Hesperidin protects against diethylnitrosamine / carbon tetrachloride-induced renal repercussions via up-regulation of Nrf 2 / HO-1 signaling and attenuation of oxidative stress

Article history: Received on: 05/09/2017 Accepted on: 29/10/2017 Available online: 30/11/2017 The kidney plays a central role in detoxification and excretion of toxic metabolites, and therefore is susceptible to toxicity by xenobiotics. Hesperidin is a citrus flavonoid with multiple beneficial therapeutic effects. Here, we investigated the possible modulatory effect of hesperidin on diethylnitrosamine (DEN)/carbon tetrachloride (CCl4)-induced nephrotoxicity in rats, pointing to the role of Nrf2/HO-1 signaling. Male Wistar rats received a single intraperitoneal injection of DEN. Two-weeks later, the DEN-induced rats received a subcutaneous injection of 3 ml/kg CCl4 once/week for 16 weeks. DEN/CCl4-induced rats were treated with 50 and 100 mg/kg hesperidin throughout the experiment. After 18 weeks, DEN/CCl4-induced rats showed renal injury evidenced by the significant increase in circulating kidney function markers as well as histopathological alterations. Concurrent supplementation of hesperidin significantly ameliorated kidney function markers and prevented renal tissue damage induced by DEN/CCl4. In addition, hesperidin treatment suppressed collagen deposition, lipid peroxidation and nitric oxide, and enhanced the antioxidant defenses in the kidney of DEN/CCl4-induced rats. Hesperidin up-regulated the expression of Nrf2 and HO-1 in the kidney of DEN/CCl4-induced rats. In conclusion, hesperidin prevents DEN/CCl4-induced nephrotoxicity via attenuation of oxidative stress and alleviation of the antioxidant defense system. These effects are mediated via up-regulation of Nrf2/ARE/HO-1 signaling pathway.


INTRODUCTION
The kidney contains many xenobiotic metabolizing enzymes and plays a central role in metabolizing drugs and foreign compounds in the body.Kidney diseases represent a public health issue worldwide and can range from mild infection to dangerous kidney failure (Nasri, 2014).The kidneys receive approximately 25% of the cardiac output, and renal tubules have a high tendency for drugs uptake via transporter proteins or endocytosis.This can result in high intracellular levels of various drugs and substances that then metabolized, leading to formation of reactive oxygen species (ROS) and toxic metabolites (Perazella, 2009).Excessive ROS production and oxidative stress have been demonstrated to play a role in drug-induced renal damage and tubular necrosis (Lopez-Novoa et al., 2011;Mahmoud et al., 2014Mahmoud et al., , 2015;;Abd El-Twab et al., 2016).N-nitrosamines are chemical compounds produced by reactions of nitrosating compounds and organic amines (Rostkowska et al., 1998).High concentrations of nitrosamines have been reported in processed meat because of the addition of nitrite to prevent the growth of Clostridium botulinum (Cho and Bratzler, 1970).Diethylnitrosamine (DEN) is a potent carcinogen found in soybean, cheese, tobacco smoke, processed meats, and a wide variety of foods (Verna et al., 1996).The metabolism of DEN generates high levels of ROS leading to mutagenicity and carcinogenesis (Pradeep et al., 2007;Ahmed et al., 2015;Mahmoud et al., 2017a).Therefore, attenuation of ROS generation can protect against DEN-induced cellular and tissue damage.
Activation of the basic leucine zipper (bZIP) protein nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a major mechanism in the defense against oxidative stress.Nrf2 activates the expression of genes whose products are antioxidant and cytodefensive proteins involved in the detoxication and elimination of electrophilic agents and reactive oxidants (Nguyen et al., 2009).We have recently demonstrated that activation of Nrf2 protects against drug-induced nephrotoxicity (Abd El-Twab et al., 2016), hepatotoxicity (Mahmoud and Al Dera, 2015;Kamel et al., 2016;Mahmoud et al., 2017b,c,d) and hepatocarcinogenesis (Mahmoud et al., 2017a), and oxidative stress induced by palmitate (Mahmoud et al., 2017e,f) and ammonium chloride (Mahmoud et al., 2017g).In this study, we demonstrated the protective effect of hesperidin against DEN/carbon tetrachloride (CCl 4 )-induced nephrotoxicity and pointed to the role of Nrf2/heme oxygenase 1 (HO-1) signaling.Hesperidin is a citrus flavonoid belongs to a class of non-nutritive dietary substances that are widely distributed in vegetables, fruits and beverages (Mahmoud, 2012).Previous work from our laboratory showed multiple beneficial effects of hesperidin, including anti-oxidant (Mahmoud et al., 2012), anti-inflammatory (Mahmoud, 2013(Mahmoud, , 2014)), anti-diabetic (Mahmoud et al., 2012) and hepatoprotective (Mahmoud, 2014).We have also demonstrated the nephroprotective effect of hesperidin in DEN/phenobarbital (PB)induced rats (Ahmed et al., 2015).Other studies have showed the protective effect of hesperidin against nephrotoxicity induced by several agents (Sahu et al., 2013;Kamel et al., 2014;Siddiqi et al., 2015).However, the mechanisms underlying the nephroprotective effect of hesperidin are not fully elucidated.This study is a step toward exploring the protective mechanism of hesperidin against xenobiotics-induced renal injury.

Experimental design and treatments
Thirty adult male Wistar rats weighing 140-160 g were used in the present study.The animals were obtained from the National Research Centre (NRC, Giza, Egypt), and were housed in well-aerated cages at normal atmospheric temperature and normal 12-hour light/dark cycle.The animals had free access to water and a standard diet of known composition.All efforts were done to reduce the number and suffering of animals and all animal procedures were in accordance with the recommendations of the animal ethics committee of Beni-Suef University (Egypt).
The experimental animals were divided into five groups as follows: Group I (Control): rats received one intraperitoneal injection of 0.9% NaCl.Two weeks later, the animals received mineral oil subcutaneously (once/week) for 16 weeks.In addition, rats received 0.5% carboxymethyl cellulose (CMC) via oral gavage daily for 18 weeks.
Group II (100 mg HES): rats received 0.9% NaCl and mineral oil as Group I, and 100 mg/kg hesperidin (Ahmed et al., 2015) dissolved in 0.5% CMC via oral gavage for 18 weeks.
At the end of the experiment, rats were sacrificed under mild anesthesia and blood samples were obtained for serum preparation.Kidneys were rapidly excised and immediately perfused with ice-cold saline.Samples from the kidney were homogenized in cold phosphate buffered saline (10% w/v), centrifuged and clear homogenate was separated and stored at -20 o C. Other kidney samples were kept at -80 o C for Western blotting while others were collected on 10% neutral buffered formalin for histological processing.

Assay of kidney function markers
Serum levels of creatinine, urea and uric acid were assayed using reagent kits purchased from Biosystems (Spain) according to the methods of Young (1995), Kaplan (1984) and Fossati et al (1980) respectively.

Assay of lipid peroxidation, nitric oxide (NO) and antioxidant defenses
Lipid peroxidation was determined in the kidney homogenate by assaying the level of malondialdehyde (MDA) according to the method of Preuss et al (1998).NO level was assayed following the method of Grisham et al (1996).GSH content was determined following the method of Beutler et al (1963) while superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were measured according to the methods of Marklund and Marklund (1974), Matkovics et al (1998) and Mannervik and Gutenberg (1981) respectively.

Histology
Kidney samples fixed in 10% neutral buffered formalin were dehydrated in ascending ethanol series, cleared in xylene, and embedded in paraffin wax.Blocks were prepared, and 4-μm thick sections were cut by a sledge microtome.The paraffin embedded sections were deparaffinized, washed, and stained with hematoxylin and eosin (H&E) for histopathological examination under light microscope.To demonstrate collagen deposition in the kidney, paraffin sections were stained with Masson's trichrome.

Western blotting
Kidney samples were homogenized in RIPA buffer supplemented with proteinase inhibitors and the protein content was determined using Bradford reagent.Thirty µg proteins were separated on SDS-PAGE, electrotransferred to nitrocellulose membranes and then blocked in 5% skimmed milk.The membranes were incubated with primary antibodies against Nrf2, HO-1 and β-actin (Santa Cruz Biotechnology, USA), followed by washing and incubation with the secondary antibodies.The blots were developed using enhanced chemiluminescence kit (BIORAD, USA), scanned and quantified using ImageJ (NIH, USA).

Statistical Analysis
Statistical analysis was performed using SPSS (v.16).
Results were expressed as mean ± standard deviation (SD) and all statistical comparisons were made by means of the one-way analysis of variance (ANOVA) test followed by Tukey's test post hoc analysis.A P value less than 0.05 was considered significant.

Effect of Hesperidin on kidney function markers in control and DEN/CCl 4 -induced rats
Data summarized in Table 1 show the effect of hesperidin on kidney function markers in control and DEN/CCl 4induced rats.The administration of DEN and CCl 4 produced a significant increase in serum creatinine (P<0.01),urea (P<0.001) and uric acid (P<0.001) when compared with the control rats.Concurrent treatment of the DEN/CCl 4 -induced rats with either 50 or 100 mg/kg hesperidin significantly ameliorated serum creatinine, urea and uric acid levels.Rats received 100 mg/kg hesperidin for 18 weeks showed non-significant (P>0.05)changes in serum levels of creatinine, urea and uric acid when compared with the control rats (Table 1).

Hesperidin prevents kidney injury induced by DEN/CCl 4 in rats
Microscopic examination of the kidney sections showed normal histological structure of the renal tissue in both control (Fig. 1A) and 100 mg/kg hesperidin supplemented rats (Fig. 1B).Rats received DEN and CCl 4 showed multiple histopathological changes, including congestion of glomerular tuft, atrophy of glomerular tuft, edema, cloudy swelling in convoluted tubules, thickening of the blood vessels, pyknosis, fibrosis and inflammatory cells infiltration (Fig. 1C-1F).Concomitant treatment of the DEN/CCl 4 -induced rats with 50 mg/kg hesperidin showed marked amelioration in kidney tissues, however, congestion of glomerular tuft was observed (Fig. 1G).DEN/CCl 4induced rats treated with 100 mg/kg hesperidin showed nearly normal structure of the renal corpuscles and renal tubules (Fig. 1H).

Hesperidin prevents collagen deposition in kidney of DEN/CCl 4 -induced rats
Kidney sections of control (Fig. 2A) and 100 mg/kg hesperidin supplemented rats (Fig. 2B) stained with Masson's trichrome revealed normal amount and distribution of collagen fibers in the interstitium of renal cortex.In contrast, kidney sections of DEN/CCl 4 -induced rats showed a massive accumulation of collagen fibers in between the glomeruli and the renal tubules (Fig. 2C & 2D).DEN/CCl 4 -induced rats treated with 50 (Fig. 2E) and 100 mg/kg hesperidin (Fig. 2F) showed markedly decreased collagen when compared with the DEN/CCl 4 -induced rats.

Hesperidin diminishes lipid peroxidation and NO in kidney of DEN/CCl 4 -induced rats
DEN/CCl 4 -induced rats exhibited a significant (P<0.001)increase in kidney lipid peroxidation levels when compared with the control rats as depicted in Figure 3A.Treatment with 50 and 100 mg/kg hesperidin significantly (P<0.001)decreased lipid peroxidation in the kidney of DEN/CCl 4 -induced rats.Similarly, DEN/CCl 4 -induced rats showed a marked (P<0.001) increase in kidney NO levels when compared with the control rats, an effect that was significantly (P<0.001)reversed following treatment with both doses of hesperidin (Fig. 3B).
Oral supplementation of 100 mg/kg hesperidin didn't affect levels of lipid peroxidation (Fig. 3A) and NO (Fig. 3B) in the kidney of normal rats.

Hesperidin enhances antioxidant defenses in kidney of DEN/CCl 4 -induced rats
GSH content in the kidney homogenate of DEN/CCl 4induced rats showed a significant (P<0.001)decrease when compared with the control group as represented in Figure 4A.On the other hand, both doses of hesperidin markedly ameliorated GSH content in the kidney of DEN/CCl 4 -induced rats.The activity of SOD (Fig. 4B), GPx (Fig. 4C) and GST (Fig. 4D) was significantly (P<0.001) declined in the kidney of DEN/CCl 4induced rats when compared with the corresponding control rats.Concurrent treatment of the DEN/CCl 4 -induced rats with either dose of hesperidin significantly (P<0.001)alleviated the activity of SOD and GPx.The lower dose of hesperidin didn't affect the activity of GST which was significantly (P<0.001)increased in DEN/CCl 4 -induced rats treated with the higher hesperidin dose.
Rats received 100 mg/kg hesperidin showed nonsignificant (P>0.05)changes in kidney GSH, SOD, GPx and GST when compared with the control rats.

DISCUSSION
The kidney is highly susceptible to damage by toxicants because of the high volume of blood flowing through it and the filtration of large amounts of toxins which can be concentrated in the kidney tubules (Perazella, 2009).Herein, we demonstrated the protective effect of the citrus flavonoid hesperidin against DEN/CCl 4 -induced nephrotoxicity in rats.Our results showed the ability of hesperidin to prevent DEN/CCl 4 -induced kidney injury and oxidative stress via up-regulation of the Nrf2/HO-1 signaling pathway.
DEN/CCl 4 -induced rats in the present study showed renal damage as evidenced by the significant increase in circulating levels of creatinine, urea and uric acid.Serum creatinine level has been reported to reveal glomerular function and its increase is an indicator of renal failure (Stevens and Levey, 2005).Urea is a byproduct of protein metabolism and is used as a marker of kidney injury (Stevens and Levey, 2005), and uric acid has been proposed as a potential risk factor for new-onset kidney diseases (Kanda et al., 2015).These findings are in agreement with our previous studies where we reported increased serum levels of creatinine, urea and uric acid in DEN/PB-induced rats (Ahmed et al., 2015;Mahmoud et al., 2015).Renal injury induced by DEN/CCl 4 was further confirmed by the observed histological alterations, including congestion and atrophy of glomerular tuft, edema, cloudy swelling in convoluted tubules, thickening of the blood vessels, pyknosis, fibrosis and inflammatory cells infiltration.In this context, we have shown dysplastic renal tubules with karyomegalic nuclei, atrophy of glomerular tuft, and inflammatory cells infiltration in the kidney of rats treated with DEN/PB (Ahmed et al., 2015;Mahmoud et al., 2015).Treatment of the DEN/CCl 4 -induced rats with hesperidin significantly prevented kidney damage.These findings support our previous study where hesperidin prevented DEN/PB-induced nephrotoxicity in rats (Ahmed et al., 2015).The nephroprotective effect of hesperidin has been previously reported in animal models of cisplatin and trichloroethylene-induced rats (Sahu et al., 2013;Kamel et al., 2014;Siddiqi et al., 2015).
Renal fibrosis caused by excessive accumulation of collagen in the kidney is considered the principal process involved in the progression of chronic kidney diseases (Pradère et al., 2008).The presence of kidney fibrosis seemed mostly to be viewed as an endpoint or marker of tissue or organ failure and loss of function (Cohen, 1995).In addition, Khubchandani et al (2010) reported that the normal glomerular basement membrane composed of type IV collagen which has an important function in the process of filtration, therefore, increased collagen production plays a key role in the development and progression of glomerular sclerosis.Moreover, Yang et al (2010) stated that the development of interstitial fibrosis was a secondary process that resulted from defective epithelial repair and could be regarded as a default mechanism of inadequate regeneration.Here, kidney sections of DEN/CCl 4 -induced rats showed a massive accumulation of collagen fibers in the interstitium of renal cortex.To the best of our knowledge, reports showing the fibrogenic effect of DEN in kidney of rats are scarce, but several studies demonstrating increased collagen deposition after CCl 4 administration (Ogeturka et al., 2005;Hamed et al., 2012) add support to our findings.Interestingly, treatment with hesperidin prevented excessive collagen deposition in the kidney of DEN/CCl 4 -induced rats, demonstrating the anti-fibrogenic activity of hesperidin.The antifibrotic activity of hesperidin against liver fibrosis in rats has been reported in the studies of Elshazly and Mahmoud (2014) and Pérez-Vargas et al (2014).
DEN has been reported to induce the generation of ROS and eventually resulting in oxidative stress and cellular injury (Pradeep et al., 2007;Ahmed et al., 2015;Mahmoud et al., 2017a).Trichloromethyl free radicals, produced from CCl 4 metabolism, combine with cellular lipids and proteins in the presence of oxygen forming trichloromethyl peroxyl radicals which attack lipids in the membrane of endoplasmic reticulum faster than trichloromethyl free radical.Thus, trichloromethyl peroxyl free radical elicit lipid peroxidation and destruct Ca 2+ homeostasis, resulting in cell death (Matei et al., 2008).ROS provoke peroxidation of native membrane lipids, leading to altered membrane functions through decreasing its fluidity and changing the activity of its bounding enzymes and their receptors.Since ROS have been implicated in the mechanisms that lead to tubular necrosis (Lopez-Novoa et al., 2011), reduction of oxidative stress may be a good target for prevention and treatment of renal toxicity.This explains the significantly increased lipid peroxidation levels in the kidney of DEN/CCl 4 -induced rats as we previously demonstrated (Ahmed et al., 2015;Mahmoud et al., 2015).
In contrast, DEN/CCl 4 -induced rats showed a significant decline in kidney GSH content.The declined GSH might be due to nicotinamide adenine dinucleotide phosphate (NADPH) depletion or increased consumption of GSH in non-enzymatic removal of oxygen-radicals (Gumieniczek, 2005).In addition, reduced activity of the antioxidant enzymes SOD, GPx and GST was observed in the kidney of the DEN/CCl 4 -induced rats.The reduced activity of antioxidant enzymes could be explained in terms of increased consumption of these enzymes to act upon the free radicals.Therefore, the induced renal damage in this study occurred via both stimulation of oxidative stress and abolishment of antioxidant defense system.In agreement with these findings, we have reported increased lipid peroxidation and NO along with declined GSH, SOD, GPx and GST in kidney and liver of DEN/PB-induced rats (Ahmed et al., 2015;Mahmoud et al., 2015Mahmoud et al., , 2017a)).
Treatment of the DEN/CCl 4 -induced rats with hesperidin significantly attenuated lipid peroxidation, decreased NO and enhanced antioxidant defenses in the kidney.Therefore, we assume that hesperidin exerts a nephroprotective effect against DEN and CCl 4 via its antioxidant potential.In this context, we have reported the ability of hesperidin to attenuate oxidative stress in animal models of diabetes (Mahmoud et al., 2012), nephrotoxicity (Ahmed et al., 2015) and hepatotoxicity (Mahmoud, 2014).In the study of Tirkey et al (2005), hesperidin successfully attenuated the effects of CCl 4 on GSH content and SOD activity in kidney tissues.
To explore the mechanism behind the attenuated oxidative stress and enhanced antioxidants in DEN/CCl 4 -induced rats treated with hesperidin, we determined the expression levels of Nrf2 and HO-1.Nrf2 is a basic leucine zipper protein that activates the expression of antioxidant and defensive proteins involved in the detoxication and elimination of electrophilic agents and reactive oxidants (Nguyen et al., 2009).Under homeostatic conditions, Nrf2 is kept at low levels via binding to a homodimer of Kelch-like ECH-associated protein 1 (Keap1) through the DC domain of either one of the Keap1 subunits.This binding leads to Cullin3/Rbx1-catalyzed polyubiquitination followed by proteasomal degradation of Nrf2 (Cullinan et al., 2004;Katoh et al., 2005).In cases of oxidative/electrophilic or xenobiotic stress, the Cullin3/Rbx1-dependent polyubiquitination of Nrf2, resulting in the accumulation of Nrf2 and its translocation to the nucleus (Cullinan et al., 2004).Within the nucleus, Nrf2 binds to the antioxidant response element (ARE) and activates the expression of genes of antioxidant proteins, including HO-1, nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1), SOD, GPx and GST (Hayes and Dinkova-Kostova, 2014).Therefore, activation of Nrf2 enhances the antioxidant defenses and subsequently prevent oxidative stress.
In this study, DEN/CCl 4 -induced rats exhibited declined Nrf2 and HO-1 expression, an effect that was significantly reversed in hesperidin treated groups.Although Nrf2 is induced by oxidative/xenobiotic stress, DEN/CCl 4 -induced rats showed a decrease in Nrf2 expression.This decline is a direct result of excessive ROS production as we have demonstrated in different models of surplus ROS production (Mahmoud and Al Dera, 2015;Kamel et al., 2016;Abd El-Twab et al., 2016;Mahmoud et al., 2017b-g).In addition, we have recently reported decreased expression of Nrf2 and HO-1 in the liver of DEN/PB-induced rats (Mahmoud et al., 2017a).Interestingly, DEN/CCl 4 -induced rats treated with hesperidin showed a significant increase in kidney Nrf2 and HO-1 expression.In support of our findings, hesperidin has been reported to up-regulate Nrf2 expression in a model of gentamicin-induced nephrotoxicity (Subramanian et al., 2015).However, our study is the first to show the ability of hesperidin to up-regulate Nrf2/HO-1 signaling in the kidney of DEN/CCl 4induced rats.
In conclusion, our results demonstrate the protective efficacy of hesperidin against DEN/CCl 4 -induced oxidative stress, kidney injury and fibrosis in rats.Hesperidin diminished lipid peroxidation and enhanced the antioxidant defenses GSH, SOD, GPx and GST in the kidney of DEN/CCl 4 -induced rats.These effects are mediated via up-regulation of Nrf2/ARE/HO-1 signaling pathway.

Fig 2 :
Fig 2: Hesperidin prevents collagen deposition in kidney of DEN/CCl4-induced rats.Photomicrographs of Masson's trichrome-stained kidney sections of (A) control and (B) hesperidin treated normal rats showing normal content of collagen fibers in the interstitium of renal cortex, (C&D) DEN/CCl4-induced rats showing heavy accumulation of collagen fibers in the interstitium of renal

Table 1 :
Effect of hesperidin on kidney function markers in control and DEN/CCl4-induced rats.