Aldose reductase mediates endothelial cell dysfunction induced by high uric acid concentrations
Huang et al. Cell Communication and Signaling
Aldose reductase mediates endothelial cell dysfunction induced by high uric acid concentrations
Zhiyong Huang 0
Quan Hong 0
Liyuan Wang 0
Shaoyuan Cui 0
Zhe Feng 0
Yang Lv 0
Guangyan Cai 0
Xiangmei Chen 0
Di Wu 0
0 Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center of Kidney Diseases , Beijing 100853 , People's Republic of China
Background: Uric acid (UA) is an antioxidant found in human serum. However, high UA levels may also have pro-oxidant functions. According to previous research, aldose reductase (AR) plays a vital role in the oxidative stress-related complications of diabetes. We sought to determine the mechanism by which UA becomes deleterious at high concentrations as well as the effect of AR in this process. Method: Endothelial cells were divided into three groups cultured without UA or with 300 μM or 600 μM UA. The levels of total reactive oxygen species (ROS), of four ROS components, and of NO and NOX4 expression were measured. Changes in the above molecules were detected upon inhibiting NOX4 or AR, and serum H2O2 and vWF levels were measured in vivo. Results: Increased AR expression in high UA-treated endothelial cells enhanced ROS production by activating NADPH oxidase. These effects were blocked by the AR inhibitor epalrestat. 300 μM UA decreased the levels of the three major reactive oxygen species (ROS) components: O2 -, OH, and 1O2. However, when the UA concentration was increased, both O2 - levels and downstream H2O2 production significantly increased. Finally, an AR inhibitor reduced H2O2 production in hyperuricemic mice and protected endothelial cell function. Conclusions: Our findings indicate that inhibiting AR or degrading H2O2 could protect endothelial function and maintain the antioxidant activities of UA. These findings provide new insight into the role of UA in chronic kidney disease.
Aldose reductase; Endothelial cell dysfunction; Uric acid; Reactive oxygen species; Hyperuricemia; CKD
Uric acid (UA) is the final enzymatic product in the
degradation of purine nucleosides and free bases in humans
and the great apes [1–3]. UA is a powerful antioxidant
that scavenges singlet oxygen (1O2) molecules, oxygen
radicals, and peroxynitrite (ONOO−) molecules. UA also
chelates transition metals to reduce ion–mediated
ascorbic acid oxidation [4–7]. UA is responsible for
approximately 50% of serum antioxidant activity [2, 4].
However, in vivo and cellular studies have demonstrated
that depending on its chemical microenvironment, UA
may also be a pro-oxidant . Strong epidemiological
evidence suggests that the prevalence of gout and
hyperuricemia is increasing worldwide . High UA levels are
strongly associated with and often predict the
development of hypertension, visceral obesity, insulin resistance,
dyslipidemia, type II diabetes, kidney disease, and
cardiovascular events [10, 11]. Although endothelial
dysfunction generally occurs in the initial stages of these
diseases, few studies on the effect of UA on human
endothelial cells have been performed . UA
dosedependently decreases nitric oxide (NO) production in
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intact bovine aortic endothelial cells , and
hyperuricemia induces endothelial dysfunction via mitochondrial Na
+/Ca2+ exchange-mediated mitochondrial calcium
overload . However, how the effects of UA become
deleterious at high concentrations is unknown. Although the
pathogenesis of these diseases is extremely complex and
incompletely understood, oxidative stress clearly plays a
central role. The urate oxidant-antioxidant paradox led us
to investigate the point at which urate becomes an oxidant
and the pathway through which this occurs. Although UA
may have protective effects under certain conditions [15,
16], it cannot scavenge all radicals. Additionally, UA and/
or its downstream radicals can trigger intracellular oxidant
production via the ubiquitous NADPH oxidase-dependent
pathway, thereby resulting in oxidative stress.
Aldose reductase (AR) is the rate-limiting enzyme in
the polyol pathway, and NADPH acts as a cofactor .
AR plays an important role in the pathogenesis of
diabetic complications  and atherosclerosis . In
diabetic rats, AR expression was increased, and inhibiting
AR ameliorated renal function . The AR inhibitor
epalrestat suppresses the progression of diabetic
complications such as retinopathy, nephropathy and
neuropathy . Genetic AR deficiency also prevented the
progress of diabetic nephropathy . The elevation of
AR may be related to higher oxidative stress levels in
diabetic rats . In our previous research, AR
expression was increased in endothelial cells treated with high
uric acid concentrations . Increased substrate flux
via AR leads to increased ROS production, cell injury,
apoptosis, altered ion regulation, and mitochondrial
dysfunction [25–31], and increased ROS production is
associated with NADPH oxidase activation [32–34].
In this study, we detected the different ROS types
generated in HUVECs cultured with different UA concentrations
as well as the changes in ROS production upon transfecting
HUVECs with siRNA against AR. We then investigated the
role of AR in UA-induced endothelial injury in vitro and in
vivo. Our results suggest a novel mechanism underlying the
endothelial dysfunction caused by high UA levels.
Cell culture and uric acid preparation
Human umbilical vein endothelial cells (HUVECs) were
purchased from YRbio (Cat#NC006, Changsha, China)
and cultured in RPMI-1640 media supplemented with
10% fetal bovine serum (FBS) at 37 °C in a humidified
incubator in a 5% CO2 atmosphere.
Uric acid was purchased from Sigma–Aldrich
(Carlsbad, CA). Uric acid powder was dissolved in a 1 mol/L
NaOH solution at a concentration of 40 mmol/L. Then,
the uric acid solution was added to the serum containing
medium at a final concentration and at a pH 7.2–7.4.
Intracellular reactive oxygen species (ROS) assays
Cells were seeded onto 35-mm confocal dishes (with a
cover glass) and classified into control, normal uric acid
(UA), and high uric acid (HUA) groups (n = 3). Cells in
the UA and HUA groups were cultured in medium
containing 300 or 600 μM uric acid, respectively, for 24 h
followed by incubation with the total oxidative stress
indicator chloromethyl derivative
dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Beyotime, Nanjing
China, 5 μM) for 30 min in the dark at 37 °C. After three
washes in PBS, green fluorescence was visualized using a
laser scanning confocal microscope at an excitation
wavelength of 488 nm and an emission wavelength of
Before HUA treatment, the HUA group was pretreated
with 0.5 mM apocynin (Santa Cruz Biotechnology) or
0.1 μM epalrestat (Santa Cruz Biotechnology). Pyocyanin
(100 μM; Sigma) was used as a positive control in the
comparisons of ROS production.
Detection of intracellular ROS components
For the total intracellular ROS levels, method was
referenced previously by the oxidant-sensing fluorescent
probe CM-H 2 DCFDA . Briefly, this probe was
loaded into previously subcultured HUVEC-Cs at a final
concentration of 10 μmol/L, and the cells were then
cultured for 30 min at 37 °C. After incubation, the culture
medium was washed twice with PBS and analyzed by
laser confocal microscopy with an excitation wavelength
of 488 nm and an emission wavelength of 515 nm.
The detection of intracellular ROS components
occurred as follows. For O•2−(Mitochondrial): cells were
incubated with 4 μmol/L Mito-SOX Red (Invitrogen) in
the dark at 37 °C for 10 min, and red fluorescence was
observed at an excitation wavelength of 510 nm and an
emission wavelength of 580 nm. For H2O2: cells were
incubated with 30 μmol/L BES-H2O2 (Seebio, China) in
the dark at 37 °C for 1 h, and green fluorescence was
observed at an excitation wavelength of 485 nm and an
emission wavelength of 515 nm. For · OH: cells were
incubated with 100 μmol/L proxylfluorescamine
(Invitrogen) in the dark at 37 °C for 30 min, and green
fluorescence was observed at an excitation wavelength of
488 nm and an emission wavelength of 520 nm. For 1O2:
cells were incubated with 20 μmol/L
trans-1-(2′-methoxyvinyl)pyrene (Invitrogen) in the dark at 37 °C for
10 min, and blue fluorescence was observed at an
excitation wavelength of 405 nm and an emission wavelength
of 460 nm. For ONOO−: cells were incubated with
10 μmol/L dihydrorhodamine123 (Santa Cruz) in the
dark at 37 °C for 30 min, and green fluorescence was
observed at an excitation wavelength of 488 nm and an
emission wavelength of 520 nm.
Generation of inducible and stable cell lines
Reverse transcription was carried out on human kidney
RNA with Superscript II reverse transcriptase, according to
the manufacturer’s instructions. The full-length human
NOX4 was amplified by PCR using the primers NOX4_F,
CATGGCTGTGTCCTGGAGG-3′, and NOX4_R, 5′-GGG
AAAGACTCTTTATTGTATTC-3′. The PCR product was
cloned into a pcDNA3.1 vector according to the
manufacturer’s instructions, obtained pcDNA-NOX4. HUVECs were
transfected with the pcDNA-NOX4 plasmid using
Lipofectamine 2000 (Invitrogen). Clones were selected 10–
16 days after transfection using 400 μg/ml neomycin (G418)
to obtain HUVECs stably expressing NOX4.
Measurement of nitric oxide levels in culture
supernatants or serum
Before treatment with chemicals or UA, media were
replaced with Dulbecco’s modified Eagle’s medium (DMEM).
The supernatant or serum was centrifuged and subjected to
NO level evaluation using the Nitric Oxide Assay Kit
(Applygen Technologies, China) according to the
manufacturer’s instructions. The end-point measured
mula was NO.2.
RNA was extracted from tissues and cells using TRIzol
reagent (Invitrogen) and reverse transcribed into cDNA using
M-MLV reverse transcriptase (Invitrogen). The cDNA was
used as a template in quantitative real-time PCR reactions
performed using SYBR green I PCR Master Mix and an
ICycler system (Bio-Rad). The following primers were
designed from the full-length AR and Nox4 mRNA sequences
and synthesized by SBS Biotechnology Corporation
(Beijing, China): AR sense, 5′- CCTATGGCCAAGGACA
CACT-3′ and antisense, 5′-CTGGTCTCAGGCAAGGAA
AG-3′; NOX4 sense, 5′-TTGCCTGGAAGAACCCAAGT
-3′ and antisense, 5′- TCCGCACAATAAAGGCACAA-3′.
As an internal control, mouse GAPDH was amplified using
the following primers: sense,
5′-GGCATGGACTGTGGTCATGAG-3′ and antisense,
5′-TGCACCACCAACTGCTTAGC-3′. Relative expression (fold change vs. control) was
quantified using the 2-ΔΔCt method.
For Western blotting, proteins were extracted from
tissues or cells using RIPA lysis buffer (50 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet
P-40, 0.1% SDS, 1 mM PMSF, and protease cocktail at
1 μg/ml). Protein concentrations were measured using a
BCA kit (Pierce). Protein samples (60 μg per lane) were
separated by 12% SDS-PAGE and transferred to
nitrocellulose (NC) membranes. After staining with Ponceau S, the
membranes were incubated overnight at 4 °C in 5% non-fat
milk followed by incubation with a primary antibody
against AR (Santa Cruz Biotechnology) or β-actin (Sigma).
Immunoreactive bands were visualized using ECL reagent
(Santa Cruz Biotechnology) according to the
manufacturer’s instructions and were then exposed to X-ray film.
Protein band intensities were quantified using the Quantity
One software (Bio-Rad). The assay was repeat 3 times.
Aldose reductase activity assays
AR activity was measured spectrophotometrically as
previously described [35, 36]. Briefly, AR activity was
measured as the decrease in the absorbance of NADPH at
340 nm using DL-glyceraldehyde as the substrate. The
assay mixture contained 30 mM potassium phosphate
buffer (pH 6.5), 5 mM DL-glyceraldehyde, 0.2 M
ammonium sulfate, and 1.0 mM NADPH. The results are
presented as μmol NADPH · min-1 · g-1 protein. All
reagents were from Sigma. The assay was repeat 3 times.
Establishment of hyperuricemic mouse models
Hyperuricemic mouse models were established as
described by Yang et al.  with slight modifications. The
animal protocol was reviewed and approved by the
Institutional Animal Care and Use Committee of the Chinese
PLA General Hospital. Wild-type C57BL/6 mice obtained
from the Experimental Animal Center of the Academy of
Military Medical Sciences (China) were used as controls.
The mice were housed in temperature-controlled cages on
a 12-h light-dark cycle and given free access to water and
normal chow. After one week of breeding for adaptation,
the mice were grouped into control (n = 6) and
hyperuricemic model (n = 24) groups. Mice were intraperitoneally
injected with 250 mg/Kg · d oxonic acid potassium salt
(Sigma) and 250 mg/Kg · d uric acid (Sigma). After
receiving intraperitoneal injections for 3 days, the hyperuricemic
model group was sub-classified into hyperuricemic mice
(n = 6), hyperuricemic mice treated with epalrestat
(100 mg/Kg · d, Dyne, China) (n = 6), and hyperuricemic
mice treated with polyethylene glycol catalase
(PEG-catalase, 12000 U/Kg · d, Sigma) (n = 6). The antioxidant
treatments PEG-catalase and epalrestat were intragastrically
administered. After 10 days of modeling, the levels of UA,
NO, H2O2, and von Willebrand factor (vWF) in the blood
Measurement of serum UA, H2O2 and vWF levels
The serum UA level was assayed using an enzymatic
method that measures the end production of quinonimine
using an automatic biochemical analyzer (Hitachi, Japan).
Serum H2O2 levels were assayed using a hydrogen peroxide
assay kit (NJJCbio, Nanjing, China) with the end formula
Mn2+. vWF was detected using a von Willebrand Factor
All data are expressed as the means ± SD. Mean
comparisons among multiple groups were conducted using
oneway analysis of variance (ANOVA). Comparisons of the
means between two groups were conducted using
randomized controlled t-tests. A p value < 0.05 was
considered statistically significant.
Fig. 1 Effect of different UA concentration on intracellular oxidative stress, NO release and AR activity. Endothelial cells were cultured in the
presence of 300, 600 μM UA or without UA for 24 h. a Total ROS was reduced in cells treated with 300 μM UA (#P < 0.05 vs. control, n = 3), but
increased in cells treated with 600 μM UA (*P < 0.05 vs. control, n = 3). b Compared to the control, the NO level was not significantly different in
the 300 μM UA-treated group, but was reduced in the 600 μM UA-treated group (*P < 0.05 vs. control, n = 6). c Cells were treated with 600 μM
UA at 6, 12, 24, and 48 h; total ROS production increased (*P < 0.05 vs. control, n = 6), ROS production was saturated at 24–48 h (#P < 0.05 vs. 6 h
or 12 h, n = 6), and (d) NO levels decreased at 6 h (*P < 0.05 vs. control, n = 6). e AR expression increase at 24 h (*P < 0.05 vs. control, n = 3) in the
high concentration of uric acid. There is no significate change at the 6 h or 12 h. f, g After intraperitoneal injection with oxonic acid potassium
salt and UA for 10 days, serum UA levels in wild-type C57BL/6 mice increased significantly (*P < 0.05 vs. control, n = 6), whereas serum NO levels
declined (*P < 0.05 vs. control, n = 6). g AR activity increased in endothelial cells cultured in the presence of 600 μM UA for 24 h, but there was
no change upon treatment with 300 μM UA (*P < 0.05 vs. control, n = 6)
HUVECs UA (300 μM) reduced total ROS levels in
endothelial cells, whereas high UA (600 μM) treatment
increased intracellular ROS production (Fig. 1a). NO
release was reduced after high UA treatment in vitro with
the turning point of 500 μmol/L, An additional file
shows this in more detail [see Additional file 1] but
unchanged after UA treatment (Fig. 1b). Additionally, total
ROS production increased and NO levels decreased in a
time-dependent manner in cells treated with high UA,
AR protein expression increased at 24 h and 48 h of
high concentration uric acid treated (Fig. 1c, d and e). In
male hyperuricemic C57BL/6 mice after modeling in
vivo serum UA levels significantly increased (Fig. 1f ),
whereas NO levels decreased (Fig. 1g). Furthermore, AR
activity increased in endothelial cells (Fig. 1h). Our
results showed that AR activity increased upon treatment
with high UA concentrations but not with normal UA
High UA increased AR expression via p38/MAPK pathway
In order to assay how UA trigger AR expression. P38
and extracellular signal–regulated kinase (ERK) 42/44
MAPK phosphorylation are involved in the UA-induced
cell proliferation and activation in the UA-induced
HUVEC . Therefore we determined the effect of
blocking p38 and ERK44/42 MAPK in UA-induced AR
expression using specific inhibitors of the p38
(SB203580, 5 μM) and ERK44/42 (PD 98059, 10 μM)
MAPK pathways respectively. Also, we used the organic
anion transporter inhibitor, probenecid (Sigma-Aldrich,
St. Louis, MO, USA), to block the uric acid transport
into cells. We found AR expression increased when
HUVECs were treated with high UA for 48 h, p38 and
p-ERK42/44 were activated simultaneously [Fig. 2a].
When blocking p38 and MAPK by specific inhibitor
SB203580 and PD 98059 respectively, or blocking the
organic anion transporter that could transport uric acid
into intracellular by probenecid, AR protein expression
decreased [Fig. 2b].
Increased AR expression enhances ROS production by
activating NADPH oxidase
NOX4 is the main NOX as well as the main source of
ROS in endothelial cells under oxidative stress [38–40].
NOX4 mRNA and protein expression levels increased in
endothelial cells following challenge with high UA
(Fig. 3a and b), but not NOX2, An additional file shows
this in more detail [see Additional file 2]. However, when
pretreated with the NOX inhibitor apocynin, ROS
production induced by high UA levels was reduced (Fig. 3c).
However, NOX4 expression was downregulated
following pretreatment with the AR inhibitor epalrestat before
high UA treatment, (Fig. 3a and b), suggesting that the
enhanced AR expression induced by high UA activates
NOX, thereby upregulating ROS expression and
ultimately impairing endothelial cells. The AR inhibitor
enhanced NO production compared with that in the high
UA group (Fig. 3d), suggesting that inhibiting ROS
production protected endothelial cells. However, when
NOX4 was overexpressed in combination with AR
knockdown, high UA treatment significantly decreased
ROS production compared with that of cells
overexpressing NOX4 alone. Furthermore, NO secretion
concomitantly increased (Fig. 3e–g).
Fig. 2 High UA increased AR expression via p38/MAPK pathway. Effect of UA on mitogen-activates protein kinase (MAPK) pathway activation (a).
UA activated p38 and extracellular signal–regulated kinase (ERK) 44/42 MAPK pathway in HUVEC. Western blots shown are representative of four
experiments for phosphorylated and total p38 and phosphorylated and total ERK44/42. Effect of co-stimulation of UA with MAPK inhibitors and
probenecid on AR protein expression (B). UA-induced expression of AR (600 μM, 48 h) was blocked by inhibitors of p38 (SB203580, 5 μM), ERK42/
44 (PD 98059, 10 μM), MAPK, and probenecid (PN)
Fig. 3 (See legend on next page.)
(See figure on previous page.)
Fig. 3 The AR inhibitor alleviated oxidative stress and impaired HUVECs by inhibiting NADPH oxidase activity. a and b Nox4 production was
up-regulated in endothelial cells cultured in the presence of 600 μM UA for 24 h (*P < 0.05 vs. control, n = 6), but down-regulated in Epalrestat
+ HUA cells pretreated with epalrestat (0.1 μM) for 30 min, followed by incubation with UA (600 μM) for 24 h (#P < 0.05 vs. HUA group, n = 6).
c ROS production increased in endothelial cells incubated with UA (600 μM) for 24 h (*P < 0.05 vs. control, n = 6), was similar to that in cells
treated with pyocyanin, decreased in endothelial cells when pretreated with apocynin/epalrestat (# P < 0.05 vs. HUA group, n = 6), and was
reduced significantly in cells pretreated with the AR inhibitor epalrestat (†P < 0.05, Apocynin + HUA group vs. Epalrestat + HUA group, n = 6). d
NO levels in supernatant in the Epalrestat + HUA group were enhanced compared to those in the high UA group after endothelial cells were
pretreated with epalrestat for 30 min followed by UA (600 μM) for 24 h (*P < 0.05 vs. HUA group, n = 6). e–g Cells were transfected with siRNA
or pcDNA3-NOX4, and treated with high uric acid for 24 h. AR siRNA knocked-down AR protein expression and downregulated NOX4
expression (*P < 0.05 vs. siCon group, n = 6). ROS production decreased and NO production increased (*P < 0.05 vs. siCon group, n = 6).
However, overexpression of NOX4 did not affect AR. If cells were treated with AR RNAi and overexpressed NOX4, ROS production and NO
concentration significantly decreased and increased, respectively, compared to the NOX4 overexpression group (▲P < 0.05, n = 6)
High UA impaired endothelial cells by enhancing H2O2
production but inhibited other ROS components
We then assessed the production of four ROS
components after treatment with various UA concentrations.
UA partially eliminated superoxide anion (O•2−), 1O2, and
hydroxyl radical (·OH) production and subtly increased
H2O2 production. However, high UA treatment
increased O•2− and H2O2 production but reduced 1O2 and ·
OH production (Fig. 4a).
After high UA treatment, O•2− and H2O2 levels
increased. Although O•2− is highly dynamic, it soon became
disproportionate to H2O2. Therefore, we inferred that
H2O2 is the major ROS contributor to endothelial cell
impairment induced by high UA treatment. H2O2 levels
significantly decreased in endothelial cells pretreated
with epalrestat followed by high UA treatment, but
ONOO− levels did not change (Fig. 4b). When
PEGcatalase was applied to eliminate intracellular H2O2,
total intracellular ROS levels were reduced, and NO
levels increased compared with the high UA group that
did not receive PEG-catalase treatment (Fig. 4c, d).
PEG-catalase and AR inhibitor epalrestat reduce H2O2
production in hyperuricemic mice and effectively protect
mouse endothelial cell function
Elevated H2O2 can lead to vascular endothelial
dysfunction . H2O2 production increased in endothelial cells
after high UA treatment in vitro (Fig. 3a). Upon
endothelial dysfunction, cells release more vWF, which is a marker
of endothelial dysfunction [42–44]. To estimate vascular
endothelial function in vivo, we detected serum vWF
concentrations. Serum H2O2 and vWF (Fig. 5b ~ d) levels
increased in the hyperuricemic model, while the NO
concentration decreased. We concluded that AR, O•2−, and
H2O2 are the major contributors to impaired endothelial
cell function. Inhibiting these could reduce the number of
impaired endothelial cells induced by high UA treatment.
After treatment with PEG-catalase or epalrestat, serum
H2O2 (Fig. 5b) and vWF (Fig. 5c) levels decreased
compared with those of the untreated group, while NO
concentration increased (Fig. 5d). Based on the PEG-catalase
treatment results, H2O2 may be the final ROS product
responsible for endothelial cell impairment. Our data
suggest that PEG-catalase and epalrestat decrease H2O2
production, thereby protecting endothelial function.
Uric acid (UA), which is generated in mammalian
systems as an end product of purine metabolism, is the
most abundant antioxidant in human plasma and
possesses free radical scavenging properties. In humans and
other higher primates, uric acid is the final compound of
purines catabolism, but all other mammals converts uric
acid to allantoin with enzyme uricase which is deficient
in humans and other higher primates , and is the
main reason why serum UA levels in adult males are
350 μmol/L, compared with the majority of mammals
who have UA levels <30–60 mg/dl . Evidence has
demonstrated Western diet could elevate serum uric
acid . However, it may also act as a pro-oxidant
under oxidative stress conditions. Markedly increased
UA levels cause gout and nephrolithiasis , and high
UA concentrations are also associated with an increased
risk of developing cardiovascular disease (CVD),
particularly hypertension, obesity/metabolic syndrome, and
kidney disease [10, 11, 48–51]. Jia et al. verified
hyperuricemia is related with the development of obesity/
metabolic cardiomyopathy . However, the role of UA
in CVD pathogenesis is still debated. UA is one of the
most important antioxidants in body fluids and
effectively eliminates ROS . Other risk factors exist in
CVD patients in addition to the superoxide generation
that accompanies UA production by xanthine
oxidoreductase . Whether UA is a causative risk factor or
plays a protective role with respect to its antioxidant
properties is not known [54, 55]. The mechanism (s) by
which UA acts as a “double-edged sword” remain to be
Mounting evidence indicates that hyperuricemia
induces heart and kidney injury by promoting free radical
generation and subsequent endothelial dysfunction ,
which are regulated by NO bioavailability and activity
Fig. 4 (See legend on next page.)
(See figure on previous page.)
Fig. 4 H2O2 production increased in endothelial cells treated with various concentrations of UA, and impaired endothelial cells were reduced
after elimination of H2O2 (A) O2−, 1O2, ·OH, and ONOO− levels decreased in endothelial cells (*P < 0.05 vs. control, n = 6), but H2O2 was
upregulated in endothelial cells treated with 300 μM UA for 24 h (*P < 0.05 vs. control, n = 6). 1O2 and · OH levels were down-regulated in endothelial
cells treated with 600 μM UA for 24 h (#P < 0.05 vs. control, n = 6), whereas O2−, H2O2, and ONOO− levels were up-regulated in endothelial cells
treated with 600 μM UA (#P < 0.05 vs. control, n = 6). b H2O2 production decreased in epalrestat + HUA cells that were pretreated with epalrestat
for 30 min followed by treatment with 600 μM UA for 24 h (*P < 0.05 vs. HUA group, n = 6), but ONOO− levels in Epalrestat + HUA cells did not
change compared to the HUA group. c Tntracellular H2O2 and total ROS levels were reduced by using PEG-catalase in the high UA cells, and NO
production was enhanced in PEG-catalase + high UA cells pretreated with PEG-catalase for 30 min followed by treatment with 600 μM UA for
24 h (*P < 0.05 vs. HUA group, n = 6)
changes [57, 58]. UA possesses the potential to
downregulate NO production and induce endothelial injury
through at least three mechanisms, namely modulating
the eNOS phosphorylation status, potentiating arginase
activity, and increasing intracellular superoxide levels
. Because UA is a powerful free radical scavenger, we
first investigated changes in levels of the major free
radicals in the presence of different UA concentrations in
the endothelium. The four major cellular ROS
components are Mito-O•2-, ·OH, 1O2, and H2O2, all of which
can be interconverted [38, 60–63]. Here, we observed
that under normal UA concentrations (300 μM), UA
suppresses O•2-, ·OH, and 1O2 release and slightly
increases H2O2. The slight increase in H2O2 levels (Fig. 4a)
may not be harmful because low H2O2 concentrations
can protect endothelial function [64, 65] and affect
vasodilation . When the UA concentration was increased
to 600 μM, 1O2 and · OH release remained suppressed,
whereas O•2- and H2O2 levels significantly increased.
Because H2O2 is generated from reduced O•2- by superoxide
dismutase (SOD), high UA levels likely did not suppress
O•2- release but rather stimulated O•2- generation. This
Fig. 5 The AR inhibitor reduced H2O2 production and protected endothelial function. a After intraperitoneal injection with oxonic acid potassium salt
and UA for 10 days, serum UA levels in wild-type C57BL/6 mice increased significantly (*P < 0.05 vs. control, n = 6), (b) Serum H2O2 levels increased in
non-treated hyperuricemic mice (*P < 0.05 vs. control, n = 6). AfterPEG-catalase or epalrestat treatment, serum H2O2 levels declined significantly
compared to the non-treated hyperuricemic group (#P < 0.05 vs. HUA group, n = 6). c Serum vWF levels increased in the hyperuricemic mouse model
(*P < 0.05 vs. control, n = 6). After treatment with PEG-catalase or epalrastat, serum vWF levels declined significantly compared to the non-treated
hyperuricemic group (#P < 0.05 vs. HUA group, n = 6). d NO levels decreased in the hyperuricemic model (*P < 0.05 vs. control, n = 6). After treatment
with PEG- catalase or epalrastat, serum NO levels increased significantly compared to the non-treated hyperuricemic group (# P < 0.05 vs. HUA
group, n = 6)
result is similar to previous reports [3, 67]. Although it
is not a free radical, we also measured ONOO− levels, −
which is a highly toxic molecule that can cause harmful
effects. Under normal conditions, the ONOO− level
remains low due to the low level of O•2- generation.
However, if O•2- generation is enhanced, ONOO− production
also increases [68–70]. Because UA can scavenge
ONOO−, ONOO− levels decreased in the presence of
normal UA concentrations. The increase in ONOO−
levels resulting from high UA treatment was likely due
to elevated O•2- levels. These results suggest that
enhanced O•2- generation plays a central role in high
UAinduced endothelial dysfunction.
Our previous study demonstrated high concentration
uric acid could induce AR mRNA and protein
expression level of HUVECs . Johnson et al. reported that
p38 and ERK44/42 MAPK pathways are activation in rat
VSMC incubated with uric acid [71, 72]. Later, they
confirmed the activation of p38 and ERK44/42 MAPK
pathways were involved in HVSMC and endothelial cells
treated with uric acid . In order to try to clarify
whether high UA mediate AR expression increase via
above signal pathway. We observed AR expression could
be repressed when using p38 or MAPK inhibitor
respectively, or the organic anion transporter blocker
probenecid. It implied that high UA might mediated AR
expression via p38/MAPK pathway (Fig. 6). Researchers
demonstrated p38 could activate osmotic response
enhancerbinding protein (OREBP/TonEBP), transcriptional
factors, which bind AR promoter then induce its expression
. We also measured the activity of the two protein.
Yet in the high concentration uric acid environment, the
Fig. 6 The mechanism of uric acid inducing endothelial dysfunction.
OAT: organic anion transporter. AR: aldose reductase, NOX4:
nicotinamide adenine dinucleotide phosphate oxidase 4, ROS:
reactive oxygen species, NO: nitric oxide
expression or the activity of OREBP/TonEBP did not
increase (data was not shown). The mechanism of the
elevated AR expression and activity induced by uric acid
does not depend on the p38-OREBP/TonEBP. Nishikawa
et al. considered that ROS production also can activated
AR expression . We observed that ROS produced at
early time but AR expression obviously changed at 24 h.
Our previous study proved high concentration of uric
acid caused abnormal sodium-calcium exchanger of
mitochondria then induced the ROS production ,
which will further increase AR expression and these may
be locked into a destructive cycle. There might be other
pathway mediate ROS production in the high
concentration of uric acid.
When an AR inhibitor was added to the high
UAtreated HUVECs, total ROS levels significantly
decreased, and NO levels recovered. AR-induced ROS
production is associated with NADPH [32, 33]; our results
also suggested the involvement of NOX4 activation, but
not NOX2 (Additional file 1). Upon blocking NOX4
with apocynin, ROS levels decreased, and NO levels
recovered. When NOX4 was overexpressed In
ARknockdown HUVECs overexpressing NOX4 treated with
high UA, ROS production and NO levels were the
inverse of those resulting from NOX4 overexpression
alone. Additionally, the AR inhibitor epalrestat affected
H2O2 but not ONOO− levels and increased NO levels.
These results confirm that AR activation plays an
important role in high UA-stimulated HUVECs.
The above results suggest that the high UA-induced
increased O•2- generation was associated with the switch
of UA functioning as an antioxidant to a pro-oxidant in
vitro. Because O•2- is catalyzed to H2O2 by SOD in vivo,
H2O2 is likely the major contributor to endothelial
dysfunction. Additionally, serum UA levels correlate with
plasma H2O2 in preeclampsia . Therefore, blocking
AR, reducing H2O2, or decreasing O•2- would protect the
endothelium from high UA-induced injury. In this study,
epalrestat and PEG-catalase recovered NO secretion
levels and decreased vWF levels.
Previously, we measured blood pressure in
hyperuricemic wild-type mice and reported no difference between
the two groups (data not shown). Endothelial dysfunction
resulting from hyperuricemia would not impact blood
pressure in the two-week model. However, if the duration
of exposure to high UA concentrations was extended, the
accumulation of endothelial dysfunction would cause
artery dysfunction and dysarteriotony. These data are similar
to the report by Johnson et al. [51, 76].
Our study confirmed that the levels of ROS components
changed in HUVECs cultured in media containing
different UA concentrations. In particular, H2O2
significantly increased in the high UA group compared
with the control group. The elevated ROS levels were
reversed when AR was inhibited in vivo or in vitro. Thus,
the pro-oxidant activity of UA when present at high
concentrations likely plays an important role in endothelial
dysfunction via AR.
1O2: Singlet oxygen; AR: Aldose reductase; H2O2: Hydrogen peroxide;
HUVEC: Human umbilical vein endothelial cells; NO: Nitric oxide;
NOX: NADPH oxidase; O2-: Superoxide anion; OH: Hydroxyl radical; ONOO
−: Peroxynitrite; ROS: Reactive oxygen species; SOD: Superoxide dismutase;
UA: Uric acid; VWF: von Willebrand factor
Zhiyong Huang and Quan Hong: Acquisition of data, drafting of the article;
Xueguang Zhang, Wenzhen Xiao, and Liyuan Wang: Analysis and
interpretation of the data, and statistical expertise; Guangyan Cai, Xiangmei
Chen and Di Wu: Conception and design of the study. Quan Hong,
Xiangmei Chen and Di Wu: obtaining of funding; Shaoyuan Cui, Zhe Feng,
Yang Lv: Administrative, technical, or logistic support; Shaoyuan Cui and
Quan Hong: Provision of study materials; All of authors: final approval of the
Consent for publication
All of authors content to publication.
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