Dict 500

Blood and spot urines were collected as previously described^{71 (link)}. Glucose measurements as a measure of recent feeding were taken immediately after removal of the pups from the mother at euthanasia using a handheld glucometer (Accu-Chek, Roche, Switzerland). Creatinine levels were measured by HPLC/MS/MS commercially from serum samples collected at euthanasia (Antech, Fountain Valley, CA, USA). BUN was measured from serum samples using kit 0580 (Stanbio, Boerne, TX, USA). The COBAS Mira Plus automated chemistry analyzer (Roche Diagnostics, Pleasanton, CA, USA) was used to determine plasma and urine inorganic phosphorus. Urinary creatinine was measured by colorimetric assay (DICT-500; BioAssay Systems, Hayward, CA, USA). Concentrations of serum intact fibroblast growth factor (FGF)-23 protein were determined using mouse FGF-23 ELISA assays (60–6500; Immutopics, San Clemente, CA, USA).

Mouse blood samples collected from heart puncture at sacrifice or from the tail vein were clotted and centrifuged to collect serum. Serum creatinine concentration was measured by the picric acid method with a commercial kit (DICT-500, BioAssay Systems), following the manufacturer’s instructions. BUN was measured with a commercial kit (DIUR-500, BioAssay Systems) following the manufacturer’s instructions.

Trunk blood was collected right after the animal sacrifice and allowed to clot for 30 min at room temperature, followed by 5000 g centrifugation for 10 minutes at 4° C to collect the serum fraction [19 (link), 73 (link)]. Serum creatinine and ALT levels were measured using the following assay kits respectively: DICT-500 (BioAssay Systems) and MAK052 (Sigma-Aldrich). Assays were performed according to manufacturer instructions.