INTRODUCTION

Paracetamol (APAP) induced combined hepatonephrotoxicity is common in both humans and experimental animal models after accidental or intentional ingestion of an overdose of the drug (^{McGill et al., 2012}; ^{Karaali et al., 2018}). About 50 % of acute liver failure admitted cases in the United States of America are caused by APAP poisoning (^{Ostapowicz et al., 2002}; ^{Larson et al., 2005}). It is the most common agent of intentional self-harm and APAP poisoning claimed the life of 284 persons aged 12 years and over between 1993-1996 in England and Wales, UK (^{Hawton et al., 2004}). APAP is metabolised in the liver and hepatotoxic metabolites that represents about 10 % of the whole metabolites are rapidly inactivated by glutathione (GSH) to protect the hepatocytes (^{James et al., 2003}). But, with the drug overdose for example, the elevated levels of liver toxic metabolites, mainly N-acetyl-p-benzoquinoimine (NAPQI) rapidly deplete GSH and covalently modify cellular proteins leading to the generation of high levels of reactive oxygen species (ROS) and depletion of the ATP, which results in mitochondrial damage and hepatocyte and kidney injuries (^{Hinson et al., 2004}). However, it was postulated that depletion of 90 % of GSH in hepatocytes is critically necessary for the development of cell necrosis (^{Henderson et al., 2000}). In addition, hepatic inflammatory cytokines are also reported to be involved in APAP-induced liver injury (^{Blazka et al., 1996}).

Quercetin and resveratrol are polyphenolic antioxidants found in fruits, vegetables, and grains (^{Burda & Oleszek, 2001}; ^{Cudmore et al., 2012}). They have been widely known to have potent cardiovascular protective and therapeutic effect via scavenging ROS (^{Hung et al., 2000}), anti-inflammatory effects (^{Rogerio et al., 2007}; ^{Al-Ani, 2013}), inhibit lipid peroxidation (^{Frankel et al., 1993}), and liver and kidney protection (^{Faghihzadeh et al., 2015}; ^{Zhang et al., 2017}). The combination of resveratrol and quercetin has not been used before to study the protection of kidney tissue upon paracetamol intoxication in an animal model. Therefore, this study was designed to investigate the degree of protection by resveratrol and quercetin given in combination against APAP intoxication to the glomerular and renal tubular structure, and compare it with the level of protection provided by these agents to known kidney injury biomarkers.

MATERIAL AND METHOD

Reagents and assay kits. Quercetin (C15H10O7, CAS Number 117-39-5) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was prepared daily and freshly by dissolving in a normal saline solution (0.9 % NaCl) to the final concentration of 50mg/ml. Resveratrol (C14H12O3, Cat No. R5010) was also purchased from Sigma-Aldrich (St. Louis, MO, USA) and was prepared daily and freshly by dissolving in a saline solution (0.9 % NaCl) containg 20 % hydroxypropyl cyclodextrin (American Maize-Products Co., Hammond, IN, USA) to a final concentration of 30 mg/ kg. Assay kits for determination of malondialdehyde (MDA, Cat No. NWK-MDA01) were purchased from NWLSS (Vancouver, BC, Canada). Superoxide dismutase (SOD) assay kit was purchased from Cayman Chemical, Cat. No.706002. ELISA kits for determination the levels of IL-6 (Cat No. ELR-IL6-001) was purchased from RayBio, GA, USA. ELISA kits for determination of TNF-α (Cat No. ab46070) was purchased from Abcam, Cambridge, UK.

Animals. All experimental procedures were approved by the medical research ethical committee at King Khalid University and according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No.85-23, revised 1996). Sprague Dawley rats (n=18) weighing 170-200 g were used in this study. All rats were bred and housed in the research center of King Khalid University, College of Medicine (Abha, Saudi Arabia), at temperature of 23 ± 1 °C and a 12 h light: 12 h dark cycle. Rats had free access to tap water and fed standard laboratory chow during the acclimatization period.

Experimental design. After a one week adaptation period, rats were randomly assigned into 3 groups (n = 6; each) and were distributed in their corresponding cages and classified as follows: (1) Control group: rats received normal saline daily for 7 days; (2) APAP intoxicated group (Model group): rats received normal saline for 7 consecutive days and then given a single dose of APAP (2 g/kg, orally); and (3) RES+QUR+APAP group: rats were pre-treated with RES (30 mg/kg) and 50 mg/kg QUR for 7 consecutive days and then administered with a single dose of APAP (2 g/kg, orally). APAP was administered to the desired groups one hour after the last dose of treatment on day 7 and all treatment in all groups were administered i.p. in a final volume of 1 ml. All animals were sacrificed at day 8.

Determination of tissue levels of MDA, SOD, and TNFα, and IL-6. At day 8, animals were sacrificed and tissue levels of MDA, SOD, TNF-α, and IL-6 were determined using ELISA kits according to the manufacturer’s instructions.

Histological examination. As we previously reported (AlHashem et al., 2019), kidney specimens were immediately fixed in 10 % formal saline for 24 hours. Paraffin blocks were prepared,and 5 µm thick sections were subjected to hematoxylin and eosin (H&E) stain to elucidate the status of kidney architecture and the structural changes.

Statistical and morphometric analysis. The data were expressed as mean ± standard deviation (SD). Data were processed and analyzed using the SPSS version 10.0 (SPSS, Inc., Chicago, Ill., USA). One-way ANOVA was done followed by Tukey’s post hoc test. Pearson correlation statistical analysis was done for detection of a probable significance between two different parameters. Results were considered significant if p ≤ 0.05.

Using "Leica Qwin 500 C" image analyzer (Cambridge, UK), the diameters of glomeruli space were obtained in 10 non overlapping high power fields/ rat of H&E-stained sections. Quantitative data were tabulated as means and standard deviations (SD) and compared using analysis of variance (ANOVA) followed by post-Hoc analysis (Tukey test). Significant difference was considered when P-value <0.05. Calculations were made on SPSS software (version 19).

RESULTS

Resveratrol plus quercetin protect kidney tissue against APAP-induced injury. Acute kidney injury was induced in the model group of rats by a toxic dose (2 g/kg body weight) of APAP. Harvested kidney tissues from all animal groups at day 8 were stained with H&E and examined under light microscopy. Compared to normal tissue histological structure of renal corpuscles or Malpighian renal corpuscles (MC), proximal (P) and distal (D) convoluted tubules in the control group (Figs. 1A and 1B), APAP substantially damaged the kidney tissue as demonstrated by widening of glomeruli space (arrowhead), tubular dilatation (curved arrow), numerous cellular debris in the renal tubules with tubular epithelial degeneration, vacuolization (wavy arrows), and prominent desquamation of the lining epithelium (Figs. 1C and 1D). Pretreatment with resveratrol plus quercetin preserved normal structure of renal corpuscles and convoluted tubules (Figs. 1E and 1F). However, few mildly dilated glomeruli space were noted in some fields. Furthermore, quantitative analysis of the mean diameter of glomeruli space revealed substantial protection (p<0.0001) by resveratrol plus quercetin but still significant (p=0.0012) to the control group (Fig. 1G).

Fig. 1 Resveratrol and quercetin protect against APAPinduced acute kidney injury in rats. H&E stained kidney sections (A, C, E x200; B, D, F x400) obtained at the end of the experiment in different groups of rats used in this study; Control group (A and B), APAP group (C and D), and RES+QUR+APAP group (E and F). Quantitative analysis of the mean diameter of Glomeruli’s space is shown in (G). Abbreviations: MC, renal corpuscles or Malpighian renal corpuscles; P, proximal convoluted tubule; D, distal convoluted tubule. 

Resveratrol plus quercetin protect the modulation of biomarkers of kidney tissue injury induced by APAP. High blood and tissue levels of inflammation and oxidative stress are known to be involved in the pathology of acute kidney injury in animal models and humans (^{Askari et al., 2018}; ^{Ravarotto et al., 2018}). To investigate the level of inhibition of modulation of these biomarkers by combined injections of resveratrol and quercetin for 7 days prior to APAP intoxication; we measured tissue levels of inflammation (Figs. 2A and 2B) and oxidative stress and antioxidant (Figs. 3A and 3B) in all rat groups. As shown in Figures 2A and 2B, acute kidney injury induced by APAP significantly (p<0.05) increased TNF-α and IL-6, which were substantially protected by RES+QUR. In addition, APAP significantly (p<0.05) augmented the oxidative stress biomarker MDA (Fig. 3A) and ameliorated the antioxidant SOD (Fig. 3B), which were also substantially protected by RES+QUR.

Fig. 2 Resveratrol and quercetin inhibit APAP-induced inflammatory biomarkers in rats. Kidney homogenates levels of TNF-a (A) and IL-6 (B) were measured at the end of the experiment in different groups of rats used in this study; Control group, APAP group, and RES+QUR+APAP group. Results represent the mean (±SD); n=6 for each group. Experiments were performed in triplicate. *p<0.05 versus control, **p<0.05 versus APAP. 

Fig. 3 Resveratrol and quercetin protect against APAP-induced modulation of oxidative stress and antioxidant biomarkers in rats. Kidney homogenates levels of MDA (A) and SOD (B) were measured at the end of the experiment in different groups of rats used in this study; Control group, APAP group, and RES+QUR+APAP group. Results represent the mean (±SD); n=6 for each group. Experiments were performed in triplicate. *p<0.05 versus control, **p<0.05 versus APAP. 

Positive correlation between renal corpuscle injury scoring and biomarkers of inflammation and oxidative stress. We determined the correlation between the mean diameter of glomeruli capsule space and the tissue levels of inflammation and oxidative stress biomarkers in all animal groups in order to further confirm and characterize that the role of the antioxidants and anti-inflammatory agents, resveratrol plus quercetin are stable and useful “drugs” in kidney injury rats, and to further support the link between acute kidney injury and inflammation and oxidative stress. As shown in Figures 4A-C, a positive correlation was shown between acute kidney injury and these biomarkers; glomeruli space versus TNF-α (r = 0.8899) (p<0.0001), glomeruli space versus IL-6 (r = 0.8986) (p<0.0001), and glomeruli space versus MDA (r = 0.8552) (p<0.0001). In addition, a negative correlation was shown between glomeruli space and the antioxidant SOD (r = - 0.7870) (p<0.0001) (Fig. 4D).

Fig. 4 Correlation between Glomeruli’s space and biomarkers of inflammation, oxidative stress, and antioxidant. The mean diameter of Glomeruli’s space was measured in all groups of rats on day 8 and the relationship between Glomeruli’s space and TNF-α, IL-6, MDA, and SOD are shown in (A - D), respectively. 

DISCUSSION

The main objective of our study was to investigate the potential protective effect of the combined two antioxidants, resveratrol and quercetin on kidney damage induced by APAP in a rat model of acute kidney injury using light microscopy. In addition, our protective approach using both agents was also used to assess levels of kidney injury biomarkers, oxidative stress, antioxidant, and inflammation using ELISA approach. Therefore, rats were pre-treated for one week with resveratrol plus quercetin prior to the induction of the disease by a toxic dose of APAP, and the histological and biochemical parameters were monitored and confirmed the beneficial effects of these agents (Figs. 1-4). Resveratrol plus quercetin markedly inhibit kidney injury and effectively inhibit the modulation of MDA, SOD, TNFα, and IL-6 in APAP-induced acute nephrotoxicity in rats. Our data that point to the beneficial effects of resveratrol plus quercetin that ameliorate the deleterious effects of paracetamol are in agreement with the previously published studies that showed resveratrol protects against several types of renal injury induced by several methods such as diabetic nephropathy, drug-induced injury, and aldosterone-induced renal injury (^{Kitada & Koya, 2013}), and quercetin improved renal function and protected the kidney in a rat model of adenine-induced chronic kidney disease (^{Yang et al., 2018}). However, conflicting data on the protective / treatment effects of resveratrol alone against APAP-induced acute liver injury in rats have been reported (^{Wojnarová et al., 2015}; ^{Elbe et al., 2018}). A recently published work (Elbe et al.) reported a significant inhibition of liver iNOS immunostaining and hepatocyte ultrastructure damage by RES (10 mg/kg) given 20 minutes post APAP (1 g/kg) injection in rats. Whereas, administrating triple-dose RES (30 mg/kg) at the same time with APAP (1 g/kg) to rats caused a weak inhibition of biomarkers of liver injury, ALT and no effect on the ALT levels in cultured hepatocytes prepared from these animals (Wojnarová et al.). In addition, the same group (Wojnarová et al.) found no significant pathological changes in the liver tissue of the model group (APAP) stained with H&E. Whereas, our H&E images demonstrated a substantial kidney damage induced by APAP (Figs. 1C and 1D). Our treatment protocol (2 g/kg APAP) versus their treatment protocol (1 g/ kg APAP) could be the reason of such differences.

Collectively, our data support the conclusion that pretreatment with resveratrol plus quercetin can effectively protect against acute kidney injury and inhibits inflammatory and oxidative stress biomarkers in a rat model of APAP overdose-induced nephrotoxicity.