Diclofenac-Induced Alterations in Renal Antioxidants and Cytokines in Male Wistar Rats

ABSTRACT


INTRODUCTION
Diclofenac is an analgesic and belongs to the non-steroidal anti-inflammatory drug (NSAID) that is widely used to treat pain and inflammation.It works by inhibiting the production of prostaglandins, which are responsible for inflammation.[1,2] However, diclofenac use has been associated with a number of side effects, including renal toxicity.Nephrotoxicity is a major concern, as it can lead to kidney failure.[1,3,4] The exact mechanism of diclofenac-induced renal toxicity is not fully understood.However, it is thought to be mediated by inflammation, oxidative stress, and depletion of renal antioxidant molecules such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx).[3,[5][6][7] Oxidative stress is a condition in which there is an imbalance between the production of free radicals and the body's ability to neutralize them.Free radicals are unstable molecules that can damage cells and tissues.Renal damage associated with diclofenac has also been linked with inflammation, a complex biological process that is characterized by the release of inflammatory cytokines that play a significant role in the immune response.Tumour necrotic factor alpha and Interleukin-1β (TNF-α and IL-1β) are involved in the recruitment of inflammatory cells to the site of infection or injury.They also promote the release of other pro-Corresponding Author: Samuel Dada inflammatory cytokines.This study was designed to investigate the effects of diclofenac on renal antioxidants and cytokine in male Wistar rats.

MATERIALS AND METHODS
In this study, 15 male Wistar rats were selected.These rats are known for their docile nature, ease of handling, and genetic homogeneity, making them an ideal choice for controlled experiments.The rats were randomly divided into 3 groups of equal number, housed in separate compartments of plastic cages and allowed to acclimatize for a week.Standard laboratory setting with a natural light/dark cycle and room temperature and humidity were maintained and they were fed standard rat pellets and distilled water ad libitum.Experimental design: The rats were randomly divided into three groups: control group, low-dose diclofenac group (10mg/kg/day), and high-dose diclofenac (30mg/kg/day).
Drugs and vehicle: Diclofenac potassium tablet (Cataflam® 50mg by Novartis) was purchased from a private pharmaceutical outlet.The drug was dissolved in distilled water and the calculated dosages was administered orally for 10 successive days according to the study protocol using rat cannula.The dose of diclofenac that is sufficient to cause renal damage was determined from previous reports.[9][10][11] Collection of tissue and blood samples for biochemical analysis At the end of the experiment, the rats were sacrificed by cervical dislocation method.Whole blood samples were collected from each rat separately using cardiac puncture technique with sterile needles and syringes into clean anticoagulant-free bottles.The blood was then allowed to coagulate for about 30 minutes before the serum in each tube was separated by cold centrifuge at 4000rpm for 10 minutes to obtain sera.The serum was collected with the aid of a Pasteur pipette in plain bottles and kept in ice-cold pack for analysis.Also, the right kidneys were harvested immediately and rinsed in ice-cold physiological saline.About 10% of each kidney tissue was homogenized using a mortar and pestle in 100 mM potassium phosphate buffer at pH 7.4 and centrifuged at 10,000 rpm for 10 minutes at 4°C in a cold centrifuge.The supernatant was collected and used for the determination of enzyme activities in renal homogenates and marker of lipid peroxidation using appropriate method; Superoxide dismutase (SOD) [12], Malondialdehyde (MDA) [13] and Fortress kit (Fortress Diagnostic Limited, United Kingdom) was used for the quantitative determination of total Glutathione peroxidase (GPx) according to the manufacturer's instructions.ELISA kits (Fortress) were used for the determination of KIM-1 and renal cytokine (TNF-α) according to the manufactural instructions.Serum creatinine was analysed using the method described by Bartels and Bohmer. [14]

Statistical analysis
The data obtained were entered and analysed with GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA, USA.Values of all measured parameters were presented as the mean and standard deviations and were analysed using oneway analysis of variance followed by Tukey's multiple comparison test.The P values of less than or equal to 0.05 were considered statistically significant.

Effect of treatment on kidney function.
The serum creatinine level was significantly higher in highdose DCF treated rats compared to the control (2.65±0.43versus 0.80±0.11),P = <0.0001,(Figure1a).There was no significant difference between the control group and low dose DCF group, (0.80±0.11versus 1.28±0.37),P= 0.0630, Table 1.As shown in figure 1b and Table 1, the level of KIM-1 in the high-dose DCF treated group was significantly elevated compared to the control group, P=<0.0001.we observed no significant difference in the KIM-1 level between the lowdose DCF (10mg/kg) group and the control group.

Effect of treatment on renal cytokine.
A significant increase in the mean value of the TNF-α was observed in the high-dose diclofenac-treated group compared to the control (1276.0±90.18versus 222.90±38)P=<0.000, (Figure 2, Table 1).

Effect of treatment on the level of antioxidants and markers of oxidative stress.
The effect of the low-dose diclofenac (10mg/kg) is shown in Tables 2. There was no significant difference in the mean level of these markers between the control and the low-dose DCF treated group.Compare with the control group, the highdose DCF (30mg/kg) caused a significant increase in the level of MDA (2.62±0.29 versus 8.74±4.34),p<0.001 with a concomitant reduction in the level of SOD (0.78±0.11 versus 0.61±0.

DISCUSSION
Nonsteroidal anti-inflammatory drug such as diclofenac are a class of drugs commonly used for their analgesic and antiinflammatory effects.
[1] In the medical literature, the use of diclofenac has been associated with different forms of kidney damage in both experimental animals and humans.
In this study, we found that administration of diclofenac at a dose of 30mg\kg\day resulted into deranged renal function, altered renal antioxidant, increase cytokine level in addition to elevated marker of lipid peroxidation.Non-steroidal antiinflammatory drug has been linked to altered redox reaction, inflammation and development of renal injury.[1,9] Nonetheless, low levels of reactive oxygen species (ROS) and reactive nitrogen specie (RNS) are important for normal redox signalling in the kidney.This is essential for renal vasoreactivity, cell growth and survival while also providing sensor for hypoxia.[5-7, 11, 18, 19] Diclofenac was found to be a strong inducer of oxidative stress, as evidenced by the dose-dependent increase in MDA levels and reduction in SOD and GPx activities in our study.This finding is in agreement with the work of Hickey et al., who demonstrated the nephrotoxicity of diclofenac and documented the drug to be a strong inducer of oxidative stress in male ICR mice.
[3] The role of oxidative stress, particularly production of reactive oxygen species (ROS) and antioxidant system dysfunction with administration of diclofenac has been well established.[2,5,9,10,17,[20][21][22] Diclofenac produced ROS mainly through targeting two subcellular organelles, microsomes and mitochondria.Mitochondria are essential organelles in the kidney they produce cellular energy for metabolic processes.During mitochondrial metabolism, ROS are produced which function as secondary messengers that induce post-translational modifications (PTM) in proteins in addition to activating or deactivating different cell signalling pathways.
[23] However, in the presence of nephrotoxic substances, ROS overproduction may lead to oxidative stress inducing dysregulation of redoxsensitive signalling pathways, mitochondrial dysfunction and altered metabolism.[24,25] These imbalances can ultimately lead to changes in the cell redox-sensitive signalling pathways, causing inflammation and apoptosis cell death.

CONCLUSION
This study provides evidence that diclofenac may induce alterations in renal antioxidants, mediates oxidative stress and Figure 1a.Effect of treatments on level of serum creatinine.Data are expressed as mean ± SD. (n = 5/group).DCF=diclofenac, *p< 0.05 versus control group.+p>0.05DCF low dose versus control

Table 2 . Level of renal Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in control and experimental groups of rats.
*: Significant different from control group at p <0.05 + : non-significant difference from control group, p>0.05.