Twenty-five (25) RHTN subjects  [15 non-T2D and 10 T2D, 15 (60%) females and 10 (40%) males], regularly followed in the ambulatory service of cardiovascular clinical pharmacology, complying with pharmacological prescription for HTN and T2D, were recruited to participate in this transversal study. The diagnosis of resistant hypertension required a good office blood pressure measurement technique and ambulatory blood pressure monitoring (ABPM) to confirm persistently elevated blood pressure levels . Pseudoresistance cases, including lack of blood pressure control secondary to poor medication adherence, were properly observed and excluded . White coat hypertension (WCH) was excluded by ABPM . Regarding obstructive sleep apnea (OSA), only patients classified as "low risk" by Berlin sleep questionnaire were enrolled . Resistant hypertension include patients whose blood pressure is uncontrolled with use of more than three medications or patients whose blood pressure is controlled, but required four or more medications to achieve blood pressure goals . All subjects provided written informed consent and the study was approved by the local ethics committee.
The exclusion criteria comprised: acute or moderate-severe renal dysfunction, non-complied pharmacological prescription, use of beta-blockers within the last six months, severe obesity (body mass index ≥ 35 kg/m2), heart failure (ejection fraction < 50%), valvular heart disease, cardiomyopathies, primary hyperaldosteronism [aldosterone:PRA ratio > 20 ng per 100 mL per ng.ml(-1)h(-1)], sleep apnea (classified as "high risk" by the Berlin sleep questionnaire), atrial fibrillation, sick sinus syndrome, supraventricular and ventricular tachycardias, aortic disease (Marfan's syndrome, coarctation of the aorta, aneurysms or aortic surgery, etc), history of coronary artery disease or proven coronary artery disease by coronary angiography or noninvasive tests, familial hyperlipidemia, asthma or chronic obstructive lung disease, pregnancy or oral contraceptive use, connective tissue disorders, neurological problems, malignancies, psychiatric diseases, other than T2D endocrinological diseases, smoking, alcohol use and drug abuse.
Blood pressure measurements
Blood pressure was assessed by considering the orientations of the last guideline on hypertension of the European Society of Cardiology . Blood pressure (SBP - systolic blood pressure/DBP - diastolic blood pressure) was measured three times for each subject using a digital sphygmomanometer (Omron HEM-711DLX) on the right upper arm in the sitting position after a 10-minute rest. The average of two consecutive measurements was used with a variation lower than 5 mmHg.
All subjects underwent the following laboratory tests: hemogram, serum fasting glucose, glycolized hemoglobin (HbA1c), serum urea and creatinine, serum total cholesterol, serum LDL-cholesterol fraction, serum HDL-cholesterol fraction, serum triglycerides, serum uric acid, serum sodium and potassium, and plasma adiponectin levels (Quantikine® Human total adiponectin/Acrp30 Immunoassay DRP 300, R&D Systems, Inc., Minneapolis, USA).
All subjects were submitted to standard transthoracic echocardiography in the left lateral decubitus position by using a Vivid 7 Pro machine with a 2.5 Mhz probe (General Electric, Florida, USA). Standard transthoracic views were used to determine end-diastolic and end-systolic volumes, stroke volume index, left ventricular ejection fraction (LVEF), transmitral E and A waves velocities, E/A ratio, tissue doppler velocity of the mitral annulus and left ventricular mass index (LVMI). The left ventricular diastolic dysfunction (LVDD) was assessed by the Omnen SR and Nishimura RA algorithm. The echocardiographic examination was performed by only one experienced cardiologist examiner. There was no intra or inter-observer measurement variability.
Heart rate variability
Heart rate variability (HRV) parameters were derived from the recording of 24-hour Holter monitoring and analyzed in time and frequency domains. Measures were stratified into three time periods for time domain: 24 hour period (24 h), day time period (DT), 1 p.m. to 5 p.m. and night time period (NT), from 2 a.m to 6 a.m. Frequency domain measures were stratified into two periods of one hour each at 3 a.m. (night time period - NT) and 3 p.m. (day time period - DT). A three-channel, 24-hour Holter recording was obtained from each subject using the Cardio light digital 24-hour recorder device and the CardioSmart Institutional CS 550 software (Cardio Sistema Comércio e Indústria Ltda, São Paulo, SP, Brazil).
Time domain HRV parameters included the following measures [6
rMSSD (ms): Square root of the mean of the squares of differences between successive RR intervals.
SDNN (ms): Standard deviation of all normal RR intervals in 24-hour Holter recording.
SDANN (ms): Standard deviation of RR intervals means in all 5-minute segments of 24-hour recording.
pNN50 (%): Percentage of differences between successive RR intervals that are greater than 50 ms.
Frequency domain measures were calculated using the Fast Fourier Transform (FFT) to break down the time series to its underlying periodic function. Frequency domain HRV parameters included the following measures [6
Low frequency (LF) and high frequency (HF) measured in normalized units, which represent the relative value of each power component in proportion to the total power minus the very low frequency (VLF) component. Normalized LF (LF nu) was calculated as LF power in normalized units LF/(total power-VLF) × 100, and normalized HF (HF nu) as HF power in normalized units HF/(total power-VLF) × 100. Low frequency (LF) and high frequency (HF). LF nu and HF nu denote the energy in the heart period power spectrum between 0.04 and 0.15 Hz (which is due to the joint action of the vagal and sympathetic components on the heart, with a predominance of the sympathetic ones) and 0.15 and 0.40 Hz (which corresponds to the respiratory modulation and is an indicator of the performance of the vagus nerve on the heart), respectively. "Day time" and "night time" were established at 3:00 p.m. and 3:00 a.m., respectively, in order to collect HRV data during wake and sleep periods.
Data were expressed as mean (μ) and standard deviation (SD) or mean (μ) and standard error of the mean (SEM) for HRV measures. Unpaired groups were compared using Mann-Whitney U test while correlation analysis were performed using Spearman's rank test. Fisher's exact test was used to determine whether a certain group had significantly different proportion of a particular characteristic. The level of statistical significance accepted was less than 0.05. All data were entered into a spreadsheet program (MS Excel Microsoft Corp, Phoenix, Arizona, USA) for statistical analysis. Analytical statistics were performed by Analyse-it version 2.21 Excel 12+ (Analyse-it Software Ltd., Leeds, UK), a statistical add-in program for Excel (MS Excel Microsoft Corp, Phoenix, Arizona, USA).