| Objectives: Chronic kidney disease (CKD) has been emerged as a worldwide epidemic with the aging population increase. Renal fibrosis is a common endpoint of CKD which will lead to functional impairment ultimately resulting in end-stage renal diseases (ESRD). A large number of studies have demonstrated the importance of various profibrogenic and proimflamotary cytokines in renal fibrogenesis. Emerging evidence suggests some urinary cytokines excretion were associated with the progression of CKD. However, the application of urinary cytokines in predicting or monitoring CKD progression remains an open question. In the present study, we applied a novel proteomic technology, antibody array, to analyze urinary cytokine profiles in patients with CKD.Materials and 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 eGFR calculated using MDRD formula: CKD I group (eGFR≥80ml/min/1.73m2, n=3) and CKD II group(eGFR≤40 ml/min/1.73m2, n=4). Urine sample from healthy volunteers (n=3) served as control. Differential excretion of urinary cytokines was determined by using of human cytokine antibody array (Raybiotech Norcross GA). The antibody arrays were used according to manufacter's manual. The data were expressed as a normalized value of the density value of the cytokine spot(mean value of duplicated cytokine spot)divided by the density value of the positive control spot. Then, normalized value was converted into the fold change. A two-fold change in spot intensity compared to controls was considered significant. In order to check the reliability of the data obtained by antibody array, we quantified MCP-1 and TNF-αusing ELISA. The procedure was completed according to manufacter's manual.Results: A total of 15 cytokines varied significantly in urinary samples from patients with CKD, comparing with normal controls. It was shown that MCP-1, RANTES, TIMP-1,TNF-α, VEGF, E-selectin, Fas, ICAM-1, IL-2, MMP-2, TGF-βincreasing in two-five folds in CKD patients with normal renal function(CKD I group), while the excretion of MCP-1, RANTES, TIMP-1, TNF-αand VEGF is further increased with the declining of renal function. A decreasing excretion of urinary VCAM-1 and PDGF was found in patients with renal failure (CKD II group). Impressively, the urinary MMP-9 excretion was 492-fold increase in CKD I group and 198-fold increase in CKD II group compared to normal controls. The expression of MCP-1 and TNF-αis significantly correlated with the relative 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-α, respectively.Conclusion: Antibody array could serve as a fast, high-throughput and sensitive tool for detecting levels of urinary cytokines. The relative levels obtained by antibody array correlated quite well with the actual levels of selected cytokines by ELISA. Our study firstly demonstrated a significant change of urinary cytokine profiles in patients with CKD compared with normal controls by using of the novel antibody array technology, which may provide important information regarding the role of cytokines in the progression of CKD. It is possible that the novel proteomic technology will become valuable tool to diagnosis and monitor the progression of CKD in the future. |
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