Abstract
Aims
Nebivolol is a selective β1-receptor antagonist with vasodilating properties. In patients with essential hypertension, we tested the hypothesis that nebivolol increases systemic and renal nitric oxide (NO) availability using L-NG-monomethyl arginine (L-NMMA) as an inhibitor of NO production.
Methods
In a randomized, placebo-controlled, crossover study, patients with essential hypertension were treated with nebivolol for five days, along with a standardized diet and fluid intake. We examined the acute effects of systemic NO synthase inhibition with L-NMMA on brachial blood pressure (bBP), pulse wave velocity (PWV) and central blood pressure (cBP) estimated by applanation tonometry, glomerular filtration rate (GFR), fractional excretion of sodium (FENa), urinary excretion of both aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ), and plasma concentrations of nitrate/nitrite (p-NOx) and vasoactive hormones after five days' treatment with placebo and nebivolol.
Results
Nebivolol significantly reduced PWV, bBP, cBP and plasma renin, angiotensin II and aldosterone concentrations. The renal parameters, p-NOx and plasma arginine vasopressin concentration were not changed by nebivolol. There was no difference between nebivolol and placebo in the response to L-NMMA, with LMMA inducing a similar increase in PWV, bBP and cBP and a similar decrease in GFR, uAQP2 and u-ENaCγ and FENa [mean change −0.62% (95% confidence interval {CI} −0.40 to −0.84) during placebo vs. −0.57% (95% CI −0.46 to −0.68; P = 0.564) during nebivolol treatment]. Vasoactive hormones were changed to a similar extend by L-NMMA during administration of nebivolol and placebo.
Conclusions
Nebivolol did not change p-NOx, and inhibition of NO synthesis induced the same response in blood pressure, GFR, renal tubular function and vasoactive hormones during nebivolol and placebo. Thus, the data did not support the hypothesis that nebivolol changes vascular and renal NO availability in patients with essential hypertension.
Keywords: arterial stiffness, blood pressure, essential hypertension, fractional excretion of sodium, nebivolol, nitric oxide
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT
Nebivolol decreases blood pressure in patients with hypertension.
Nebivolol is a selective β1-receptor blocker with vasodilator properties which are at least partly thought to be mediated through an increase in nitric oxide production.
The effect of nebivolol on renal nitric oxide is unknown.
WHAT THIS STUDY ADDS
Nebivolol treatment decreased blood pressure, pulse wave velocity and plasma renin concentration, but renal parameters and plasma nitrate/nitrite was unchanged.
All responses to nitric oxide inhibition were similar, indicating no change by nebivolol in vascular and renal nitric oxide in patients with essential hypertension.
Introduction
The clinical benefits of β-blockers are well established in the treatment of hypertension and cardiovascular disease. However, in treatment of essential hypertension, concerns have been raised regarding efficacy and adverse events with the use of traditional β-blockers when compared with other drug classes such as angiotensin-converting enzyme inhibitors and thiazide diuretics [1–3]. Nebivolol is a third-generation β-blocker which reduces blood pressure (BP) in a dose-dependent manner in hypertensive patients [4,5]. It has a high β1 selectivity without sympathomimetic activity [6], combined with vasodilator effects mediated through endothelium-derived nitric oxide (NO) [7–10]. The increased NO production caused by nebivolol may be mediated through β2- or β3-receptors [11,12]. In hypertensive patients with impaired endothelial function, nebivolol was found to increase both basal and stimulated endothelium-derived NO release [13]. In the kidney, NO promotes natriuresis and diuresis [14]. In diabetic rats, nebivolol was found to increase not only vascular, but also renal NO [15], but the effect of this agent on renal NO in essential hypertension has not previously been investigated.
In the present study of patients with essential hypertension, we hypothesized that nebivolol increases renal and systemic NO availability. NO synthesis can be inhibited by L-NG-monomethyl arginine (L-NMMA) [16,17]. Systemic infusion of L-NMMA reduces renal plasma flow, fractional excretion of sodium (FENa), urine output (UO) and renin secretion, and increases BP and arterial stiffness [18–22]. During treatment with placebo and nebivolol, we examined the acute effects of systemic NO synthase (NOS) inhibition by L-NMMA on: (i) renal tubular sodium and water transport; (ii) urinary excretion of aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ); (iii) plasma nitrate/nitrite (p-NOx); (iv) brachial blood pressure (bBP), central blood pressure (cBP) and pulse wave velocity (PWV); and (v) vasoactive hormones.
Materials and methods
Patients
Screening examination included medical history, physical examination, renography, 24-h BP measurement, electrocardiography, clinical biochemistry and urine albumin analysis. Inclusion criteria included: both men and women; age 40–70 years; and 24-h BP >135 mmHg systolic and/or 85 mmHg diastolic during amlodipine treatment of at least 5 mg. Exclusion criteria included: renography with suspicion of renovascular hypertension or urinary tract obstruction; p-metanephrine >100 ng l–1; p-normetanephrine >200 ng l–1, a history or clinical signs of phaeochromocytoma or diseases in the heart and lungs, thyroid gland or central nervous system; diabetes mellitus; malignancies; estimated glomerular filtration rate (eGFR) Modification of Diet in Renal Disease (MDRD) <30 ml min–1; urinary albumin >1.5 g l–1; clinically important hypokalaemia; lower urinary tract symptoms; breastfeeding; pregnancy; alcohol or medical abuse, allergy or intolerance towards nebivolol or amlodipine; and unwillingness to participate. Withdrawal criteria included: development of exclusion criteria or a sustained increase in bBP above 170/105 mmHg on amlodipine treatment. During L-NMMA infusion, a BP up to 200/115 mmHg was considered acceptable.
Recruitment and examinations took place between 13 August 2012 and 12 September 2013.
Design
The study was designed as a placebo-controlled, randomized, placebo-controlled, double-blinded, crossover trial.
Randomization
After inclusion, patients were allocated via computer-generated randomization in blocks of six to receive nebivolol or placebo for 5 days in a random order. Participants sequentially received a numbered bottle containing either nebivolol or matching placebo capsules. Treatment periods were separated by a washout phase of at least 2 weeks. The randomization was performed by the hospital pharmacy. The randomization code was kept in a sealed envelope until after the final visit of the last participant. Investigators, participants and other study personnel were blinded to treatment assignment for the duration of the study.
Study drugs
Nebivolol (5 mg; Menarini, Florence, Italy) and placebo were covered by a gelatine capsule, and were identical in appearance. They were given orally at 7:00 AM. L-NMMA (Bachem, Weil am Rhein, Germany) was dissolved in isotonic saline solution. Usual antihypertensive treatment was discontinued, and patients were given amlodipine 5 mg as sole antihypertensive treatment throughout the study.
Ethics
The study was approved by the Regional Committee on Biomedical Research Ethics (case number: 1-10-72-241-12) and Danish Health and Medicines Authority (EudraCT number: 2012-001113-31). It was carried out in accordance with the Declaration of Helsinki. A signed informed consent form was obtained from each patient.
Effect variables
The main effect variable was FENa. Other effect variables were 24-h BP, central diastolic blood pressure (cDBP), central systolic blood pressure (cSBP), PWV, augmentation index (Aix), plasma concentration of renin (PRC), plasma concentration of angiotensin II (p-AngII), plasma concentration of aldosterone (p-Aldo), plasma concentration of arginine vasopressin (p-AVP), free water clearance (CH2O), GFR, fractional excretion of potassium (FEK), urinary albumin, u-AQP2 and u-ENaCγ.
Recruitment
Patients were consecutively recruited by advertisements in local newspapers.
Number of patients
With a power of 90% and a significance level of 5%, a total of 18 patients were needed to detect a 0.002 difference in FENa [standard deviation (SD) 0.003]. Because incomplete voiding was expected in some patients, it was estimated that 25 patients should be included in the trial.
Experimental procedure
Examinations were carried out on the last day of the 5-day treatment period at the University Clinic in Nephrology and Hypertension, Department of Medical Research, Holstebro Hospital. Drug treatment was accompanied by a standard diet, as previously described [23,24]. The diet comprised three main meals and three minor meals. The diet was composed of 55% carbohydrates, 15% protein and 30% fat, and contained 11 000 kJ day–1. The sodium content was 150 mmol day–1. Patients were instructed to eat variedly from the diet until satiated. Daily fluid intake was 2.5 l; up to two cups of tea or coffee each day were allowed. No consumption of alcohol was allowed.
Before each examination, 24-h urine collection and 24-h BP were performed, and 24-h urine was analysed for creatinine, osmolality, sodium, potassium, albumin, AQP2 and ENaCγ.
After an overnight fast, patients arrived at 7:45 AM. The study drug was taken at 7:00 AM on the day of examination, while usual medication, including amlodipine, was taken after examination. Two indwelling catheters for blood sampling and administration of 51Cr-ethylene diamine tetra-acetic acid (EDTA) and L-NMMA were placed in both arms. Every 30 min after arrival, participants received an oral water load of 0.175 l of tap water. Urine was collected by voiding in the sitting or standing position. At all other times, patients were kept in a supine position in a quiet, temperature-controlled room (22–25 °C). At 11:00 AM, a bolus L-NMMA was given (4.5 mg kg–1 over 3 min) followed by a continuous infusion for 60 min (3 mg kg–1 h–1) [19].
Blood and urine samples were collected every 30 min from 9:30 AM to 1:00 PM, and analysed for 51Cr-EDTA, sodium, potassium, creatinine and osmolality. The first three clearance periods from 9:30 AM to 11:00 AM were defined as the baseline period. The baseline period was followed by four 30-min clearance periods. Blood samples were drawn at 11:00 AM, 12:00 AM and 1:00 PM for determination of PRC, p-Ang II, p-Aldo and p-AVP.
Measurements of cBP, AIx and PVW were performed before and during L-NMMA infusion (at 10:40 AM and 11:40 AM, respectively).
Blood pressure measurements
Twenty-four-h BP was measured using Kivex TM-2430 (Kivex, Hoersholm, Denmark). Measurements were taken every 15 min during the day and every 30 min overnight. During examinations, BP was recorded using the semiautomatic oscillometric device, Omron 705IT (Omron Matsusaka CO. Ltd., Matsusaka City, Japan).
Applanation tonometry
Recordings of carotid–femoral PWV and Pulse wave velocity (PWA) were obtained by applanation tonometry (SphygmoCor® CPV system®, AtCor Medical, Sydney, NSW, Australia) as double recordings by trained observers. An operator index of 80 or more was required to accept recordings of a peripheral pulse-wave form. Only duplicate recordings meeting the quality requirements were included in the final analysis [21–23].
Biochemical analyses
Blood samples for measurements of vasoactive hormones were centrifuged for 10 min at 2200 G and 4 °C. Plasma was separated from blood cells and kept frozen until assayed [22]. p-Aldo was determined by radioimmunoassay using a kit from Demeditec Diagnostics GmbH, Kiel, Germany. The minimal detection level was 25 pmol l–1. The coefficients of variation were 8.5% (intra-assay) and 9.0% (inter-assay). PRC was determined using an immunoradiometric assay from CIS Bio International, Gif-Sur-Yvette, France. The minimal detection level was 1 pg ml–1. The coefficients of variation were 3.7–5.0% (inter-assay) and 0.9–3.6% (intra-assay) in the range 4–263 pg ml–1. p-Ang II and p-AVP were extracted from plasma using C18 Sep-Pak (Water associates, Milford, MA, USA), and subsequently determined by radioimmunoassay [25,26]. The antibody against Ang II was obtained from the Department of Clinical Physiology, Glostrup Hospital, Denmark. The minimal detection level was 2 pmol l–1. The coefficients of variation were 8% (intra-assay) and 12% (inter-assay). The antibody against AVP was a gift from Professor Jacques Dürr, Miami, FL, USA. The minimal detection level was 0.2 pmol l–1. The coefficients of variation were 9% (intra-assay) and 13% (inter-assay).
The plasma concentrations of NO metabolites (NO3– + NO2–, NOx) were measured using a commercially available assay from R&D Systems, Minneapolis, MN, USA. The assay involves the conversion of NO3– to NO2– by the enzyme nitrite reductase. After conversion of NO3– to NO2–, the spectrophotometric measurement of NO2– is accomplished by using the Griess reaction. The detection of total nitrite is then determined as a coloured azo-dye product of the Griess reaction that absorbs visible light at 540 nm. The minimal detection level was 0.25 µmol l–1. The coefficients of variation were 1.8 % (intra-assay) and 3.9 % (inter-assay).
Urine samples were kept at –20 °C until assayed. U-ENaCγ was measured by radioimmunoassay as previously described [20,22,24]. Antibodies were raised against the synthetic ENaCγ peptide in rabbits and affinity purified as described previously [27]. The minimal detection level was 48 pg per tube. The coefficients of variation were 6.7% (intra-assay) and 14% (inter-assay). U-AQP2 was measured by radioimmunoassay as described previously [22,28,29]. Antibodies were raised in rabbits to a synthetic peptide corresponding to the 15 COOH-terminal amino acids in human AQP2, to which was added an NH2-terminal cysteine for conjugation and affinity purification. The minimal detection level was 34 pg per tube. The coefficients of variation were 5.9% (intra-assay) and 11.7% (inter-assay). The anti-AQP2 antibody was a gift from Soren Nielsen, Department of Biomedicine, Aarhus University, Denmark.
GFR was estimated using the constant infusion clearance technique with 51Cr-EDTA as reference substance. More than 15% variation in GFR in the three clearances that defined baseline led to the exclusion of clearance-related analysis.
Plasma and urine concentration of creatinine, sodium and potassium and were determined by routine methods at the Department of Clinical Biochemistry.
Calculations
FENa and FEK were calculated according to the formula FEX = (Xu * V/Xp)/GFR, where V is urine flow in ml min–1 and Xu and Xp are urine and plasma concentrations, respectively, of X. CH2O was calculated according to the formula CH2O = UO – Cosm, where Cosm is osmolar clearance.
Statistics
When normality was present, data are presented as means ± SD; if it was not present, data are shown as medians, with 25% and 75% percentiles in brackets. The major effect variable, FENa, is also presented as mean with the 95% confidence interval (CI) in brackets. The Student's paired t-test or Wilcoxon signed-rank test was used to perform paired comparison between groups at baseline and to perform paired comparison in responses to L-NMMA between the groups. A general linear model for repeated measures (GLM) was performed to test deviation in effect variables during the experimental procedure. If data did not show normality, they were logarithmic transformed before the GLM. Friedman's test was used to test deviations of vasoactive hormones within treatment, during the experimental procedure. A multiple regression analysis was performed to examine if an interaction between independent variables [age, weight, body mass index (BMI), baseline creatinine and baseline SBP and DBP] and the primary effect variable (change in FENa) was present. Statistical significance was defined as P < 0.05. Statistical analyses were performed using PASW version 20.0.0 (SPSS Inc.; Chicago, IL, USA).
Results
Demographics
Thirty-four patients were screened for participation in the trial (Figure1). Nine patients were not included because of their 24-h BP being below the level allowed in the inclusion criteria (5), withdrawal of consent (3) or unilateral hydronephrosis (1). Thus, 25 patients were included in the trial. One patient withdrew consent in the first treatment period and was withdrawn from the trial. The 24 patients (10 females, 14 males) who completed the trial, had a mean BMI 26.4 ± 3.4 kg m–2, age 60 ± 7 years, 24-h BP 142/86 ± 8/5 mmHg, estimated GFR (MDRD) 83 ± 16 ml min–1, p-creatinine 78 ± 14 µmol l–1, urine albumin 4 (1; 9) mg l–1, p-metanephrine 40 (29; 52) ng l–1 and p-normetanephrine 54 (50; 68) ng l–1. One patient was an active smoker, 11 were former smokers and 12 were nonsmokers.
Figure 1.
Flow chart showing patient flow in the study and the reasons for exclusion of the excluded patients. BP, blood pressure
Effect of nebivolol and L-NMMA on blood pressure
Nebivolol reduced bBP and heart rate in 24-h BP measurements (Table 1). Heart rate and bBP were both reduced to a similar extent during the day and the night (Table 1). Nebivolol reduced bBP during the examination day (Figure2). In both groups, bBP peaked rapidly after the 3-min L-NMMA bolus infusion, and then gradually declined over the first 15 min of infusion (Figure2). During the remaining 50 min of infusion, the bBP changes were the same in both groups (P = 0.884 for bSBP and P = 0.439 for bDBP with the GLM). An average from this 50 min plateau period was then compared with the baseline BP. L-NMMA caused a significant increase in bSBP (14 ± 7 mmHg in placebo vs. 15 ± 7 mmHg in nebivolol) and bDBP (8 ± 4 mmHg vs. 8 ± 3 mmHg). The increases were not significantly different between treatments (P = 0.261 for bSBP and P = 0.495 for bDBP). Heart rate dropped significantly in both treatments, in response to L-NMMA, and the reduction was more pronounced during placebo (–5 ± 3 vs. -3 ± 1, P < 0.001).
Table 1.
Effect of nebivolol on 24-h ambulatory blood pressure and 24-h urine collection in 24 patients with essential hypertension
| Placebo | Nebivolol | Pvalue | |
|---|---|---|---|
| 24-h SBP (mmHg) | 139 ± 12 | 129 ± 10 | < 0.001 |
| Daytime SBP (mmHg) | 144 ± 12 | 133 ± 11 | < 0.001 |
| Night-time SBP (mmHg) | 122 ± 12 | 114 ± 10 | < 0.001 |
| 24-h DBP (mmHg) | 86 ± 7 | 78 ± 6 | < 0.001 |
| Daytime DBP (mmHg) | 89 ± 7 | 80 ± 7 | < 0.001 |
| Night-time DBP (mmHg) | 74 ± 7 | 68 ± 7 | < 0.001 |
| 24-h heart rate (beats per minute) | 66 ± 8 | 56 ± 8 | < 0.001 |
| Daytime heart rate (beats per minute) | 68 ± 9 | 58 ± 8 | < 0.001 |
| Night-time heart rate (beats per minute) | 57 ± 7 | 50 ± 7 | < 0.001 |
| Urine output (ml min–1) | 1.98 ± 0.49 | 1.94 ± 0.46 | 0.632 |
| CH2O (ml min–1) | -0.20 ± 0.52 | -0.30 ± 0.55 | 0.295 |
| U-Na (mmol 24 h–1) | 114 ± 34 | 120 ± 26 | 0.349 |
| FENa (%) | 0.46 ± 0.13 | 0.50 ± 0.11 | 0.103 |
| U-K (mmol 24 h–1) | 75 ± 17 | 72 ± 19 | 0.378 |
| FEK (%) | 11.1 ± 2.3 | 10.6 ± 2.1 | 0.365 |
| Creatinine clearance (mmol ml–1 m–2) | 110 ± 15 | 107 ± 16 | 0.211 |
| UAER (mg/24 h–1) | 6 (4;10) | 7 (5;9) | 0.109 |
| U-AQP2 min–1 (ng min–1) | 1.25 ± 0.25 | 1.32 ± 0.25 | 0.025 |
| U-AQP2/creatinine (ng mmol–1) | 135 ± 17 | 142 ± 20 | 0.020 |
| U-ENaCγ (ng min–1) | 0.74 ± 0.30 | 0.74 ± 0.29 | 0.989 |
| U-ENaCγ/creatinine (ng mmol–1) | 83 ± 41 | 81 ± 32 | 0.745 |
24-h ambulatory systolic (SBP) and diastolic (DBP) blood pressure, 24-h BP divided into daytime and night-time values, urine output, free water clearance (CH2O), urine excretion of sodium (U-Na) and potassium (U-K), fractional excretion of sodium (FENa) and potassium (FEK), creatinine clearance, urinary excretion rates of albumin (UAER), aquaporin-2 (u-AQP2 min–1) and γ-fraction of the epithelial sodium channel (u-ENaCγ min–1), and in relation to creatinine (u-AQP2/creatinine, u-ENaCγ/creatinine). 24-h BP was monitored and urine collected from 7:00 AM the day before the examination to 7:00 AM on the examination day. Data are shown as means ± standard deviation in brackets, or medians with 25 and 75 percentiles in brackets. Statistics were performed using the Student's paired t-test or Wilcoxon signed-rank test
Figure 2.
Effect of systemic nitric oxide inhibition with L-NG-monomethyl arginine (L-NMMA) during nebivolol treatment on brachial mean arterial blood pressure (MAP) in a randomized, placebo-controlled crossover study of 24 patients with essential hypertension. Baseline brachial blood pressure (bBP) was defined as a mean of the four measurements 30 min prior to L-NMMA infusion. A stable bBP was achieved for the last 45 min of the L-NMMA infusion. A mean of the seven bBP measurements from the last 45 min of the L-NMMA infusion was used to calculate changes from baseline. Values represent mean ± standard error of the mean.
Effect of nebivolol and L-NMMA on arterial stiffness and central BP
The effect of nebivolol and L-NMMA on PWV, AIx, cDBP and cSBP are shown in Table 2. Baseline PWV, cDBP and cSBP were significantly lower during nebivolol treatment than during placebo. Baseline AIx was not different between treatments. During L-NMMA infusion, PWV, AIx, cDBP and cSBP increased significantly but there was no difference between treatments.
Table 2.
Effect of nebivolol (5-day) and nitric oxide synthase inhibition on central BP and PWV in a randomized, placebo-controlled, crossover study of 24 patients with essential hypertension (n = 16)
| Before L-NMMA | During L-NMMA | P value | |
|---|---|---|---|
| PWV, m s–1 | |||
| Placebo | 8.3 ± 1.6 | 8.7 ± 1.6* | 0.171 |
| Nebivolol | 7.7 ± 1.2† | 8.3 ± 1.2* | |
| AIx, % | |||
| Placebo | 22 ± 11 | 25 ± 8 | 0.244 |
| Nebivolol | 20 ± 9 | 25 ± 8* | |
| cSBP, mmHg | |||
| Placebo | 138 ± 15 | 155 ± 17* | 0.612 |
| Nebivolol | 131 ± 10† | 148 ± 13* | |
| cDBP, mmHg | |||
| Placebo | 89 ± 8 | 97 ± 9* | 0.421 |
| Nebivolol | 83 ± 7† | 92 ± 8* | |
Pulse wave velocity (PWV), augmentation index (AIx), and central diastolic (cDBP ) and systolic (cSBP) blood pressure. Measurements are performed just before initiation of the L-NG-monomethyl arginine (L-NMMA) infusion (at 10:40 AM) and during the L-NMMA infusion (at 11:40 AM).Values are means ± standard deviation. The P values represent the probability of a difference in the response to L-NMMA between treatments. Statistics were performed using the Student's paired t-test or Wilcoxon signed-rank test. Statistically significant difference from baseline:
P < 0.05; statistically significant difference from placebo:
P < 0.05.
GFR and tubular function during baseline conditions (24-h urine)
In the 24-h urine collection made prior to examination, FENa [means (95% CI) 0.46% (0.41%; 0.52%) during placebo vs. 0.50% (0.46%; 0.55%) during nebivolol; P = 0.103], UO, CH2O, urinary excretion of sodium and potassium, FEK, creatinine clearance, urinary albumin excretion rate (UAER) and u-ENaCγ were not significantly different between treatments (Table 1). U-AQP2 was significantly increased by nebivolol.
GFR and tubular function during L-NMMA
Table 3 shows the effect of nebivolol on L-NMMA-induced changes in GFR, UO, CH2O, FENa, FEK and UAER. Using a GLM, the same response pattern was seen during nebivolol and placebo administration. During examination, FENa was similar at baseline during placebo and nebivolol [means (95% CI) 1.64% (1.27%; 2.01%) vs. 1.51% (1.16%; 1.85%); P = 0.144] and decreased to a similar extent during placebo and nebivolol [means (95% CI) –0.62% (0.40%; –0.84%) vs. –0.57% (–0.46%; –0.68%); P = 0.564]. GFR, OU, CH2O and FEK all decreased, and UAER increased to the same extend during placebo and nebivolol, during L-NMMA infusion. There was no interaction between the change in FENa and age, weight, BMI, baseline SBP and DBP, and baseline creatinine (P > 0.05 for all variables).
Table 3.
Effect of nebivolol and L-NMMA on GFR and tubular function in a randomized, placebo-controlled, crossover study of 24 patients with essential hypertension
| Period | Baseline | L-NMMA infusion | Post-L-NMMA | |||
|---|---|---|---|---|---|---|
| 0–90 min | 90–120 min | 120–150 min | 150–180 min | 180–210 min | P value(using a GLM) | |
| GFR (51Cr-EDTA clearance) | ||||||
| Placebo | 88.0 ± 11.9 | 77.5 ± 10.4* | 80.0 ± 11.3* | 81.8 ± 12.9* | 83.1 ± 14.8* | 0.567 |
| Nebivolol | 85.6 ± 11.1 | 73.4 ± 12.1* | 74.5 ± 9.3* | 79.9 ± 10.1* | 77.5 ± 12.8* | |
| P (GLM for nebivolol vs. placebo) | 0.252 | |||||
| Urine output (ml min–1) | ||||||
| Placebo | 6.5 ± 1.4 | 3.3 ± 1.3* | 3.0 ± 1.1* | 4.3 ± 1.3* | 5.1 ± 1.2 | 0.946 |
| Nebivolol | 6.4 ± 1.4 | 3.0 ± 1.5* | 2.9 ± 1.5* | 4.3 ± 1.7* | .5.0 ± 1.5 | |
| P (GLM for nebivolol vs. placebo) | 0.758 | |||||
| CH2O (ml min–1) | ||||||
| Placebo | 3.2 ± 1.1 | 1.1 ± 0.8* | 0.9 ± 0.7* | 2.0 ± 1.1* | 2.7 ± 1.1* | 0.943 |
| Nebivolol | 3.3 ± 1.0 | 1.0 ± 1.0* | 1.0 ± 1.0* | 2.0 ± 1.2* | 2.7 ± 1.1* | |
| P (GLM for nebivolol vs. placebo) | 0.985 | |||||
| FENa (%) | ||||||
| Placebo | 1.49 (1.11; 2.11) | 1.01 (0.53; 1.19)* | 0.87 (0.45; 1.19)* | 1.07 (0.65; 1.38)* | 1.13 (0.78; 1.65)* | 0.737 |
| Nebivolol | 1.43 (0.97; 1.97) | 0.74 (0.58; 1.06)* | 0.73 (0.37; 1.06)* | 1.09 (0.55; 1.32)* | 1.22 (0.78; 1.43)* | |
| P (GLM for nebivolol vs. placebo) | 0.517 | |||||
| FEK (%) | ||||||
| Placebo | 29.7 ± 9.4 | 21.7 ± 8.1* | 20.2 ± 8.7* | 20.0 ± 9.2* | 19.6 ± 9.2* | 0.724 |
| Nebivolol | 27.2 ± 8.8 | 20.1 ± 7.4* | 18.3 ± 7.4* | 17.2 ± 7.7* | 17.8 ± 8.7* | |
| P (GLM for nebivolol vs. placebo) | 0.413 | |||||
| UAER (mg h–1) | ||||||
| Placebo | 0.0 (0.0; 0.1) | 0.3 (0.1; 0.7)* | 0.2 (0.1; 0.7)* | 0.3 (0.2; 0.5)* | 0.3 (0.0; 0.4)* | 0.768 |
| Nebivolol | 0.0 (0.0; 0.1) | 0.3 (0.2; 0.6)* | 0.3 (0.2; 0.5)* | 0.2 (0.0; 0.3)* | 0.0 (0.0; 0.3)* | |
| P (GLM for nebivolol vs. placebo) | 0.986 | |||||
Glomerular filtration rate (GFR), urine output, fractional excretion of sodium (FENa), fractional excretion of potassium (FEK), free water clearance (CH2O) and urinary albumin excretion rate (UAER). Urine was collected every 30 min in the 90-min baseline period, during 60 min of L-NG-monomethyl arginine (L-NMMA) infusion, and 60 min after cessation of L-NMMA infusion. Data from three baseline periods are pooled and shown as one period. Data are presented as means ± standard deviation, or medians with 25 and 75 percentiles in brackets. Statistics were performed using a general linear model (GLM), the Student's paired t-test or the Wilcoxon signed-rank test. Data for FENa, FEK, UAER and UACR were logarithmically transformed before the GLM was performed. Statistically significant difference from baseline:
P < 0.05.
Table 4 shows the effect of nebivolol on L-NMMA-induced changes in u-AQP2 and u-ENaCγ. U-AQP2 and u-ENaCγ fell to the same extent during L-NMMA infusion, when measured as secretion rates. When adjusted to urinary creatinine excretion, only u-AQP2 decreased significantly in response to L-NMMA, whereas u-ENaCγ was unchanged. However, using a GLM, the same response patterns to L-NMMA in u-AQP2 and u-ENaCγ during nebivolol and placebo were demonstrated. Thus, the responses to L-NMMA in u-AQP2 and u-ENaCγ were not changed by nebivolol.
Table 4.
Effect of nebivolol and L-NMMA on the excretion of proteins from epithelial sodium channels and aquaporin-2 channels in a randomized, placebo-controlled, crossover study of 24 patients with essential hypertension
| Period | Baseline | L-NMMA infusion | Post-L-NMMA | |||
|---|---|---|---|---|---|---|
| 0–90 min | 90–120 min | 120–150 min | 150–180 min | 180–210 min | P (GLM within) | |
| U-ENaCγ (ng min–1) | ||||||
| Placebo | 0.38 (0.30; 0.70) | 0.33 (0.25; 0.48)* | 0.32 (0.29; 0.54)* | 0.32 (0.24; 0.55)* | 0.37 (0.24; 0.49)* | 0.284 |
| Nebivolol | 0.43 (0.32; 0.52) | 0.37 (0.28; 0.49)* | 0.40 (0.28; 0.60) | 0.41 (0.28; 0.61) | 0.37 (0.28; 0.68)* | |
| P (GLM for nebivolol vs. placebo) | 0.664 | |||||
| U-ENaCγ/creatinine (ng mmol–1) | ||||||
| Placebo | 44 (33; 60) | 44 (29; 53) | 44 (34; 53) | 44 (33; 51) | 42 (34; 54) | 0.097 |
| Nebivolol | 43 (37; 55) | 40 (36; 63) | 46 (36; 65) | 45 (35; 61) | 41 (34; 59) | |
| P (GLM for nebivolol vs. placebo) | 0.446 | |||||
| U-AQP2 (ng min–1) | ||||||
| Placebo | 1.56 ± 0.38 | 1.08 ± 0.28* | 1.05 ± 0.23* | 1.11 ± 0.27* | 1.17 ± 0.26* | 0.508 |
| Nebivolol | 1.48 ± 0.33 | 1.02 ± 0.28* | 1.01 ± 0.25* | 1.14 ± 0.29* | 1.13 ± 0.32* | |
| P (GLM for nebivolol vs. placebo) | 0.672 | |||||
| U-AQP2/creatinine (ng mmol–1) | ||||||
| Placebo | 147 ± 18 | 122 ± 19* | 115 ± 18* | 120 ± 18* | 126 ± 19* | 0.896 |
| Nebivolol | 148 ± 21 | 122 ± 21* | 116 ± 17* | 121 ± 16* | 124 ± 17* | |
| P (GLM for nebivolol vs. placebo) | 0.990 | |||||
Aquaporin-2 excretion rate (u-AQP2 min–1), creatinine-adjusted u-AQP2 excretion (U-AQP2/creatinine), excretion of the γ-fraction of the epithelial sodium channel (u-ENaCγ min–1) and creatinine-adjusted u-ENACγ (U-ENaCγ/creatinine). Data are shown as means ± standard deviation, or medians with 25 and 75 percentiles in brackets. P values represent the probability of a difference in the response to L-NG-monomethyl arginine (L-NMMA) (response from baseline to L-NMMA) between treatments. Statistics were performed using a general linear model (GLM), the Student's paired t-test or the Wilcoxon signed-rank test. U-ENaCγ and u-ENaCγ/creatinine were logarithmically transformed before the GLM was performed. Statistically significant difference from baseline:
P < 0.05.
p-NOx
Nebivolol did not change p-NOx (Table 5). p-NOx decreased in response to L-NMMA, but the decrease was only significant during nebivolol treatment. However, the responses to L-NMMA were not different between treatments.
Table 5.
Effect of nebivolol and L-NMMA on the plasma concentration of nitrates and vasoactive hormones in a randomized, placebo-controlled, crossover study of 24 patients with essential hypertension
| Baseline(90 min) | After 60-min L-NMMA infusion(150 min) | 60 min post-L-NMMA infusion(210 min) | Pvalue(difference in response) | |
|---|---|---|---|---|
| p-NOx (µmol l–1) | ||||
| Placebo | 9.5 (7.0; 14.8) | 10.0 (7.0; 13.0) | 9.5 (6.3; 12.8) | 0.194 |
| Nebivolol | 9.0 (7.3; 10.8) | 9.0 (8.0; 11.0) | 8.0 (7.0; 9.8)* | |
| PRC (ng l–1) | ||||
| Placebo | 4.0 (2.6; 8.4) | 3.8 (2.5; 7.9)* | 3.6 (2.4; 7.5)* | 0.594 |
| Nebivolol | 2.3 (1.7; 5.4)† | 2.4 (1.7; 5.1)*, † | 2.4 (1.7; 4.9)† | |
| p-AngII (ng l–1) | ||||
| Placebo | 7 (3; 16) | 6 (4;17) | 8 (4;14) | 0.392 |
| Nebivolol | 6 (4; 16)† | 7 (4; 12)* | 6 (3; 12) | |
| p-Aldo (pmol l–1) | ||||
| Placebo | 95 (71; 170) | 119 (82;192)* | 104 (71;159) | 0.188 |
| Nebivolol | 86 (57; 155)† | 109 (75; 190)* | 92 (69; 163) | |
| p-AVP (ng l–1) | ||||
| Placebo | 0.30 (0.30; 0.40) | 0.35 (0.23; 0.40) | 0.35 (0.23; 0.40) | 0.119 |
| Nebivolol | 0.30 (0.30; 0.40) | 0.35 (0.30; 0.50) | 0.40 (0.30; 0.40) | |
Plasma concentrations of nitrate/nitrite (p-NOx), renin (PRC), angiotensin II (p-AngII), aldosterone (p-Aldo) and arginine vasopressin (p-AVP) were measured before L-NG-monomethyl arginine (L-NMMA) infusion, after 60 min of L-NMMA infusion and 60 min after cessation of L-NMMA infusion on the examination day. Data are shown as medians with 25 and 75 percentiles in brackets. P values represent the probability of difference in response to L-NMMA (response from baseline to L-NMMA infusion) between treatments. The Student's t-test was used to test the difference in the response to L-NMMA between treatments. The Wilcoxon signed-rank test was used to test for a statistically significant difference from baseline
(P < 0.05), and from placebo
(P < 0.05).
Vasoactive hormones in the plasma
PRC, pAng II and p-Aldo were significantly lower during nebivolol (Table 5). PRC remained lower during L-NMMA infusion. PRC, p-AngII and p-Aldo responses to L-NMMA were not different between treatments. Nebivolol did not change p-AVP, and there were no significant differences in responses to L-NMMA between treatments.
Discussion
In the present placebo-controlled, randomized, double-blinded, crossover study, we investigated the effect of 5-day nebivolol treatment on renal tubular function and systemic haemodynamics during baseline conditions and during systemic NOS inhibition in patients with essential hypertension. To our knowledge, the effect of nebivolol on renal tubular function during NO inhibition has not been investigated previously in essential hypertension. It was our main hypothesis that nebivolol increases vascular and renal NO availability. However, nebivolol did not change the responses to systemic NOS inhibition in any of the measured variables. This suggests that nebivolol did not change NO availability in the kidneys or the vasculature in essential hypertension. It should be noted that estimating NO using an indirect method like NOS inhibition by L-NMMA provides supportive, rather than definitive, evidence of the NO-mediated effects of nebivolol. However, the finding is supported by the unchanged p-NOx during nebivolol. The transient and volatile nature of NO makes it unsuitable for most convenient detection methods, and p-NOx is used as a surrogate quantitative measure of NO production in the vasculature.
The vasodilator property of nebivolol has been confirmed in previous studies. In vitro incubation of isolated vessels and in vivo infusion in forearm vasculature with nebivolol was found to induce vasodilatation, which could be blocked by inhibition of NO synthesis [7–10]. Nebivolol was also found to increase NO release in cultured endothelial cells and isolated arteries [9,30–33]. The stimulatory effect of nebivolol may be mediated through β2- or β3-receptors [11,12,33]. Oral nebivolol in combination with bendroflumethiazide for 8 weeks increased both basal and stimulated endothelium-derived NO estimated by forearm venous occlusion plethysmography and intra-arterial infusions of acetylcholine and L-NMMA [13]. Similarly, in streptozotocin-induced diabetic mice and spontaneously hypertensive rats, nebivolol treatment for 8 weeks increased NO release from isolated arteries [34,35].
The reason for the lack of differences in the responses to L-NMMA and in p-NOx during nebivolol and placebo treatment is not clear. Several explanations are possible. First, a 5-day intervention with nebivolol was chosen; although increased NO-dependent vasodilatation to a nebivolol infusion has been demonstrated in essential hypertension [10], it cannot be excluded that a longer treatment period is required to increase basal NO in patients with essential hypertension. Secondly, patients were given amlodipine throughout the study period. Improved NO availability and improved endothelial function have been reported in spontaneously hypertensive rats and patients with essential hypertension during amlodipine treatment [36,37]. Thus, it cannot be excluded that amlodipine treatment may mask the effect of nebivolol on NO availability. Thirdly, NO was estimated using indirect methods, by measuring responses to NO inhibition and by the surrogate measure p-NOx. These parameters used to estimate vascular and renal NO may not be sensitive enough to detect changes in NO induced by nebivolol. Changes in BP and PWV induced by L-NMMA may be too crude estimates to detect changes in vascular NO availability. In the kidney, sodium excretion is highly sensitive to L-NMMA [19]. If renal NO were to change during nebivolol treatment, we would expect these changes to be revealed by measuring FENa during systemic L-NMMA infusion. In addition, p-NOx reflects not only NO production, but also dietary intake [38]. The decrease in p-NOx in response to L-NMMA was expected, but was subtle and only significant during nebivolol administration. If nebivolol increases vascular NO production, the additional NOx thereby generated may not be detected if the fraction is very small compared with that from dietary intake. Hence, a large pool of p-NOx generated from dietary intake could conceal a small increase in p-NOx by nebivolol. Fourthly, L-NMMA is an inhibitor of all three isoforms of NOS [16]. Although L-NMMA causes vasoconstriction [13], the mechanisms by which it increases BP have not been fully clarified. Selective neuronal NOS inhibition caused similar changes in vascular tone and BP to those of L-NMMA, but with preserved endothelium-dependent vasodilatation responses [39]. The effects of nebivolol on NO seems to be mediated through endothelial NOS [9,11,12,30–33] and if the effects of L-NMMA on BP and PWV are – at least, partially – due to inhibition of neuronal NOS, this could explain why no changes in responses to L-NMMA during nebivolol treatment were observed. Fifthly, a stimulatory agent such as flow, that creates shear stress on endothelial cells or acetylcholine may be necessary to reveal changes in NO release during nebivolol. The use of L-NMMA enables NO release under basal circumstances to be estimated. Previously, an increased response to L-NMMA in the forearm in mildly hypertensive patients was demonstrated during nebivolol-based therapy [13]. Hence, we would expect that an increased basal NO during nebivolol could be detected, but cannot exclude that a stimulatory agent, rather than an inhibitory agent such as L-NMMA, is necessary to reveal changes in vascular and renal NO.
The BP-lowering effect of nebivolol is well established [4,5,40], and, as expected, a reduction in both central BP and bBP during nebivolol treatment was found. The reduction in PWV is probably secondary to the reduction in BP. Similar reductions have previously been observed in hypertensive patients [40,41]. PRC and p-Ang II were reduced during nebivolol treatment. Renin release is stimulated by β1-receptors [42,43], and although NO also stimulates renin release, a decrease in PRC was anticipated during nebivolol treatment. A lower PRC would lead to a subsequent reduction in p-AngII, which was also found in the present study. Our results are in accordance with observations from animal studies in which nebivolol reduced PRC and p-Ang II in spontaneously hypertensive rats [44,45]. Angiotensin II stimulates aldosterone secretion [46]. A decrease in p-Aldo was found in the present study, a possible explanation for which could be the decreased p-Ang II, although other mechanisms cannot be excluded.
U-AQP2 was slightly increased in 24-h urine by nebivolol compared with placebo, but this finding was not present on the examination day. A change in sympathetic nerve receptor activity might explain this change [47], but it was very small and unlikely to be of clinical significance. It is important to note that patients received a water load of 175 ml every 30 min during examination and 2.5 l per day 4 days prior to examination, which would tend to suppress AVP. This water load could have masked potential nebivolol-mediated changes in AVP and subsequent changes in u-AQP2. Further investigations are warranted to investigate if nebivolol changes AQP2 channel activity.
Strengths and limitations
The design as a placebo-controlled, randomized, double-blinded, crossover trial was an essential strength of the present study. In addition, the study population was a group of patients with well-defined essential hypertension, and the study was performed during standardized food and fluid intake. p-NOx reflects not only endogenous NO production, but also dietary intake. The standardized diet should minimize the confounding effects from dietary intake. Finally, during nebivolol treatment, BP was significantly reduced, which is indicative of good compliance with the study medication.
BP medication was standardized, and patients were given amlodipine during the entire study period as we did not find it ethically justified to discontinue BP medication. Amlodipine may have influenced the effects on variables and masked the effects of nebivolol treatment. The crossover design should minimize the effect of confounders such as smoking and medication. Measurement of plasma asymmetric dimethylarginine and the surrogate parameters of renal NO production, u-NOx and cyclic guanosine monophosphate might add information to help to identify the effects of nebivolol on the NO systems, but these parameters were not analysed in the present trial.
Conclusions
As expected, nebivolol treatment decreased BP, PWV and PRC. During nebivolol and placebo treatment, the inhibition of systemic NO synthesis induced the same responses in bBP, cBP, GFR, renal tubular function and vasoactive hormones. In addition, p-NOx was unchanged during nebivolol treatment. Thus, the data did not support the hypothesis that nebivolol changes vascular and renal NO availability in patients with essential hypertension.
Acknowledgments
We thank laboratory technicians Lisbeth Mikkelsen, Anne Mette Ravn, Henriette Vorup Simonsen and Kirsten Nyborg for their skilful assistance in examining the patients and performing laboratory analyses.
Competing Interests
All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare that: the project was supported by grants from the A.P. Møller Foundation for the Advancement of Medical Science; no financial relationships with any organizations that might have had an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.
Contributors
All authors contributed to the manuscript. FHM, EBP and JNB designed the project. FHM, JMJ and ST performed the experiments, FHM and ABH performed laboratory analysis, JM performed renography and FHM, JMJ, ST, JNB and EBP wrote and edited the manuscript.
References
- 1.Lindholm LH, Carlberg B, Samuelsson O. Should beta blockers remain first choice in the treatment of primary hypertension? A meta-analysis. Lancet. 2005;366:1545–53. doi: 10.1016/S0140-6736(05)67573-3. [DOI] [PubMed] [Google Scholar]
- 2.Dahlof B, Devereux RB, Kjeldsen SE, Julius S, Beevers G, de Faire U, Fyhrquist F, Ibsen H, Kristiansson K, Lederballe-Pedersen O, Lindholm LH, Nieminen MS, Omvik P, Oparil S, Wedel H. Cardiovascular morbidity and mortality in the Losartan Intervention For Endpoint reduction in hypertension study (LIFE): a randomised trial against atenolol. Lancet. 2002;359:995–1003. doi: 10.1016/S0140-6736(02)08089-3. [DOI] [PubMed] [Google Scholar]
- 3.Dahlof B, Sever PS, Poulter NR, Wedel H, Beevers DG, Caulfield M, Collins R, Kjeldsen SE, Kristinsson A, McInnes GT, Mehlsen J, Nieminen M, O'Brien E, Ostergren J. Prevention of cardiovascular events with an antihypertensive regimen of amlodipine adding perindopril as required versus atenolol adding bendroflumethiazide as required, in the Anglo-Scandinavian Cardiac Outcomes Trial-Blood Pressure Lowering Arm (ASCOT-BPLA): a multicentre randomised controlled trial. Lancet. 2005;366:895–906. doi: 10.1016/S0140-6736(05)67185-1. [DOI] [PubMed] [Google Scholar]
- 4.Weiss RJ, Weber MA, Carr AA, Sullivan WA. A randomized, double-blind, placebo-controlled parallel-group study to assess the efficacy and safety of nebivolol, a novel beta-blocker, in patients with mild to moderate hypertension. J Clin Hypertens (Greenwich ) 2007;9:667–76. doi: 10.1111/j.1524-6175.2007.06679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cleophas TJ, Agrawal R, Lichtenthal A, Makel W, Fici F. Nationwide efficacy-safety study of nebivolol in mildly hypertensive patients. Am J Ther. 2006;13:192–7. doi: 10.1097/01.mjt.0000149923.39085.44. [DOI] [PubMed] [Google Scholar]
- 6.Toblli JE, DiGennaro F, Giani JF, Dominici FP. Nebivolol: impact on cardiac and endothelial function and clinical utility. Vasc Health Risk Manag. 2012;8:151–60. doi: 10.2147/VHRM.S20669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Cockcroft JR, Chowienczyk PJ, Brett SE, Chen CP, Dupont AG, Van NL, Wooding SJ, Ritter JM. Nebivolol vasodilates human forearm vasculature: evidence for an L-arginine/NO-dependent mechanism. J Pharmacol Exp Ther. 1995;274:1067–71. [PubMed] [Google Scholar]
- 8.Parenti A, Filippi S, Amerini S, Granger HJ, Fazzini A, Ledda F. Inositol phosphate metabolism and nitric-oxide synthase activity in endothelial cells are involved in the vasorelaxant activity of nebivolol. J Pharmacol Exp Ther. 2000;292:698–703. [PubMed] [Google Scholar]
- 9.Maffei A, Vecchione C, Aretini A, Poulet R, Bettarini U, Gentile MT, Cifelli G, Lembo G. Characterization of nitric oxide release by nebivolol and its metabolites. Am J Hypertens. 2006;19:579–86. doi: 10.1016/j.amjhyper.2005.09.021. [DOI] [PubMed] [Google Scholar]
- 10.Dawes M, Brett SE, Chowienczyk PJ, Mant TG, Ritter JM. The vasodilator action of nebivolol in forearm vasculature of subjects with essential hypertension. Br J Clin Pharmacol. 1999;48:460–3. doi: 10.1046/j.1365-2125.1999.00037.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Tran QT, Rozec B, Audigane L, Gauthier C. Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta. Br J Pharmacol. 2009;156:601–8. doi: 10.1111/j.1476-5381.2009.00074.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Georgescu A, Pluteanu F, Flonta ML, Badila E, Dorobantu M, Popov D. The cellular mechanisms involved in the vasodilator effect of nebivolol on the renal artery. Eur J Pharmacol. 2005;508:159–66. doi: 10.1016/j.ejphar.2004.11.043. [DOI] [PubMed] [Google Scholar]
- 13.Tzemos N, Lim PO, MacDonald TM. Nebivolol reverses endothelial dysfunction in essential hypertension: a randomized, double-blind, crossover study. Circulation. 2001;104:511–4. doi: 10.1161/hc3001.094207. [DOI] [PubMed] [Google Scholar]
- 14.Ortiz PA, Garvin JL. Role of nitric oxide in the regulation of nephron transport. Am J Physiol Renal Physiol. 2002;282:F777–84. doi: 10.1152/ajprenal.00334.2001. [DOI] [PubMed] [Google Scholar]
- 15.Mason RP, Kubant R, Jacob RF, Malinski P, Huang X, Louka FR, Borowiec J, Mizuno Y, Malinski T. Loss of arterial and renal nitric oxide bioavailability in hypertensive rats with diabetes: effect of beta-blockers. Am J Hypertens. 2009;22:1160–6. doi: 10.1038/ajh.2009.163. [DOI] [PubMed] [Google Scholar]
- 16.Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J. 2001;357(Pt 3):593–615. doi: 10.1042/0264-6021:3570593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33:829–37. doi: 10.1093/eurheartj/ehr304. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Bech JN, Nielsen CB, Pedersen EB. Effects of systemic NO synthesis inhibition on RPF, GFR, UNa, and vasoactive hormones in healthy humans. Am J Physiol. 1996;270(5 Pt 2):F845–51. doi: 10.1152/ajprenal.1996.270.5.F845. [DOI] [PubMed] [Google Scholar]
- 19.Larsen T, Mose FH, Bech JN, Pedersen EB. Effect of nitric oxide inhibition on blood pressure and renal sodium handling: a dose-response study in healthy man. Clin Exp Hypertens. 2012;34:567–74. doi: 10.3109/10641963.2012.681727. [DOI] [PubMed] [Google Scholar]
- 20.Mose FH, Larsen T, Bech JN, Pedersen EB. Effects of atorvastatin on systemic and renal nitric oxide in healthy man. Clin Exp Hypertens. 2013;35:148–57. doi: 10.3109/10641963.2012.702835. [DOI] [PubMed] [Google Scholar]
- 21.Mose FH, Larsen T, Jensen JM, Hansen AB, Bech JN, Pedersen EB. Effect of atorvastatin on renal NO availability and tubular function in patients with stage II-III chronic kidney disease and type 2 diabetes. Scand J Clin Lab Invest. 2014;74:8–19. doi: 10.3109/00365513.2013.855942. [DOI] [PubMed] [Google Scholar]
- 22.Mose FH, Larsen T, Jensen JM, Hansen AB, Bech JN, Pedersen EB. Effects of atorvastatin on systemic and renal NO dependency in patients with non-diabetic stage II-III chronic kidney disease. Br J Clin Pharmacol. 2014;78:789–99. doi: 10.1111/bcp.12390. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Matthesen SK, Larsen T, Vase H, Lauridsen TG, Pedersen EB. Effect of potassium supplementation on renal tubular function, ambulatory blood pressure and pulse wave velocity in healthy humans. Scand J Clin Lab Invest. 2011;72:78–86. doi: 10.3109/00365513.2011.635216. [DOI] [PubMed] [Google Scholar]
- 24.Matthesen SK, Larsen T, Lauridsen TG, Vase H, Gjorup PH, Nykjaer KM, Nielsen S, Pedersen EB. Effect of amiloride and spironolactone on renal tubular function, ambulatory blood pressure, and pulse wave velocity in healthy participants in a double-blinded, randomized, placebo-controlled, crossover trial. Clin Exp Hypertens. 2012;34:588–600. doi: 10.3109/10641963.2012.681730. [DOI] [PubMed] [Google Scholar]
- 25.Pedersen EB, Danielsen H, Spencer ES. Effect of indapamide on renal plasma flow, glomerular filtration rate and arginine vasopressin in plasma in essential hypertension. Eur J Clin Pharmacol. 1984;26:543–7. doi: 10.1007/BF00543482. [DOI] [PubMed] [Google Scholar]
- 26.Pedersen EB, Eiskjaer H, Madsen B, Danielsen H, Egeblad M, Nielsen CB. Effect of captopril on renal extraction of renin, angiotensin II, atrial natriuretic peptide and vasopressin, and renal vein renin ratio in patients with arterial hypertension and unilateral renal artery disease. Nephrol Dial Transplant. 1993;8:1064–70. [PubMed] [Google Scholar]
- 27.Hager H, Kwon TH, Vinnikova AK, Masilamani S, Brooks HL, Frokiaer J, Knepper MA, Nielsen S. Immunocytochemical and immunoelectron microscopic localization of alpha-, beta-, and gamma-ENaC in rat kidney. Am J Physiol Renal Physiol. 2001;280:F1093–106. doi: 10.1152/ajprenal.2001.280.6.F1093. [DOI] [PubMed] [Google Scholar]
- 28.Pedersen RS, Bentzen H, Bech JN, Pedersen EB. Effect of water deprivation and hypertonic saline infusion on urinary AQP2 excretion in healthy humans. Am J Physiol Renal Physiol. 2001;280:F860–7. doi: 10.1152/ajprenal.2001.280.5.F860. [DOI] [PubMed] [Google Scholar]
- 29.Graffe CC, Bech JN, Pedersen EB. Effect of high and low sodium intake on urinary aquaporin-2 excretion in healthy humans. Am J Physiol Renal Physiol. 2012;302:F264–75. doi: 10.1152/ajprenal.00442.2010. [DOI] [PubMed] [Google Scholar]
- 30.Kalinowski L, Dobrucki LW, Szczepanska-Konkel M, Jankowski M, Martyniec L, Angielski S, Malinski T. Third-generation beta-blockers stimulate nitric oxide release from endothelial cells through ATP efflux: a novel mechanism for antihypertensive action. Circulation. 2003;107:2747–52. doi: 10.1161/01.CIR.0000066912.58385.DE. [DOI] [PubMed] [Google Scholar]
- 31.Evangelista S, Garbin U, Pasini AF, Stranieri C, Boccioletti V, Cominacini L. Effect of DL-nebivolol, its enantiomers and metabolites on the intracellular production of superoxide and nitric oxide in human endothelial cells. Pharmacol Res. 2007;55:303–9. doi: 10.1016/j.phrs.2006.12.008. [DOI] [PubMed] [Google Scholar]
- 32.Mason RP, Kubant R, Jacob RF, Walter MF, Boychuk B, Malinski T. Effect of nebivolol on endothelial nitric oxide and peroxynitrite release in hypertensive animals: role of antioxidant activity. J Cardiovasc Pharmacol. 2006;48:862–9. doi: 10.1097/01.fjc.0000238593.67191.e2. [DOI] [PubMed] [Google Scholar]
- 33.Mason RP, Jacob RF, Corbalan JJ, Szczesny D, Matysiak K, Malinski T. The favorable kinetics and balance of nebivolol-stimulated nitric oxide and peroxynitrite release in human endothelial cells. BMC Pharmacol Toxicol. 2013;14:48. doi: 10.1186/2050-6511-14-48. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Georgescu A, Popov D, Dragan E, Dragomir E, Badila E. Protective effects of nebivolol and reversal of endothelial dysfunction in diabetes associated with hypertension. Eur J Pharmacol. 2007;570:149–58. doi: 10.1016/j.ejphar.2007.05.031. [DOI] [PubMed] [Google Scholar]
- 35.Wang Y, Evangelista S, Liu Y, Zhang MS. Beneficial effects of nebivolol and hydrochlorothiazide combination in spontaneously hypertensive rats. J Int Med Res. 2013;41:603–17. doi: 10.1177/0300060513477576. [DOI] [PubMed] [Google Scholar]
- 36.Mason RP, Jacob RF, Corbalan JJ, Kaliszan R, Malinski T. Amlodipine increased endothelial nitric oxide and decreased nitroxidative stress disproportionately to blood pressure changes. Am J Hypertens. 2014;27:482–8. doi: 10.1093/ajh/hpt202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.He Y, Si D, Yang C, Ni L, Li B, Ding M, Yang P. The effects of amlodipine and s(–)-amlodipine on vascular endothelial function in patients with hypertension. Am J Hypertens. 2014;27:27–31. doi: 10.1093/ajh/hpt138. [DOI] [PubMed] [Google Scholar]
- 38.Bailey JC, Feelisch M, Horowitz JD, Frenneaux MP, Madhani M. Pharmacology and therapeutic role of inorganic nitrite and nitrate in vasodilatation. Pharmacol Ther. 2014;144:303–20. doi: 10.1016/j.pharmthera.2014.06.009. [DOI] [PubMed] [Google Scholar]
- 39.Melikian N, Seddon MD, Casadei B, Chowienczyk PJ, Shah AM. Neuronal nitric oxide synthase and human vascular regulation. Trends Cardiovasc Med. 2009;19:256–62. doi: 10.1016/j.tcm.2010.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Koumaras C, Tziomalos K, Stavrinou E, Katsiki N, Athyros VG, Mikhailidis DP, Karagiannis A. Effects of renin-angiotensin-aldosterone system inhibitors and beta-blockers on markers of arterial stiffness. J Am Soc Hypertens. 2014;8:74–82. doi: 10.1016/j.jash.2013.09.001. [DOI] [PubMed] [Google Scholar]
- 41.Mahmud A, Feely J. Beta-blockers reduce aortic stiffness in hypertension but nebivolol, not atenolol, reduces wave reflection. Am J Hypertens. 2008;21:663–7. doi: 10.1038/ajh.2008.156. [DOI] [PubMed] [Google Scholar]
- 42.Kurtz A. Renin release: sites, mechanisms, and control. Annu Rev Physiol. 2011;73:377–99. doi: 10.1146/annurev-physiol-012110-142238. [DOI] [PubMed] [Google Scholar]
- 43.Hackenthal E, Paul M, Ganten D, Taugner R. Morphology, physiology, and molecular biology of renin secretion. Physiol Rev. 1990;70:1067–116. doi: 10.1152/physrev.1990.70.4.1067. [DOI] [PubMed] [Google Scholar]
- 44.Wang Y, Zhang MS, Liu Y. Nebivolol treatment improves resistant arterial function and reduces ventricular hypertrophy and angiotensin II in spontaneously hypertension rats. J Renin Angiotensin Aldosterone Syst. 2013;14:146–55. doi: 10.1177/1470320312470580. [DOI] [PubMed] [Google Scholar]
- 45.Varagic J, Ahmad S, Voncannon JL, Moniwa N, Simington SW, Jr, Brosnihan BK, Gallagher PE, Habibi J, Sowers JR, Ferrario CM. Nebivolol reduces cardiac angiotensin II, associated oxidative stress and fibrosis but not arterial pressure in salt-loaded spontaneously hypertensive rats. J Hypertens. 2012;30:1766–74. doi: 10.1097/HJH.0b013e328356766f. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Hattangady NG, Olala LO, Bollag WB, Rainey WE. Acute and chronic regulation of aldosterone production. Mol Cell Endocrinol. 2012;350:151–62. doi: 10.1016/j.mce.2011.07.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Lee J, Yoo K, Kim SW, Jung KH, Ma SK, Lee YK, Kim WY, Kim J, Choi KC. Decreased expression of aquaporin water channels in denervated rat kidney. Nephron Physiol. 2006;103:170–8. doi: 10.1159/000092918. [DOI] [PubMed] [Google Scholar]