Renal function was determined by measuring creatinine levels by high-performance liquid chromatography (HPLC) in serum samples (n = 5/group) as previously reported (Baudoux et al. 2012 (link)). A calibration curve with standards containing 13.3–88.4 µmol/l creatinine (Sigma-Aldrich) was set up. The creatinine peak was used to quantify the amount of creatinine (mg/dl) within the serum samples.
LAP enzyme activity was measured with a spectrofluorometric assay in urine samples (n = 3/group). Urinary excretion of this enzyme is used as an indicator of the integrity of the proximal tubular brush border (Lebeau et al. 2005 (link)). Urine samples (100 µl with glycerol) were diluted 1:30 and 1:60 with 50 mM Tris–HCl buffer (pH 7.6). The substrate leucine-7-amido-4-methyl coumarin (Leu-AMC; Bachem) was incubated with the diluted samples at 37 °C for 60 min. The reaction was terminated by heating the samples at 95 °C for 5 min. Fluorescence of free AMC was measured at 367 nm (excitation) and 440 nm (emission). LAP activity was normalised against urine creatinine (U/g urine creatinine). creatinine levels in urine (100 µl) were measured using the Jaffé method (creatinine Diagnostic Kit, Sigma-Aldrich).
LAP enzyme activity was measured with a spectrofluorometric assay in urine samples (n = 3/group). Urinary excretion of this enzyme is used as an indicator of the integrity of the proximal tubular brush border (Lebeau et al. 2005 (link)). Urine samples (100 µl with glycerol) were diluted 1:30 and 1:60 with 50 mM Tris–HCl buffer (pH 7.6). The substrate leucine-7-amido-4-methyl coumarin (Leu-AMC; Bachem) was incubated with the diluted samples at 37 °C for 60 min. The reaction was terminated by heating the samples at 95 °C for 5 min. Fluorescence of free AMC was measured at 367 nm (excitation) and 440 nm (emission). LAP activity was normalised against urine creatinine (U/g urine creatinine). creatinine levels in urine (100 µl) were measured using the Jaffé method (creatinine Diagnostic Kit, Sigma-Aldrich).