Biomolecules & Therapeutics 2023; 31(6): 599-610
Synergistic Renoprotective Effect of Melatonin and Zileuton by Inhibition of Ferroptosis via the AKT/mTOR/NRF2 Signaling in Kidney Injury and Fibrosis
Kyung Hee Jung1,†, Sang Eun Kim1,†, Han Gyeol Go1, Yun Ji Lee1, Min Seok Park1, Soyeon Ko1, Beom Seok Han1, Young-Chan Yoon1, Ye Jin Cho1, Pureunchowon Lee1, Sang-Ho Lee2, Kipyo Kim3,* and Soon-Sun Hong1,*
1Department of Medicine, College of Medicine, and Program in Biomedical Science & Engineering, Inha University, Incheon 22332,
2Division of Nephrology, Department of Internal Medicine, College of Medicine, Kyung Hee University, Seoul 02453,
3Divison of Nephrology and Hypertension, Department of Internal Medicine, Inha University Hospital, Inha University College of Medicine, Incheon 22332, Republic of Korea
*E-mail: (Hong SS), (Kim K)
Tel: +82-32-890-3683 (Hong SS), +82-32-890-3246 (Kim K)
Fax: +82-32-890-2462 (Hong SS), +82-32-890-2462 (Kim K)
The first two authors contributed equally to this work.
Received: March 23, 2023; Revised: April 7, 2023; Accepted: April 17, 2023; Published online: May 15, 2023.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
According to recent evidence, ferroptosis is a major cell death mechanism in the pathogenesis of kidney injury and fibrosis. Despite the renoprotective effects of classical ferroptosis inhibitors, therapeutic approaches targeting kidney ferroptosis remain limited. In this study, we assessed the renoprotective effects of melatonin and zileuton as a novel therapeutic strategy against ferroptosis-mediated kidney injury and fibrosis. First, we identified RSL3-induced ferroptosis in renal tubular epithelial HK-2 and HKC-8 cells. Lipid peroxidation and cell death induced by RSL3 were synergistically mitigated by the combination of melatonin and zileuton. Combination treatment significantly downregulated the expression of ferroptosis-associated proteins, 4-HNE and HO-1, and upregulated the expression of GPX4. The expression levels of p-AKT and p-mTOR also increased, in addition to that of NRF2 in renal tubular epithelial cells. When melatonin (20 mg/kg) and zileuton (20 mg/kg) were administered to a unilateral ureteral obstruction (UUO) mouse model, the combination significantly reduced tubular injury and fibrosis by decreasing the expression of profibrotic markers, such as α-SMA and fibronectin. More importantly, the combination ameliorated the increase in 4-HNE levels and decreased GPX4 expression in UUO mice. Overall, the combination of melatonin and zileuton was found to effectively ameliorate ferroptosis-related kidney injury by upregulating the AKT/mTOR/ NRF2 signaling pathway, suggesting a promising therapeutic strategy for protection against ferroptosis-mediated kidney injury and fibrosis.
Keywords: Ferroptosis, RSL3, Kidney injury, Fibrosis, GPX4

Kidney diseases pose a tremendous burden to patients and healthcare facilities globally (Luyckx et al., 2018). A recent report indicates that over 700 million individuals are affected by chronic kidney disease (CKD) worldwide (Sundström et al., 2022). According to time frames, acute kidney injury (AKI) and CKD have a substantial impact on mortality and other health-related consequences, including end-stage kidney disease (ESKD) and cardiovascular events (Kellum et al., 2021; Kovesdy, 2022; Matsushita et al., 2022). Despite the recent development of promising therapeutics, such as sodium-glucose co-transporter 2 inhibitors, disease-specific therapies for diverse kidney diseases are still lacking (van Asbeck et al., 2020).

Various forms of cell death including apoptosis, necroptosis, autophagy, and ferroptosis are involved in different types of kidney injuries (Priante et al., 2019; Maremonti et al., 2022). Among these cell death processes, non-apoptotic cell death is considered a potential therapeutic target. Regulated necrosis such as ferroptosis and necroptosis is a more immunogenic form of cell death that triggers the innate immune system by releasing damage-associated molecular patterns (Mulay et al., 2016). Ferroptosis is a potential cell death mechanism induced by lipid peroxidation in an iron-dependent manner. Notably, recent studies have suggested the crucial role of ferroptosis in kidney diseases (Dixon et al., 2012; Feng et al., 2022; Zhang et al., 2022). Mice deficient in GPX4, a key regulator of ferroptosis, show extensive tubular cell death characterized by ferroptosis by lipid peroxidation (Friedmann Angeli et al., 2014). Subsequent studies demonstrated that ferroptosis is a major mechanism in kidney tubular cell death in ischemia-reperfusion injury (IRI) and folic acid-induced kidney injury models (Linkermann et al., 2014; Martin-Sanchez et al., 2017). Ferroptotic stress also contributes to fibrosis and the transition to CKD and plays a pathogenic role in diabetic nephropathy (Ide et al., 2021; Kim et al., 2021). However, despite recent advances in understanding of kidney ferroptosis, therapeutic agents targeting ferroptosis have been limited to experimental agents, such as ferrostatin-1 (Fer-1) or deferoxamine (DFO), and have not been clinically tested.

Zileuton is an inhibitor of 5-lipoxygenase (5-LOX), an enzyme involved in leukotriene biosynthesis. It is used to treat asthma by blocking the production of leukotrienes and inflammatory molecules that can cause airway constriction in the lungs (Berger et al., 2007). 5-lipoxygenase (5-LOX) is an enzyme involved in the metabolism of arachidonic acid and fatty acid that is present in cell membranes, which may contribute to lipid peroxidation and induce ferroptosis (Shah et al., 2018). Also, it is involved in the regulation of inflammation, immune response, and other physiological processes (Mashima and Okuyama, 2015). Therefore, the inhibition of 5-LOX has been reported to protect against various inflammatory diseases, including asthma, rheumatoid arthritis, and kidney injury diseases involving inflammation and oxidative stress (Israel et al., 1990; Lin et al., 2014; Montford et al., 2019). In particular, zileuton has been reported to clear free radicals and decrease the level of lipid oxidation, which protects the retinal pigment epithelium and neuronal cells from ferroptosis (Czapski et al., 2012; Liu et al., 2015; Lee et al., 2022). According to a recent study, zileuton decreases renal fibrosis in chronic kidney disease (CKD) (Montford et al., 2019); however, its effect and role in the regulation of ferroptosis in kidney injury are not known.

Melatonin (N-acetyl-5-methoxytryptamine) has many physiological effects, including regulation of sleep-wake rhythm, circadian cycle, immune defense, cardiovascular function, and bone metabolism (Mahmood, 2019). It is also known to regulate fundamental cellular functions by exhibiting antioxidant, anti-aging, cytoprotective, and immunomodulatory activities in various diseases (Montilla et al., 2001; Ferlazzo et al., 2020). In animal studies of CKD, melatonin was found to exert renoprotective effects by lowering oxidative stress and inflammation (Quiroz et al., 2008), of which role and underlying mechanisms have not been fully elucidated. Recent evidence indicates the anti-ferroptotic effects of melatonin beyond its antioxidant activity. Melatonin effectively inhibits hemin-induced ferroptosis and lipid peroxidation in platelets (NaveenKumar et al., 2019). Melatonin also exerts anti-ferroptotic activity against PM2.5-induced lung injury and osteoporosis (Ma et al., 2020; Guohua et al., 2021). As ferroptosis is inhibited by free radical scavengers, a potent antioxidant, melatonin, and zileuton, which has free radical scavenging capacity, may be promising therapeutic candidates for kidney injury induced by ferroptosis. Therefore, in this study, we investigated the combination effect of melatonin and zileuton on the inhibition of kidney ferroptosis and its related mechanisms in a unilateral ureteral obstruction (UUO) mouse model.


Cell culture and reagents

The human renal proximal cell lines HKC-8 and HK-2 were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). HKC-8 and HK-2 were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) or Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cell lines were maintained at 37°C in an incubator with a controlled humidified atmosphere composed of 95% air and 5% CO2. RSL3, zileuton, and melatonin were purchased from Selleckchem (Houston, TX, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

Cell viability assay

Cell viability was determined using the Alamar Blue assay (Invitrogen, Carlsbad, CA, USA, DAL1025). HKC-8 (3×103 cells/well) and HK-2 (5×103 cells/well) cells were seeded in 96-well plates and incubated for 24 h. Cells were treated with combinations of RSL3 (0.05-1 µM) and melatonin (0.1-2 mM), zileuton (1-50 µM), ferrostatin-1 (1 µM), deferoxamine (100 µM), Z-VAD-FMK (20 µM), necrostatin-1 (2 µM), or N-acetyl cysteine (5 mM). After 12 h of incubation, 100 µL of Alamar blue diluted in the medium was added to each well and incubated for 2 h at 37°C. The plate was measured using a microplate reader (Thermofisher, Waltham, MA, USA) at 570-600 nm. The assay was conducted in triplicate.

Cell death assay

Cell death was assessed using SYTOX Green (Invitrogen, S34860) that stains dying cells. HKC-8 (5×104 cells/well) cells were seeded in a 12-well plate and treated with RSL3 (1 µM), melatonin (1 mM), zileuton (5 µM), ferrostatin-1 (1 µM), or deferoxamine (100 µM), and the diluted SYTOX green was also treated. Fluorescence expression was observed using the JuLI™ Stage Real-Time Cell History Recorder (NanoEn Tek, Seoul, Korea). SYTOX green-positive cells were counted.

Western blotting assay

Cells were lysed with RIPA buffer (Biosesang, Seoul, Korea) containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Proteins quantified by the BCA assay were separated using 8-10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) for 1 h. The membranes were blocked with 5% skim milk at room temperature for 1 h and incubated overnight with primary antibodies at 4°C. The blots were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) at room temperature for 1 h. Protein expression was detected using an enhanced chemiluminescence (ECL) reagent (Bio-Rad, Hercules, CA, USA) and X-ray film. The following primary antibodies were used: 4-HNE (Abcam, Cambridge, MA, USA, ab46545), GPX4 (Santa Cruz Biotechnology, Dallas, TX, USA, SC-166570), HO-1 (Cell Signaling, Danvers, MA, USA, 43966S), NRF2 (Santa Cruz Biotechnology, SC-722), p-AKT (Cell Signaling Technology, 4060S), p-mTOR (Cell Signaling Technology, 2971S), and β-actin (A5441; Sigma-Aldrich). The secondary antibodies were purchased from Cell Signaling Technology.

Lipid peroxidation

BODIPY C11 (Invitrogen, D83861) staining was performed to detect lipid peroxidation in cells. Briefly, the cells were seeded in a 12 well-plate coated with collagen and treated with RSL3 (2 µM), melatonin (1 mM), and zileuton (5 µM) for 30 min. Thereafter, the cells were stained with BODIPY C11 diluted with PBS at 37°C for 30 min and counterstained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. BODIPY C11 stained cells were observed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan) at 488 nm and 568 nm. For FACS analysis of BODIPY C11, the cells were treated with RSL3 (1 µM) and ferrostatin-1 (1 µM) for 1 h, trypsinized, and labeled with BODIPY C11 for 30 min. BODIPY C11-labeled cells were analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA) at a wavelength of 488 nm.

Iron measurement

FerroOrange (Dojindo, Rockville, MD, USA, F374) staining was performed to measure ferrous iron in cells. Briefly, the cells were seeded in 12-well plates coated with collagen and treated with RSL3 (2 µM), melatonin (1 mM), and zileuton (5 µM) for 30 min. Thereafter, the cells were stained using FerroOrange diluted with HBSS at 37°C for 30 min and visualized with 4,6-diamidino-2-phenylindole (DAPI). FerroOrange-stained cells were analyzed using a confocal laser- scanning microscope (Olympus) at 488 and 568 nm.

UUO mouse model

Six-week-old male BALB/c mice were purchased from Orient Bio Animal Inc. (Seongnam, Korea). The animal care and experimental procedures were conducted in accordance with the approval and guidelines of the INHA Institutional Animal Care and Use Committee (INHA 201228-740-2) of Inha University (Incheon, Korea). The left ureter of 50 mice was ligated to establish the UUO models. An incision was made with operating scissors in the position of the mouse kidney, vertically downwards for ~1 cm. The ureter was exposed after removal from around the ureter and ligated with a double ligature of black silk 5-0 (AILEE, Busan, Korea). After UUO, the skin of each mouse was sutured. Mice were randomly divided into five groups. One group did not undergo UUO surgery (Normal), the second group was administered saline (UUO), the third group was administered melatonin 20 mg/kg (UUO+Mel), the fourth group was administered zileuton 20 mg/kg (UUO+Zil), and the fifth group was administered both melatonin and zileuton (UUO+Mel+Zil) once per day for one week, through intraperitoneal injection. The body weights of mice were measured 3 times during the experiment. At the end of the experiment, mice were sacrificed and their kidney tissues were harvested.

Histopathological analysis

Mouse tissues were fixed in 10% paraformaldehyde and embedded in paraffin. The tissues were sectioned into 3-μm thick slices and stained with hematoxylin and eosin (H&E) for histopathological analysis. The degree of tubular injury was scored based on the percentage of tubular necrosis, tubular atrophy, tubular dilation, and tubulointerstitial damage: score 0: normal, score 1: <10%, score 2: 10-25%, score 3: 26-50%, score4: 51-75% and score 5: >75%. The glomerular size was expressed as a percentage by measuring the area using ImageJ 1.41 (National Institutes of Health, MD, USA).


Paraffin-embedded kidney tissues were deparaffinized and heated in citrate buffer (PH 6.0) for 30 min. The tissue sections were permeabilized with 0.5% Triton X-100 for 10 min. After endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide, the tissue sections were blocked using CAS block solution (Life Technologies, Carlsbad, CA, USA) for 1 h, incubated overnight with the primary antibody (1:50 dilution) at 4°C, and then with a biotinylated secondary antibody (1:100 dilution) at room temperature for 1 h. The sections were visualized using diaminobenzidine tetrahydrochloride substrate (DAB) and hematoxylin after an avidin-biotin peroxidase complex (ABC) reaction (ABC kit, Vector Laboratories, Burlingame, CA, USA). At least three random fields in each section were examined at 200 × magnification.

Statistical analysis

Statistical calculations were performed using the SPSS software for Windows (version 10.0, SPSS, Chicago, IL, USA). The results are expressed as mean ± standard deviation (SD) and were considered statistically significant at *p<0.05, **p<0.01, and ***p<0.001.


RSL3 induces ferroptosis-mediated cell death and lipid peroxidation in renal tubular cell lines

To characterize ferroptosis-dependent cell death and compare it with apoptosis-dependent cell death, we first identified the expression of cleaved caspase-3 and cleaved PARP after treatment with RSL3 and erastin (ferroptosis inducer), and staurosporine (STS), which is widely used to induce apoptotic cell death (Sulzmaier et al., 2012). STS induced an increase in cleaved caspase-3 and cleaved PARP expression in HKC-8 cells, while RSL3 and erastin did not result in such increase, suggesting the involvement of non-apoptotic pathway (Fig. 1A). To determine whether the cell death induced by RSL3 was due to ferroptosis, the cells were treated with inhibitors of different cell death types including apoptosis (the caspase inhibitor Z-VAD), necroptosis (necroptosis inhibitor, Nec-1), ferroptosis [ferroptosis inhibitor, ferrostatin (Fer-1) and deferoxamine (DFO)], and ROS accumulation [(ROS inhibitor, N-acetyl cysteine (NAC)]. RSL3 significantly induced cell death by ferroptosis, which was suppressed by the ferroptosis inhibitors Fer-1 and DFO, but not by Z-VAD or Nec-1 (Fig. 1A). To confirm this result, RSL3 was treated to HK-2 and HK-8 cells at various concentrations (0-1 µM) and tested for recovery by ferroptosis inhibitor (Fer-1 and DFO). As shown in Fig. 1B, RSL3 reduced cell viability similarly in a dose-dependent manner in both cell lines, which was recovered by Fer-1 and DFO. Consistently, RSL3 triggered prominent cell death by SYTOX green-stained cells in a dose-dependent manner (Fig. 1C). Also, lipid peroxidation as determined by BODIPY C11 staining using flow cytometry, was increased in RSL3-treated cells, which was suppressed by Fer-1 treatment (Fig. 1D), indicating that RSL3 induced ferroptosis-mediated cell death and lipid peroxidation in the kidney epithelial cell lines.

Figure 1. RSL3-induced ferroptosis of human renal tubular cells. (A) HKC-8 cells were treated with 1 µM RSL3 (6 h), 10 µM erastin (24 h), and 1 µM staurosporine (STS 2 h). Cleaved caspase-3 and cleaved PARP expressions were analyzed by western blotting. After HKC-8 cells were treated with RSL3 and inhibitors for 6 h, cell death was measured using Alamar blue assay. Fer-1: 1 µM ferrostatin-1; DFO: 100 µM deferoxamine; Z-VAD: 20 µM Z-VAD-FMK; Nec-1: 2 µM necrostatin-1; NAC: 5 mM N-acetyl cysteine. (B) HKC-8 and HK-2 cells were treated with RSL3 (1 µM) and ferroptosis inhibitors for 12 h; thereafter, the cell viability was assessed using the Alamar blue assay. (C) HKC-8 cells were treated with RSL3 (1 µM) and ferroptosis inhibitors and then stained with SYTOX green for 12 h. SYTOX green stains ferroptosis-induced cells. Scale bar=50 μm. (D) HKC-8 cells were treated with RSL3 (1 µM) and ferrostatin-1 (1 µM) for 1 h and then stained with BODIPY C11 for 30 min. BODIPY C11 stained cells were analyzed using a CytoFLEX Flow Cytometer. Data are represented as the mean ± SD for three separate experiments (*p<0.05, **p<0.01 and ***p<0.001).

Combination of melatonin and zileuton effectively suppresses ferroptosis induced by RSL3 in vitro

We evaluated the anti-ferroptosis effects of melatonin and zileuton in HKC-8 renal tubular cells. As shown in Fig. 2A, the increased cell death induced by RSL3 was significantly reduced when cells were treated with 1mM or higher of melatonin. Zileuton also inhibited RSL3-induced cell death in a dose-dependent manner. Combination treatment with melatonin (1 mM) and zileuton (5 µM) exerted synergistic effects on ferroptotic-cell death, similar to Fer-1 (Fig. 2B). In addition, SYTOX green and BODIPY C11 staining revealed that combination treatment significantly ameliorated cell death and lipid peroxidation (Fig. 2C, 2D) compared with individual treatment.

Figure 2. Synergistic cytotoxic effect of combination of melatonin and zileuton in human renal tubular cells. (A) HKC-8 cells were treated with RSL3 (1 µM), melatonin (0-2 mM), or zileuton (0-50 µM) for 12 h. Cell viability was measured using Alamar blue assay (n=3). (B) HKC-8 cells were treated with RSL3 (1 µM), melatonin (1 mM), and zileuton (5 µM) for 6 h. Alamar blue assay was performed to determine the combination effects of melatonin and zileuton (n=3). (C) HKC-8 cells were treated with RSL3 (1 µM), melatonin (1 mM), and zileuton (5 µM) for 6 h. The synergistic cytotoxic effect of melatonin and zileuton in human renal tubular cells was confirmed by SYTOX green staining. Scale bar=25 μm. (D) HKC-8 cells were treated with RSL3 (2 µM), melatonin (1 mM), and zileuton (5 µM) for 30 min and then stained with BODIPY C11 for 30 min. BODIPY C11-stained cells were analyzed with a confocal laser-scanning microscope (Olympus) at 488 and 568 nm. Scale bar=40 μm. Data are presented as means ± SD (*p<0.05, **p<0.01 and ***p<0.001).

Combination of melatonin and zileuton inhibits ferroptosis through the AKT/mTOR/NRF2 signaling pathway

In addition to lipid peroxidation, ferroptosis is also characterized by the lethal iron accumulation. The accumulation of ferrous iron products participates in RSL3-induced ferroptosis (Li et al., 2020). Thus, we determined the levels of intracellular iron in RSL3-induced renal tubular cells using a Fe2+ iron probe known as FerroOrange. The cells treated with RSL3 emitted markedly stronger orange fluorescence, which was synergistically decreased by the combination of melatonin and zileuton (Fig. 3A). In addition, RSL3 increased the expression of 4-HNE, an product of lipid peroxidation, and decreased the expression of GPX4, a key regulator of ferroptosis, in a time-dependent manner, indicating the involvement of ferroptosis. However, the combination of melatonin and zileuton decreased 4-HNE and HO-1 expression, and increased GPX4 expression in RSL3-treated HKC-8 renal tubular cells (Fig. 3B). To investigate the signaling pathway involved, we determined the expression of p-AKT, p-mTOR, and NRF2 in RSL3-induced ferroptosis as previous studies revealed that ferroptosis is associated with activation of the PI3K/AKT/mTOR and PI3K/AKT/NRF2 pathways (Yi et al., 2020; Liu et al., 2022b). As a result, we found that RSL3 induced the decreased expression of p-AKT and p-mTOR and the increased expression of NRF2. However, these changes were remarkably reversed by the combination of melatonin and zileuton (Fig. 3B), suggesting that this combination activated AKT/mTOR signaling, which promoted the nuclear translocation of NRF2 and increased the intracellular antioxidant response, thereby conferring inhibition of ferroptosis.

Figure 3. Inhibition of ferroptosis by the combination of melatonin and zileuton via AKT/mTOR/NRF2 signaling pathway. (A) HKC-8 cells were treated with RSL3 (2 µM), melatonin (1 mM), and zileuton (5 µM) for 30 min and then stained with FerroOrange for 30 min. FerroOrange stained cells were analyzed with a confocal laser-scanning microscope (Olympus) at 488 and 568 nm. Scale bar=50 μm. (B) HKC-8 cells were treated with RSL3 (1 µM), melatonin (1 mM), and zileuton (5 µM) for 12 and 24 h. The expression levels of 4-HNE, GPX4, HO-1, p-AKT, p-mTOR, and Nrf2 were determined using western blotting analysis. Data are presented as means ± SD (***p<0.001).

Combination of melatonin and zileuton alleviates tubular injury in UUO animal models

To correlate our in vitro findings in animal model, the effect of a combination of melatonin and zileuton on ferroptosis-induced kidney injury was evaluated in UUO mouse models (Fig. 4A). As expected, mice treated with either melatonin or zileuton had significantly low tubular injury scores than UUO-induced mice (Fig. 4B). Combination treatment resulted in the lowest tubular injury score compared with other treatment groups. Similar findings were found for glomerular injury; glomerular size was greater in combination of melatonin and zileuton group. In addition, no significant adverse effects or changes in body weight, liver damage, and ALT were observed in any group (Fig. 4C). Immunohistochemical staining revealed that 4-HNE expression was increased and GPX4 expression was decreased in the kidney tissue of the UUO group. However, these altered expression levels were significantly recovered by the combination of melatonin and zileuton treatment (Fig. 5A). The UUO group also showed an increase in 5-LOX expression, which was reduced by administering melatonin and/or zileuton. Additionally, we found that melatonin and/or zileuton treatment significantly decreased the expression of profibrotic markers, such as α-SMA and fibronectin expression (Fig. 5B). Consistently, tubulointerstitial fibrosis by Sirius Red staining was significantly lower in the treatment groups than in the UUO group. Furthermore, the expression levels of p-AKT, p-mTOR, and NRF2 were markedly increased in the combination treatment group. These findings suggest that the combination of melatonin and zileuton upregulates the AKT/mTOR/NRF2 signaling pathway, thereby ameliorating ferroptosis-related kidney injury in UUO mice.

Figure 4. Kidney injury inhibition by the combination of melatonin and zileuton in the UUO mouse model. (A) Mice were intraperitoneally administered 20 mg/kg melatonin and 20 mg/kg zileuton, once per day for one week (n=10). (B) Histological analysis of UUO mice model tissues based by hematoxylin and eosin (H&E) staining; tubular injury scoring: score 0: normal, score 1: <10%, score 2: 10-25%, score 3: 26-50%, score4: 51-75% and score 5: >75% and glomerular size measurement using the ImageJ 1.41. (C) Drug toxicity was assessed by body weight, serum AST concentration, and liver histological analysis (n=8). Scale bar=50 μm. Data are presented as means ± SD (*p<0.05, **p<0.01 and ***p<0.001).

Figure 5. Renoprotective effects of melatonin and zileuton in the UUO mouse model. (A) Expression levels of ferroptosis-related proteins (4-HNE, 5-LOX, and GPX4) in kidney tissue were determined by immunohistochemistry. (B) Expression levels of fibrosis-related proteins including α-SMA and fibronectin in kidney tissue were confirmed by immunohistochemistry and Sirius red staining. (C) Expression levels of AKT/mTOR/NRF2 signaling pathway-related proteins in kidney tissue were determined by immunohistochemistry. Data are presented as means ± SD (*p<0.05, **p<0.01 and ***p<0.001). Scale bar=50 μm.

In this study, we demonstrated the protective effect of a combination of melatonin and zileuton on UUO-induced kidney injury and subsequent fibrosis by inhibiting ferroptosis through the activation of the AKT/mTOR/NRF2 signaling pathway. Melatonin ameliorated lipid peroxidation and downregulated profibrotic markers in the UUO-kidney injury models and consistently inhibited RSL3-induced ferroptosis in kidney cell lines. The combination of melatonin and zileuton also synergically enhanced the anti-ferroptotic effects in UUO-kidney injury and RSL3-induced ferroptosis, suggesting its therapeutic potential in targeting kidney ferroptosis.

The role of ferroptosis in tubular cells in kidney disease has been emphasized in recent years. Linkermann et al. reported that kidney tubular cell necrosis in IRI and oxalate crystal-induced kidney injury models is mainly driven by ferroptosis and not by necroptosis (Linkermann et al., 2014; Zhao et al., 2020). UUO-induced kidney injury involves inflammation, oxidative stress, and tubular cell death, eventually leading to kidney fibrosis (Martínez-Klimova et al., 2019). Consistent with our results, UUO kidneys displayed ferroptotic changes, including increased lipid peroxidation, by-product generation, and changes in the expression of ferroptosis regulators, such as GPX4 (Zhang et al., 2021; Zhou et al., 2022). Therefore, targeting ferroptosis may be a good therapeutic strategy for alleviating interstitial inflammation and renal fibrosis. Indeed, a recent study revealed that UUO-induced inflammation and fibrosis are effectively alleviated by the classical ferroptosis inhibitor Fer-1 or DFO, used in preclinical studies (Zhou et al., 2022). Several studies have shown that melatonin improves kidney function and ameliorates histological damage in 5/6 nephrectomized rats and IRI mice (Kurcer et al., 2007; Li et al., 2023). Although the efficacy of melatonin in various kidney diseases has been reported, few studies have revealed that it reduces the renal damage and fibrosis by inhibiting ferroptosis induced by RSL3. In addition, as LOXs contribute to the generation of lipid peroxide that triggers the initiation of ferroptosis, and its overexpression is more susceptible to RSL3-induced ferroptosis (Shah et al., 2018), we speculated that zileuton, a 5-LOX inhibitor, could also inhibit ferroptosis-mediated renal cell death and damage in vitro and in vivo. Therefore, we evaluated whether the combination of melatonin and zileuton synergistically improved ferroptosis-mediated renal injury. As expected, we found that melatonin and zileuton acted as potent anti-ferroptotic agents in UUO-induced kidney injury. Indeed, the combination of melatonin and zileuton effectively reversed the downregulation of GPX4 increased by RSL3 and the upregulation of 4-HNE and HO-1, showing the synergistic effects on lipid peroxidation and tissue fibrosis. These results were confirmed by those obtained with the UUO animal model. More importantly, therapeutic efficacy of zileuton was demonstrated at a very low dose in this study (in vitro, in vivo; 5 µM, 20 mg/kg) compared with other studies (in vitro, and in vivo; 100-150 µM, 100 mg/kg) when combined with melatonin (Liu et al., 2015; Ou et al., 2016; Wenzel et al., 2017; Lee et al., 2022), suggesting that the combination of melatonin and zileuton may be a potential treatment to increase the effect of low drug doses against kidney-injured diseases without side effects.

NRF2 is a well-known transcription factor that plays a key role in antioxidation and is considered an important regulatory factor in ferroptosis (Song and Long, 2020). Under oxidative stress, NRF2 translocates to the nucleus and binds to the antioxidant response element (ARE), thereby initiating the transcription of antioxidant enzymes (Suzuki et al., 2013). Thus, NRF2 also serves as a factor that inhibits ferroptosis. And the PI3K/AKT/mTOR pathway is an important pathway that regulates cell proliferation. AKT which is a downstream target of PI3K, plays a pivotal role in several cellular processes, including cell proliferation, apoptosis, cell migration, and transcription (Manning and Toker, 2017). AKT activity is impaired in several types of CKD, leading to the development of glomerular lesion, podocyte injury, and CKD progression. Also, it regulates neuronal ferroptosis in brain injury (Liu et al., 2022a). Recent evidence also suggests that mTOR is a key regulator of ferroptosis (Lei et al., 2021). Its overexpression showed protective effects against ferroptosis induced by erastin and RSL3 in cardiomyocytes, and RSL3 suppressed mTOR activation during ferroptosis (Baba et al., 2018; Liu et al., 2021). Importantly, NRF2 is activated by the PI3K/Akt pathway and is involved in regulating cell damage (Zou et al., 2013; He et al., 2020).

Recent studies have shown that the effect of melatonin on ferroptosis is depending on NRF2, and it has a modulatory effect on the NRF2 signaling pathway (Ahmadi and Ashrafizadeh, 2020; Ma et al., 2020). And it inhibits ferroptosis by activating the PI3K/AKT/mTOR signaling pathway (Li et al., 2022). Therefore, we investigated whether the expressions of AKT, mTOR, and NRF2 were altered by melatonin combined with zileuton. Our study showed that NRF2 expression was markedly enhanced by combination treatment and individual treatment with melatonin. Also, AKT/mTOR expression was downregulated in response to RSL3 treatment but remarkably enhanced by the combination treatment. Considering a previous study showing that melatonin activated AKT/mTOR signaling and increased NRF2 translocation into the nucleus, melatonin may improve the anti-ferroptosis effect of zileuton through the AKT/mTOR/NRF2 signaling axis. As the role of the AKT/mTOR/NRF2 pathway axis in kidney ferroptosis has rarely been addressed, our findings may provide mechanistic insights.

Collectively, we revealed the renoprotective effects of the combination of melatonin and zileuton in UUO-induced kidney injury and fibrosis models through their anti-ferroptotic action via the AKT/mTOR/NRF2 pathway (Fig. 6). Our findings provide new insights into potential therapeutic strategies for kidney injury and fibrosis, and lay the foundation for the future development of clinical regimens.

Figure 6. Schematic diagram of the novel ferroptosis-targeted therapeutic strategy by combination of melatonin and zileuton. The scheme shows that the combination of melatonin and zileuton increases cell survival by inhibiting the ferroptosis signaling pathway in renal injury.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government Ministry of Science and Information and Communication Technologies (NRF-2021R1A5A2031612, 2021R1A2B5B03086410, 2022R1G1A1011829, 2022R1A2C1092933) and Inha University Research Grant.


The authors declare that they have no competing interests.

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