M 1 cell line

The M-1 cell line derived from the CD of mice^{28 (link)}, was purchased from American Type Culture Collection (ATCC, Manassas, VA). M-1 cells are composed by principal and intercalated cells, this cell model has been validated previously^{29 (link)–31 (link)}. Prior to the experiments, M-1 cells were maintained in Dulbecco′s modified Eagle’s medium (DMEM)/F-12 medium (LIFE TECHNOLOGIES, Carlsbad, CA) containing 10% fetal bovine serum (FBS), growth factors (transferrin, insulin, and sodium selenite at physiological concentrations), 100 nmol/L dexamethasone and 5 mM glucose. When the cells reached approximately 70–75% confluence, they were dissociated using trypsin and split into 4-wells chambers and dishes for the trafficking assays.

Mouse kidney epithelial cells (M-1 cell line) were purchased from the American Type Culture Collection (ATCC, CRL-2038). The cell line was established from normal renal tissue taken from a mouse transgenic for the SV40 early region (tg(SV40E)Bri7). The cells retain many characteristics of cortical collecting duct (CCD) cells including morphology and antigens.
Cells were cultured at 37 °C in a humidified atmosphere of 95% air/ 5% CO₂, and maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium with 2.5 mM L-glutamine (1:1 DMEM/F12) adjusted to contain 15 mM HEPES, 0.5 mM sodium pyruvate and 1.2 g/L sodium bicarbonate supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 5% fetal bovine serum. Cells were subcultured using trypsin–EDTA at a ratio of 1:3 to 1:4 as recommended by the manufacturer.

Bradykinin (B3259), H89 (PKA inhibitor, B1427), calphostin C (PKC inhibitor, C6303), L‐NAME (NOS inhibitor, N5751), Icatibant (HOE 140 B₂R antagonist, H157) were purchased from Sigma‐Aldrich (Saint Louis, MO). For detection of prorenin and renin, we used a rabbit anti‐renin polyclonal IgG H‐105 antibody (sc‐22752), B₂R was detected using anti‐B₂R goat polyclonal IgG antibody (sc‐15050) and β‐actin was detected using a mouse anti‐β‐actin monoclonal IgG antibody (sc‐4778) all from Santa Cruz Biotechnology, Santa Cruz, CA. Anti‐rat aquaporin‐2 (AQP2) antibody was purchased from Abcam (Cambridge, UK). For Western blot experiments the secondary antibodies used were the IR Dye 800CW or 650 anti‐goat, mouse and rabbit according to the primary antibody chosen (Li‐Cor Bioscience, NE) and for immunofluorescence the secondary Alexa fluor antibodies (Alexa fluor‐488 or ‐594) were purchased from Life Technologies (Carlsbad, CA). M‐1 cell line was obtained from American Type Culture Collection (ATCC, CRL‐2038, Manassas, VA).