Abstract
Diabetic nephropathy (DN) is the main reason for end-stage renal disease. Microalbuminuria as the non-invasive available diagnosis marker lacks specificity and gives high false positive rates. To identify and validate biomarkers for DN, we used in the present study urine samples from four patient groups: diabetes without nephropathy, diabetes with microalbuminuria, diabetes with macroalbuminuria and proteinuria without diabetes. For the longitudinal validation, we recruited 563 diabetic patients and collected 1363 urine samples with the clinical data during a follow-up of 6 years. Comparative urinary proteomics identified four proteins Apolipoprotein A-I (APOA1), Beta-2-microglobulin (B2M), E-cadherin (CDH1) and Lithostathine-1-alpha (REG1A), which differentiated with high statistical strength (p < 0.05) between DN patients and the other groups. Label-free mass spectrometric quantification of the candidates confirmed the discriminatory value of E-cadherin and Lithostathine-1-alpha (p < 0.05). Immunological validation highlighted E-cadherin as the only marker able to differentiate significantly between the different DN stages with an area under the curve (AUC) of 0.85 (95%-CI: [0.72, 0.97]). The analysis of the samples from the longitudinal study confirmed the prognostic value of E-cadherin, the critical increase in urinary E-cadherin level was measured 20 ± 12.5 months before the onset of microalbuminuria and correlated significantly (p < 0.05) with the glomerular filtration rate measured by estimated glomerular filtration rate (eGFR).