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

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          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 ≧80 ml/min/1.73 m<sup>2</sup>, n = 3) and group B (eGFR ≤40 ml/min/1.73 m<sup>2</sup>, 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) α 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-α, vascular endothelial growth factor (VEGF), E-selectin, Fas, intercellular adhesion molecule 1, interleukin 2, matrix metalloproteinase (MMP) 2 and transforming growth factor β 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-α 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-α 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-α, 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.

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          Most cited references 18

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          Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit.

          Serum creatinine is not an ideal marker of renal function in patients with acute kidney injury (AKI). Previous studies demonstrated that urinary IL-18 is increased in human AKI. Thus, whether urine IL-18 is an early diagnostic marker of AKI was investigated. A nested case-control study was performed within the Acute Respiratory Distress Syndrome (ARDS) Network trial. AKI was defined as an increase in serum creatinine by at least 50% within the first 6 d of ARDS study enrollment. A total of 400 urine specimens that were collected on study days 0, 1, and 3 of the ARDS trial were available from 52 case patients and 86 control patients. The data were analyzed in a cross-sectional manner and according to the time before development of AKI. The median urine IL-18 levels were significantly different at 24 and 48 h before AKI in case patients as compared with control patients. On multivariable analysis, urine IL-18 values predicted development of AKI 24 and 48 h later after adjustment for demographics, sepsis, Acute Physiologic Assessment and Chronic Health Evaluation (APACHE) III score, serum creatinine, and urine output. Urine IL-18 levels of >100 pg/ml are associated with increased odds of AKI of 6.5 (95% confidence interval 2.1 to 20.4) in the next 24 h. On diagnostic performance testing, urine IL-18 demonstrates an area under the receiver operating characteristic curve of 73% to predict AKI in the next 24 h. The urine IL-18 values were also significantly different between survivors and nonsurvivors (P < 0.05), and on multivariable analysis, the urine IL-18 value on day 0 is an independent predictor of mortality. Urinary IL-18 levels can be used for the early diagnosis of AKI. Urine IL-18 levels also predict the mortality of patients who have ARDS and are in the intensive care unit.
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            Progress in protein and antibody microarray technology.

            The success of genome sequencing projects has led to a shift from the description of single molecules to the characterisation of complex samples. At the same time, there is growing interest not only in studying organisms at the genomic level, but in the characterization of their proteome. Such a task would not be possible without the availability of appropriate technologies. Protein and antibody microarray technologies are, in addition to two-dimensional gel electrophoresis followed by mass spectrometry, two of the most propitious technologies for the screening of complex protein samples. Nevertheless, to succeed, protein and antibody microarrays have to overcome their current limitations. This review aims to introduce these new technologies and highlights their current prospects and limitations.
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              Multiplexed sandwich assays in microarray format.

              Antibody arrays have evolved into powerful tools for quantifying proteins and qualifying their state of activation in complex biological samples. This level of analysis holds tremendous promise as part of a diagnostic or prognostic platform. In particular, multiplex sandwich ELISA assays performed with microarrays in the wells of multi-well plates enable high-throughput analysis of multiple samples with multivariate readouts. Here, we compare and review recent advances in antibody microarray technology and describe its promises towards the systematic analysis of complex biological samples, ranging from profiling patient sera to studying intracellular signaling.

                Author and article information

                Am J Nephrol
                American Journal of Nephrology
                S. Karger AG
                December 2006
                19 December 2006
                : 26
                : 5
                : 483-490
                Institute of Nephrology, Zhong Da Hospital, Southeast University, Nanjing, China
                96871 Am J Nephrol 2006;26:483–490
                © 2006 S. Karger AG, Basel

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                Page count
                Figures: 2, Tables: 5, References: 26, Pages: 8
                Self URI (application/pdf):
                Original Report: Patient-Oriented, Translational Research


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