INTRODUCTION

Acute kidney injury (AKI) is characterized by acute tubular cell damage and renal dysfunction, and may develop due to septic, toxic or ischemic causes (1). One of the most important risk factors for the development of AKI is severe bacterial infection (2). AKI-associated Gram-negative bacterial infections continue to be a cause of high mortality in patients (3). While a specific method for the treatment of AKI has not been developed yet, the development of new agents in the treatment of septic AKI has a great potential to reduce mortality and morbidity in patients (4).

Lipopolysaccharide (LPS), which is an important pathogenic factor in the cell membrane of Gram-negative bacteria, consists of lipid and polysaccharide linked by covalent bond (5). Experimental studies have shown that it is an endotoxin that provides a strong immune response (6,7).

Various histopathological and biochemical changes have been reported in kidney tissue in LPS-induced experimental models. These are renal tubular damage, increase in blood urea nitrogen (BUN) and creatinine levels, inflammatory cell infiltration, pathological changes such as necrosis in tubular cells, and increased oxidative stress (8,9).

LPS stimulation induces the activation of nuclear factor-kappa beta and may activate the Toll-like receptor-4 pathway, which stimulates the release of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin 1-beta (IL-1b) and transforming growth factor-beta 1 (TGF-β1) (10,11). Excessive production of inflammatory cytokines may cause kidney damage by causing the migration of neutrophils and monocytes to the glomerulus (11,12). Therefore, suppression of inflammatory cytokine production is an important strategy in the prevention of kidney damage (13).

Some natural flavonoids and polyphenols have broader biological effects and targets than chemical-synthetic agents, which usually have a specific target, especially in diseases with multifactorial pathogenesis (12-14).

Hesperetin (3¢,5,7-trihydroxy-4-methoxyflavanone) is a natural flavonoid found abundantly in citrus fruits such as lemon, orange, grapefruit, and mandarin (15). Flavonoids are polyphenolic compounds with various biological activities, and the free hydroxyl groups in their molecular structures function as free radical scavengers (16). Previous studies have shown that hesperetin has potent anti-inflammatory and antioxidative properties, both in vivo and in vitro (17,18).

It has been reported that application of hesperetin in acrolein-induced experimental lung injury model suppresses oxidative stress and supports tissue structure by preventing apoptosis in cells (19). It has been reported that application of hesperetin in experimental rat ischemia/reperfusion-induced retinal injury model prevents retinal thinning and apoptosis in the retina (18).

These experimental studies show that hesperetin reduces damage by supporting cell and tissue structure in different tissues and organs.

When studies in which hesperetin was used as a treatment agent in different kidney damage models were examined, it was reported that it reduced lipid peroxidation, suppressed oxidative stress, decreased the release of inflammatory cytokines, and helped to preserve tissue structure by reducing histopathological changes (20,21).

The LPS-induced AKI model is the most commonly used in vivo experimental model to investigate the histopathological changes caused by sepsis in kidney tissue and the mechanisms underlying potential treatments (11). In the literature review, no study that examined the effects of hesperetin in the LPS-induced AKI model was found. In this study, it was aimed to examine the efficacy of hesperetin in the LPS-induced AKI model by histological, immunohistochemical and TUNEL methods.

METHODS

Experiment Model and Applications

The ethics committee approval required for this experimental study was obtained from the Tekirdağ Namık Kemal University Animal Experiments Local Ethics Committee (decision no: 4, date: 31.10.2018), and all applications were carried out in accordance with the Laboratory Animal Care and Guidelines. Our study was carried out at Tekirdağ Namık Kemal University Experimental Animals Application and Research Center (DHUAM) between March and May 2019. Eighteen healthy adult male Wistar albinos (3 months old, 250-300 g) were included in the study. The rats were fed ad libitum at 22±2 °C temperature, 12 hours light/12 hours dark cycle and 50-60% humidity during the experiment. Rats were divided into 3 groups, respectively (n=6), as control, LPS, and LPS + hesperetin groups. Escherichia coli (O26:B6 serotype, Sigma-Aldrich) was administered intraperitoneally to the LPS group at a dose of 10 mg/kg (22). In the LPS + hesperetin group, hesperetin (SantaCruz, dissolved in 0.5 mL saline) was administered orally at a dose of 100 mg/kg, following endotoxin administration (14). At the end of 24 hours following LPS administration, the rats were opened from the midline under ketamine-xylazine (90-10 mg/kg) anesthesia. The kidney tissue was dissected and the animals were sacrificed after drawing blood from the heart. The kidney tissue was fixed in 10% formalin solution.

Biochemical Analysis

The blood samples were centrifuged at 4500 rpm for 5 minutes and serum samples were obtained. BUN and creatinine levels were determined from the obtained serum samples with the help of a biochemistry autoanalyzer (Cobas-501, RocheDiagnostic).

Histopathological Examination

Following the 24-hour fixation of the tissues in formalin, they were washed by keeping them under running tap water for a night. After washing, kidney tissues were exposed to alcohol series with increasing concentrations (60-70-80-90-96-100) and tissue blocks were obtained by embedding kidney tissues first in a soft paraffin-toluene mixture and then in hard paraffin (Slee, MPS). These blocks were used in histological, immunohistochemical and TUNEL examinations.

Hematoxylin-eosin (H&E) staining method was used for histopathological examination. Sections (5 µm) obtained using a semi-automatic microtome (Sleecut, 5061) were kept in an oven (37 °C) overnight to ensure complete adhesion to the slide surface. These sections were taken in toluene and dewaxed (5 min x 2 times). Afterwards, they were put into water after passing through alcohol series at decreasing concentrations (100-96-90-80-70-60%). After the hydration process, they were stained with hematoxylin for 3 minutes and after washing, with eosin for 5 minutes.

Afterwards, the slides were passed through a series of increasing concentrations of alcohol and placed in toluene. They were covered with enthallan following the transparentizing process.

Obtained H&E stained kidney tissue sections were examined using a light microscope (Olympus CX40) and image analysis program (Kameram, GEN III). The histopathological changes that occurred were detected and photographed.

TGF-β1 Immunohistochemical Staining

Avidin-biotin peroxidase complex technique was used for TGF-β1 immunohistochemical marking. Paraffin-free slides were passed through an alcohol series at decreasing concentrations and washed with phosphate buffer. Tissue areas on the slide were delimited with a hydrophobic pen. For antigen retrieval, sections were boiled in a microwave oven for 5 min in citrate buffer. The procedure of endogenous peroxide suppression was performed with hydrogen peroxide. In order to prevent non-specific binding, primary antibody application (TGF-β1, sc-52893) was carried out in a humid box for one hour at 37 °C, following a one-hour block serum step at laboratory temperature. After the application of secondary antibodies with biotin and streptavidin, which were applied for 20 minutes after the primary antibody application, the coloring process was carried out with chromogen (AEC; 3-amino-9-ethyl carbazole, Thermoscientific). Sections stained with Mayer’s hematoxylin were examined under a light microscope. TGF-β1 staining intensity was graded semiquantitatively (0; no staining, 1; weak, 2; moderate, 3; strong).

Apoptotic Examination

TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP nickend-labelling) method was used to determine apoptosis in kidney tissue. The procedure for using the kit was applied to the deparaffinized slides. DAB (3,3’-diaminobenzidine tetrahydrochloride) was used as chromogen. After ground staining with Mayer’s hematoxylin, the sections were examined under a light microscope. The apoptotic index was calculated by measuring the number of TUNEL-positive cells stained dark brown in the cell nucleus in photographs obtained from 10 different areas of each kidney section (TUNEL positive cell number/total number of cells counted x 100) (23).

Statistical Analysis

The obtained data were analyzed using SPSS (PASW, 18.0.0) statistical program. The data of the groups were evaluated using the non-parametric Kruskal-Wallis test. Differences between the groups were measured with the Mann-Whitney U test. When the difference between the groups was less than p<0.05, it was considered statistically significant.

RESULTS

In the histopathological evaluation, normal histological structure was observed in the kidney tissue of the control group. The structure of proximal and distal tubules, interstitial tissue and kidney bodies were found to be normal (Figure 1a). In the LPS group, shedding especially in tubular epithelial cells and pycnotic nuclei in some tubular epithelial cells were observed (Figure 1b). Although the tubule epithelial shedding continued in the group treated with hesperetin, it was found to be less frequent compared to the LPS group (Figure 1c).

When immunohistochemical TGF-β1 staining was examined, it was observed that positive immune reactivity was intense in tubular epithelial cells (Figure 2a-c). TGF-β1 semiquantitative staining scores were 0.3±0.5, 2.5±0.54, and 1.66±0.51 in the control, LPS and LPS + hesperetin groups, respectively. It was determined that there was an increase in TGF-β1 immune reactivity in the LPS group compared to the control group (p<0.05). It was found that hesperetin administration provided a significant decrease in TGF-β1 immune reactivity (p<0.05).

In our study, apoptosis occurring in kidney tissue was determined by the TUNEL method based on the detection of apoptotic cells by marking broken DNA ends (Figure 3). In the control, LPS and LPS + hesperetin groups, the apoptotic index was calculated as 5.3±1.8, 29.5±5.2, and 15.8±5.3, respectively. While a significant increase was observed in the LPS group compared to the control group (p<0.05), a significant decrease was detected in the LPS + hesperetin group compared to the LPS group (p<0.05).

Serum BUN and creatinine levels are presented in Table 1. LPS administration caused a significant increase in serum BUN and creatinine levels compared to the control group (p<0.05). It was determined that hesperetin administration provided a significant decrease in kidney function tests BUN and creatinine levels compared to the LPS group (p<0.05).

DISCUSSION

Although the LPS-induced sepsis model does not represent all the features of sepsis encountered in the clinic, it is frequently used in the literature to create an AKI model because the findings of AKI caused by it are similar (24).

Although septic AKI is a complication that can be seen frequently in intensive care units and has a high mortality rate, there is no specific treatment method yet (25). In these patients, especially kidney transplantation is an effective treatment method (26). Today, studies examining the possible treatment efficacy of agents with known anti-inflammatory, anti-apoptotic and antioxidant properties against AKI damage are frequently encountered in the literature.

The findings obtained in our study demonstrated the renal protective effect of hesperetin in the rat LPS-induced AKI model with histological and biochemical data for the first time in the literature. Preservation of the renal histological structure by hesperetin administration may have been achieved by inhibiting TGF-β1 immunoreactivity and the activity of the inflammatory cytokine cascade and preventing apoptosis. There are many studies in the literature showing that hesperetin reduces/inhibits tissue damage in different experimental models due to its antioxidative, anti-inflammatory and anti-apoptotic properties (27,28).

Duran and Karaboğa (28) reported that administration of hesperetin prevented liver damage following blunt chest trauma. In the study of Trivedi et al. (29), it was revealed that heart damage caused by doxorubicin can be alleviated by the administration of hesperetin.

Kumar et al. (20) reported the therapeutic effect of hesperetin against kidney damage caused by cisplatin, a frequently used anticancer drug. In the aforementioned study, hesperetin treatment provided a significant decrease in histopathological changes caused by cisplatin application, serum BUN and creatinine levels, oxidative stress markers of superoxide dismutase, glutathione peroxidase, malondialdehyde, nitric oxide and inflammatory cytokines of TNF-a, IL-1b and IL-6 levels in kidney tissue.

It was determined that AKI-like findings occurred in the endotoxemia model created using LPS. Hesperetin administration can prevent the development of kidney damage by inhibiting the activation of the proinflammatory cytokine cascade and the expression of TGF-β1 and also by suppressing apoptosis in tubular epithelial cells.

There are some limitations in our study. Hesperetin treatment was applied following LPS administration and its effect was evaluated for a short time (24 h). This is not consistent with patient management in a clinical setting. In addition, hesperetin treatment was administered as a single dose. These limitations can be improved in new studies evaluating the effect of hesperetin application at different doses and longer than our study on kidney damage.

CONCLUSION

The results of our study show that hesperetin, a natural flavonoid, should be evaluated not only as a preventative but also for its therapeutic potential in diseases with kidney damage. More extensive in vivo and in vitro studies are needed to fully explain the treatment mechanism of hesperetin in the LPS-induced AKI model.

ETHICS

Ethics Committee Approval: The ethical aproval required to conduct this study was obtained from Tekirdağ Namık Kemal University Animal Experiments Local Ethics Committee (HADYEK- T2018-115) (decision no: 4, date: 31.10.2018).

Informed Consent: Animal experiment study.

Authorship Contributions

Surgical and Medical Practices: S.K., İ.K., Concept: S.K., İ.K., Design: S.K., İ.K., Data Collection or Processing: S.K., İ.K., Analysis or Interpretation: S.K., İ.K., Literature Search: S.K., İ.K., Writing: S.K., İ.K.

Conflict of interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.