ABC | Volume 112, Nº4, April 2019

Original Article de Faria et al Inflammatory score and resistant hypertension Arq Bras Cardiol. 2019; 112(4):383-389 NO synthase. 10 It down regulates TNF- α production from macrophage by inhibiting nuclear transcription factor NF‑kappa B. 11,12 On the other hand, IL-6 inhibits adiponectin expression and secretion in 3T3-L1 adipocytes in vitro . 13 Additionally TNF- α increases the secretion of leptin, 14 which in turn stimulates the SNS. 15 Since cytokines and adipokines have interconnected roles, we aimed with this study (1) to develop an integrated measure of several circulating cytokines/adipokines among subjects with RHTN and mild to moderate hypertension (HTN), and (2) to assess the potential impact of this inflammatory score (IS) on resistance to antihypertensive treatment. Population and methods A convenience sample of 112 subjects diagnosed with RHTN attending the Specialized Outpatient Clinic in RHTN of the University of Campinas (UNICAMP, Campinas, Brazil) and 112HTNattending theHypertensionClinic of Valinhos (Valinhos, Brazil) were consecutively enrolled in this cross‑sectional study. RHTN was defined according to American Heart Association Statement as either (1) the subjects whose BP levels remain above goal (≥ 140/90 mmHg) despite concurrent use of three or more antihypertensive drugs of different classes, or (2) those with controlled BP levels using four or more antihypertensive medication. Ideally, one of the agents should be a diuretic, and all agents should be prescribed at optimal doses. 16 Patients with controlled BP using three or less antihypertensive drugs, or not yet controlled using two or less of these medications were classified as having HTN (grade 1 and grade 2 hypertension). 17 A 6-month period follow-up for screening and exclusion of secondary causes of hypertension was performed to guarantee a precise diagnosis for HTN and “true” RHTN. The exclusion criteria were compounded with renal artery stenosis, coarctation of the aorta, pheochromocytoma, primary hyperaldosteronism (aldosterone to renin ratio > 20 ng.dL -1 per ng.mL -1 .h -1 ), Cushing syndrome, obstructive sleep apnea‑hypopnea syndrome (patients withprevious polysomnographic diagnosis, or classified as high risk by the Berlin questionnaire). This period also included pill count to exclude the lackof BPcontrol due topoormedicationadherence, 18 and ambulatory BP monitoring (ABPM) to exclude white coat hypertension. We also excluded patients with symptomatic ischemic heart disease, impaired renal function, chronic kidney disease (creatinine clearance < 30 mL/min/1.73m 2 ) and liver disease (medical history, and platelet and transaminase levels). Inclusion criterion was age over 18 years old. Blood pressure measurements Office systolic BP (SBP) and diastolic BP (DBP) were assessed by a trained health professional according to the European Society of Hypertension guidelines for the management of arterial hypertension. 17 We used a validated digital sphygmomanometer (HEM-907XL, OMRON Healthcare Inc., Bannockburn, IL, USA). Ambulatory BP measurement was performed using an automatic oscillometric monitor (Spacelabs90207, SpacelabsInc, Redmon, WA, USA). Patients were instructed to maintain normal daily activities and record their 24-hour activities in a personal diary. Body composition The body compositionwas determinedby theBioimpedance Analyser 450 device (Biodynamics Corporation, Seattle, WA, USA) to assess fat-free mass and fat mass (FM). Briefly, the method is based on tetrapolar bioelectrical impedance (electrodes on feet and hands) to estimate mass and fluid compartments of the body. The measurements were performed after an 8-hour fast, and patients were instructed to avoid physical activity and smoking prior to the exam. Biochemical tests Blood samples were collected at morning after an 8-hour fast from patients in the sitting position. Plasma levels of aldosterone and renin were measured by radioimmunoassay (Immunotech SAS, Marseille, France), while the cytokines and adipokines – TNF-alpha, IL-6, IL-8, IL-10, leptin and adiponectin – were measured using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., Minneapolis, USA), according to the manufacturer’s instructions. Creatinine clearance was calculated from 24h-urine creatinine level, urine flow rate, and plasma creatinine concentration as the removal rate per minute divided by plasma creatinine concentration. Statistical analyses Continuous variables were expressed as mean and standard deviation or median (1 st and 3rd quartiles), according to data distribution assessed by the Kolmogorov–Smirnov test. Unpaired Student’s t-test or the Mann Whitney test was applied to compare continuous data between the RHTN and HT. Categorical variables were presented in frequencies and percentages compared by chi-square or Fisher’s exact test. Pearson or Spearman tests was used to assess correlation of continuous data. Multiple logistic regression analyses were performed to evaluate the association of IS with resistance to antihypertensive treatment, adjusting for potential confounders. For IS calculation, the values of plasma cytokine/adipokine (TNF-alpha, IL-6, -8, -10, leptin and adiponectin) were divided into tertiles, and a score ranging from 1 (lowest tertile) to 3 (highest tertile) was assigned to them. The IS was considered as the sum of each pro-inflammatory cytokine score (TNF‑alpha, IL-6, IL-8 and leptin) from which adiponectin and IL-10 – both anti-inflammatory cytokines – scores were subtracted for each subject. The analyses were performed using the software SigmaPlot (version 12, Systat Software, Inc., San Jose, CA USA, www. systatsoftware.com) and GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad. com). The level of significance accepted was alpha 0.05. Results General characteristics of both hypertensive groups are described in Table 1. Body fat parameters (body mass index - BMI, waist circumference - WC and FM) revealed to be increased in the RHTN subjects, as well as lipid profile, glycated hemoglobin and aldosterone levels compared to their counterparts. Compared with HTN, RHTN individuals used 384