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Exclusion criteria included active malignancy, severe heart, lung or liver disease, stroke, chronic infection, e.g. tuberculosis within 1 year of starting the study, and any immunological or inflammatory disorders. A complete medical history and physical examination were performed on all subjects. All subjects fasted for at least 12 ADAMTS12 h overnight before all blood drawing. Complete blood counts, blood urea nitrogen (BUN), serum creatinine and comprehensive serum chemistries were measured. The serum concentration of creatinine was measured using the enzymatic method. The glomerular filtration rate (GFR) was estimated from calibrated serum creatinine with the 2009 CKD-EPI creatinine equation [12]. The study was approved by the Institutional Review Boards of the Royal Thai Army Medical Department, Bangkok, Thailand. All participants gave their written and informed consent. Urinary biomarker for kidney injury Urinary albumin and creatinine concentrations were measured and expressed as the urinary albumin creatinine ratio (UACR). Diabetic nephropathy status was determined by measuring UACR in at least two of the last three urine specimens. Patients were divided into three groups according to levels of UACR and urine protein: normoalbuminuria (UACR Alpelisib datasheet (UACR 30�C300 mg albumin/g creatinine) and overt nephropathy (UACR >300 mg albumin/g creatinine and/or persistent proteinuria). The urine angiotensinogen was performed using a commercially available ELISA assay (Angiotensinogen ELISA JP27412 Kit; Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan) that specifically detects human angiotensinogen in serum, urine and cell culture supernatants. The assay was performed according to the manufacturer's protocols. Statistical analysis Data are given as mean �� SD for continuous variables or as a percentage in categorical variables. Statistical analysis was performed using SPSS, version 15. Either http://www.selleckchem.com/products/SB-431542.html the two-sample t-test or Mann�CWhitney rank-sum test was used for continuous variables. For multiple comparisons, ANOVA was used followed by the least significance difference test. Spearman correlation coefficients were used as appropriate to test correlations between urine angiotensinogen and other variables. A multivariate model using a stepwise regression analysis was performed to correct for confounders. Receiver-operating characteristics (ROC) analysis was used to calculate the area under the curve (AUC) for angiotensinogen and to find the best cut-off values for identifying diabetic nephropathy. A P