https://doi.org/10.52973/rcfcv-e34323
Received: 20/09/2023 Accepted: 13/10/2023 Published: 13/01/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34323
ABSTRACT
In this study, it was aimed to investigate Adropin levels in kidney tissues
after Methotrexate (MTX) administration to identify potential changes
potential. Twenty four adult rats male albino Wistar rats were used
in this study, and randomly divided into four groups. Control: These
rats did not receive any treatment during the 14–day (d) experiment.
N–acetylcysteine (NAC): These rats were administered 100 mg·kg
-1
·day
-1
NAC intraperitoneally (i.p.) for 14 d. MTX: A single dose of 20 mg·kg
-1
MTX was administered i.p. at the beginning of the study. MTX+ NAC: A
single dose of 20 mg·kg
-1
MTX was administered i.p. at the beginning
of the study, and the rats were given 100 mg·kg
-1
·day
-1
NAC i.p. for
Total antioxidant, and serum Adropin levels were found to be the
lower in the MTX group than in the MTX+NAC group (P<0.001). TUNEL
were observed.
on the role of Adropin in the development of kidney failure following
MTX administration.
Key words:
o
RESUMEN
En este estudio, el objetivo fue investigar los niveles de adropina
en los tejidos renales después de la administración de MTX para
se utilizaron 24 ratas adultas, ratas Wistar albinas macho, y se
dividieron aleatoriamente en cuatro grupos. Estas ratas no recibieron
ningún tratamiento durante los 14 días (d) del experimento. NAC: A
estas ratas se les administró 100 mg·kg
-1
·día
-1
de N–acetilcisteína
(NAC) por vía intraperitoneal (i.p.) durante 14 d. MTX: Se administró
una dosis única de 20 mg·kg
-1
de MTX i.p. al inicio del estudio. MTX+
NAC: Se administró una dosis única de 20 mg·kg
-1
de MTX i.p. al
comienzo del estudio, y las ratas recibieron 100 mg·kg
-1
·día
-1
de NAC
i.p. durante 14 d. Se encontró que los niveles de antioxidantes totales
y de adropina sérica eran los más bajos en el grupo de MTX, mientras
el grupo de MTX que en el grupo de MTX+NAC (P<0,001). La positividad
de TUNEL fue similar entre los grupos y no se observaron diferencias
la administración de MTX.
Palabras clave:
Evaluating Adropine levels in kidney tissue after Methotrexate treatment in
rats: a prospective experimental study
Evaluación de los niveles de adropina en el tejido renal después del tratamiento
con Metotrexato en ratas: un estudio experimental prospectivo
Karakeci Ahmet
1
, Kuloglu Tuncay
2
, Acisu Tutku Can
3
* , Keles Ahmet
4
, Ozan Tunc
1
, Vural Osman
2
,
Orhan Irfan
1
, Sabaz Karakeci Emel
5
1
Firat University, School of Medicine, Department of Urology. Elazig, Türkiye.
2
Firat University, School of Medicine, Department of Histology and Embryology. Elazig, Türkiye.
3
Firat University, Faculty of Veterinary Medicine, Department of Reproduction and Articial Insemination. Elazig, Türkiye.
4
Istanbul Medeniyet University, School of Medicine, Department of Urology. Istanbul, Türkiye.
5
Health Sciences University, Elazig Fethi Sekin City Hospital, Department of Physical Therapy and Rehabilitation. Elazig, Türkiye.
Corresponding Author: tcacisu@rat.edu.tr
Adropin levels after Methotrexate treatment in renal tissue / Ahmet et al. __________________________________________________________
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INTRODUCTION
The kidney is one of the most important organs and takes part in
several vital bodily functions, particularly blood pressure regulation
and the maintenance of electrolyte balance [1]. However, its role in
the concentration and excretion of drugs and toxic materials often
exposes the kidney to toxic chemicals [2]. Consequently, the use of
drugs, particularly chemotherapeutic agents such as Methotrexate
(MTX), can lead to nephrotoxicity and the subsequent progression
of acute and chronic kidney diseases (CKD). This occurs because
of MTX–induced oxidative stress, which increases reactive oxygen
species (ROS), leading to cytotoxicity [3].
MTX blocks Deoxyribonucleic acid (DNA) synthesis by inhibiting
dihydrofolate reductase. Clinically, it is used in high doses in the
treatment of various neoplastic diseases and in low doses in the
treatment of autoimmune diseases [4, 5]. However, the side effects
of MTX limit its clinical usefulness. MTX administration is associated
with hepatotoxicity, gastrointestinal disturbances, neurotoxicity,
and hematological abnormalities. Moreover, the drug is linked to
acute kidney failure at an occurrence rate of 2–12%. MTX has also
been reported to reduce the liver’s Glutathione level and increase
lipid peroxidation. This leads to defects in the antioxidant protection
mechanism [6]. No precise mechanisms are currently known that
explain how MTX causes kidney damage, but oxidative stress and
nephrotoxicity [7, 8].
MTX activates the mitochondrial pathway of apoptosis and
by decreasing the homocysteine remethylation rate. MTX also
stimulates neutrophil recruitment by decreasing the intracellular
levels of nicotinamide adenine dinucleotide phosphate (NADPH) [9,
10]. Recent studies have shown that endothelial damage is also an
important factor in the toxicity that arises from MTX use [11].
Endothelial function is regulated at least in part by Adropin,
a hormone expressed in the vascular endothelial cells that takes
part in the regulation of lipid metabolism and in the maintenance of
energy homeostasis [12]. The level of Adropin is regulated by nutrient
intake and is detected in the liver as well as in the kidney glomerulus,
peritubular capillaries, pancreatic tissue, and heart endocardium,
myocardium, and epicardium [13, 14]. Adropin has a positive impact
capillary density, and angiogenesis, but decreases endothelial
permeability due to the stimulation of endothelial nitric oxide synthase
(eNOS)–nitric oxide (NO) signal pathways [12]. Plasma Adropin levels
have been found to be low in chronic diseases and this condition is
closely related to endothelial dysfunction [15, 16].
In this study, it was aimed to investigate Adropin levels in kidney
tissues after MTX administration to identify potential changes
potential, as well as to explore the potential usefulness of Adropin
levels after MTX treatment in the diagnosis of kidney failure.
MATERIALS AND METHODS
All procedures included in the study were performed according to
the National Institutes of Health’s (NIH) Guidelines for the Care and Use
of Animals. After obtaining the necessary approval (decision number:
31.03.21/2021.06) from the local animal experiments ethics committee,
it was began this prospective experimental study at the Animal
Laboratory of Firat University, Elazig, Türkiye, between April 2021
and December 2022. Furthermore, the PubMed and Web of Science
esearches.
Animals and groups
In the current study, 24 mature male albino rats wistar (Ratus
norvegicus) were kept in conditions of 22–25 °C with 12 h of light
(7:00 am–7:00 pm) and 12 h of darkness (7:00 pm–7:00 am). Rat food
pellets and water were available at all times. The rats were randomly
divided into four groups of six, as follows: Group 1 (Control): These
rats did not receive any treatment during the 14–day (d) experiment.
Group 2 (NAC): These rats were administered 100 mg·kg
-1
·day
-1
NAC
(Bilim Pharmaceuticals, Kocaeli, Türkiye) intraperitoneally (i.p.)
for 14 d [17]. Group 3 (MTX): A single dose of 20 mg·kg
-1
MTX (Abdi
Ibrahim, Istanbul, Türkiye) was administered i.p. at the beginning of
the study [18]. Group 4 (MTX + NAC): A single dose of 20 mg·kg
-1
MTX
was administered i.p. at the beginning of the study, and the rats were
given 100 mg·kg
-1
·day
-1
NAC i.p. for 14 d.
At the conclusion of the experiment, all rats received Ketamine
(75 mg·kg
-1
) (Alfamine 10%, Ege Vet, Türkiye) + Xylazine (10 mg·kg
-1
)
(Rompun 2%, Bayer, Türkiye) i.p. and were decapitated under
anesthesia. Blood samples were taken and centrifuged (NF1200R,
maintained at -80°C until assayed. Adropin levels, total antioxidant
status (TAS), and total oxidant status (TOS) were determined using
enzyme–linked immunoassay (ELISA) kits. Kidney tissues were also
embedding.
Assessment of total antioxidant status and total oxidant status
The TAS of the rat serum samples was detected with an ELISA
Shanghai, China) according to the manufacturer’s instructions. The
kit’s measuring range was 1–300 pg·mL
-1
[the in–assay variation
<12%, and the sensitivity was 0.54 pg·mL
-1
]. The plates were cleaned
with a BioTek ELX50 automated washer (BioTek Instruments, USA). The
absorbance readings were made at wavelengths of 630 nm and 450
nm with a ChroMate P4300 Microplate Reader (Awareness Technology
s pg·mL
-1
.
The TOS of the rat serum samples was detected by ELISA kits
Shanghai, China) according to the manufacturer’s instructions. The
kit’s measuring range was 0.02–60 U·mL
-1
(the intra–assay CV value
was <10%, the inter–assay CV value was <12%, and the sensitivity
was 0.013 U·mL
-1
). The plates were washed, and the absorbance was
measured at wavelengths of 630 nm and 450 nm, as described for
TAS assays. The test results were presented in U·mL
-1
.
The serum adropin levels were measured by ELISA (Rel Assay Rat
Ltd., Gaziantep, Türkiye) following the manufacturer’s instructions.
The kit had an intra–assay CV value of <8%, an inter–assay CV value
of <10%, and a sensitivity of 0.02 ng·mL
-1
. The plates were washed
as described for TAS assays, and absorbance readings were taken
using a Bio–Tek ELX800 ELISA reader (BioTek Instruments, USA) at
a wavelength of 450 nm. The serum contents were determined by
comparison to a standard curve prepared from known concentrations
of adropin and expressed as ng·mL
-1
.
FIGURE 1. Total Antioxidant Status (TAS), Total Oxidant Status (TOS), and
Apoptotic Index (%) of the groups. NAC: N–acetylcysteine, MTX: Methotrexate
FIGURE 2. Serum Adropin levels, and Immunohistochemistry scores. NAC:
N– acetylcysteine , MTX: Methotrexate
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Immunohistochemistry and TUNEL assessment
Sections 4–6 µm thick were cut from kidney tissues embedded
immunohistochemical study was planned and prepared with the
[19, 20].
During the staining, the immunohistochemistry score was
immunohistochemistry score equals prevalence × intensity.
Plus Peroxidase in Situ Apoptosis Detection Kit (Chemicon, catalogue
no: S7101, USA) according to the manufacturer’s instructions. TUNEL
evaluation was performed using a method previously described in
the literature [19, 20].
Statistical analysis
SPSS version 22 was used for statistical analysis. The quantitative
measures were reported as median and minimum–maximum values.
To assess the normal distribution of the data, the Kolmogorov–
Smirnov test was utilized. The Mann–Whitney U test was used to
compare quantitative data between two groups that did not have
a normal distribution, and the Kruskal–Wallis test was used for
general comparisons between more than two groups. The Bonferroni
adjustment was used for paired–group comparisons. A P<0.05 value
RESULTS AND DISCUSSION
biological indicators of antioxidants’ protective capacity against
oxidative damage.
2 regarding serum TAS levels (P=0.535). TAS levels were higher in
Groups 1 and 4 than in Group 3 (P<0.001) (FIG. 1).
Groups 1 and 2 (P=0.805). TOS level was higher in Group 3 than in
Groups 1 and 4 (P<0.001) (FIG. 1).
Serum Adropin levels were similar in Groups 1 and 2 (P=0.151). Group
and 4 (P<0.008) (FIG. 2).
TUNEL positivity was observed in the tubular cells in the kidney
tissues (black arrow). TUNEL positivity was similar among the groups
(P=0.845).
Adropin immunoreactivity was observed in the glomeruli of the
kidney tissue (black arrow). The immunoreactivity was similar in
Groups 1 and 2 (FIGS. 4a and b) (P
in Group 3 than in Groups 1 and 4 (P<0.002) (FIGS. 4c and d).
diseases and various malignancies. However, besides its therapeutic
impact, it may also cause harm to many organs. The mechanism
underlying the dysfunction caused in the kidneys is still unclear [21
however, damage to the kidneys following MTX administration occurs
known to increase the level of ROS in various organs and tissues while
also decreasing the level of antioxidants, such as Glutathione, [22]
an endogenous antioxidant that plays a critical role in dissipating
reactive oxygen molecules. Oxidative stress decreases the level
of Glutathione, but this can be reversed with NAC treatment [23].
oxidative stress in kidneys caused by exposure to MTX [24]. The
oxidant capacity was more pronounced in the MTX–treated rats(
Ratus norvegicus) (Group 3) than in the control rats (Group 1), and
the antioxidant capacity was correspondingly decreased. The total
antioxidant capacity was similarly decreased after MTX treatment in
treatment.
The parameters of all four groups are summarized in TABLE I.
FIGURE 3. Comparison of TUNEL positivity of tubular cells in kidney tissues according to groups. a: Control group, b: NAC group, c: MTX group, and d: MTX+NAC group.
TUNEL: Terminal deoxynucleotidyl transferase dUTP transferase–mediated nick end–labeling, NAC: N–acetylcysteine, MTX: Methotrexate (Mag. 400X)
3a 3b
3c 3d
Adropin levels after Methotrexate treatment in renal tissue / Ahmet et al. __________________________________________________________
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Oxidative stress plays a known role in the activation of various signal
pathways, leading to transcription factor activation, gene expression,
apoptosis induction, and tissue necrosis. The administration of
MTX in rats increases the expression of Bax, which is an indicator
of apoptosis [25]. MTX–mediated apoptosis is believed to be activated
by the excess oxygen radical production that causes DNA damage.
TABLE I
Total Antioxidant Status, Total Oxidant Status, Apoptotic Index (%), serum adropin levels, and immunohistochemistry scores
TAS
Mean (min–max)
TOS
Mean (min–max)
Apoptotic Index (%)
Mean (min–max)
Serum Adropin levels
Mean (min–max)
Immunohisto–chemistry score
Mean (min–max)
CONTROL 1.59 (1.42–1.72) 17.42 (16.83–18.05) 2.14 (1.00–4.00) 29.56 (26.45–33.15) 0.73(0.60–0.90)
NAC 1.65 (1.31–1.91) 17.06 (15.18–18.61) 2.28 (1.00–4.00) 32.39 (29.78–34.16) 0.70 (0.45–0.90)
MTX 1.24 (1.01–1.39)
a
22.71 (21.18–24.81)
a
2.28 (1.00–4.00) 20.69 (19.05–22.86)
a
0.28 (0.20–0.40)
a
MTX+NAC 1.53 (1.35–1.66)
b
18.84 (17.93–19.61)
b
1.85 (1.00–3.00) 25.30 (24.56–26.23)
b
0.69 (0.45–0.90)
b
a
:Compared to the control group,
b
:Compared to the MTX group (P<0.05), MTX+NAC group: NAC: N–acetylcysteine, MTX: Methotrexate
Increased oxidative stress may decrease Bcl–2 expression, regulate
Bax levels, and activate the mitochondrial apoptosis pathway.
and TOS levels observed in the rats that were administered MTX were
in line with those reported in many other studies, yet the number of
FIGURE 4. Comparison of Adropin immunoreactivity in the kidney tissues according to groups. a: Control group, b: NAC group, c: MTX group, and d: MTX+NAC group.
NAC: N–acetylcysteine, MTX: Methotrexate (Mag. 400X)
4a 4b
4c 4d
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apoptotic cells did not increase. This suggests that different signal
pathways may trigger apoptosis, and that the sudden response to
that undergo apoptosis. Recent studies have claimed that apoptosis
induction may be a long–term process that depends on the prolonged
presence of oxygen free radicals in the environment [26]. Short–
term MTX use in intermittent doses is known to mediate adenosine
anti–apoptotic pathways, suggesting that MTX may have indirect
ects [27].
in these MTX effects. Adropin is a peptide hormone coded by a gene
many organs, particularly in the liver, brain, and heart. In kidney
tissue, Adropin immunoreactivity is localized in the peritubular
interstitial cells, peritubular capillary endothelium of the cortex, and
the glomeruli [20]. Lovren et al. [12] showed that Adropin activates
vascular endothelial growth factor receptors and increases the level
of eNOS. Decreasing Adropin levels would consequently decrease
eNOS enzyme activity and cause a decline in NO, which acts as a
vasodilator and is excreted by the endothelium. Therefore, Adropin
may potentially have the capacity to protect endothelium tissues.
Current evidence now indicates that endothelium dysfunction is
closely related to low plasma Adropin levels [28]. Adropin treatment has
been shown to partially restore endothelial cell proliferation, migration,
and capillary tube generation, and to reduce cell permeability and
apoptosis induced by tumor necrosis factor alpha [12]. Experimental
occurs via eNOS activation, and that Adropin decreases oxidative
damage. For example, in one study, the brains of old rats showed
decreased levels of total and phosphorylated eNOS [29].
Chen et al. [30] evaluated the impact of Adropin on antioxidant
reactions in preventing the progression of nonalcoholic steatohepatitis
Adropin levels after Methotrexate treatment in renal tissue / Ahmet et al. __________________________________________________________
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and the clearance of ROS. They found lower Glutathione levels that did
not contain Adropin in rats, as well as increased levels of oxygen free
radicals, caspase–3 (an indicator of Malondialdehyde production), and
apoptosis. Furthermore, they found that Adropin treatment increased
the level of nuclear erythroid 2–related factor (Nrf2), which is involved
in the induction of antioxidant reactions, especially Glutathione.
Adropin treatment also increased the expression of antioxidative
osis [30].
in Group 3 than in Groups 1 and 2. It was believed that this result, in
line with the literature, is related to the ability of Adropin to balance
the processes involved in oxidative stress. Furthermore, the serum
higher in Group 4 than in Group 3. It was believed that these results
support the presence of an inverse relationship between the severity
of oxidative stress, Adropin levels in circulation, and/or Adropin
shed some light on the role of Adropin in the development of kidney
failure following MTX administration.
CONCLUSION
In the present study supports a role for Adropin as a protective
peptide against the kidney failure that occurs due to MTX–induced
oxidative stress. However, this study was limited by its small
sample size, and the small number of kidney tissues analyzed.
Therefore, larger, and independent studies are needed to verify the
d results.
Conicts of interest
interest.
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