Evaluation of the effects of empagliflozin on acute lung injury in rat intestinal ischemia–reperfusion model

Empagliflozin is a selective sodium–glucose co-transporter (SGLT2) inhibitor that is approved for the treatment of type 2 diabetes. The beneficial effects of empagliflozin on other organ systems including the heart and kidneys have been proven. The aim of this study is to evaluate the role of empagliflozin on acute lung injury induced by intestinal ischemia–reperfusion (I/R). A total of 27 male Wistar albino rats were divided into three groups: sham, I/R, and I/R + empagliflozin; each group containing nine animals. Sham group rats underwent laparotomy without I/R injury. Rats in the I/R group underwent laparotomy, 1 h of after ischemia–reperfusion injury (superior mesenteric artery ligation was followed by 2 h of reperfusion). Rats in I/R were given empagliflozin (30 mg/kg) by gastric gavage for 7 days before the ischemia–reperfusion injury. All animals were killed at the end of reperfusion and lung tissue samples were obtained for immunohistochemical staining and histopathological investigation in all groups. Serum glucose, AST, ALT, creatinine, native thiol, total thiol, and disulfide levels and disulfide–native thiol, disulfide–total thiol, and native thiol–total thiol ratios as well as the IMA levels were analyzed and compared among the groups. While intestinal I/R significantly increases serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine levels; did not cause any change in homeostasis parameters and IMA level. Empagliflozin treatment had no significant effect on biochemical parameters. Empagliflozin treatment induced a significant decrease in positive immunostaining for IL-1, IL-6, TNF-alpha, caspase 3, caspase 8, and caspase 9 compared to the I/R group in lung tissue samples. Intestinal I/R caused severe histopathological injury including edema, hemorrhage, increased thickness of the alveolar wall, and infiltration of inflammatory cells into alveolar spaces. Empagliflozin treatment significantly attenuated the severity of intestinal I/R injury. It was concluded that empagliflozin treatment may have beneficial effects in acute lung injury, and, therefore, has the potential for clinical use.


Introduction
Intestinal ischemia-reperfusion may occur in conditions such as ischemic colitis, acute mesenteric ischemia, septic shock, hemorrhagic or traumatic shock, small intestine transplantation, and severe burns. Systemic inflammation and multi-organ failure play an important role in the pathogenesis of intestinal I/R [1]. Acute respiratory failure is the most important component of multi-organ dysfunction after intestinal damage, and it is an important cause of morbidity and mortality in critically ill patients [2].
Lung ischemia is a rapid complex sterile inflammatory condition with reperfusion injury, edema, and defective gas exchange resulting from damage to endothelial and epithelial barriers. In I/R injury, there is a strong innate immune response involving alveolar macrophages, natural killer cells, and neutrophils, as well as the release of cytokines and injury-related molecules. The majority of these responses are led by rapidly and strongly produced reactive oxygen products. Gielis and Chatterjee showed that acute oxidative stress products occur in peripheral blood in their lung I/R models in rats [3].
Modified albumin is a sensitive marker for determining oxidative stress after ischemia. The oxidant-antioxidant balance plays a critical role in vascular homeostasis. Accordingly, antioxidant mechanisms that neutralize oxidative free radicals may be critical for cell survival. Thiol/disulfide homeostasis is an important antioxidant in the defense mechanism against reactive oxygen radicals [4].
Programmed cell death, including apoptosis, also plays an important role in lung tissue damage after ischemia-reperfusion. Apoptosis is regulated by enzymes involving the caspase cascade. Caspase 8 and caspase 9 are the initiator of apoptosis; caspase 6 is from the effector caspase family [5,6].
Empagliflozin is a selective sodium-glucose cotransporter (SGLT2) inhibitor that is accepted for the treatment of type 2 diabetes. Recent studies showed the beneficial effects of empagliflozin beyond glycemic control. Empagliflozin reduced the risk of hospitalization for heart failure and cardiovascular deaths in patients with or without type 2 diabetes (T2DM) in large-scale clinical trials. Empagliflozin also has renoprotective effects in chronic kidney disease [7]. The mechanism of action of empagliflozin in these organs is not clear yet. Empagliflozin reduces the production of free oxygen radicals and upregulates antioxidant defense mechanisms. The antioxidant effects of empagliflozin depend on its effects on zinc transporters, matrix metalloproteinase, and oxidative stress. In addition, in animal studies, it has been observed that empagliflozin has an anti-apoptotic effect at the lung tissue level, as well as an antiinflammatory effect by suppressing IL-6 and TNF-alpha expression [8][9][10]. Caspase inhibition may reduce ischemia-reperfusion injury and help improve lung function. Studies have shown that empagliflozin reduces hepatocyte apoptosis by inhibiting caspase 8 expression in the liver; It is not known whether it has a similar effect on lung tissue [5,6,11]. There is no study examining the effect of empagliflozin on apoptosis by affecting caspase 9 and 6 levels in lung tissue.
In this study, we aimed to investigate the effects of empagliflozin on lung tissue and whether it reduces acute lung injury following ischemia-reperfusion (I/R) of the superior mesenteric artery in rats.

Materials and methods
Animals Male albino Wistar albino rats (200-250 g) were used in the present study. All the animals were kept under optimum conditions (21 + 1C, 40-70% humidity, 12/12 dark-light cycle) and were fed ad libitum with a standard pellet diet and water. The experimental protocol was approved by the local ethic committee for Animal Research. The study was achieved in Ankara Training and Research Hospital animal experiment laboratory (Ankara, Turkey) by the National Laboratory Animal Use and Care Guidelines.

Experimental groups
A total of 27 male Wistar albino rats were divided into 3 groups: sham, I/R, and I/R + empagliflozin; each group contains 9 rats. Animals were pretreated with empagliflozin by gastric gavage (in a dose of 30 mg/kg body weight) for 7 days before intestinal I/R as described for I/R + empagliflozin group. In the I/R group, saline solution was given as a placebo by gastric lavage. Rats were weighed daily from the beginning of the experiment.

The technique of intestinal I/R
Feeding of the animals was stopped 12 h before the start of the intestinal I/R procedure and they received only water. The rats were anesthetized with ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (10 mg/kg) intraperitoneally (i.p.) and their temperature was regulated using a lamp light bulb during the test. Intestinal I/R was induced as follows: the rats were placed in the supine position and secured in the dissection tray. The abdominal region was shaved and cleaned with antiseptic solutions. The intestinal region was reached using midline laparotomy. The superior mesenteric artery was subjected with care and occluded with an atraumatic microvascular clamp, thus intestinal ischemia was created in 1 h when the existence of pulseless or pale color of the intestine was recognized. The abdominal region was then closed. Following ischemia, the clamp was removed and 2 h reperfusion was induced. The return of the pulses and the re-establishment of the pink color were assumed to be due to the reperfusion of the intestine. At the end of reperfusion, the rats were killed by drawing blood will be drawn from the right carotid artery. The lungs of the killed rats were taken out and placed in formalin.

Histological analysis
To remove the fixed tissue samples from formalin, they were washed in running water overnight. Then, it was subjected to routine pathological tissue follow-up and passed through graded alcohol (50, 75, 96, 100%) and xylol series and blocked in paraffin. 5 μ thick sections from the prepared blocks were taken on slides with Leica RM 2125 RT microtome (Leica, Biosystems, Nussloch, GmbH, Nussloch, Baden-Württemberg, Germany) the first three sections, and every tenth section. The prepared preparations were passed through alcohol and xylol series and stained with hematoxylin-eosin (HE). All samples were examined under a high-resolution light microscope (Olympus DP-73 camera, Olympus BX53-DIC microscope; Tokyo, Japan). The slides were examined for the presence of peribronchial inflammatory cell infiltration (PICI), alveolar septal infiltration (ASI), alveolar edema (AED), alveolar exudate (AEX), and interstitial fibrosis (IF). These changes were scored according to the 4-point scale used by Takil et al. (2003; Table 1). All the changes detected in tissue structures were noted and graded according to the presence and severity of any particular finding as in some pre-studies: 0: none, 1: mild, 2: moderate, and 3: severe. These changes were scored according to the 4-point scale used by Takil et al. 2003 [12]; Table 1 Immunohistochemical stainings For the immunohistochemical studies, 4 μm-thick sections were obtained from the paraffin-embedded tissue blocks and placed on poly-L-lysine-coated glass slides. They were stained with the streptavidin-biotin-peroxidase complex (ABC) technique after routine deparaffinization and rehydration procedures. Antigen retrieval was performed in a microwave oven with citrate buffer (pH 6.0) (700 W, 20 min). Endogenous peroxidase activation in the tissues was blocked for 15 min with 0.3% hydrogen peroxide in 0.01 mol/l PBS in methanol at room temperature. Before applying the primary antibody, the tissues were incubated for 20 min with 5% normal goat serum for protein blocking. Then, the sections were incubated with caspase-3 (5 μg/mL, PA5-16335; Invitrogen, California, USA), Caspase-8 (ab4052; Abcam, USA), Caspase-9 (ab52298; Abcam, USA), TNF-alpha (1:50, ab6671; Abcam, Cambridge, USA), IL-1β (1:200, sc-52012, Santa Cruz Bıotechnology, Inc. Texas, USA) and IL-6 (10 μg/mL, RPA079Ga01-Recombinant Interleukin 6; Cloud-Clone Corp. (CCC, USA)) primer antibodies for 1 h at room temperature. Sections were then reacted with a biotinylated secondary antibody for 30 min after removing the unbound primary antibody. Then, the sections were reacted with horseradish peroxidase streptavidin for 30 min after washing with PBS, the sections were treated and incubated with DAB (3,3'-Diaminobenzidine, Dako, Glostrup, Denmark) for 5 min. Finally, the background of the tissue sections was stained with hematoxylin. For negative controls, PBS was used instead of the primary antibody. All staining steps were carried out at 37 °C and in humidity cabinets. PBS solution was used as a wash-away solution during all the staining steps. Staining indexes were calculated as follows on the basis of the percentages of the stained nuclei for these three markers; negative: 0 (< 1% positive); weak: 1 (1-25% positive); moderate: 2 (> 25-75% positive); and strong: 3 (> 75% positive).

Laboratory analysis
The samples were centrifuged at 3000 rpm for 10 min to separate the plasma and serum. The serum was immediately frozen and stored at 80 C until the analysis was performed for the Thiol/Disulfide homeostasis and IMA. Serum levels of native and total thiol and the ratio of disulfide to native and total thiol were measured using a simple novel fully automated colorimetric method in the same manner as the method developed by Erel and Neselioglu [13]. In this method, the reducible dynamic disulfide bonds (-S-S-) were reduced to functional thiol groups (-SH) using NaBH4. Following this procedure, the residual NaBH4 materials were entirely removed from the biochemical environment with formaldehyde. Ellman's reagent was used to measure the amount of total thiol in the sample. The dynamic disulfide content was calculated by taking half of the difference between the amount of total thiol and native thiol. In this way, the native thiol, total thiol, and disulfide levels were measured, and the percentage of disulfide-native thiol, disulfide-total thiol, and native thiol-total thiol were calculated in all subjects. Serum IMA levels were measured using the colorimetric assay method previously described by Bar-Or et al. [14]. This colorimetric method is based on the biochemical properties of albumin to bind exogenous cobalt. In brief, 200 lL of a subject serum was added to 50 lL of 0.1% cobalt II chloride (CoCl2, 6H2O) (Sigma-Aldrich Chemie GmbH Riedstrasse 2, Steinheim, Germany) followed by mixing and 10 min of incubation in the dark at 37 C to allow for cobalt albumin cobalt binding. Then, a total of 50 lL dithiothreitol (DTT) were added as a coloring agent. After 2 min of incubation, 1 mL of 0.9 sodium chloride was added to reduce the binding capacity. The blank was prepared similarly with distilled water instead of DTT. The absorbance of samples was measured at 470 nm using a spectrophotometer (Jenway 6315 UV/visible Scanning Spectrophotometers, United Kingdom). IMA results were expressed in absorbance units (ABSUs). Each sample was measured in duplicate and the mean value was reported.

Statistical analysis
The Shapiro-Wilk test was used to understand that the distribution was normal. Normally distributed data were reported using mean ± standard deviation (SD) and non-normally distributed data using descriptive statistical methods such as median (min-max). χ 2 or Fisher's exact test was used to compare categorical variables. A comparison of continuous variables was performed using one-way ANOVA for normally distributed data and Kruskal Wallis test for nonnormally distributed data. The differences between the continuous variables were evaluated by a two-tailed t test. All statistical analyses were done using SPSS, v.26 program. p < 0.05 value was considered statistically significant.

Biochemical findings
All rats survived until the end of the study period. Demographics and laboratory characteristics of participants and statistical differences of these variables are shown in Table 2. There was no significant difference between the groups in terms of first and final weights and serum glucose levels of the rats (p = 0.84, p = 0.23, p = 0.11, respectively).
Intestinal I/R significantly increased the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine levels (p < 0.001, p < 0.001, p < 0.009, respectively) when compared to the sham group. There was no statistically significant difference between the I/R + empagliflozin group and the I/R group in terms of these laboratory variables (p = 0.89, p = 0.54, p = 0.06). Comparison of thiol/ disulfide homeostasis parameters and IMA levels among the groups were similar in all groups (p > 0.05). Data are shown in Table 3.

Histopathologic findings
The histopathological examinations of the study groups stained with hematoxylin and eosin are shown in Fig. 1.
Intestinal I/R resulted in the characteristic features of pulmonary injury, whereas rats in the sham group showed normal lung architecture (Fig. 1a). In the intestinal ischemia-reperfusion group, severe edema in the lung tissue, alveolar hemorrhage, an increase in alveolar wall thickness, many inflammatory cells infiltrating the interstitium and alveoli, and fibrosis were observed (Fig. 1b). The pathological damage was apparently less in the empagliflozin pretreatment group in comparison to the I/R group. The interstitium of the lungs appeared thinner and the number of inflammatory cells and fibrosis is apparently reduced (Fig. 1c). Histopathological results of study groups are presented in Table 4. Histopathological parameters including PICI, ASI, AED, AEX and IF were decreased significantly in all treated intestinal I/R with empagliflozin groups compared to I/R groups (p < 0.001).

Immunohistochemical findings
Intestinal ischemia-reperfusion induced a significant increase in positive immunostaining for IL-1, IL-6, TNFalpha, caspase-3, caspase-8, caspase-9 compared to the control group (Fig. 2), Immunohistochemical findings were significantly improved in the empagliflozin I/R group when compared to the I/R group (Fig. 2). A comparison of immunohistochemical staining of lung tissue between groups is given in Table 5. There was a statistically significant difference between the groups in terms of immunohistochemical staining of IL-1, IL-6, TNF-alpha, caspase 3, caspase 8, and caspase 9 in the lung tissue (p < 0.001). The number of immunohistochemically stained cells was significantly higher in the I/R group compared to the sham group (p < 0.001). There was a statistically significant decrease in the number of cells stained for IL-1, IL-6, TNF-alpha, caspase 3, caspase 8, and caspase 9 in I/R empagliflozin compared to the I/R group (p < 0.001). Immunohistochemical findings were similar between the sham group and the I/R empagliflozin group.

Discussion
Acute respiratory failure is the most important component of multi-organ dysfunction (MODS) after intestinal damage and is an important cause of morbidity and mortality in critically ill patients. Intestinal ischemia-reperfusion causes a widespread systemic inflammatory response, resulting in MODS with subsequent acute lung injury. Disturbance in the intestinal epithelial barrier following intestinal ischemia causes activation of pro-inflammatory cytokines and circulating leukocytes [2]. TNF-alpha, reactive oxygen substrate(ROS), and IL-6 are present in tissue damage that occurs during ischemia-reperfusion, these toxic molecules cause changes in the structure of cellular proteins, lipids, and ribonucleic acids that cause cell dysfunction or death [5]. Tissue hypoperfusion secondary to ischemia results in an increase in adhesion molecules on the endothelial cell surface in addition to these changes. The interaction between activated leukocytes and endothelial cells causes the migration of leukocytes and the production of proteases and ROS. Failure to control this inflammatory response causes tissue damage [2]. In our study, there were significant elevations of AST, ALT, and creatinine due to damage to the liver and kidney tissue in rats treated with I/R. Reperfusion following intestinal ischemia is associated with acute lung injury characterized by increased microvascular permeability, histological evidence of alveolar-capillary endothelial cell damage, and lung deposition of neutrophils. Typical histological features in the I/R group were edema, hemorrhage, increased alveolar wall thickness, and inflammatory cell infiltration in the alveolar spaces of the lung tissue.
In clinical and experimental studies, it has been shown that there is a rapid release of pro-inflammatory cytokines in ischemia-reperfusion of solid organs such as the lung. TNF-alpha, ROS, and IL-6 are present in tissue damage that occurs during ischemia-reperfusion, these toxic molecules cause changes in the structure of cellular proteins, lipids, and ribonucleic acids that cause cell dysfunction or death [15].
Free radicals formed during ischemia-reperfusion are very important. Apoptosis occurs through two pathways, the mitochondrial-dependent intrinsic pathway activated by ROS, and the extrinsic pathway linked to inflammatory molecules such as TNF-alpha. The intrinsic pathway is activated in the early phase of reperfusion; the extrinsic pathway is activated a few hours after reperfusion. Both pathways accelerate the activation of caspases and proteases responsible for the clearance of specific cellular substrates that cause cell death [5]. Using an experimental lung ischemia-reperfusion model, Forgiarini et al. showed that there are a large number of apoptotic cells with increased caspase 3 activity after ischemia [16]. It has been shown that caspase 3, 8, and 9 activities are increased in lung tissue samples in programmed cell damage after ischemia-reperfusion injury in lung transplantation [6]. In our study, significantly increased IL-1, IL-6, TNF-alpha staining, and diffuse caspase 3,8, and 9 activity were observed in the lung tissue after I/R. Many treatment options have been tried to prevent or minimize cell death that occurs during ischemia-reperfusion. Sodium-glucose co-transporter 2 (SGLT2) inhibitors-, have been shown to have antiinflammatory, antioxidant, and antifibrotic properties in cardiovascular diseases and renal damage. In lung infections, it has been shown to effectively reduce Pseudomonas infection and increase antibiotic effectiveness in diabetic rats. Therefore, the idea that SGLT2 inhibitors may show potential benefits in lung diseases has arisen. In the study of Lina et al. canagliflozin successfully reduced inflammatory cell infiltration, congestion, and edema in the lung [17]. Kıngır et al. reported that dapagliflozin provided mild histological improvement Table 4 Histopathological evaluation of pulmonary tissue between groups Data without normal distribution (PICI, ASI, AED, AEX, IF) were presented as median (min-max). A comparison of continuous variables was performed using the Kruskal-Wallis test for non-normally distributed data PICI peribronchial inflammatory cell infiltration, ASI Alveolar septal infiltration, AED alveolar edema, AEX alveolar exudate, IF ınterstitial fibrosis, I/R ıschemia-reperfusion, I/R + Empa ischemia-reperfusion + empagliflozin a Significance comparison between sham and I/R group b Significance comparison between sham and I/R + Empagliflozin group c Significance comparison between I/R group and I/R + Empagliflozin group Sham (n = 9) I/R group (n = 9) I/R + Empa group (n = 9) p PICI 0.00 (0.00-0.00) 3.00 (3.00-3.00) 0.00 (0.00-1.00) < 0.001 < 0.001 a ,0.53 b , < 0.001 c ASI 0.00 (0.00-0.00) 3.00 (2.00-3.00) 0.00 (0.00-1.00) < 0.001 < 0.001 a ,0.54 b , < 0.001 c AED 0.00 (0.00-0.00) 2.00 (2.00-3.00) 0.00 (0.00-1.00) < 0.001 < 0.001 a ,0.75 b , < 0.001 c AEX 0.00 (0.00-1.00) 3.00 (2.00-3.00) 0.00 (0.00-1.00) < 0.001 < 0.001 a ,0.77 b , < 0.001 c IF 0.00 (0.00-0.00) 3.00 (2.00-3.00) 0.00 (0.00-1.00) < 0.001 < 0.001 a ,0.75 b , < 0.001 c by reducing oxidative stress and inflammation (TNF-α) in lung tissue [18].
There are few studies in the literature on the pulmonary protective effect of empagliflozin. Ojima et al. (2015) also reported that empagliflozin exerts its antiinflammatory and antifibrotic effects by inhibiting pro-inflammatory cytokine expression and by suppressing advanced glycosylation products and receptor axis [8]. In the study of Hess et al. it was shown that the number of pro-angiogenic CD133 + progenitor cells in the circulation increased, pro-inflammatory granulocyte precursors decreased, and the antiinflammatory M2 polarization of monocytes increased with 6-month  [8,21]. In our study, we observed that the lung damage developed after I/R in rats pretreated with empagliflozin was significantly milder compared to the placebo group that was not given empagliflozin. In the immunohistochemical staining of the lungs of rats given empagliflozin, it was shown that the number of cells showing inflammatory cytokines such as IL-1, IL-6, and TNF-alpha and caspase 3, 8, and 9 activity was lower. The relationship between lung injury due to ischemia-reperfusion injury and oxidative stress was investigated in our study. No significant decrease in thiol-related antioxidant capacity  and an increase in the level of ischemia-modified albumin (IMA), the oxidant marker was observed. The absence of the expected change led to the assumption that the main change after ischemia-reperfusion occurred in the precursor molecules that we could not measure, or that a different oxidant-antioxidant system was effective. Empagliflozin may have exerted its suppressive effect on inflammation and apoptosis in acute lung injury through the inhibition of sodium-hydrogen exchange at the cellular level which has been proven as a cardiorenal protective mechanism in previous studies. Na + /H exchangers (NHE) are integral membrane ion transporters and play a very important role in intracellular pH homeostasis. The expression pattern of NHE in lung tissue and its role in alveolar fluid homeostasis is unknown. It has been previously shown that NHE mutation in the airway epithelium is a risk factor for airway infection in patients with cystic fibrosis, and loss of NHE function may lead to impaired mucociliary clearance [22]. While NHE, a transmembrane protein, has many isoforms, NHE1 and NHE2 were found to be expressed in all lung tissue samples; There is also evidence that NHE3s are regulatory in the respiratory system [23]. In addition to NHE1, NHE5 and NHE8 are also expressed in pulmonary vascular structures. In recent years, the NHE8 isoform has been isolated from the apical part of alveolar epithelial cells in rats. NHE tightly regulates intracellular pH, cell proliferation, migration, and volüme [24]. Correlation between alkalization and cell proliferation was initially observed in fibroblasts, and then in pulmonary artery smooth muscle cells (PASMC) with the effect of platelet-derived growth factor (PDGF) or epidermal growth factor. NHE1 is also effective in PASMC migration. Activation of NHE causes neutrophil activation, and chemotaxis [23]. In our study, empagliflozin may have exerted its positive effects on lung tissue through NHE inhibition. In addition, inhibition of the extracellular-signal-related kinases (ERKS)1 and 2 signaling pathways, which are protein kinases involved in the phosphorylation of NHE1, of empagliflozin may also contribute to its protective effect in the lung [25].
We conclude that empagliflozin therapy causes morphologic improvement in acute lung injury in rats after intestinal I/R injury. This effect of empagliflozin has led to the idea that it may be a promising agent in the treatment of sterile inflammation-mediated I/R injury, which causes graft dysfunction and mortality after lung transplantation [3]. We believe that further preclinical research into the utility of empagliflozin may indicate its usefulness as a potential treatment for pulmonary damage after intestinal I/R injury in rats.
Author contributions PG: substantial contributions to the conception, acquisition, analysis, or interpretation of data for the work drafting of the work, or revising it critically for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SMK: substantial contributions to the conception, drafting the work, final approval of the version to be published, agreement to be accountable for all aspects of the work. GK: design of the work, revising for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work. CEO: analysis of data for the work,, drafting the work, final approval of the version to be published, agreement to be accountable for all aspects of the work. NY: revising for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work. OE: substantial contributions to the conception, final approval of the version to be published, agreement to be accountable for all aspects of the work. ASN: substantial contributions to the conception, final approval of the version to be published, agreement to be accountable for all aspects of the work. CC: interpretation of data for the work,, revising for important intellectual content, final approval of the version to be published, agreement to be accountable for all aspects of the work. All authors read and approved the manuscript and all data were generated in-house and that no paper mill was used.
Funding None.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflict of interest
The authors declare there are no conflicts of interest.

Ethical approval
The study was conducted according to the ethical standards specified in the 1964 Declaration of Helsinki. Research and publication ethical rules were followed in our study. Our study was approved by the ethics committee of Ankara Training and Research Hospital Animal Experiments Local Ethics Committee, dated 26.07.2021, with a 0066 meeting and 668 decision number.
Informed constent In our animal study titled 'Evaluation of the effects of empagliflozin on acute lung injury in rat intestinal ischemia-reperfusion model', no informed consent is required.