Twenty-Four-Hour Metabolic Cage Experiments

Mice were randomized to acute application, via oral gavage, of vehicle or Npt2a-I (30 mg/kg). Because mice underwent several rounds of acute metabolic cage experiments, mice were not further acclimatized for 24-hour metabolic cage experiments. After their bladders were emptied, mice were placed in metabolic cages at 9:00 for quantitative urine collection for 24 hours with free access to food and water. Urine was collected at 3, 6, 12, and 24 hours.²⁰ After each collection period, the bladders were emptied, as described above, to assure complete collection. Urine was analyzed as described below.