Naunyn-Schmiedeberg's Arch Pharmacol
DOI 10.1007/s00210-016-1239-1

ORIGINAL ARTICLE

Febuxostat, a novel xanthine oxidoreductase inhibitor, improves
hypertension and endothelial dysfunction in spontaneously
hypertensive rats
Takashi Shirakura 1 & Johji Nomura 1 & Chieko Matsui 1 &Tsunefumi Kobayashi 1 & Mizuho Tamura 1 & Hiroaki Masuzaki 2

Received: 11 February 2016 / Accepted: 28 March 2016
# The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract Xanthine oxidase (XO) is an enzyme responsible
for the production of uric acid. XO produces considerable
amount of oxidative stress throughout the body. To date,however, its pathophysiologic role in hypertension and endothelial dysfunction still remains controversial. To explore thepossible involvement of XO-derived oxidative stress in thepathophysiology of vascular dysfunction, by use of a selective
XO inhibitor, febuxostat, we investigated the impact of pharmacological inhibition of XO on hypertension and vascularendothelial dysfunction in spontaneously hypertensive rats
(SHRs). Sixteen-week-old SHR and normotensive WistarKyoto (WKY) rats were treated with tap water (control) or
water containing febuxostat (3 mg/kg/day) for 6 weeks.
Systolic blood pressure (SBP) in febuxostat-treated SHR(220 ± 3 mmHg) was significantly (P < 0.05) decreased compared with the control SHR (236 ± 4 mmHg) while SBP infebuxostat-treated WKY was constant. Acetylcholineinduced endothelium-dependent relaxation in aortas fromfebuxostat-treated SHR was significantly (P < 0.05) improved
compared with the control SHR, whereas relaxation in response to sodium nitroprusside was not changed. Vascular XO
activity and tissue nitrotyrosine level, a representative indicator of local oxidative stress, were considerably elevated in thecontrol SHR compared with the control WKY, and this increment was abolished by febuxostat. Our results suggest thatexaggerated XO activity and resultant increase in oxidative
stress in this experimental model contribute to the hypertension and endothelial dysfunction, thereby supporting a notionthat pharmacological inhibition of XO is valuable not only for
hyperuricemia but also for treating hypertension and related
endothelial dysfunction in human clinics.
Keywords Xanthine oxidase . Febuxostat . Endothelial
dysfunction . Hypertension

Abbreviations
ACh
Acetylcholine
NAD Nicotinamide adenine dinucleotide
NO
Nitric oxide
ROS
Reactive oxygen species
SBP
Systolic blood pressure
SHR
Spontaneously hypertensive rat
SNP
Sodium nitroprusside
WKY Wistar-Kyoto
XDH Xanthine dehydrogenase
XO
Xanthine oxidase
XOR Xanthine oxidoreductase* Takashi Shirakurat.shirakura@teijin.co.jp

Introduction
1

Pharmaceutical Development Research Laboratories, Teijin Institutefor Bio-Medical Research, Teijin Pharma Ltd., 4-3-2, Asahigaoka,
Hino 191-852, Tokyo, Japan

2

Division of Endocrinology, Diabetes and Metabolism, Hematology,
Rheumatology (Second Department of Internal Medicine), Graduate
School of Medicine, University of the Ryukyus, Nishihara, Okinawa,
Japan

Xanthine oxidoreductase (XOR) catalyzes the oxidation of
hypoxanthine to xanthine and on to uric acid, which are thefinal reactions of purine catabolism in humans. Under physiological conditions, the enzyme functions mainly as a dehydrogenase (XDH) and uses NAD+ as the electron acceptor.
Noticeably, under a variety of pathologic conditions such as

Naunyn-Schmiedeberg's Arch Pharmacol

tissue ischemia and tissue damage (Harrison 2002), it can beconverted into an oxidase (xanthine oxidase (XO)) using molecular oxygen as the electron acceptor.
Because of its ability to generate reactive oxygen species(ROS), the role of XOR has long been investigated in a wide
variety of ROS-related diseases (Boueiz et al. 2008).
Hypertension is a major risk for cardiovascular diseases. A
large body of evidence suggests that locally exaggerated oxidative stress is profoundly involved in the pathophysiology ofhypertension (Nickenig and Harrison 2002; Ceriello 2008;
Cutler et al. 2008; Grossman 2008). In accordance with thisnotion, augmented oxidative stress has been implicated in
vascular dysfunction of several models of experimental hypertension ((Fukui et al. 1997; Kerr et al. 1999; Tanito et al. 2004;
Touyz 2004). In particular, endothelial dysfunction is a hallmark of hypertension (Lockette et al. 1986; Morawietz et al.
2001; Landmesser et al. 2003), which is also evoked by oxidative stress. As one of underlying mechanisms, it is suggested that oxidative stress reduces bioavailability and bioactivity of nitric oxide (NO), a potent endothelium-derived
relaxing factor (Li et al. 2013).
Based on the notion that XO produces ROS, it is reasonable
to speculate that XO would ameliorate hypertension and associated endothelial dysfunction. However, conflicting results have been reported depending on the experimental hypertensive models (Tian et al. 2005; Zhang et al. 2005;
Yamamoto et al. 2006; Ong et al. 2007; Viel et al. 2008). It
also should be noted that most of previous studies used allopurinol to attain the pharmacological XO inhibition.
Importantly, allopurinol is a classic suicide inhibitor, as its
binding to and reduction of the Mo cofactor induce selfoxidation to form oxypurinol, an active inhibitory metabolite.
Reduction of the Mo cofactor by allopurinol leads to ROS
production (Galbusera et al. 2006). On the other hand, allopurinol is known to exert radical scavenging effect due to its
chemical structure (Augustin et al. 1994). For these reasons,it seems to be hard to precisely evaluate the pharmacologicaleffect of XO inhibition by allopurinol. In contrast, febuxostat
is a novel, selective, and potent XO inhibitor with non-purine
structure for treating gout in human clinics. Unlike allopurinol, febuxostat does not produce or scavenge ROS by its
chemical structure, because it is neither a suicide XO inhibitor
nor a radical scavenger (Okamoto et al. 2003). In this sense,
febuxostat would be better tool to resolve the issue whether
pharmacological XO inhibition reduces blood pressure andimproves the endothelial dysfunction in experimental models.
To explore the possible involvement of XO-derived oxidative stress in the pathophysiology of vascular
dysfunction, we evaluated the impact of febuxostat on hypertension and vascular endothelial dysfunction in spontaneously hypertensive rats (SHRs), representative animalmodel of human essential or primary hypertension(Zicha and Kunes 1999).

Materials and methods
Animals and treatments
Male SHRs and age-matched Wistar-Kyoto (WKY) rats
weighing 200–300 g were purchased from Charles River
Japan (Yokohama, Japan) and were maintained under standard conditions until the experiments were done. All studies
were performed in accordance with procedures approved by
the Animal Ethics Committee of the Teijin Institute for BioMedical Research Institute, Teijin Pharma Limited, Tokyo,
Japan.
Four groups of rats were used in this study: (1) WKY
control rats (WKY-C), (2) febuxostat-treated WKY rats
(WKY-Fx), (3) control SHR (SHR-C), and (4) febuxostattreated SHR (SHR-Fx). WKY-C and SHR-C groups were
given tap water. The WKY-Fx and SHR-Fx groups were given
febuxostat dissolved in drinking water ad libitum at a concentration of 0.03 mg/L. At this concentration, the dose offebuxostat per day calculated by the daily water intake andbody weight was approximately 3 mg/kg/day. As a matter offact, we recently found that single oral administration offebuxostat at 1, 3, and 10 mg/kg to rats showed dosedependent inhibition of XO activity in plasma and aorta (datanot shown). It should be noted that repeated administration of
XO inhibitor at high dose in rodents results in death, partly
because xanthine calculi caused renal impairment due to inhibition of XOD/XDH (Isa et al. 1968). According to this notion, oral administration of febuxostat at 10 mg/kg for 28 days
caused calculi in 1 of 30 animals (Horiuchi et al. 1999). In thiscontext, to achieve the maximal inhibition of XO and avoid
possible renal damages, we carefully selected 3 mg/kg/day as
a dose of febuxostat. The febuxostat treatment was started at
16 weeks of age and carried out for 6 weeks. Systolic bloodpressures (SBPs) were measured before and 2, 4, and 6 weeks
during the drug treatment. Vascular function, XO activity, andother biochemical parameters were determined at 6 weeks.
Blood pressure measurement
SBP was measured using a tail-cuff sphygmomanometer(Visitech BP2000, Visitech Systems, Apex, NC). All animals
were acclimated for blood pressure measurements 1 week
before drug treatment.
Xanthine oxidase activity
Plasma and tissue XO activities were measured by the pterinbased assay. In brief, frozen tissues were homogenized withpotassium phosphate buffer, pH 7.4, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitors.
The homogenates were centrifuged 12,000 rpm for 15 min
at 4 °C, and supernatants were co-incubated with 50-μM

Naunyn-Schmiedeberg's Arch Pharmacol

pterin solution to assay XO activity. After 60-min incubation
at 37 °C, fluorometric assays were performed to calculate theproduction of isoxanthopterin. Activity of purified XO derived from buttermilk (Calbiochem) and protein concentration
were measured and used to normalize the sample activity.

Statistical calculations for significant differences were performed by using Student’s t test. Significance was accepted at
P < 0.05.

Results
In vitro organ bath studies
General conditions of animals
The animals were sacrificed at the end of the 6-week study bydecapitation under pentobarbital anesthesia. The thoracic aorta was isolated carefully and cut into 3-mm-length ring. The
ring segments were mounted in organ baths containing 5 mLof Krebs-Henseleit solution aerated with 95 % O2 and 5 %CO2 and warmed at 37 °C. The composition ofKrebsHenseleit solution is as follows (mM): NaCl (119), KCl(4.7), CaCl2 (2.5), MgSO4 (1.2), NaHCO3 (25), KH2PO4(1.2), and glucose (10); pH7.4. Each ring was connected toa force transducer (FD, NIHON KODEN, Tokyo, Japan) for
isometric force recording. The rings were stretched to 2 g of
optimal tension and equilibrated for 60 min until a stablebaseline tone was obtained.
Following equilibration, the rings were repeatedly exposed
to 60 mM KCl, and then, the presence of endothelium wasverified by the ability of acetylcholine (ACh, 1 × 10−6 M) torelax phenylephrine (PE, 1 × 10−7 M)-induced contraction.
After washout, endothelium-mediated relaxation was measured as a concentration-response curve to ACh (1 × 10−10–
1 × 10−4 M) in rings contracted with the submaximal dose ofPE (5 × 10−7 M). Endothelium-independent relaxation was
also measured as a concentration-response curve to sodium
nitroprusside (SNP) (1 × 10−10–1 × 10−4 M).
Oxidative stress measurement
Tissues were weighed and homogenized with 0.05 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA andprotease inhibitors. Homogenates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected and used for assay. The measurement of nitrotyrosine
was performed by using a commerciallyavailable
nitrotyrosine assay kit (Northwest Life Science, CA, USA)according to the manufacturer’s instructions.

Febuxostat treatment did not alter the body weight in both
WKY rats and SHRs. The plasma uric acid levels were similar
in WKY-C and SHR-C groups (Table 1). Febuxostat treatment
significantly decreased plasma uric acid levels in both WKY
rats and SHRs to the same extent (0.9 ± 0.1 mg/dL in WKY-C,
0.5 ± 0.1 mg/dL in WKY-Fx, 1.0 ± 0.1 mg/dL in SHR-C, and
0.5 ± 0.1 mg/dL in SHR-Fx; Table 1).
Systolic blood pressure
SBP in the SHR-C group was significantly higher comparedto that in the WKY-C group. Febuxostat treatment significantly (P < 0.05) decreased SBP in the SHRs (236 ± 4 mmHg in
SHR-C, 220 ± 3 mmHg in SHR-Fx, Fig. 1), while it did notalter SBP in the WKY rats after 6-week treatment (164
± 7 mmHg in WKY-C, 156 ± 9 mmHg in WKY-Fx, Fig. 1).
Vascular and plasma xanthine oxidase activity
XO activity in the thoracic aorta of SHRs was significantly
higher than that of WKY rats (Fig. 2a). The treatment withfebuxostat lowered this activity in both strains (957 ± 214 μU/
mg protein in WKY-C, 463 ± 114 μU/mg protein in WKY-Fx,
2549 ± 427 μU/mg protein in SHR-C, and 805 ± 73 μU/mg
protein in SHR-Fx, Fig. 2a). Similarly, the level of XO activity
in plasma of SHRs was significantly higher than that of WKY
rats (Fig. 2b). Treatment with febuxostat reduced this activity
in both strains (80.5 ± 2.2 mU/mL in WKY-C, 19.0 ± 2.5 mU/
mL in WKY-Fx, 113.4 ± 6.4 mU/mL in SHR-C, and 31.7
± 2.1 mU/mL in SHR-Fx, Fig. 2b).
Vascular oxidative stress

Plasma uric acid levels were determined by an enzymaticme tho d b ase d on t he ur ica se- per ox i da se sys t em
(PUREAUTO® S CRE-N, Sekisui Medical Co. Ltd., Japan).

To investigate the therapeutic effect of febuxostat on oxidative
stress, we examined the tissue nitrotyrosine level.
Nitrotyrosine, a marker of nitro-oxidative stress in the thoracic
aorta of SHRs, was significantly higher than that of WKY rats
(Fig. 3). The treatment with febuxostat lowered nitrotyrosine
concentration in both strains (Fig. 3).

Statistical analysis

Vascular reactivity

Data are expressed as mean ± SEM. The responses to ACh and
SNP are expressed as percentages of PE contraction.

Finally, we evaluated the effect of febuxostat on endothelial
function. ACh-induced endothelium-dependent relaxation in

Plasma uric acid level

Naunyn-Schmiedeberg's Arch Pharmacol
Table 1

Body weight and plasma uric acid

Improvement in hypertension and endothelial dysfunction

WKY-C

WKY-FxSHR-C

SHR-FxBW (g)

376 ± 5370 ± 6384 ± 4387 ± 4

Uric acid (mg/dL)

0.9 ± 0.1

0.5 ± 0.1**

1.0 ± 0.1NS

0.5 ± 0.1##

Values are means ± SEM (n = 9∼10 per group)
NS not significant WKY-C vs SHR-C, Student’s t test**P < 0.01 WKY-C vs WKY-Fx, ##P < 0.01 SHR-C vs SHR-Fx

the thoracic aorta was attenuated in SHR compared to that of
WKY rats (P < 0.01, Fig. 4a). Febuxostat treatment restored
the endothelium-dependent relaxation in comparison to theuntreated SHRs (P < 0.05, Fig. 4a). The endotheliumindependent relaxation measured using SNP, a direct NO donor, was similar in SHRs and WKY rats (Fig. 4b). Febuxostat
treatment did not alter the relaxation in response to SNP in
both WKY rats and SHRs.

Discussion
By using a novel, selective xanthine oxidase inhibitor
febuxostat, the present study aimed to investigate the therapeutic effects of pharmacological inhibition of XO on hypertension and endothelial dysfunction in SHRs. Our data demonstrated that the therapeutic dose of febuxostat appropriate
for hyperuricemia considerably decreased the SBP, reduced
the vascular and plasma XO activity, suppressed the vascular
nitrotyrosine level, and improved endothelial

dysfunction in

SHR.

Our results showed that the therapeutic dose offebuxostat appropriate for hyperuricemia significantly
ameliorated hypertension in SHR. On the other hand, acouple of studies showed that chronic treatment withallopurinol, a classic type of XO inhibitor, failed tolower blood pressure in SHRs (Trachtman et al. 1991;
Laakso et al. 1998, 2004; Yamamoto et al. 2006). With
respect to the possible mechanisms for the inconsistent
results between the two XO inhibitors, a couple of possibilities are raised as follows. First, allopurinol does
produce oxidative stress when metabolized to oxypurinol
as described above (Galbusera et al. 2006), while
febuxostat does not. Second, both allopurinol andoxypurinol showed the limitation to inhibit theendothelial-binding xanthine oxidase (Kelley et al.
2004; Malik et al. 2011). Third, allopurinol has beenshown to be more nephrotoxic in SHR than in WKY,
thereby masking its beneficial effect on hypertension(Trachtman et al. 1991). These may explain, at least inpart, the difference in impact on hypertension of SHR
between allopurinol and febuxostat.
XO inhibitors such as tungsten and allopurinol have beenreported to improve endothelial dysfunction in several animalmodels and human diseases such as atherosclerosis and coronary heart disease (Schroder et al. 2006; Dopp et al. 2011;
George et al. 2006; Yiginer et al. 2008). Our recent work
demonstrated that febuxostat improved endothelial dysfunction also in high-fat diet-induced obese diabetic mice
(Masuzaki et al., manuscript submitted). Based on our resultsin various experimental hypertension models, XO inhibitors
may exert favorable effects in several types of endothelial
dysfunction.
Mechanism of action of XO inhibition

Systolic blood pressure(mm Hg)

300

SHR-C

WKY-C

SHR-FxWKY-Fx

350

**

200

*
NS

150

100
0

2

4

6

Time (weeks)

Fig. 1 Systolic blood pressure (SBP) in untreated and febuxostat-treated(3 mg/kg/day) spontaneously hypertensive rats and WKY rats. Values aremeans ± SEM (n = 4∼5 per group). *P < 0.05, **P < 0.01 SHR-C vsSHR-Fx, NS not significant WKY-C vs WKY-Fx, Student’s t test

In the present study, XO activities in both aorta and plasma
from SHRs were significantly elevated as compared to thatof WKY rats. Importantly, treatment of febuxostat substantially lowered aorta and plasma XO activities in bothstrains. Circulating XO binds to glycosaminoglycan residues on the surface of endothelium in a partially heparinreversible manner and subsequently translocates to intracellular compartments (Radi et al. 1997; Houston et al.
1999). Although mechanisms whereby plasma XO activity
is elevated in SHRs are not yet entirely clarified, it has
been reported that XO is released from several organs into
systemic circulation in a line of pathophysiology including
hepatitis, hemorrhagic shock, sickle cell disease, and ischemia reperfusion (Yokoyama et al. 1990; Tan et al. 1998;
Aslan et al. 2001). SHRs represent a variety of organ damages related to severe hypertension (Liu et al. 2011; BoonNaunyn-Schmiedeberg's Arch PharmacolFig. 2 Xanthine oxidase activity
in aorta (a) and plasma (b)obtained from spontaneously
hypertensive rats and WKY rats.
Values are means ± SEM (n = 6
per group). tP < 0.05, ttP < 0.01
WKY-C vs WKY-Fx, **P < 0.01
WKY-C vs SHR-C, ##P < 0.01
SHR-C vs SHR-Fx, Student’s t
test

et al. 2013). Therefore, XO derived from damaged organs
may contribute to the elevation of plasma and vascular XO
activities in SHRs.
Our data demonstrated that endothelial relaxation by
ACh was improved in febuxostat-treated SHRs comparedwith the vehicle-treated SHRs, while the response to SNP in
aorta was unaltered. Enhanced ACh-induced relaxation in
vascular endothelium by febuxostat is attributed, at least
partly, to the increase in NO response, resulting from thescavenge of NO by ROS, which reacts rapidly with NO toform peroxynitrite (Trujillo et al. 1998). To assess the involvement of NO scavenge in this experimental paradigm,
we measured vascular nitrotyrosine levels. Protein
nitrosylation is a representative indicator of peroxynitrite
formation in vascular tissues. As expected, SHRs showedsignificantly elevated nitrotyrosine levels in the aortic homogenate as compared to WKY rats, which were normalized by the treatment of febuxostat. Thus, the data suggestthat febuxostat attenuates NO scavenge and subsequently
increases the availability of NO. Furthermore, peroxynitrite
per se induces endothelial and tissue injuries in varioustpathophysiological conditions (Pacher et al. 2002a, b,
2005; Szabo et al. 2002a, b, 2004; Ungvari et al. 2005).
Hence, suppression of peroxynitrite by febuxostat is also
beneficial to ameliorate endothelial dysfunction.
It is well documented that elevation of serum uric acidper se is a potent risk for dysfunction of kidney and vascular system, resulting in hypertension (Sundstrom et al.
2005; Galbusera et al. 2006; Krishnan et al. 2007; Grayson
et al. 2011). In animal models, hyperuricemia in rats leads
to increase in blood pressure, while the alleviation of hyperuricemia with urate-lowering drugs including
benzbromarone leads to the reversal of hypertension(Mazzali et al. 2001; Nakagawa et al. 2003; SanchezLozada et al. 2008a, b). In clinical settings, reduction ofuric acid with allopurinol in hyperuricemic, hypertensive
adolescents did ameliorate blood pressure profile (Feig etal. 2008). In contrast to these findings, the present study
used SHRs and the control normotensive WKY rats andcompared the results to minimize the possible influenceof uric acid-dependent actions, because the serum uric acidlevel in SHRs is similar to WKY rats (Trachtman et al.
1991; Durante et al. 2010). Treatment of febuxostat
equipotently lowers plasma uric acid levels in both WKY
rats and SHRs and ameliorated hypertension in SHRs, suggesting that the BP-lowering effects of febuxostat arecaused by the suppression of ROS production independentof the lowering effect on plasma uric acid. Comparison
study using XO inhibitors and other hypouricemic agents
such as benzbromarone would be required to test thishypothesis.
Study limitations

Fig. 3 Effect of febuxostat on vascular nitrotyrosine levels in untreated
and febuxostat-treated (3 mg/kg/day) spontaneously hypertensive ratsand WKY rats. Values are means ± SEM (n = 6 per group). tP < 0.05
WKY-C vs WKY-Fx, **P < 0.01 WKY-C vs SHR-C, ##P < 0.01 SHRC vs SHR-Fx, Student’s t test

In the present study, the dose of febuxostat was limited toabout 3 mg/kg/day, because the higher dose of febuxostat
highly causes xanthine calculi as described (Isa et al.
1968). As a matter of fact, febuxostat at 3 mg/kg/day could
not attain the complete inhibition of XO systemically inthis study. Therefore, we could not assess the maximal

Naunyn-Schmiedeberg's Arch Pharmacol
SNP

ACh

b

0

Relative Relaxation (%)

Relative Relaxation (%)

a

25

50

**
#
75

100
-11

0

WKY-C
WKY-Fx25

SHR-C
SHR-Fx

50

75

100
-10

-9

-8

-7

-6

-5

-4

-3

-11

-10

Log [ACh(M)]

-9

-8

-7

-6

-5

-4

-3

Log [SNP(M)]

Fig. 4 Concentration-response curves to a acetylcholine and b sodium
nitroprusside in aortic rings isolated from untreated and febuxostattreated spontaneously hypertensive rats and WKY rats. Values aremeans ± SEM (n = 4∼5 per group). **P < 0.01 WKY-C vs SHR-C,#P < 0.05 vs SHR-C, Student’s t test

effects of febuxostat on the hypertension and endothelial
dysfunction in the current experimental setting. The present study used SHR as a representative animal model ofhypertension and demonstrated the potential antihypertensive effects by pharmacological XO inhibition.
Recently, Boban et al. reported that circulating xanthineoxidase activity was increased in patients with essential
hypertension (Boban et al. 2014), which is consistent with
our results that plasma XO activity was considerably elevated in SHR. Therefore, XO inhibitor may be beneficial
for the treatment of some forms of hypertension in humans.
However, there is little information about the relationshipbetween plasma XO activity and other forms of hypertension such as renal hypertension and pulmonary hypertension. In this context, further studies are warranted to clarify
the clinical implication of XO inhibition in various types of
diseases.
With respect to the prediction of clinical efficacy of XO
inhibition, we should be careful about extrapolating the resultsin experimental animals to humans. Importantly, unlike
humans, almost all animals including rodents have uricase, aurate oxidase enzyme. Although few animal species that mimic uric acid metabolism in humans are known (Oda et al. 2002;
Bannasch et al. 2008), there seems no available model ofhypertension in these species. Therefore, clinical studies are
required to verify the effects of XO inhibition on thehypertension and endothelial dysfunction in humanpathophysiology.
In conclusion, present study demonstrates that exaggerated
XO activity and resultant increase in oxidative stress in SHRs
contribute, at least partly, to their hypertension and endothelial
dysfunction. This notion was endorsed by the finding thatchronic treatment of selective XO inhibitor febuxostat
improves a line of vascular phenotypes. Our data suggest thatpharmacological inhibition of exaggerated XO may bevaluable not only for hyperuricemia but also for treating someforms of hypertension and related endothelial dysfunction inhumans.
Compliance with ethical standards
Funding This research was supported by Teijin Pharma Limited.
Conflict of interest Dr. Masuzaki declares no conflicts of interest.
Dr. Masuzaki is supported in part by Grants-in-Aid from the Ministryof Education, Culture, Sports, Science and Technology of Japan (MEXT)
KAKENHI [Grant 24591338, (2012∼2014)] and by Special AccountBudget for Education and Research (2011∼2015) granted by the Japan
Ministry of Education, Culture, Sports, Science and Technology of Japan.
Ethical approval All procedures performed in studies involving animals were in accordance with the ethical standards of the Teijin Institutefor Bio-Medical Research Institute, Teijin Pharma Limited, Tokyo, Japan.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.

References
Aslan M, Ryan TM, Adler B, Townes TM, Parks DA, Thompson JA,
Tousson A, Gladwin MT, Patel RP, Tarpey MM, Batinic-Haberle I,
White CR, Freeman BA (2001) Oxygen radical inhibition of nitricoxide-dependent vascular function in sickle cell disease. Proc Natl
Acad Sci U S A 98:15215–15220. doi:10.1073/pnas.221292098
Augustin AJ, Boker T, Blumenroder SH, Lutz J, Spitznas M (1994) Free
radical scavenging and antioxidant activity of allopurinol andoxypurinol in experimental lens-induced uveitis. Invest
Ophthalmol Vis Sci 35:3897–3904
Bannasch D, Safra N, Young A, Karmi N, Schaible RS, Ling GV (2008)
Mutations in the SLC2A9 gene cause hyperuricosuria and hyperuricemia in the dog. PLoS Genet 4:e1000246

Naunyn-Schmiedeberg's Arch Pharmacol
Boban M, Kocic G, Radenkovic S, Pavlovic R, Cvetkovic T, Deljanin-Ilic
M, Ilic S, Bobana MD, Djindjic B, Stojanovic D, Sokolovic D,
Jevtovic-Stoimenov T (2014) Circulating purine compounds, uricacid, and xanthine oxidase/dehydrogenase relationship in essential
hypertension and end stage renal disease. Ren Fail 36:613–618
Boon CM, Ng MH, Choo YM, Mok SL (2013) Super, red palm and palm
oleins improve the blood pressure, heart size, aortic media thickness
and lipid profile in spontaneously hypertensive rats. PLoS One 8,
e55908. doi:10.1371/journal.pone.0055908
Boueiz A, Damarla M, Hassoun PM (2008) Xanthine oxidoreductase inrespiratory and cardiovascular disorders. Am J Physiol Lung Cell
Mol Physiol 294:L830–840. doi:10.1152/ajplung.00007.2008
Ceriello A (2008) Possible role of oxidative stress in the pathogenesis of
hypertension. Diabetes Care 31(Suppl 2):S181–184. doi:10.2337/
dc08-s245
Cutler JA, Sorlie PD, Wolz M, Thom T, Fields LE, Roccella EJ (2008)
Trends in hypertension prevalence, awareness, treatment, and control rates in United States adults between 1988–1994 and 1999–
2004. Hypertension 52:818–827. doi:10.1161/
HYPERTENSIONAHA.108.113357
Dopp JM, Philippi NR, Marcus NJ, Olson EB, Bird CE, Moran JJ,
Mueller SW, Morgan BJ (2011) Xanthine oxidase inhibition attenuates endothelial dysfunction caused by chronic intermittent hypoxia in rats. Respir Int Rev Thorac Dis 82:458–467
Durante P, Chavez M, Perez M, Romero F, Rivera F (2010) Effect of uricacid on hypertension progression in spontaneously hypertensive
rats. Life Sci 86:957–964. doi:10.1016/j.lfs.2010.05.004
Feig DI, Soletsky B, Johnson RJ (2008) Effect of allopurinol on bloodpressure of adolescents with newly diagnosed essential hypertension: a randomized trial. JAMA J Am Med Assoc 300:924–932.
doi:10.1001/jama.300.8.924
Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Q, Taylor WR,
Harrison DG, de Leon H, Wilcox JN, Griendling KK (1997)
p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res 80:45–51
Galbusera C, Orth P, Fedida D, Spector T (2006) Superoxide radical
production by allopurinol and xanthine oxidase. Biochem
Pharmacol 71:1747–1752. doi:10.1016/j.bcp.2006.02.008
George J, Carr E, Davies J, Belch JJ, Struthers A (2006) High-dose
allopurinol improves endothelial function by profoundly reducing
vascular oxidative stress and not by lowering uric acid. Circulation
114:2508–2516
Grayson PC, Kim SY, LaValley M, Choi HK (2011) Hyperuricemia andincident hypertension: a systematic review and meta-analysis.
Arthritis Care Res 63:102–110. doi:10.1002/acr.20344
Grossman E (2008) Does increased oxidative stress cause hypertension?
Diabetes Care 31(Suppl 2):S185–189. doi:10.2337/dc08-s246
Harrison R (2002) Structure and function of xanthine oxidoreductase:where are we now? Free Radic Biol Med 33:774–797
Horiuchi H, Ota M, Kobayashi M, Kaneko H, Kasahara Y, Nishimura S,
Kondo S, Komoriya K (1999) A comparative study on the
hypouricemic activity and potency in renal xanthine calculus formation of two xanthine oxidase/xanthine dehydrogenase inhibitors:
TEI-6720 and allopurinol in rats. Res Commun Mol Pathol
Pharmacol 104:307–319
Houston M, Estevez A, Chumley P, Aslan M, Marklund S, Parks DA,
Freeman BA (1999) Binding of xanthine oxidase to vascular endothelium. Kinetic characterization and oxidative impairment of nitricoxide-dependent signaling. J Biol Chem 274:4985–4994
Isa M, Hayashi G, Kowa Y (1968) Effects of xanthine oxidase inhibitor,
4-hydroxypyrazolo-(3, 4-d)-pyrimidine(zyloric) on the nucleic acidmetabolism and its pharmacological actions. 1. Nihon Yakurigaku
Zasshi 64:108–122
Kelley EE, Trostchansky A, Rubbo H, Freeman BA, Radi R, Tarpey MM(2004) Binding of xanthine oxidase to glycosaminoglycans limits

inhibition by oxypurinol. J Biol Chem 279:37231–37234. doi:10.
1074/jbc.M402077200
Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, Hamilton
CA (1999) Superoxide anion production is increased in a model ofgenetic hypertension: role of the endothelium. Hypertension 33:
1353–1358
Krishnan E, Kwoh CK, Schumacher HR, Kuller L (2007) Hyperuricemia
and incidence of hypertension among men without metabolic syndrome. Hypertension 49:298–303. doi:10.1161/01.HYP.
0000254480.64564.b6
Laakso J, Mervaala E, Himberg JJ, Teravainen TL, Karppanen H,
Vapaatalo H, Lapatto R (1998) Increased kidney xanthine oxidoreductase activity in salt-induced experimental hypertension.
Hypertension 32:902–906
Laakso JT, Teravainen TL, Martelin E, Vaskonen T, Lapatto R (2004)
Renal xanthine oxidoreductase activity during development of hypertension in spontaneously hypertensive rats. J Hypertens 22:
1333–1340
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM,
Mitch WE, Harrison DG (2003) Oxidation of tetrahydrobiopterin
leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111:1201–1209. doi:10.1172/JCI14172
Li H, Horke S, Forstermann U (2013) Oxidative stress in vascular disease
and its pharmacological prevention. Trends Pharmacol Sci 34:313–
319
Liu W, Wang W, Song SW, Gu XF, Ma XJ, Su FY, Zhang H, Liu AJ, Su
DF (2011) Synergism of telmisartan and amlodipine on blood pressure reduction and cardiorenal protection in hypertensive rats. J
Cardiovasc Pharmacol 57:308–316. doi:10.1097/FJC.
0b013e3182073e41
Lockette W, Otsuka Y, Carretero O (1986) The loss of endotheliumdependent vascular relaxation in hypertension. Hypertension 8:
II61–66
Malik UZ, Hundley NJ, Romero G, Radi R, Freeman BA, Tarpey MM,
Kelley EE (2011) Febuxostat inhibition of endothelial-bound XO:
implications for targeting vascular ROS production. Free Radic Biol
Med 51:179–184. doi:10.1016/j.freeradbiomed.2011.04.004
Mazzali M, Hughes J, Kim YG, Jefferson JA, Kang DH, Gordon KL, Lan
HY, Kivlighn S, Johnson RJ (2001) Elevated uric acid increases
blood pressure in the rat by a novel crystal-independent mechanism.
Hypertension 38:1101–1106
Morawietz H, Weber M, Rueckschloss U, Lauer N, Hacker A, Kojda G(2001) Upregulation of vascular NAD(P)H oxidase subunit
gp91phox and impairment of the nitric oxide signal transduction
pathway in hypertension. Biochem Biophys Res Commun 285:
1130–1135. doi:10.1006/bbrc.2001.5312
Nakagawa T, Mazzali M, Kang DH, Kanellis J, Watanabe S, SanchezLozada LG, Rodriguez-Iturbe B, Herrera-Acosta J, Johnson RJ(2003) Hyperuricemia causes glomerular hypertrophy in the rat.
Am J Nephrol 23:2–7
Nickenig G, Harrison DG (2002) The AT(1)-type angiotensin receptor inoxidative stress and atherogenesis: part I: oxidative stress and atherogenesis. Circulation 105:393–396
Oda M, Satta Y, Takenaka O, Takahata N (2002) Loss of urate oxidase
activity in hominoids and its evolutionary implications. Mol Biol
Evol 19:640–653
Okamoto K, Eger BT, Nishino T, Kondo S, Pai EF (2003) An extremely
potent inhibitor of xanthine oxidoreductase. Crystal structure of the
enzyme-inhibitor complex and mechanism of inhibition. J Biol
Chem 278:1848–1855. doi:10.1074/jbc.M208307200
Ong SL, Vickers JJ, Zhang Y, McKenzie KU, Walsh CE, Whitworth JA(2007) Role of xanthine oxidase in dexamethasone-induced hypertension in rats. Clin Exp Pharmacol Physiol 34:517–519. doi:10.
1111/j.1440-1681.2007.04605.x
Pacher P, Liaudet L, Mabley J, Komjati K, Szabo C (2002a)
Pharmacologic inhibition of poly(adenosine diphosphate-ribose)Naunyn-Schmiedeberg's Arch Pharmacol
polymerase may represent a novel therapeutic approach in chronic
heart failure. J Am Coll Cardiol 40:1006–1016
Pacher P, Mabley JG, Soriano FG, Liaudet L, Komjati K, Szabo C(2002b) Endothelial dysfunction in aging animals: the role ofpoly(ADP-ribose) polymerase activation. Br J Pharmacol 135:
1347–1350. doi:10.1038/sj.bjp.0704627
Pacher P, Schulz R, Liaudet L, Szabo C (2005) Nitrosative stress andpharmacological modulation of heart failure. Trends Pharmacol
Sci 26:302–310. doi:10.1016/j.tips.2005.04.003
Radi R, Rubbo H, Bush K, Freeman BA (1997) Xanthine oxidase binding
to glycosaminoglycans: kinetics and superoxide dismutase interactions of immobilized xanthine oxidase-heparin complexes. Arch
Biochem Biophys 339:125–135. doi:10.1006/abbi.1996.9844
Sanchez-Lozada LG, Tapia E, Bautista-Garcia P, Soto V, Avila-Casado C,
Vega-Campos IP, Nakagawa T, Zhao L, Franco M, Johnson RJ(2008a) Effects of febuxostat on metabolic and renal alterations inrats with fructose-induced metabolic syndrome. Am J Physiol Ren
Physiol 294:F710–718. doi:10.1152/ajprenal.00454.2007
Sanchez-Lozada LG, Tapia E, Soto V, Avila-Casado C, Franco M, Zhao
L, Johnson RJ (2008b) Treatment with the xanthine oxidase inhibitor febuxostat lowers uric acid and alleviates systemic and glomerular hypertension in experimental hyperuricaemia. Nephrol, Dial,
Transplant Off Publ Eur Dial Transplant Assoc Eur Ren Assoc 23:
1179–1185. doi:10.1093/ndt/gfm783
Schroder K, Vecchione C, Jung O, Schreiber JG, Shiri-Sverdlov R, vanGorp PJ, Busse R, Brandes RP (2006) Xanthine oxidase inhibitor
tungsten prevents the development of atherosclerosis in ApoE
knockout mice fed a Western-type diet. Free Radic Biol Med 41:
1353–1360
Sundstrom J, Sullivan L, D’Agostino RB, Levy D, Kannel WB, Vasan
RS (2005) Relations of serum uric acid to longitudinal blood pressure tracking and hypertension incidence. Hypertension 45:28–33.
doi:10.1161/01.HYP.0000150784.92944.9a
Szabo C, Mabley JG, Moeller SM, Shimanovich R, Pacher P, Virag L,
Soriano FG, Van Duzer JH, Williams W, Salzman AL, Groves JT(2002a) Part I: pathogenetic role of peroxynitrite in the development
of diabetes and diabetic vascular complications: studies with FP15, a
novel potent peroxynitrite decomposition catalyst. Mol Med 8:571–580
Szabo C, Zanchi A, Komjati K, Pacher P, Krolewski AS, Quist WC, LoGerfo
FW, Horton ES, Veves A (2002b) Poly(ADP-Ribose) polymerase isactivated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity. Circulation 106:2680–2686
Szabo C, Pacher P, Zsengeller Z, Vaslin A, Komjati K, Benko R, Chen M,
Mabley JG, Kollai M (2004) Angiotensin II-mediated endothelial
dysfunction: role of poly(ADP-ribose) polymerase activation. Mol
Med 10:28–35
Tan S, McAdams M, Royall J, Freeman BA, Parks DA (1998)
Endothelial injury from a circulating mediator following rat liverischemia. Free Radic Biol Med 24:427–434

Tanito M, Nakamura H, Kwon YW, Teratani A, Masutani H, Shioji K,
Kishimoto C, Ohira A, Horie R, Yodoi J (2004) Enhanced oxidative
stress and impaired thioredoxin expression in spontaneously hypertensive rats. Antioxid Redox Signal 6:89–97. doi:10.1089/
152308604771978381
Tian N, Thrasher KD, Gundy PD, Hughson MD, Manning RD Jr (2005)
Antioxidant treatment prevents renal damage and dysfunction andreduces arterial pressure in salt-sensitive hypertension.
Hypertension 45:934–939. doi:10.1161/01.HYP.0000160404.
08866.5aTouyz RM (2004) Reactive oxygen species, vascular oxidative stress, andredox signaling in hypertension: what is the clinical significance?
Hypertension 44:248–252. doi:10.1161/01.HYP.0000138070.
47616.9d
Trachtman H, Valderrama E, Futterweit S (1991) Nephrotoxicity of allopurinol is enhanced in experimental hypertension. Hypertension 17:
194–202
Trujillo M, Alvarez MN, Peluffo G, Freeman BA, Radi R (1998)
Xanthine oxidase-mediated decomposition of S-nitrosothiols. J
Biol Chem 273:7828–7834
Ungvari Z, Gupte SA, Recchia FA, Batkai S, Pacher P (2005) Role ofoxidative-nitrosative stress and downstream pathways in various formsof cardiomyopathy and heart failure. Curr Vasc Pharmacol 3:221–229
Viel EC, Benkirane K, Javeshghani D, Touyz RM, Schiffrin EL (2008)
Xanthine oxidase and mitochondria contribute to vascular superoxideanion generation in DOCA-salt hypertensive rats. Am J Physiol Heart
Circ Physiol 295:H281–288. doi:10.1152/ajpheart.00304.2008
Yamamoto Y, Ogino K, Igawa G, Matsuura T, Kaetsu Y, Sugihara S,
Matsubara K, Miake J, Hamada T, Yoshida A, Igawa O,
Yamamoto T, Shigemasa C, Hisatome I (2006) Allopurinol reduces
neointimal hyperplasia in the carotid artery ligation model in spontaneously hypertensive rats. Hypertens Res Off J Jpn Soc Hypertens
29:915–921. doi:10.1291/hypres.29.915
Yiginer O, Ozcelik F, Inanc T, Aparci M, Ozmen N, Cingozbay BY,
Kardesoglu E, Suleymanoglu S, Sener G, Cebeci BS (2008)
Allopurinol improves endothelial function and reduces oxidant-inflammatory enzyme of myeloperoxidase in metabolic syndrome.
Clin Res Cardiol Off J Ger Card Soc 97:334–340
Yokoyama Y, Beckman JS, Beckman TK, Wheat JK, Cash TG, Freeman
BA, Parks DA (1990) Circulating xanthine oxidase: potential mediator of ischemic injury. Am J Phys 258:G564–570
Zhang Y, Chan MM, Andrews MC, Mori TA, Croft KD, McKenzie KU,
Schyvens CG, Whitworth JA (2005) Apocynin but not allopurinol
prevents and reverses adrenocorticotropic hormone-induced hypertension in the rat. Am J Hypertens 18:910–916. doi:10.1016/j.
amjhyper.2005.02.017
Zicha J, Kunes J (1999) Ontogenetic aspects of hypertension development: analysis in the rat. Physiol Rev 79:1227–1282