Urinary albumin

At 10 weeks post-TAC, mice were weighed and placed individually into metabolic cages for 24 h urine collection (Tan et al., 2018 ). Mice were acclimatized to the metabolic cages by housing for a short period of daylight prior to the 24 h collection. An ELISA kit was used for the measurement of urinary albumin (Bethyl Laboratories, Montgomery, United States). At 10.5 weeks, glomerular filtration rate (GFR) was measured by plasma clearance of intravenously injected FITC-sinistrin (10 mg/100 g body weight dissolved in 0.9% NaCl). Briefly, FITC-sinistrin was retro-orbitally injected under brief isoflurane anesthesia (4%; 0.5–1.0 L/min, O₂) and blood samples were collected from the tail vein in conscious mice at 3-, 5-, 7-, 10-, 15-, 35-, 56-, and 75-min post-injection. Plasma was fluorometrically measured (excitation 470 nm and emission 530 nm) and quantification achieved using a FITC-sinistrin standard curve of known concentrations. GFR was calculated using a two-compartment model (Rieg, 2013 ; Mullins et al., 2020 (link)).

Blood urea nitrogen (BUN) was assayed in accordance with the kit manufacturer’s instructions (BioAssay Systems, Hayward, CA). Mouse urine was collected before the animals were euthanized. Urinary albumin was measured in accordance with the manufacturer’s instructions (Bethyl Laboratories, Montgomery, TX).

Two weeks prior to sacrifice mice were transferred to metabolic cages for 20h urine collection (12:00 p.m. day 1 until 8:00 a.m. day 2). Mice had ad libitum access to food and water. Samples were collected and spun down at 15.000rpm, urine was transferred and volume was determined. Urinary albumin (Bethyl laboratories, Montgomery, TX, USA), NGAL (R&D systems) and KIM-1 (R&D systems) concentration were determined by ELISA, and adjusted for urinary volume.

N_ε‐(Carboxymethyl)lysine (CML) concentrations in plasma and urine were measured by an in‐house indirect ELISA as previously described.³⁰ Urinary albumin (Bethyl Laboratories, United States) was normalized to the 24‐h flow rate of urine. Serum and urinary creatinine were measured spectrophotometrically at 505 nm (Roche/Hitachi 902 Analyzer, Roche Diagnostics GmBH) using the Jaffe method.