Acta Pharmacologica Sinica (2009) 30: 1601–1606
© 2009 CPS and SIMM All rights reserved 1671-4083/09 $32.00
Adenosine 5’-triphosphate (ATP) is not only an important
source of energy for cells, but also an important extracellular
signaling molecule that modulates various cell functions. The
release of ATP from the cell occurs by conductive transporta-
tion, facilitated transport and exocytosis. After leaving the
cell, ATP can bind to two subclasses of purinergic P2 receptors
on the cell membrane. P2X receptors are non-selective cation
channels while P2Y are G-protein-coupled receptors. Both
these receptors are composed of several subtypes[3, 4]. In the
extracellular space, ATP is rapidly degraded into adenosine
diphosphate (ADP), adenosine monophosphate (AMP) and
adenosine by multiple ecto-enzymes, including ectonucleotide
triphosphate diphosphohydrolases, ectonucleotide pyrophos-
phatases/phosphodiesterases and alkaline phosphatases.
Therefore, ATP is thought of as a local mediator that acts in
an autocrine or paracrine manner within tissues and tissue
Extracellular ATP in the kidney, which is produced by
tubular epithelial cells, endothelial cells, smooth muscle cells,
platelets and perivascular nerves, can act on glomeruli, renal
tubules, collecting ducts and renal blood vessels by binding
to either P2X or P2Y receptors[1, 6–9]. ATP has been shown to
regulate renal blood flow, glomerular filtration rate, water and
salt reabsorption and tubuloglomerular feedback[1, 7, 10, 11].
Mesangial cells play a critical role in the initiation and pro-
gression of diabetic nephropathy and their abnormal prolif-
eration and secretion of extracellular matrix (ECM) promotes
glomerular sclerosis. High glucose levels induce the genera-
tion of reactive oxygen species (ROS) by mesangial cells; ROS,
Adenosine 5′-triphosphate stimulates the increase
of TGF-β1 in rat mesangial cells under high-glucose
conditions via reactive oxygen species and ERK1/2
Lin-ping QU1, Hong XUE1, Ping YUAN1, Li ZHOU1, Tai YAO1, Yu HUANG2, Li-min LU1,*
1Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China; 2Department
of Physiology and Institute of Vascular Medicine, The Chinese University of Hong Kong, Hong Kong, China
Aim: To investigate the role of adenosine 5′-triphosphate (ATP)-induced generation of reactive oxygen species (ROS) and phosphoryla-
tion of extracellular signal-regulated kinase 1/2 (ERK1/2) in the production of transforming growth factor-β1 (TGF-β1) in cultured rat
glomerular mesangial cells under high-glucose conditions.
Methods: Subconfluent glomerular mesangial cells were serum-starved for 24 h and pretreated with suramin, diphenylenechloride
iodonium (DPI) or PD98059 followed by stimulation with a high concentration of glucose (30 mmol/L D-glucose) or ATP (300 μmol/L).
Extracellular and total ATP and ROS production were detected using commercially available kits. Phosphorylation of ERK1/2 was eval-
uated by Western blot. TGF-β1 mRNA expression was examined by real-time PCR.
Results: Suramin had a dose-dependent inhibitory effect on the generation of ROS induced by high glucose. Extracellular ATP produc-
tion by mesangial cells increased markedly after a 2-h incubation with high glucose. ROS production was upregulated in mesangial
cells after 5 min incubation with 300 μmol/L ATP and was sustained for 120 min. ERK1/2 was significantly activated after 5 min
incubation of mesangial cells with ATP, this activation was partially inhibited by DPI. The effects of high glucose on TGF-β1 mRNA were
markedly inhibited by suramin, DPI or PD98059.
Conclusion: Our results suggest that a high concentration of glucose increases the extracellular levels of ATP in mesangial cells within
a short time-frame. ATP, in turn, activates ERK1/2, an effect which is at least partially dependent on ROS, which results in the upregu-
lation of TGF-β1.
Keywords: diabetic nephropathy; reactive oxygen species; adenosine 5′-triphosphate; extracellular signal-regulated kinase 1/2;
transforming growth factor-β1; mesangial cells
Acta Pharmacologica Sinica (2009) 30: 1601–1606; doi: 10.1038/aps.2009.155
* To whom correspondence should be addressed.
Received 2009-07-15 Accepted 2009-09-16
Qu LP et al
Acta Pharmacologica Sinica
in turn, activate mitogen-activated protein kinase (MAPK)
in mesangial cells, leading to their secretion of transforming
growth factor-β1 (TGF-β1) and fibronectin. In addition,
extracellular ATP has been shown to stimulate ROS genera-
tion in macrophages and microglia[14, 15]. As there is evidence
showing that mesangial cells express almost all P2 receptor
subtypes, it became interesting to us to determine whether,
under high-glucose conditions, ATP and P2 receptors mediate
the production of ROS by mesangial cells.
This study was aimed at evaluating the role of extracellu-
lar ATP-induced generation of ROS and phosphorylation of
extracellular signal-regulated kinase 1/2 (ERK1/2) in TGF-β1
synthesis under high-glucose conditions in cultured rat renal
Materials and methods
Drugs and chemicals
Low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM),
D-glucose, mannitol, adenosine 5’-triphosphate (ATP),
suramin, diphenylenechloride iodonium (DPI) and PD98059
were purchased from Sigma (St Louis, MO, USA). Rabbit
anti-rat p44/42 MAP Kinase (ERK1/2) and phospho-p44/42
MAP Kinase (p-ERK1/2) antibodies were purchased from
Cell Signaling Technology (Danvers, MA, USA). Horserad-
ish peroxidase-conjugated goat anti-rabbit IgG antibody was
purchased from Zhongshan Golden Bridge Biotechnology
(Beijing, China). Proteinase inhibitor was purchased from
Upstate (Waltham, MA, USA). Oligo dT, dNTP and Rnasin
were purchased from Shenergy Biocolor BioScience and Tech-
nology (Shanghai, China), M-MLV reverse transcriptase was
purchased from Promega (WI, USA), SYBR Green quantitative
real-time (qRT)-PCR Master Mix was purchased from Toyobo
(Osaka, Japan). Primers for RT-PCR were synthesized by San-
gon Biological Engineering Technology and Services (Shang-
hai, China). All other chemicals and reagents were of analyti-
The rat glomerular mesangial cell line (HBZY-1) purchased
from China Center for Type Culture Collection (Wuhan,
China) was cultured in low-glucose DMEM (5.5 mmol/L
D-glucose) supplemented with 10% neonatal bovine serum in
an atmosphere of 95% O2 and 5% CO2 at 37 °C. Before each
experiment, cells were pre-incubated with DMEM supple-
mented with 1% neonatal bovine serum for 24 h. All antago-
nists were added 30 min in advance of changing to high-
glucose culture media or ATP addition. High-glucose culture
media was made by supplementing low-glucose DMEM (5.5
mmol/L D-glucose) with additional D-glucose for a final
D-glucose concentration of 30 mmol/L.
Reactive oxygen species assay
Experiments were performed using the reactive oxygen spe-
cies assay kit (Beyotime, Haimen, China) according to the
manufacturer’s instructions. Briefly, cells seeded in 96-well
plates were incubated with 10 μmol/L DCFH-DA probes
(100 μL/well) at 37 °C for 30 min and then washed with PBS
3 times in order to remove residual probes. The fluorescence
intensity at 488 nm excitation wavelength and 525 nm emis-
sion wavelength was measured using a luminometer (Tecan,
ATP detection assay
ATP production was analyzed using the luminescence ATP
detection assay kit (Perkin Elmer, Boston, MA, USA) accord-
ing to the manufacturer’s instructions. The principle of
this assay is based on the production of light caused by the
reaction of ATP with added luciferase and D-luciferin. Rat
mesangial cells seeded in 96-well plates were incubated with
the substrate solution (luciferase and D-luciferin, 50 μL/well),
shaken for 5 min and placed in the dark for 10 min. The cells
were then placed into the test chamber of a luminometer and
the emission of light was recorded and used to estimate the
relative levels of extracellular ATP. In order to determine the
relative levels of total ATP, the cells were further treated with
lysis solution (50 μL/well), shaken for 5 min and placed in
dark for 10 min, then measured in the luminometer.
Rat mesangial cells were lysed in 1×sodium dodecyl sulfate
(SDS) supplemented with proteinase inhibitor at a dilution of
1:25. Protein concentrations were measured using the BCA
protein assay kit (Shenergy Biocolor BioScience and Technol-
ogy, Shanghai, China). Thirty micrograms of protein lysate
was electrophoresed on a 12% polyacrylamide SDS gel and
transblotted onto a PVDF membrane at 270 mA for 90 min.
After transfer, the membranes were blocked with 5% skim
milk in Tris-buffered saline (TBS) and 0.1% Tween (TBS/
Tween) for 1 h at room temperature with gentle rocking. The
membranes were then incubated with rabbit anti-rat phospho-
p44/42 MAPK antibodies (1:1000 in 5% skim milk in TBS/
Tween) and incubated overnight at 4 °C. The following
day, the membranes were washed 3 times (15 min per wash)
with TBS/Tween and incubated with secondary anti-rabbit
antibody (1:2000) for 1 h at room temperature. The mem-
branes were then washed again 3 times, developed using the
enhanced chemiluminescent (ECL) detection kit (Pierce Bio-
technology, IL, USA) according to the manufacturer’s instruc-
tions and exposed to X-ray film (Kodak, Rochester, NY, USA)
for 0.5–5 min as necessary to visualize signals. Additionally,
the membranes were treated with stripping buffer (0.5 mol/L
NaCl, 0.5 mol/L acetic acid) for 1 h at room temperature.
After three 15-min washes, the membranes were re-blocked
with 5% skim milk in TBS/Tween for 1 h at room tempera-
ture. Afterwards, the membranes were incubated with rabbit
anti-rat p44/42 MAPK antibodies (1:1000), washed, incubated
with secondary antibody and developed as described above.
The relative intensity of the bands exposed on the films was
quantified using Smart viewer software (Furi Technology Co,
Shanghai, China). The results are expressed as a ratio of phos-
phorylated to total ERK1/2.
Qu LP et al
Acta Pharmacologica Sinica
Isolation of total RNA and synthesis of cDNA
Rat mesangial cells were lysed in TRIZOL and total RNA was
isolated. The amount of RNA isolated was determined by
measuring the specific absorbance at 260 nm. One microgram
of total RNA was used for cDNA synthesis in a 20 μL reaction
mixture that contained 1 μg Oligo dT, 10 mmol/L dNTP, 20 U
RNase inhibitor and 200 U M-MLV reverse transcriptase. A
1-μL aliquot of the resulting single-strand cDNA was used for
Quantitative Real-time PCR
TGF-β1 and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) primer sequences are listed in Table 1. SYBR
Green qRT-PCR was used to quantify the relative abundance
of target mRNA in the samples. qRT-PCR procedures were
performed according to the manufacturer’s instructions. The
accumulated fluorescence was detected using the iCycler iQ
RT-PCR detection system (Bio-Rad, Hercules, CA, USA). The
PCR amplification conditions were: 95 °C for 10 min and 40
cycles of 95 °C for 30 s, 62 °C for 45 s and 72 °C for 1 min. In
addition, the amplified products were subjected to a stepwise
increase in temperature from 55 °C to 95 °C and dissociation
curves were constructed. Target mRNA levels were quantified
by measuring the threshold cycle (when fluorescence is
statistically significant above the background) and comparing
it against a calibration curve. The relative amount of each
mRNA was normalized to the housekeeping gene, GAPDH.
Each sample was run and analyzed in triplicate.
The results are expressed as the mean±SEM. Data were ana-
lyzed using the one-way ANOVA with the Bonferroni cor-
rection for all pairwise comparisons. A P-value of <0.05 was
considered statistically significant.
Effect of suramin, a nonspecific P2 receptor antagonist, on ROS
generation induced by high glucose
Suramin exhibited a dose-dependent inhibitory effect on the
increased generation of ROS induced by high glucose for 2 h
in rat mesangial cells; suramin at 100 μmol/L had an obvious
effect on the diminishment of ROS generation (P<0.05 vs HG,
high glucose; Figure 1).
Effect of high glucose on extracellular ATP levels
Extracellular ATP levels increased markedly after incubation
of rat mesangial cells in high-glucose media for 2 h (P<0.05 vs
low-glucose control), but ATP levels did not change in cells
cultured in media supplemented with 24.5 mmol/L mannitol
for the same time period as the iso-osmolar (Figure 2A). Total
ATP levels were measured after lysing rat mesangial cells.
Table 1. PCR primer pairs used to amplify TGF-β1 and GAPDH cDNA
Target Oligonucleotide sequence Tm ºC bp
TGF-β1 F 5′-TGG CGT TAC CTT GGT AAC C-3′
R 5′-GGT GTT GAG CCC TTT CCA G-3′
GAPDH F 5′-CCC TTC ATT GAC CTC AAC TAC ATG-3′
R 5′-CTT CTC CAT GGT GGT GAA GAC-3′
F, forward primer; R, reverse primer; Tm, melting temperature.
Figure 1. Effect of suramin on the generation of ROS induced by high
glucose in mesangial cells. Rat mesangial cells were cultured under high
glucose (30 mmol/L D-glucose) conditions for 2 h. In order to explore the
involvement of extracellular ATP in the generation of ROS, the cells were
pretreated with suramin, a nonspecific P2 receptor antagonist, at the
indicated concentrations for 30 min. n=4–6. Mean±SEM. aP>0.05 vs
control; bP<0.05 vs control; eP<0.05 vs HG (high glucose).
Figure 2. Effect of high glucose on extracellular and total ATP levels in
mesangial cells. Rat mesangial cells were cultured under low-glucose
(5.5 mmol/L D-glucose), high-glucose (HG, 30 mmol/L D-glucose), or
iso-osmolar (5.5 mmol/L D-glucose+24.5 mmol/L mannitol) conditions
for 2 h, extracellular (A) and total (B) ATP levels were measured. n=6.
Mean±SEM. bP<0.05 vs control.
Qu LP et al
Acta Pharmacologica Sinica
There were no obvious differences in total ATP levels between
low-glucose and high-glucose groups. As the intracellular
ATP levels showed at least 10 000-fold higher than extracelluar
levels[1,5], the possiblity of the difference in cell numbers could
be excluded (Figure 2B).
Effect of extracellular ATP on ROS generation
ATP at 300 μmol/L induced an obvious increase in the genera-
tion of ROS (P<0.05 vs control), while ATP at 100 μmol/L only
slightly enhanced ROS generation (P>0.05 vs control). Lower
concentration of ATP (10 μmol/L) had almost no effect on the
generation of ROS (Figure 3A). In order to determine whether
the effect of ATP was long-lasting, cells were exposed to 300
μmol/L ATP for 5, 15, 30, 60, or 120 min. We observed that
ROS generation was upregulated after exposure of the cells
to ATP for 5 min and was followed by a sustained increase
in ROS generation at 120 min (P<0.05 vs control); the largest
amount of ROS was observed after 15 min exposure to ATP
Effect of extracellular ATP on ERK1/2 phosphorylation
Phosphorylation of ERK1/2, one of the downstream targets of
ROS, was assessed by measuring the level of p-ERK1/2 pro-
tein and comparing it to total level of ERK1/2 in rat mesan-
gial cells. ERK1/2 was largely activated by exposure to 300
μmol/L ATP for 5 min (P<0.05 vs control). Longer exposure
to ATP led to a gradual decrease in ERK1/2 phosphorylation.
After a 60 min exposure time, the levels of p-ERK1/2 were
much less than those in the control (P<0.05 vs control; Figure
4A). In mesangial cells, ROS are mostly generated by NADPH
oxidase. Therefore, we used the NADPH oxidase inhibi-
tor DPI to assess the effect of ROS inhibition on ATP-induced
ERK1/2 phosphorylation. We found that 1 µmol/L DPI
partially inhibited ATP-induced phosphorylation of ERK1/2
(P<0.05 vs control, P<0.05 vs ATP; Figure 4B).
TGF-β1 mRNA levels under high-glucose conditions
Increased renal TGF-β1 bioactivity during high-glucose con-
ditions is an important factor in the pathogenesis of diabetic
Figure 3. Effect of extracellular ATP on ROS production in mesangial cells
cultured under low-glucose conditions. (A) ROS levels were measured in
rat mesangial cells treated with ATP for 5 min at 10, 100, or 300 μmol/L.
n=5–6. (B) The effective concentration of 300 μmol/L ATP was selected
to treat mesangial cells for 5–120 min in order to observe the continuous
effect of ATP on ROS production. n=6. Mean±SEM. bP<0.05 vs control.
Figure 4. Effect of extracellular ATP on ERK1/2 phosphorylation in
mesangial cells cultured under low-glucose conditions. Western blot
analysis of p-ERK1/2 in rat mesangial cells (normalized to total ERK1/2).
(A) Cells were treated with 300 μmol/L ATP for 5 to 60 min. (B) Cells were
pretreated with the NADPH oxidase inhibitor DPI (1 μmol/L) for 30 min
in order to explore whether ROS were involved in ATP-induced ERK1/2
phosphorylation. n=4. Mean±SEM. aP>0.05 vs control; bP<0.05 vs
control; eP<0.05 vs ATP.
Qu LP et al
Acta Pharmacologica Sinica
nephropathy. TGF-β1 mRNA levels were much higher when
rat mesangial cells were cultured in high-glucose media
than when cultured in low-glucose media (P<0.05 vs con-
trol). However, this increase was attenuated by suramin (100
μmol/L), DPI (1 μmol/L) and the ERK kinase (MEK)-specific
inhibitor PD98059 (50 μmol/L) (Figure 5).
Diabetic nephropathy is one of the most common complica-
tions associated with diabetes and is a major cause of pathol-
ogy in chronic renal dysfunction. In diabetic nephropathy,
mesangial cells make a major contribution to glomerular scle-
rosis by producing increased amounts of ECM in response to
hyperglycemia; TGF-β1 is considered to be a major cytokine
that modulates the increased production of ECM since it
promotes matrix synthesis and reduces its degradation. In
addition, ROS and ERK1/2, both of which signal upstream
of TGF-β1, play a significant part in the kidney damage that
results from hyperglycemia.
Previously, it was reported that mesangial cells produce
increased levels of ATP when exposed to high-glucose condi-
tions leading to the stimulation of TGF-β1 production and
ECM secretion. However, the role of ROS and ERK1/2 in
this process is not known.
Our studies suggest that high-glucose levels promote the
production of extracellular ATP level in rat mesangial cells.
In contrast to previous studies, we cultured rat mesangial
cells in high-glucose media for 2 h rather than 10–15 d. We
found that extracellular ATP levels were increased within 2 h
in response to high glucose, suggesting that high glucose can
increase extracellular ATP levels in a relatively short period
of time. This effect may depend on the increased release of
ATP, decreased ecto-enzyme activity or both. It has been
largely shown that the release of ATP into the extracellular
milieu is mediated by ATP-permeable release channels, which
are similar to voltage-dependent anion channels of the outer
mitochondrial membrane, adenine nucleotide transporters
or adenine nucleotide/nucleoside exchangers and ATP-filled
vesicles. In addition, several secondary messengers have
been shown to modulate ATP release by elevating intracellular
Ca2+ levels and activating the phosphatidylinositol-3- (PI-3K)
and Rho kinases. These three signaling pathways have also
been shown to be activated under high-glucose conditions in
renal mesangial cells, which may result in the stimulation of
ATP release. We were not able to determine ecto-enzyme
activity in response to high glucose levels after 2 h; therefore,
these studies will be further assessed in future studies. Incu-
bation of rat mesangial cells with iso-osmolar mannitol did
not lead to an increase in extracellular ATP, indicating that the
effect induced by high glucose is not dependent on increased
In human eosinophils, production of reactive oxygen meta-
bolites increases in response to ATP in a dose-dependent
manner, with the maximum concentration of metabolites
being reached after 5 min of exposure to ATP. Primary
rat microglial cells stimulated with ATP rapidly generate
hydrogen peroxide (H2O2) through the activation of NADPH
oxidase. H2O2 production was sustained for at least 90 min
after stimulation. Additionally, ATP-induced apoptosis in
murine macrophages is mediated in part by ROS produced
by NADPH oxidase. Our results show that exposure of rat
mesangial cells to 300 μmol/L ATP results in rapid and contin-
uous generation of ROS; non-specific antagonist to P2 receptor
suramin inhibits high glucose-induced ROS production. These
results suggested a role for ATP in high glucose-induced ROS
generation in rat mesangial cell. The concentration of ATP
used here is similar to that previously reported[14, 15, 18] and
similar to that which has been previously shown to induce
proliferation and abnormal secretion of TGF-β1 and ECM in
mesangial cells[17, 19, 20]. Studies have shown that the generation
of ROS induced by ATP is associated with an increase in intra-
cellular Ca2+ levels[14, 15, 18]. The ATP receptors P2X and P2Y can
mediate Ca2+ upregulation; however, while P2X is dependent
on Ca2+ influx, P2Y is dependent on mobilization from intrac-
ellular Ca2+ stores[21, 22]. ATP mediates the generation of ROS
via either P2X or P2Y in different types of cell. It was difficult
to determine from our results which subclass or subtype of P2
receptor was responsible for the observed ATP-induced effects
in rat mesangial cells. Further studies using specific agonists
and antagonists for P2 receptor subtypes will help us identify
the appropriate receptor.
We observed that p-ERK1/2 levels in rat mesangial cells
reached their maximum after 5 min of exposure to ATP and
decreased gradually during a subsequent period of time. This
result suggests that extracellular ATP induces rapid and tran-
sient phosphorylation of ERK1/2, consistent with previous
studies[12, 19, 20, 23]. However, in contrast to previous studies, we
found that stimulation of rat mesangial cells with ATP for 60
min resulted in decreased p-ERK1/2 levels when compared
with the control. Distinct cell species, differences in cell types
or different experimental conditions may account for the dif-
ferences observed between studies; therefore, further studies
Intracellular ROS are generated through both the NADPH
Figure 5. qRT-PCR analysis of TGF-β1 mRNA levels. Cells were pretreated
with suramin (100 μmol/L), DPI (1 μmol/L) or PD98059 (50 μmol/L) for
30 min before being cultured in high glucose (30 mmol/L D-glucose)
media for 2 h. n=4. Mean±SEM. bP<0.05 vs control; eP<0.05 vs HG (high
Qu LP et al
Acta Pharmacologica Sinica
oxidase system and mitochondrial metabolism. By contrast,
the increased production of ROS by mesangial cells under
high-glucose conditions is predominantly, although not
exclusively, mediated by NADPH oxidase. In addition, the
NADPH inhibitor DPI has been shown to effectively block
the generation of ROS induced by high glucose in mesangial
cells[13, 16]. In our study, DPI only partially inhibited the ATP-
induced increase in ERK1/2 phosphorylation suggesting that,
in addition to ROS, ATP might activate ERK1/2 through other
ROS-independent pathways. It is important to note, however,
that NADPH oxidase is at least partially involved in ERK1/2
activation by ATP.
Studies using neutralizing antibodies against TGF-β1 have
provided convincing evidence that the prosclerotic and hyper-
trophic effects of high glucose levels are largely mediated by
the production and activation of TGF-β1 by glomerular mesan-
gial cells. Activated ERK can activate Elk-1, a member of the
ternary complex factors that enhances the expression of c-fos
and promotes subsequent DNA binding of the transcription
factor AP-1. An increase in AP-1 binding to DNA has been
reported to mediate the expression of TGF-β1. Here, we
show without a doubt that DPI and PD98059 decrease TGF-β1
mRNA levels under high-glucose conditions. Suramin had a
similar effect, suggesting that extracellular ATP plays a part
in the activation of downstream pathways in response to high
glucose in mesangial cells.
Taken together, our results suggest that, in rat mesangial
cells, high-glucose conditions increase the extracellular levels
of ATP within a short period of time. ATP, in turn, activates
ERK1/2, an effect which is at least partially dependent on
ROS, leading to the upregulation of TGF-β1. As it is known
that there are two subclasses of P2 receptors expressed by
mesangial cells and each has several subtypes, future studies
will be aimed at determining which subtypes of P2 receptors
are involved in this pathway.
This research was financially supported by the Shanghai Nat-
ural Science Foundation (No 09ZR1404200) and the National
Natural Science Foundation of China (No 3047627).
Lin-ping QU, Li-min LU, and Yu HUANG designed research;
Lin-ping QU, Hong XUE, Ping YUAN, Li ZHOU performed
research; Lin-ping QU, Li ZHOU analyzed data; Lin-ping QU,
Tai YAO, Yu HUANG, and Li-min LU wrote the paper.
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