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

  • Marianna Ranieri
  • Grazia Tamma
  • Tommaso Pellegrino
  • Vanessa Vezzi
  • Caterina Ambrosio
  • Cristina Grò
  • Annarita Di Mise
  • Tommaso Costa
  • Giovanna ValentiEmail author
  • Susanna CotecchiaEmail author
Molecular and cellular mechanisms of disease
Part of the following topical collections:
  1. Molecular and cellular mechanisms of disease


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.


V2 receptor AQP2 NSIAD 



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 information

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.”

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Birnbaumer M (2000) Vasopressin receptors. Trends Endocrinol Metab 11:406–410CrossRefGoogle Scholar
  2. 2.
    Bouley R, Hasler U, Lu HA, Nunes P, Brown D (2008) Bypassing vasopressin receptor signaling pathways in nephrogenic diabetes insipidus. Semin Nephrol 28:266–278. CrossRefGoogle Scholar
  3. 3.
    Boone M, Deen PM (2008) Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption. Pflugers Arch 456:1005–1024. CrossRefGoogle Scholar
  4. 4.
    Olesen ET, Moeller HB, Assentoft M, MacAulay N, Fenton RA (2016) The vasopressin type 2 receptor and prostaglandin receptors EP2 and EP4 can increase aquaporin-2 plasma membrane targeting through a cAMP-independent pathway. Am J Physiol Renal Physiol 311:F935–F944. CrossRefGoogle Scholar
  5. 5.
    Tamma G, Lasorsa D, Trimpert C, Ranieri M, Di Mise A, Mola MG, Mastrofrancesco L, Devuyst O, Svelto M, Deen PM, Valenti G (2014) A protein kinase A-independent pathway controlling aquaporin 2 trafficking as a possible cause for the syndrome of inappropriate antidiuresis associated with polycystic kidney disease 1 haploinsufficiency. J Am Soc Nephrol 25:2241–2253. CrossRefGoogle Scholar
  6. 6.
    Bichet DG (2006) Diabètes insipides néphrogéniques (Nephrogenic diabetes insipidus). Nephrol Ther 2:387–404. CrossRefGoogle Scholar
  7. 7.
    Morello JP, Bichet DG (2001) Nephrogenic diabetes insipidus. Annu Rev Physiol 63:607–630. CrossRefGoogle Scholar
  8. 8.
    Ranieri M, Di Mise A, Tamma G, Valenti G (2019) Vasopressin-aquaporin-2 pathway: recent advances in understanding water balance disorders. F1000Research 8:8. CrossRefGoogle Scholar
  9. 9.
    Feldman BJ, Rosenthal SM, Vargas GA, Fenwick RG, Huang EA, Matsuda-Abedini M, Lustig RH, Mathias RS, Portale AA, Miller WL, Gitelman SE (2005) Nephrogenic syndrome of inappropriate antidiuresis. N Engl J Med 352:1884–1890. CrossRefGoogle Scholar
  10. 10.
    Bartter FC, Schwartz WB (1967) The syndrome of inappropriate secretion of antidiuretic hormone. Am J Med 42:790–806CrossRefGoogle Scholar
  11. 11.
    Rosenthal W, Seibold A, Antaramian A, Gilbert S, Birnbaumer M, Bichet DG, Arthus MF, Lonergan M (1994) Mutations in the vasopressin V2 receptor gene in families with nephrogenic diabetes insipidus and functional expression of the Q-2 mutant. Cell Mol Biol (Noisy-le-Grand) 40:429–436Google Scholar
  12. 12.
    Carpentier E, Greenbaum LA, Rochdi D, Abrol R, Goddard WA 3rd, Bichet DG, Bouvier M (2012) Identification and characterization of an activating F229V substitution in the V2 vasopressin receptor in an infant with NSIAD. J Am Soc Nephrol 23:1635–1640. CrossRefGoogle Scholar
  13. 13.
    Erdelyi LS, Mann WA, Morris-Rosendahl DJ, Gross U, Nagel M, Varnai P, Balla A, Hunyady L (2015) Mutation in the V2 vasopressin receptor gene, AVPR2, causes nephrogenic syndrome of inappropriate diuresis. Kidney Int 88:1070–1078. CrossRefGoogle Scholar
  14. 14.
    Tiulpakov A, White CW, Abhayawardana RS, See HB, Chan AS, Seeber RM, Heng JI, Dedov I, Pavlos NJ, Pfleger KD (2016) Mutations of vasopressin receptor 2 including novel L312S have differential effects on trafficking. Mol Endocrinol 30:889–904. CrossRefGoogle Scholar
  15. 15.
    Decaux G, Vandergheynst F, Bouko Y, Parma J, Vassart G, Vilain C (2007) Nephrogenic syndrome of inappropriate antidiuresis in adults: high phenotypic variability in men and women from a large pedigree. J Am Soc Nephrol 18:606–612. CrossRefGoogle Scholar
  16. 16.
    Rochdi MD, Vargas GA, Carpentier E, Oligny-Longpre G, Chen S, Kovoor A, Gitelman SE, Rosenthal SM, von Zastrow M, Bouvier M (2010) Functional characterization of vasopressin type 2 receptor substitutions (R137H/C/L) leading to nephrogenic diabetes insipidus and nephrogenic syndrome of inappropriate antidiuresis: implications for treatments. Mol Pharmacol 77:836–845. CrossRefGoogle Scholar
  17. 17.
    Tenenbaum J, Ayoub MA, Perkovska S, Adra-Delenne AL, Mendre C, Ranchin B, Bricca G, Geelen G, Mouillac B, Durroux T, Morin D (2009) The constitutively active V2 receptor mutants conferring NSIAD are weakly sensitive to agonist and antagonist regulation. PLoS One 4:e8383. CrossRefGoogle Scholar
  18. 18.
    Kocan M, See HB, Sampaio NG, Eidne KA, Feldman BJ, Pfleger KD (2009) Agonist-independent interactions between beta-arrestins and mutant vasopressin type II receptors associated with nephrogenic syndrome of inappropriate antidiuresis. Mol. Endocrinol (Baltimore, Md) 23:559–571. CrossRefGoogle Scholar
  19. 19.
    Iolascon A, Aglio V, Tamma G, D'Apolito M, Addabbo F, Procino G, Simonetti MC, Montini G, Gesualdo L, Debler EW, Svelto M, Valenti G (2007) Characterization of two novel missense mutations in the AQP2 gene causing nephrogenic diabetes insipidus. Nephron Physiol 105:p33–p41. CrossRefGoogle Scholar
  20. 20.
    Jung HJ, Kwon TH (2016) Molecular mechanisms regulating aquaporin-2 in kidney collecting duct. Am J Physiol Renal Physiol 311:F1318–F1328. CrossRefGoogle Scholar
  21. 21.
    Kwon TH, Frokiaer J, Nielsen S (2013) Regulation of aquaporin-2 in the kidney: a molecular mechanism of body-water homeostasis. Kidney Res Clinic Pract 32:96–102. CrossRefGoogle Scholar
  22. 22.
    Nedvetsky PI, Tamma G, Beulshausen S, Valenti G, Rosenthal W, Klussmann E (2009) Regulation of aquaporin-2 trafficking. Handb Exp Pharmacol:133–157.
  23. 23.
    Noda Y, Sasaki S (2006) Regulation of aquaporin-2 trafficking and its binding protein complex. Biochim Biophys Acta 1758:1117–1125. CrossRefGoogle Scholar
  24. 24.
    Valenti G, Procino G, Tamma G, Carmosino M, Svelto M (2005) Minireview: Aquaporin 2 trafficking. Endocrinology 146:5063–5070. CrossRefGoogle Scholar
  25. 25.
    Ando F, Sohara E, Morimoto T, Yui N, Nomura N, Kikuchi E, Takahashi D, Mori T, Vandewalle A, Rai T, Sasaki S, Kondo Y, Uchida S (2016) Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway. Nat Commun 7:13636. CrossRefGoogle Scholar
  26. 26.
    Cheung PW, Terlouw A, Janssen SA, Brown D, Bouley R (2019) Inhibition of non-receptor tyrosine kinase Src induces phosphoserine 256-independent aquaporin-2 membrane accumulation. J Physiol 597:1627–1642. CrossRefGoogle Scholar
  27. 27.
    Olesen ET, Fenton RA (2017) Aquaporin-2 membrane targeting: still a conundrum. Am J Physiol Renal Physiol 312:F744–F747. CrossRefGoogle Scholar
  28. 28.
    Barak LS, Oakley RH, Laporte SA, Caron MG (2001) Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus. Proc Natl Acad Sci U S A 98:93–98. CrossRefGoogle Scholar
  29. 29.
    Costa T, Cotecchia S (2005) Historical review: negative efficacy and the constitutive activity of G-protein-coupled receptors. Trends Pharmacol Sci 26:618–624. CrossRefGoogle Scholar
  30. 30.
    Scheer A, Costa T, Fanelli F, De Benedetti PG, Mhaouty-Kodja S, Abuin L, Nenniger-Tosato M, Cotecchia S (2000) Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation. Mol Pharmacol 57:219–231Google Scholar
  31. 31.
    Steinberg EA, Moss M, Buchanan CL, Goebel J (2018) Adherence in pediatric kidney transplant recipients: solutions for the system. Pediatr Nephrol 33:361–372. CrossRefGoogle Scholar
  32. 32.
    Tamma G, Procino G, Strafino A, Bononi E, Meyer G, Paulmichl M, Formoso V, Svelto M, Valenti G (2007) Hypotonicity induces aquaporin-2 internalization and cytosol-to-membrane translocation of ICln in renal cells. Endocrinology 148:1118–1130. CrossRefGoogle Scholar
  33. 33.
    Procino G, Barbieri C, Tamma G, De Benedictis L, Pessin JE, Svelto M, Valenti G (2008) AQP2 exocytosis in the renal collecting duct—involvement of SNARE isoforms and the regulatory role of Munc18b. J Cell Sci 121:2097–2106. CrossRefGoogle Scholar
  34. 34.
    Ranieri M, Tamma G, Di Mise A, Russo A, Centrone M, Svelto M, Calamita G, Valenti G (2015) Negative feedback from CaSR signaling to aquaporin-2 sensitizes vasopressin to extracellular Ca2. J Cell Sci 128:2350–2360. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Marianna Ranieri
    • 1
  • Grazia Tamma
    • 1
    • 2
  • Tommaso Pellegrino
    • 1
  • Vanessa Vezzi
    • 3
  • Caterina Ambrosio
    • 3
  • Cristina Grò
    • 3
  • Annarita Di Mise
    • 1
  • Tommaso Costa
    • 3
  • Giovanna Valenti
    • 1
    • 2
    • 4
    Email author
  • Susanna Cotecchia
    • 1
    Email author
  1. 1.Department of Biosciences, Biotechnologies and BiopharmaceuticsUniversity of BariBariItaly
  2. 2.Istituto Nazionale di Biostrutture e BiosistemiRomeItaly
  3. 3.Department of PharmacologyIstituto Superiore di SanitàRomeItaly
  4. 4.Center of Excellence in Comparative Genomics (CEGBA)University of BariBariItaly

Personalised recommendations