ORIGINAL PAPER
Nebivolol Effects on Nitric Oxide Levels, Blood Pressure, and Renal
Function in Kidney Transplant Patients
Alfonso H Santos, Jr, MD; Michael J. Casey, MD, MS; Charles M. Bucci, PA-C; Shehzad Rehman, MD; Mark S. Segal, MD, PhD
From the Division of Nephrology, Hypertension and Renal Transplantation, Department of Medicine, College of Medicine, University of Florida,
Gainesville, FL
In hypertensive kidney transplant recipients, the effects of
nebivolol vs metoprolol on nitric oxide (NO) blood level,
estimated glomerular filtration rate (eGFR), and blood pres-
sure (BP) have not been previously reported. In a 12-month
prospective, randomized, open-label, active-comparator
trial, hypertensive kidney transplant recipients were treated
with nebivolol (n=15) or metoprolol (n=15). Twenty-nine
patients (nebivolol [n=14], metoprolol [n=15]) completed the
trial. The primary endpoint was change in blood NO level
after 12 months of treatment. Secondary endpoints were
changes in eGFR, BP, and number of antihypertensive drug
classes used. After 12 months of treatment, least squares
mean change in plasma NO level in the nebivolol kidney
The success of modern immunosuppression in improv-
ing short-term graft outcome after kidney transplanta-
tion primarily through reduction of acute rejection rates
has not been matched with as much progress in
prolonging long-term graft or patient survival.1,2 The
most common cause of kidney transplant loss is death
with a functioning graft primarily caused by cardiovas-
cular diseases.3,4 Therefore, in conjunction with pre-
vention of rejection through immunosuppression, risk
reduction for and treatment of cardiovascular diseases is
paramount in ensuring prolonged overall graft survival.
Hypertension occurs in up to 80% to 90% of kidney
transplant recipients and is identified as a significant risk
factor for allograft vasculopathy, peripheral artery
disease, and decreased survival and allograft.4–11
Endothelial dysfunction with deficiency of nitric oxide
(NO) is a common pathophysiologic mechanism under-
lying aging, atherosclerosis, diabetes mellitus, renal
disease, vascular disease, and hypertension.12–15 The
progression of chronic kidney disease has been associ-
ated with NO deficiency.16 After successful kidney
transplantation, endothelium-dependent vasorelaxation
increases with the reversal of the NO deficiency that
characterizes end-stage renal disease.12,13 Unfortu-
nately, calcineurin inhibitors (CNIs), the cornerstone
of current antirejection immunosuppression, can negate
these beneficial effects through various mechanisms
Address for correspondence: Alfonso H. Santos, Jr., M.D., Kidney
Transplantation, Box 100224, MSB, Room NG-04, 1600 SW Archer Road,
Gainesville, FL 32610
E-mail: alfonso.santos@medicine.ufl.edu
Manuscript received: August 31, 2015; revised: October 14, 2015;
accepted: October 14, 2015
DOI: 10.1111/jch.12745
transplant recipient group younger than 50 years was higher
by 68.19% (99.17% confidence interval [CI], 13.02–123.36),
69.54% (99.17% CI, 12.71–126.37), and 66.80% (99.17%
CI, 12.95–120.64) compared with the metoprolol group
younger than 50 years, the metoprolol group 50 years and
older, and the nebivolol group 50 years and older, respec-
tively. The baseline to month 12 change in mean arterial BP,
eGFR, and number of antihypertensive drug classes used
was not significantly different between the treatment groups.
In hypertensive kidney transplant recipients, nebivolol use in
patients younger than 50 years increased blood NO. J Clin
Hypertens (Greenwich). 2016;18:741–749. ª 2015 Wiley
Periodicals, Inc.
including elaboration of vasoconstrictor cytokines and
reduction in vasodilator prostaglandins and NO.3,5,17–20
CNIs are also implicated in the pathogenesis of post-
transplant hypertension through activation of the sym-
pathetic nervous and renin-angiotensin-aldosterone
systems, intense afferent arteriolar vasoconstriction,
and sodium retention.21–23
In the absence of a robust, evidence-based guideline
on the management of post-transplant hyperten-
sion,11,24 clinicians have a wide latitude in selecting
drugs for treating hypertension in transplant recipients.
Thus, the recognition that a certain agent can control
blood pressure (BP) and concomitantly improve vascu-
lar endothelial NO generation would make it the agent
of choice for renal transplantation, once its benefits in
improving graft and patient outcomes are proven.
Nebivolol, a third-generation b-blocker with BP-
lowering effects comparable with other b-blockers, has
been effective in treating hypertension, preventing
vascular thrombosis, decreasing endothelin secretion,
and increasing NO formation in human endothelial
cells.25–27 Animal studies have demonstrated that
nebivolol can cause a dose-dependent reduction in renal
perfusion pressure and increase NO release with vasodi-
lation of the renal vasculature.28,29 Models of partial
renal ablation have shown that nebivolol decreases
levels of collagen type 1 expression resulting in reduced
glomerular and interstitial fibrosis.30
We sought to answer the question of whether
nebivolol’s benefits in treating hypertension and improv-
ing NO levels shown in previous studies would also apply
in renal transplant patients given that most of them are
taking a CNI-containing immunosuppression regimen.31
We also sought to determine whether exposure to high or
low tacrolimus (FK-506) levels has different effects on the
The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016 741
Nebivolol in Kidney Transplant Patients | Santos et al.
change in plasma NO level of the treatment groups. For
the purpose of this study, we used 10 ng/mL, the midpoint
of the reference range for FK-506 (5–15 ng/mL) in
categorizing exposure to tacrolimus; although, in clinical
practice, a target FK-506 level is determined based on
certain factors such as age of the transplant.32
Previous studies have shown that the age-related
decline in NO level is evident by the third decade of life
and progressively worsens from the fourth through the
sixth decades of life.16,33,34 The Scientific Registry of
Transplant Recipients report in 2009 (the year preced-
ing recruitment of patients for the study), showed that
the ages of adult kidney transplant recipients in the
United States were close to being evenly distributed
between two ranges: 18–49 years and 50+ years.35
Thus, we applied these ranges in categorizing age and
using it as a predictor variable on the relevant linear
models of the study.
Within the setting of our university-based kidney
transplant clinic, we conducted this randomized open-
label trial comparing the effects of nebivolol and a more
commonly used b-blocker, metoprolol, on the plasma
NO level, BP, and renal function of recent kidney
transplant recipients who were being followed regularly
according to our center’s post-transplant care protocol.
After 12 months of treatment, we did not find a
significant difference in the effects of nebivolol and
metoprolol on BP and renal function in kidney trans-
plant recipients. However, we found that nebivolol, but
not metoprolol, increased NO levels in kidney trans-
plant recipients younger than 50 years and nebivolol
combined with low to moderate trough levels of
tacrolimus significantly increased NO plasma levels.
Our findings have not been previously reported, and
large randomized trials will be needed to confirm our
conclusions and determine the clinical implications of
increased NO from nebivolol use in kidney transplant
recipients.
PATIENTS AND METHODS
This was a prospective randomized open-label com-
parator study. Hypertensive kidney transplant patients
were randomly assigned to receive oral treatment with
either nebivolol or metoprolol. The trial, which con-
sisted of 14 study visits over a 12-month period, was
undertaken at the University of Florida and Shands
Hospital Renal Transplant Clinic. The study protocol
was approved by the Western Institutional Review
Board (Olympia, WA). The trial is registered at www.
clinicaltrials.gov (NCT01157234). The primary end-
point of the study was change in plasma NO levels.
Secondary endpoints were changes in renal function
based on the Modification of Diet of Renal Disease
(MDRD) estimated glomerular filtration rate (eGFR)36;
systolic BP (SBP), diastolic DP (DBP), and mean arterial
pressure (MAP); quantity of BP medication classes used
to control BP; markers of oxidative stress in serum
including asymmetric dimethylarginine (ADMA) and
dimethylarginine dimethylaminohydrolase (DDAH);
742 The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016
and serum arginine levels. Inclusion criteria included
men or women at least 18 years of age who were
recipients of a solitary kidney or combined kidney-
pancreas transplant within 24 months of enrollment,
current diagnosis of hypertension, normal hepatic
enzyme levels, and estimated creatinine clearance
≥30 mL/min. Exclusion criteria included uncontrolled
hypertension defined as an SBP ≥210 mm Hg or a DBP
≥120 mm Hg, symptomatic hypotension, previous
intolerance to b-blockers, cerebrovascular accident
within 3 months of randomization, bradycardia (heart
rate <60 beats per minute), greater than first-degree
heart block, decompensated cardiac failure, sick sinus
syndrome (unless a permanent pacemaker is in place),
severe hepatic impairment (defined as elevation of
aspartate aminotransferase, alanine aminotransferase,
or bilirubin levels to three times the upper limit of
normal reference range), severe peripheral arterial
circulatory disorder, history of bronchospasm and/or
asthma, regular use of inhaled bronchodilators, or any
medical condition that may interfere with the partici-
pant’s ability to safely complete the protocol.
Patients who signed informed consent were screened
based on the above inclusion and exclusion criteria,
and those who qualified were enrolled in the study. A
computer-generated list of random numbers was used
to allocate participants to study drug. Baseline health
information, 12-lead electrocardiography, heart rate,
BP measurements, and 12-hour fasting laboratory tests
including plasma NO concentration and serum crea-
tinine levels were obtained. In each subsequent study
visit, heath information, heart rate, BP measurements,
and 12-hour fasting laboratory tests including plasma
NO level and serum creatinine were again obtained.
Patients were randomly assigned to receive oral
treatment with either nebivolol or metoprolol with
dose titration to a target BP of at least <140/90 mm
Hg. If the target BP of at least <140/90 mm Hg was
not achieved following a study drug titration period of
up to 10 weeks after randomization, the treating
physicians were allowed to add other antihypertensive
medications.
PROCEDURES
Measurement of BP
BP measurements were made using an automatic BP
monitor (Omron Healthcare, Inc, Lake Forest, IL). Two
separate measurements 2 minutes apart were taken with
the patient quietly seated in a chair for at least
5 minutes with both feet on the floor and the arm
supported at heart level. The average of systolic and
diastolic BPs were used for data analysis. MAP was
calculated as 1/3 (SBP) + 2/3 (DBP).37
Laboratory Analyses
Measurement of NO. Sample: A 12-hour overnight fast
and 20 to 30 minutes of rest in a sitting position was
observed prior to obtaining blood samples. Blood was

obtained by phlebotomy and collected into an EDTA
tube and the samples centrifuged for 10 minutes at
4°C at 1000 9 g and then the plasma stored at 80°C
until analyzed.
NO2 Determination: Standard solution of 100 mM
NO2 was freshly prepared by weighing NaNO2 and
diluting it in nitrite-free, deionized water. Standards
prepared by serial dilution were stored in the dark at
4°C until analysis. After obtaining a stable baseline, the
solutions were injected into an NO analyzer (NOA 280i
Sievers; GE Analytical Instruments, Boulder, CO) and
the amount of NO was calculated on the basis of the
peak area from each injection and used to plot the
calibration curves (1–100 pmol injected, r2=0.9999).
Plasma samples were thawed on ice, an antifoaming was
agent added, the sample was injected as above, and the
basis of the peak area was determined.
Measurement of Other Blood Tests. Blood samples
were collected for measurements of serum creatinine
(mg/dL) and whole blood trough level of tacrolimus
(FK-506 in ng/mL). The eGFR in mL/min was calcu-
lated according to the MDRD study group equation.
Due to unforeseen technical limitations in the labora-
tory, markers of oxidative stress in serum including
ADMA and DDAH and serum arginine levels were not
measured as originally planned.
Safety
Adverse Event and Serious Adverse Event. Patients
were monitored for possible adverse events associated
with the study drugs. Information about all adverse
events were collected, recorded, and followed as appro-
priate. All serious adverse events were reported to Forest
Global Drug Safety.
STATISTICAL ANALYSIS
Statistical analyses were performed in the intention-to-
treat (ITT) population using general linear models with
continuous outcome and concomitant (covariate)
variables and categorical predictor variables. Primary
and secondary endpoints were analyzed by comparison
of least squares (LS) mean change derived from the
difference in baseline and last-visit observations
adjusted for the baseline measurement. For missing
data, values were imputed using the next-observation-
carried-backward or last-observation-carried-forward
method. Paired comparisons with Bonferroni-adjusted
P values were used when an interaction of main factor
effects was shown in the analyses of primary and
secondary outcomes. Exploratory analyses using general
linear models were performed to determine: (1) the
effect of study drugs and baseline FK-506 trough level
categories (≤10 ng/mL or >10 ng/mL) on the change in
NO level; (2) the effect of age and FK-506 interaction
on the change in NO levels; and (3) the correlation of
nebivolol or metoprolol dose on the change in NO levels
in the age and FK-506 subgroups described above.
Statistical evaluations were performed using an online
Nebivolol in Kidney Transplant Patients | Santos et al.
statistical package (StatsToDo Trading Pty Ltd, Queens-
land, Australia).38,39 In all analyses, an actual or
adjusted P value equivalent to <.05 was considered
significant.
RESULTS
The first patient was randomized on July 8, 2010, and
the follow-up of the last patient ended on July 21, 2014.
Recruitment to the trial was stopped before reaching the
enrollment target of 50 patients due to changes in the
funding entity. In total, 32 patients were screened, of
whom 30 met the eligibility criteria and were random-
ized (15 nebivolol, 15 metoprolol), forming the ITT
safety and efficacy population. A total of 29 of 30
patients (96.7%) completed the 12-month study, with
14 (48.3%) taking nebivolol and 15 (51.7%) taking
metoprolol (Figure 1). One patient in the nebivolol
group withdrew from the study after the eighth week
because of BP normalization. The mean study duration
was 11.9 months (standard deviation [SD], 0.73). The
baseline characteristics of the study population are
shown in Table I. The mean ages of the patients in the
nebivolol and metoprolol groups were 53 years (SD,
15.9) and 46.4 years (SD, 13.8), respectively (P=.23).
The mean duration from kidney transplantation to
study enrollment was 56.7 days (SD, 111.7) in the
nebivolol group and 46.7 days (SD, 62.7) in the
metoprolol group. All participants were taking a
tacrolimus-containing immunosuppressant regimen at
randomization and the mean 12-hour trough serum
drug levels were not significantly different between
groups (nebivolol 10.7 ng/mL [SD, 2.9] vs metoprolol
11.2 ng/mL [SD, 1.9], P=.612).
Analysis of Outcomes
Nitric Oxide. The nebivolol and metoprolol groups did
not differ in mean plasma NO levels at baseline
(52.93 nmol/L [n=15; SD, 21.09] and 48.13 nmol/L
[n=15; SD, 15.37], respectively; P=.482). After
12 months of treatment, the LS mean plasma NO level
of the nebivolol group was 50.07 nmol/L (standard
error [SE] 3.93) vs 38.13 (SE 3.93) nmol/L of the
metoprolol group (difference, 11.94 nmol/L, P=.04;
95% CI, 0.48–23.40). There was a significant interac-
tion between treatment and age effects on the plasma
NO level (F [1, 25], 7.22; P=.013), (Figure 2). Manda-
tory subgroup analyses showed that the nebivolol
younger than 50 years age subgroup had achieved a
significantly higher LS mean plasma NO level than any
of the metoprolol subgroups and the nebivolol 50 years
and older subgroup (Figure 2). None of the patients in
the treatment groups were diagnosed with an acute
allograft rejection at the time of NO testing.
The 12-month change in LS mean plasma NO was
+11.47% (standard error of the mean [SEM] 9.20) in
the nebivolol group and 17.27% (SEM 9.20) in the
metoprolol group (difference in LS means, 28.73%
[SEM 13.01], P=.038; 95% CI, 1.93–55.53)
(Figure 3). Statistically, there was a significant interac-
The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016 743
Nebivolol in Kidney Transplant Patients | Santos et al.
FIGURE 1. Trial profile.
tion in the effects of treatment and age (categorized <50
years or ≥50 years) on the LS mean percent change in
plasma NO level (F [1, 25], 7.66; P=.011). To clarify
this interaction, post hoc multiple subgroup compar-
isons were employed (Figure 3). Among the four sub-
groups categorized based on drug treatment and age,
only the nebivolol younger than 50 years subgroup had
a significant increase in plasma NO level from baseline:
LS mean +51.55% (95% CI, 23.03–80.07) (Figure 3).
Results of mandatory post hoc subgroup comparisons
(Figure 3) demonstrated that nebivolol treatment in
kidney transplant recipients younger than 50 years
resulted in a significant increase in plasma NO levels
compared with: (1) metoprolol treatment in kidney
transplant recipients younger than 50 years (difference
of LS means, +68.19%; 99.17% CI, 13.02–123.36); (2)
metoprolol treatment in kidney transplant recipients
50 years and older (difference of LS means, +69.54%;
99.17% CI, 12.71–126.37); and (3) nebivolol treatment
in kidney transplant recipients 50 years and older
(difference of LS means, +66.80%; 99.17% CI, 12.95–
120.64). Exploratory analysis demonstrated that the use
of nebivolol with the rejection prophylaxis drug
tacrolimus at a baseline trough level ≤10 ng/mL resulted
in a significant increase in plasma NO level from
baseline (LS mean change, +14.49 nM; 95% CI, 1.94–
27.04) (Figure 4). The use of nebivolol or metoprolol
with tacrolimus at baseline trough level above 10 ng/mL
resulted in a significant decrease in plasma NO level
744 The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016
from baseline (Figure 4). The change in plasma NO
level from baseline was not affected by the interaction
effect of age and FK-506 level (F [1, 25], 2.73; P=.11).
Drug doses did not correlate with the change in NO
from baseline in the age subgroups (<50 years: nebivo-
lol, t [6] = 2.78, P=.56; metoprolol, t [6] = 2.45, P=.87)
and (≥50 years: nebivolol, t [6] = 0.17, P=.87; meto-
prolol, t [5] = 0.47, P=.66). Similarly, drug doses did
not correlate with the change in NO from baseline in the
FK-506 level subgroups (≤10 ng/mL: nebivolol, t
[4] = 1.80, P=.14; metoprolol, t [2] = 1.5, P=.27)
and (>10 ng/mL: nebivolol, t [7] = 0.46, P=.66;
metoprolol, t [9] = 0.14, P=.89).
Renal Function
The endpoint for renal function is the change from
baseline to 12-month visit eGFR in the nebivolol and
metoprolol groups stratified by the kidney donors’ age
(<50 or ≥50 years). The baseline eGFR of the nebivolol
and metoprolol groups were similar: 53.13 mL/min
(SD, 14.15) and 54.47 mL/min (SD, 14.65), respectively
(P=.80). After 12 months of treatment, the LS mean
GFR of the nebivolol group was 53.10 mL/min (SE
3.89) vs 58.57 (SE 3.89) mL/min in the metoprolol
group (difference, 5.47 mL/min; 95% CI, 5.87 to
16.81 [P=.33]).
The 12-month change in LS mean eGFR was not
significantly different between the nebivolol and meto-
prolol groups (8.27% [SE 10.13]; 95% CI, 12.56 to
 Nebivolol in Kidney Transplant Patients | Santos et al.

TABLE I. Baseline Characteristics

Baseline Characteristics Nebivolol Cohort (n=15) Metoprolol Cohort (n=15) P Value

Age, y 53 (15.90) 46.40 (13.80) .24

Sex, No. (%) .69
Male 10.00 (66.70) 11.00 (73.30)
Female 5.00 (33.30) 4.00 (26.70)
Race, No. (%) .27
White 6.00 (40.00) 10.00 (66.70)
Black 8.00 (53.30) 5.00 (33.30)
Hispanic 1.0 (6.70) 0 (0)
Days post-transplant at screening 56.70 (111.70) 46.70 (62.70) .77
b-Blockers at enrollment 15.00 (100.00) 15.00 (100.00)
Mean blood pressure medications at enrollment, No. 2.00 (0.84) 1.80 (0.68) .43
Transplant kidney donor .78
Type
Living unrelated 3.00 (20.00) 4.00 (26.70)
Living related 2.00 (13.00) 3.00 (20.00)
Deceased 10.00 (66.70) 8.00 (53.00)
Age, y 43.80 (40.0) 34.30 (14.90)
≤50 y 7.00 (46.60) 12.00 (80.00) .08
Sex
Female 8.00 (53.30) 9.00 (60.00) .12
Male 7.00 (46.70) 6 (40.00) .71
Weight, kg 88.40 (22.50) 82.50 (15.80) .41
Height, cm 167.60 (11.10) 170.10 (9.70) .69
Systolic average, sitting 132.93 (15.79) 131.33 (18.14) .79
Diastolic average, sitting 84.07 (8.59) 86.33 (8.62) .48
Mean arterial pressure, sitting 100.20 (8.06) 101.47 (10.76) .72
Heart rate average, sitting 72.7 (9.7) 72.6 (9.8) .97
Nitric oxide, nmol/L 52.90 (21.10) 48.10 (15.4) .48
Serum creatinine, mg/dL 1.51 (0.45) 1.45 (0.41) .73
Glomerular filtration rate, mL/minute/1.73 m2 53.13 (14.15) 54.57 (14.65) .80
Tacrolimus at baseline, µg/L 10.70 (2.90) 11.20 (1.90) .61
Month-12 LS Mean Serum Nitric Oxide level, nMol

70 66.46

60

50.07
50

39.13 38.13 38.16 38.1

40

30

20

10

0
Neb, All Neb < 50 yr Neb > =50 yr Met, All Met < 50 yr Met > =50 yr

95% CI 42.37 to 57.77 54.28 to 78.66 29.17 to 49.09 30.43 to 45.83 27.59 to 48.73 26.77 to 49.43
SEM 3.93 6.22 5.08 3.93 5.39 5.78
Comparisons Difference in LS means SE Confidence Interval**
Neb, all vs. Met, all * 11.94 nMol 5.56 (95%) 0.48 to 23.40
Age<50 vs. >=50 * 11.61 nMol 5.56 (95%) 0.15 to 23.07
Neb <50 vs. Neb >=50 27.24 nMol 8.03 (99.17%) 4.22 to 50.26
Neb <50 vs. Met <50 28.31 nMol 8.23 (99.17%) 4.72 to 51.89
Neb <50 vs. Met >=50 28.37 nMol 8.48 (99.17%) 4.07 to 52.67
* Drug x Age interacon F[1,25]=7.22, p=0.013 **CI 99.17% based on Bonferroni correcon

FIGURE 2. Plasma nitric oxide achieved at month 12 of nebivolol (Neb) or metoprolol (Met) treatment corrected for pretreatment level. LS
indicates least square.

The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016 745

Nebivolol in Kidney Transplant Patients | Santos et al.
FIGURE 3. Percent change in plasma nitric oxide level from baseline to month 12 of nebivolol (Neb) or metoprolol (Met) treatment. LS indicates
least square; CI, confidence interval.
FIGURE 4. Change in plasma nitric oxide (NO) level from baseline to month 12 of treatment: effect of nebivolol (Neb) or metoprolol (Met) with
tacrolimus (FK) trough level categories <10 µg/L or >10 µg/L.
29.12 [P=.42]) (Figure 5). Recipients of kidney allo-
grafts from donors younger than 50 years had a
significant increase in LS mean GFRs from baseline
(+14.65%; 95% CI, 2.19–27.11), while recipients of
kidney allografts from donors 50 years old and older
had trends of decrease in their LS mean GFRs from
baseline ( 8.13%, 95% CI, 24.52 to 8.26). Between
the younger than 50 years and 50 years and older donor
age categories, the mean change of LS mean eGFR from
baseline differed by 22.77% (95% CI, 1.13–44.41;
P=.04) in favor of the younger than 50 years donors.
BP Control and Medication Requirements
The baseline and change from baseline SBP, DBP, and
MAP were compared between treatment groups and no
significant differences were found (Tables I and II).
Similarly, the average number of BP medication classes
746 The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016
used at the time of randomization (Table I) and at the
end of the study were not significantly different between
the nebivolol and metoprolol treatment groups
(Table II).
Safety
A total of 60% (9 of 15) of patients in the nebivolol
group and 86% (13 of 15) in the metoprolol group had
at least one mild or moderate adverse event. There were
six serious adverse events in four patients (26.7%) in the
nebivolol group, consisting of urinary tract infection (1),
peritoneal hematoma (1), and graft dysfunction (4).
There were seven serious adverse events in five (33.3%)
patients in the metoprolol group, consisting of prostate
cancer (1), hernia repair (1), graft dysfunction (1),
nausea and vomiting (1), and cytomegalovirus infection
(1). Each adverse event was investigated and none were
 Nebivolol in Kidney Transplant Patients | Santos et al.

FIGURE 5. Percent change in glomerular filtration rate (GFR) after 12 months of nebivolol (Neb) or metoprolol (Met) treatment. Groups
stratified by age of kidney donors (<50 years or >50 years).

TABLE II. Secondary Endpoint Measures

Endpoint Measures Nebivolol (n=15) Metoprolol (n=15) Differencea 95% CI P Value

Systolic blood pressure, mm Hg

Baseline, observed mean (SD) 132.93 (15.79) 131.33 (18.14) .79
12 mo, observed mean (SD) 129.93 (16.18) 125.93 (8.89) .41
Absolute change, LS mean (SE) 2.65 (2.73) 3.88 (2.73) 1.23 (3.87) 6.74 to 9.20 .75
Diastolic blood pressure, mm Hg
Baseline, observed mean (SD) 84.07 (8.59) 86.33 (8.62) .48
12 mo, observed mean (SD) 84.47 (7.12) 82.93 (7.04) .56
Absolute change, LS mean (SE) 0.66 (1.91) 2.35 (1.91) 1.71 (2.70) 3.85 to 7.23 .54
Mean arterial blood pressure, mm Hg
Baseline, observed mean (SD) 100.20 (8.06) 101.47 (10.76) .72
12 mo, observed mean (SD) 99.67 (8.39) 97.40 (7.05) .43
Absolute change, LS mean (SE) 1.07 (2.11) 3.19 (2.11) 2.05 (2.99) 4.10 to 8.20 .48
Number of antihypertensive drug classes used
Baseline, observed mean (SD) 2.00 (0.84) 1.80 (0.68) .80
12 mo, observed mean (SD) 2.27 (0.96) 2.20 (1.21) .34
Absolute change, LS mean (SE) 0.26 (0.22) 0.01 (0.22) 0.25 (0.32) 0.40 to 0.89 .45
Percent change, LS mean (SE) 8.14 (11.58) +8.70 (11.58) 16.84 (16.38) 16.9 to 50.5 .32
Abbreviations: CI, confidence interval; SD, standard deviation; SE, standard error.
a
In estimating differences in all outcome categories, change from baseline was analyzed through the general linear regression model evaluating
differences in least-square (LS) means, fitted with treatment and age < or ≥50 years old as categorical independent variables, corrected with the baseline
measurement as continuous co-variate.

found to be related to the study drugs. There were no
deaths or graft kidney loss reported during the study.
DISCUSSION
While kidney transplantation provides significant sur-
vival benefit over maintenance dialysis, cardiovascular
disease is still among the most common causes of death
in renal transplant recipients.3,4,22 It is important to
apply any treatment that can mitigate cardiovascular
risk in transplant recipients to improve overall graft and
patient survival. Hypertension, diabetes, dyslipidemia,
and impaired kidney function––states characterized by
endothelial dysfunction and impaired NO generation––
are risk factors for increased cardiovascular complica-
tions in kidney transplant recipients.4,12,22,23,40 CNIs
seem to aggravate endothelial dysfunction and vasocon-
striction caused by the foregoing risk factors.21,22,41,42
Conversely, the potent vasodilator substance, NO, has
been shown to provide beneficial protective cardiovas-
cular effects including the prevention of atherosclerosis

The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016 747

Nebivolol in Kidney Transplant Patients | Santos et al.
and its complications.19,25 Thus, we conducted this
randomized clinical trial in order to compare the effects
of nebivolol and metoprolol on NO plasma level, BP,
and renal function in kidney transplant recipients. We
performed an exploratory analysis to study the effect of
the study drugs combined with tacrolimus, the CNI used
for immunosuppression in all of the study participants.
Our results demonstrate that during 12 months, nebi-
volol and metoprolol treatment did not have signifi-
cantly different effects on the reduction of BP, change in
renal function, or change in drug regimen for hyperten-
sion. We found a significant increase from baseline in
the plasma NO levels of the nebivolol-treated transplant
recipients younger than 50 years or those receiving
tacrolimus at a trough exposure no higher than 10 ng/
mL. We did not find any significant improvement in the
plasma NO level in patients in the metoprolol-treated
subgroups.
In addition, we did not find a significant difference in
the change in BP parameters between the treatment
groups after 12 months of observation (Table II).
Although our results are consistent with previous
findings of a comparable BP-lowering effect of nebivolol
as with other b-blockers,25 the reduction in BPs in both
treatment groups was not significant. This is likely
because the BP of the patients in this study were already
at or close to the target of <140/90 mm Hg at the time
of randomization.43 The results could also possibly be
attributable to the post-transplant improvement in renal
function, volume status, and endothelium-dependent
vasodilation.12,13,21,22,31,40,43 The uniform use of tacro-
limus rather than the more prohypertensive CNI,
cyclosporine, may have also contributed to the excellent
BP achieved in this population of kidney transplant
recipients.3,17,21,31,44,45
Our analysis did not show a difference in the effect of
nebivolol and metoprolol on the renal function of
kidney transplant recipients from baseline to the 12th
month of the study. This is consistent with the relatively
neutral physiologic effect of b-blockers on overall renal
function.46 Our analysis demonstrated that kidney
allografts from older donors had achieved lower GFRs
at the 12-month study visit and a decreased GFR
relative to the baseline visit as compared with allografts
from younger donors. These findings possibly reflect
how aging affects the kidney by increasing glomerular,
vascular, and tubular senescence resulting in decreased
renal blood flow and GFR.33,47,48 In a previous study,
these factors were implicated in the increased incidence
of delayed graft function and graft loss in kidneys from
older donors.48
Based on our results, nebivolol increased NO in
kidney transplant recipients younger than 50 years but
not in older patients. We hypothesize that the effect of
nebivolol in increasing NO generation is counterbal-
anced by the decline in NO synthesis or secretion––a
consequence of endothelial dysfunction and increased
endothelin-1 that occurs with aging.16,33
748 The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016
Tacrolimus, the cornerstone of immunosuppressive
regimens after kidney transplantation, significantly
reduces graft rejection rates but could have nephrotoxic
effects.3,17 Tacrolimus attenuates NO production,
release, and endothelium-dependent relaxation in a
dose-dependent manner.7,41 This impairment has been
implicated in the causation of kidney allograft vascu-
lopathy.49,50 In this study, nebivolol used with tacrolimus
at a trough level of ≤10 ng/mL (but not >10 ng/mL)
increased the plasma NO level. We theorize that the NO-
elevating effect of nebivolol operates when tacrolimus
level is low but can be reversed by high tacrolimus levels,
resulting in inhibition of NO generation.
Metoprolol or nebivolol used with tacrolimus at a
trough level of >10 ng/mL decreased the plasma NO
level. These findings may have implications for long-
term renal allograft outcomes, since previous studies
have shown that inhibition of NO production by all NO
synthase isoforms decreases the survival of the kidney
transplant by augmentation of the alloimmune response
or by ischemia to the graft.49,50
LIMITATIONS OF THE STUDY
This study was limited by its small sample size. We
enrolled only 15 participants in each arm instead of 25
as originally planned, and we were not able to measure
mediators of oxidative stress (ADMA and DDAH) and
arginine levels in the blood. The participants were
randomly assigned to the treatment arms but investiga-
tors and drug dispensers were not blinded to the
treatment allocation of patients. However, follow-up
of participants in our single university-based transplant
clinic using a uniform protocol minimized the variability
in patient management.
CONCLUSIONS
We report the first randomized study to directly compare
the effect of two b-blockers on NO blood levels in kidney
transplant recipients. Nebivolol but not metoprolol
increased plasma NO levels in kidney transplant recipi-
ents younger than 50 years or with lower tacrolimus
exposure. A large randomized trial is needed to confirm
our findings and determine the clinical implications of
increased plasma NO level from nebivolol treatment in
hypertensive transplant recipients.
Acknowledgments and funding: The authors would like to gratefully acknowl-
edge Elaine Whidden for her administrative management of the trial and
Briana Foerman and Linda Owens for patient recruitment and data handling.
We especially acknowledge the efforts of Dr Herwig Ulf Meier-Kriesche, who
was the original principal investigator of this study. The study was funded by
Actavis (formerly Forest Laboratories, LLC).
Disclosures: The authors of this manuscript have no conflicts of interest to
disclose.
References
1. Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal allograft
survival: have we made significant progress or is it time to rethink our
analytic and therapeutic strategies? Am J Transplant. 2004;4:1289–
1295.

2. Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B. Lack of
improvement in renal allograft survival despite a marked decrease in
acute rejection rates over the most recent era. Am J Transplant.
2004;4:378–383.
3. Artz MA, Boots JM, Ligtenberg G, et al. Improved cardiovascular risk
profile and renal function in renal transplant patients after random-
ized conversion from cyclosporine to tacrolimus. J Am Soc Nephrol.
2003;14:1880–1888.
4. Kasiske BL, Anjum S, Shah R, et al. Hypertension after kidney
transplantation. Am J Kidney Dis. 2004;43:1071–1081.
5. Schwenger V, Zeier M, Ritz E. Hypertension after renal transplan-
tation. Ann Transplant. 2001;6:25–30.
6. Opelz G, Wujciak T, Ritz E, et al. Association of chronic kidney
graft failure with recipient blood pressure. Kidney Int.
1998;53:217–222.
7. Morales JM, Dominguez-Gil B. Impact of tacrolimus and mycophe-
nolate mofetil combination on cardiovascular risk profile after kidney
transplantation. J Am Soc Nephrol. 2006;17:S296–S303.
8. Opelz G, Wujciak T, Ritz E. Association of chronic kidney graft
failure with recipient blood pressure. Collaborative Transplant Study.
Kidney Int. 1998;53:217–222.
9. Mange KC, Cizman B, Joffe M, et al. Arterial hypertension and renal
allograft survival. J Am Med Assoc. 2000;283:633–638.
10. Multicenter TU. FK506 Liver Study Group. A comparison of
tacrolimus for immunosuppression in liver transplantation. N Engl
J Med. 1994;331:1110–1111.
11. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant
Work Group. KDIGO clinical practice guideline for the care of kidney
transplant recipients. Am J Transplant. 2009;9(suppl 3):S1–S157.
12. Passauer J, Bussemaker E, Lassig G, Gross P. Kidney transplantation
improves endothelium-dependent vasodilatation in patients with end-
stage renal disease. Transplantation. 2003;75:1907–1910.
13. Zhong W, Zhou C, Xie J, et al. Serum asymmetric dimethylarginine
and endothelial function after renal transplantation. J Cent South
Univ (Med Sci). 2009;34:289–294.
14. Klahr S. The role of nitric oxide in hypertension and renal disease
progression. Nephrol Dial Transplant. 2001;16(suppl 1):60–62.
15. Bleakley C, Hamilton PK, Pumb R, et al. Endothelial function in
hypertension: victim or culprit? J Clin Hypertens (Greenwich).
2015;17:651–654.
16. Zatz R, Baylis C. Chronic nitric oxide inhibition model six years on.
Hypertension. 1998;32:958–964.
17. Artz MA, Boots JM, Ligtenberg G, et al. Conversion from cyclospor-
ine to tacrolimus improves quality-of-life indices, renal graft function
and cardiovascular risk profile. Am J Transplant. 2004;4:937–945.
18. Kandavar R, Fernandez C, Sander GE, et al. Digital plethysmography
and arginine metabolism in prehypertension—effect of nebivolol
therapy. J Clin Hypertens (Greenwich). 2015;17:14–19.
19. Van Houtte PM, Gao Y. Beta blockers, nitric oxide, and cardiovas-
cular disease. Curr Opin Pharmacol. 2013;13:265–273.
20. De Lima JJ, Xue H, Coburn L, et al. Effects of FK506 in rat and
human resistance arteries. Kidney Int. 1999;55:1518–1527.
21. Curtis JJ, Luke RG, Jones P. Hypertension in cyclosporine-treated
renal transplant recipients is sodium-dependent. Am J Med.
1988;85:134–138.
22. Ojo AO. Cardiovascular complications after renal transplantation and
their prevention. Transplantation. 2006;82:603–611.
23. Ponticelli C, Cucchiari D, Graziani G. Hypertension in kidney
transplant recipients. Transpl Int. 2011;24:523–533.
24. KDIGO clinical practice guideline for the management of blood
pressure in chronic kidney disease. Kidney Int. 2012;2(suppl 5):337–
414
25. Cheng J. Nebivolol: a third-generation b-blocker for hypertension.
Clin Ther. 2009;31:447–462.
26. Ignarro LJ. Experimental evidences of nitric oxide-dependent
vasodilatory activity of nebivolol, a third-generation B-blocker. Blood
Press. 2004;13(suppl 1):2–16.
27. Brehm BR, Wolf SC, Bertsch D, et al. Effects of nebivolol on
proliferation and apoptosis of human coronary artery smooth muscle
and endothelial cells. Cardiovasc Res. 2001;49:430–439.
Nebivolol in Kidney Transplant Patients | Santos et al.
28. Georgescu A, Pluteanu F, Flonta ML, et al. The cellular mechanisms
involved in the vasodilator effect of nebivolol on the renal artery. Eur
J Pharmacol. 2005;508:159–166.
29. Kakoki M, Hirata Y, Hayakawa H, et al. Effects of vasodilatory b-
adrenoceptor antagonists on endothelium-derived nitric oxide release
in rat kidney. Hypertension. 1999;33:467–471.
30. Pires M, Rodriquez-Pena A, Arevalo M, et al. Long-term nebivolol
administration reduces renal fibrosis and prevents endothelial dys-
function in rats with hypertension induced by renal mass reduction.
J Hypertens. 2007;25:2486–2496.
31. Kislikova M, Seras M, Monfa E, et al. Number of antihypertensive
drugs at 1 year after kidney transplantation. Transplant Proc.
2015;47:76–77.
32. Laskow DA, Vincenti F, Neylan JF, et al. An open-label, concentra-
tion-ranging trial of FK506 in primary kidney transplantation: a
report of the United States Multicenter FK506 Kidney Transplant
Group. Transplantation. 1996;62:900–905.
33. Toprakql M, 0zmen D, Mutaf I, et al. Age-associated changes in nitric
oxide metabolites nitrite and nitrate. Int J Clin Lab Res. 2000;30:83–85.
34. Gerhard M, Roddy MA, Creager SJ, Creager MA. Aging progressively
impairs endothelium-dependent vasodilation in forearm resistance
vessels of humans. Hypertension. 1996;27:849–853.
35. Program-Specific Report Archives. Scientific Registry of Transplant
Recipients. http://srtr.org/csr/archives/200912/FLUFTX1KI200912.pdf.
Accessed April 06, 2015.
36. Levey AS, Bosch JP, Lewis JB, et al. More accurate method to estimate
glomerular filtration rate from serum creatinine: a new prediction
equation. Modification of Diet in Renal Disease Study Group. Ann
Intern Med. 1999;130:461–470.
37. Sesso HD, Stampfer MJ, Rosner B, et al. Mean arterial pressure as
predictors of cardiovascular disease risk in men. Hypertension.
2000;36:801–807.
38. O’Leary TJ. Assessing and comparing the performance of molecular
diagnostic tests. J Mol Diagn. 2014;16:2.
39. Andrew K, Satomi M, Istvan B, et al. Simulated surgical-type cerebral
biopsies from post-mortem brains allows accurate neuropathological
diagnoses in the majority of neurodegenerative disease groups. Acta
Neuropathologica Communications. 2013;1:53.
40. Veenstra D, Best J, Hornberger J, et al. Incidence and long-term cost
of steroid-related side effects after renal transplantation. Am J Kidney
Dis. 1999;33:829.
41. Long C, Cook LG, Wu GY, et al. Removal of FKBP12/12.6 from
endothelial ryanodine receptors leads to an intracellular calcium leak
and endothelial dysfunction. Arterioscler Thromb Vasc Biol.
2007;27:1580–1586.
42. Mangray M, Vella JP. Hypertension after kidney transplant. Am J
Kidney Dis. 2011;57:331–341.
43. Midtvedt K, Hartman A. Hypertension after renal transplantation: are
treatment guidelines emerging? Nephrol Dial Transplant.
2002;17:1166–1169.
44. Robert N, Wong GW, Wright JM. Effect of cyclosporine on blood
pressure. Cochrane Database Syst Rev. 2010;1:CD007893
45. Textor SC, Wiesner R, Wilson DJ, et al. Systemic and renal hemo-
dynamic differences between FK506 and cyclosporine in liver trans-
plant recipients. Transplantation. 1993;55:1332–1339.
46. Tomiyama H, Yamashina A. Beta-Blockers in the management of
hypertension and/or chronic kidney disease. Int J Hypertens.
2014;2014:919256.
47. Davis JT, Pasha DN, Khandrika S, et al. Central hemodynamics in
prehypertension: effect of the b-adrenergic antagonist nebivolol. J Clin
Hypertens (Greenwich). 2013;15:69–74.
48. Alexander JW, Bennett LE, Breen TJ. The effect of donor age on
outcome of kidney transplantation. A two-year analysis of transplants
reported to the United Network for Organ Sharing Registry. Trans-
plantation. 1994;57:871–876.
49. Vos IH, Joles JA, Rabelink TJ. The role of nitric oxide in renal
transplantation. Semin Nephrol. 2004;24:379–388.
50. Watarai Y, Takeuchi I, Togashi H, et al. Nitric oxide production in
renal transplant recipients and iNOS expression in renal allografts.
Transplantation. 1999;67:S9.
The Journal of Clinical Hypertension Vol 18 | No 8 | August 2016 749