Reference intervals of urinary kidney injury biomarkers for middle-aged men and women determined by quantitative protein mass spectrometry

There is an ongoing need to recognize early kidney injury and its progression in structural chronic pathologies. The proteins neutrophil-gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), C-X-C motif chemokine 9 (CXCL9), transforming growth factor-beta 1 (TGF-β1), solute carrier family 22 member 2 (SLC22A2), nephrin, cubilin, and uromodulin (UMOD) have been proposed as early kidney injury biomarkers. To guide clinical interpretation, their urinary concentrations should be accompanied by reference intervals, which we here establish in a representative Dutch middle-aged population.The 24 h urine samples from 1443 Caucasian middle-aged men and women were analyzed for the biomarkers by quantitative LC-MS/MS. Biomarker excretion per 24 h were calculated, and urine creatinine and osmolality were measured for dilution normalization. This population was characterized by demographic and anthropometric parameters, comorbid conditions, and conventional kidney function measures.NGAL, IGFBP7, TIMP2, KIM-1, and UMOD could be quantified in this population, whereas nephrin, SLC22A2, and CXCL9 were below their detection limits. Urine creatinine and osmolality were correlated to urine volume (r = -0.71; -0.74) and to IGFBP7 (r = 0.73; 0.71) and TIMP2 (r = 0.71; 0.69). Crude and normalized biomarker concentrations were affected by sex, but not by age, body mass index, smoking, kidney function, or common comorbid conditions. The reference intervals (men; women) were 18-108; 21-131 pmol IGFBP7/mmol creatinine, 1-63; 4-224 pmol NGAL/mmol creatinine, 7-48; 7-59 pmol TIMP2/mmol creatinine, <1-9; <1-12 pmol KIM-1/mmol creatinine, and 0.1-1.2; 0.1-1.7 mg UMOD/mmol creatinine.We present dilution-normalized and sex-stratified urinary reference intervals of kidney injury biomarkers in a middle-aged Caucasian population.