Angiostatin Levels in Systolic Heart Failure Patients with Chronic Kidney Disease

42 Int J Med Biochem According to the literature, local synthesis of angiostatin following acute kidney disease suggests a possible role for angiostatin in this activity [10]. However, studies examining the relationship between plasma angiostatin and the characteristics and laboratory findings of systolic HF patients with CKD are limited. The hypothesis of this study was that an increase in angiostatin in the plasma might contribute to the impairment of angiogenesis in HF patients with CKD, and the relationship with the laboratory findings and medications of the patients were investigated. Materials and Methods Study sample and study protocol This comparative, cross-sectional study was conducted with a total of 69 individuals: HF patients with CKD (n=29) and participants without disease (n=40) who presented at the nephrology or internal medicine outpatient facilities of Gulhane Training and Research Hospital, Ankara, Turkey, between 2011 and 2013. All of the procedures applied were performed in accordance with the ethical standards of the committees on human trials (institutional and national) and the Declaration of Helsinki. This study was approved by Gulhane Education and Research Hospital local ethics council with protocol number 1491-181-12/1539-491 on February 28, 2012. The EF was less than 40% and between 40% and 50%, respectively, for the patients with HFrEF and HFmrEF. All of the patients and the control group participants were older than 18 years of age. In all, 17 of the participants in the control group were male and 23 were female. In the patient group, 17 were male, and 12 were female. The diagnosis of systolic HF was based on the symptoms, a physical examination (European Society of Cardiology, 2016), echocardiographic results, and N-terminal pro-brain natriuretic peptide (NT-proBNP) level. Patients with rheumatic disease or clinical signs of infection, atrial fibrillation, chronic obstructive pulmonary disease, high C-reactive protein (CRP) value (>5 mg/L) and/or known malignancy were excluded from the study. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to determine the glomerular filtration rate (GFR) [11]. There were 15 patients with diabetes mellitus in the patient group and all were on insulin treatment. The serum angiostatin level of the HF patients with CKD and the control group were compared. The angiostatin level and the clinical findings of demographic features, laboratory results, comorbidities, and medications in the HF patient were compared with the CKD group to assess possible relationships. After an overnight fast, baseline blood samples were drawn from an antecubital vein in each participant into a BD Vacutainer venous blood collection tube containing clot activator and gel for serum isolation (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Centrifugation at 2000 g for 10 minutes was performed to separate the serum. A portion was used for analysis of the biochemical parameters without freezing, while 2 to 3 mL of the serum sample was aliquoted and immediately frozen at −80°C for further analysis of angiostatin. High sensitivity C-reactive protein (hs-CRP) in the serum was evaluated using the immunoturbidimetric fixed rate method and an AU5800 autoanalyzer (Beckman Coulter, Inc., Brea, CA, USA). Enzymatic and colorimetric methods were used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting blood glucose, urea, and creatinine with the same autoanalyzer. An automated blood cell counter (ABX Pentra 120, Horiba Ltd., Kyoto, Japan) was used for complete blood count analysis. The angiostatin level was analyzed using quantitative enzyme-linked immunosorbent assay (ELISA) kits (Cat.No: CKE90461; Hangzhou Eastbiopharm Co., Ltd, Hangzhou, China). Measurements were made using the Bio-Tek Synergy HT ELISA plate reader (Biotek Instruments Inc., Winooski, VT, USA). The intra-assay coefficient of variation (CV) and inter-assay CV results were <10% and <12%, respectively, while the minimum detectable dose of angiostatin was less than 20 ng/mL. Statistical analysis The clinical characteristics, history of medications, and the laboratory results of the participants were compared according to the angiostatin level. SPSS for Windows, Version 16.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses. Discontinuous variables were shown as numbers and percentages for the descriptive statistics, and mean±SD or median (25th–75th percentile, interquartile range) was used for the evaluation of continuous variables. Normality of the data was evaluated with the Kolmogorov-Smirnov test. The Mann-Whitney U test was applied to assess differences between independent groups and the Spearman test was used for nonparametric correlations. P values of less than 0.05 were considered statistically significant.