https://doi.org/10.52973/rcfcv-e34419
Received: 10/03/2024 Accepted: 05/06/2024 Published: 25/08/2024
1 of 8
Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34419
ABSTRACT
Gentamicin (GM), which causes nephrotoxicity, is an aminoglycoside
antibiotic commonly prescribed to treat of gram–negative infections.
Erythropoietin (EPO), which has several biological functions including
neuroprotection, wound healing and nephroprotection, is a glycoprotein
hormone that controls erythropoiesis. Hypericum perforatum (HP) is
a medicinal herb with antibacterial and nephroprotective effects.

in GM nephrotoxicity using combined biochemical, histopathological
and immunohistochemical evaluations together. A total of 36 male
Spraque–Dawley rats were divided into as control, GM (100 mg·kg
-1

-1
three consecutive days apart)

-1
day) groups (n=6) and the experiment lasted for
9 days. GM–induced increased relative kidney weight and increased


which increased with GM application, and increased the activities

catalase (CAT). GM nephrotoxicity resulted in tubular degeneration,
vacuolization and hyaline deposits, glomerular degeneration and





effect on the negative consequences of oxidative stress.
Key words: Apoptosis; Erythropoietin; gentamicin; Hypericum
perforatum; oxidative stress
RESUMEN
La gentamicina (GM), que causa nefrotoxicidad, es un antibiótico
aminoglucósido comúnmente indicado para tratar infecciones por
gram negativos. La eritropoyetina (EPO), que tiene diferentes funciones
biológicas entre las que se incluyen neuroprotección, cicatrización de
heridas y nefroprotección, es una hormona glicoproteica que controla
la eritropoyesis. Hypericum perforatum


transgénicos utilizando evaluaciones bioquímicas, histopatológicas

Spraque–Dawley se dividieron como control, GM (100 mg·kg
-1
día),

-1
tres días consecutivos de diferencia)

-1


transgénicos; mientras que provocaron un incremento de los niveles


con la aplicación de transgénicos, y aumentaron las actividades del

(CAT). La nefrotoxicidad de los transgénicos resultó en degeneración
tubular, vacuolización y depósitos hialinos, degeneración glomerular e


inmunorreactividad de la caspasa–3, que aumentó con la aplicación de

sobre la lesión renal inducida por transgénicos y, especialmente, el

sobre las consecuencias negativas del estrés oxidativo.
Palabras clave: Apoptosis; eritropoyetina; gentamicina; Hypericum
perforatum; estrés oxidativo
Erythropoietin and Hypericum perforatum ameliorate Gentamicin–induced
nephrotoxicity in rats
La eritropoyetina y el Hypericum perforatum mejoran la nefrotoxicidad inducida
por gentamicina en ratas
Tuba Parlak Ak
1
* , Meltem Sağıroğlu
2
, Gizem Elif Korkmaz
3
, Mine Yaman
4
1
University of Munzur, Faculty of Health Sciences, Department of Nutrition and Dietetics. Tunceli, Türkiye.
2
University of Firat, Faculty of Veterinary Medicine, Department of Physiology. Elazig, Türkiye.
3
University of Munzur, Pertek Sakine Genc Vocational School, Department of Veterinary. Tunceli, Türkiye.
4
University of Firat, Faculty of Veterinary Medicine, Department of Histology and Embryology. Elazig, Türkiye.
*Corresponding author: tubaparlakak@munzur.edu.tr
Nephrotoxicity in rats / Parlak Ak et al. ______________________________________________________________________________________________

INTRODUCTION
Gentamicin (GM), a nephrotoxic agent, is an aminoglycoside
antibiotic drug widely used against diseases caused by gram–negative
bacteria [1]. Despite this negative side effect, it remains a powerful

some antibiotics []. The mechanism leading to GM nephrotoxicity

with necrosis, which is linked to oxidative stress, inflammatory
cascades, apoptosis, and lipid peroxidation [3]. After glomerular

cells and is subsequently internalised by endocytosis [4]. Gentamicin,
which generally accumulates in lysosomes and may cause lysosomal
phospholipid rupture, is distributed to various intracellular organelles
and causes their dysfunction [5

and activates the intrinsic apoptotic pathway, leading to cell death
[45
play a role in the pathogenesis of GM nephrotoxicity [6]. Erythropoietin
(EPO) is a glycoprotein hormone that stimulates erythropoiesis [7].
EPO, which has several biological functions such as neuroprotection,
wound healing and nephroprotection in addition to erythropoiesis,

capillaries in the kidney. EPO has been shown to exert nephroprotective
8] and
to stimulate renal regeneration by acting directly on damaged
tubular cells [9

10
11]. This protection is
generally attributed to its anti–apoptotic and antioxidant activities [].
Hypericum perforatum

properties [13]. This plant contains hyperforin, hypericin, quercetin,

These have antioxidant activity, inhibition of lipid peroxidation
and free radical scavenging properties [14]. Studies have shown

nephroprotective effects [15, 16, 17

-1
to

-1
and are safe to use [18]. Therefore, it is widely used in for the
cure of diabetes, wound healing burns, nephrotoxicity [13]. The aim
of this study is to demonstrate the antioxidant and antiapoptotic

biochemical, histopathological and immunohistochemical evaluations.
MATERIALS AND METHODS
Chemicals and animals


commercial companies. Thirty–six male Spraque–Dawley rats (Rattus
norvegicus) 


hours light/dark period) and were fed free. This study was initiated




Experimental design
Animals were randomly divided into six groups (n=6). Control group:
the rats received normal saline 0.5 mL·kg
-1
day intraperitoneal (ip) for
9 days. GM group: the rats received GM 100 mg·kg
-1
day ip for 9 days
[19]. GM + EPO group: the rats received GM 100 mg·kg
-1
day ip for 9

-1
ip on days 1, 5 and 9 of the study [].

-1
day ip for 9 days,

-1
day orally for 9 days []. EPO group: the rats

-1
ip on days the 1, 5 and 9 of the study.

-1
day orally for 9 days.

experiment. Blood samples were collected for serum analysis. The
kidney tissues were used for histopathological, immunohistochemical
and biochemical analyses.
Serum analysis


Co., Ogak
Biochemical analysis




-1
[
-1
[
-1
[]
and

-1
[].
Histological examination

μm
thick sections []. The slides were evaluated by histologist blindly


tubular degeneration, tubular brush border loss, tubular vacuolization,
hyaline cast in the tubules, glomerular degeneration, mononuclear cell

shown as follows: (–): null, (+): mild, (++): moderate, (+++): severe [].
Immunohistochemical analysis

immunoreactivity in the kidney tissues were determined using the
Avidin–Biotin–Peroxidase Complex (ABC) method was according to
the procedure described previously []. Counterstaining was done

the area × density formula (density; none (0), very little (0.5), little (1),

0.9 (76–100%) [].
Statistical analysis



P
TABLE I
Eect of EPO and HP on GM–induced relative kidney
weight and renal function biomarkers
Groups
Relative kidney
weight (g)
BUN
(mg·dL
-1
)
Creatinine
(mg·dL
-1
)
Urea
(mg·dL
-1
)
Control 0.44 ± 0.04
c
15.23 ± 1.80
c
0.60 ± 0.02
c
20.15 ± 0.55
c
GM 0.58 ± 0.07
a
110.40 ± 2.20
a
3.73 ± 0.08
a
103.23 ± 1.03
a
GM + EPO 0.48 ± 0.03
b
28.33 ± 1.78
b
1.36 ± 0.04
b
31.11 ± 0.93
b
GM + HP 0.47 ± 0.06
b
25.30 ± 1.50
b
1.00 ± 0.02
b
30.16 ± 0.88
b
EPO 0.46 ± 0.02
c
17.20 ± 1.42
c
0.55 ± 0.03
c
19.17 ± 0.40
c
HP 0.45 ± 0.01
c
16. 38 ± 1.20
c
0.52 ± 0.05
c
18.12 ± 0.23
c
a,b,c
: Different superscripts in the same row indicate the significant difference. GM:
Gentamicin, EPO: Erythropoietin, HP:
Hypericum perforatum and BUN: Blood urea nitrogen
TABLE II
Eect of EPO and HP on GM–induced MDA, GSH, GSH–Px and CAT
Groups
MDA
(nmol·mL
-1
)
GSH
(nmol·mL
-1
)
GSH–Px
(IU·L
-1
)
CAT
(KU·L
-1
)
Control 2.55 ± 0.07
c
0.53 ± 0.04
a
287.29 ± 3.3
a
399.95 ± 3.7
a
GM 5.18 ± 0.17
a
0.22 ± 0.01
c
164.74 ± 2.1
c
263.71 ± 2.5
c
GM + EPO 3.85 ± 0.09
b
0.41 ± 0.02
b
279.14 ± 2.7
ab
305.82 ± 2.9
ab
GM + HP 3.68 ± 0.08
b
0.45 ± 0.0
b
281.22 ± 2.5
ab
328.51 ± 2.7
ab
EPO 2.75 ± 0.06
c
0.54 ± 0.08
a
291.15 ± 2.3
a
374.96 ± 3.2
a
HP 2.90 ± 0.05
c
0.51 ± 0.06
a
295.20 ± 2.2
a
397.68 ± 3.3
a
a,b,c
: Dierent superscripts in the same row indicate the signicant dierence. GM:
Gentamicin, EPO: Erythropoietin, HP:
Hypericum perforatum, MDA: malondialdehyde,
GSH: glutathione, GSH–Px: glutathione peroxidase and CAT: catalase
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34419
3 of 8
RESULTS AND DISCUSSIONS


known, is limited due to its nephrotoxic effect []. GM–induced
nephrotoxicity is initiated by pathological mechanisms related to
30
to protect the kidney from off–target toxicity of GM is of great clinical
importance. The use of agents with different pharmacological effects
for nephroprotection is of interest in terms of safe GM practices [].


to the control group (P

P
The relative weights of the kidneys and the levels of the biomarkers of

nephrotoxicity [31
[7, 
Therefore, it can be stated that the improvement in these impaired
biological markers may depend on the agents used.
of oxidative stress–related parameters in all groups are shown in

, 37]. Another study on antioxidant agents

Px and CAT activities increase [36

due to their strong free radical scavenging activity is supported.
Oxidative stress plays an important role in the pathophysiology of
GM nephropathy [6]. Previous studies have shown that MDA levels
[33

GM application [, 33
levels through excessive production of free radicals or depletion of
sulphhydryl protein groups [34

such as CAT, and GM reduces CAT expression [35].


induced nephrotoxicity [36


the control group (P


groups compared to the GM group (P


pathways that contribute to lipid peroxidation, protein denaturation,
6

disrupting the lipid structure unity and reducing antioxidant defense
mechanisms. Therefore, tubular cytotoxicity in GM nephrotoxicity has
also been found to be associated with cellular apoptosis and necrosis
[38
vacuolization, desquamation, hyaline eruption and mononuclear cell

with GM [35, 37]. Besides, it has been declared that thickening of
the cell membrane of glomeruli and destruction of endothelial cells,
vacuolization and loss of microvilli in convoluted proximal and distal
tubule cells [39
swelling and degeneration in the glomerulus in the GM group.

epithelial cells of the proximal tubules, together with degenerated and
desquamated cells collected in the lumen of these tubules, were noted.


in the interstitial region were detected. Mild histopathological lesions

groups compared to the GM group. Degenerative, necrotic, picnotic and

reduced. The scores of the kidney tissues in all groups is exhibited in

it has been reported by Codea et al. [7] that changes such as tubular

eruptions observed in GM–induced nephropathy were alleviated by the



angiogenic, and apoptotic inhibition [7].
TABLE III
Eect of EPO and HP on histopathological score in GM–induced nephrotoxicity
Groups
Tubular
degeneration
Loss of brush
border
Tubular
vacuolization
Hyaline cast in
tubular lumen
Glomerular
degeneration
Cell
inltration
Control – – – – – –
GM +3 +3 +3 +3 +2 +2
GM + EPO +1 +2 +1 +2 – +1
GM + HP +1 +1 +1 +1 – –
EPO – – – – – –
HP – – – – – –
(–): null, (+): mild, (++): moderate, (+++): severe
Hematoxylin-Eosin
Control
GM
EPO
GM + EPO
HP
GM + HP
FIGURE 1. Histopathological changes of the kidney tissues in all groups, gl: glomerulus, dt: distal tubule, pt: proximal tubule, black thick
arrow: vacuolization, black thin arrow: picnotic cell, white thick arrow: hyaline cast, white thin arrow: loss of brush border, black star:
degenerated tubule, Bars: 100 µm
Nephrotoxicity in rats / Parlak Ak et al. ______________________________________________________________________________________________
4 of 8
Caspase–3
Control
GM
EPO
GM + EPO
HP
GM + HP
FIGURE 2. Immunohistochemical analysis of caspase–3 immunoreactivity in the kidney tissues in all groups, gl: glomerulus, dt: distal
tubule, pt: proximal tubule, Bars: 100 µm
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34419
5 of 8
GM has been reported to increase pro–apoptotic Bax and caspase–3

cells [40]. Similar to the literature, in this study found that caspase–3
immunoreactivity was increased in the renal tubules of the GM

immunoreaction was found to be reduced in the GM + EPO and GM +


histoscores is exhibited 


and ischemia–reperfusion [, 41
damage due to GM nephrotoxicity was considerably reduced by
et al. [
protection by reducing edematous damage against GM nephrotoxicity
and decreased caspase–3 activity as a protective against apoptosis.

against renal damage in nephrotoxicity induced by different agents
[17, ]. Caglar et al. [
renal damage in CCl
4

the nephroprotective activity may be due to the repair of severe
and widespread kidney damage by downregulating oxidative stress
biomark
FIGURE 3. Immunohistochemical histoscore of the kidney tissues in all groups.
a: P<0.05 for comparison between GM group and other groups, b: P<0.05 for
the comparison between the GM group and the GM + EPO and GM + HP groups,
c: P<0.05 for comparison between Control, EPO and HP groups and other groups.
GM: Gentamicin, EPO: Erythropoietin, HP: Hypericum perforatum
Nephrotoxicity in rats / Parlak Ak et al. ______________________________________________________________________________________________
6 of 8
CONCLUSIONS
Possible mechanisms of GM nephrotoxicity may be due to changes
in renal function parameters, oxidative stress markers, apoptosis

the renal damage reasoned by GM–induced these changes have


antiapoptotic and nephroprotective properties. Further studies are

agents to reduce the risk of nephrotoxicity.
Conict of Interests
 interest.
ACKNOWLEDGEMENT


work by Grant Code: MF
BIBLIOGRAPHIC REFERENCES
 
Tamarindus indica against gentamicin–induced nephrotoxicity.
https://doi.
org/gt7gmb
 


agents against aminoglycoside–induced nephrotoxicity: a

https://doi.org/gt7gmc
 
Gentamicin nephrotoxicity in animals: Current knowledge and

https://doi.org/gtnhhg
 



p. 1044–1099.
 



 
De Sarro A, Pierpaoli S, Caputi AP, Masini E, Salvemini D. A role

https://
doi.org/dwb8mc
 

O. Melatonine and erythropoietin prevents gentamicin induced

doi: https://doi.org/gt7gmd
 


https://doi.org/f588xf
 

https://doi.org/
cd7z3b
 
efect of erythropoietin and melatonin on renal ischemia


 
protective effects of erythropoietin and melatonin on renal

https://doi.org/gt7gmf
 

microvascular damage in a murine model of septic acute kidney

doi: https://doi.org/gfcfpw
[13] Shrivastava M, Dwivedi LK. Therapeutic potential of Hypericum
perforatum

[14] Keskin C. Antioxidant, anticancer and anticholinesterase activities
Hypericum amblysepalum

https://doi.org/gt7gmg
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34419
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 In–vivo
and in–vitroEchinacea purpurea and
Hypericum perforatum
54(10):1379–1383. doi: https://doi.org/b8vd5p
 

https://doi.org/cq4b4b
 

of Hypericum perforatum on kidney ischemia/reperfusion damage.
https://doi.org/gkcr83
 


 Nigella sativa against
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https://doi.org/ffvrpp
 

against cisplatin–induced nephrotoxicity in rats: antigenotoxic

35(1):89–95. doi: https://doi.org/fd5ztv
 
effect of Hypericum perforatum on dexamethasone–induced


 
Hypericum perforatum on dexamethasone–induced diabetic
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 
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 
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https://doi.org/fthsdb
 



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 


https://
doi.org/ndhk
 
phoenixin–14 and nesfatin–1 in the hypothalamo–pituitary–

https://doi.org/gt7gmm
 
Protective effect of Rotula aquatica Lour against gentamicin
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
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 


and apoptosis underlies the protective effect of low dosage of
atorvastatin in gentamicin–induced nephrotoxicity. PLoS One

 
into the impact of rebamipide on gentamicin–induced
β–catenin/cyclin

doi: https://doi.org/gt7gmn
 
Hypericum perforatum extract
attennuates gentamicin induced oxidative stress, apoptosis and

doi: https://doi.org/gt7gmp
 


1315. doi: https://doi.org/f
[34] Antar SA, Al–Karmalawy AA, Mourad A, Mourad M, Elbadry M, Saber
S, Khodir A. Protective effects of mirazid on gentamicin induced



 
KS, Bodhankar SL. Ameliorative effect of berberine against
gentamicin–induced nephrotoxicity in rats via attenuation of



 



doi: https://doi.org/gnp878
 


https://doi.org/gt7gmq
 

of Heracleum persicum L. extract on rats with gentamicin –

https://doi.org/gt7gmr
 
histological alterations by aqueous garlic extract in gentamicin
induced renal toxicity in albino rats: a histological and

https://doi.org/gt7gms
Nephrotoxicity in rats / Parlak Ak et al. ______________________________________________________________________________________________
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


https://doi.
org/gt7gmt


κβ 

https://doi.org/gt7gmv
 
A, Meydan S. Effect of Camellia sinensis, Hypericum perforatum
and Urtica dioica

doi: https://doi.org/gt7gmw