Entacapone scavenges peroxynitrite and protects against kidney and liver injuries induced by renal ischemia/reperfusion in rats

Eman Soliman1 · Samar M. Shewaikh1 · Ahmed Fahmy1 · Shimaa Elshazly1
Received: 25 September 2020 / Accepted: 24 February 2021
© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021



Acute kidney injury (AKI), secondary to renal ischemia/reperfusion (I/R), is a serious problem associated with high mortality. The pathophysiology of AKI after renal I/R involves peroxynitrite production; hence, scavenging this metabolite may rescue AKI. Entacapone is a catechol-O-methyl transferase (COMT) inhibitor which elicits antioxidant activity by scavenging peroxynitrite. Therefore, we hypothesized that the peroxynitrite scavenging activity of entacopone protects against AKI after renal I/R injury in rats.


Male Wistar rats were given either entacapone or a well-known peroxynitrite scavenger (FeTPPS) daily for 10 days before I/R procedures. I/R was induced by occluding both renal pedicles for 45 min followed by reperfusion for 24 h.


Pre-treatment with either entacapone or FeTPPS improved renal function as indicated by a significant reduction in serum creatinine and urea when compared to I/R group (P < 0.05). I/R injury increased renal levels of NO (4-folds, P < 0.05), iNOS (4-folds, P < 0.05), and 3-nitrotyrosine (5-folds, P < 0.05) compared to sham control. These effects were abrogated in animals pre-treated with entacapone or FeTPPS before being subjected to I/R (P < 0.05). In addition, entacapone or FeTPPS significantly inhibited I/R-induced elevation in renal TNF-α levels (78% and 58%, respectively) and caspase-3 activity (72% and 56%, respectively) indicating the reduction of both inflammation and apoptosis in the kidney (P < 0.05). The two drugs also improved kidney and liver functions in rats with renal I/R injury. Conclusion Our study showed that entacapone and FeTPPS protected against AKI and remote liver damage associated with renal I/R and this effect might be due to scavenging peroxynitrite and reducing nitrosative stress. Keywords : Acute kidney injury · Renal ischemia/reperfusion · Peroxynitrite · Nitrosative stress · Entacapone · Rats Introduction Acute kidney injury (AKI) is a complex disorder developed in about 7% of hospitalized patients and 25% of patients admitted to intensive care units [1]. Renal ischemia/reperfu- sion (I/R) injury is one of the most common causes of AKI. Reduction of renal blood flow due to I/R results in a reduc- tion of oxygen supply to kidneys which eventually impairs kidney function and leads to AKI. I/R-induced AKI can be seen in several cases such as shock, hepato-renal syndrome, sepsis, and surgical procedures such as kidney transplanta- tion where I/R directly affects grafts and patient survival [2]. Several mechanisms are involved in the development and progression of I/R injury, including the generation of reactive oxygen species and peroxynitrite [3]. Peroxynitrite is a strong oxidant formed by the reac- tion of superoxide anion (O2.−) and nitric oxide (NO). This reaction is 3–8 times faster than the decomposi- tion of O2.− by superoxide dismutase [4]. Peroxynitrite can damage the cell by several mechanisms. It can cause direct damage to the mitochondria, disrupting their func- tion [5]. Peroxynitrite can also alter the structure of DNA, resulting in over-activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP-1) that consumes nico- tinamide adenine dinucleotide (NAD +) and adenosine triphosphate (ATP) for (ADP-ribose) (PAR) formation, leading to apoptosis and necrosis [6]. Peroxynitrite can enhance nuclear factor kappa B (NF-KB) mediated pro- inflammatory pathways, therefore increasing inflammatory cytokines production [7]. Previous studies have shown that peroxynitrite is implicated in the pathogenesis of AKI, and the use of peroxynitrite scavengers may protect against renal I/R injury [8, 9]. Among the well-known peroxynitrite scavengers is Fe (III) 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrina- tochloride (FeTPPS). FeTPPS is a decomposition catalyst that scavenges peroxynitrite by activating its isomerization to nitrate [10, 11]. Previous studies have shown that scav- enging renal peroxynitrite by FeTPPS ameliorates nitrosa- tive stress and reduces acute renal damage [12]. Entacapone is a nitrocatechol derivative that selectively and reversibly inhibits the catechol-o-methyl transferase (COMT) enzyme [13, 14]. Entacapone is used in combi- nation with levodopa in the treatment of Parkinsonism to increase its bioavailability and activity and also to pre- vent the wearing-off phenomenon associated with levo- dopa treatment [15–18]. Previous studies have shown that entacapone has an anti-inflammatory effect and a potent peroxynitrite scavenging activity [19, 20]. It was suggested that this activity is attributed to the presence of a potent peroxynitrite scavenger moiety, catechol (1,2-dihydroxy- benzene), in the entacapone structure [21]. Given that peroxynitrite is involved in the pathogenesis of AKI and that peroxynitrite scavengers can protect against renal I/R injury [8, 9], we hypothesized that reducing peroxynitrite levels by entacapone ameliorates nitrosative stress and protects against renal I/R injury. Materials and methods Animals Forty adult male Wistar rats (180–250 g) were used in the present study. Rats were obtained from the Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. The animals were housed 4 animals/cage and kept at a controlled temperature (23 ± 2 °C), humidity (60 ± 10%), and a light/dark (12 h/12 h) cycle. Rats were supplied with a standard diet and water ad libitum. Ethical statement The experimental design and animal handling proce- dures were approved by the Ethical Committee for Ani- mal Handling at Zagazig University (approval number is P3-12-2017). Drugs and chemicals Entacapone was purchased from Al-Andalus Medical Co (Egypt). FeTPPS was from Cayman Chemical (USA). Thio- pental sodium was obtained from EPICO (10th of Rama- dan, Egypt), and Dimethyl sulfoxide (DMSO) was obtained from Tedia Company Inc., (Fairfield, OH, USA). All other chemicals used in the study were obtained from El-Nasr. Co, (Egypt) and are of high analytical grade. Experimental groups Rats were randomly divided into five experimental groups (8 animals/group). Group 1 (control group), group 2 (sham- operated), group 3 (I/R injury only), group 4 (received entacapone 10 mg/kg, orally and subjected to I/R injury), group 5 (received FeTPPS 10 mg/kg, i.p and subjected to I/R injury). Groups 1–3 received vehicle (4% DMSO, 4% Tween 80, 92% H2O). Entacapone, FeTPPS, and vehicle were administered daily for 10 days before the I/R procedure. The dose of entacapone (10 mg/kg) was selected accord- ing to preliminary experiments performed in our laboratory using different concentrations of entacapone up to 100 mg/ kg. The dose of FeTPPS (10 mg/kg) was used as previously described [22]. Induction of acute kidney injury by I/R procedure Animals were anesthetized by thiopental (50 mg/kg, i.p), placed on heating pads to maintain their temperature at 37 °C [23], and then subjected to I/R surgery as previously described [24]. In brief, abdominal areas were shaved, steri- lized, then flank incisions were made on both sides expos- ing left and right renal pedicles. Ischemia was induced by occluding renal pedicles using non-traumatic clamps for 45 min. At the end of the ischemic period, clamps were removed and reperfusion was initiated for 24 h. Sham- operated rats were subjected to the same surgical procedure without the application of clamps. Blood sampling At the end of the reperfusion period, animals were euthanized by isoflurane inhalation and then blood samples were collected from the retro-orbital plexus using non-heparinized microcap- illary tubes [25]. Blood samples were kept at room temperature for 30 min to clot and then centrifuged at 2700 × g to separate the serum. Serum samples were then aliquoted and stored at – 20 °C until the biochemical examination. Tissue sampling Kidneys and livers were collected, rinsed with saline, and divided into two parts. The first part was fixed in a 10% phosphate-buffered formalin solution for histological exami- nation and the second part was flash frozen and then stored at − 20 °C until the biochemical examination. Kidney and liver function measurements Serum creatinine, urea, AST, and ALT were measured by colorimetric methods using colorimetric assay kits (Bio diagnostic kits, Egyptian company of biotechnology, Egypt) according to the manufacturer’s instructions. Absorbance was detected using a Jenway Genova spectrophotometer (Bibby Scientific, Staffordshire, UK). Measurement of nitric oxide (NO) levels in kidney Stored tissues were homogenized in ice-cold phosphate- buffered saline (PBS, pH 7.2), centrifuged at 12,000 × g for 10 min, and the supernatant was separated. Nitric oxide (NO) content was measured using a colorimetric assay kit (Cat. No. 2533, Bio-diagnostic, Egyptian company of bio- technology, Egypt) as described by the manufacturer.Enzyme‑linked immunosorbent assay (ELISA) for measurement of 3‑nitrotyrosine, nitric oxide synthases (NOS), and tumor necrosis factor‑alpha (TNF‑α) Stored liver and kidney tissues were homogenized in ice- cold PBS (pH 7.2), centrifuged at 12,000 × g for 10 min, and the supernatant was separated. ELISA kits from MyBio- source (Hayward, USA) were used to measure 3-nitrotyros- ine (Sandwich, Cat. No. MBS732683) in liver and kidney homogenates and endothelial nitric oxide synthase (eNOS, Competitive, Cat. No. MBS721860), inducible nitric oxide synthase (iNOS, Competitive, Cat. No. MBS723326), and TNF-α (Sandwich, Cat. No. MBS355371) in kidney homogenates as described by the manufacturer. Measurement of caspase‑3 activity in kidney tissue Caspase-3 activity in kidney tissue homogenates was meas- ured using a colorimetric kit (APT131, Sigma-Aldrich, USA) as instructed by the manufacturer. Histopathological examination Formalin-fixed kidney and liver tissues were paraffin- embedded and sectioned 5 µm thick. Sections were dewaxed, gradually hydrated, and then stained with hematoxylin and eosin (H&E). Stained sections were then examined and blindly scored by expert pathologists who captured the most representative images from each group. Scoring has been performed according to the following criteria: 0 = negative (no change); 1 = weak (mild); 2 = moderate; and 3 = severe alterations [24, 26]. Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was carried out using Graph- Pad Prism version 5 (Graph Pad Software, Inc. CA, USA). One-way analysis of variance (ANOVA), followed by Tuk- ey’s multiple comparison test, was performed to compare between different groups (P < 0.05). Normality tests were performed and all data follow a normal distribution. Histo- pathology scoring data were analyzed using non-parametric ANOVA, followed by the Kruskal–Wallis test, and are pre- sented as median (25th percentile-75th percentile). Results Entacapone and FeTPPS reduced renal 3‑nitrotyrosine and nitric oxide production by I/R To determine if entacapone and the peroxynitrite scavenger, FeTPPS, reduced renal peroxynitrite, the level of a footprint of peroxynitrite-mediated protein tyrosine nitration, 3-nitro- tyrosine, was measured. I/R induced a significant elevation in renal 3-nitrotyrosine compared to control groups. Pre- treatment of rats with entacapone or FeTPPS, significantly reduced 3-nitrotyrosine when compared to the I/R group suggesting a reduction in peroxynitrite levels (Fig. 1a). The observed reduction in peroxynitrite with entacapone and FeTPPS led us to hypothesize that entacapone and FeTPPS reduced AKI-induced nitrosative stress in rats with renal I/R. To test our hypothesis, the levels of NO and the expression of nitric oxide synthases (NOSs) were measured. We observed that I/R induced a significant elevation in both renal NO production and iNOS expression when compared to control groups. As anticipated, both entacapone and FeT- PPS significantly mitigated this effect (Fig. 1b, c). On the contrary, I/R reduced renal eNOS expression and this effect was inhibited by entacapone and FeTPPS (Fig. 1d). These observations suggested that the peroxynitrite scavenging activity of entacapone and FeTPPS was associated with a reduction in renal nitrosative stress. Fig. 1 Entacapone and FeTPPS reduce renal 3-nitrotyrosine and NO production by I/R. Animals were pre-treated with entaca- pone (EN,10 mg/kg) or FeTPPS (10 mg/kg) daily for 10 days then subjected to I/R injury as indicated in the materials and methods section 3-nitrotyrosine levels (a), nitric oxide levels (b), inducible nitric oxide synthase (iNOS) expression (c), and endothelial nitric oxide synthase (eNOS) expression (d) were measured in kidney tissues. Sta- tistical analysis was done using one-way ANOVA followed by Tukey’s post hoc test. @P < 0.05 vs. control group; *P < 0.05 vs. sham-operated group; #P < 0.05 vs. I/R injury group; $P < 0.05 vs. EN group. Entacapone and FeTPPS improved kidney function Serum creatinine and blood urea nitrogen were meas- ured to evaluate the reno-protective effect of entacapone and FeTPPS. AKI induced by I/R was associated with significant elevation in serum creatinine and blood urea nitrogen when compared to control groups. As expected, entacapone and FeTPPS improved kidney functions as indicated by a significant reduction in serum creatinine and blood urea nitrogen when compared to the I/R group (Fig. 2a, b). Entacapone and FeTPPS reduced I/R‑mediated renal inflammation and apoptosis To investigate whether entacapone and FeTPPS reduce I/R-associated inflammation, renal levels of the inflamma- tory cytokine and tumor necrosis factor-alpha (TNF-α), were measured. Our results showed that induction I/R sig- nificantly elevated TNF-α in the kidney when compared to control groups. This effect was associated with a marked increase in apoptosis as indicated by increased caspase-3 activity when compared to control groups. Pre-treatment of entacapone and FeTPPS significantly reduced both TNF-α level and caspase-3 activity when compared to the I/R group (Fig. 3). These results suggest that entacapone and FeTPPS protected against I/R-induced renal inflammation and apoptosis. Fig. 2 Entacapone and FeTPPS improve kidney function.Creatinine levels (a) and blood urea nitrogen (BUN) (b) were measured in blood serum. Sta- tistical analysis was done using one-way ANOVA followed by Tukey’s post hoc test. @P < 0.05 vs. control group; *P < 0.05 vs. sham-operated group; #P < 0.05 vs. I/R injury group; $P < 0.05 vs. EN group. Fig. 3 Entacapone and FeTPPS reduce I/R-induced renal inflammation and apoptosis.Tumor necrosis factor alpha (TNF-α) (a) and caspase-3 activity (b) were measured in kidney tissues. Statisti- cal analysis was done using one-way ANOVA followed by Tukey’s post hoc test. @P < 0.05 vs. control group; *P < 0.05 vs. sham-operated group; #P < 0.05 vs. I/R injury group; $P < 0.05 vs. EN group. Renal histopathological findings Ischemia for 45 min followed by 24 h reperfusion resulted in AKI as indicated by marked histological damage in renal cortex when compared to sham. Histological changes in the I/R group included the presence of atrophic glomerulus with wide bowman’s space and pyknotic or exfoliated nuclei in some renal tubules with vacuolated cytoplasm along with intraluminal proteinaceous casts in their lumen. Pre-treat- ment with entacapone and FeTPPS showed improvement in I/R-induced histological derangements (Fig. 4 and Table 1). Fig. 4 Representative images for kidney histology (H&E, 400×). Renal cortex of control group (a) and sham-operated group (b) shows glomerulus (G) surrounded by Bowman’s space (*), proximal (PT), and distal (DT) convoluted tubules. c Renal cortex of IR group shows atrophic glomerulus (ag) with wide bowman’s space (*). Most tubules (T) lose their nuclei (bifid arrow). Intralumina proteinaceous casts (ca) are shown in the lumen of some tubules. Others have dark- stained pyknotic nuclei (arrow), vacuolated cytoplasm (v), or exfoli- ated nuclei into lumens (short arrow). d Renal cortex of entacapone treated rats shows proximal tubule (PT) and distal tubules (DT) with vesicular nuclei (arrow head), few tubules (T) with pyknotic nuclei (arrow), others with exfoliated nuclei in the lumen (short arrow), glo- merulus (G) with slightly wide bowman space (*). e Renal cortex of FeTPPS treated rats shows few tubules (T) with dark pyknotic nuclei (arrow) and vacuolated cytoplasm, others with intralumina proteinaceous casts (ca) and exfoliated cells (short arrow) into their lumens. Congested glomerulus (Cg) with wide bowman space (*) is observed. Entacapone and FeTPPS improved AKI‑associated liver dysfunction Given that remote liver injury is an inevitable consequence of AKI induced by renal I/R, we determined whether entacapone and FeTPPS protected against renal I/R-induced liver damage. Serum levels of AST and ALT along with hepatic levels of 3-nitrotyrosine were measured. Renal I/R caused a significant elevation in hepatic 3-nitrotyrosine as well as blood AST and ALT when compared to control groups. Both entacapone and FeTPPS reduced 3-nitrotyrosine levels and improved liver functions as indicated by a significant reduction in serum AST and ALT when compared to the I/R group (Fig. 5a–c). Hepatic histopathological findings The histological assessment showed that renal I/R-induced remote liver damage was associated with a disruption in liver architecture around dilated and congested central veins, a hydropic degeneration of hepatocyte cytoplasm, and a dilated blood sinusoids. Pre-treatment with entacapone and FeTPPS reduced I/R-induced histological modification in the liver (Fig. 6 and Table 1). Discussion Renal I/R is one of the most common causes of AKI [27]. Peroxynitrite is involved in the pathophysiology of AKI after renal I/R and targeting peroxynitrite may be beneficial for renal I/R [8, 9]. Entacapone is a COMT inhibitor that elic- its antioxidant activity by scavenging peroxynitrite [20]. In the present study, we reported, for the first time, that enta- capone protected against kidney damage induced by renal I/R, and that this effect was associated with a reduction in peroxynitrite and an amelioration of nitrosative stress in the kidney. Entacapone also reduced AKI-mediated remote liver damage as well as hepatic peroxynitrite. The reno- and hepato-protective activities of entacapone mimicked those of the peroxynitrite scavenger, FeTPPS. Our data suggest that scavenging peroxynitrite by entacapone may be beneficial for renal I/R injury. Our findings are supported by a previous study by Chen and his colleagues which compares the scavenging ability of entacapone to other known antioxidants. The results of their study reveal that entacapone scavenges peroxynitrite more effectively than vitamin C [20]. Peroxynitrite is known to cause nitration of tyrosine to form 3-nitrotyrosine [28]. Given that FeTPPS, which is a known scavenger of perox- ynitrite, reduced 3-nitrotyrosine levels in both kidney and liver of I/R rats, it is tempting to speculate that the reduc- tion of 3-nitrotyrosine levels by entacapone was due to its peroxynitrite scavenging activity. In renal I/R injury, peroxynitrite is formed after the rep- erfusion due to the interaction between oxygen radicals and NO and contributes to acute kidney failure [9, 29]. Although adequate levels of NO is essential for preserving renal glo- merular function, excessive production is deleterious to renal tissue [30–32]. NO is produced in the kidney by two main enzymes, iNOS and eNOS. In the present study, we observed that renal I/R injury increased NO, peroxynitrite, and iNOS expression. However, eNOS expression was reduced. These observations are not surprising because upregulation of iNOS in AKI contributes to NO production; while, the downregulation of eNOS contributes to renal hemodynamic dysregulation [30, 33, 34]. Interestingly, the use of entaca- pone or peroxynitrite scavenger, FeTPPS, improved renal function, reduced NO, peroxynitrite, and iNOS levels in the kidney. Our findings are in line with the previous studies showing that impeding nitric oxide (NO) synthesis and scav- enging peroxynitrite ameliorate nitrosative stress and may improve renal functions in rats subjected to renal I/R [9]. Fig. 5 Entacapone and FeTPPS reduce hepatic levels of 3-nitro- tyrosine and improve liver func- tion. a 3-nitrotyrosine levels were measured in liver tissues. b AST levels and b ALT levels were measured in blood serum. Statistical analysis was done using one-way ANOVA fol- lowed by Tukey’s post hoc test. Peroxynitrite-induced nitrosative stress causes lipid per- oxidation, DNA destruction, and protein nitration [35–37]. This damage eventually results in cell apoptosis and tissue inflammation [4, 7]. In the current study, we reported an increase in TNFα expression and caspase 3 activation in rats with renal I/R injury, indicating the presence of inflam- mation and apoptosis along with impaired kidney function. We also observed that entacapone reduced I/R-induced TNFα expression and caspase 3 activation. Entacapone also improved the kidney structure and function of I/R rats. This effect mimics peroxynitrite scavenger, FeTPPS suggesting that reducing peroxynitrite by entacapone may play role in its reno-protective activity. Acute kidney injury induced by renal I/R is usually asso- ciated with damage to distant vital organs including the liver [38, 39]. Previous studies have reported that AKI-mediated systemic inflammation is involved in remote liver damage [40]. This inflammation is attributed to over-production and reduced clearance of cytokines by the kidney [41]. Using an experimental mouse model, Khastar and colleagues found that renal I/R induces hepatic oxidative stress and inflam- mation [42]. In line with these findings, the current study showed that renal I/R caused elevation of blood levels of liver enzymes ALT and AST, indicating hepatic dysfunction, which has been verified by histological changes in hepatic tissues of the I/R group. Interestingly, rats pre-treated with entacapone and FeTPPS showed improvement in hepatic structure and function. This hepatoprotective effect might be attributed to their peroxynitrite scavenging activity since the two drugs reduced renal I/R-induced elevation of perox- ynitrites in the liver. Conclusion Entacapone showed, for the first time, reno- and hepato- protective activities in rats subjected to renal I/R injury. Both entacapone and FeTPPS reduced 3-nitrotyrosine lev- els in the kidney and liver of I/R rats, suggesting the pos- sible role of peroxynitrite scavenging in the prophylactic activity of entacapone. Even though future studies are still needed to verify this mechanism, the current study provides a promising approach to protect liver and kidney in patients undergoing renal IR. Fig. 6 Representative images for liver histology (H&E, 400×). a Liver section of control group shows tightly packed cords of hepato- cytes with rounded vesicular nuclei and acidophilic cytoplasm (arrow heads) radiating from the central vein (CV) and separated by blood sinusoids (S) with intact endothelial cells. b Liver section of sham- operated animals shows normal hepatocytes with rounded vesicular nuclei and acidophilic cytoplasm (arrow heads) radiating from the central vein (CV) and separated by slightly dilated and congested blood sinusoids (S). c Liver section of renal I/R animals shows dis- rupted liver architecture around the dilated and congested central Acknowledgements The authors would like to thank Dr. Amira Ibra- him (Pathology department, Faculty of Medicine, Zagazig University, Egypt) for performing the histological analysis. Author contribution SE and AF conceived and designed research. SM conducted experiments. ES and SM analyzed data. ES and SM wrote the manuscript. 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