Application of Antibody Array Technology in the Analysis of Urinary Cytokine Profiles in Patients with Chronic Kidney Disease

Aims: Emerging evidence suggests that the urinary excretion of cytokines is associated with the progression of chronic kidney disease (CKD). However, detection of urinary cytokines in high throughput is still a problem in clinical practice. In this cross-sectional study, we applied a novel proteomic technology, antibody array, to analyze urinary cytokine profiles in patients with CKD.

Methods: A total of 10 subjects including 7 CKD patients and 3 normal controls were studied. These patients with CKD were divided into two groups according to the levels of estimated glomerular filtration rate (eGFR): group A (eGFR >or=80 ml/min/1.73 m(2), n = 3) and group B (eGFR <or=40 ml/min/1.73 m(2), n = 4). Urine samples taken from age and gender-matched healthy volunteers (n = 3) served as control. Differential excretion of urinary cytokines was determined by human cytokine antibody array (Raybiotech, Norcross, Ga., USA). A 2-fold change in spot intensity compared to controls was considered as significant. In order to check the reliability of the data obtained by antibody array, urinary monocyte chemoattractant protein (MCP) 1 and tumor necrosis factor (TNF) alpha were determined by using enzyme-linked immunosorbent assay.

Results: A total of 15 cytokines varied significantly in urinary samples obtained from patients with CKD compared with normal controls. It was shown that the levels of MCP-1, RANTES, tissue inhibitor of metalloproteinase (TIMP) 1, TNF-alpha, vascular endothelial growth factor (VEGF), E-selectin, Fas, intercellular adhesion molecule 1, interleukin 2, matrix metalloproteinase (MMP) 2 and transforming growth factor beta in patients with CKD at stage 1-2 (group A) were 2- to 5-fold higher than those in normal controls, while the excretion of MCP-1, RANTES, TIMP-1, TNF-alpha and VEGF was further increased in the patients with renal insufficiency (group B). However, a lower excretion of urinary vascular cell adhesion molecule 1 and platelet-derived growth factor was found in patients with CKD compared with normal controls. Impressively, the urinary MMP-9 excretion was 492-fold higher in group A and 198-fold higher in group B than that in normal controls. The levels of MCP-1 and TNF-alpha correlated well with the relative n-fold change seen in the antibody array experiments. The rank correlation coefficients (r values) were 0.976 (p < 0.001) and 0.939 (p < 0.001) for MCP-1and TNF-alpha, respectively.

Conclusion: Antibody array could serve as a fast, high-throughput and sensitive tool for detecting the excretion of urinary cytokines. Our study is the first to provide an important information regarding the utility of antibody array in evaluating the role of cytokines in the initiation and progression of CKD.