This study examined the effects of prolonged simvastatin (SIM) treatment on ischemia-reperfusion (I/R) induced acute kidney injury in rats. Rats were divided into four groups: sham, ischemia, I/R, and I/R+SIM treated. The I/R group showed intense inflammation, necrosis, and apoptosis in kidney tissue. The I/R+SIM group showed reduced inflammation and tissue damage. Biochemical analysis found increased oxidative stress and inflammation markers in the ischemia and I/R groups compared to control, but levels in the I/R+SIM group were similar to control. Histological analysis also showed more damage in ischemia and I/R groups versus control, while the I/R+

1. 167
OBJECTIVE: Ischemia-reperfusion (I/R) leads to reac-
tive oxygen species formation and cell death in kidney
tissue with injury and organ transplantation. Simvas-
tatin (SIM) is an antioxidant, anti-inflammatory, and
anticoagulant agent. Alterations in I/R-induced acute
kidney injury model with SIM treatment were analyzed.
STUDY DESIGN: Wistar rats (n=28) were grouped
into Sham, Ischemia, I/R, and I/R+SIM treated. Left rat
kidney renal vessels were clamped for 60 minutes for
ischemia, and the I/R group had 6 hours of reperfusion.
10 mg/kg SIM was given orally for 28 days. MDA,
GSH, and MPO were analyzed. Kidney tissues were
paraffin embedded, and primary antibodies TNF-α and
caspase-3 were applied for immunohistochemistry.
RESULTS: In the I/R group, intense inflammatory cell
infiltration around the vessels and necrosis in the glo-
merular structures were observed. In the treated group,
proximal and distal tubular cells were found to be close
to normal. Immunoexpression of caspase-3 in the ische-
mia group was positive in degenerative glomeruli. In
the treated group, TNF-α expression was negative in
the glomerular structures. MDA and MPO levels were
significantly increased in ischemia and I/R.
CONCLUSION: We suggest that SIM treatment im-
proved kidney tissue structure and function in a model
of I/R injury. (Anal Quant Cytopathol Histpathol
2021;43:167–173)
Keywords: caspase-3; immunohistochemistry; is-
chemia/reperfusion; kidney; MPO; simvastatin.
Ischemia-reperfusion (I/R) is a situation when the
blood supply to an organ is restricted—ischemia—
and then reoxygenation of tissues occurs—reper­
fusion. Following the reperfusion procedure, when
oxygen increases, reactive oxygen species (ROS)
generate and cause cell death.1 Statins, specific inhi-
bitors of 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase, are used to lower the cho-
lesterol level for a long time.2 Statins have pleiotro-
pic effects: antioxidant, inhibiting of thrombogenic
responses, and healing of endothelial function.3
Statins inhibit the formation of proinflammatory
cytokines and decrease inflammation.4 Statins used
after major abdominal, cardiac, thoracic, and vas­
cular surgery decrease the development of acute
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4304-0167/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Prolonged Simvastatin Treatment Provided a
Decrease in Apoptotic, Inflammatory, and
Oxidative Stress Markers in Ischemia-
Reperfusion–Induced Acute Kidney Injury
Model of Rats
Alper Kafkasli, M.D., and Ebru Gokalp Ozkorkmaz, Ph.D.
From the Urology Clinic, Lutfi Kirdar Training and Research Hospital, Istanbul; and the Department of Histology and Embryology,
Faculty of Medicine, Dicle University, Diyarbakır, Turkey.
Alper Kafkasli is Assistant Professor, Urology Clinic, Lutfi Kirdar Training and Research Hospital (Orcid ID: 0000-0002-0362-8165).
Ebru Gokalp Ozkorkmaz is Assistant Professor, Department of Histology and Embryology, Faculty of Medicine, Dicle University (Orcid
ID: 0000-0002-1967-4844).
Address correspondence to: Ebru Gokalp Ozkorkmaz, Ph.D., Department of Histology and Embryology, Faculty of Medicine, Dicle
University, 21010 Diyarbakır, Turkey (ebrug76@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. kidney damage.5,6 Simvastatin (SIM) is a statin
group compound that induces angiogenesis and
promotes endothelial cell growth.7 Tumor necrosis
factor (TNF-α) leads to a destruction in the sur-
rounding tissue.8 Apoptosis is the primary mode
of cell death in renal I/R, triggered with TNF-α
expression.9 Caspases are promoters of apoptosis,
inflammation, and cell death. Caspase-3, a major
executional caspase, acts downstream in the apop-
tosis pathway involved in the process of proteo-
lytic cleavage.10 Here we aimed to observe the
alterations in tissue structure and apoptotic, in-
flammatory, and oxidative stress biomarkers in
I/R-induced acute kidney injury model with pro-
longed orally given SIM treatment.
Materials and Methods
Experimental Design
All of the experimental protocols were approved
by the Local Ethical Committee of Health Sciences,
Dicle University, Diyarbakır, Turkey (2020/19).
Male Wistar albino rats (n=28), 3 to 4 months
old and weighing 180 to 240 g, were used for the
study. They were kept under standard conditions
(light/dark cycles of 12 h/12 h with 50–70% hu­
midity, at 25±3°C) and fed with standard pellet
diet and water ad libitum. Surgical procedures were
performed under anesthesia with sodium pentobar-
bital (45 mg/kg, i.p.).
Group 1. Sham group (n=7). Rats were submitted to
all surgical steps except the renal ischemia.
Group 2. Ischemia (I) group (n=7). Renal vessels of
the left rat kidney were clamped for 60 minutes for
renal occlusion with renal clips to create ischemia.
Group 3. I/R group (n=7). Renal vessels of the left
rat kidney were clamped for 60 minutes and had
6 hours of reperfusion after the renal clips were
removed.
Group 4. SIM treated I/R (n=7). Renal vessels of
the left rat kidney were clamped for 60 minutes,
had 6 hours of reperfusion, and were treated with
Simvastatin (10 mg/kg per day) (Wako Pure Chem.
Industries, Japan). The SIM was diluted by physi-
ological saline solution and administrated via oral
route using a gastric tube for 28 days.
Unilateral vessel clamping was preferred so as
not to lose subjects as bilateral injury with longer
clamp times may cause permanent tubular injury
and subjects can die within the first 3 days after
injury due to severe renal insufficiency.11 All rats
were euthanized with an intraperitoneal injection
of 30 mg/kg Ketamine (Ketalar, Eczacıbası, Turkey)
and 10 mg/kg xylazine hydrochloride (Rompun,
Bayer, Turkey) at the end of the experiment, and
kidney tissues were quickly removed.
Biochemical Analyses
Tissue samples were homogenized with ice-cold,
0.15 mM KCl buffer for malondialdehyde (MDA)
and glutathione (GSH) analyses. MDA was as-
sayed via a thiobarbituric acid reaction according
to the method by Ohkawa et al.12 GSH level was
also determined using spectrophotometric meth-
od, with the use of Ellman’s reagent, and the re-
sults were given as mmol/g. Tissue myeloperox-
idase (MPO) activities were analyzed according
to Hillegass et al,13 and the results were given as
U/g.
Histopathology and Immunohistochemistry
Procedures
Kidney tissues were immediately fixed with 10%
neutral formalin, and routine paraffin tissue follow-
up protocol was applied. 4-6 µm sections were
obtained from paraffin blocks with a microtome
(Leica, Germany). Sections were stained with rou­
tine hematoxylin and eosin, and remaining were
incubated for 3×5 minutes in PBS for immuno­
staining. Samples were incubated in Ultra V block
(catalog no. TA-015UB, Thermo Fisher, USA) for
8 minutes. Blocking solution was removed from
the sections and allowed to incubate overnight at
+4°C with primary antibodies TNF-α (lot #ab6671,
Abcam, USA) and caspase-3 (lot #ab208161, Ab-
cam). Secondary antibody (TP-015-BN, Thermo
Fisher) was applied for 20 minutes. The sections
were exposed to streptavidin-peroxidase (TS-015-
HR, Thermo Fisher) for 20 minutes and allowed
to react with DAB (TA-001-HCX, Thermo Fisher),
and then examined under a light microscope (Zeiss
Imager A2, Germany).14
Statistical Analyses
Statistical analyses were prepared using SPSS Ver-
sion 20.0 (IBM Corp., Released 2011. IBM SPSS
Statistics for Windows, Armonk, New York, USA).
The data were represented as the mean±SD and
evaluated using one-way analysis of variance
(ANOVA) with Tukey post-hoc tests, and p<0.05
168 Analytical and Quantitative Cytopathology and Histopathology®
Kafkasli and Gokalp Ozkorkmaz

3. and p<0.01 were considered as significant. Histo­
pathological assay of preparations for tubular de-
generation, vascular dilation, congestion, and in­
flammation were performed in a blind manner
with a semi-quantitative scoring system (from 0 to
4). Immunohistochemical analyses of groups was
performed according to the brownish staining dis­
tribution of caspase-3 and TNF-α immunoexpres-
sions.
Results
Histopathological observation results are given in
Figure 1A–D. Immunohistochemistry results are
also depicted as caspase-3 (Figure 2A–D) and
TNF-α immunoexpressions (Figure 3A–D). Statis-
tical data of biochemical analyses (MDA [nmol/g],
GSH [µmol/g], and MPO [U/g]) are given in Ta-
ble I and in Figures 4–5. All values except for the
GSH value were significantly increased in the is-
Volume 43, Number 4/August 2021 169
Effects of Simvastatin on Kidney Injury
Figure 1
(A) Control group section.
Regular shape of squamous
cells in the parietal and
visceral sections, regular
proximal tubular cells.
(B) Ischemia group.
Degeneration in the visceral
cells of the glomerular
structure (asterisk), and
apoptotic cells, pyknosis, and
degeneration in the nuclei of
proximal and distal tubular
cells. (C) I/R group. Intense
inflammatory cell infiltration
around the vessels (asterisk),
apoptosis in tubular cells, and
significant degeneration.
(D) I/R+SIM group. Pyknotic nuclei in visceral cells of glomerular structures, reduced dilation and congestion in the blood vessels, small
number of inflammatory cells in the solitary form. HE staining. Bar = 500 µm.
Figure 2
(A) Control group. Negative
caspase-3 expression in
glomerular visceral and
parietal cells. (B) Ischemia
group. Positive caspase-3
reaction in degenerative
glomeruli cells, blood vessel
endothelial cells (asterisks).
(C) I/R group. Positive
caspase-3 expression in the
intense inflammatory cells
around the glomeruli.
(D) I/R+SIM group. Positive
caspase-3 reaction in some of
the glomeruli and tubule cells.
Caspase-3 immunostaining.
Bar = 500 µm.

4. chemia and I/R groups as compared to the con-
trol group (p<0.05 and p<0.01). The I/R+SIM
group values were similar to those of the control
group for all parameters (p<0.05). Histopatho-
logical scoring results are shown in Table I and in
Figure 6.
Discussion
I/R is a pathological change induced by decreased
oxygen delivery due to limited blood supply in-
ducing an exchange from aerobic to anaerobic glu-
cose metabolism. Insufficient oxygen causes a de-
crease in intracellular ATP levels, and this process
results with the destabilization of lysozyme mem­
brane which leaks and hydrolases disrupt the cell
structure. I/R may cause cell damage via several
cellular pathways such as cell death, microvascular
dysfunction, and immune system disorders. Patho-
logical conditions such as acute kidney injury and
failure in organ transplantation may occur due to
I/R in a broad perspective.15,16 Researchers have
indicated that kidney injury is a complex patho­
physiological process including a variety of fac-
tors. Herein, in the ischemia group, acute tubular
epithelial cell damage with apoptosis and necrotic
appearance in glomeruli, infiltration of interstitial
inflammatory cells, and pyknosis and degenera­
tion in the nuclei of proximal and distal tubular
cells were observed. The I/R group depicted con-
gestion in the blood vessels around the glomeruli
in the cortex, intense inflammatory cell infiltration
around the vessels, necrosis in the glomerular
structures, apoptosis in tubular cells, and signifi-
cant degenerations. However, in the SIM-treated
group, pyknotic nuclei in visceral cells of glomer­
ular structures, small number of inflammatory cells
170 Analytical and Quantitative Cytopathology and Histopathology®
Kafkasli and Gokalp Ozkorkmaz
Figure 3
(A) Control group. Negative
TNF-α expression in the
glomerular structures and
proximal distal tubular cells.
(B) Ischemia group. Positive
TNF-α expression in the
inflammatory cells (asterisk)
and in solitary scattered
macrophage cells. (C) I/R
group. Increased TNF-α
expression in intensely
accumulated inflammatory
cells (asterisk), in the
degenerative tubular cells.
(D) I/R+SIM group. Negative
TNF-α expression in
glomerular and tubular cells.
TNF-α immunostaining.
Bar = 500 µm.
Table I Statistical Analyses of Tubular Degeneration, Vascular Dilation and Congestion, Inflammation, MDA, GSH, and MPO Results of
the Groups
Control Ischemia I/R I/R+SIM
group
group group group
Degeneration in tubular cells 0.6±0.16 3.4±0.16** 3.6±0.16** 1.1±0.18
Vascular dilation and congestion 0.3±0.15 3.7±0.15** 3.0±0.21** 0.8±0.13
Inflammation 0.6±0.16 3.4±0.22** 3.7±0.15** 0.9±0.18
MDA (nmol/g) 34.56±1.02 51.67±0.59* 52.40±0.80* 35.99±1.20
GSH (µmol/g) 1.10±0.03 0.69±0.05 0.74±0.04 1.11±0.03
MPO (U/g) 3.78±0.15 7.16±0.23* 7.70±0.12* 3.98±0.14
*p value <0.05 and **p value <0.01 were considered significant as compared to the control group.
Results are shown as mean±SEM.

5. in the solitary form, and regular arrangement of
tubular cells around the lumen were observed in
our histopathological examinations. Statistical ana­
lyses of histopathological findings indicated in-
creased degeneration in tubular cells and inflam-
mation in the ischemia and I/R. Nesić et al17
performed acute pretreatment with a single dose
of SIM (1 mg/kg, i.v., 30 min before ischemia) on
renal dysfunction in the rat I/R injury (45 min
ischemia+4 h reperfusion with saline). SIM im-
proved glomerular and tubular dysfunction and
histological score. In another study of the same
group, a single i.v. injection of SIM (1 mg/kg)
protected the rat kidney injured by I/R (45 min+
6 h).18 We preferred to give SIM via the oral route
for 28 days, a prolonged treatment for rats. SIM is
well absorbed from the gastrointestinal tract but
is highly extracted by the liver, and only 7% of
the dose reaches the circulation. The peak inhibi-
tion of HMG-CoA reductase activity is observed
within 2 to 4 hours.19 According to Yang et al,20
who studied I/R-induced acute kidney injury,
caspase-3 in regulating microvascular endothelial
cell apoptosis and renal fibrosis after I/R injury
has been explained with the findings of endothe-
lial peritubular cell death in caspase-3–deficient
mice with I/R injury. We observed in ischemia and
I/R groups that caspase-3 reaction was positive
in degenerative glomerular cells, endothelial cells,
and inflammatory cells around the glomeruli but
negative in the I/R+SIM–treated group. We can
suggest that SIM inhibits caspase-3 activation and
possibly prevents apoptotic cell death in the kid-
ney after reperfusion. In a previous study, it was
pointed out that long-term exposure to higher
doses of SIM itself may cause both apoptosis and
Volume 43, Number 4/August 2021 171
Effects of Simvastatin on Kidney Injury
Figure 4
Graph of MDA levels of
groups. MDA levels were
significantly increased in
the Ischemia and I/R groups.
*p<0.05.
Figure 5
Statistical graph of GSH and
MPO analyses of groups. GSH
and MPO levels increased
significantly in the Ischemia
and I/R groups (p<0.05). The
I/R+SIM group was found to
be similar to the control group.
*p>0.05.

6. necrosis of renal mesangial cells.21 SIM seems to
reduce post-reperfusion oxidative stress and con­
tribute to the improvement of renal structure and
function. TNF-α was positively expressed in the
inflammatory cells in the ischemia group and in
I/R group, in inflammatory cells around the blood
vessels, and in the degenerative tubular cells as
a sign of the ongoing inflammatory process. SIM
treatment provided a reduction in TNF-α immu-
noexpression with inflammation in the injured
tissue. Data from biochemical analyses of MDA,
GSH, and MPO exhibited an increment in ische-
mia and I/R groups; however, SIM treatment has
achieved values similar to that of the control group,
supporting other findings. Finally, SIM was found
to be effective in reducing the cellular outcomes of
renal ischemia and I/R such as apoptosis, inflam-
mation, and oxidative stress.
References
1. Salvadori M, Rosso G, Bertoni E: Update on ischemia-
reperfusion injury in kidney transplantation: Pathogenesis
and treatment. World J Transplant 2015;5:52-67
2. Elavarasu S, Suthanthiran TK, Naveen D: Statins: A new era
in local drug delivery. J Pharm Bioallied Sci 2012;4:248-251
3. Kaya Y, Kaya A, Bekataş O: Beyond the Lipid-lowering Effe-
cts of Statins: Renal Effects. Koşuyolu Heart J 2017;20(3):240-
247
4. Mason JC: Statins and their role in vascular protection. Clin
Sci 2003;105:251-266
5. Molnar AO, Coca SG, Devereaux PJ, Jain AK, Kitchlu A,
Luo J, Parikh CR, Paterson JM, Siddiqui N, Wald R, Walsh
M, Garg AX: Statin use associates with a lower incidence of
acute kidney injury after major elective surgery. J Am Soc
Nephrol 2011;22:939-946
6. Dormuth CR, Hemmelgarn BR, Paterson JM, James MT,
Teare GF, Raymond CB, Lafrance JP, Levy A, Garg AX,
Ernest P; Canadian Network for Observational Drug Effect
Studies: Use of high potency statins and rates of admission
for acute kidney injury: Multicenter retrospective observa­
tional analysis of administrative databases. BMJ 2013;346:880
7. Jain MK, Ridker PM: Anti-inflammatory effects of statins:
Clinical evidence and basic mechanisms. Nat Rev Drug Dis-
cov 2005;4:977-987
8. Ricci-Vitiani L, Casalbore P, Petrucci G, Lauretti L, Montano
N, Larocca LM, Falchetti ML, Lombardi DG, Di Giorgio
Gereveni V, Cenciarelli C, D’Allessandris QG, Fernandez E,
De Maria R, Maira G, Peschle C, Parati E, Pallini R: Influence
of local environment on the differentiation of neural stem
cells engrafted onto the injured spinal cord. Neurol Res 2006;
28:488-492
9. Yune TY, Chang MJ, Kim SJ, Lee YB, Shin SW, Rhim H,
Kim YC, Shin ML, Oh YJ, Han CT, Markelonis GJ, Oh TH:
Increased production of tumor necrosis factor-alpha induces
apoptosis after traumatic spinal cord injury in rats. J Neuro­
traumatol 2003;20:207-219
10. McIlwain DR, Berger T, Mak TW: Caspase functions in cell
death and disease. Cold Spring Harb Perspect Biol 2013;5(4):
a008656
11. Skrypnyk NI, Harris RC, de Caestecker MP: Ischemia-reper-
fusion model of acute kidney injury and post injury fibrosis
in mice. J Vis Exp 2013;78:e50495
12. Ohkawa H, Ohishi H, Yagi K: Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Analytic Bio­
chem 1979;95(2):351-358
13. Hillegass LM, Griswold DE, Brickson B, Albrightson-
Winslow C: Assessment of myeloperoxidase activity in
whole rat kidney. J Pharmacol Methods 1990;24(4):285-295
14. Zeytun H, Gökalp Özkorkmaz E: Effects of carvacrol in an
experimentally induced esophageal burn model: Expression
of VEGF and Caspase-3 proteins. J Invest Surg 2021;34(4):
408-416
15. Philipponnet C, Aniort J, Garrouste C, Kemeny J, Heng AE:
Ischemia reperfusion injury in kidney transplantation: A
case report. Medicine (Baltimore) 2018;97(52):e13650
16. Fuquay R, Renner B, Kulik L, McCullough JW, Amura C,
Strassheim D, Pelanda R, Torres R, Thurman JM: Renal
ischemia-reperfusion injury amplifies the humoral immune
response. J Am Soc Nephrol 2013;24:1063-1072
17. Nesić Z, Todorović Z, Stojanović R, Basta-Jovanović G,
172 Analytical and Quantitative Cytopathology and Histopathology®
Kafkasli and Gokalp Ozkorkmaz
Figure 6
Statistical results graph of the
groups’ scores of degeneration
in tubular cells, vascular
dilation and congestion, and
inflammation. In the Ischemia
and I/R groups, a significant
increase was detected in all 3
scores (p<0.05).

7. Radojević-Skodrić S, Velicković R, Chatterjee PK, Thiemer-
mann C, Prostran M: Single-dose intravenous SIM treat-
ment attenuates renal injury in an experimental model of
ischemia-reperfusion in the rat. J Pharmacol Sci 2006;102(4):
413-417
18. Todorovic Z, Nesic Z, Stojanović R, Basta-Jovanović G,
Radojevic-Skodrić S, Velicković R, Chatterjee PK, Thiemer-
mann C, Prostran M: Acute protective effects of simvastatin
in the rat model of renal ischemia-reperfusion injury: It is
never too late for the pretreatment. J Pharmacol Sci 2008;
107(4):465-470
19. Mauro VF: Clinical pharmacokinetics and practical ap-
plications of simvastatin. Clin Pharmacokinet 1993;24:195–
202
20. Yang B, Lan S, Dieudé M, Sabo-Vatasescu JP, Karakeussian-
Rimbaud A, Turgeon J, Qi S, Gunaratnam L, Patey N, Hébert
MJ: Caspase-3 is a pivotal regulator of microvascular rare­
faction and renal fibrosis after ischemia-reperfusion injury.
J Am Soc Nephrol 2018;29(7):1900-1916
21. Heusinger-Ribeiro J, Fischer B, Goppelt-Struebe M: Differ­
ential effects of simvastatin on mesangial cells. Kidney Int
2004;66:187-195
Volume 43, Number 4/August 2021 173
Effects of Simvastatin on Kidney Injury