Nephelometric assay

Covariates were selected based on prior knowledge about the factors that could confound the associations of uromodulin with UTI and were ascertained at the CHS clinic visit in 1996-1997. These included age, gender, BMI, diabetes (defined by use of hypoglycemic agents, fasting plasma glucose >126 mg/dL or non-fasting glucose ≥ 200 mg/dL), baseline eGFR, urinary albumin, and urinary creatinine. The eGFR was estimated using the 2008 CKD-EPI cystatin C equation (eGFR = 127.7 × [nonstandardized CysC]^1.17 × age^0.13 × 0.91 [if female] × 1.06 [if black]).^{17 (link)} Cystatin C was measured by a Siemens nephelometric assay.^{18 (link)}

Urinary uromodulin, β2-microglobulin (β2m), and α1-microglobulin (α1m) were measured at the Laboratory for Clinical Biochemistry Research at the University of Vermont. All urine specimens were in continuous storage at −80°C until biomarker measurement without prior thaw. Laboratory personnel performing the biomarker assays were blinded to clinical information about the participants. Urine β2m and uromodulin were performed by multiplex assays (Meso Scale Diagnostics, Rockville, Maryland, USA) with detectable ranges of 1.2–5020 ng/mL and 0.6–2510 ng/mL, respectively. Urine α1m was measured by a nephelometric assay (Siemens, Tarrytown, NY) with a detectable range of 5–480 mg/L. Inter-assay coefficients of variation for the urine measurements were 13–16% for uromodulin, 15–16% for β2m and, 3.5–8.8% for α1m. Biomarkers were measured in duplicate and averaged to increase precision. Urine creatinine was measured by an enzymatic procedure (Roche, Indianapolis, IN) and urine albumin by a nephelometric method (Siemens, Tarrytown, NY). Inter-assay coefficients of variation for urine creatinine and albumin measurements were 1.5–4.3% and 2.2–6.9%, respectively. Samples with biomarker values below the limit of detection were assigned a value equivalent to the lower limit of detection divided by the square root of two.²³

All urine biomarkers were measured at the Laboratory for Clinical Biochemistry Research at the University of Vermont. Urine specimens were stored at −80°C until biomarker measurement, without prior thaw. To minimize analytic drift in repeated biomarker measurements, urine samples from baseline and year 4 visits were measured at the same time. Laboratory personnel measuring the biomarker assays were blinded to clinical information. For each urine sample, all biomarkers were measured in duplicate, and results were averaged to increase precision. Urine β2m and uUMOD measurements were performed using a multiplex assay on a MESO Scale Diagnostics (MSD) platform (Rockville, Maryland, USA). The analytic ranges were 1.2–5020 ng/ml and 0.6–2510 ng/ml, respectively, and the inter-assay coefficients of variation (CVs) were 15–16% and 13–16%, respectively. Urine α1m was measured using a Siemens nephelometric assay with a detectable range from 5–480 mg/L and inter-assay CV of 3.5–8.8%.

All urine specimens were stored at −80°C until time of measurement in 2018. IL-18, KIM-1, MCP-1, and YKL-40 were measured together using a multiplex assay on a MESO Scale Diagnostics platform (Rockville, MD). Interassay CVs were 4.9% to 13.7%, 6.1% to 13.0%, 7.1% to 12.0%, and 6.5% to 11.1%, respectively. The analytic ranges were 2 to 10,000 pg/mL for IL-18, 4 to 200,000 pg/mL for KIM-1, 3 to 10,000 pg/mL for MCP-1, and 10 to 500,000 ng/mL for YKL-40. α-1 microglobulin (A1M) was measured using a Siemens nephelometric assay (Tarrytown, NY) with interassay CV of 3.5% to 8.8% and detectable range of 5 to 480 mg/g. β-2 microglobulin (B2M), Umod, and NGAL were measured on a multiplex assay on a MESO Scale Diagnostics platform (Rockville, MD) with interassay CVs of 15% to 16%, 13% to 16%, and 11% to 19%, respectively. The ranges of detection were 1.2 to 5020 ng/mL for B2M, 0.6 to 2510 ng/mL for uromodulin, and 6 to 251000 ng/mL for NGAL.^{13 (link)} Urine albumin (mg/L) and urine creatinine were measured using a nephelometric method (Siemens, Tarrytown, NY),^{24 (link)} and by an enzymatic procedure (Roche, Indianapolis, IN), respectively.