Measurement of Urinary Parameters in Water-Deprived Mice

Mice were acclimated for few days in metabolic cages (3600M021; Techniplast, West Chester, PA, USA). Following acclimation and baseline measurements of urinary parameters, mice were water deprived for 24 hours, as we did previously [25 (link)]. Tails were clipped with sterilized scissors to collect 50 μl blood into heparinized micro-hematocrit capillary tubes (Fischer, Cat. # 22-362-566) before and after water deprivation. Urine and plasma osmolarity was measured in duplicates using freezing point depression osmometer Model 3320 (Advanced Instruments, Norwood, MA, USA). Urinary creatinine concentration was assessed with QuantiChrom Creatinine Assay Kit (DICT-500; BioAssay Systems, Hayward, CA, USA) utilizing the improved Jaffe method, as we did before [30 (link)]. Urinary AVP levels were assayed with Vasopressin Arg⁸-Vasopressin ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) following the manufacturer’s protocols. Urinary Ca²⁺ concentration was measured using a Jenway PFP7 Flame photometer (Bibby Scientific, Burlington, NJ, USA).