Urine collection and assay methods for nephrin, protein and creatinine

All spot urine samples were coded and processed within 2 hours of collection. Urine samples were transferred to tubes and centrifuged at 700× g for 5 min. Urinary supernatant was stored at −20°C until measurement of protein, creatinine and nephrin levels. Protein and creatinine concentrations were measured using a Protein Assay Rapid Kit Wako and Laboassay Creatinine (Wako Pure Chemical Industries, Osaka, Japan), respectively. Nephrin concentration was measured using an ELISA kit (Exocell, Philadelphia, Pennsylvania, USA). Urine samples were diluted in the range of 1:10–1:50 depending on proteinuria in the sample. The intra-array and interassay coefficients of variation for nephrin were <10%. Since the detection limits were 0.26 ng/mL for nephrin and 5 μg/mL for protein, we assumed that samples with undetectable levels contained 0.13 ng/mL nephrin and 2.5 μg/mL protein. Protein and nephrin concentrations in the urine were corrected by urine creatinine concentration and expressed as PCR (mg/mg) and nephrin:creatinine ratio (NCR, ng/mg).