Clinical data collection
All clinical data were collected at the Nagoya University hospital. Hypertonic saline infusion tests were performed and assessed as previously described 15.
The generation of FNDI mice heterozygous for the mutant Avp gene (Cys98stop) has been previously described 13. Wild-type (WT) mice (C57BL/6J) were purchased from Japan SLC, Inc. (Shizuoka, Japan). In all animal experiments, 2-month-old male FNDI and WT mice were used. Mice were maintained under controlled conditions (23.0±0.5°C, lights on from 09:00 to 21:00).
Measurement of urine volumes, urine AVP concentrations, urine osmolality, and serum potassium levels
WT mice and FNDI mice were implanted with osmotic minipumps (Alzet model 2002; Muromachi Kikai Co., Tokyo, Japan) containing: 1) dimethyl sulfoxide (DMSO) (WT or FNDI + vehicle group); 2) aldosterone (A9477; Sigma–Aldrich, St. Louis, MO, USA; 0.025 µg/g body weight [BW]/day) in DMSO (WT or FNDI + aldosterone group); or 3) dexamethasone (D2915; Sigma–Aldrich; 0.2 µg/g BW/day) in DMSO (WT or FNDI + dexamethasone group). All groups had free access to water and food. Mice were housed in metabolic cages, and the 24-h pooled urine was collected for 5 days. Urine AVP concentrations were measured using the Arg8-Vasopressin ELISA Kit (Enzo Life Sciences, Farmingdale, NY, USA) following the instructions provided by the manufacturer. Urine osmolality was determined using the cryoscopic method (Oriental Yeast Co., Ltd., Tokyo, Japan). The levels of potassium in the serum were determined using the ion-selective electrode method (Oriental Yeast Co., Ltd.).
Western blotting analysis
Homogenates of the inner medullae separated from the kidneys of mice were prepared. Semiquantitative immunoblotting was carried out to assess the relative expression levels of proteins of interest, as previously described 20. The blots were probed with the following primary antibodies: rabbit anti-aquaporin-2 (anti-AQP2, SPC-503D; StressMarq Biosciences, Victoria, British Columbia, Canada; 1:1,000) and rabbit anti- glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; ab181602; Abcam, Cambridge, UK; 1:10,000). Horse radish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG) (NA934; GE Healthcare, Little Chalfont, UK; Research Resource Identifier: AB_772206) was used as the secondary antibody. Can Get Signal Immunoreaction Enhancer Solution (TOYOBO, Osaka, Japan) was used for the dilution of the primary and secondary antibodies. ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) was used to detect signals. The intensities of bands in western blots were quantified with the ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
Immunohistochemistry and immunofluorescence analysis
Kidneys were fixed by perfusion (through the left ventricle) with periodate lysine (0.2 M) and paraformaldehyde (2%) in phosphate-buffered saline. Tissue samples were soaked for several hours in 20% sucrose in phosphate-buffered saline, embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan), and stored at −80°C until sectioning. Kidneys were cut into sections (thickness: 10 μm) using a cryostat at −20°C, thaw mounted on Superfrost Plus microscope slides (Matsunami, Tokyo, Japan), and stored at −80°C until immunohistochemical analysis, which was performed as previously described 21. Briefly, the sections were incubated with the primary antibody anti-AQP2 (ab199975; Abcam; 1:3,000) overnight at 4°C. The sections were rinsed and incubated with the secondary antibody Alexa Fluor 488-conjugated goat anti-mouse IgG (H +L) highly cross-adsorbed (1:1,000; A11029; Invitrogen, San Diego, CA, USA) for 2 h at room temperature. Immunofluorescence images were captured by a laser-scanning confocal microscope (LSM 5 Pascal; Carl Zeiss, Oberkochen, Germany). The distribution of AQP2 was semiquantitatively analyzed. The fluorescence intensities of AQP2 in the kidneys were quantified with the ImageJ software, according to previous studies 22–24. The AQP2 intensities were measured from the apical to the basolateral membrane. These data were obtained using principal cells constructing tubule segments (n=9 collecting ducts from three animals per group).
The statistical significance of differences among groups was analyzed using the unpaired t-test, one-way analysis of variance (ANOVA), or two-way ANOVA, with repeated measures followed by the Bonferroni test as appropriate. Results are expressed as the mean ± standard error, and P-values <0.05 denoted statistically significant differences.