3. Urinary YKL-40 measurements

Samples for measuring urinary YKL-40 levels were obtained from the children upon routine urine collection for culture. Samples were centrifuged at 4℃ for 15 minutes at 3,000×g within 30 minutes of collection and stored at -80℃ until final analyses were conducted. Urinary levels of YKL-40 were measured using ELISA kits for human CHI3L1 (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. All samples were run in duplicate, and mean values were presented. The respective inter- and intra-assay precision values were 4.3–4.7% and 5.3–6.9%, respectively. The standard curve range for the assay was 62.5–4,000 pg/mL, and the detection limit for YKL-40 was 3.55 pg/mL. While the majority of samples were within the range of the standard curve, urinary YKL-40 levels for some control subjects were below the detectable range, which is consistent with a previous report [16]. Accordingly, YKL-40 values below the detection limit were approximated to 1.775 pg/mL (the mean value between 0 and the lower limit of detection), and urinary levels of YKL-40 were normalized to the urinary Cr level. The urinary concentrations of biomarkers, including urinary YKL-40, is inversely proportional to the urinary flow rate for a given amount of a total excreted biomarker. Because urinary Cr excretion is relatively constant in the case of normal renal function, the urinary Cr level reflects the urinary flow rate. Therefore, we expressed the concentration of urinary YKL-40 as the urinary YKL-40-to-Cr ratio (urinary YKL-40/Cr) [17,18]. The study was approved by the institutional review board at Bucheon St. Mary's Hospital, the Catholic University of Korea, and written informed consent was obtained from the parents of all participating children.