Protective Effects of Orexin A in a Murine Model of Cisplatin-Induced Acute Kidney Injury

Cisplatin is a chemotherapeutic agent widely used in the treatment of various cancers, but its application is often limited due to complications such as acute kidney injury (AKI). Orexins are hypothalamic neuropeptides that modulate the sleep-wake cycle, neuroendocrine function, and the autonomic nervous system. Emerging evidence suggests that orexin A (OXA) has anti-inflammatory and neuroprotective effects in animal models of neuroinflammatory diseases of the central nervous system. However, the effect of OXA on kidney diseases has not been examined. Here, we investigated whether OXA has a protective effect in a murine model of cisplatin-induced AKI. Intraperitoneal administration of OXA ameliorated renal dysfunction, and histological abnormalities in mice injected with cisplatin. OXA inhibited cisplatin-induced oxidative stress through the modulation of prooxidant and antioxidant enzymes. This peptide reduced apoptotic cell death by inhibiting the p53-mediated pathway in mice injected with cisplatin. OXA also alleviated cisplatin-induced cytokine production and macrophage infiltration into injured kidneys. Taken together, these results showed that OXA ameliorates cisplatin-induced AKI via antioxidant, anti-apoptotic, and anti-inflammatory actions. This peptide could be a potential therapeutic agent for cisplatin-induced AKI.


Introduction
The kidneys are vital organs that play a major role in maintaining homeostatic regulation of the body's water and ion balance [1]. They are susceptible to various forms of acute injury due to their role as the major eliminator of exogenous drugs and toxins. Acute kidney injury (AKI) is defined by a sudden decrease in kidney function and results from various types of insults, such as decreased renal perfusion and exposure to nephrotoxins [1]. Among nephrotoxic drugs, cisplatin is a chemotherapy drug used to treat many types of cancer [2]. However, the major drawback of cisplatin treatment is that it often leads to treatment failure due to serious side effects, including nephrotoxicity [2][3][4]. Thus, the development of new pharmacological agents for cisplatin-induced AKI has great clinical significance.
Orexins (orexin A and orexin B) are hypothalamic neuropeptides that regulate the sleep-wake cycle [5]. The activity of orexin neurons increases during wakefulness and decreases during sleep. The loss of orexin neurons in humans has been known to be associated with narcolepsy, which is a chronic sleep disorder characterized by excessive daytime sleepiness [5]. Orexins also play an important role in the regulation of the neuroendocrine and autonomic nervous systems [5,6]. Orexins act on two G-protein-coupled receptors, orexin receptor type 1 and type 2 [7]. Orexin receptors are present in the central nervous system (CNS) and many peripheral tissues [8]. Currently, orexin receptor antagonists are used for the treatment of insomnia [5]. In addition, accumulating evidence suggests that the administration of orexin A (OXA) exerts anti-inflammatory and neuroprotective effects in animal models of neuroinflammatory diseases of the CNS [9][10][11][12][13]. Furthermore, OXA also exhibited anti-inflammatory action in endotoxin shock [14] and ulcerative colitis [15]. However, the effect of OXA on peripheral tissue inflammation remains largely unknown. Here, we investigated the potential effect of OXA on cisplatin-induced AKI and explored the underlying mechanism.

Animal Experiments
Seven-week-old male C57BL/6 mice were purchased from HyoSung Science (Daegu, Republic of Korea). Before starting experiments, the mice were acclimated for 1 week under 20-24 • C on a 12/12 h light/dark cycle. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Daegu Catholic University Medical Center (DCIAFCR-180312-20-Y). The mice were randomly grouped into four groups (n = 8 in each group): (1) control (Con) group; (2) OXA group; (3) Cisplatin (CP) group; (4) cisplatin plus OXA (CP + OXA) group. The CP group and the CP + OXA group were injected intraperitoneally with cisplatin [15 mg/kg in 0.9% saline; Cayman Chemical, Ann Arbor, MI, USA]. The OXA group and the CP + OXA group were given an intraperitoneal injection of OXA (1 µmol/kg in phosphate-buffered saline (PBS); Tocris Bioscience, Bristol, UK) daily for 4 consecutive days, starting from 1 day prior to 0.9% saline or cisplatin injection. An equal volume of PBS was injected intraperitoneally into the CP group. The doses of cisplatin and OXA were chosen based on previous studies [16][17][18][19]. At 72 h after cisplatin injection, the mice were sacrificed.

Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from tissues using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA). The reverse transcription of extracted RNA was conducted for cDNA synthesis. Then, Real-time RT-PCR was performed using the specific primers (Table 1) in the Thermal Cycler Dice Real Time System III (TaKaRa, Tokyo, Japan). Relative expression was calculated using 2 −∆∆CT method, using GAPDH as an internal control.

TdT-Mediated dUTP Nick End Labeling (TUNEL) Staining
Apoptosis was detected in tissues using a TUNEL assay kit (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer s protocol. Briefly, the sections were deparaffinized, permeabilized, and incubated in the TUNEL reaction mixture. Positive cells were counted in 10 random fields per sample.

Statistical Analysis
Data were expressed as the mean ± SEM. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests using Prism version 5 (GraphPad Software, San Diego, CA, USA). A p-value less than 0.05 was considered significant.

Discussion
In the present study, we investigated the effect of OXA on cisplatin-induced AKI in mice. Systemic administration of OXA ameliorated renal dysfunction and histopathological alterations in mice injected with cisplatin. OXA alleviated cisplatin-induced oxidative stress through the regulation of prooxidant and antioxidant enzymes. Apoptotic cell death in mice injected with cisplatin was also attenuated by OXA. This peptide inhibited cytokine production and macrophage infiltration.
Although OXA is known to modulate the sleep-wake cycle, recent studies have shown that this peptide has anti-inflammatory effects in murine models of several inflammatory diseases [9][10][11][12][13][14][15]. In this study, we found that OXA reduced serum levels of creatinine and BUN, established markers of kidney function, in cisplatin-injected mice, suggesting that OXA protects mice from cisplatin-induced renal dysfunction. In addition, OXA attenuated cisplatin-induced structural damage, as evidenced by a decrease in the tubular injury score, an increase in the area of positive staining for LTL, and a decrease in NGAL expression. Because LTL is a marker for the brush borders of the proximal tubules [24,25], a decrease in the area of LTL staining indicates a brush border loss. The inhibitory effect of OXA on the tubular injury was also confirmed by decreased expression of the tubular injury marker NGAL. Collectively, these results demonstrate the protective effect of OXA on cisplatin-induced AKI.
Oxidative stress is a hallmark of cisplatin-induced AKI [2][3][4]. Therefore, we next investigated changes in oxidative stress to study the mechanism for the protective effect of OXA on cisplatin-induced AKI. The amounts of 4-HNE and MDA were measured to assess lipid peroxidation, and the amount of 8-OHdG was evaluated to assess DNA oxidation. As previously reported [29,37], cisplatin injection markedly increased lipid peroxidation and DNA oxidation in mice. Moreover, the GSH/GSSG ratio, an established indicator of oxidative stress [38], was decreased after cisplatin injection. However, OXA significantly inhibited cisplatin-induced oxidative stress. To further evaluate the mechanisms underlying the inhibitory effect of OXA on oxidative stress, we examined the expression or activity of prooxidant and antioxidant enzymes. Cisplatin injection increased NOX4 expression and decreased the expression and activity of catalase, SOD, and GPx. It has been shown that NOX4 is a major source of intracellular reactive oxygen species (ROS) and plays an important role in cisplatin-induced AKI [31][32][33]. In addition, it has been shown that cisplatin injection reduces the expression and activity of several antioxidant enzymes in rodents, exacerbating cisplatin-induced oxidative damage [2][3][4]. However, OXA significantly reversed cisplatin-induced changes in prooxidant and antioxidant enzymes. These findings suggest that OXA inhibits cisplatin-induced oxidative stress through the regulation of prooxidant and antioxidant systems. Consistent with our findings, a recent study showed that OXA inhibited NOX4 expression and ROS production in high glucose-exposed human endothelial cells [26]. Activation of the antioxidant defense system was also observed in OXA-treated human endothelial cells [27].
Tubular cell apoptosis plays an important role in cisplatin-induced AKI [2][3][4]. Previous studies have shown that OXA exerts a neuroprotective effect by inhibiting neuronal apoptosis [39][40][41]. These results prompted us to investigate the effect of OXA on apoptosis in mice injected with cisplatin. In this study, apoptotic cells were identified using the TUNEL staining. As previously reported [38,42], cisplatin injection largely increased the number of TUNEL-positive apoptotic cells in the kidney. This is also confirmed by the activation of the key executioner caspase, caspase-3. However, OXA significantly attenuated cisplatininduced apoptosis. Accumulating evidence suggests that the p53 protein is a key player in the cisplatin-induced apoptosis of tubular epithelial cells [43]. This protein is a transcription factor that induces apoptosis through transcriptional regulation of apoptosis-related genes such as Bax. We found that cisplatin injection increased p53 and Bax expression but was inhibited by OXA. Altogether, these results suggest that OXA inhibits cisplatin-induced apoptosis by inhibiting the p53-mediated pathway.
Inflammation is the main process in cisplatin-induced AKI [2][3][4]. Cisplatin-induced inflammatory responses are characterized by excessive production of proinflammatory cytokines and infiltration of immune cells [2][3][4]. In this study, increased serum and renal concentrations of TNF-α, IL-6, and IL-1β were significantly reduced by OXA. In line with our findings, a recent study reported that OXA suppressed inflammatory responses in murine models of colitis [15]. Production of proinflammatory cytokines in immune cells and intestinal epithelial cells was inhibited by OXA [15]. We found that this peptide also alleviated macrophage infiltration in cisplatin-injected mice. Among proinflammatory cytokines, TNF-α has been known to play an essential role in cisplatin-induced inflammation [44]. Pharmacological inhibition or genetic ablation of TNF-α attenuated cisplatin-induced AKI [44]. TNF-α stimulates the production of other proinflammatory cytokines and promotes immune cell infiltration [2][3][4]. Macrophages are one of the cellular sources of proinflammatory cytokines, including TNF-α, and exacerbate cisplatin-induced kidney injury [43]. In this study, serum and renal levels of MCP-1 were reduced by OXA. MCP-1 is a major chemokine that plays a key role in macrophage recruitment [16]. Previous studies have reported that cisplatin injection increased macrophage infiltration along with MCP-1 upregulation in rodents [29,45]. Altogether, our findings suggest that OXA attenuates cisplatin-induced inflammation by inhibiting cytokine production and macrophage infiltration.
It has been known that OXA cannot normally penetrate the blood-brain barrier (BBB) [5]. However, recent studies have shown that OXA can penetrate into the CNS due to BBB dysfunction under the condition of systemic inflammation [13,14]. Peripheral administration of OXA ameliorated inflammatory responses and improved survival in mice with endotoxin shock through its central action [14]. Multiple pathways, including the neuroendocrine and autonomic nervous systems, are involved in the mechanisms underlying the beneficial effect of OXA on septic shock through the CNS [14,46]. Uremia can induce disruption of BBB integrity, and thus AKI has been shown to be associated with BBB dysfunction [47]. Therefore, the protective effect of OXA on cisplatin-induced AKI is likely due to the central action of OXA through the neuroendocrine and autonomic nervous systems. However, because orexin receptors are also widely present in peripheral tissues [8], the possibility that OXA may act directly on the kidney cannot be excluded. Future studies will be needed to elucidate the exact mechanism of the protective effect of OXA against kidney diseases.

Conclusions
In conclusion, our findings demonstrated that OXA has a protective effect on cisplatininduced AKI by inhibiting oxidative stress, apoptosis, and inflammation. These results suggest that OXA has the potential to be used as a therapeutic agent for cisplatin-induced AKI.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data supporting the findings of this study are available within the article.

Conflicts of Interest:
The authors declare no conflict of interest.