Nephroprotective effects of febuxostat and/ or mirtazapine against gentamicin-induced nephrotoxicity through modulation of ERK 1/2,
NF-B and MCP1
KEYWORDS : Gentamicin; Nephrotoxicity; Mirtazapine; Febuxostat; NF- κβ; ERK 1/2; MCP-1
1. Introduction
Gentamicin is an aminoglycoside antibiotic used to treat var- ious bacterial infections, particularly gram-negative bacteria. Nephrotoxicity is a significant risk in its clinical use. Many antioxidant products have been used to protect against the renal toxicity of gentamicin [1].
Nephrotoxic effect of gentamicin is associated with oxida- tive stress, induction of apoptosis, and necrosis [2,3]. Further, it is characterized morphologically by epithelial edema, tubular necrosis, proximal tubule epithelial desquamation, and glo- merular hypertrophy. In the kidney, gentamicin increased the level of reactive oxygen species (ROS) and decreased the activity of renal antioxidant enzymes, such as superoxide dis- mutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH) [4].
Febuxostat is a nonpurine xanthine oxidase inhibitor used in the management of hyperuricemia. It prevents the synthesis of uric acid in the body, decreasing its concentration [5]. It has been reported that febuxostat, in addition to gout therapy, can inhibit inflammation and tissue injury in patients with chronic kidney disease [6,7]. Febuxostat is a promising protec- tive agent against doxorubicin-nephrotoxicity through improving biochemical, inflammatory, histopathological, and immunohistochemical alterations induced by doxorubicin [8]. Mirtazapine (MTZ) is a serotonin and noradrenergic antide- pressant drug that is used to treat primary depression. MTZ can exert prominent antioxidant and cytoprotective actions in several experimental models through activation of enzymatic and nonenzymatic antioxidant mechanisms and inhibition of some toxic oxidant mechanisms [9,10]. MTZ was found to be efficacious to prevent nephrotoxicity due to methotrexate (MTX) shows that MTX can be used safely in higher doses and for a longer course in combination with MTZ in cancer chemotherapy [11].
This study aimed to investigate the nephroprotective effects of febuxostat, MTZ, and their combination against nephrotoxicity of gentamicin in rats. Further, the underlying molecular mechanisms will be identified.
2. Materials and methods
2.1. Drugs
Gentamicin was purchased from EIPICO, Egypt. Febuxostat was purchased from HIKMA company, Egypt. MTZ was purchased from Mash Premiere, Egypt. Febuxostat and MTZ were freshly prepared in carboxymethylcellu- lose (CMC).
2.2. Experimental design and animals
The experimental protocol was accepted by the Ethical Committee of the Faculty of Pharmacy, Tanta University (Egypt). Fifty-six adult male Albino rats weighing 180–200 g were purchased from the Faculty of Veterinary Medicine,Tanta, Egypt. Rats were housed in wire cages for 1 week under identical environmental conditions for acclimation, with free access to food and water. The ethical code is PT00025.
After an acclimatization period, all the rats were randomly divided into seven groups and were treated according to the following protocol: Group I (negative control group): rats received 0.5% CMC [12]. Group II (Gentamicin group): rats were given the vehicle for 14 days and then received gentamicin in a dose of 100 mg/kg, I.P. once daily for 7 days [13]. Group III (Febuxostat 5 mg/kg): rats were P.O. administered with 5 mg/kg febuxostat daily for 14 days, then concomitantly with gentamicin for addi- tional 7 days [14]. Group IV (Febuxostat 10 mg/kg): rats were P.O. administered with 10 mg/kg febuxostat daily for 14 days, then concomitantly with gentamicin for additional 7 days [14]. Group V (MTZ 15 mg/kg): rats were P.O. administered with 15 mg/kg MTZ daily for 14 days, then concomitantly with gentamicin for addi- tional 7 days [15]. Group VI (MTZ 30 mg/kg): rats were P.O. administered with 30 mg/kg MTZ daily for 14 days, then con- comitantly with gentamicin for additional 7 days [15]. Group VII (combination group): rats were P.O. administered MTZ (30 mg/kg) and febuxostat (10 mg/kg) daily for 14 days and then concomi- tantly with gentamicin for additional 7 days.
2.3. Sample collection
All rats were put in a closed 9”× 4” jar, containing three paper towels saturated with 40 mL of diethyl ether at the end of the experiment [16]. The blood was then withdrawn via cardiac puncture. Serum was collected and stored at −20°C until analysis. Rats were killed by cervical dislocation, and their kidneys were removed immediately. For histopathological stu- dies, the left kidneys were fixed in 10% buffered formalin solution at room temperature. For biochemical and further analysis, the right kidneys were cut and stored at −80°C.
2.4. Determination of kidney biomarkers
The following biomarkers were estimated using commercial kits (Biodiagnostic Co., Giza, Egypt). Urease colorimetric and Allen methods were used for measuring BUN and serum crea- tinine levels, respectively [3,17].
2.5. Determination of oxidative stress markers in kidney tissue
2.5.1. Superoxide dismutase
SOD activity was measured by the method of Nishikimi et al. using the kit obtained from Biodiagnostic Co. (Giza, Egypt) [18]. Kidney tissue was homogenized in 5–10 mL cold buffer (50 mM potassium phosphate, pH 7.5, 1-mM EDTA) per gram tissue. The assay is based on the enzyme’s ability to prevent nitroblue tetrazolium dye from being reduced by phenazine methosulphate. The increase in absorbance was measured at 560 nm for 5 min for control (A control) and sample (A sample) at 25 °C. The enzyme activity was expressed as U/gm tissue.
2.5.2. Lipid peroxides (measured as malondialdehyde) Malondialdehyde (MDA) was determined according to Ohkawa et al. [19]. TBA reactive substance is formed when thiobarbituric acid reacts with MDA in an acidic medium at 95° C for 30 minutes. The absorbance of the resultant pink pro- duct can be measured at 534 nm using the kit obtained from Biodiagnostic Co. (Giza, Egypt). MDA in the sample was deter- mined colorimetrically and expressed as nmol/gm tissue.
2.5.3. Glutathione peroxidase enzyme activity
GPx was assayed according to the method described by Palglia et al. using the kit obtained from Biodiagnostic Co. (Giza, Egypt) [20]. The assay is a GPx activity indirect measure. The enzyme glutathione reductase (GR) is recycled oxidized glutathione (GSSG) to its reduced state.
For assaying GPx, tissue homogenate is added to a solution containing NADPH, GSH, and GSH reductase. The enzyme reaction is started by adding the substrate and hydrogen peroxide, and the absorbance at 340 nm is recorded. The rate of decrease in the A340 is directly proportional to GPx activity in the sample. The enzyme activity was measured in (mU/gm tissue).
2.6. Assessment of inflammatory markers
2.6.1. Tumor necrosis factor-alpha (TNF-α)
100 mg of kidney tissue was homogenized into 1 mL of phosphate-buffered saline (PBS). The homogenates were cen- trifuged at 5000 × g for 5 minutes at 2–8°C. The supernatant was removed and assayed immediately using the Cusabio ELISA kit (USA). Finally, the intensity of the yellow color is measured at 450 nm. It is proportional to the amount of rat TNF-α in the specimen. The concentration of TNF-α in the test sample was expressed as ng/gm tissue using a standard curve.
2.6.2. Monocyte chemoattractant protein (MCP-1) and nuclear factor-kappa B (NF-κB)
Total RNA was extracted from kidney tissue using a Qiagen tissue extraction kit (Qiagen, USA). The RNA purity was verified spectro- photometrically at 260/280 nm. Total RNA was used for cDNA conversion using high-capacity cDNA reverse transcription kit Fermentas, USA. The SYBR Green PCR master mix (iNtRON Biotechnology, Korea) was used for quantitative PCR of obtained cDNA as described by the manufacturer using the RT-PCR system (Thermo Fisher Scientific, Pikoreal 5100, Finland).
Primers were purchased from Biosearch Technologies Co. (USA) and prepared according to Vásquez-Loarte et al. and Bottero et al., using an Applied Biosystem with software version 3.1 (StepOne™, USA) [21,22]. The real-time PCR instru- ment’s temperature was set to 50°C for 40 cycles and 2 hours (95°C for 15 min for denaturation, 60°C for 1 h for annealing, and 72°C for 1 h for extension). The Ct values (threshold cycle) of the sample and the relative content of the gene amplifica- tion product were calculated using the 2−∆∆Ct method [23].
2.7. Extracellular signal regulated protein kinase 1/2 (ERK1/2) concentrations
ERK1/2 was measured in renal tissue using the ERK1/2 Simple Step ELISA® Kit. The intensity of color was measured at 450 nm. The concentration of ERK1/2 in the test sample was expressed as µg/gm tissue using a standard curve using a typical recombinant protein standard curve.
2.8. Histopathological examination of kidney tissue
2.8.1. Hematoxylin and eosin
Using microtome (Leica RM2135, Germany), kidney tissue was cut into 3-µm thick sections, deparaffinized, and hydrated in a descending series of ethyl alcohol. The section was stained with hematoxyline (H) and eosin (E) stains, then dehydrated and cleared in xylene [24]. Using a light microscope (Olympus Electron Microscope, Olympus, Japan), the slides were then examined blindly for the tubular and glomerular structure by an expert pathologist and photo- graphed. A minimum of six fields were examined for each kidney section.Table 1
2.8.2. Immunohistochemical examination of renal caspase-3
Fixed samples were permeabilized in PBS/0.1% Triton X-100 for 5 minutes at room temperature, then washed for 5 min- utes three times in phosphate-buffered saline solution (PBS) at room temperature. The slides were drained, and 200 µL of blocking buffer was added. The slides are then laid flat for 1–2 hours in a humidified chamber at room temperature and rinsed once in PBS. Hundred microliters of the active caspase-3 antibody diluted 1/200 was added in blocking buffer. A slide with no active caspase 3 was prepared as a negative control. The slides were then incubated in a humidified chamber overnight at 4°C. The slides were washed three times in PBS/0.1% Tween 20 at room tem- perature the next day, each time for 10 minutes. After draining the slides, 100 µL of goat anti-rabbit conjugate diluted 1:500 in PBS was added. The light-protected slides were laid flat in a humidified chamber and incubated at room temperature for 1–2 hours before being washed three times in PBS/0.1% Tween 20 for 5 minutes per time. The liquid was drained from the slides, which were then placed in a permanent or aqueous mounting medium and exam- ined under a fluorescence microscope.
The sections were examined in randomly selected micro- scopic fields at a magnification of X400. Digital images from immune-stained sections were obtained using the Olympus BX41 microscope. The intensity of caspase-3 immunostaining was analyzed for mean area percent (MA%) using NIH Image J (v1.46 r) [25].
2.9. Statistical analysis
Results were expressed as the mean ± SEM. A one-way analy- sis of variance (ANOVA) was used to compare various groups, followed by Tukey multiple comparison tests. A significant difference was accepted when P < 0.05. Graphpad prism 5.0 Demo (Graphpad software, San Diego, CA, USA) was used for the statistical analysis of different groups [26].
3. Results
3.1. Effects on BUN and creatinine levels
Serum BUN and creatinine levels were increased significantly (p > 0.05) in the gentamicin group compared to the negative control group.
Compared to the gentamicin group, the febuxostat (5 mg/kg, 10 mg/kg) and MTZ (15 mg/kg, 30 mg/kg) treated groups had significantly lower BUN and creatinine levels. The combination group significantly reduced BUN and creatinine levels compared to the gentamicin group (p > 0.05) (Figure 1(a,b) and Table 2).
3.2. Effects on MDA, SOD enzyme activity, and glutathione peroxidase activity
As shown in Figure 2(a), the tissue MDA contents were signifi- cantly increased in the gentamicin group compared to the nega- tive control group (p > 0.05). On the other hand, febuxostat (5 mg/kg, 10 mg/kg) and MTZ (15 mg/kg, 30 mg/kg) treated groups significantly decreased tissue MDA contents compared to the gentamicin group. Compared to the gentamicin group, the highest significant decrease in the tissue content of MDA occurred in the rats treated with the combination group (Table 3). SOD and GPx activities in the gentamicin group were signifi- cantly lower than in the negative control group (p > 0.05). In contrast to the gentamicin group, febuxostat (5 mg/kg, 10 mg/ kg) and MTZ (15 mg/kg, 30 mg/kg) treated groups had signifi- cantly higher tissue GPx activity and SOD activity. The rats treated with the combination group had the largest statistical increase as compared to the gentamicin group (Figure 2(b) and Table 3).
3.3. Effects on TNF-α content
Gentamicin treatment resulted in a significant increase in the renal content of TNF-α as compared to the negative control group (p > 0.05). Treatment with febuxostat (5 mg/kg, 10 mg/ kg) and MTZ (15 mg/kg, 30 mg/kg) or their combination significantly reduced the renal content of TNF-α compared to the gentamicin group (Figure 3(a) and Table 4).
3.4. Effects on ERK1/2 concentration
As shown in Figure 3(c), it is apparent that gentamicin sig- nificantly increased ERK1/2 concentration compared to the negative control group (p ˂ 0.05). On the other hand, ERK1/2 concentration in febuxostat (5 mg/kg, 10 mg/kg) and MTZ (15 mg/kg, 30 mg/kg) treated rats was significantly decreased compared to the gentamicin group (p ˂ 0.05). The combination group decreased ERK1/2 concentration to about the same level as the control group (Table 4).
Figure 1. Effect of febuxostat and mirtazapine and their combination treatment on (a) blood urea nitrogen (BUN) and (b) serum creatinine. Values are mean ± SD, set at p > 0.05 was significance, n = 8 per group. *:Significant compared negative control; @:Significant compared gentamicin group.
Figure 2. Effect of febuxostat and mirtazapine and their combination treatment of (a) renal lipid peroxides content measured as malondialdehyde (MDA), (b) level of superoxide dismutases (SOD), and(c) level of glutathione peroxidase activity (GPx) values are mean ± SD, set at p > 0.05 was significance, n = 8 per group. *:Significant compared to negative control; @:Significant compared to gentamicin group; a: Significant compared to febuxostat 5 mg group; b: Significant compared to febuxostat 10 mg group; c: Significant compared to mirtazapine 15 mg group; d: Significant compared to mirtazapine 30 mg group.
Figure 3. Effect of febuxostat and mirtazapine and their combination treatment with (a) TNF–α, (b) monocyte chemoattractant protein (MCP-1) using PCR, (c) renal ERK1/2Concentration, and (d) renal nuclear factor-kappa B (NF-κB) using PCR . Values are mean ± SD, set at p > 0.05 was significance, n = 8 per group. *:Significant compared to negative control.; @:Significant compared to gentamicin group; a: Significant compared to febuxostat 5 mg group; b: Significant compared to febuxostat 10 mg group; c: Significant compared to mirtazapine 15 mg group; d: Significant compared to mirtazapine 30 mg group.
Figure 4. Kidney section showing (a) normal histological features of kidney cortex from negative normal group. Renal corpuscle (RC) is seen. proximal tubules (P) is seen lined with eosinophilic cuboidal cells, narrow lumen and apparent brush border. Distal tubules (D) are apparent with wide lumen and lined with eosinophilic cuboidal epithelium. (b) Gentamicin group showing extensive degenerative changes in form of congested glomeruli (G). Distal tubular cells (black arrow) are seen with extensive cytoplasmic vacuolation. Tissue debris is seen in the lumen. (c) Gentamicin group showing cytoplasmic vacuolation is seen in proximal tubular cells (P) with loss of brush border. Nuclear changes are noticed in form of irregular nucleus margination of the nucleolus. (d) Feboxustate group (5 mg/kg) showing evident congestion in glomeruli (G). Cytoplasmic vacuolation is seen in most of proximal (P) and distal (D) tubular cells. Cellular infilteration (black arrow) is noticed. (e) Feboxustate group (10 mg/kg), showing mild congested glomeruli (G), minimal cytoplasmic vacuolation in proximal (P) and distal (D) tubular cells and hydropic swelling of tubular cell (black arrow). (f) MTZ group (15 mg/kg), showing residual congestion in glomeruli (G), widely spread cytoplasmic vacuolation is seen in proximal, distal tubular cells (black arrow) some nuclei appear with irregular outline (n), others show nucleolar margination (n1). (g) Mirtazapine group (30 mg/kg) showing some congestion in the glomeruli (G) Some tubular cells show limited cytoplasmic vacuolation. (h) Combination group showing almost normal appearance of renal corpuscle (RC). Few proximal (P) and distal (D) tubular cells show limited cytoplasmic vacuolation. Other tubular cells (black arrow) appear almost normal. Most nuclei are vesicular.
3.6. Effect on histopathological examination of kidney tissues
Histological features of the kidney cortex in the negative control group were consistent. Renal corpuscle (RC) was seen. Eosinophilic cuboidal cells lined the proximal tubules (P), which had a narrow lumen and an apparent brush border. Distal tubules (D) were apparent with the wide lumen and lined with eosinophilic cuboidal epithelium (Figure 4(a)).
In the gentamicin group, rats have shown extensive degen- erative changes in the form of congested glomeruli. Distal tubular cells (black arrow) were seen with extensive cytoplas- mic vacuolation. Tissue debris was seen in the lumen (Figure 4 (b)). Cytoplasmic vacuolation was also seen in proximal tubu- lar cells with loss of brush border. Nuclear changes are noticed in irregular nucleus margination of the nucleolus (Figure 4(c)). On the other hand, kidney sections from rats treated with febuxostat(5 mg/kg) have shown some change in histological features. Evident congestion was seen in the glomeruli (G). Cytoplasmic vacuolation was found in most proximal (P) and distal (D) tubular cells. A cellular infiltration (black arrow) was noticed (Figure 4(d)).
Kidney sections from rats given feboxustat (10 mg/kg) dis- played mildly congested glomeruli (G), minimal cytoplasmic vacuolation in the proximal (P) and distal (D) tubular cells, and hydropic swelling (black arrow) (Figure 4(e)). Kidney sections from rats given MTZ (15 mg/kg) have shown some improvement in histological features, with resi- dual congestion in the glomeruli (G) and widespread cytoplasmic vacuolation in the proximal and distal tubular cells (black arrow) (Figure 4(f)). Some nuclei had an irregular outline (n), while others had nucleolar margination (n1).
Improvement of histological features was noticed in most renal cortical tubules in kidney sections from rats treated with MTZ (30 mg/kg). Some congestion was detected in the glo- meruli (G). Some tubular cells have shown limited cytoplasmic vacuolation (Figure 4(g)).
Kidney sections from rats treated with the combination therapy exhibited a great improvement in histological fea- tures. The renal corpuscle (RC) had a nearly normal appear- ance. Few tubular cells, both proximal (P) and distal (D), revealed cytoplasmic vacuolation. The appearance of other tubular cells (black arrow) was almost normal. The majority of nuclei were vesicular (Figure 4(h)).
3.7. Effect on immunohistochemical examination of caspase-3 activity
The proposed anti-apoptotic effect was assessed immunohis- tochemically (IHC) by determining the apoptotic index by measuring the mean area percent (MA %) of the IHC apoptotic marker, caspase-3. Generally, the immunoreaction of kidney tissues to caspase-3 was mainly cytoplasmic. Figure 5(a) depicts the differences in MA % of caspase-3 apoptotic mar- kers between classes.
Immunohistochemical staining of kidney sections of the negative control group showed a mild cytoplasmic reaction to caspase-3, with MA% of 12.45 ± 0.95 (Figures 5(b-1)).Immunohistochemical staining of kidney sections from the gentamicin group revealed intense immunostain for caspase- 3, indicating high apoptotic activity. The majority of cells in several sections had dense cytoplasmic brown coloration, with the MA% being the highest ever recorded (Figures 5 (b-2)).
Figure 5. (a) Analysis of the intensity of caspase-3 immunostaining for mean area percent (MA%) using NIH Image J (v1.46 r). Values are mean ± SD, set at p > 0.05 was significance, n = 8 per group. (b) Kidney sections from (1) negative control group showing mild immunoreaction to caspase-3. (2) Gentamicin group showing intense immunoreaction to caspase-3. (3) Rats treated with febuxostat (5 mg/kg), showing limited areas of cytoplasmic brown coloration for caspase-3. (4) Rats treated with Febuxostat (10 mg/kg) showing moderate cytoplasmic immunoreaction to caspase-3. (5) Rats treated with Mirtazapine (15 mg/kg) showing mild reaction for caspase-3. (6) Rats treated with Mirtazapine (30 mg/kg) showing very limited areas of brownish coloration for caspase-3. (7) Rats treated with combination group showing limited focal areas of brownish color staining for caspase-3 almost similar to normal. *:Significant compared to negacve control; @:Significant compared to gentamicin group; a: Significant compared to febuxostat 5 mg group; b: Significant compared to Febuxostat 10 mg group; c: Significant compared to mirtazapine 15 mg group;d: Significant compared to Mirtazapine 30 mg group.
On the other hand, kidney sections from rats treated with both doses of febuxostat (5 mg/kg and 10 mg/kg) demon- strated moderate cytoplasmic immunoreactions for caspase-3. The MA% revealed a significant decrease (Figures 5(b-3,b- 4)). Similarly, there are significant decreases in the MA% of caspase-3 from rats treated with MTZ (15 mg/kg) and MTZ (30 mg/kg). Moderate cytoplasmic brown coloration was seen in scattered areas of kidney tissues retrieved from both MTZ groups (Figure 5(b-5,b-6), respectively).
The mild immunoreaction was found in kidney sections from rats treated with a combination therapy, with almost negative areas for caspase-3. The MA% decreased the most among the treated groups, approaching normal levels, indicat- ing a superior anti-apoptotic effect of combined febuxostat and MTZ (Figure 5(b-7)).
4. Discussion
More than any other organ in the body, the kidney is particu- larly vulnerable to toxicity due to the high toxins being deliv- ered to it [27]. The nephroprotective efficacy of the two drugs, febuxostat (5 mg/kg, 10 mg/kg) and MTZ (15 mg/kg, 30 mg/ kg) and their combination against gentamicin nephrotoxicity in rats, was examined in this study, and the underlying mole- cular mechanisms were clarified.
The current study found that gentamicin has a major nephrotoxic effect evidenced by increased serum creatinine and BUN levels. The histopathological observations of hyper- cellular, lobulated glomeruli with wide Bowman spaces and diffuse dense mononuclear cellular infiltration, suggesting nephritis, corroborated these biochemical changes. Glomerular atrophy and extreme brush border loss were also found in the gentamicin group.
Several studies indicated that the generation of free radicals and induction of oxidative stress is an important pathway of gentamicin-induced nephrotoxicity [28–30]. The reduction of anti- oxidant defense is linked with the overproduction of ROS resulting in a potential damage to proximal renal tubules, which subse- quently develop into tubular damages and lipid peroxidation [30]. These findings are consistent with our results as gentamicin was linked to low SOD and GSH-Px enzyme activity, and high MDA content in renal tissue and glomerular dysfunction, indi- cating acute oxidative stress. Furthermore, gentamicin has been shown to increase oxidant generation, such as hydroxyl radicals, superoxide anions, hydrogen peroxides, and reactive nitrogen species (RNS), resulting in functional and structural
degradation of kidneys [31,32].
Gentamicin induced-nephrotoxicity is associated with renal overexpression of p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor kappa B (NFkB) pathways via inter- ference with mitochondrial function [33]. Gentamicin induces apoptosis and cellular necrosis, leading to disruption of the respiratory chain, reducing the ATP synthesis, inhibiting some membrane protein carriers, decreasing GFR via tubular blockage, and increasing Bowman’s capsule pressure [34]. Besides, lysoso- mal phospholipidosis and apoptosis play a vital role in gentami- cin-induced nephrotoxicity [35]. Therefore, in experimental studies, drugs with direct or indirect antioxidant and anti- inflammatory properties were found to avoid gentamicin nephrotoxicity.
In the present research, the co-administration of febuxostat with gentamicin significantly improved renal function as reflected by reducing BUN and serum creatinine, indicating a renoprotective effect of febuxostat. These results mirror those of the previous studies, which showed that febuxostat reduced serum uric acid, creatinine levels, and improved renal function [36–38]. Treatment with febuxostat specifically tar- gets XO, which can prevent in vivo UA formation [39].
In this present study, febuxostat demonstrated potential anti-inflammatory and antioxidant effects on gentamicin- induced-nephrotoxicity by reducing MDA contents and restoration of the antioxidant enzymes SOD and GSH-Px that is consistent with other studies [8,40]. Febuxostat suppresses both reduced and oxidized forms of XO, which may be related to its superiority in diminishing oxidative stress [41]. Febuxostat is effective in controlling hyperuricemia and in preventing Tac-induced renal injury, via a reduction of oxida- tive stress. Therefore, febuxostat may be a useful approach in the management of the progression of nephropathy in renal transplant patients treated with Tac [14].
Febuxostat helps to reduce inflammatory responses in the body. Furthermore, it has been found that febuxostat prevents renal damage primarily by reducing inflammatory mediators, such as IL 1 and TNF-α, due to its inhibitory action on XO [8]. Further, it has been reported that febuxostat treatment reduced caspase-3 [40].
The ability of febuxostat to reduce caspase-3 reactivity in the kidney demonstrated anti-apoptotic properties in this study. Febuxostat’s anti-apoptotic effect can be attributed to its reduction of oxidative stress, according to Wang et al. [42], febuxostat also inhibited JNK phosphorylation in macro- phages, implying that it affects the MAPK pathway [43].
The present results also showed that febuxostat had sig- nificantly lower levels of NF-κB, MCP1, and ERK1/2 than the control group. To the best of our knowledge, no other study had discussed these effects on the kidney. It was found that MTZ significantly decreased serum crea- tinine and BUN, which were increased due to gentamicin. Serum BUN and creatinine are critical biochemical parameters for measuring renal functions. Increases in creatinine levels and serum BUN are indicators of kidney injury, such as the loss of nephron activity. MTZ inhibited the rise of BUN and creatinine levels, which is one proof of its protective role against nephrotoxicity. These findings are in line with those of previous studies of Uzkeser et al. [11]. Nephroprotective effect of MTZ is not generated from its antioxidant property alone but also MTZ blocks 5-HT2 and 5-HT3 receptors [11].
The results suggested that the biochemical results are har- monious with histopathological findings. It has been indicated that MTZ‘s protective effect on gentamicin nephrotoxicity can be attributed to its antioxidant activity.MTZ also blocks 5-HT3 and 5-HT2 receptors [11]. Stimulation of 5-HT2 and 5-HT3 receptors was accompanied by toxic side effects. Therefore, the function of these receptors can also be related to the MTZ nephroprotective effect. Also, MTZ resulted in cellular protection of cisplatin-induced testi- cular damage in adult male albino rats [44].
According to the literature, MTZ‘s efficacy stems from its ability to suppress proinflammatory cytokines and their anti- oxidant properties. In conclusion, MTZ was found to be influ- ential in preventing nephrotoxicity induced by gentamicin.It has been demonstrated that apoptosis is vital as a cell death mechanism during gentamicin nephrotoxicity. The role of free oxygen radicals in cell death related to gentamicin administration has been shown by Khaksari et al. [45]. MTZ is known as an antioxidative and antiapoptotic agent [46]. The current results also revealed that MTZ showed significantly lower levels of TNF alpha, NF-κB, MCP1, and ERK1/2 than the gentamicin group.
Gupta et al. suggested that MTZ is effective and well tolerated in severely depressed patients [47]. Treatment response is combined with raising in serum brain-derived neurotrophic factor and lowering in serum TNF-α levels. Pro- inflammatory cytokines stimulate neuronal mitogen- activated protein kinase (MAPK) pathways, increasing mono- amine transporter expression and activity in general, result- ing in increased presynaptic reuptake of not only serotonin but also other neuroactive amines [48,49]. MTZ has clear anti- inflammatory effects, acting against the high inflammatory state on the symptoms and signs of depression.
MTZ also exerted an antifibrotic effect by reducing protein kinase C and transforming growth factor-β1 (TGF-β1) and the expression of phosphorylated-Smad3 (p-Smad) and phos- phorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) liver content [50]. Thus, the previous findings sup- port the results of the current study.
Furthermore, the current research revealed that the com- bined effect of febuxostat and MTZ had a stronger effect than either febuxostat or MTZ alone. Depending on the particular parameters that are affected, this combination can be syner- gistic. It led to reduced inflammatory markers and oxidative stress related to significant nephroprotective effect as reflected by decreasing of BUN and serum creatinine levels compared with gentamicin group. Paradoxically, the combina- tion group did not show the highest decline in BUN or serum creatinine. Interestingly, it was postulated that these para- meters need more time to be significantly affected [8]. These results are consistent with a recent study that found that febuxostat to have a potential therapeutic impact in alleviat- ing renal damage in STZ-induced diabetic rats and a hypouricemic effect by inhibiting renal tissue fibrosis [39]. By analogy, MTZ also improves kidney function by attenuating oxidative stress-induced nephrotoxicity due to methotrex- ate [11].
While they were used together, they had a greater impact than when they were used separately. This is the first study to publish these interesting findings. Nevertheless, the current findings need further prospective preclinical and clinical stu- dies on the use of MTZ and febuxostat to elucidate the issue.
Conclusion
In conclusion, the current study found that MTZ and/or febuxostat treatment reduced nephrotoxicity markers and inflammatory mediators induced by gentamicin, restored nor- mal oxidative stress biomarker values, and inhibited renal caspase-3 expression. They also help to reverse gentamicin- induced histological changes. TNF-α, NF-B, MCP1, and ERK1/2, elevated by gentamicin, were reduced by MTZ and febuxostat. Accordingly,NF-κΒ activator 1 combination therapy appears to be a successful protective agent against gentamicin-induced nephrotoxicity.