World Journal of Cardiovascular Diseases, 2013, 3, 561-568 WJCD
http://dx.doi.org/10.4236/wjcd.2013.39088 Published Online December 2013 (http://www.scirp.org/journal/wjcd/)
The gene expression of adenosine receptors in the processes
of contrast induced nephropathy in mouse kidney
Luyu Yao1, Cynthia Zhao2, Xin Gu2, Gopi K. Kolluru2, Christopher G. Kevil2, Wayne W. Zhang1*
1Department of Surgery, Louisiana State University Health Sciences Center, Shreveport, USA
2Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, USA
Email: *wzhan2@lsuhsc.edu
Received 24 September 2013; revised 26 October 2013; accepted 15 November 2013
Copyright © 2013 Luyu Yao et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Objective: Contrast induced nephropathy (CIN) is
the third leading cause of hospital acquired renal
failure. The mechanism of CIN is not fully under-
stood. The objectives of this study were to investigate
the expression changes of the four subtypes of adeno-
sine receptors (A1AR, A2AAR, A2BAR, and A3AR)
following administration of contrast media in mice.
Methods: C57BL/6J mice were randomized into
treatment and control groups. Iodixanol (IDX) was
administered to two treatment groups through retro-
orbital injection at two different dosages, 0.75 gI/kg
and 2.75 gI/kg. Phosphate buffered saline (PBS) was
given to the control group. Mice kidneys were har-
vested at day 3 and day 7 after Iodixanol administra-
tion. Kidney injuries and function were evaluated
according to Hematoxylin and eosin stain, Ki67 pro-
tein expression, and TUNEL assay of paraffin em-
bedded kidney sections, and plasma creatinine assay.
RNA and protein were extracted from the kidney
specimens. A1AR, A2AAR, A2BAR, and A3AR RNA
and protein level of the samples were assessed using
qRT-PCR and Western blotting, with GAPDH as an
endogenous control. Results: H&E staining showed
no significant histopathology injuries after Iodixanol
administration. No evidence of kidney injury and
functional impairment was found. However, there
was an increased number of A1AR, A2AAR, A2BAR,
and A3AR RNA transcripts detected in the kidney 3
days after Iodixanol injection. The RNA levels in all
the four subtypes of adenosine receptors were in-
creased 2 - 3 fold in the day 3 specimens and back to
normal at day 7. Western blot demonstrated that
A1AR, A2AAR, and A3AR expression increased 1.5 to
2 fold at day 3 and day 7 following Iodixanol injection.
A2BAR baseline expression was low in normal phy-
siological conditions and no significant change was
detected by Western blot. Conclusions: Iodixanol sig-
nificantly increases adenosine receptors gene expres-
sion in mice. This suggests that adenosine receptors
may play a role in the development of CIN.
Keywords: Contrast Induced Nephropathy; Adenosine
Receptor; Iodixanol
1. INTRODUCTION
In clinical radiologic examinations, including coronary
angiography, peripheral angiography and computed to-
mography angiography (CTA) contrast media have to be
used. It is also required for mini-invasive interventional
and endovascular procedures. However, contrast media
may cause acute kidney dysfunction, especially in pa-
tients with preexisting underlying diseases, such as hy-
pertension, diabetes mellitus, or renal insufficiency. This
acute renal function deterioration following intravascular
contrast imaging study is called contrast induced nephro-
pathy (CIN).
CIN continues to be the third leading cause of hospital
acquired renal failure (ARF) for over three decades. It
not only excludes numbers of patients from important
imaging studies, such as CTA and digital subtraction
angiogram, but also deprives the patients of less invasive
endovascular procedures. Although it was suggested that
contrast media may cause reduced vasodilatation and in-
creased vasoconstriction resulting in kidney local ische-
mia and hypoxia [1-3], the mechanisms of CIN have not
been fully understood.
Adenosine, a metabolite of ATP hydrolysis, is a potent
renal vasoconstrictor which plays a role in signal trans-
mission of tubuloglomerular feedback [4]. In the kidney,
during states of increased energy metabolism that occurs
during increased tubular transport, i.e., contrast media
administration, ATP is hydrolyzed to adenosine diphos-
*Corresponding author.
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L. Y. Yao et al. / World Journal of Cardiovascular Diseases 3 (2013) 561-568
562
phate and 5’-AMP. Then 5’-AMP is hydrolyzed by 5’-
nucleotidase to adenosine [2,4]. There are reports sug-
gesting that adenosine receptor antagonists may prevent
CIN [5-7]. Thus adenosine release and adenosine recep-
tor stimulation may play a role in CIN development [2].
There are reports indicating that the adenosine recep-
tors’ expression is changed during ischemic, hypoxic,
and inflammation conditions [8-13]. We were curious
whether contrast media might alter the adenosine recep-
tors’ expression. The objectives of this study were to in-
vestigate the expressions of the four subtypes of adeno-
sine receptors (A1AR, A2AAR, A2BAR, and A3AR) fol-
lowing administration of contrast media in mice.
2. MATERIALS AND METHODS
2.1. Animals
Normal C57BL/6J mice, JAX stock number 000664,
were utilized. Seven-week-old male mice were obtained
and accommodated in the central animal facility at Loui-
siana State University Health Sciences Center Shreveport
for one week before experiments. All animals were from
The Jackson Laboratory. They were caged with free ac-
cess to food and water under conditions of 21˚C ± 1˚C
and 50% to 80% relative humidity at all times in an alter-
native 12-hour light-dark cycle. All animal maintenan-
ce and treatment protocols were in compliance with the
Guide for Care and Use of Laboratory Animals as adop-
ted and promulgated by the National Institutes of Health,
and were approved by the Animal Care and Use Com-
mittee at the Louisiana State University Health Sciences
Center Shreveport.
2.2. Iodixanol Administration
Eight-week-old C57BL/6J mice were anesthetized with
150 mg/kg Ketamine and 10 mg/kg Xylazine. Then Io-
dixanol (IDX) was retro-orbitally injected into the mice
at doses of 0.75 gI/kg (n = 3) and 2.75 gI/kg (n = 3). A
0.9% saline solution instead of Iodixanol was injected
into the mice in the control group (n = 3). Three days and
7 days after Iodixanol administration, the animals were
sacrificed. Their kidneys were harvested, cut sagittally,
and placed into labeled cassettes with 10% neutral for-
malin fixation for 24 hours at room temperature. The
kidney sections were then embedded into paraffin. The
other parts of the kidneys used for protein extraction
were snap frozen in liquid nitrogen and stored in an
80˚C refrigerator. The kidney parts used for RNA ex-
traction were put into 10 volumes of RNAlater (Am-
bion) at 4˚C overnight and then stored in an 80 ˚C refri-
gerator.
2.3. Histopathology and Histology Scoring
Histopathology evaluation was performed as described
by Hoffmann et al. [14]. Briefly, the embedded tissues
were cut into 5-µm sections and stained with Hemato-
xylin and eosin stain (H&E stain). The histology damage
of the sections was evaluated under a light microscope
by a pathologist, who was blind to this study. A severity
score based on grading scales of 0 - 3 was used to grade
pathological damages of the kidney: 0, none to minimal
damage; 1, mild acute tubular injury manifested by mild
tubular distension, a low epithelial lining, and nonspeci-
fic degenerative changes of the epithelial cells; 2, mode-
rate acute tubular injury, with frequent single cell necro-
sis within the epithelial layer; and 3, severe acute tubular
injury demonstrated by the presence of widespread epi-
thelial cell necrosis, low epithelial lining, and accu-
mulation of cellular debris in the tubule lumens.
2.4. Immunohistochemical (IHC) Procedures
Paraffin-embedded specimens were cut into 5-µm sec-
tions. The slides were deparaffinized and dehydrated. For
antigen retrieval purposes, slides were put into a vessel
containing sodium citrate buffer (10 mM sodium citrate,
0.05% Tween 20, pH 6.0) and were heated approaching
100˚C for about 20 minutes. Endogenous peroxidase was
inhibited with 0.3% peroxide in 50% methanol (in PBS)
for 30 minutes at room temperature. The slides were
washed with PBS three times, and then blocked for 20
minutes with 2.5% normal goat serum. Next, the primary
antibody, rabbit anti mouse Ki67 antibody (Abcam,
Cambridge, MA), was diluted 100 times in 0.1% normal
goat serum PBS solution and put onto the slides and
incubated for 1 hour at room temperature. Slides were
washed with PBS five times, and then the secondary
antibody, goat anti rabbit IgG-HRP (Abcam, Cambridge,
MA) diluted 500 times in 0.1% normal goat serum PBS
solution, was added onto the slides and incubated for 1
hour at room temperature. The slides were again washed
with PBS three times. Following this, Chromogen 3,3’-
Diaminobenzidine (DAB) (Abcam, Cambridge, MA)
was added onto the slides prior to incubating for another
7 minutes. After a PBS rinse, the slides were counter
stained with hematoxylin (Sigma-Aldrich, St. Louis, MO)
for 2 minutes and rinsed under tap water for 5 minutes.
Slides were then dehydrated. Finally, cover slips were
mounted onto the slides with permount.
2.5. Terminal Deoxynucleotidyl Transferase
Mediated dUTP Nick end Labeling (TUNEL)
Assay Procedures
Paraffin-embedded specimens were cut into 5-µm sec-
tions. The slides were deparaffinized and dehydrated.
Apoptosis of samples were identified using an In Situ
Cell Death Detection Kit POD (Roche Applied Science,
Mannheim, Germany), based on labeling of DNA strand
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L. Y. Yao et al. / World Journal of Cardiovascular Diseases 3 (2013) 561-568 563
bricks (TUNEL technology). Briefly, endogenous pero-
xidase was inhibited with 0.3% peroxide in 50% metha-
nol (in PBS) for 30 minutes at room temperature after
dehydration. The slides were washed with PBS three
times, and then permeabilized by 20 µg/ml Proteinase K
(in 10 mM Tris/HCl, pH 7.4 - 8.0) for 30 minutes at
room temperature. Slides were washed with PBS three
times, then samples were incubated with the TUNEL
reaction mixture, containing fluorescein (FITC) labeled
dUTP and terminal deoxynucleotidyl transferase, in a
humidified chamber for 1 hour at 37˚C. Control slides
were incubated in the absence (negative control) of ter-
minal deoxynucleotidyl transferase, or presence (positive
control) of DNase I. After PBS washes three times, slides
were blocked by 2.5% sheep serum in PBS for 20 minu-
tes in room temperature. Slides were washed with PBS
three times, and then converter-POD (Anti-fluorescin
antibody conjugated with horseradish peroxidase from
sheep) was added onto each slide and slides were incu-
bated in a humidified chamber for 30 minutes at 37˚C.
The slides were again washed with PBS three times.
Following this, Chromogen 3,3’-Diaminobenzidine
(DAB) (Abcam, Cambridge, MA) was added onto the
slides prior to incubating for another 10 minutes at room
temperature. After a PBS rinse, the slides were counter
stained with hematoxylin (Sigma-Aldrich, St. Louis, MO)
for 2 minutes and rinsed under tap water for 5 minutes.
Slides were then dehydrated. Finally, cover slips were
mounted onto the slides with permount.
2.6. Serum Creatinine Level Analysis
Mouse serum was collected at day 3 and day 7 time point.
Plasma creatinine level was measured by creatinine assay
kit (BioVision, Milpitas, CA) following the product in-
structions. Results are represented as means ± standard
deviation (SD).
2.7. Quantitative Real-Time PCR
Kidney samples were taken out from the RNAlater, and a
slice from each sample was cut off and weighed. The
total RNA of the specimen was isolated using an RNeasy
mini kit (QIAGEN, Valencia, CA). First-strand cDNAs
were synthesized from total RNA by reverse transcrip-
tion (1 µg total RNA in 20 μl of reaction volume) using
the iScript cDNA Synthesis Kit (Bio-Rad Laboratories,
Hercules, CA) primed with oligo dT and random hexa-
mer primers. SsoFast EvaGreen Supermix (Bio-Rad La-
boratories, Hercules, CA) was used for dye-based detec-
tion. Reactions were conducted in a CFX96 TouchTM
Real-Time PCR Detection System (Bio-Rad Laboratories,
Hercules, CA). Fluorescence was monitored during
every PCR cycle at the annealing step. The primers for
adenosine receptors were: A1, 5’-AGC CTA CTA TTA
GGT GTT-3’, 5’-GCA TAT CCA TTT CTC TCT AT-3’;
A2A, 5’-AAC AGC CTC ATG GTC TTC-3’, 5’-GCG
TTT CTT CTC TTC TCT TAA-3’; A2B, 5’-ATG TCT
CTT TGA GAA TGT-3’, 5’-TAG ATC ACC AGC ATT
ATG-3’; A3, 5’-CAG TCT GTG TCT ATG ATG-3’,
5’-GGT GAA GGC AAT AAT GTT-3’; GADPH:
5’-GAG TCA ACG GAT TTG GTC GT-3’, 5’-TTG
ATT TTG GAG GGA TCT CG-3’. PCR conditions were
as follows: 95˚C, 30 s; (95˚C, 2 s; 55˚C, 2 s) × 40 cycles;
60˚C, 1 min; Melt curve: 65˚C - 95˚C in 0.5˚C interval, 2
s/step. Results were analyzed with a Bio-Rad CFX mana-
ger. Results for all experiments represent triplicate deter-
minations. Results are represented as means ± SD.
2.8. Western Blot Analysis
Kidney samples were homogenized in RIPA buffer (150
mM NaCl, 1% Triton × 100, 0.5% Sodium deoxycholate,
0.1% SDS, 50 mM Tris-Cl pH 8.0 supplemented with 2
µg/ml of aprotinin, 2 µg/ml of leupeptin, 40 µg/ml of
phenylmethylsulfonyl fluoride). Protein of the samples
were extracted. Approximately 10 - 20 μg of total pro-
teins were separated on a 10% SDS-polyacrylamide gel
and transferred to a polyvinylidene difluoride membrane
(Bio-Rad Laboratories, Hercules, CA). The membrane
was incubated for 1 hour at room temperature in a block
buffer (TBS containing 0.03% Tween 20% and 5% fat-
free milk power). The blots were probed with antibodies
specific for A1 (Abcam, Cambridge, MA), A2A (Biorbyt
Limited, Cambridge, United Kingdom), A2B (Santa Cruz
Biotechnology, Santa Cruz, CA), A3 (Biorbyt Limited,
Cambridge, United Kingdom) receptors or GAPDH
(Santa Cruz Biotechnology, Santa Cruz, CA) at 4˚C
overnight respectively. Membranes were washed three
times at 10 minutes per washing with 0.03% Tween 20 in
TBS, and were then incubated with the secondary anti-
body for 1 hour at room temperature. Membranes were
washed again three times and developed using ECL
Western Blotting Substrate (Thermo Scientific, Waltham,
MA).
2.9. Statistical Methods
Statistical analysis of changes in adenosine receptors
RNA and protein expression were performed with Stu-
dent’s t-test using Prism software (GraphPad Software,
Inc., La Jolla, CA). A P value of 0.05 or less was con-
sidered statistically significant. Data are expressed as
means ± SD.
3. RESULTS
3.1. Histopathology Injuries of Kidney after
Iodixanol Administration in Normal
C57BL/6J Mice
H&E staining showed no significant histopathological
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L. Y. Yao et al. / World Journal of Cardiovascular Diseases 3 (2013) 561-568
Copyright © 2013 SciRes.
564
damages after Iodixanol administration in normal
C57BL/6J mice. Our results of H&E staining (Figure 1)
and histology scoring demonstrated that all the PBS
treated and Iodixanol treated mice had no to minimal
injury, with a score of 0. There was no significant diffe-
rence between control groups and the treatment groups
of the mice treated with Iodixanol, i.e., there was no sig-
nificant histological evidence of CIN in Iodixnaol treated
mice.
administration.
3.5. Adenosine Receptors Expression Changes
after Iodixanol Administration in Normal
C57BL/6J Mice
The qRT-PCR results (Figure 4) showed that Iodixanol
induced transcription of all the four adenosine receptor
subtypes, which was more obvious in the group treated
with the dosage of 2.75 gI/kg day 3 after injection. The
RNA levels in all the four subtypes of adenosine recep-
tors were increased 2 - 3 fold at day 3, but were back to
normal at day 7. The Western blot results (Figure 5)
showed that A1AR, A2AAR, A3AR expression increased
1.5 to 2 fold at day 3 and day 7 following Iodixanol in-
jection. The baseline A2BAR expression was very low
and no significant change was detected by Western blot.
3.2. Ki67 Expression after Iodixanol
Administration in Normal C57BL/6J Mice
The IHC results (Figure 2) revealed that Iodixanol did
not add any further damage to the kidneys of the normal
C57BL/6J mice, as there was no increase of Ki67 expres-
sion after Iodixanol administration. There was no eviden-
ce of renal cell proliferation which indicates renal injury
in Iodixnaol treated mice. 4. DISCUSSION
Adenosine is a tissue hormone that is locally generated in
many organs. It binds to the cell surface of adenosine
receptors to mediate a vast array of physiological func-
tions, including cardiac rate and contractility, smooth
muscle tone, neurotransmitter release, sedation, lipolysis,
white blood cell function, platelet function, and renal
function [15]. There are four different adenosine recep-
tors (AR) named A1, A2A, A2B, and A3. They are G pro-
tein coupled receptors. The A2A and A2B preferentially
interact with members of Gs family of proteins and the
A1 and A3 interact with Gi/o proteins [16].
3.3. TUNEL Assay Results after Iodixanol
Administration in Normal C57BL/6J Mice
Our TUNEL data does not show any significant apopto-
sis in Iodixanol treated samples (Figure 3). There was no
evidence of renal cell apoptosis in Iodixnaol treated
mice.
3.4. Creatinine Assay Results after Iodixanol
Administration in Normal C57BL/6J Mice
The mouse plasma creatinine data does not show any
significant increase in Iodixanol treated samples (Table
1). CIN was not induced in normal mice after Iodixanol
Adenosine release and adenosine receptor stimulation
were proposed to be pathophysiologic factors in the de-
Figure 1. Representative images of H&E-stained kidney histology sections of PBS treated versus Iodixanol treated
mice. A, PBS control, day 3. B, 0.75 gI/kg Iodixnaol, day 3. C, 2.75 gI/kg Iodixnaol, day 3. D, PBS control, day 7. E,
0.75 gI/kg Iodixnaol, day 7. F, 2.75 gI/kg Iodixnaol, day 7. No evidence of kidney injuries was found (×200).
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L. Y. Yao et al. / World Journal of Cardiovascular Diseases 3 (2013) 561-568 565
Figure 2. Representative images of Ki67 IHC results of kidney histology sections in PBS
treated versus Iodixanol treated mice. A, PBS control, day 3. B, 0.75 gI/kg Iodixnaol, day 3.
C, 2.75 gI/kg Iodixnaol, day 3. D, PBS control, day 7. E, 0.75 gI/kg Iodixnaol, day 7. F,
2.75 gI/kg Iodixnaol, day 7. G, Ki67 expression level calculated according to Ki67 IHC
results. No significant differences between PBS and Iodixanol treated groups (×200).
Figure 3. Representative images of TUNEL results of kidney histology sections in PBS
treated versus Iodixanol treated mice. A, PBS control, day 3. B, 0.75 gI/kg Iodixnaol,
day 3. C, 2.75 gI/kg Iodixnaol, day 3. D, PBS control, day 7. E, 0.75 gI/kg Iodixnaol, day
7. F, 2.75 gI/kg Iodixnaol, day 7. G, positivie control. H, negative control. No significant
differences between PBS and Iodixanol treated groups (×200).
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L. Y. Yao et al. / World Journal of Cardiovascular Diseases 3 (2013) 561-568
Copyright © 2013 SciRes.
566
Figure 4. mRNA levels of A, A1AR, B, A2AAR, C, A2BAR, D, A3AR, in PBS treated versus
Iodixanol treated mice kidney. *P < 0.01 (n = 3), **P < 0.05 (n = 3).
Figure 5. Representative Western blot results of A, A1AR, B, A2AAR, C, A3AR, in PBS treated versus Iodixanol treated
mice kidney. *P < 0.01 (n = 3), **P < 0.05 (n = 3).
Table 1. Mouse plasma creatinine level (each group n = 3).
Group Control Day 3 IDX 0.75 gI/kg
Day 3
IDX 2.75 gI/kg
Day 3 Control Day 7 IDX 0.75 gI/kg
Day 7
IDX 2.75 gI/kg
Day 7
Creatinine (±SD) (mg/dL) 0.578 ± 0.080 0.509 ± 0.156 0.652 ± 0.087 0.633 ± 0.068 0.586 ± 0.182 0.527 ± 0.062
velopment of CIN. In the kidney, adenosine modulates
vascular and tubular function primarily via A1 and A2A
adenosine receptors, which are distributed throughout the
kidney with a predominance of A1 receptors presenting
on the afferent arteriole and renal tubules. A2A receptors
are primarily located on the efferent arteriole. This dis-
tribution pattern decides the responses of renal blood
vessels to adenosine. Although stimulation of A2A recep-
tors, which are coupled to Gs protein, causes vasodila-
tion, the adenosine itself induces vasoconstrictions of the
afferent glomerular arteriole via A1 receptors and overall
decreases renal blood flow [2]. In this study, we investi-
gated the expression changes of the four subtypes of
adenosine receptors (A1AR, A2AAR, A2BAR, and A3AR)
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L. Y. Yao et al. / World Journal of Cardiovascular Diseases 3 (2013) 561-568 567
following administration of contrast media in normal
mice. Our histological, Ki67, TUNEL and creatinine
assay results showed no evidence of CIN caused by Io-
dixanol, but Iodixanol did increase the expression of the
four adenosine receptor subtypes in these young, normal
mice.
It has been documented that patients with preexisting
renal diseases are vulnerable to CIN [17-20], but healthy
people are not [21,22]. Similarly, it is understandable
that CIN with obvious renal cell apoptosis and proli-
feration may be difficult to be induced in normal healthy
mice. The useful CIN animal model is not available at
present [23]. Although these mice treated with Iodixanol
did not develop CIN, the observation of the responses of
normal kidney to contrast media can still provide us with
useful information for further study in CIN mechanisms.
Previous studies suggest that contrast media can cause
the increase of adenosine levels [2,4,24], and adenosine
accumulation reduces renal blood flow via A1 receptor
stimulation, which is believed to be one of the mecha-
nisms of CIN [2]. Our results demonstrated that the ade-
nosine receptors’ expression was significantly up-regu-
lated by contrast media in a healthy mouse, which means
that the renal cells will be more sensitive to adenosine
signal, because the higher the number of receptors, the
more potent or efficacious the agonist will be. The rather
low levels of endogenous adenosine presenting under
basal physiological conditions have the potential of acti-
vating receptors where they are abundant, but not where
they are sparse [16]. This may exacerbate the effect of
adenosine release and makes the kidney more vulnerable
to CIN [2].
As we mentioned earlier, healthy people’s kidneys are
resistant to CIN [21,22]. Although our data showed that
Iodixanol failed induce CIN, it did increase the expres-
sion of adenosine receptors. The possible explanations
are: 1) Iodixanol, as a new generation of iso-osmolar
contrast agent, may be less toxic to the kidney; 2) CIN
may only present with transient renal dysfunction with-
out significant histological damages to the normal kid-
neys in healthy mice. Since adenosine receptor expres-
sion is changed during ischemic, hypoxic, and inflamma-
tion conditions [8-13], the pre-existing kidney diseases
may be also required in the development of CIN. Further
investigation using db/db and hypertension mice will be
carried out in our research lab.
Our data also demonstrated that the adenosine receptor
transcription was elevated at day 3 following Iodixanol
administration, and recovered to normal at day 7. This
trend is consistent with the renal function changes in cli-
nical patients, in whom serum creatinine usually reaches
the peak level within 3 - 5 days and start recovering after
5 - 7 days following contrast imaging studies.
5. CONCLUSION
Iodixanol, an intravenous contrast media, significantly
increases adenosine receptor gene expression in normal
mice. Since the adenosine release and adenosine receptor
stimulation were proposed to be pathophysiologic factors
in the development of CIN [2], although the CIN was not
induced in the normal mice, we presume that the adeno-
sine receptor expression increase we found in normal
mice may play a role in the CIN development by exac-
erbating adenosine-induced vasoconstriction. Further stu-
dy should focus on whether this alteration really plays a
role in CIN development using animal models that actu-
ally developed CIN. We plan to use db/db and hyperten-
sion mice treated with Iodixanol or other contrast media
to do this further study.
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