la stimulation du transport de sodium par l’EGF. L’effet de l’EGF sur le transport semble résulter d’un compromis entre l’activation de la voie de la PI 3-kinase qui l’augmente et l’activation de la voie des MAPK qui le diminue.En conclusion, une augmentation de PIP3, soit par addition de PIP3 exogène, soit par augmentation endogène sous l’effet de l’insuline ou d’autres agents stimulant la PI 3-kinase, augmente le transport du sodium tandis qu’une diminution de PIP3 endogène (par surexpression de PTEN) le diminue. L’importance de l’activation de la PI 3-kinase est quantitativement corrélée avec l’importance de l’augmentation du transport du sodium. La PI 3-kinase est donc un médiateur-clef de la régulation rénale de ce transport.Doctorat en sciences biomédicalesinfo:eu-repo/semantics/nonPublishe

Physiology and pathophysiology of SLC12A1/2 transporters.

The electroneutral Na(+)-K(+)-Cl(-) cotransporters NKCC1 (encoded by the SLC12A2 gene) and NKCC2 (SLC12A1 gene) belong to the Na(+)-dependent subgroup of solute carrier 12 (SLC12) family of transporters. They mediate the electroneutral movement of Na(+) and K(+), tightly coupled to the movement of Cl(-) across cell membranes. As they use the energy of the ion gradients generated by the Na(+)/K(+)-ATPase to transport Na(+), K(+), and Cl(-) from the outside to the inside of a cell, they are considered secondary active transport mechanisms. NKCC-mediated transport occurs in a 1Na(+), 1K(+), and 2Cl(-) ratio, although NKCC1 has been shown to sometimes mediate partial reactions. Both transporters are blocked by bumetanide and furosemide, drugs which are commonly used in clinical medicine. NKCC2 is the molecular target of loop diuretics as it is expressed on the apical membrane of thick ascending limb of Henle epithelial cells, where it mediates NaCl reabsorption. NKCC1, in contrast, is found on the basolateral membrane of Cl(-) secretory epithelial cells, as well as in a variety of non-epithelial cells, where it mediates cell volume regulation and participates in Cl(-) homeostasis. Following their molecular identification two decades ago, much has been learned about their biophysical properties, their mode of operation, their regulation by kinases and phosphatases, and their physiological relevance. However, despite this tremendous amount of new information, there are still so many gaps in our knowledge. This review summarizes information that constitutes consensus in the field, but it also discusses current points of controversy and highlights many unanswered questions.info:eu-repo/semantics/publishe

Selection of in-phase or out-of-phase synchronization in a model based on global coupling of cells undergoing metabolic oscillations.

On the basis of experimental observations, it has been suggested that glycolytic oscillations underlie the pulsatile secretion of insulin by pancreatic beta cells, with a periodicity of about 13 min. If beta cells within an islet are synchronized through gap junctions, the question arises as to how beta cells located in different islets of Langerhans synchronize to produce oscillations in plasma levels of insulin. We address this question by means of a minimal model that incorporates the secretion of insulin by cells undergoing glycolytic oscillations. Global coupling and synchronization result from the inhibition exerted by insulin on the production of glucose, which serves as the substrate for metabolic oscillations. Glycolytic oscillations are described by a simple two-variable model centered on the product-activated reaction catalyzed by the allosteric enzyme phosphofructokinase. We obtain bifurcation diagrams for the cases in which insulin secretion is controlled solely by the product or by the substrate of the metabolic oscillator. Remarkably, we find that the oscillating cells in these conditions synchronize, respectively, in phase or out of phase. Numerical simulations show that in-phase and out-of-phase synchronization can sometimes coexist when insulin release is controlled by both the substrate and the product of the metabolic oscillator. The results provide an example of a system in which the selection of in-phase or out-of-phase synchronization is governed by the nature of the coupling between the intracellular oscillations and the secretion of the biochemical signal through which the oscillating cells are globally coupled.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Physiologie générale :Travaux pratiques

MED2, VETE2, BIME2, DENT2, MEDI-G-204info:eu-repo/semantics/published

Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin stimulation of sodium transport in A6 cell monolayers. In this study, we investigate whether stimulation of the PI 3-kinase by other agents also provoked an increase in sodium transport. Both epidermal growth factor (EGF) and H2O2 provoked a rise in sodium transport that was inhibited by LY-294002, an inhibitor of PI 3-kinase activity. PI 3-kinase activity was estimated in extracts from A6 cell monolayers directly by performance of a PI 3-kinase assay. We also estimated the relative importance of the PI 3-kinase pathway by two different methods: 1) coprecipitation of the p85 regulatory subunit with anti-phosphotyrosine antibodies and 2) phosphorylation of PKB on both Ser 473 and Thr 308 residues observed by Western blotting. Since the mitogen-activated protein kinase (MAPK) pathway has also been implicated in the regulation of sodium transport, we also investigated whether this pathway is turned on by insulin, H2O2, or EGF. Phosphorylation of ERK1/2 was increased only transiently by insulin and H2O2 but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2 stimulation of sodium transport but increased substantially the stimulation induced by EGF. The latter effect was associated with an increase in PKB phosphorylation, thus suggesting that the stimulation of the MAPK pathway prevents, in part, the stimulation of the PI 3-kinase pathway in the transport of sodium stimulated by EGF.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers.

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport (Markadieu N, Crutzen R, Blero D, Erneux C, Beauwens R. Am J Physiol Renal Physiol 288: F1201-F1212, 2005). We therefore questioned whether insulin would produce an intracellular burst of H2O2 leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate was already detected after 2 min of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase, indicating that H2O2 is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide (O2(*-); nitro blue tetrazolium, 100 microM) or H2O2 (50 microM ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 microM; phenylarsine oxide, 1 microM and plumbagin, 30 microM) prevented both insulin-stimulated H2O2 production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of the p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin-stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase, inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H2O2 production. Taken together, our data suggest that insulin induces an acute burst of H2O2production which participates in an increase in phosphatidylinositol 3,4,5-trisphosphate production and subsequently stimulation of sodium transport.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Hypotonicity induces insulin release by opening volume-sensitive anion channels in BRIN-BD11 ß cells

FLWNOinfo:eu-repo/semantics/publishedComm. Meeting of the Belgian Society of Fundamental and Clinical Physiology and Pharmacology - Antwerp, 19.11.200

Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells.

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP(3) and PI(3,4)P(2) but not PI(4,5)P(2) stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP(3) levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP(3) was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP(3) also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP(3) and PI(3,4)P(2) mediate the effect of insulin on sodium transport across A6 cell monolayers.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe