Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells

BACKGROUND: Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. METHODS: AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. RESULTS: We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+. CONCLUSION: Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation

Un cadre méthodologique pour évaluer l'équivalence entre pertes et gains de biodiversité induits par les projets d'aménagement et leurs mesures compensatoires

In France, the Mitigation hierarchy aims to achieve the "no net loss" (NNL) of biodiversity at the development projects scale. One of the key issues to achieve this goal is to demonstrate the ecological equivalence between the gains associated with offsets and the losses caused by the impacts. Despite regulatory improvements, the French law does not include a method to follow for determining equivalence, and none is unanimously recognized. This leads to heterogeneous practices and difficulty in reaching the NNL. In this context, we have developed a methodological framework for assessing equivalence adapted to the French regulatory and ecological context and combining three challenges: operationality, scientific basis and comprehensiveness. This methodological framework makes it possible 1 / to evaluate the biodiversity found on impacted and compensating sites by taking into account ordinary biodiversity and the one of interest, with a focus on functionalities; 2 / to estimate the value of the indicators after impact and MC, in the short and long term, taking into account associated uncertainties; and 3 / calculating losses and gains leading to a quantitative and transparent equivalence assessment. The use of the methodological framework favors dialogue between actors and also allows monitoring of offsets over time.En France, la séquence « Eviter Réduire Compenser » (ERC) a pour objectif d'atteindre « l'absence de perte nette (APN) » de biodiversité à l'échelle des projets d'aménagement. Un des enjeux clé pour y arriver consiste à démontrer l'équivalence écologique entre les gains associés aux mesures compensatoire (MC) et les pertes occasionnées par les impacts. Malgré les avancées règlementaires, le cadre français n'inclut pas de méthode à suivre pour déterminer l'équivalence et aucune n'est unanimement reconnue. Cela amène à des pratiques hétérogènes et une difficulté d'atteindre l'APN. Dans ce contexte, nous avons développé un cadre méthodologique d'évaluation de l'équivalence adapté au contexte règlementaire et écologique français, répondant à trois défis : opérationnalité, bases scientifiques et exhaustivité. Ce cadre méthodologique permet 1/ d'évaluer la biodiversité des sites impactés et compensatoires en tenant compte de la biodiversité ordinaire et à enjeu en insistant sur les fonctionnalités, 2/ d'estimer la valeur des indicateurs après impact et MC à court et long terme, en prenant en compte les incertitudes associées et 3/ de calculer les pertes et des gains, aboutissant ainsi à une évaluation quantitative et transparente de l'équivalence. L'utilisation du cadre méthodologique favorise la concertation entre acteurs et permet également un suivi des MC dans le temps

The optimisation of stochastic grammars to enable cost-effective probabilistic structural testing

Abstract The effectiveness of statistical testing, a probabilistic structural testing strategy, depends on the characteristics of the probability distribution from which test inputs are sampled. Metaheuristic search has been shown to be a practical method of optimising the characteristics of such distributions. However, the applicability of the existing search-based algorithm is limited by the requirement that the software’s inputs must be a fixed number of ordinal values. In this paper we propose a new algorithm that relaxes this limitation and so permits the derivation of probability distributions for a much wider range of software. The representation used by the new algorithm is based on a stochastic grammar supplemented with two novel features: conditional production weights and the dynamic partitioning of ordinal ranges. We demonstrate empirically that a search algorithm using this representation can optimise probability distributions over complex input domains and thereby enable cost-effective statistical testing, and that the use of both conditional production weights and dynamic partitioning can be beneficial to the search process

Circadian Integration of Glutamatergic Signals by Little SAAS in Novel Suprachiasmatic Circuits

Neuropeptides are critical integrative elements within the central circadian clock in the suprachiasmatic nucleus (SCN), where they mediate both cell-to-cell synchronization and phase adjustments that cause light entrainment. Forward peptidomics identified little SAAS, derived from the proSAAS prohormone, among novel SCN peptides, but its role in the SCN is poorly understood.Little SAAS localization and co-expression with established SCN neuropeptides were evaluated by immunohistochemistry using highly specific antisera and stereological analysis. Functional context was assessed relative to c-FOS induction in light-stimulated animals and on neuronal circadian rhythms in glutamate-stimulated brain slices. We found that little SAAS-expressing neurons comprise the third most abundant neuropeptidergic class (16.4%) with unusual functional circuit contexts. Little SAAS is localized within the densely retinorecipient central SCN of both rat and mouse, but not the retinohypothalamic tract (RHT). Some little SAAS colocalizes with vasoactive intestinal polypeptide (VIP) or gastrin-releasing peptide (GRP), known mediators of light signals, but not arginine vasopressin (AVP). Nearly 50% of little SAAS neurons express c-FOS in response to light exposure in early night. Blockade of signals that relay light information, via NMDA receptors or VIP- and GRP-cognate receptors, has no effect on phase delays of circadian rhythms induced by little SAAS.Little SAAS relays signals downstream of light/glutamatergic signaling from eye to SCN, and independent of VIP and GRP action. These findings suggest that little SAAS forms a third SCN neuropeptidergic system, processing light information and activating phase-shifts within novel circuits of the central circadian clock

Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption

To prevent dehydration, terrestrial animals and humans have developed a sensitive and versatile system to maintain their water homeostasis. In states of hypernatremia or hypovolemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary and binds its type-2 receptor in renal principal cells. This triggers an intracellular cAMP signaling cascade, which phosphorylates aquaporin-2 (AQP2) and targets the channel to the apical plasma membrane. Driven by an osmotic gradient, pro-urinary water then passes the membrane through AQP2 and leaves the cell on the basolateral side via AQP3 and AQP4 water channels. When water homeostasis is restored, AVP levels decline, and AQP2 is internalized from the plasma membrane, leaving the plasma membrane watertight again. The action of AVP is counterbalanced by several hormones like prostaglandin E2, bradykinin, dopamine, endothelin-1, acetylcholine, epidermal growth factor, and purines. Moreover, AQP2 is strongly involved in the pathophysiology of disorders characterized by renal concentrating defects, as well as conditions associated with severe water retention. This review focuses on our recent increase in understanding of the molecular mechanisms underlying AVP-regulated renal water transport in both health and disease