The patient was born as sibling 6 out of 7. His father, grandmother, and three of his siblings reported symptoms of polydipsia and polyuria. None of them were aware of any disease in the family. The mother experienced no symptoms. X-linked inheritance can be excluded based on the pedigree with father-to-son transmission of the disease, and similar polyuric-polydipsic symptoms in both sexes in two generations indicate autosomal dominant inheritance (Figure 1A). As a child, the patient suffered from enuresis until the age of 10 years and reported since a continuous need to drink and urinate several times at night. At age of 23 years, the patient was admitted to hospital with similar symptoms, and a water deprivation test was carried out. The conclusion was psychogenic polydipsia, and the patient was recommended to reduce water intake. At age of 28 years, the patient was hospitalized for one week for reevaluating the continuous symptoms. Gradually, water intake was reduced under constant monitoring of serum electrolytes and physical well-being. At termination, the patient was discharged from the hospital with water intake at 2.5 l/day and normal electrolytes, creatinine, and body weight. The only symptom the patient experienced was headache. The diagnosis psychogenic polydipsia was maintained. The patient experienced continuous symptoms and could not diminish water intake without side effects. The patient, 36-year-old when examined in the present case story, was admitted to the outpatient clinic of the Department of Nephrology with symptoms of polydipsia and polyuria and a suspected diagnosis of NDI. He was a non-smoker and did not take any regular medication. At presentation, the patient was physically normal, body weight was 65.9 kg (BMI 22.8). Ultrasonographic examination of both kidneys was normal. The patient had 24 h urinary volumes of 10-13 l/day and an equivalent large water intake. Baseline plasma parameters (Supplemental Table 1) were all within normal ranges including p-sodium at 141 mmol/l (Figure 1B). Baseline urinary osmolality was 111 mmol/kg (Figure 1C) with corresponding urinary sodium Figure 1B). Despite a high and increasing p-AVP concentration in response to water deprivation (3.9-5.3 pmol/l) (Figure 1B) and a concomitant increase in p-sodium, a continued urinary output between 100 to 360 ml/h was registered (Figure 1D). Urinary osmolality increased from 111 mmol/kg to 398 mmol/kg (Figure 1C). The weight loss matched diuresis (65.9 kg to 64.4 kg at termination, Figure 1D). Blood pressure increased slightly from 95/61 mmHg one hour after water deprivation to 128/70mmHg at the end with a stable heart rate (Figure 1E). Prolonged treatment with desmopressin was tested by incremental doses and 24h urine sampling (Figure 1F). There was a gradual response where 24 h urinary output was reduced from 11 l/day to 6.8 l/day on the maximal tested dose of desmopressin at 240 μg x 3/day. The proband complained about headache, a known side effect of desmopressin, and he terminated the medication which resulted in a parallel and gradual increase in 24 h urinary volume (Figure 1F).

Figure 1(A) Pedigree of the family showing autosomal dominant inherited congenital nephrogenic diabetes insipidus. (B-E) Diagrams showing the effects on water deprivation and dDAVP administration in the patient expressing the sequence variant AQP2-R267G. The patient was water-deprived for 8 h and the indicated parameters were determined before and after initiation of desmopressin treatment. Desmopressin was administered from 6th h. (F) During prolonged treatment (18 months) with incremental doses of desmopressin, 24 h urinary output was reduced. Termination of desmopressin resulted in a gradual increase in 24 h urinary volume.

Functional analysis of AQP2-R267G on AQP2 glycosylation and cellular traffickingTo determine the effects of the sequence variant on the AQP2 protein, Western blotting was carried out to analyze the expression level and glycosylation. For this, wild-types (WT) or mutant (MUT) AQP2 was transiently expressed in MDCK and HEK293 cells (Figure 2A+B). Wild-type AQP2 is expressed as non-glycosylated, complex glycosylated and high mannose forms. Mutant AQP2 showed a significant increase in the complex-glycosylation form compared to wild-type AQP2 in HEK293 cells. AQP2 was detected with both an antibody directed against the C terminus (E-2) or the N terminus (N-20) of AQP2.The mutation did not affect the recognition of the C-terminal epitope by the antibody. Glycosylated AQP2 was not detectable in our MDCK cells (not shown) although a weak glycosylation in MDCK cells has previously been reported7Deen P.M. Rijss J.P. Mulders S.M. et al.Aquaporin-2 transfection of Madin-Darby canine kidney cells reconstitutes vasopressin-regulated transcellular osmotic water transport.. However, in SDS-PAGE the elongated mutant form migrated at a higher molecular weight than the wild-type, as was the case in HEK293 cell lysates.

Figure 2(A) The complex-glycosylation of mutant AQP2-R267G2 compared to wild-type AQP2 is increased in HEK293 cells. Cells were transfected to transiently express wild-type AQP2 (WT) or mutant AQP2 (Mut) or were left untransfected (UT). Cell lysates were prepared and complex-glycosylated (cg), high mannose (hm), and nonglycosylated (ng) forms of AQP2 and Hsp90 (loading control) were detected by Western blotting using specific antibodies directed against the N terminus (N-20) or the C terminus (E-2) of AQP2. Antibody E-2 can still detect the mutant form with C-terminal elongation, and it was used for further experiments and for quantifying the glycosylation. (B) Signals emerging from the cg form of AQP2 in HEK293 cell lysates were semi-quantitatively analyzed by densitometry and normalized to total AQP2 and the loading control. Statistically significant differences are indicated (mean ± SEM; **P<0.01). (C) The mutant AQP2-R267G is retained in the ERGIC, the ER-Golgi-intermediate compartment, in MDCK cells. MDCK cells were transfected to transiently express wild-type AQP2 or mutant AQP2 or were left untransfected. Cells were left untreated or treated with forskolin (+Fsk). AQP2 (red) and ERGIC-53/p58 (green) were detected by immunofluorescence microscopy using specific primary and fluorophore-coupled secondary antibodies. Nuclei were stained with DAPI (blue). Shown are representative images from three independent experiments. Scale bar 30 μm. (D) Plasma membrane and perinuclear immunofluorescence AQP2 signal intensities were determined and the ratios of plasma membrane to perinuclear fluorescence signal intensities were calculated. Ratios >1 indicate a predominant localization at the plasma membrane. Statistical analysis was carried out using one-way ANOVA and Kruskal-Wallis test multiple-comparison Shown are means ± SD of three independent experiments with a total of 7 cells per condition. Statistically significant differences are indicated, *, p<0.1, ***, p<0.001.

Immunofluorescence microscopy was used to analyze the effect of the R267G substitution on the localization of AQP2 (Figure 2C). For semiquantitative analysis, the AQP2 fluorescence signals at the plasma membrane and in the perinuclear region of the cells were determined and the ratios of plasma membrane to perinuclear AQP2 fluorescence intensities were calculated. Ratio >1 indicated a predominant localization at the plasma membrane, ratios 8Baltzer S. Bulatov T. Schmied C. et al.Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells.,9Vukićević T. Hinze C. Baltzer S. et al.Fluconazole Increases Osmotic Water Transport in Renal Collecting Duct through Effects on Aquaporin-2 Trafficking.. A fraction of the mutant AQP2, as the wild-type, was found at the plasma membrane in untreated cells. Forskolin did not affect the localization of the mutant AQP2. Further, co-localization studies revealed an accumulation of the mutant AQP2 in the ER-Golgi intermediate compartment (ERGIC), (Figure 2C +2D).