Formononetin Upregulates Nrf2/HO-1 Signaling and Prevents Oxidative Stress, Inflammation, and Kidney Injury in Methotrexate-Induced Rats

Acute kidney injury (AKI) is a serious complication of methotrexate (MTX). This study explored the protective effect of the isoflavone formononetin (FN) against MTX nephrotoxicity with an emphasis on oxidative stress, inflammation, and nuclear factor (erythroid-derived 2)-like 2/heme oxygenase 1 (Nrf2/HO-1) signaling. Rats received FN (10, 20, and 40 mg/kg) for 10 days and a single dose of MTX on day 7. MTX induced kidney injury was characterized by increased serum creatinine and urea, kidney injury molecule-1 (Kim-1), and several histological alterations. FN ameliorated kidney function and inhibited the renal tissue injury induced by MTX. Reactive oxygen species (ROS), lipid peroxidation (LPO), nitric oxide, and 8-Oxo-2′-deoxyguanosine were increased, whereas antioxidant defenses were diminished in the kidney of MTX-administered rats. In addition, MTX upregulated renal iNOS, COX-2, TNF-α, IL-1β, Bax, caspase-9, and caspase-3, and decreased Bcl-2, Nrf2, and HO-1. FN suppressed oxidative stress, LPO, DNA damage, iNOS, COX-2, proinflammatory cytokines, and apoptosis, and boosted Bcl-2, antioxidants, and Nrf2/HO-1 signaling in MTX-administered rats. In conclusion, FN prevents MTX-induced AKI by activating Nrf2/HO-1 signaling and attenuates oxidative damage and inflammation. Thus, FN may represent an effective adjuvant that can prevent MTX nephrotoxicity, pending further mechanistic studies.


Introduction
Methotrexate (MTX), a folic acid antagonist, is a potent chemotherapeutic agent used in the treatment of malignancies and inflammatory diseases [1]. However, some restrictions have been made on its clinical applications because of its nephrotoxicity and other adverse effects [2][3][4].

Determination of HO-1 Activity
Total HO-1 activity was measured following the method of Abraham et al. [39]. Briefly, tissue samples were mixed with 2 mM glucose-6-phosphate, 0.8 mM NADPH, 20 µM hemin, and 0.2 U glucose-6-phosphate dehydrogenase in a total volume of 1.2 mL. After incubation at 37 • C for 1 h, the absorbance was measured at 464 nm, and the activity was normalized to the control group.

Determination of ATP
Adenosine triphosphate (ATP) content in the renal homogenate was measured using a kit supplied by Sigma (St. Louis, MO, USA). In this test, ATP is determined by phosphorylating glycerol, and the product is proportional to the amount of ATP in the sample and is determined calorimetrically at 570 nm.

Histological Examination of Kidney Sections
Specimens from kidney fixed in 10% buffered formalin were dehydrated, embedded in paraffin wax and cut into 5-µm sections. Following deparaffinization and rehydration, the sections were processed for hematoxylin and eosin (H&E) staining and then examined.

Gene Expression Analysis
The effect of MTX and FN on the mRNA expression levels of inducible nitric oxide synthase (iNOS), BAX, BCL-2, cyclooxygenase-2 (COX-2), IL-1β, TNF-α, Nrf2, HO-1, and caspase-3 was quantified using qRT-PCR, as previously reported [40][41][42]. Isolation of RNA from the frozen kidney samples was performed using TRIzol reagent (Invitrogen, Waltham, MA, USA). Following treatment with RNase-free DNase (Qiagen, Düsseldorf, Germany), RNA was quantified on a nanodrop, and samples with A260/A280 nm > 1.7 were reverse transcribed into cDNA. PCR amplification of the cDNA was carried out using SYBR Green master mix and the primers listed in Table 1. The amplification data were analyzed by the 2 −∆∆Ct method [43] and normalized to β-actin. Table 1. Primers used for qRT-PCR.

Western Blotting
Samples from the kidney were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors, centrifuged, and protein content was determined in the supernatant using Bradford reagent. Forty micrograms of proteins were subjected to 10% SDS-PAGE followed by electrotransfer to a nitrocellulose membrane, which was blocked and probed with anti-Nrf2 and anti-β-actin. After overnight incubation at 4 • C and washing, the secondary antibodies were added, and the blots were developed. The obtained bands were scanned, and intensity was quantified using ImageJ (version 1.32 j, NIH, USA). The results were normalized to β-actin and presented as a percent of control. All antibodies were provided by Novus Biologicals (Centennial, CO, USA).

Assessment of the Impact of FN on MTX Cytotoxicity in HepG-2 Cells
HepG2 cells were grown in PRMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin/streptomycin (100 U/mL) at 37 • C and 5% CO 2 . Upon confluency, the cells were trypsanized and seeded in 96-well plates (10 4 cells/well). The cells were treated with different doses of FN for 24 h, followed by MTX for 48 h. The cells were stained with 5 mg/mL MTT, incubated for 2 h at 37 • C. The medium was replaced by 100 µl DMSO, and the absorbance was read at 570 nm after 10 min.

Statistical Analysis
The significance value of the obtained data was analyzed by one-way (ANOVA) followed by Tukey's test using GraphPad Prism 7 (La Jolla, CA, USA). All results were presented as mean ± standard error of the mean (SEM). A p-value < 0.05 was considered significant.

FN Prevents Renal Dysfunction and Injury in MTX-Administered Rats
MTX increased creatinine, urea, and Kim-1 significantly (p < 0.001) as depicted in Figure 1A-C. FN attenuated the MTX-induced kidney dysfunction, without altering the kidney function markers in normal rats ( Figure 1A-C).

Statistical Analysis
The significance value of the obtained data was analyzed by one-way (ANOVA) followed by Tukey's test using GraphPad Prism 7 (La Jolla, CA, USA). All results were presented as mean ± standard error of the mean (SEM). A p-value < 0.05 was considered significant.

FN Prevents Renal Dysfunction and Injury in MTX-Administered Rats
MTX increased creatinine, urea, and Kim-1 significantly (p < 0.001) as depicted in Figure 1A-C. FN attenuated the MTX-induced kidney dysfunction, without altering the kidney function markers in normal rats ( Figure 1A-C).
The renoprotective efficacy of FN was supported by the histological examination ( Figure 1D). While the control and FN-supplemented animals showed normal renal tubules and corpuscles, MTX caused multiple alterations, including interstitial hemorrhage, glomerular atrophy, infiltration of leukocytes, and others. In contrast, rats which received 10, 20, and 40 mg/kg FN showed noticeable improvement in the kidney structure where all doses remarkably prevented MTX-induced tissue injury ( Figure 1D).   The renoprotective efficacy of FN was supported by the histological examination ( Figure 1D). While the control and FN-supplemented animals showed normal renal tubules and corpuscles, MTX caused multiple alterations, including interstitial hemorrhage, glomerular atrophy, infiltration of leukocytes, and others. In contrast, rats which received 10, 20, and 40 mg/kg FN showed noticeable improvement in the kidney structure where all doses remarkably prevented MTX-induced tissue injury ( Figure 1D).

FN Prevents Oxidative Stress and DNA Damage in MTX-Induced Rats
The effect of FN on oxidative stress and DNA damage induced by MTX was assessed by determining ROS, LPO, NO, and 8-Oxo-dG. MTX triggered a significant increase (p < 0.001) in renal ROS (Figure 2A), LPO ( Figure 2B), and NO ( Figure 2C) levels. 8-Oxo-dG, a marker of DNA damage, was markedly increased in the kidney following MTX injection ( Figure 2D). Rats received FN before MTX exhibited noticeable amelioration of renal ROS, LPO, NO, and 8-Oxo-dG levels.

FN Prevents Oxidative Stress and DNA Damage in MTX-Induced Rats
The effect of FN on oxidative stress and DNA damage induced by MTX was assessed by determining ROS, LPO, NO, and 8-Oxo-dG. MTX triggered a significant increase (p < 0.001) in renal ROS (Figure 2A), LPO ( Figure 2B), and NO ( Figure 2C) levels. 8-Oxo-dG, a marker of DNA damage, was markedly increased in the kidney following MTX injection ( Figure 2D). Rats received FN before MTX exhibited noticeable amelioration of renal ROS, LPO, NO, and 8-Oxo-dG levels.
In addition to suppressing ROS generation and oxidative DNA damage elicited by MTX, FN boosted renal antioxidant defenses. MTX diminished renal GSH ( Figure 3A), SOD ( Figure 3B), CAT ( Figure 3C), and GPx ( Figure 3D) in rats. In contrast, animals received 10, 20, or 40 mg/kg FN before MTX exhibited markedly alleviated renal antioxidants. Of note, normal rats received FN showed no changes in both oxidative stress markers and antioxidants.

FN Upregulates Nrf2/HO-1 Signaling in Kidney of MTX-Administered Rats
To explore the mechanism underlying the antioxidant efficacy of FN, Nrf2 expression, HO-1 both expression and activity, along with mRNA expression of SOD and CAT, were assessed. MTX downregulated renal Nrf2 in both the mRNA and protein in rats; an effect that was significantly attenuated by FN ( Figure 4A,B). These data highlighted the MTX-induced suppression of Nrf2 signaling. This notion was confirmed by the diminished HO-1 mRNA ( Figure 4C) and activity ( Figure  4D), and mRNA abundance of SOD ( Figure 4E) and CAT ( Figure 4F) in the kidney of rats following MTX injection. FN (10, 20, and 40 mg/kg) prevented the deleterious effect of MTX on HO-1, SOD, and CAT. Although FN had no effect on Nrf2, SOD, and CAT in normal rats, it increased renal HO-1, both gene expression and activity.

FN Upregulates Nrf2/HO-1 Signaling in Kidney of MTX-Administered Rats
To explore the mechanism underlying the antioxidant efficacy of FN, Nrf2 expression, HO-1 both expression and activity, along with mRNA expression of SOD and CAT, were assessed. MTX downregulated renal Nrf2 in both the mRNA and protein in rats; an effect that was significantly attenuated by FN ( Figure 4A,B). These data highlighted the MTX-induced suppression of Nrf2 signaling. This notion was confirmed by the diminished HO-1 mRNA ( Figure 4C) and activity ( Figure 4D), and mRNA abundance of SOD ( Figure 4E) and CAT ( Figure 4F) in the kidney of rats following MTX injection. FN (10, 20, and 40 mg/kg) prevented the deleterious effect of MTX on HO-1, SOD, and CAT. Although FN had no effect on Nrf2, SOD, and CAT in normal rats, it increased renal HO-1, both gene expression and activity.

FN Increases Renal ATP Levels in MTX-Induced Rats
Owing to the role of MTX in inducing mitochondrial dysfunction, we investigated the effect of MTX and FN on ATP content in the kidney. MTX diminished renal ATP (p < 0.001) and FN treatment prior to MTX restored mitochondrial function ( Figure 5). FN alone had no effect on renal ATP of normal rats.

FN Increases Renal ATP Levels in MTX-Induced Rats
Owing to the role of MTX in inducing mitochondrial dysfunction, we investigated the effect of MTX and FN on ATP content in the kidney. MTX diminished renal ATP (p < 0.001) and FN treatment prior to MTX restored mitochondrial function ( Figure 5). FN alone had no effect on renal ATP of normal rats.

FN Suppresses Renal Inflammation in MTX-Induced Rats
Analysis of the mRNA expression in the kidney of rats which received MTX revealed significant upregulation of COX-2, iNOS, TNF-α, and IL-1β ( Figure 6). The inflammatory response following MTX administration was evidenced by the increased circulating levels of TNF-α and IL-1β. FN significantly attenuated the expression and circulating levels of the assayed inflammatory mediators and its effect on TNF-α and IL-1β mRNA and serum TNF-α was dose-dependent ( Figure 6). All determined proinflammatory markers showed no changes in rats received 40 mg/kg FN.

FN Suppresses Renal Inflammation in MTX-Induced Rats
Analysis of the mRNA expression in the kidney of rats which received MTX revealed significant upregulation of COX-2, iNOS, TNF-α, and IL-1β ( Figure 6). The inflammatory response following MTX administration was evidenced by the increased circulating levels of TNF-α and IL-1β. FN significantly attenuated the expression and circulating levels of the assayed inflammatory mediators and its effect on TNF-α and IL-1β mRNA and serum TNF-α was dose-dependent ( Figure 6). All determined proinflammatory markers showed no changes in rats received 40 mg/kg FN.

FN Prevents MTX-Induced Apoptosis
Oxidative damage, mitochondrial dysfunction, and inflammation can drive cell death. Given that MTX administration was associated with these processes, we determined its effect as well as the ameliorative potential of FN on apoptosis. MTX-mediated apoptosis was evidenced by the increased mRNA abundance of Bax (p < 0.001; Figure 7A) with a concomitant decline in Bcl-2 expression ( Figure 7B) along with an increased Bax/Bcl-2 ratio ( Figure 7C). Moreover, caspase-3 mRNA ( Figure 7D) and activities of caspase-9 ( Figure 7E) and caspase-3 ( Figure 7F) were significantly increased following MTX injection. Remarkably, FN protected against MTX-induced apoptosis by diminishing Bax and caspases and enhancing Bcl-2 in rat kidney. FN had no effect on all apoptotic markers in normal animals (Figure 7).

FN Does Not Interfere with the Antitumor Activity of MTX
To assess the effects of FN on the antitumor properties of MTX, we investigated the cytotoxic effects of MTX alone and in combination with FN in HepG2 cells. Treatment of HepG2 cells with either MTX or FN resulted in cytotoxicity. Per-treatment of HepG2 cells with FN did not interfere with the cytotoxic action of MTX (Figure 8).

FN Does Not Interfere with the Antitumor Activity of MTX
To assess the effects of FN on the antitumor properties of MTX, we investigated the cytotoxic effects of MTX alone and in combination with FN in HepG2 cells. Treatment of HepG2 cells with either MTX or FN resulted in cytotoxicity. Per-treatment of HepG2 cells with FN did not interfere with the cytotoxic action of MTX (Figure 8).

Discussion
MTX is an effective anticancer and immunosuppressive agent; however, its serious side effects limit its clinical applications. The mechanism of MTX-induced nephrotoxicity involves oxidative injury and inflammation [6,7]. Despite the accumulating knowledge about MTX nephrotoxicity, efficient pharmacotherapies hampering this serious complication are unavailable. Hence, it is becoming more urgent to find novel therapeutic approaches to prevent and/or treat MTX nephrotoxicity. Herein, we investigated the protective effect of FN, a natural isoflavone with promising pharmacological activities, against MTX-induced AKI in rats. Our findings demonstrated that FN can effectively prevent renal oxidative injury, inflammation, and apoptosis in MTXadministered rats, possibly through augmenting Nrf2 signaling.
AKI in MTX-administered rats was evidenced by the elevated serum creatinine and urea, and renal Kim-1. Creatinine and urea are commonly measured as indices of glomerular function [44,45]. Kim-1 is a transmembrane protein markedly upregulated in renal injuries, particularly renal proximal tubules injury [46,47]. Therefore, elevated levels of these kidney injury biomarkers indicate renal dysfunction. AKI, induced by MTX, was further confirmed by the histological alterations. The examination of kidney sections revealed leukocyte infiltration, degenerative changes of glomeruli and tubular epithelial cells, interstitial hemorrhage, and others. These results were supported by previous studies where MTX administration was associated with altered renal function and histological structures [6][7][8]. Kidney injury is caused by the precipitation of MTX and its 7-hydroxy metabolite in the renal tubules, resulting in tubular obstruction and compromised renal clearance

Discussion
MTX is an effective anticancer and immunosuppressive agent; however, its serious side effects limit its clinical applications. The mechanism of MTX-induced nephrotoxicity involves oxidative injury and inflammation [6,7]. Despite the accumulating knowledge about MTX nephrotoxicity, efficient pharmacotherapies hampering this serious complication are unavailable. Hence, it is becoming more urgent to find novel therapeutic approaches to prevent and/or treat MTX nephrotoxicity. Herein, we investigated the protective effect of FN, a natural isoflavone with promising pharmacological activities, against MTX-induced AKI in rats. Our findings demonstrated that FN can effectively prevent renal oxidative injury, inflammation, and apoptosis in MTX-administered rats, possibly through augmenting Nrf2 signaling.
AKI in MTX-administered rats was evidenced by the elevated serum creatinine and urea, and renal Kim-1. Creatinine and urea are commonly measured as indices of glomerular function [44,45]. Kim-1 is a transmembrane protein markedly upregulated in renal injuries, particularly renal proximal tubules injury [46,47]. Therefore, elevated levels of these kidney injury biomarkers indicate renal dysfunction. AKI, induced by MTX, was further confirmed by the histological alterations. The examination of kidney sections revealed leukocyte infiltration, degenerative changes of glomeruli and tubular epithelial cells, interstitial hemorrhage, and others. These results were supported by previous studies where MTX administration was associated with altered renal function and histological structures [6][7][8]. Kidney injury is caused by the precipitation of MTX and its 7-hydroxy metabolite in the renal tubules, resulting in tubular obstruction and compromised renal clearance [3,5]. Remarkably, FN afforded protection and alleviated kidney function in MTX-intoxicated rats, indicating a potent renoprotective efficacy. FN effectively reduced creatinine, urea, and Kim-1 and prevented histological alterations induced by MTX. In accordance, FN has shown renoprotective effects in type 2 diabetes [29], and rhabdomyolysis- [48] and cisplatin-induced toxicity [24].
Oxidative stress is one of the main mechanisms through which MTX induces tissue damage [6,7,11,49]. MTX has been reported to increase ROS production by suppressing homocysteine remethylation, depletion of NADPH, stimulation of neutrophils, activation of NADPH oxidase, and mitochondrial dysfunction [9][10][11]13]. In turn, ROS can cause cell injury through oxidizing lipid and proteins, inactivating antioxidant enzymes, and provoking DNA damage, leading to a dysfunctional cellular protective response [50]. LPO can lead to loss of membrane integrity by altering its fluidity and permeability and inactivating membrane-bound receptors and enzymes. ROS also promote the oxidation of amino acid side chains and the formation of protein-protein cross-linkages, increasing the havoc throughout the cell [51]. Consistent with several previous studies [6,7], MTX increased renal ROS, LPO, and NO, and reduced GSH and antioxidant enzymes. NO, being derived from iNOS induction, can react with superoxide anion, forming peroxynitrite that, among other effects, modifies purine and pyrimidine bases, resulting in DNA breaks [52]. In accordance, our findings showed an increase in 8-Oxo-dG, a marker of DNA damage. Moreover, several lines of evidence suggested direct and indirect mitochondria-related toxicity as a common effector mechanism of nephrotoxicity. ROS can cause mitochondrial damage which, in turn, results in the formation of mitochondrial permeability transition pore, further resulting in altered oxidative phosphorylation and progressive depletion of ATP, which may eventually culminate in cell death [53]. Our results showed that MTX administration diminished ATP content in the kidney. Therefore, maintenance of the cellular redox balance can represent an effective strategy to prevent MTX-induced AKI.
FN has been reported to exert antioxidant actions in several preclinical models of a variety of pathological conditions associated with oxidative stress [24,26,29]. Hence, we assumed that the nephroprotective efficacy of FN could be attributed to its antioxidant activity. Our results showed attenuation of ROS generation and DNA damage, enhancement of cellular antioxidants, and restoration of mitochondrial function in MTX-administered rats. Thus, FN prevented MTX-provoked AKI via suppressing oxidative injury and restoring antioxidant defenses and mitochondrial function.
In addition to oxidative stress, MTX-induced ROS generation can elicit various stress signaling, such as NF-κB, which promotes the expression of TNF-α, IL-1β, COX-2, and iNOS [6][7][8]49]. Here, MTX upregulated COX-2 and iNOS, and this explained the increase in NO levels. MTX has also increased both gene expression and serum TNF-α and IL-1β, demonstrating an inflammatory response. During glomerular injury and tubulointerstitial diseases, NF-κB activation in podocytes, mesangial cells, and tubular cells has been reported [54,55]. Recent work from our lab demonstrated the activation of ROS/NF-κB/NLRP3 inflammasome axis in the kidney of rodents received MTX [8,49]. FN diminished the expression and circulating levels of proinflammatory mediators in MTX-intoxicated rats, demonstrating an anti-inflammatory activity. Consistently, FN attenuated neuroinflammatory reaction through downregulating TNF-α and IL-1β and upregulating IL-10 in a rat model of traumatic brain injury [56]. In rat insulinoma cell line, FN blocked IL-1β-induced NF-κB activation and consequent iNOS expression and NO production [28]. FN has also suppressed cognitive impairment in diabetic mice, possibly through the downregulation of TLR4/NF-κB signaling and NLRP3 inflammasome [56]. Given the recently reported role of ROS/NF-κB/NLRP3 inflammasome axis in MTX AKI [8,49], it could be assumed that suppression of this signaling pathway has a role in the anti-inflammatory effect of FN against MTX nephrotoxicity.
Accumulating evidence has pointed to the role of oxidative injury and inflammation in provoking apoptosis in MTX-induced AKI [6][7][8]49]. In this study, increased ROS and inflammation were associated with oxidative DNA damage and upregulated Bax and caspases. Bax is a proapoptotic protein that elicits cytochrome c release from the mitochondria and consequent activation of caspases. In addition, excessive mitochondrial ROS production during MTX metabolism can damage the mitochondrial membrane, resulting in the loss of membrane potential and consequently the release of cytochrome c which ultimately culminates in renal apoptosis by activating caspase-3 [13]. Thus, inhibition of MTX-mediated ROS generation and proinflammatory cytokines production can protect against apoptosis. Given its dual ability to attenuate oxidative injury and inflammation, FN prevented apoptosis in MTX-administered rats as shown by the diminished expression and/or activity of Bax and caspases and upregulation of the antiapoptotic Bcl-2. In the same context, previous studies have shown that FN suppressed apoptosis in rhabdomyolysis- [48] and cisplatin-induced nephrotoxicity [24] in rodents and cisplatin-induced LLC-PK1 cells [57].
To gain more insight into the potential underlying mechanism of the renoprotective effect of FN, we investigated the role of Nrf2/HO-1 signaling in mediating the effects of FN. Extensive evidence indicated that Nrf2 signaling plays comprehensive cytoprotective roles through activating the expression of several genes encoding detoxification, antioxidant, and anti-inflammatory proteins [6, 14,17,[58][59][60]. Therefore, pharmacological activation of the Nrf2 signaling may provide an additional protective strategy against chemotherapy-induced AKI. Herein, MTX diminished Nrf2 signaling as evidenced by the diminished Nrf2, HO-1, and other antioxidant enzymes. Consistently, we have recently demonstrated reduced renal Nrf2 and HO-1 expression in MTX-intoxicated rats [6,7]. Although exposure to moderate oxidative stress leads to Nrf2 activation, excessive and sustained ROS generation can diminish Nrf2 signaling in the kidney [6-8,49] and liver [4,18] of rats challenged with MTX. Thus, the diminished Nrf2/ HO-1 pathway is a direct consequence of the sustained ROS generation induced by MTX.
FN upregulated renal Nrf2 and consequent induction of HO-1, CAT, and SOD in rats challenged with MTX. Interestingly, FN increased HO-1 mRNA and activity in normal rats. Upregulated Nrf2 signaling by FN resulted in enhanced antioxidants and diminished ROS and oxidative damage. Furthermore, activation of Nrf2 had a key role in the anti-inflammatory and antiapoptotic effects of FN. Nrf2 and HO-1 can directly inhibit NF-κB signaling and proinflammatory cytokines, and activate the anti-inflammatory cytokines, thereby regulating the inflammatory cascade [61]. In this context, the lack of Nrf2 aggravated inflammation through activation of NF-κB and downstream proinflammatory mediators in murine cultured astrocytes [62], and the asperity of drug hepatotoxicity in mice [63]. The role of Nrf2 in mediating the anti-inflammatory and antiapoptotic efficacies of FN in MTX-administered rats was supported by previous reports which demonstrated increased expression of Nrf2 and suppressed inflammation in acetaminophen-induced hepatotoxicity [24], traumatic brain injury [64], and rhabdomyolysis-induced renal injury [48] in rodents. Another interesting finding in this study was the significant increase in HO-1 mRNA and activity in the kidney of normal rats.
To further investigate whether the above-mentioned beneficial effects of FN would interfere with the antitumor activity of MTX, we evaluated its effect on MTX-induced cytotoxicity in HepG2 cells. Per-treatment of HepG2 cells with FN did not interfere with the cytotoxic action of MTX. Indeed, several molecular mechanisms have been involved in the anticancer activity of MTX; however, oxidative/nitrative stress does not seem to be implicated. Efficient antioxidant strategies, which counteract the toxicity of chemotherapies, do not interfere with their antitumor action. For example, the cardioprotective agent dexrazoxane that reduces doxorubicin cardiotoxicity is a potent antioxidant [65]. Here, the renoprotective effect of FN did not interfere with the antitumor efficacy of MTX. In addition, FN by itself has also been demonstrated to exert various antitumor properties, including inhibition of AKT phosphorylation and induction of cervical cancer cell line HeLa apoptosis in a dose-dependent manner [66].

Conclusions
Our findings indicate that the natural isoflavone FN prevented AKI induced by MTX. FN suppressed excess ROS, oxidative injury, and improved mitochondrial function. Consequently, FN attenuated inflammation and inhibited cell death in the kidney of rats and hence, possesses a therapeutic benefit against MTX toxicity. Activation of Nrf2/HO-1 signaling and enhancement of the antioxidant defenses represent the main mechanism underlying the nephrotprotective effect of FN (Figure 9). These results provide new information on the protective effects of FN against MTX-induced AKI. In addition, the reported antineoplastic properties of FN in various malignancies are particularly encouraging from the therapeutic point of view. However, the exact mechanism underlying the renoprotective action of FN undoubtedly deserves further exploration in upcoming studies. particularly encouraging from the therapeutic point of view. However, the exact mechanism underlying the renoprotective action of FN undoubtedly deserves further exploration in upcoming studies.

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