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.