Identification, characterization and rescue of a novel vasopressin-2 receptor mutation causing nephrogenic diabetes insipidus

Abstract

Objective: X-linked nephrogenic diabetes insipidus (XNDI), caused by mutations in the V2 vasopressin receptor (V2R), is clinically distinguished from central diabetes insipidus (CDI) by elevated serum vasopressin (AVP) levels and unresponsiveness to 1-desamino-8-d-arginine vasopressin (DDAVP). We report two infants with XNDI, and present the characterization and functional rescue of a novel V2R mutation.

Patients: Two male infants presented with poor growth and hypernatraemia. Both patients had measurable pretreatment serum AVP and polyuria that did not respond to DDAVP, suggesting NDI. However, both also had absent posterior pituitary bright spot on MRI, which is a finding more typical of CDI.

Methods: The AVPR2 gene encoding V2R was sequenced. The identified novel missense mutation was re-created by site-directed mutagenesis and expressed in HEK293 cells. V2R activity was assessed by the ability of transfected cells to produce cAMP in response to stimulation with DDAVP. Membrane localization of V2R was assessed by fluorescence microscopy.

Results: Patient 1 had a deletion of AVPR2; patient 2 had the novel mutation L57R. In transiently transfected HEK293 cells, DDAVP induced detectable but severely impaired L57R V2R activity compared to cells expressing wild-type V2R. Fluorescence microscopy showed that myc-tagged wild-type V2R localized to the cell membrane while L57R V2R remained intracellular. A nonpeptide V2R chaperone, SR121463, partially rescued L57R V2R function by allowing it to reach the cell membrane.

Conclusions: L57R V2R has impaired in vitro activity that can be partially improved by treatment with a V2R chaperone. The posterior pituitary hyperintensity may be absent in infants with XNDI.

Figure 1

Pre-contrast sagittal T1-weighted images of the brain demonstrate a normal-appearing pituitary gland. No posterior pituitary bright spot is seen within the pituitary gland or in an ectopic location. The infundibulum demonstrates a normal thickness without evidence of abnormal enhancement after contrast administration. The suprasellar cistern is unremarkable. The hypothalamus demonstrates no evidence of mass. Insets are higher magnification of the sella. Lower panels show recurrence of the posterior pituitary bright spot in each patient, 6 weeks and 3 months after the initial diagnosis, respectively.

Figure 2

Maximum DDAVP-stimulated receptor activities. HEK293 cells expressing cAMP-inducible luciferase, Renilla luciferase and WT or L57R V2R were treated with 10⁻⁷ M DDAVP with and without 10⁻⁶ M SR121463. Luciferase activity, a measure of intracellular cAMP generation, is normalized to Renilla luciferase activity and expressed as percentage of maximum WT V2R activity. Data are mean +/− SEM of 2 experiments, each performed in triplicate. NS = not significant; * = P < 0.05; *** = P < 0.001.

Figure 3

Dose-response curves. Basal (no DDAVP) and stimulated (10⁻¹³ to 10⁻⁷ M DDAVP) WT V2R and L57R V2R activities expressed as percentage of maximum WT V2R activity are plotted versus log of DDAVP concentration. The EC₅₀ values for WT and mutant are significantly different (P = 0.002). Each point represents the mean +/− SEM of 3 experiments, each performed in triplicate.

Figure 4

Membrane expression of V2R. Nonpermeabilized HEK 293 cells expressing myc-tagged WT or L57R V2R and farnesylated mCherry were incubated with and without 10⁻⁶ M SR121463 for 48h and imaged by fluorescence microscopy. Red fluorescence identifies the cell membrane of all transfected cells. Green fluorescence identifies the WT or mutant receptor when it is present at the cell membrane.