Spironolactone

THP-1 and RAW 264.7 cells (106/ml) were pretreated with the indicated inhibitors 1 hour prior to aldosterone (10⁻⁶ M) treatment. After 24 hours, the cell culture supernatants were collected for galectin-3 detection by EIA. The inhibitors were PD98059 (Sigma, St. Louis, MO, USA; ERK inhibitor), LY294002 (Sigma, St. Louis, MO, USA; phosphoinositide 3-kinase inhibitor), SB203580 (Sigma, St. Louis, MO, USA; p38 inhibitor), Ro318220 Sigma, St. Louis, MO, USA; protein kinase C inhibitor), and SP600125 (Sigma, St. Louis, MO, USA; Jun N-terminal kinase inhibitor).
For nuclear factor (NF)-κB pathway analysis, THP-1 and RAW-264.7 cells were serum-starved for 24 hours and then pre-treated with 10⁻⁷ M of spironolactone, LY294002 or BAY117082 (Sigma, St. Louis, MO, USA; NF-κB inhibitors) for 1 hour before 10⁻⁶ M aldosterone treatment. Nuclear and cytosolic proteins were collected after 1 hour, and the level of the NF-κB p65 subunit was determined by Western blotting.

For ATP release assay, BMDNs were left to equilibrate in Tyrode’s buffer (124 mM NaCl, 2.4 mM KCl, 10.8 mM NaHCO₃, 0.4 mM NaH₂PO₄*H₂O, 0.9 mM MgCl₂*6H₂O, 1.8 mM CaCl₂*6H₂O, pH 7.35). Panx1 channel mediated ATP release was activated by reducing osmolarity of the Tyrode’s buffer by decreasing NaCl to 30.24 mM. WT and Panx1^−/− BMDNs were stimulated with appropriate controls for 5 min. Cells were centrifuged at 1200 rpm for 5 minutes and supernatants were collected for ATP measurement using the ATP bioluminescent assay kit (#FLAA, Sigma Aldrich), according to the manufacturer’s instructions.
For dye uptake assay, BMDNs were seeded in 384-well plate and maintained in a physiological solution (136 mM NaCl, 4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 2.5 mM glucose, buffered to pH 7.4 with 10 mM HEPES-NaOH solution). YO-PRO-1 Iodide (5 µM, ThermoFisher Scientific, #Y3603) was added to equilibrate cells. Hypo-osmotic shock was induced to maximally open Panx1 channels. Inhibitors of Panx1 BB-FCF dye-Erioglaucine disodium salt (Sigma Aldrich, #80717)^{24 (link),25 (link)} and spironolactone (Sigma Aldrich, #S3378)^{39 (link)} were prepared in DMSO and used at 1μM and 100 μM, respectively. Global quantification of YO-PRO-1 dye uptake per well was performed using the FDSS/µCELL Functional Drug Screening System (Hamamatsu Photonics).

Mdx mice were treated using water bottles containing 250 mg/L spironolactone (Sigma-Aldrich, S3378) or 6.7 mg/L prednisolone (Sigma-Aldrich, P6004), the active metabolite of prednisone dissolved in MediDrop containing sucralose (ClearH2O, 75-01-1001). Mice were treated during the peak of inflammation for 10 days for cytokine- and chemokine-level analysis to allow detection of differences in protein expression (4–5.5 weeks of age), for 7 days (3.5–4.5 weeks of age) for flow cytometry, RNA-Seq, and diaphragm IHC or for 14 days (3.5–5.5 weeks of age) for quadriceps IHC and histology. Approximate dosages for spironolactone and prednisolone treatment were 37.5 and 1 mg/kg × day, respectively (8 (link), 33 (link)). The mdx mice given MediDrop vehicle were used as controls for all experiments. Mice were housed 5 per cage and euthanized via cervical dislocation.

Dosing for these animals was calculated using the Food and Drug Administration's 2005 allometric scaling calculations as described by Reagon‐Shaw et al. (2008). To deliver a human equivalent dose of 50 mg/day of spironolactone (Sigma‐Aldrich), 4.41 mg kg day⁻¹ was administered to each rat. Experimental animals were dosed orally each day from 10 weeks of age (Day 1), and to enhance acceptance, spironolactone was mixed into a caramel syrup (Quaterpast, Shott Beverages Ltd.).