Characterization of Diclofenac-induced Renal Damage in Normotensive and Hypertensive Rats: A Comparative Analysis

Abstract

Background Diclofenac is the non-steroidal anti-inflammatory drug (NSAID) mostly prescribed worldwide, but it is highly associated with hypertension and acute kidney injury. Despite that, little information is available about the renal effects of diclofenac in hypertensive individuals, which led us to carry out this comparative study between the renal effects of this NSAID in normotensive (NTR) and spontaneously hypertensive rats (SHR).

Methods Male Wistar NTR and SHR were orally treated with vehicle (V: 10 mL/kg) or diclofenac sodium (D: 100 mg/kg) once a day for 3 days. Urine volume, electrolytes excretion (Na⁺, K⁺, Cl^-, and Ca²⁺), urea, creatinine, pH, and osmolarity were evaluated. Furthermore, blood samples and renal tissue were collected to perform biochemical and histological analysis.

Results Diclofenac increased the renal corpuscle and bowman’s space in the SHR, while no microscopic changes were observed in the renal tissue of NTR. Regarding the urinary parameters, diclofenac reduced urine volume, pH, osmolarity, and all electrolytes excretion, followed by decreased urea and creatinine levels in both lineages. Moreover, it also induced hyponatremia, hypokalemia, and hypocalcemia in SHR, while reduced glutathione-S-transferase activity, lipid hydroperoxides, and nitrite levels in renal tissue.

Conclusions The data presented herein demonstrated that diclofenac induces renal damage and impaired renal function in both NTR and SHR, but those effects are exacerbated in SHR, as seen by the histological changes and electrolytes balance disturbance, therefore, reinforcing that diclofenac may increase the risks of cardiovascular events in hypertensive patients.

Keywords

diclofenac sodium - kidney - renal corpuscle - hypertension - diuresis

Introduction

Diclofenac is the non-steroidal anti-inflammatory drug (NSAID) mostly prescribed worldwide. It was synthesized in 1973 and approved by FDA for the treatment of acute and chronic pain associated with inflammatory conditions, such as osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis, however, it cannot reverse or prevent chronic joint damage seen in these musculoskeletal disorders. Besides, diclofenac has been used to treat biliary colic, corneal abrasion, fever, gout, migraine, myalgia, and post-episiotomy pain [1] [2].

The mechanism of action of diclofenac involves the inhibition of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), reducing downstream arachidonic acid metabolite production, such as prostaglandin (PG)-E2, prostacyclin, and thromboxane, especially thromboxane-B2 (TXB2) [1]. Its adverse effects are more associated with the risk of cardiovascular events, such as myocardial infarction, heart failure, and stroke, and less with gastrointestinal events because diclofenac appears to act more selectively on COX-2 inhibition [3]. Moreover, the kidney plays a crucial role in the synthesis and metabolism of prostaglandins [4]; thus, NSAIDs can reduce renal blood flow and cause tubular obstruction inducing acute kidney injury [5] [6].

NSAIDs are very likely the drug that most raises the blood pressure of people in the United States, even because most patients who use them are older people at increased risk for developing hypertension. In addition, many people use this medication independently without informing the medical team. Indeed, the use of NSAID in patients with hypertension is frequent, being highly recommended for close monitoring of their blood pressure and kidney functions [7]. Despite that, little information is available about the renal effects of diclofenac sodium in hypertensive individuals, which led us to carry out this comparative study between the renal effects of this NSAID in normotensive and spontaneously hypertensive rats.

Materials and Methods

Drugs

Diclofenac sodium and other drugs and reagents were commercially obtained from Merck (Darmstadt, Germany). Diclofenac was prepared as a suspension in 1% carboxymethylcellulose.

Animals

Male Wistar normotensive rats (NTR) and spontaneously hypertensive rats (SHR) 3–4 months old were provided by Universidade do Vale do Itajaí (UNIVALI) after approval by the institutional ethics committee of the University (Process 007/19p1.). The animals were housed under standard laboratory conditions, 12 h light/dark cycle, and constant temperature (22±2°C), with free access to chow (Nuvilab Produtos Agropecuários LTDA, Colombo, Brazil) and water. All the experiments were conducted under international standards and ethical guidelines on animal welfare.

Experimental protocol

NTR and SHR were divided into two groups of six rats each that were orally treated with vehicle (V: 1% carboxymethylcellulose; 10 mL/kg), or diclofenac sodium (D: 100 mg/kg, 10 mL/kg), once a day, for 3 days. Forty-eight hours later, all animals were placed in metabolic cages with free access to water for urine collection. Volume measurement and collection were performed after 8 h, and urinary content of electrolytes (Na⁺, K⁺, Ca^2+, and Cl⁻), urea, creatinine, and pH values were determined at the end of the experiment. The osmolality values were calculated using the formula [2(Na⁺+K⁺)+(Urea x 0.16651)+(Glucose x 0.055)] [8]. After urine collection, all experimental groups were anesthetized with a solution of xylazine (10 mg/kg) and ketamine (80 mg/kg), and blood samples were collected. Moreover, renal tissue was removed, immediately homogenized, and frozen at − 80°C to perform biochemical analyses, and another part was fixed to perform histological analysis.

Analytical procedures

The urinary and blood levels of Na⁺ and K⁺ were measured by flame photometer, model BFC-300 by Benfer (São Paulo, Brazil), as previously described [9]. Colorimetric tests were used to evaluate the content of Cl⁻, Ca²⁺ (Gold Analisa, Belo Horizonte, MG, Brazil), creatinine, and urea, or the blood levels of glucose (Bioclin, Belo Horizonte, MG, Brazil) following the manufacturer’s instructions.

Biochemical analysis

The renal tissue was homogenized with 200 mM potassium phosphate buffer (pH 6.5) and subsequently used to determine the levels of reduced glutathione (GSH) and lipid hydroperoxides (LOOH). The remaining homogenate was centrifuged at 11,000 rpm for 20 min at 4 °C, and the supernatant was used for further determination of superoxide dismutase (SOD), and glutathione-S-transferase (GST) activity following the protocols previously described [10]. The myeloperoxidase (MPO) activity and nitrite and protein levels were determined following protocols previously described [11].

N-acetyl-beta-D-glucosaminidase (NAG) activity was evaluated following the method described by Mendes et al. [12] with some modifications. Briefly, 25 µl of each sample was mixed with 25 µl of NAG solution (2.24 mM in Milli-Q water) and 100 µl of 0.1 M citrate buffer (pH 4.5) and incubated 1 h at 37°C. The reaction was stopped by the addition of 100 μl of 0.2 M glycine buffer (pH 10.6). The absorbance values were measured at 400 nm. NAG activity was expressed as a change in OD per mg of protein.

Histological analysis

For histological examination, renal tissues were fixed in ALFAC solution (85% ethanol, 10% formaldehyde, and 5% acetic acid) and then dehydrated with alcohol and xylene, embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin/eosin (H&E). The material was photographed using a stereomicroscope (Olympus CBA microscope) with a magnification of 40+×+. The thickness (µm) of the renal corpuscle and glomerulus, as well as the bowman’s space, were measured using the ImageJ® program.

Statistical analysis

A blinded investigator to the experimental conditions analyzed the data. The results were expressed as mean±SD. One-way ANOVA followed by Dunnett's multiple comparisons test was applied to verify the differences between means using GraphPad Prism 7.00 (GraphPad Software, La Jolla, CA, USA). Values of p<0.05 were considered to show significant differences between means.

Results and Discussion

The kidney plays a crucial role in the synthesis and metabolism of the PGs [4]; thus, NSAIDs such as diclofenac sodium promote the reduction of PGs by inhibiting COX-1 and 2 activities are known for inducing kidney injury [5] [6]. Hassid et al. [13] have shown that PGs are synthesized in both the renal medulla and cortical area, and important cortical functions, such as renin secretion and control of renal blood flow, may be influenced by PGs produced locally. Moreover, it is well known that the adverse effects of diclofenac are highly associated with cardiovascular and renal events [3], which led us to carry out this comparative study between the renal effects of diclofenac in NTR and SHR.

The SHR lineage was developed by Okamoto and Aoki [14] to create an animal model for the study of hypertension that resembles human pathogenesis. These animals show ventricular hypertrophy, changes in morphology and vascular function, hemodynamic changes, and renal dysfunction [15]. Indeed, Ofstad and Iversen [16] demonstrated that SHR displays severe glomerulosclerosis, tubular damage, widespread interstitial fibrosis, and glomerular adherences induced by variations of capillary and tubular wall stretch caused by everyday variations of the systemic blood pressure, which corroborates with the increased size of renal glomerulus and decreased bowman’s space observed in SHR-vehicle-treated animals compared to NTR-vehicle presented in [Fig. 1a, c and d].

Fig. 1 Effect of diclofenac on microscopic morphology of renal tissue. (a) Representative images of histological analysis of the kidney sections were obtained from normotensive rats (NTR) and hypertensive rats (SHR) treated for 3 days with Vehicle (V: 10 ml/kg) or diclofenac (D: 100 mg/kg). (b) Renal corpuscle (µm); (c) Renal glomerulus (µm), and (d) Bowman’s space (µm). The values show the mean±SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons tests. *p<0.05 when compared with the V-SHR group. #p<0.05 when compared with the V-NTR group.

Interestingly, we have shown that diclofenac treatment for 3 days induced significant histological renal changes ([Fig. 1a, b, c], and d) in the SHR animals that were not detected in NTR. As seen in [Fig. 1d], the SHR group treated with diclofenac showed significantly increased bowman’s space compared to SHR vehicle, resulting in increased renal corpuscle as well ([Fig. 1a]). Reduced bowman’s space in SHR-vehicle is probably related to the increased capillary volume and glomerular adherences already described in this lineage [17]; thus, diclofenac's ability to reduce vasodilation by altering renal blood flow may explain this effect. The findings of the NTR align with those reported by Stormer [18]. Their study demonstrated that diclofenac at 100 mg/kg over three consecutive days resulted in an exacerbation of renal injury with upregulation of the pro-fibrotic marker fibronectin only in context of subclinical acute kidney injury induced by ischemia and reperfusion, but not in sham animals.

This data shows that hypertension, a well-known risk factor for chronic kidney disease (CKD) is intensified by diclofenac. Hypertension and CKD are intricately connected physiological conditions. Prolonged hypertension can contribute to the deterioration of kidney function, while a gradual decline in kidney function can reciprocally result in impaired blood pressure regulation [19]. In addition, diclofenac sodium by inhibiting COX-1 and 2 activities [5] [6] compromises renal function and hemodynamics of hypertensive individuals by inducing vasoconstriction due to decreased prostaglandin synthesis, which can compromise renal blood flow and glomerular filtration rate [20].

Diclofenac affected rat urine production by decreasing urine volume compared to the vehicle by 38% and 33%, respectively, in both NTR and SHR ([Fig. 2a]). According to the results presented in [Fig. 2b], diclofenac reduced the urinary pH (to 5.9 and 6.3 in NTR and SHR, respectively); however, these values remain within the normal range and, therefore, without biological relevance. However, urine osmolarity was also reduced by 74% and 70% in NTR and SHR, respectively ([Fig. 2b and c]) compared to the vehicle group, a reflection of the disturbance of electrolyte excretion seen in [Fig. 3 a, b, c, and d].

Fig. 2 Effect of diclofenac on urinary parameters. Effect of diclofenac on urine volume (a), pH (b), and osmolarity (c) in normotensive rats (NTR) and hypertensive rats (SHR). Vehicle (V: 10 ml/kg), Diclofenac (D: 100 mg/kg). The values show the mean±SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons tests. *p<0.05 when compared with each respective V group. #p<0.05 when compared with the V-NTR group.

Fig. 3 Effect of Diclofenac on electrolytes excretion. Panels a, b, c, and d represent the levels of Na⁺, K⁺, Cl⁻ and Ca²⁺; panels E and F represent the levels of urea and creatinine from the urine of normotensive rats (NTR) and hypertensive rats (SHR). Vehicle (V: 10 ml/kg), diclofenac (D: 100 mg/kg). The values show the mean±SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons tests. *p<0.05 when compared with each respective V group. #p<0.05 when compared with the V-NTR group.

Although changes in the renal corpuscle of SHR compared to NTR are evident, no differences were observed in urine production between the lineages; however, significant differences were observed between them in the electrolytes’ excretion. As observed in [Fig. 3] a and [3] d, the vehicle-only treated SHR group showed decreased Na⁺ and Ca²⁺ excretion compared to the NTR-vehicle group (30% and 27%, respectively). On the other hand, groups treated with diclofenac exacerbated this reduction. Interestingly, the plasma levels of these electrolytes ([Fig. 4a and d]) also showed that diclofenac induced hyponatremia and hypocalcemia, but only in SHR. Furthermore, NTR and SHR treated with diclofenac showed a marked reduction in the excretion of all evaluated electrolytes, pointing out that this NSAID can induce in the NTR and exacerbate in the SHR, the retention of sodium and water, which is a known risk for increasing peripheral vascular resistance and blood pressure, and also kidney failure [21]

Fig. 4 Effect of diclofenac on the plasmatic levels of electrolytes. Panels a, b, c, and D represent the levels of Na⁺, K⁺, Cl⁻ and Ca²⁺; panels E and F represent the levels of urea and creatinine from the urine of normotensive rats (NTR) and hypertensive rats (SHR). Vehicle (V: 10 ml/kg), Diclofenac (D: 100 mg/kg). The values show the mean±SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons tests. *p<0.05 when compared with each respective V group. #p<0.05 when compared with the V-NTR group.

Besides, the diclofenac also reduced the urinary excretion of other electrolytes not disturbed only by the pathophysiology of SHR, such as K⁺ and Cl^- ([Fig. 3c and d]). In fact, Cl^- levels suffered a slight increase in the plasma ([Fig. 4c]), especially in SHR. Changes related to Cl^- excretion in urine may be dependent and directly related to Na⁺ excretion or independent and associated with acid-base imbalance [17]. Although we did not measure bicarbonate values in urine and blood, we can suggest that Cl^- excretion changes are related to the reduction of glomerular filtration and Na⁺ excretion since urinary pH was little affected and the increase in plasma levels of Cl^- was quite discrete. Moreover, plasma K⁺ levels were significantly decreased by diclofenac in SHR ([Fig. 4b]). Considering that renal K⁺ excretion is also reduced; we can assume that transcellular shifts are altered. It is known that cellular uptake of K⁺ can be promoted by alkalemia, insulin, beta-adrenergic stimulation, and aldosterone [22]. Although we cannot suppose the cause of this change, it can be inferred that diclofenac increases the risk of SHR developing cardiac arrhythmias.

In addition, diclofenac-induced renal dysfunction was confirmed by the reduced excretion of urea and creatinine, the routine markers used to analyze renal function [23] ([Fig. 3e and f]). However, the plasmatic levels of these markers were not relevant altered ([Fig. 4e and f]), probably because the consequences of renal damage are initial and further studies evaluating more extended treatment periods are needed.

The oxidative imbalance in the renal tissue of SHR has been demonstrated in several studies [24] [25] following the data presented in [Fig. 5] that shows an increase in LOOH, a marker of lipid peroxidation [26], and a decrease in SOD activity, an essential antioxidant enzyme responsible for catalyzing the dismutation of the superoxide anion to hydrogen peroxide [27].

Fig. 5 Effect of diclofenac on renal markers of oxidative stress and inflammation. (a) Lipid hydroperoxides (LOOH) content, (b) reduced glutathione (GSH) levels, (c) superoxide dismutase (SOD) activity, (d) glutathione S-transferase (GST) activity, (e) myeloperoxidase (MPO) activity, (f) N-acetyl-beta-glucosaminidase (NAG) activity, and (g) nitrite levels evaluated in kidney samples obtained from normotensive rats (NTR) and hypertensive rats (SHR) treated for 3 days with Vehicle (V: 10 ml/kg) or Diclofenac (D: 100 mg/kg). The values show the mean±SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons tests. *p<0.05 when compared with the respective V group. #p<0.05 when compared with the V-NTR group.

Treatment with diclofenac appeared to reduce oxidative damage to SHR cell membranes but not to NTR, as seen by decreased lipid peroxidation levels ([Fig. 5a]). In contrast with another authors that found increased levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂)/ROS in smaller doses [28], NTR treated diclofenac 100 mg/kg did not increase LOOH herein. However, as we do not directly quantify reactive oxygen species, we cannot infer the absence of oxidative damage in the NTR.

Moreover, previous studies have suggested that diclofenac may restrict the progression of oxidative stress through its anti-inflammatory action [29] [30] Besides, diclofenac increased GSH levels in both lineages ([Fig. 5b]). GSH is a non-enzymatic antioxidant that acts as a co-factor for many cytoplasmic enzymes, including GST and GPX (glutathione peroxidase) [31]; therefore, it is expected that it increases during an oxidative imbalance or may be stimulated by diclofenac's actions. On the other hand, diclofenac decreased SOD activity ([Fig. 5c]) in both lineages and GST activity in SHR ([Fig. 5d]). One possibility for these alterations may be that these reductions in the groups treated with diclofenac occur due to its anti-inflammatory action and lower progression of oxidative stress, as discussed above.

In addition, decreased MPO activity and nitrite levels, markers of neutrophil infiltration, and nitric oxide (NO) production, respectively, are also observed ([Fig. 5g]). In fact, endothelial dysfunction in SHR decreases NO production in tissue and plasma compared to NTR [29] [30]. The role of NO and prostaglandins in the autoregulation of renal blood flow and glomerular filtration rate (GFR) in spontaneously hypertensive rat (SHR) have been studied [20] [32]. The autoregulation of RBF in aging SHR has been found to be dependent on prostaglandins [32]. Moreover in hypertensive rats with renal hypertensive damage, the GFR autoregulation is strongly NO dependent, thus it is possible that the impact of diclofenac in NO and PGs levels can alter GFR and contribute to the renal damage observed [20]. One aspect not addressed in our study that still requires investigation is the direct effect of diclofenac on blood vessels of NTR and SHR animals.

Furthermore, MPO activity, which is a marker of neutrophil infiltration, was reduced in both SHR groups ([Fig. 5e]), while NAG, which is a marker of macrophage infiltration was reduced by diclofenac in NTR, but not in SHR ([Fig. 5f]). These data suggest that neutrophils are already affected in the SHR group, which may be due to the pathophysiological process already installed in the kidneys. Through these last data, the complexity of the actions arising from the treatment with diclofenac is evident, which despite the known anti-inflammatory benefits, causes several other alterations at the tissue level, which needs to be duly deepened in future studies.

Conclusion

In conclusion, the data presented herein demonstrated that diclofenac induces renal damage and impaired renal function in both NTR and SHR by decreasing kidney function. Even more relevantly, we have seen that diclofenac evoked renal corpuscle size and bowman's space increase in SHR, besides inducing hyponatremia, hypokalemia, and hypocalcemia. Therefore, the data presented here support the importance of assessing the risks of cardiovascular and renal events in hypertensive patients using diclofenac.

Author contributions

T.B. contributed to the acquisition of the data, analysis and writing of the manuscript; A.B.F.L., M.E.B., L.N.B.M., L.M.S., and R.C.V.S. contributed to the acquisition and analysis of data; and P.D.S. contributed to the acquisition of the data, analysis, writing, and revision of the study. All authors approved the final version of the manuscript.

Ethical approval

This study was performed under international standards and ethical guidelines on animal welfare. Approval was granted by the Ethics Committee of Universidade do Vale do Itajaí (UNIVALI) (Process 007/19p1).

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Publication History

Received: 02 December 2023

Accepted: 26 February 2024

Article published online:
19 March 2024