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Gain-of-function mutations of the V2 vasopressin receptor in nephrogenic syndrome of inappropriate antidiuresis (NSIAD): a cell-based assay to assess constitutive water reabsorption

  • Molecular and cellular mechanisms of disease
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Abstract

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently identified chromosome X-linked disease associated with gain-of-function mutations of the V2 vasopressin receptor (V2R), a G-protein-coupled receptor. It is characterized by inability to excrete a free water load, hyponatremia, and undetectable vasopressin-circulating levels. Hyponatremia can be quite severe in affected male children. To gain a deeper insight into the functional properties of the V2R active mutants and how they might translate into the pathological outcome of NSIAD, in this study, we have expressed the wild-type V2R and three constitutively active V2R mutants associated with NSIAD (R137L, R137C, and the F229V) in MCD4 cells, a cell line derived from renal mouse collecting duct, stably expressing the vasopressin-sensitive water channel aquaporin-2 (AQP2). Our findings indicate that in cells expressing each active mutant, AQP2 was constitutively localized to the apical plasma membrane in the absence of vasopressin stimulation. In line with these observations, under basal conditions, osmotic water permeability in cells expressing the constitutively active mutants was significantly higher compared to that of cells expressing the wild-type V2R. Our findings demonstrate a direct link between activating mutations of the V2R and the perturbation of water balance in NSIAD. In addition, this study provides a useful cell-based assay system to assess the functional consequences of newly discovered activating mutations of the V2R on water permeability in kidney cells and to screen the effect of drugs on the mutated receptors.

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Acknowledgments

We are grateful to Dr. Paola Molinari (Istituto Superiore di Sanità, Rome) for her advice in signal transduction experiments and Prof. Francesca Fanelli (University of Modena-Reggio Emilia) for helpful discussions. We thank Prof. Michel Bouvier (University of Montreal, Canada) for providing the cDNA of the V2 receptors.

Funding

The financial support of Telethon - Italy (Grant Number GGP13227) is gratefully acknowledged. Marianna Ranieri is a postdoctoral research fellow supported by “Intervento cofinanziato dal Fondo di Sviluppo e Coesione 2007-2013–APQ Ricerca Regione Puglia, Programma Regionale a Sostegno della Specializzazione Intelligente e della Sostenibilità Sociale ed Ambientale–FutureInResearch.”

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Authors and Affiliations

  1. Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125, Bari, Italy

    Marianna Ranieri, Grazia Tamma, Tommaso Pellegrino, Annarita Di Mise, Giovanna Valenti & Susanna Cotecchia

  2. Istituto Nazionale di Biostrutture e Biosistemi, 00136, Rome, Italy

    Grazia Tamma & Giovanna Valenti

  3. Department of Pharmacology, Istituto Superiore di Sanità, 00161, Rome, Italy

    Vanessa Vezzi, Caterina Ambrosio, Cristina Grò & Tommaso Costa

  4. Center of Excellence in Comparative Genomics (CEGBA), University of Bari, 70125, Bari, Italy

    Giovanna Valenti

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  1. Marianna Ranieri
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Ranieri, M., Tamma, G., Pellegrino, T. et al. Gain-of-function mutations of the V2 vasopressin receptor in nephrogenic syndrome of inappropriate antidiuresis (NSIAD): a cell-based assay to assess constitutive water reabsorption. Pflugers Arch - Eur J Physiol 471, 1291–1304 (2019). https://doi.org/10.1007/s00424-019-02307-x

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