Urine albumin

Blood was collected from the inferior vena cava at euthanasia. BUN was measured on the Beckman Coulter AU 680 analyzer. Blood counts were performed on the IDEXX Procyte DX analyzer. Both procedures were performed with the assistance of the Weill Cornell Medicine Laboratory of Comparative Pathology. Serum iron levels were measured using SFBC kits (Biolabo) following manufacturer’s instructions. We used ELISA to measure serum cystatin C (R&D Systems, Bio-Techne), serum soluble TfR (MyBioSource), and urine albumin (Abcam) following manufacturers’ instructions.

Complete blood counts were measured by the Hemavet® 950 automated processor (Drew Scientific, Oxford, CT). Colorimetric methods were used to assay serum and urine phosphate (StanBio Labs, Boerne, TX), serum iron (Genzyme, Cambridge, MA), serum urea nitrogen (BioAssay Systems, Hayward, CA), and serum and urine creatinine (Crystal Chem, Elk Grove Village, IL). Commercially available enzyme-linked immunosorbent assays (ELISA) kits were used to measure urine albumin (Abcam, Cambridge, MA), serum erythropoietin (R&D Systems, Minneapolis, MN), serum amyloid A (R&D Systems), serum interleukin 1β (R&D Systems), serum interleukin 6 (R&D Systems), plasma parathyroid hormone (PTH) (Quidel, San Diego, CA), plasma C-terminal (total) fibroblast growth factor 23 (FGF23) (Quidel), and plasma intact FGF23 (Quidel). Whereas the total FGF23 assay detects both full-length, intact FGF23 and C-terminal FGF23 proteolytic fragments, the intact FGF23 assay detects only the full-length form. Percentage intact FGF23 was calculated as follows: ( [intact FGF23] / [total FGF23] ) × 100%. In-house ELISAs were used to measure serum hepcidin^{73 (link)} and serum erythroferrone^{74 (link)}, as previously described.