Effects of Dapagliflozin on Experimental Sepsis Model in Rats

Abstract

Background:
The aim of this study was to evaluate the possible protective effects of dapagliflozin in an experimental sepsis model in rats.

Methods:
Saline (1 mL/kg, p.o.) or dapagliflozin (10 mg/kg, p.o.) was administered to Sprague-Dawley rats for 5 days prior to the surgical procedures. Under anesthesia, sepsis was induced by cecal ligation puncture, while sham control groups underwent laparotomy only. Blood urea nitrogen, creatinine, and glucose levels were measured in serum samples and the levels of malondialdehyde (MDA), glutathione (GSH), myeloperoxidase (MPO), tumor necrosis factor alpha, interleukin 1 beta, caspase 8, and caspase 9 were determined in tissue samples (kidney, liver, and lung). Histological evaluation was also performed.

Results:
The administration of dapagliflozin in a sepsis model reduced oxidative stress (MDA), increased antioxidant levels (GSH), and reduced inflammation (MPO) in the kidney (p<0.05). Dapagliflozin also decreased oxidative stress (MDA) in lung tissue and decreased inflammation (MPO) in lung and liver tissue (p<0.05). Caspase 8 and 9 levels in kidney, lung, and liver tissue were increased (p<0.05) in the dapagliflozin group compared with the sepsis group. According to the histopathological results, sepsis was moderately improved in renal tissue and slightly attenuated in lung and liver tissue with the administration of dapagliflozin.

Conclusion:
Dapagliflozin had a preventive effect on sepsis-induced kidney damage, but the protective effect was mild in lung and liver tissue in the present study.

Full text

Effects of dapagliflozin in experimental sepsis model in rats
Zehra Betül Kıngır, M.Sc.,1 Zarife Nigar Özdemir Kumral, Ph.D.,2 Muhammet Emin Çam, Ph.D.,3
Özlem Tuğçe Çilingir, Ph.D.,4 Turgut Şekerler, M.Sc.,5 Feriha Ercan, Ph.D.,4
Özlem Bingöl Özakpınar, Ph.D.,5 Derya Özsavcı, Ph.D.,5 Mesut Sancar, Ph.D.,1 Betül Okuyan, Ph.D.1
1Department of Clinical Pharmacy, Marmara University Faculty of Pharmacy, İstanbul-Turkey
2Department of Physiology, Marmara University Faculty of Medicine, İstanbul-Turkey
3Department of Pharmacology, Marmara University Faculty of Pharmacy, İstanbul-Turkey
4Department of Histology and Embryology, Marmara University Faculty of Medicine, İstanbul-Turkey
5Department of Biochemistry, Marmara University Faculty of Pharmacy, İstanbul-Turkey
ABSTRACT
BACKGROUND: The aim of this study was to evaluate the possible protective eects of dapagliflozin in an experimental sepsis
model in rats.
METHODS: Saline (1 mL/kg, p.o.) or dapagliflozin (10 mg/kg, p.o.) was administered to Sprague-Dawley rats for 5 days prior to the
surgical procedures. Under anesthesia, sepsis was induced by cecal ligation puncture, while sham control groups underwent laparo-
tomy only. Blood urea nitrogen, creatinine, and glucose levels were measured in serum samples and the levels of malondialdehyde
(MDA), glutathione (GSH), myeloperoxidase (MPO), tumor necrosis factor alpha, interleukin 1 beta, caspase 8, and caspase 9 were
determined in tissue samples (kidney, liver, and lung). Histological evaluation was also performed.
RESULTS: The administration of dapagliflozin in a sepsis model reduced oxidative stress (MDA), increased antioxidant levels (GSH),
and reduced inflammation (MPO) in the kidney (p<0.05). Dapagliflozin also decreased oxidative stress (MDA) in lung tissue and de-
creased inflammation (MPO) in lung and liver tissue (p<0.05). Caspase 8 and 9 levels in kidney, lung, and liver tissue were increased
(p<0.05) in the dapagliflozin group compared with the sepsis group. According to the histopathological results, sepsis was moderately
improved in renal tissue and slightly attenuated in lung and liver tissue with the administration of dapagliflozin.
CONCLUSION: Dapagliflozin had a preventive eect on sepsis-induced kidney damage, but the protective eect was mild in lung
and liver tissue in the present study.
Keywords: Apoptosis; dapagliflozin; inflammation; oxidative stress; sepsis.
pulmonary system, coagulation mechanism, central nervous
system, gastrointestinal system, and renal failure are com-
mon problems that can increase mortality.[3] Acute renal
damage associated with sepsis and the deleterious eects
of organic waste, such as uremic toxins, cause oxidative
stress, inflammation, and insulin resistance, and these con-
sequences also aect morbidity and mortality in sepsis.[4]
Cecal ligation and puncture (CLP) is a well-designed, easy,
and inexpensive polymicrobial septic shock model in ex-
perimental animals that also conforms to the human sepsis
model.[5–7]
EXPERIMENTAL STUDY
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3 213
INTRODUCTION
Sepsis is defined as serious clinical syndrome which occurs
as a consequence of an impaired inflammation response
against infection characterized by an abnormal physiological,
biological, and biochemical process.[1] According to a retro-
spective analysis of international databases, the global inci-
dence rate between 1995 and 2015 was 437 out of 100,000
for sepsis and 270 out of 100,000 for severe sepsis.[2] During
the management of sepsis, organ failure must be carefully
evaluated because it is well established that damage to the
Cite this article as: Kıngır ZB, Özdemir Kumral ZN, Çam ME, Çilingir ÖT, Şekerler T, Ercan F, et al. Eects of dapagliozin in experimental sepsis
model in rats. Ulus Travma Acil Cerrahi Derg 2019;25:213-221.
Address for correspondence: Betül Okuyan, Ph.D.
Marmara Üniversitesi Eczacılık Fakültesi, Klinik Eczacılık Anabilim Dalı, İstanbul, Turkey.
Tel: +90 216 - 414 05 45 E-mail: betulokuyan@yahoo.com
Ulus Travma Acil Cerrahi Derg 2019;25(3):213-221 DOI: 10.5505/tjtes.2018.82826 Submitted: 03.04.2018 Accepted: 18.09.2018 Online: 20.05.2019
Copyright 2019 Turkish Association of Trauma and Emergency Surgery

Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
In many studies, sodium-glucose co-transporter 2 (SGLT2)
inhibitors, such as dapagliflozin, have been observed to have
antioxidant eects by increasing antioxidant enzymes and
reducing oxidative stress markers. Furthermore SGLT2 in-
hibitors have been reported to reduce inflammatory markers,
suppress apoptosis in the cell and have a potential eect on
cell healing.[8–12]
A literature review revealed that dapagliflozin, a new antidi-
abetic agent, has not been investigated for its eects on ox-
idative stress, inflammation, and apoptosis in an experimental
sepsis model. Therefore, the objective of this study was to
investigate the antioxidant, anti-inflammatory, and antiapop-
totic eects of dapagliflozin in rats in a cecal binding and
puncture sepsis model.
MATERIALS AND METHODS
Experimental Animals
Sprague-Dawley rats (250–350 g) of both sexes supplied by
the Experimental Animal Implementation and Research Cen-
ter of Marmara University were housed in relative humidity
(65–70%) and a temperature-controlled room (22±2°C) with
standardized light/dark (12-hour) cycles. The rats were fed
with standard rat pellets and had free access to water. This
study protocol was approved by Marmara University Animal
Experiments Local Ethical Committee (Ethical approval num-
ber and date: 115.2016.mar; 12.12.2016).
Experimental Design
The experimental sepsis model was developed using a CLP
procedure. It has been established that the cecum contains a
high concentration of Gram-positive and Gram-negative bac-
teria. This polymicrobial content spreads to the peritoneum
in the CLP model and causes sepsis. The rats were randomly
divided into a sham or a CLP group, each of which included
subjects of both sexes. Anesthesia was provided with a com-
bination of ketamine 100 mg/kg and xylazine 10 mg/kg. After
the midline laparotomy, the cecum was gently pulled out and
ligated above the ileocecal valve to maintain bowel passage,
3 perforations were made on the antimesenteric side with
a 21-gauge needle, and feces expression was allowed. The
sham-operated control group (n=16) underwent a laparo-
tomy without ligature or punctures and the abdomen was
closed appropriately. Data obtained from previous studies
indicate that CLP-induced sepsis mortality occurs in the first
3 days.[13] The rats were decapitated 24 hours after the op-
eration.[5,6] The survival rate of the experimental animals was
recorded throughout the process.[14–16]
Four days before the CLP surgery, saline (n=8) or dapagliflozin
(n=8; 10 mg/kg, 10 mL/kg; Forxiga, AstraZeneca, Cambridge,
UK) was administered to the subjects in the sham and da-
pagliflozin groups. Orogastric gavage was performed in the
dapagliflozin-treated CLP group and the sham-operated rats
were injected with saline.[9,14,15,17] On day 5, sepsis was in-
duced using the CLP model and 24 hours later the rats were
sacrificed. There were 4 female rats (250–300 g) and 4 male
rats (300–350 g) in each group. Serum and tissue (kidney,
lung, liver) samples were obtained and preserved (-80°C or
10% buered formalin) in order to be used for further bio-
chemical and histological analysis (Fig. 1).
Biochemical Analyses
Measurement of Serum Blood Urea Nitrogen,
Creatinine, and Glucose
The serum blood urea nitrogen (BUN), creatinine, and glu-
cose levels were determined using an auto analyzer according
to the manufacturer’s instructions (Cobas Integra 400 plus;
Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland).
Measurement of Malondialdehyde and Glutathione
The level of malondialdehyde (MDA), a byproduct of lipid
peroxidation, was measured based on thiobarbituric acid re-
active substance formation in kidney, lung, and liver tissues.
[18] Tissue samples were homogenized in 10% trichloroacetic
acid solution using 10-fold dilutions. The homogenized tis-
sue samples were then centrifuged at 2000 g for 15 minutes
at 4°C; the supernatant was removed and re-centrifuged
at 41,400 g for 8 minutes. The upper organic liquid layer
was separated and was measured with a spectrophotome-
ter (Epoch; BioTek Instruments, Inc., Winooski, VT, USA)
at 532 nm. Thiobarbituric acid reactive substance forma-
tion was measured[19] and the lipid peroxidation measure-
ment was provided in terms of MDA equivalents using an
extinction coecient of 1.56×105 M−1 cm−1 and expressed
as nmoL MDA/g tissue.
GSH levels were measured in kidney, lung, and liver tissues
according to the method developed by Beutler[20] using a
modification of the Ellman procedure. The experimental
principle is to measure the colored product resulting from
the reaction of the sulphydryl groups with 5-5 ‘dithiobis 1-2
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3214
Figure 1. Schematic representation of the experimental design.
Saline-treated sham group
Dapagliozin (10 mg/kg) - treated CLP group
Cecal ligation and puncture
(CLP) model
- Oral gavage treatments
(10 ml/kg)
Dapagliozin (10 mg/kg) - treated sham group
Saline-treated CLP group
Decapitation and
collection
- Blood
- Kidney
- Lung
- Liver tissues
1st
day
6th
day
2nd 3rd 4th 5th

Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
nitrobenzoic acid in the spectrophotometer at 412 nm. The
results were expressed as µmol GSH/g tissue.
Measurement of Myeloperoxidase Activity
Myeloperoxidase (MPO) is a member of the peroxidase
family. MPO activity in the lysates of kidney, lung, and liver
tissues was assessed using a commercial enzyme-linked im-
munosorbent assay (ELISA) kit (Catalog No: LS-F4305, Lot
No: 103692; LifeSpan BioSciences, Inc., Seattle, WA, USA).
The results were measured as U/g tissue.
Measurement of Interleukin 1 Beta and Tumor
Necrosis Factor-Alpha
Interleukin 1 beta (IL-1β) and tumor necrosis factor alpha
(TNF-α) levels were measured in all tissues (kidney, lung,
liver) with commercial ELISA kits (Catalog No: BMS630, Lot
No: 143373023; eBio-science, Inc., San Diego, CA, USA;
Catalog No: KRC3011, Lot No: 1818268a; Thermo Fisher
Scientific, Inc., Waltham, MA, USA, respec-tively). The results
were measured as ng/mL.
Measurement of Tissue Caspase 8 and Caspase 9
The kidney, lung, and liver lysates were analyzed to deter-
mine caspase 8 and 9 levels with a commercial kit (Catalog
No: APT171, Lot No: 2829013; Cat. No: APT173, Lot No:
2841705, respectively; MilliporeSigma, Burlington, MA, USA).
The p-nitroaniline absorbance in non-apoptotic specimens
and apoptotic specimens was compared and the caspase 8
and 9 activity was calculated as fold increase.
Histological Evaluation
The samples obtained from kidney, liver, and lung tissues were
fixed in 10% neutral buered formalin for 48 hours and then
examined with routine histological processing. Approximately
4 µm-thick paran sections were stained with hematoxylin
and eosin. Periodic acid-Schi staining was applied to assess
the basal membrane and proximal tubules of kidney samples.
The sections were examined and photographed using a light
microscope (BX51; Olympus Corp., Tokyo, Japan) attached to
a digital camera (DP72; Olympus Corp., Tokyo, Japan). Histolo-
gists evaluated the glomerular structure and Bowman’s capsule,
proximal and distal tubules, interstitial bleeding, and vascular
congestion in kidney tissue; damaged hepatocytes with vac-
uoles and pyknotic nuclei, sinusoidal congestion, and increase
of activated Kuper cells in liver tissue; and alveolar morphol-
ogy, interstitial bleeding, and vascular congestion in lung tissue.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 5.0
(GraphPad Software, Inc. La Jolla, CA, USA). All data are
expressed as mean±SEM. Relationships within groups were
measured using one-way analysis of variance followed by
Tukey’s post hoc test. P<0.05 was considered statistically sig-
nificant. The odds ratio (OR) was calculated based on a chi-
square test for survival rate.
RE SULTS
Survival Rate
The 24-hour survival rate was 75% (6/8 rats) in the saline-
treated CLP group, whereas the survival rate was 100% for
the other groups. There was no statistically significant dier-
ence in survival between the dapagliflozin-treated CLP group
and the saline-treated CLP group (OR: 0.75, 95% confidence
interval [CI]: 0.50–1.12; p>0.05).
Blood Urea Nitrogen, Creatinine and Glucose Levels
The CLP surgery groups developed kidney dysfunction and
had higher plasma BUN (p<0.01) and creatinine (p<0.01–
0.001) levels in comparison with the saline-treated sham
group (Fig. 2).
The glucose level was significantly higher in the dapagliflozin-
treated sham group compared with the saline-treated sham
group (p<0.05). A significant decrease was observed in the
saline-treated CLP group when it was compared with the
saline-treated sham group (p<0.01) (Fig. 2c).
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3 215
Figure 2. The level of blood urea nitrogen (BUN) and creatinine was increased, and the blood glucose level was decreased in the cecal lig-
ation and puncture (CLP) groups, but dapagliozin use did not demonstrate a change in the level of these parameters. (a) Serum BUN level
after dapagliozin treatment. (b) Serum creatinine level after dapagliozin treatment. (c) Body glucose level after dapagliozin treatment.
The data are presented as the mean±SEM. One-way analysis of variance and post hoc Tukey-Kramer multiple comparison tests were
used. *p<0.05, **p<0.01, ***p<0.001 vs saline-treated sham group. Each group consisted of 6–8 samples.
150 300
Saline Saline
Dapagliozin Dapagliozin
BUN (mg/dl)
Glucose (mg/dl)
Control ControlCLP CLP
***
**
*
***
100
50 100
200
0 0
1.0 Saline
Dapagliozin
Creatinine (mg/dl)
Control CLP
***
**
0.8
0.4
0.2
0.6
0.0
(a) (b) (c)

Malondialdehyde and Glutathione Levels
The MDA level in all tissues was significantly elevated in the
saline-treated CLP group when compared with the saline-
treated sham group (p<0.05–0.01; Fig. 3). The MDA level in
the dapagliflozin-treated CLP group was significantly lower in
all tissues than that of the saline-treated CLP group (p<0.05–
0.01; Fig. 3). Interestingly, the MDA level in the dapagliflozin-
treated sham group was significantly higher in kidney tissue
than that of the saline-treated sham group (p<0.05) (Fig. 3a).
The GSH level was significantly lower in the kidney and he-
patic tissue of the saline-treated CLP group when compared
with the saline-treated sham group (p<0.050–001; Fig. 3b and
f), while an increase in GSH in kidney tissue was observed
with administration of dapagliflozin (p<0.001, Fig. 3).
Myeloperoxidase, Tumor Necrosis Factor Alpha,
and Interleukin 1 Beta Levels
MPO activity was found to be significantly high in all of the tis-
sue samples of the saline-treated CLP group when compared
with the saline-treated sham group (p<0.01–0.001; Fig. 4).
CLP-induced elevation in MPO activity were only significantly
decreased in renal tissue in the dapagliflozin-treatment group
(p<0.05; Fig. 4).
TNF-α and IL-1β levels demonstrated a statistically signifi-
cant increase in the saline-treated CLP group when compared
with the saline-treated sham group in lung and liver tissues
(p<0.05–0.001). Administration of dapagliflozin significantly
diminished these alterations in the saline-treated CLP group
(p<0.01–0.001; Fig. 4).
Caspase 8 and 9
In the saline-treated CLP group, caspase-8 and 9 activity in
kidney, lung, and liver tissues was significantly greater when
compared with the saline-treated sham group (p<0.01–
0.001; Fig. 5). Dapagliflozin administration did not alleviate
CLP-induced apoptosis and there was a significant increase
(p<0.001) in caspase-8 activity in kidney tissue samples when
compared with the saline-treated CLP group.
Histological Evaluation
Regular morphology of the interstitial space, Bowman’s space
and glomeruli, proximal and distal tubules were seen in kidney
tissues obtained from the saline-treated sham and dapaglifloz-
in-treated sham groups (Fig. 6a, b). In the saline-treated CLP
group, interstitial bleeding, glomerular congestion, significant
dilation of Bowman’s space, and tubular degeneration were ob-
served (Fig. 6c). Regression in the dilation of Bowman’s space,
tubular degeneration, moderate glomerular congestion, mild
interstitial bleeding, and vascular congestion were observed in
the dapagliflozin-treated CLP group (Fig. 6d).
Regular parenchyma morphology was also seen in liver tis-
sue collected from the saline and dapagliflozin treated sham
groups (Fig. 7a, b). In the saline-treated CLP group, signifi-
cant sinusoidal congestion, degenerated hepatocytes, and an
increased number of activated Kuper cells were observed
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3216
Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
Figure 3. Dapagliozin reduced the level of malondialdehyde (MDA) in kidney, lung and liver tissue, and increased the level of glutathione
(GSH) in kidney tissue. (a) Kidney MDA level after dapagliozin treatment. (b) Kidney GSH level after dapagliozin treatment. (c) Lung
MDA level after dapagliozin treatment. (d) Lung GSH level after dapagliozin treatment. (e) Liver MDA level after dapagliozin treatment.
(f) Liver GSH level after dapagliozin treatment. The data are presented as the mean±SEM. One-way analysis of variance and post hoc
Tukey-Kramer multiple comparison tests were used. *p<0.05, **p<0.01, ***p<0.001 vs saline-treated sham group. Each group consisted
of 6–8 samples.
40
5
40
2.0
Dapagliozin
MDA (nmol/g tissue)GSH (mol/g tissue)
GSH (mol/g tissue) MDA (nmol/g tissue)
Control
Control
Control
Control
Kidney Lung
CLP
CLP
CLP
CLP
*
*
*
***
**
**
++
+++
+
30
4
30
1.5
20
3
2
20
1.0
10
1
10
0.5
0
0
0
0.0
Saline
(a)
(b)
(c)
(d)
40
2.0
MDA (nmol/g tissue)MDA (nmol/g tissue)
Control
Control
Liver
CLP
CLP
*
*** ***
***
+
30
1.5
20
1.0
10
0.5
0
0.0
(e)
(f)

(Fig. 7c). Sinusoidal congestion, and degenerated hepatocytes
and activated Kuper cells were reduced in the dapagliflozin-
treated CLP group (Fig. 7d).
Regular parenchyma morphology was viewed in lung tissue ob-
tained from the saline-treated sham and dapagliflozin-treated
sham groups (Fig. 8a, b). Severe interstitial bleed-ing and vas-
cular congestion, cellular debris in the alveolar lu-men, and
degenerated alveolar structures were observed in the saline-
treated CLP group (Fig. 8c). Moderate interstitial bleeding and
vascular congestion, partial degeneration of alveolar struc-
tures, cellular debris in the lumen of a number of alveoli, and
in some regions, alveoli with regular morphology were seen in
the dapagliflozin-treated CLP group (Fig. 8d).
DISCUSSION
Dapagliflozin reduced oxidative stress (MDA) and inflamma-
tion (MPO), but conversely, increased the level of antioxidants
(GSH) in the kidney. Recovery of histological features of renal
injury was demonstrated. In addition, dapagliflozin treatment
decreased oxidative stress (MDA) and inflammation (TNF-α)
in lung tissue. A slight recovery in the histological features of
lung injury was noted. Additionally, dapagliflozin treatment
lowered inflammation (TNF-α, IL-1β), but there was only a
limited decrease in the level of oxidative stress in the liver. A
slight recovery in the histological features of liver injury was
observed.
Immune system function declines with diabetes mellitus. This
altered immune response can lead to the progression toward
sepsis through the growth of microorganisms. In experimental
animal models and diabetic human studies, deficiencies in im-
munoreactivity have been shown to increase susceptibility to
sepsis and other infections. In patients with Type 1 diabetes,
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3 217
Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
Figure 4. Dapagliozin reduced the level of myeloperoxidase (MPO) only in kidney tissue, and decreased the level of tumor necrosis fac-
tor alpha (TNF-α) in lung and liver tissue and the level of interleukin 1beta (IL-1β) in liver tissue. (a) Kidney MPO level after dapagliozin
treatment. (b) Kidney TNF-α level after dapagliozin treatment. (c) Kidney IL-1β level after dapagliozin treatment. (d) Lung MPO level
after dapagliozin treatment. (e) Lung TNF-α level after dapagliozin treatment. (f) Lung IL-1β level after dapagliozin treatment. (g) Liver
MPO level after dapagliozin treatment. (h) Liver TNF-α level after dapagliozin treatment. (i) Liver IL-1β level after dapagliozin treatment.
The data are presented as the mean±SEM. One-way analysis of variance and post hoc Tukey-Kramer multiple comparison tests were
used. *p<0.05, **p<0.01, ***p<0.001 vs saline-treated sham group. Each group consisted of 6–8 samples.
2.0
0.8
2.5
2.0
1.5
1.0
0.5
1.5
0.6
MPO (U/g tissue)
TNFα (ng/ml)
IL-1β (ng/ml)
Control
Control
Control
Lung
CLP
CLP
CLP
***
*
**
**
*
1.0
0.4
0.5
0.2
0.0
0.0
0.0
(d)
(e)
(f)
+++
50
0.8
2.5
1.5
0.5
1.0
0.6
2.0
0.4
0.2
40 Dapagliozin
MPO (U/g tissue)
TNFα (ng/ml)
IL-1β (ng/ml)
Control
Control
Control
Kidney
CLP
CLP
CLP
**
+
30
20
10
0
0.0
0.0
Saline
(a)
(b)
(c)
15
0.8
2.5
0.6
2.0
0.4
1.5
0.2
1.0
0.5
10
MPO (U/g tissue)
TNFα (ng/ml)
IL-1β (ng/ml)
Control
Control
Control
Lung
CLP
CLP
CLP
*** **
***
***
***
5
0.0
0.0
0.0
(g)
(h)
(i)
+
+++

neutrophil function (chemotaxis, phagocytosis, and cell death),
reactive oxygen species production, bacteremia, and sepsis
may occur as a result of the impairment in bacterial control
at the infection site. The prognosis of patients with sepsis is
more pronounced in patients with type 2 diabetes compared
with type 1 diabetes. The mortality rate is significantly high in
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3218
Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
Figure 5. Dapagliozin increased the level of caspase 8 only in kidney tissue. No changes were observed in the level of caspase 9. (a)
Kidney caspase 8 level after dapagliozin treatment. (b) Kidney caspase 9 level after dapagliozin treatment. (c) Lung caspase 8 level after
dapagliozin treatment. (d) Lung caspase 9 level after dapagliozin treatment. (e) Liver caspase 8 level after dapagliozin treatment. (f)
Liver caspase 9 level after dapagliozin treatment. The data are presented as the mean±SEM. One-way analysis of variance and post hoc
Tukey-Kramer multiple comparison tests were used. *p<0.05, **p<0.01, ***p<0.001 vs saline-treated sham group. Each group consisted
of 6–8 samples.
2.5 2.0
2.5
Caspase 8
(fold increase)
Caspase 8
(fold increase)
Caspase 8
(fold increase)
Caspase 8
(fold increase)
Control
Control
Control
Control
Kidney Lung
CLP
CLP
CLP
CLP
***
**
***
**
**
***
***
**
***
+
2.0
2.0
1.5
1.5
1.5
1.5
2.0
1.0
1.0
1.0
1.0
0.5
0.5
0.5
0.5
0.0
0.0
0.0
0.0
Dapagliozin
Saline
(a)
(b)
(c)
(d)
2.0
2.5
2.0
Caspase 8
(fold increase)
Caspase 8
(fold increase)
Control
Control
Liver
CLP
CLP
***
***
***
***
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
(e)
(f)
(a) (b) (c) (d)
Figure 6. Representative photomicrographs of kidney tissue in the experimental groups. Regular kidney morphology in saline-treated
sham control (a) and dapagliozin-treated sham control (b) groups. Interstitial bleeding (arrowhead) and glomerular congestion, dilation
of Bowman’s space (*), degenerated tubules (arrow) seen in saline-treated cecal ligation and puncture (CLP) group (c) and Bowman’s
capsule with regular morphology (*), mild glomerular congestion (*, upper inset), vascular congestion (arrowhead), and a few damaged
tubules (arrow) in the dapagliozin-treated CLP group (d). H&E staining, A and B insets, C and D below insets: PAS staining, scale bars:
50 µm (x20), inset: 20 µm (x40).
Figure 7. Representative photomicrographs of liver tissue in the experimental groups. Regular liver parenchyma in saline-treated sham
control (a) and dapagliozin-treated sham control (b) groups with severe sinusoidal dilation and congestion (*), numerous damaged hep-
atocytes (arrow) and activated Kupffer cells (arrowhead), and in the saline-treated cecal ligation and puncture (CLP) group (c), mild sinu-
soidal congestion (*), a few damaged hepatocytes (arrow), and activated Kupffer cells (arrowhead) in the dapagliozin-treated CLP group
(d). H&E staining, scale bar: 20 µm (x40).
(a) (b) (c) (d)

sepsis patients and sepsis can be a common consequence in
diabetic patients. Therefore, new treatments are needed.[21]
Dapagliflozin, a new drug used in the treatment of diabetes,
is an inhibitor of SGLT2.[9] A review of the literature did not
reveal any studies of dapagliflozin and sepsis as yet. However,
consistent with the results of the present study, it has been
reported that dapagliflozin (10 mg/kg/day) decreased BUN
and creatinin levels in a renal ischemic reperfusion model in
rats and had a protective eect on renal tubular cells.[9] In ad-
dition, it has been shown that dapagliflozin reduced apoptotic
cell death by inducing hypoxia inducible factor 1 in ischemic
renal tissue and ischemic tubular cell cultures and decreasing
Bax/BcL2 ratio and terminal dUTP nick-end labeling-positive
cells.[9]
Depending on the characteristics of a critical illness (sepsis,
burns, etc.), acute hyperglycemia and insulin resistance can
develop even if there is no history of diabetes mellitus in the
patient. This condition has been assessed as a stress response.
The development of this response may involve the release of
inflammatory cytokines such as TNF-α and IL-6; an increase
in stress hormones, such as cortisol; and the use of drugs
such as corticosteroids. In the case of sepsis or septic shock,
hyperglycemia occurs at an early stage and hypoglycemia has
a late stage development.[22,23] In our study, the decrease in
blood glucose levels in the sepsis group when compared with
the control suggests late-stage sepsis. In a study of mice using
a sepsis model (CLP), it was demonstrated that blood glu-
cose levels decreased in the sepsis group and that the glucose
levels increased in the group given pioglitazone, which is also
an anti-diabetic agent.[24] In our study, there was a decrease
in blood glucose levels in the CLP group, consistent with
other research available in the literature.[23–27] In the present
study, although glucose levels were significantly increased in
the dapagliflozin-treated sham group, there was no significant
change in the glucose levels of the dapagliflozin-treated CLP
group. Therefore, the positive findings of the current study
do not suggest that dapagliflozin makes a strong contribution
to glucose control.
In CLP studies performed on rats, serum TNF-α, IL-6, BUN,
and creatinine levels were higher in the sepsis group com-
pared with the control group. Increases in MDA and MPO
values, a decrease in GSH values, and an increase in apopto-
sis were observed in the kidney, and histological examination
showed renal tissue damage.[28–34] Similar findings were ob-
tained in the sepsis model applied in our study. It was deter-
mined that there was no significant dierence in the serum
creatinine value between the groups, but a significant increase
was noted in the CLP group after 72 hours. In addition, oxida-
tive stress parameters and apoptotic (caspase 3) values were
greater in the CLP group compared with the controls and
more tissue damage was determined in the kidney and lung
tissue in the histological examination.[35] Serum BUN and cre-
atinine values in our study were consistent with the findings
of sepsis (CLP method) in the literature.[8,35] In another study,
TNF-α, IL-1β, IL-6, MDA, and MPO levels of lung tissue were
greater in the sepsis group compared with the control group.
GSH levels were lower.[28,36–40] In the histopathological evalua-
tion, severe tissue damage was reported.[38] These results are
also consistent with the findings we obtained in our sepsis
model. It has also been reported that there were increases in
the levels of caspase 3 and Bax/BcL 2 in the lungs.[40]
The MDA level in liver tissue was elevated, whereas the GSH
level decreased in the sepsis group compared with the con-
trols.[31,39] These results are consistent with our sepsis model.
In another study, rats were evaluated 24 hours after CLP, and
when the sepsis group was compared with the control group,
the MDA level was significantly higher in the liver tissue, but
there was no increase in the kidney or lung tissues. MPO
values increased in the lung; however, there was no significant
dierence in the liver or kidney tissues. In the same study,
plasma cytokines (TNF-α, IL-1β, etc.), BUN, and creatinine
levels were greater in the CLP group, and there was no sig-
nificant decline in glucose levels.[25] Other research indicated
that there was an increase in kidney MPO values as well as
MDA values in kidney and liver tissue, but there was no sig-
nificant dierent in liver MPO level.[41]
The eect of long-term use of dapagliflozin on glucose home-
ostasis and diabetic nephropathy has previously been inves-
tigated. Dapagliflozin has improved hyperglycemia and albu-
minuria, depending on the dose (0.1–1.0 mg/kg), and reduced
macrophage infiltration, gene expression of inflammatory
cytokines, and oxidative stress in diabetic mice. There was
Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3 219
Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
Figure 8. Representative photomicrographs of lung tissue in the experimental groups. Lung parenchyma with regular morphology in the
saline-treated sham (a) and dapagliozin-treated sham (b) groups and severe interstitial bleeding (*) and vascular congestion, degener-
ation in alveolar structure (arrow) in the saline-treated cecal ligation and puncture (CLP) group (c) with mild interstitial bleeding (*) and
vascular congestion, partly degeneration of alveolar structure (arrow), and regular alveolar morphology (arrow, inset) in the dapagliozin-
treated CLP group in some places (d). H&E staining, scale bars: 50 µm (x20).
(a) (b) (c) (d)

Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3220
Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats
no significant dierence in BUN or serum creatinine levels.[8]
These results are consistent with those of our study. Other
researchers found that dapagliflozin reduced oxidative stress
and apoptosis induced by high glucose in type 1 diabetic mice,
alleviated diabetic nephropathy, and reduced macrophage in-
filtration.[42] In another study, the eect of SGLT inhibition
on polycystic kidney dysfunction was investigated in rats. Da-
pagliflozin (10 mg/kg) was found to unexpectedly cause an
increase in cyst volume with albuminuria, hyperfiltration, and
polycystic kidney failure in rats.[43]
Additional studies should be conducted to develop new al-
ternatives for prophylactic treatment of sepsis and to investi-
gate the dierent eects of this new medicine, dapagliflozin.
The results of our study suggest that diabetic patients using
dapagliflozin may also benefit from the eect on oxidative
stress. As a result, the findings of this study highlighted a pos-
sible protective eect of dapagliflozin in renal damage. Da-
pagliflozin also slightly alleviated lung and liver injury.
Acknowledgement
This work was supported by Marmara University Scientific
Research Projects Committee (SAG-C-YLP-090217-0040),
İstanbul, Turkey.
Conflict of interest: None declared.
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Ulus Travma Acil Cerrahi Derg, May 2019, Vol. 25, No. 3 221
OLGU SUNUMU
Dapaglozn’n sıçanlarda deneysel sepss model üzerne etkler
Zehra Betül Kıngır,1 Dr. Zarife Nigar Özdemir-Kumral,2 Dr. Muhammet Emin Çam,3
Dr. Özlem Tuğçe Çilingir,4 Turgut Şekerler,5 Dr. Feriha Ercan,4 Dr. Özlem Bingol-Özakpınar,5
Dr. Derya Özsavcı,5 Dr. Mesut Sancar,1 Dr. Betül Okuyan1
1Marmara Üniversitesi Eczacılık Fakültesi, Klinik Eczacılık Anabilim Dalı, İstanbul
2Marmara Üniversitesi Tıp Fakültesi, Fizyoloji Anabilim Dalı, İstanbul
3Marmara Üniversitesi Eczacılık Fakültesi, Farmakoloji Anabilim Dalı, İstanbul
4Marmara Üniversitesi Tıp Fakültesi, Histoloji ve Embriyoloji Anabilim Dalı, İstanbul
5Marmara Üniversitesi Eczacılık Fakültesi, Biyokimya Anabilim Dalı, İstanbul
AMAÇ: Bu çalışmada, sıçanlarda dapagiflozinin deneysel sepsis modeli üzerindeki olası koruyucu etkilerinin değerlendirilmesi amaçlandı.
GEREÇ VE YÖNTEM: Sprague-Dawley sıçanlara cerrahi operasyonların 5 gün öncesinde serum fizyolojik (1 mL/kg, p.o.) veya dapagliflozin (10
mg/kg, p.o.) verilmeye başlandı. Sepsis, anestezi altında, çekal ligasyon ve perforasyon modeli ile oluşturulurken, sham kontrol gruplarına sadece
laparotomi yapıldı. Serumda BUN, kreatinin ve glukoz düzeyleri; dokularda (böbrek, karaciğer ve akciğer) ise MDA, GSH, MPO, TNF-α, IL-1β,
kaspaz 8 ve kaspaz 9 düzeyleri belirlendi. Bu dokularda histolojik değerlendirme de yapıldı.
BULGULAR: Sepsiste dapagliflozin uygulaması, böbrek dokularında oksidatif stresi (MDA) azalttı, antioksidan düzeyleri (GSH) arttırdı ve enflamas-
yonu (MPO) azalttı (p<0.05). Sepsiste dapagliflozin uygulaması akciğer dokularında oksidatif stresi (MDA) azalttı, akciğer ve karaciğer dokularında
ise enflamasyonu (MPO) azalttı (p<0.05). Ayrıca, böbrek, akciğer ve karaciğer dokularında kaspaz 8 ve 9 düzeylerini arttırdı (p<0.05). Histopatolo-
jik sonuçlara göre, dapagliflozin uygulaması böbrek dokularında orta derece; akciğer ve karaciğer dokularında ise hafif iyileştirdi.
TARTIŞMA: Bu çalışmada, dapagliflozinin deneysel sepsis modelinde böbrek hasarını önleyici etkisinin olduğu gösterilmesine rağmen akciğer ve
karaciğer dokuları üzerine hafif koruyucu etkisi olduğu bulunmuştur.
Anahtar sözcükler: Apoptoz; dapagliflozin; enflamasyon; oksidatif stres; sepsis.
Ulus Travma Acil Cerrahi Derg 2019;25(3):213-221 doi: 10.5505/tjtes.2018.82826
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Kıngır et al. Eects of dapagliflozin in experimental sepsis model in rats