1.1.  Experimental animals and ethical approval

The current study was confirmed by Atatürk University Experimental Animal Ethics Committee (protocol no: 02.03.2018-52). Experimental steps of the study were carried out at Atatürk University Experimental Animals Research and Application Center and rats were acquired from the same center. They were housed in appropriate laboratory conditions including humidity, light/darkness cycle, and temperature. Rats had access to both water and food ad libitum. Twelve hours before the experiment, they were debarred from food but were allowed to drink water. This study was conducted in accordance with the Declaration of Helsinki.

1.2.  Groups and experimental design

The animals that exposed to surgical procedure were fixed in face-down positioning (FDP), shaved, and disinfected. Povidone-iodine was preferred for disinfection. And also, anesthesia was applied to animals before the surgical intervention. 10 mg/kg xylazine hydrochloride (Rompun®, Bayer, Istanbul) and 60 mg/kg ketamine (Ketalar®, Pfizer, Istanbul) were preferred for the anesthesia (intraperitoneally, i.p.). EA was purchased from Sigma Aldrich, USA. Twenty-four male Wistar Albino rats at 220-260 g weights were grouped as;

1.  Sham group: No intervention was carried out on animals. Only the back region was incised and sutured via 3/0 silk suture.

2.  I/R Group: Same steps were followed by the sham group. After incision, renal arteries and veins were occluded by using microvascular clamps, and ischemia was made for 60 minutes. Then, the clamps were released and the renal blood flow was recovered for 24 hours.

3.  I/R+EA 75 mg/kg Group (EA 75 mg/kg Group): All procedures were performed as described in the I/R group. In addition, a single dose of 75 mg/kg EA was applied i.p. to the animals prior to reperfusion.

Following the experiment, the animals were sacrificed by anesthesia and renal tissues were removed.

1.3.  Biochemical analysis

Each renal tissue sample was weighed as 100 mg and 2 mL of phosphate buffer solution (PBS) was used for the homogenization. After the homogenization, samples were centrifuged and kept at -80 0C. MDA measurement depends on the determination of the compound which occurs due to the reaction between thiobarbituric acid (TBA) and MDA [16]. Total antioxidant status (TAS) and total oxidant status (TOS) were gauged with appropriate kits (Rel Assay Diagnostics). Oxidative stress index (OSI) means the rate of TOS to TAS. Tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) values were gauged via appropriate kits (Elabscience, Wuhan, China). Myeloperoxidase (MPO) was determined with the measurement of the complex which results from the reaction between o-dianisidine and MPO [17]. The activity of SOD was gauged with the inhibition degree of formazan dye formation [18].

1.4.  Immunohistochemical (IHC) examination

Kidney tissue samples were placed in 10% neutral formalin for 2-4 days and embedded into paraffin blocks. 5-µm thick sections were stained as immunohistochemical and investigated under a microscope. Following the deparaffinization by xylene and rehydrated in graded alcohols, sections were incubated for 10 minutes in 0.3% H2O2 to quench the activity of endogenous peroxidase. Sections were heated to reveal antigens in the tissue in an antigen retrieval solution for ten minutes. They were subjected to anti-hepatitis A virus cellular receptor 1 (HAVCR1) (Cat. no.bs-2713R, dilution 1/200; Bioss, USA). After washing in PBS, incubation was done with exposing mouse and rabbit specific HRP/DAB detection IHC kit (Abcam: ab80436), the secondary antibody. As a chromogen, 3,3′diaminobenzidine (DAB) was applied and sections were counterstained with hematoxylin. Positive cells in immunostaining sections were examined under a light microscope. Immunoreactivity was evaluated as 0 (none), 1 (mild), 2 (moderate), and 3 (severe).

Figure 1. Effect of EA on RIR-induced changes in proinflammatory cytokines: (a) TNF-α and (b) IL-1β levels. Results were expressed as Mean±S.E. n=8. Data were analyzed by one-way ANOVA followed by Tukey as post-test. ap<0.001 compared to the sham group, bp<0.001 compared to the I/R group.

Ischemia causes permanent damage in tissues through decreased blood flow [19]. Reperfusion also leads to severe tissue injury [20]. I/R-induced AKI is a serious clinical condition that influences about %10 of hospitalized patients [21,22]. I/R-induced renal injury is one of the most common reasons for acute renal insufficiency and results from several conditions including renal transplantation and shock [23,24]. ROS related cellular damage ends up with I/R injury [25]. Lipid peroxidation is closely related to I/R-induced tissue injury and MDA is used to point out lipid peroxidation and to reflect the oxidative stress [26]. MDA occurs due to lipid peroxidation and frequently preferred to show ROS activity indirectly [27,28]. Here, MDA showed an increase in the I/R group while there was a significant decrease in the EA treatment group. MDA is a part of various studies about renal I/R [29,30]. Antioxidant systems including SOD take a role in the protection of tissues against ROS and oxidative damage. In the current study, the SOD level increased with EA treatment. If the oxidant system overcomes the antioxidant activity, this leads to oxidative stress. OSI reflects the oxidative stress degree [31,32]. TAS measurement demonstrates the entire antioxidant activity in a biological sample [33]. The oxidative balance improved in favor of antioxidants in the current study.

The immune system plays a role in renal I/R injury pathophysiology besides hypoxic injury [34]. Enhanced inflammatory reactions, generation of TNF-α, IL-1β, and other proinflammatory cytokines contribute to I/R injury [35,36]. In the current study, the level of TNF-α, IL-1β elevated in the I/R group and decreased with EA treatment. MPO is a pro-inflammatory enzyme and stored in neutrophils with high levels [37]. ROS generation is associated with the injury of lipid membrane structure which enhances MPO production [38]. MPO takes a role in the pathogenesis of renal I/R injury and is mostly used as an indicator of an inflammatory response [39].

Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein in proximal tubule cells [40]. KIM-1 is also called T cell immunoglobulin mucin-1 (TIM-1) or hepatitis A virus cellular receptor 1 (HAVCR1) [41]. KIM-1 production on tubular renal epithelial cells elevates during renal injuries [42,43]. KIM-1 is strongly related to renal tubular injury [44]. Therefore, KIM-1 may be an important parameter for kidney diseases. The current study demonstrated HAVCR1 levels increased in the I/R group and it was reversed with EA treatment.

EA demonstrates several biological features such as antioxidant activity [45]. EA both acted on ROS and lipid peroxidation by diminishing them in a previous study [46]. EA therapy prevented cisplatin-induced nephrotoxicity and declined plasma creatinine and urea levels [47]. Similar to these data, here, EA demonstrated antioxidant and anti-inflammatory activities in the renal I/R model in rats. In the I/R group, antioxidant activity (TAS and SOD) declined while the oxidant and inflammatory system (MDA, MPO, TNF-α, IL-1β, TOS, OSI) parameters elevated. EA treatment reversed these parameters.

 Conclusion

Treatment with EA significantly attenuated renal injury and showed protection against I/R-induced renal injury. Further researches may be useful to investigate the different protective mechanisms on I/R-induced renal tissue damage.

Conflict of interest

The authors declare that they have no conflict of interest.

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