Novel signalling pathways in nephrogenic syndrome of inappropriate antidiuresis: functional implication of site‐specific AQP2 phosphorylation

Abstract

AbstractNephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X‐linked disease caused by gain‐of‐function mutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp‐In T‐REx Madin‐Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2‐wt or non‐phosphorylatable AQP2‐S269A/AQP2‐S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2‐wt and significantly higher basal water permeability. In addition, V2R‐R137L/C showed significantly higher activity of Rho‐associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269‐AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R‐R137L/C mutants and AQP2‐S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269‐AQP2 phosphorylation in the gain‐of‐function phenotype. Conversely, V2R‐R137L/C mutants retained the gain‐of‐function phenotype when AQP2‐S256A was co‐expressed. In contrast, cells expressing the F229V mutant and the non‐phosphorylatable AQP2‐S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain‐of‐function V2R‐R137L/C mutants causing NSIAD is protein kinase A independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation. imageKey points Nephrogenic syndrome of inappropriate antidiuresis is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R‐F229V) and the other vaptan resistant (V2R‐R137C/L). In renal cells, all three activating arginine vasopressin receptor 2 (V2R) variants display constitutive AQP2 plasma membrane expression and high basal water permeability. In cells expressing V2R‐R137L/C mutants, disruption of the AQP2‐S269 phosphorylation site caused the loss of the gain‐of‐function phenotype, which, in contrast, was retained in V2R‐F229V‐expressing cells. Cells expressing the V2R‐F229V mutant were instead sensitive to disruption of the AQP2‐S256 phosphorylation site. The serine/threonine kinase Rho‐associated kinase (ROCK) was found to be involved in AQP2‐S269 phosphorylation downstream of the V2R‐R137L/C mutants. These findings might have clinical relevance for patients with nephrogenic syndrome of inappropriate antidiuresis.