Approximately 10% of the population is affected by chronic kidney disease (CKD), which is becoming more common due to rising rates of diabetes and hypertension. In 2015, CKD was ranked as the tenth greatest cause of mortality [1]. End-stage renal disease is the final outcome of chronic kidney disease, and kidney transplantation is the best treatment option since it provides a higher quality of life than dialysis. Renal ischemic-reperfusion injury (CLP), a temporary decrease of blood flow to the kidneys with the subsequent restoration of blood flow and re-oxygenation, is a significant side effect of renal transplantation. Following kidney transplantation, CLP is the primary pathogenic mechanism driving graft rejection and dysfunction [2], and it is considered the leading cause of acute kidney injury (AKI), affecting around thirteen million worldwide, with roughly 1.7 million deaths each year [3].

During infections, renal tissues are associated with an exacerbation of tissue injury and a profound inflammatory response that further aggravates cell damage. Reactive oxygen species (ROS) and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-\(\alpha\)), play a key role in the pathophysiology of renal injury [4]. Toll-like receptors (TLRs) are an evolutionarily conserved family of cell membrane receptors that are part of the innate immunity system, playing an important role as a first response to tissue injury. TLR2 and TLR4 are constitutively expressed on renal epithelium, and their expression is enhanced following renal injury induced by sepsis [5]. TNF-\(\alpha\) is a significant proinflammatory cytokine with pleiotropic effects on immunity, inflammation, cell differentiation, and apoptosis. TNF receptors, membrane potential, block the respiratory chain, and reduce mitochondrial DNA (mtDNA) in animal cells. Etanercept (ENT), a TNF receptor blocker, was developed as a treatment for disorders whose pathogenesis involves TNF-\(\alpha\). It binds to and neutralizes TNF-\(\alpha\) in individuals with renal injury. According to several research studies, ENT lessens cellular damage by lowering TNF-\(\alpha\) and free radical levels [6]. The aim of the current study was to test the hypothesis that etanercept (ENT) has a renoprotective effect in an in vivo rat model after renal injury induced by CLP.

Animals

Sprague-Dawley (SD) rats (weighing 180 to 200 g, average age 8-12 months) were utilized in this study. The rats were maintained in the animal shelter at Al-Zahrawi University College (Iraq) at 25\(^o\)C and humidity of 60-65%, with a cycle of daylight 12 hours: dark 12 hours. The rats were allowed three weeks of acclimatization with free access to food and water.

Study Design and Sample Collection

Ketamine and xylazine were administered intraperitoneally (i.p.) at doses of 80-100 mg/kg and 8-10 mg/kg, respectively, to the animals to induce general anesthesia [7]. After shaving the abdomen and rinsing it with 80% ethanol, an i.p. injection was used to administer NaCl or etanercept (5 mg/kg dissolved in 0.9% NaCl). After 60 minutes, an abdominal incision was made to reveal the renal pedicles [8]. The study was conducted by randomly dividing the rats into four groups (n=6 per group): (1) Sham group received anesthesia intraperitoneally (i.p.) with operated laparotomy surgery but did not have CLP; (2) control group was subjected to operated laparotomy surgery but had CLP but did not receive any drug; (3) vehicle group: rats were subjected to the same surgical procedure as the control group but received only the vehicle of etanercept, NaCl, i.p. 60 minutes before surgery; (4) etanercept group: rats were subjected to the surgical procedure as the control group but they received etanercept (5 mg/kg, i.p.) 60 minutes before surgery. The animals were anesthetized, killed, and the samples’ organs were retrieved after 24 hours. The kidney was cut into two parts, cleaned with 0.9% saline, put in 0.9% NaCl or 10% formalin, and kept at -20\(^\circ\)C until additional examination [3].

Histopathology

After being preserved in 10% formalin, the extracted kidneys were embedded in paraffin wax. Slices of the tissues were made that were 5 \(\mu\)m thick. Hematoxylin-eosin (H&E) was then used to stain the slices so that histological analysis could be performed. The following pathological grading scale was used in this study: Slight (1): edema or eosinophilic neurons, moderate (2): edema, an eosinophilic neuronal population, and a few RBC populations, severe (3): necrosis, edema, eosinophilic neurons, and RBC populations. Normal (no injury) (0): edema, RBCs, and eosinophilic neurons don’t exist [9].

Immunohistochemistry

TLR2 and TLR4 levels were assessed using immunohistochemistry (IHC). According to the manufacturer’s instructions, tissues from the treated and untreated groups were taken to count the number of cells tagged with TLR2 and TLR4 antibodies [10].

\[QQ = P \times I,\] where Q is the quick score, P is the percentage of positive cells, and I is the intensity.

ELISA

Modulation of NF-\(\kappa\)B p65: We used the ELISA technique to evaluate the NF-\(\kappa\)B p65 level in renal tissue from all groups at the conclusion of the experiment.

Modulation of NF-\(\kappa\)B p65

Our findings demonstrated that, in comparison to the sham group, the control and vehicle groups significantly (p < 0.05) increased the concentration of NF-\(\kappa\)B p65. However, NF-\(\kappa\)B p65 levels were considerably lower (p < 0.05) in the etanercept-treated group compared to the control group and the vehicle-treated group. NF-\(\kappa\)B p65 levels did not change significantly (p > 0.05) between the etanercept-treated group and the control groups (Figure 1).

Modulation of TLR2 and TLR4

Increased levels of TLR2 and TLR4 were observed in an IHC examination. TLR expression was induced in the control and vehicle-treated groups but not in the etanercept-treated group or the sham group (Figure 2). In control positive tissue (normal spleen tissue), TLR expression was significantly enhanced (Figure 2).

Histological Evaluation of Renal Tissue Damage

In the sham group, no renal tissue injury (necrosis, edema, dark eosinophilic neuron, or bleeding) was observed (Figure 3). In contrast, the control group and the group that received vehicle treatment showed abnormal structures (edema, necrosis, dark eosinophilic neuron, and hemorrhage) (Figures 3 and 4). Rats given etanercept treatment experienced less renal damage compared to the control and vehicle-treated groups (Figures 3 and 4).

The histopathological scores of renal tissues in the control and vehicle groups significantly increased compared to the sham group. When comparing the control group and the vehicle group, no discernible difference was observed (p > 0.05). However, in the etanercept-treated group, the scores were considerably reduced (p < 0.05, 65%) compared to the control group and the vehicle-treated group. The data showed no significant difference (p > 0.05) between the sham group and the etanercept-treated group (Figure 4).

TNF-\(\alpha\) is a potent proinflammatory cytokine and a key contributor to tissue damage caused by inflammation. There exists a positive correlation between the severity of renal damage and high blood TNF-\(\alpha\) levels [11]. In our investigation, CLP significantly elevated serum TNF-\(\alpha\) concentration levels over the course of 24 hours. Similar data has been previously reported, demonstrating that the CLP group had TNF-\(\alpha\) levels elevated by approximately three times compared to the sham group [12, 13]. ENT dramatically reduced TNF-\(\alpha\) levels. These data demonstrate the effectiveness of ENT in reducing TNF-\(\alpha\). This observation aligns with ENT’s mechanism of action, which suppresses the production of TNF-\(\alpha\) and renders it biologically inert.

According to the results of the current investigation, ENT exerts its anti-inflammatory effects by controlling pro-inflammatory mediators. The research also indicated that ENT therapy helped mitigate CLP-induced septic kidney damage. The induction of renal damage, as evidenced by tissue necrosis and inflammation, was confirmed through H&E staining of renal tissues from the CLP model group. Treatment with ENT helped to ameliorate this damage. These findings on the effects of exogenous ENT on sepsis-induced organ failure were corroborated by this data. ENT’s anti-inflammatory properties may slow the progression of AKI.

Reports suggest that ENT prevents sepsis-induced kidney damage by inhibiting the NF-\(\kappa\)B signaling pathway. The activation of NF-\(\kappa\)B has been shown to lead to increased production of inflammatory cytokines, including TNF-\(\alpha\), IL-1, and IL-6, in renal cells [14, 15]. Our findings suggest that etanercept reduces inflammatory responses following renal damage by suppressing NF-\(\kappa\)B p65 and TLR levels. Furthermore, tissue damage due to CLP was significantly reduced in the etanercept-treated group. Our observations may indicate that etanercept suppresses inflammation and ameliorates acute renal damage.

The authors would like to acknowledge Al-Zahrawi University College for providing the facilities to conduct this research. Additionally, the authors express their gratitude to Dr. Hassan for animal care and management.

This research paper received no external funding.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

Verbal informed consent was obtained from all participates in the study as needed.

1. Badalova, A. T., Aliyev, E. M., Gasimova, A. S., Huseynova, S. M., Hajiyeva, S. I., & Ahmedzade, U. I. (2020). Antioxidant and lymphostimulating activities of blackberry extract (Rubus caucasicus F.) in experimental diabetes. Azerbaijan Pharmaceutical and Pharmacotherapy Journal, 20(1), 19-23.
2. Sohrabi, C., Alsafi, Z., O'neill, N., Khan, M., Kerwan, A., Al-Jabir, A., ... & Agha, R. (2020). World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). International Journal of Surgery, 76, 71-76.
3. Freitas, F., & Attwell, D. (2022). Pericyte-mediated constriction of renal capillaries evokes no-reflow and kidney injury following ischemia. eLife, 11, e74211.
4. Lee, B. S., Lee, C., Yang, S., Ku, S. K., & Bae, J. S. (2019). Renal protective effects of zingerone in a mouse model of sepsis. BMB Reports, 52(4), 271-277.
5. Jha, A. K., Gairola, S., Kundu, S., Doye, P., Syed, A. M., Ram, C., ... & Sahu, B. D. (2021). Toll-like receptor 4: An attractive therapeutic target for acute kidney injury. Life Sciences, 271, 119155.
6. Kim, C., Ryu, SH., Kim, N., Lee, W., & Bae, J. S. (2022). Renal protective effects of sparstolonin B in a mouse model of sepsis. Biotechnology and Bioprocess Engineering, 27(2), 157-162.
7. Gadimli, A. I. (2021). The study of antioxidant activity of willow gentian (Gentiana asclepiadea L.) extracts. Azerbaijan Pharmaceutical and Pharmacotherapy Journal, 21(2), 29-33.
8. Plaeke, P., De Man, J. G., Smet, A., Malhotra-Kumar, S., Pintelon, I., Timmermans, J. P., ... & De Winter, B. Y. (2020). Effects of intestinal alkaline phosphatase on intestinal barrier function in a cecal ligation and puncture (CLP)-induced mouse model for sepsis. Neurogastroenterology & Motility, 32(3), e13754.
9. Qiu, J., Kulkarni, S., Chandrasekhar, R., Rees, M., Hyde, K., Wilding, G., ... & Khoury, T. (2010). Effect of delayed formalin fixation on estrogen and progesterone receptors in breast cancer: a study of three different clones. American Journal of Clinical Pathology, 134(5), 813-819.
10. Wan, B., Xu, W. J., Chen, M. Z., Sun, S. S., Jin, J. J., Lv, Y. L., ... & Song, Y. (2020). Geranylgeranyl diphosphate synthase 1 knockout ameliorates ventilator-induced lung injury via regulation of TLR2/4-AP-1 signaling. Free Radical Biology and Medicine, 147, 159-166.
11. Al-Huseini, L. A., Al-Mudhaffer, R., Hassan, S., & Hadi, N. (2019). DMF ameliorating cerebral ischemia/reperfusion injury in male rats. Systematic Reviews in Pharmacy, 10(1), 206-213.
12. Cao, Y. Z., Tu, Y. Y., Chen, X., Wang, B. L., Zhong, Y. X., & Liu, M. H. (2012). Protective effect of Ulinastatin against murine models of sepsis: Inhibition of TNF-a and IL-6 and augmentation of IL-10 and IL-13. Experimental and Toxicologic Pathology, 64(6), 543-547.
13. Hassan, S. M. (2022). DMF attenuates Ciprofloxacin-Induced Nephropathy in Rats via Nrf2 Pathway. Journal of Pharmaceutical Negative Results, 13(2), 87-91.
14. DeGrado, J., Babcock, K., Anger, K., & Szumita, P. (2011). Etanercept for the treatment of pulmonary complications after hematopoietic stem cell transplantation. Critical Care Medicine, 39(12), 95-99.
15. Dorokhova, L. P., & Dorokhov, A. V. (2021). Pharmaceutical service in a pharmacy: The role of staff and quality of service. Azerbaijan Pharmaceutical and Pharmacotherapy Journal, 21(2), 42-48.