Measurement of Renal Na+ and K+ Excretion

Animals were anesthetized with a peritoneal injection of Inactin at 100 mg/kg, and mice were placed on a heated small blanket to maintain body temperature at 37°C. The trachea was cannulated to clear mucus, and a carotid artery was catheterized with PE-10 tubing for blood collection. A jugular vein was also cannulated for intravenous infusion, and the bladder was exposed for urine collections (catheterized via a suprapubic incision with a 10-cm piece of PE-10 tubing). After the completion of surgery, isotonic saline was given intravenously for 4 h (0.25–0.3 mL/1 h and total 1.0–1.2 mL of 0.9% saline) to replace surgical fluid losses and to maintain hemodynamics. Urine collections started 1 h after the infusion of 0.3 mL saline, and six total collections (every 30 min) were performed [two collections before and four collections after hydrochlorothiazide (HCTZ) at 30 mg/kg body wt]. To measure the basal level of urinary K⁺ excretion, urine samples were collected every 30 min for six total collections, and samples were pooled. Plasma and urine Na⁺ and K⁺ concentrations were measured using a dual-channel flame photometer with an internal lithium standard (Cole-Parmer Instrument, Vernon Hills, IL).