Abstract
Background and purpose:
Paraquat (PQ) is a toxic herbicide to humans and animals that causes kidney damage. Ketotifen (KTF) is a first-generation antihistamine and a potent mast cell stabilizer with antioxidant and anti-inflammatory properties. This study aimed to evaluate the protective effect of KTF against PQ-induced nephrotoxicity in mice by suppressing inflammation, oxidative stress, and inducible nitric oxide synthase (iNOS).
Experimental approach:
Thirty-five male NMRI mice were randomly divided into 5 groups (n = 7) as follows: control group receiving normal saline as vehicle (10 mL/kg/day, IP), KTF, PQ, PQ + KTF 2, and PQ + KTF 4 groups. PQ was administered as a single dose (30 mg/kg, IP) on day 4. The mice received KTF at the doses of 2 and 4 mg/kg/day, IP, for seven days. On the eighth day of the study, the mice were anesthetized, and their kidneys were removed. Then, oxidative stress and inflammation factors and iNOS protein expression were evaluated, and histological examinations were performed.
Findings/Results:
KTF resulted in a significant decrease in blood urea nitrogen, creatinine, thiobarbituric acid-reactive substances, tumor necrosis factor alpha, and iNOS protein expression, and a significant increase in albumin, total thiol, and the activity of antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase in PQ-induced nephrotoxicity compared with the PQ group, which were confirmed by histopathological findings.
Conclusion and implications:
The results of this study showed that KTF could improve PQ-induced renal injury in mice by reducing oxidative stress, inflammation, and iNOS protein expression.
Keywords: Inflammation, Ketotifen, Nephrotoxicity, Oxidative stress, Paraquat.
INTRODUCTION
Despite the environmental problems caused by herbicides, these compounds continue to be used as essential components of integrated weed management worldwide. N, N-dimethyl 4,4-bipyridyl is a non-selective contact herbicide belonging to the quaternary ammonium compounds known as quats. The most famous quats are paraquat (PQ) and diquat (1,2). Peak plasma concentrations of PQ occur 30-90 min after gastrointestinal exposure. This semi-volatile compound is excreted unchanged by the kidneys and is usually detectable in urine 6-24 h after exposure (3). The mitochondrial and microsomal redox systems are implicated in the production of superoxide anion in PQ toxicity, leading to the production of reactive oxygen species (ROS) (4). The free radicals also lead to lipid peroxidation, mitochondrial membrane disruption, damage to DNA and protein, depletion of cellular and mitochondrial antioxidant stores (e.g., decreased activity of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) enzymes, and increased glutathione oxide), and ultimately mitochondrial swelling and decreased ATP synthesis (5). PQ poisoning can lead to multi-organ failure, including kidney failure. Approximately 90% of metabolized PQ is excreted in the urine by the kidney. PQ can cause significant damage to the cells of the proximal tubules of the kidney, ultimately leading to acute kidney injury (AKI). These cells are critical for filtering and reabsorbing substances in the kidney. Damaged proximal tubules can impair the excretion of PQ from the kidney, leading to its accumulation and worsening kidney damage (6). In some cases, PQ poisoning can lead to Fanconi syndrome, a condition in which the proximal tubules are unable to reabsorb essential substances such as glucose, phosphate, and amino acids, resulting in these essential substances being excreted in the urine (7). Although PQ poisoning is one of the most lethal poisonings in hospital emergency departments, PQ is still widely used in agriculture due to its broad spectrum of effects and low cost. Mortality rates of up to 75% have been reported, the highest among herbicides (8). No specific treatment has been reported for PQ poisoning. Conventional treatments include hemoperfusion and hemodialysis, and the use of immunosuppressants and antioxidants (9). PQ is a pneumotoxic agent that exerts its toxic effects by increasing intracellular levels of superoxide. PQ poisoning leads to increased expression of inducible nitric oxide synthase (iNOS) in the vascular endothelial layer and, while increasing nitric oxide (NO) production, leads to a systemic inflammatory response (10). Also, the high levels of tumor necrosis factor alpha (TNF-α) in PQ poisoning led to increased expression of iNOS and increased NO production in endothelial cells, which can affect vascular responses (11). Mast cells are innate immune cells that release mediators such as cytokines, chemokines, and leukotrienes (12). Research suggests that mast cells play a key role in drug-induced nephrotoxicity and are significantly increased in conditions of renal injury (13). Ketotifen (KTF) is a tricyclic benzocycloheptathiophene derivative and a potent mast cell stabilizer that is widely used to control certain diseases such as allergy, asthma, and inflammatory disorders (14). KTF can significantly block H1 receptors, reduce the number of mast cells, and inhibit their activity (15). KTF also leads to a decrease in the infiltration of B lymphocytes, T lymphocytes, macrophages, neutrophils, and eosinophils, and inhibits the release, proliferation, and apoptosis of IgE. KTF reduces oxidative stress and inhibits the expression of proteins associated with mast cell and macrophage aggregation (16). Given the availability and the high mortality rate of herbicide PQ due to accidental or intentional poisoning, as well as the lack of a definitive treatment for this toxic agent, this study aimed to investigate the protective effects of KTF on the PQ-induced nephrotoxicity in mice through the suppression of oxidative stress and inflammation.
MATERIALS AND METHODS
Chemicals and reagents
KTF (purity > 98%, CAS: 34580-14-8) and PQ (purity 96%, CAS: 75365-73-0) were prepared by Sigma-Aldrich Company, USA. Blood urea nitrogen (BUN), creatinine (Cr), and albumin (Alb) assay kits were prepared by Pars Azmoon, Iran. SOD, CAT, and GPx assay kits were prepared by the ZellBio Company, Germany. The TNF-α ELISA kit was prepared by the Carmania Company, Iran.
Animals
In this study, 35 male NMRI mice (weighing 23 ± 2 g) were purchased
from the Laboratory Animal Breeding Center of Ahvaz Jundishapur University of Medical Sciences (AJUMS, Ahvaz, Iran) and housed in an animal room under a light/dark cycle of 12 h/12 h, a temperature of 25 ± 2 °C, relative humidity, and free access to sufficient water and food. All ethical principles for the use and care of animals, as well as all experimental procedures, were approved by the Ethics Committee of AJUMS with Ethical No. IR.AJUMS.ABHC.REC.1401.077.
Experimental design
Thirty-five male NMRI mice were randomly divided into 5 groups (n = 7) as follows: control group treated with normal saline (10 mL/kg/day) as vehicle intraperitoneally (IP); KTF group treated with KTF at the dose of 4 mg/kg/day for seven days; PQ group treated with PQ at the single dose of 30 mg/kg on day 4; PQ + KTF 2 group treated with KTF at the dose of 2 mg/kg/day for seven days and PQ at the single dose of 30 mg/kg on day 4; PQ + KTF 4 group treated with KTF at the dose of 4 mg/kg/day for seven days and PQ at the single dose of 30 mg/kg on day 4. Both PQ and KTF were dissolved in saline and administered IP. The duration of the study was seven days. The experimental protocol including duration, the doses of PQ (17), KTF (18), and the day of PQ injection (19) was selected based on previous studies. On the eighth day, mice were anesthetized with ketamine (90 mg/kg, IP)/xylazine (10 mg/kg, IP), and blood samples were collected directly from the heart and stored in gel tubes. After centrifuging at a speed of 4000 × g for 10 min, the serum samples were separated and stored at -20 °C until measurement. The kidneys were extracted and washed with normal saline. The right kidney was placed in formalin for histopathological examination. In addition, the left kidney was kept at -70 °C to measure oxidative and inflammatory factors and iNOS protein expression. A schematic diagram of the study design is illustrated in Fig. 1.
Fig. 1.
Schematic diagram of the study design to investigate the effect of the KTF on the PQ-induced nephrotoxicity in mice. Experimental groups included control, KTF 4, PQ, PQ + KTF 2, and PQ + KTF 4. Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. KTF, Ketotifen; PQ, paraquat; IP, intraperitoneally.
Serum biochemical markers
BUN, Cr, and Alb serum levels were determined with Pars Azmoon kits using a BT 3000 auto analyzer (Biotecnica, Italy). The value s were expressed as mg/dL.
Preparation of kidney tissue homogenate
For this purpose, one part of the kidney tissue was homogenized in phosphate buffer (10% w/v, pH 7.4). After centrifuging at a speed of 12000 × g at 4 °C for 20 min, the supernatant was sep arate d and stored at -70 °C to measure tissue factors including thiobarbituric acid-reactive substances (TBARS), total thiol, CAT, SOD, GPx, TNF-α, and iNOS protein expression.
Protein content
The Bradford’s protein assay method was used to determine protein concentration in the kidney tissue supernatant (20).
Total thiol
The total thiol level was measured using Ellman’s reagent (5,5’-dithiobis-2-nitrobenzoic acid, DTNB). DTNB was dissolved in ethanol at 8 mg/mL to create a concentrated stock. Then, 20 μL of homogeneous tissue was mixed with 230 μL of phosphate buffer and 50 μL of DTNB (10 mM). Yellow TNB absorbance was read at 412 nm, and the determined values were expressed as nmol/mg protein (21).
TBARS
The level of TBARS was measured using the method reported in Satoh’s study (22). For this purpose, 0.5 mL of homogeneous tissue supernatant was mixed with 0.5 mL of 10% trichloroacetic acid and centrifuged at 4000 × g for 10 min. Then, the supernatant was mixed with 0.5 mL of 0.67% thiobarbituric acid and incubated. Finally, the absorbance of the resulting solution was measured with a microplate reader at 532 nm, and the values were reported as nmol/mg protein.
Activity of antioxidant enzymes
The activity of antioxidant enzymes, including SOD, CAT, and GPx, was measured with ZellBio kits. To measure SOD enzymatic activity, 10 μL of sample was diluted in 10 μL of assay buffer and added to the wells. Then, 50 μL of substrate was added. After adding 25 μL of xanthine oxidase reagent, the plate was incubated for 20 min at room temperature. Finally, absorbance was read at 450 nm, and the values were reported as U/mg protein (23).
To measure CAT activity, 25 μL of sample was diluted in 25 μL of assay buffer and added to the well. Then, 25 μL of hydrogen peroxide reagent was added to each well and incubated for 30 min at room temperature. Next, 25 μL of substrate and 25 μL of horseradish peroxidase (HRP) reagent were added to each well and incubated for 15 min at room temperature. Finally, absorbance was read at 560 nm, and the values were expressed as U/mg protein (24).
To measure GPx enzymatic activity, 125 μL of sample was added to a test tube including 80 μL of reagent 1, 20 μL of reagent 2, 40 μL of reagent 3, 40 μL of reagent 4, and 20 μL of reagent 5. The mixture was incubated for 5 min at 37 °C. After adding 30 μL of reagent 6, the mixture was centrifuged for 10 min. Finally, 10 μL of supernatant and 200 μL of reagent were mixed and incubated for 5 min at room temperature. Absorbance was read at 412 nm, and the values were expressed as U/mg protein (25).
TNF-α
TNF-α level was evaluated by an enzyme-linked immunosorbent assay (ELISA) kit according to the instructions of the manufacturer. The absorbance was measured at 450 nm and reported as pg/mg protein.
Expression of iNOS protein
Western blotting method was used to measure iNOS protein expression. For this purpose, kidney samples were homogenized and centrifuged. An amount of 20 μg protein for each group was electrophoresed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk for 2 h at 4 °C. The membranes were probed with the primary antibodies of iNOS (1:500, Cat No. 2982, Cell Signaling Technology, USA) and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH, 1:500, Cat No. 5174, Cell Signaling Technology, USA) for 1 h. Membranes were treated with mouse anti-rabbit IgG-HRP (1:1000, Cat No. sc-2357, Santa Cruz Biotechnology, USA) for 1 h. An electrochemiluminescence (ECL) kit (USA) was used to visualize protein bands, and Image Lab™ Touch software (BioRad, USA) was used to quantify the bands. GAPDH was used as a loading control.
Kidney index
The percentage of kidney weight to body weight ratio, reported as the kidney index, was calculated using the following equation:
where, KW and BW are instead of kidney weight and body weight, respectively.
Histopathological examination
The right kidney tissue of the mice was fixed in 10% formalin and embedded in paraffin. Then, sections of 4-6 μm were prepared and stained with hematoxylin and eosin (H&E). For each animal, three microscopy slides were examined for assessment of histopathological changes such as inflammation, hyperemia, and proximal tubule damage (PTD).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.5.1, and data were expressed as mean ± SEM. The normality of the data was confirmed using the Kolmogorov-Smirnov test. The data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. P-values < 0.05 were considered significant.
RESULTS
Effect of KTF on the kidney index
The effect of KTF on the kidney index is shown in Fig. 2. Accordingly, the kidney index was significantly lower in the PQ group compared to the control group. The kidney index increased in both PQ + KTF 2 and PQ + KTF 4 groups compared to the PQ group, significantly. No significant difference was observed in kidney index in the group receiving KTF alone compared to the control group.
Fig. 2.
Effect of KTF on the kidney index in the PQ-induced nephrotoxicity in mice. Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. Data were expressed as mean ± SEM, n = 7. ***P < 0.001 indicates significant difference compared to the control group; #P < 0.05 and ###P < 0.001 versus the PQ group; $$$P < 0.001 versus PQ + KTF 2 group. KTF, Ketotifen; PQ, paraquat.
Effect of KTF on the biochemical markers of renal function
The effect of KTF on the biochemical markers of renal function, including BUN, Cr, and Alb, is shown in Fig. 3. Accordingly, BUN and Cr serum levels in the PQ group significantly increased compared to the control group (Fig. 3A and B). Also, Alb serum level showed a significant decrement in the PQ group compared to the control group (Fig. 3C). Treatment with KTF resulted in a significant decrease in the Cr serum level in the PQ + KTF 2 and PQ + KTF 4 groups compared to the PQ group (Fig. 3B), while the only PQ + KTF 4 group showed this significant decrement in the serum level of BUN (Fig. 3A). In addition, there was a significant increase in the serum level of Alb in the PQ + KTF 2 and PQ + KTF 4 groups compared to the PQ group (Fig. 3C). The results revealed that KTF at the dose of 4 mg/kg was significantly more effective than that of 2 mg/kg against PQ-induced nephrotoxicity. No significant difference was observed in the group receiving KTF alone compared to the control group in all parameters (Fig. 3).
Fig. 3.
Effect of KTF on the biochemical markers of (A) BUN, (B) Cr, and (C) Alb in the PQ-induced nephrotoxicity in mice. Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. Data were expressed as mean ± SEM, n = 7. ***P < 0.001 indicates significant difference compared to the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the PQ group; $P < 0.05 and $$P < 0.01 versus KTF 2 group. KTF, Ketotifen; PQ, paraquat; BUN, blood urea nitrogen; Cr, creatinine; Alb, albumin.
Effect of KTF on the oxidative stress markers
The results related to the effect of KTF on the antioxidant levels of total thiol, SOD, CAT, and GPx, and the oxidant factor TBARS are shown in Fig. 4. The activities of SOD, CAT, and GPx enzymes and total thiol level significantly decreased, while the level of TBARS significantly increased in the PQ group compared to the control group. KTF led to an increase in the activities of antioxidant enzymes SOD, CAT, and GPx and total thiol level and a decrease in the level of the oxidative factor TBARS in the PQ + KTF 2 and PQ + KTF 4 groups compared to the PQ group, significantly (Fig. 4). Although the PQ + KTF 2 group showed no significant increase in SOD activity compared to the PQ group. The results revealed that KTF at the dose of 4 mg/kg was significantly more effective on PQ-induced oxidative stress markers in comparison to that of 2 mg/kg. No significant difference was observed in the group receiving KTF alone compared to the control group in all parameters (Fig. 4).
Fig. 4.
Effect of KTF on the oxidative stress markers of (A) total thiol, (B) TBARS, (C) SOD activity, (D) CAT activity, and (E) GPx activity in the PQ-induced nephrotoxicity in mice. Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. Data were expressed as mean ± SEM, n = 7. ***P < 0.001 indicates significant difference compared to the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the PQ group; $P < 0.05 and $$$P < 0.001 versus KTF 2 group. KTF, Ketotifen; PQ, paraquat; TBARS, thiobarbituric acid reactive substances; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase.
Effect of KTF on the inflammatory factor of TNF-α
The results related to the effect of KTF on the inflammatory factor of TNF-α are shown in Fig. 5. Accordingly, the level of TNF-α significantly increased in the PQ group compared to the control group. KTF could significantly decrease the level of TNF-α in the PQ + KTF groups compared to the PQ group. The results showed that KTF at the dose of 4 mg/kg was significantly more effective on the level of TNF-α than that of 2 mg/kg. No significant difference was observed in the level of TNF-α in the group receiving KTF alone compared to the control group.
Fig. 5.
Effect of KTF on the inflammatory factor of TNF-α in the PQ-induced nephrotoxicity in mice. Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. Data were expressed as mean ± SEM, n = 7. *** P < 0.001 indicates a significant difference compared to the control group; #P < 0.05 and ###P < 0.001 versus the PQ group; $P < 0.05 versus the PQ + KTF 2 group. KTF, Ketotifen; PQ, paraquat; TNF-α, tumor necrosis factor alpha.
Effect of KTF on the iNOS protein expression
The results related to the effect of KTF on the iNOS protein expression are shown in Fig. 6. According to the Western blot shown in Fig. 6A, iNOS protein expression significantly increased in the PQ group compared to the control group. KTF significantly decreased iNOS protein expression in the PQ + KTF 4 group compared to the PQ group. The results showed that KTF at the dose of 4 mg/kg was significantly more effective on the iNOS protein expression than that of 2 mg/kg. No significant difference was observed in the iNOS protein expression in the group receiving KTF alone compared to the control group (Fig. 6B).
Fig. 6.
Effect of KTF on the iNOS protein expression in the PQ-induced nephrotoxicity in mice. (A) Western blotting of iNOS protein expression and (B) iNOS/GAPDH expression (fold change of control). Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. Data were expressed as mean ± SEM, n = 7. ***P < 0.001 indicates significant difference compared to the control group; ###P < 0.001 versus the PQ group; $$P < 0.01 versus PQ + KTF 2 group. KTF, Ketotifen; PQ, paraquat; iNOS, inducible nitric oxide synthase.
Effect of KTF on the renal histological lesions
The renal histology images stained by the H&E method and the quantitative results of proximal renal tubular injury in all experimental groups are illustrated in Fig. 7A and B, respectively. Accordingly, the glomerular structure and proximal tubule arrangement were normal in the control and KTF 4 groups. In the PQ group, proximal tubules and glomeruli became irregular, and the urinary space of Bowman’s capsule increased due to the shrinkage of the glomeruli. The treatment of KTF 4 mg/kg with PQ resulted in a reduction of abnormal structures in the proximal tubules (Fig. 7A). A quantitative analysis of the histopathological results showed that the histological structure of the kidney in the control and KTF 4 groups was standard. The degree of proximal tubular injury in the PQ group significantly increased compared with the control group. KTF treatment in both doses of 2 and 4 mg/kg resulted in a decrease in PQ-induced proximal tubular injury compared with the PQ group. Therefore, KTF at the doses of 2 and 4 mg/kg was significantly effective against PQ-induced tissue damage. No significant difference was observed in the group receiving KTF alone compared to the control group in proximal tubular injury.
Fig. 7.
Effect of KTF on the histopathological lesions by (A) hematoxylin and eosin staining and (B) quantitative extent of proximal tubular damage in PQ-induced nephrotoxicity. Mice were treated with KTF at the doses of 2 and 4 mg/kg/day for 7 days. PQ was administered as a single dose of 30 mg/kg on day 4 of the study. The control group received normal saline as vehicle. Data were expressed as mean ± SEM, n = 3. ***P < 0.001 indicates significant difference compared with the control group; #P < 0.05 and ###P < 0.001 versus with the PQ group. KTF, Ketotifen; PQ, paraquat; GL, glomerulus; PT, proximal tubule; PTD, proximal tubule damage; US, urinary space.
DISCUSSION
In this study, the protective effects of KTF at the doses of 2 and 4 mg/kg against nephrotoxicity induced by the single dose of PQ (30 mg/kg) were investigated in mice by suppressing oxidative stress and inflammation and reducing iNOS protein expression. In this study, kidney weight, body weight, and kidney index were reduced in the PQ group. This weight loss may be linked to decreased food and water intake, as well as diarrhea and anorexia induced by PQ (26). Consistent with current findings, Onur’s study reported that PQ administration decreased the physiological parameters of body weight and kidneys in mice compared to the control group, indicating that PQ causes nephrotoxicity (26). On the other hand, KTF can cause weight gain, possibly due to increased appetite. This effect may be dose-dependent, with higher doses potentially leading to more significant weight gain (27). KTF has been shown to have a protective effect against kidney damage, particularly in drug-induced kidney injury. Accordingly, one study investigated the role of KTF in methotrexate (MTX)-induced nephrotoxicity in rats and showed that the oral administration of KTF was successful in reversing MTX-induced nephrotoxicity to negligible levels compared to the control group (28). Also, another study showed that KTF can reverse cisplatin-induced AKI (29). While KTF can protect against kidney damage, there is less evidence that it directly affects kidney weight (30). In this study, KTF 4 mg/kg led to an increase in the kidney index. PQ poisoning significantly affects the levels of BUN, Cr, and Alb due to its nephrotoxic effects. PQ poisoning often leads to AKI, which directly affects the levels of these biomarkers, leading to increased BUN and Cr and decreased Alb. The severity of the increase in BUN and Cr is often related to the extent of kidney damage. This increase is a result of decreased kidney function, as the kidneys are responsible for filtering these waste products from the blood. Other mechanisms, such as increased production of creatine and Cr to provide energy during oxidative stress, can also contribute to increased Cr level (31,32). PQ poisoning can lead to decreased Alb level in the blood because damaged kidneys leak Alb into the urine, resulting in decreased serum Alb concentration. In severe cases, kidney damage can lead to nephrotic syndrome, which is characterized by significant proteinuria (increased Alb in the urine) and hypoalbuminemia (33). The antiinflammatory properties of KTF, due to its mast cell stabilizing and antihistamine effects, may play a role in reducing renal injury and subsequently affecting BUN and Cr levels. In a study, KTF reduced oxidative stress and inhibited NF-κB and p38 MAPK related to mast cells and macrophages accumulation (34). The ability of KTF to reduce renal injury could have a positive indirect effect on BUN and Cr by improving overall renal function (35). In this study, KTF 4 mg/kg resulted in a decrease in BUN and Cr and an increase in Alb.
PQ leads to the production of hydroxyl radicals, H2O2, and superoxide anions. The radicals lead to oxidative stress by inhibiting the production of adenosine triphosphate. PQ leads to a decrease in glutathione concentration and ultimately the production of ROS (36). In this study, PQ led to an increase in the lipid peroxidation index TBARS and a significant decrease in the total thiol level and the activity of antioxidant enzymes SOD, CAT, and GPx in kidney tissue. An antioxidant defense network consisting of enzymatic and nonenzymatic antioxidants exists in the body. SOD, CAT, and GPx are the first-line enzymatic antioxidants that defend the body against superoxide anion radicals produced in the mitochondrial energy production pathway (37). KTF, a mast cell stabilizer and antihistamine, can affect both oxidant and antioxidant factors and often exhibits antioxidant properties. Also, KTF can reduce oxidative stress and modulate the associated inflammatory response by scavenging free radicals. KTF can affect the activity of the antioxidant enzymes CAT, SOD, and GPx, potentially increasing their levels or activity. By modulating enzyme activity and scavenging free radicals, the body’s overall antioxidant capacity is increased, helping to protect against oxidative damage (38). In animal studies, KTF has been shown to reverse the negative effects of oxidative stress induced by various agents, such as cisplatin or MTX, by restoring the activity of antioxidant enzymes and reducing lipid peroxidation (28,29). In this study, KTF 4 mg/kg led to a decrease in the lipid peroxidation index TBARS and a significant increase in the total thiol level and the activity of antioxidant enzymes SOD, CAT, and GPx in kidney tissue.
TNF-α is a cytokine used by the immune system for cellular signaling, and its increased activity is associated with chronic inflammation (39). The inhibition of TNF-α by antioxidants can reduce renal apoptosis (40). PQ induces inflammation and nephrotoxicity by increasing TNF-α inflammatory marker levels in the body (41). According to a previous study, PQ at a concentration of 1 mmol/L can damage macrophages and significantly increase TNF-α levels (42). In our study, PQ led to an increase in the TNF-α inflammatory factor level. Mast cells play a key role in drug-induced nephrotoxicity. The number of mast cells is significantly increased by renal injury. The cells reduce kidney damage in mice by reducing TNF-α (43). Aldossary’s study has shown that KTF reduces TNF-α levels in immune cells and can reduce inflammation (44). KTF also significantly reduces the release of inflammatory factors compared to dexamethasone (45). KTF helps reduce TNF-α levels in cisplatin-induced kidney damage and improves the inflammatory state (29), which confirms that KTF 4 mg/kg led to a decrease in TNF-α levels in PQ-damaged kidney tissue, as observed in the current study.
The two molecules, TNF-α and iNOS, interact in a coordinated and regulated manner in the immune system and inflammation. TNF-α increases iNOS expression by activating signaling pathways (46). NO produced by iNOS can inhibit the secretion of TNF-α through negative feedback mechanisms to prevent excessive inflammation (47) . Mast cell granules reduce NO and TNF-α production by inhibiting iNOS and TNF-α expression in macrophages (48).
Aires et al. showed a 4-fold increase in iNOS expression in PQ-intoxicated animals compared to controls. PQ exposure can lead to increased expression of iNOS, particularly in vascular endothelial cells. This increase in iNOS expression is associated with PQ-induced vascular dysfunction and increased NO production. This effect is thought to be mediated by TNF-α, as treatment with TNF-α blockers can reduce iNOS expression and improve vascular function in PQ-intoxicated animals (11). Similarly, our study found that PQ led to an increase in the iNOS protein expression compared to the control group. KTF reduces NO activity and ultimately reduces iNOS protein expression (49), which is consistent with the current results. This study revealed that KTF 4 mg/kg led to a decrease in iNOS protein expression in the kidney tissue.
PQ primarily damages the kidneys, and the renal proximal tubules are the primary target of its toxicity. PQ is actively taken up by proximal tubule cells, where it undergoes redox cycling and produces ROS. The ROS cause oxidative damage to lipids, proteins, and DNA, ultimately leading to cell damage and death (50). Renal histopathological examination in this study showed normal histology in the structure of glomeruli and the arrangement of proximal tubules in the groups without receiving PQ. While the PQ group exhibited pathological changes in kidney tissue, as follows: the proximal tubules and glomeruli became irregular, and due to the shrinkage of glomeruli, the urinary space of Bowman’s capsule increased. On the other hand, the treatment of KTF significantly reduced the extent of PTD compared to the PQ group. The current results indicated that KTF reduced the development of oxidative stress and nephrotoxicity induced by PQ in mice. KTF 4 mg/kg led to enhanced antioxidant defense, reduced lipid peroxidation, inflammation, and iNOS protein expression.
This study had some limitations, including: 1) only male mice were used in this study; 2) PQ was administered as a single dose; 3) the measurement of mast cell degranulation (via toluidine blue or tryptase) and plasma or urine KTF concentrations was not performed, and the protocol was precautionary.
CONCLUSION
The results of this study showed that KTF could significantly improve PQ-induced renal injury in mice through inhibiting inflammation, reducing oxidative stress, and modulating the signaling pathway of iNOS. Future clinical trials are warranted to evaluate the efficacy and safety of KTF as an adjuvant therapy in human PQ poisoning.
Authors’ contributions
M.J. Khodayar, M.S. Badiee, and A. Vadizadeh jointly developed the conceptualization and design of study as well as carried out formal analysis and investigative procedures; M.J. Khodayar and M.S. Badiee established methodological frameworks; M.J. Khodayar, M.S. Badiee, and F. Fakhredini prepared the initial manuscript draft; M.J. Khodayar, M.S. Badiee, H. Kalantar, A.H. Rahmani, and M. Mahdavinia performed reviewing and editing; M.J. Khodayar and M.S. Badiee managed funding acquisition and provided resources; M.J. Khodayar, F. Fakhredini, A.H. Rahmani, M. Mahdavinia, H. Kalantar, and S. Rafiei Asl supervised the study.
All authors have read and approved the finalized article. Each author has fulfilled the authorship criteria and affirmed that this article represents honest and original work. The authors alone are responsible for the content of the paper.
Conflict of interest statements
The authors declared no conflict of interest in this study.
AI declaration
The authors did not use AI-based technologies in this manuscript.
Data availability
The data supporting the findings of this study are documented and available from the corresponding author upon reasonable request. Researchers interested in accessing the data should contact the corresponding author to discuss the terms and conditions of access.
Acknowledgments
This research was financially supported by the Student Research Committee of AJUMS with Grant No. 01s60. We would like to express our sincere gratitude to the Deputy Director of Research of AJUMS for supporting this research.
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Data Availability Statement
The data supporting the findings of this study are documented and available from the corresponding author upon reasonable request. Researchers interested in accessing the data should contact the corresponding author to discuss the terms and conditions of access.