Insulin

BMSCs were isolated from long bones by crushing in PBS, and plated at 0.5 to 1 × 10⁶ cells/cm². Adipogenic differentiation was induced with 10⁻⁸ M dexamethasone, 5 μg/mL insulin, and where indicated, 1 μM rosiglitazone (Cayman Chemical, Ann Arbor, MI, USA). Calvarial osteoblasts were harvested by serial collagenase digestion as described in Yang and colleagues.^{(57 (link))} For cellular differentiation assays, calvarial osteoblasts were plated at 5 to 10 × 10³ cells/cm². For inhibition of Wnt signaling, cells were treated with 20 nM of LiCl (or NaCl as a control) or 1 μM 6-bromoindirubin-3′-oxime (BIO). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

BP was recorded by tail-cuff plethysmography (IITC Inc, Life Science, Woodland Hills, CA) in conscious animals at week 4 of both experiments 1 and 2. All animals were placed in metabolic cages for 24 hours for urine collection and water or 1.8% saline intake estimation at the end of week 4, after which the rats were euthanized by abdominal exsanguination under deep anesthesia with ketamine 100 mg/kg and xylasine 10 mg/kg. Erythrocytes were used for the measurement of Na/K-ATPase activity.^{27 (link)} Plasma was collected for insulin (Cayman Chemical, Ann Arbor, MI), MBG, and creatinine measurements. MBG was estimated in 24-hour urine using the competitive immunoassay based on 4G4 monoclonal anti-MBG antibody.^{13 (link)} Concentration of urinary Na⁺ was measured with Roche-Hitachi 917 flame photometry (Roche, Vienna, Austria). Urinary creatinine was measured with a creatinine assay kit (Cayman Chemical). Plasma electrolytes and creatinine were measured by an i-Stat analyzer (Abbott Laboratories, Abbott Park, IL). Fractional Na⁺ excretion FENa was calculated as follows: FENa = uNa × pCr × 100/(pNa × uCr), where uNa and pNa are urine Na⁺ and plasma Na⁺ concentrations (mmol/L), uCr and pCr are urine creatinine and plasma creatinine concentrations (mmol/L), and expressed as percent.