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The longest duration for serum sample storage was 9?months. Blood and urine samples were studied by biochemists with Human NGAL sandwich ELISA kit (NGAL, B?OPORTO Diagnostics Immunoassay kit, BioPorto Diagnostic A/S, Gentofte, Denmark). First, 100?��l samples were put into wells coated with anti-NGAL monoclonal antibody and incubated. Then bound NGAL incubated with another antibody marked with biotin was washed again, and as such this process removed extra antibody. Later, 100?��l streptavidin conjugated with horse radish peroxidase was put into each well, incubated and washed once more. The first three incubations were performed with the shaker speed 200/min for 60?min. Finally, 100?��l chromogen substrate (tetramethyl benzidine) was put into each well and incubated for 10?min. The reaction was stopped with stop solution http://www.selleck.cn/products/PD-0332991.html and read via ELISA reader (Rayto RT�C2100 C) at a wavelength of 450?nm within 30?min. The venous blood samples underwent centrifugation (at 4��C) at 3500 rpm for 10?min. The samples were stored http://www.selleckchem.com/products/a-1155463.html at ?80��C until analysis. Serum aldosterone levels were studied via manual ELISA method (Aldosterone DiaMetra kit, DiaMetra Company, Milano, Italy). Plasma Renin activity was studied via Radio Immuno Assay method (RIAZEN ZenTech kit, ZenTech s.a, Angleur, Belgium). All data were analysed using SPSS statistics version 17 (SPSS Inc., Chicago, IL, USA). The student's t-test was used for the analysis of the binary group of normally distributed variables. Mann�CWhitney U-test was used for non-parametric binary assessments. Pearson correlation analysis was performed for correlation analysis. One-way anova test for multi-group variance analysis was used. Intergroup comparisons were performed by post-hoc Bonferroni analysis. Values were given as mean?��?SD. Prevalence of patients' outcomes according to plasma NGAL categorical variables (>1000 and http://www.selleckchem.com/products/cb-5083.html calculated by Kaplan�CMeier and Log-Rank tests. A receiver operator characteristics curve (ROC curve) analysis was performed to identify the sensitivity and specificity of cut-off values of biomarkers (pNGAL, uNGAL) and Child-Turcot-Pugh (CTP) score to predict mortality. Between groups comparison of area under the ROC curve (AUC) was performed with the method developed by DeLong et?al. [20]. The value of P?