Sinapic acid ameliorate cadmium-induced nephrotoxicity: In vivo possible involvement of oxidative stress, apoptosis, and inflammation via NF-κB downregulation

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Sinapic acid ameliorate cadmium-induced nephrotoxicity: In vivo possible involvement of oxidative stress, apoptosis, and inflammation via NF-␬B downregulation Mushtaq Ahmad Ansari a,∗ , Mohammad Raish b,∗ , Ajaz Ahmad c , Khalid M. Alkharfy c , Sheikh Fayaz Ahmad a , Sabry M. Attia a,d , Abdulaziz M.S. Alsaad a , Saleh A. Bakheet a a

Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia c Department of Clinical Pharmacy, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia d Pharmacology and Toxicology Department, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt b

a r t i c l e

i n f o

Article history: Received 21 December 2016 Received in revised form 7 February 2017 Accepted 13 February 2017 Available online xxx Keywords: Cadmium Sinapic acid Nephrotoxicity Oxidative/nitrosative stress NF-␬B

a b s t r a c t Cadmium (CD), an environmental and industrial pollutant, generates reactive oxygen species (ROS) and NOS responsible for oxidative and nitrosative stress that can lead to nephrotoxic injury, including proximal tubule and glomerulus dysfunction. Sinapic acid (SA) has been found to possess potent antioxidant and anti-inflammatory effects in vitro and in vivo. We aimed to examine the nephroprotective, anti-oxidant, anti-inflammatory, and anti-apoptotic effects of SA against CD-induced nephrotoxicity and its underlying mechanism. Kidney functional markers (serum urea, uric acid, creatinine, LDH, and calcium) and histopathological examinations of the kidney were used to evaluate CD-induced nephrotoxicity. Oxidative stress markers (lipid peroxidation and total protein), renal nitrosative stress (nitric oxide), antioxidant enzymes (catalase and NP-SH), inflammation markers (NF-␬B [p65], TNF-␣, IL-6, and myeloperoxidase [MPO]), and apoptotic markers (caspase 3, Bax, and Bcl-2) were also assessed. SA (10 and 20 mg/kg) pretreatment restored kidney function, upregulated antioxidant levels, and prevented the elevation of lipid peroxidation and nitric oxide levels, significantly reducing oxidative and nitrosative stress. CD upregulated renal cytokine levels (TNF-␣, IL-6), nuclear NF-␬B (p65) expression, NF-␬B-DNA-binding activity, and MPO activity, which were significantly downregulated upon SA pretreatment. Furthermore, SA treatment prevented the upregulation of caspase 3 and Bax protein expression and upregulated Bcl-2 protein expression. SA pretreatment also alleviated the magnitude of histological injuries and reduced neutrophil infiltration in renal tubules. We conclude that the nephroprotective potential of SA in CDinduced nephrotoxicity might be due to its antioxidant, anti-inflammatory, and anti-apoptotic potential via downregulation of oxidative/nitrosative stress, inflammation, and apoptosis in the kidney. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Cadmium (CD) is a prevalent environmental pollutant that is severely toxic to various organs, such as the heart, kidney, liver, testis, and lung, in humans and experimental animals. In particular, renal toxicity instigated by chronic CD exposure is well documented (Fujiwara et al., 2012; Prozialeck and Edwards, 2012). CD environmental pollution is widespread owing to metal mining, refining, smelting, fossil fuel combustion, and waste incineration

∗ Corresponding authors. E-mail addresses: [email protected] (M.A. Ansari), [email protected] (M. Raish).

(Nordberg, 2004; Satarug et al., 2003). Major sources of exposure include foods, drinking water, inhaled cigarette smoke or air, and assimilation of contaminated soil and dust produced by both humans and animals that had previously accumulated CD in their bodily tissues (Talas et al., 2008; Teeyakasem et al., 2007). Several efforts and policies by global organizations and countries have been made to decrease CD usage, but it remains a chief public health risk, particularly in developing nations where environmental standards are either lax or ignored. CD is a toxic and non-biodegradable metal, with a long biological half-life, that is non-beneficial to plants, animals, and humans (Jarup and Akesson, 2009; Satarug et al., 2010). Low levels of CD exposure in the kidney result in toxicity characterized by calciuria, proteinuria, glycosuria, and tubular necrosis (Satarug et al., 2010). This may

http://dx.doi.org/10.1016/j.etap.2017.02.014 1382-6689/© 2017 Elsevier B.V. All rights reserved.

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lead to end-stage renal disease, diabetic complications, and osteoporosis. CD has an affinity for the proximal tubule of the kidney, resulting in accumulation and nephrotoxicity. Several reports indicate that CD-induced nephrotoxicity is mediated by the release of CD-metallothionein complexes from injured Kupffer cells that are filtered by the glomerulus, endocytosed by the proximal tubular cells, and degraded by lysosomes subsequent to the discharge of CD (Morales et al., 2006b). Free CD is known to generate reactive oxygen species (ROS) and cause inflammation, leading to the accumulation of pro-inflammatory cytokines and apoptosis of renal tissue cells (Fujiwara et al., 2012; Kayama et al., 1995). Antioxidant and anti-inflammatory agents were found to be effective in mitigating CD-induced nephrotoxicity (Erboga et al., 2016; Fouad and Jresat, 2011; Manna et al., 2009; Nazima et al., 2015). Sinapic acid (SA) is a hydroxycinnamic acid-derived polyphenol with 3,5-dimethoxyl and 4-hydroxyl replacements in the phenyl group of cinnamic acid. SA can be widely obtained from fruits and vegetables (Andreasen et al., 2001), and has been observed to possess potent antioxidant and anti-inflammatory effect in vitro and in vivo (Ansari et al., 2016; Chen, 2016; Lu et al., 2016) To the best of our knowledge, there has been no report on the protective effects of SA against CD-induced nephrotoxicity in a rodent model. Therefore, we hypothesize that SA can offer protection against CD-induced nephrotoxicity, and proposed the present study to evaluate the mechanisms involved in CD-induced nephrotoxicity with respect to SA, including the roles of oxidative and nitrosative stress, inflammatory mediators, and apoptotic markers (caspase 3, Bax, and Bcl2)

2. Materials and methods 2.1. Drugs and chemicals SA and cadmium chloride (CdCl2 ) were obtained from Fluka Analytical (Sigma-Aldrich, Buchs, Switzerland). Antibodies against NF-␬B (p65), I␬B-␣ (p65), caspase 3, Bax, Bcl-2, and ␤-actin, as well as horseradish peroxidase (HRP)-conjugated secondary antibody, were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The NE-PER Nuclear Protein Extraction Kit was obtained from Pierce Biotechnology (Rockford, IL, USA). The NF-␬B (p65) Transcription Factor Assay Kit was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Rat TNF-␣, IL-6, and MPO ELISA kits were procured from R&D Systems, Inc. (Minneapolis, MN, USA). All other chemicals were of analytical grade.

2.2. Experimental design Adult male Wistar rats (224–240 g) were procured from the Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The study design was approved by the Ethics Committee of the Experimental Animal Care Society, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Rats were acclimatized for 1 week prior to the study. In the present study, CD was administered as CdCl2 via oral intubation at a dose of 5 mg/kg body weight (BW) per day for 24 days. Rats were divided into four groups (n = 6 animals/group) as follows: Group I, 0.5 mL isotonic saline orally daily for 24 days (control); Group II, cadmium chloride (5 mg of CdCl2 /kg BW) in isotonic saline orally daily for 24 days (Adefegha et al., 2015; Shati, 2011); Groups III and IV, 10 or 20 mg/kg BW of SA along with CD (5 mg of CdCl2 /kg BW) in isotonic saline orally daily for 24 days, respectively. At 24 h after the last dose, blood samples were collected from all groups and serum separation was performed at 5000 rpm for 15 min. Serum was stored at −20 ◦ C until analysis. All rats were killed under anesthesia; the kidneys

were dissected and used for serum, molecular, and histopathological examinations. 2.3. Serum analysis Creatinine (Tietz, 1986), uric acid (Fossati et al., 1980), urea (Trinder, 1969), LDH, and total protein levels were estimated in ® the serum using the Reflotron Plus Analyzer and Roche kits (Roche Diagnostics, Basel, Switzerland). Calcium was analysed using Calcium Detection Kit (Colorimetric) (ab102505). All tests were performed according to the manufacturer’s protocol. 2.4. Catalase, non-protein sulfhydryls (NP-SH), and total protein content Protein content was determined using the Lowry method (Lowry et al., 1951). The activity of catalase was assessed using a kit (Crescent Diagnostics, Jeddah, Saudi Arabia) as per the manufacturer’s protocol. Total renal non-protein sulfhydryls were assessed as per the protocol of Sedlak and Lindsay (Sedlak and Lindsay, 1968). 2.5. Lipid peroxidation The levels of lipid peroxidation (MDA) in renal tissues were assessed following the protocol of Niehaus and Samuelson (Niehaus and Samuelsson, 1968). 2.5.1. Pro-inflammatory cytokine levels TNF-␣ and IL-6 contents were estimated in the kidney tissue homogenates by using commercially available kits obtained from R&D Systems. Absorbance was read at 450 nm. The protein levels of tissue homogenates were assessed, and the TNF-␣ and IL-6 contents were expressed as pg/mg of protein. 2.6. Nitric oxide (NO) and myeloperoxidase (MPO) levels Nitrate was converted to nitrite by nitrate reductase, and total nitrite was measured using the Griess reaction (Green et al., 1982). Myeloperoxidase (MPO) is an enzyme expressed in neutrophils that can be utilized as an indirect measure of neutrophil content in tissue samples based on the methods of Krueger et al. (Krueger et al., 1990). MPO concentrations in kidney tissue samples were estimated using commercially available MPO ELISA kits (MBS704859) obtained from MyBioSource (San Diego, CA, USA) as per the manufacturer’s protocol. Absorbance was read at 450 nm. 2.7. Preparation of nuclear and total protein extracts Total protein was acquired by homogenizing kidney tissues in ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 2500g for 20 min at 4 ◦ C. The upper layer was collected and used to assess protein expression. Similarly, nuclear and cytosol extracts were prepared using the NE-PER Kit (Pierce Biotechnology) following the kit instructions. The total protein levels were determined according to the Lowry method based on a standard curve using bovine serum albumin. 2.8. Western blot analysis, NF-B (p65) and NF-B DNA-binding activity An immunoblot analysis was performed as per the methods of Towbin et al. (Towbin, 2009). A detailed description of the method has been published in a previous report (Ansari et al., 2016). NF-␬B

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LDH (230.42 ± 7.24–138.35 ± 5.37 and 104.77 ± 1.09, P < 0.05); creatinine (1.87 ± 0.08–0.95 ± 0.04 and 0.72 ± 0.07, P < 0.05), uric acid (2.46 ± 0.15–1.54 ± 0.07 and 1.11 ± 0.04, P < 0.001); urea (152.72 ± 2.25–107.94 ± 1.23 and 107.94 ± 1.23, P < 0.05) (Table 1). 3.3. Effect of SA on lipid peroxidation Oral CD administration increased MDA levels by 81% (82.67 ± 3.29–149.86 ± 5.87 nmol/g tissue), which is indicative of oxidative stress, when compared to control. SA pretreatment at 10 and 20 mg/kg BW caused a significant dose-dependent decrease in CD-induced oxidative stress with regards to MDA levels (149.86 ± 5.87–118.54 ± 2.28 [20.90%] and 93.53 ± 2.97 [37.59%] nmol/mg tissue, P < 0.01) in comparison to CD-intoxicated nephrotoxic rats (Fig. 2A). 3.4. Effect of SA on endogenous antioxidant defense system

Fig 1. Effect of SA on the kidney-body weight ratio.

DNA-binding activity was examined using the NF-␬B (p65) Transcription Factor ELISA Kit (Cayman Chemical Company) as per the manufacturer’s instructions. 2.9. Histopathological examination Renal tissues were fixed with 12% paraformaldehyde. They were then dehydrated with 75% alcohol and embedded in paraffin. The blocks were cut into 4-␮m-thick sections, stained with hematoxylin and eosin (H&E), and observed under a light microscope (×400). 2.10. Statistical analysis All results are given as arithmetic means ± SEM. Data were statistically evaluated using one-way analysis of variance followed by Dunnett’s tests. P-values less than 0.05 were considered statistically significant. 3. Results 3.1. Effect of SA on body and kidney weight of the control and CD-induced nephrotic rats The effect of SA on the kidney-body weight ratio of the control, CD-induced nephropathic, and SA-treated nephropathic rats (P < 0.05) as compared with control rats is illustrated in Fig. 1. Treatment with 10 or 20 mg/kg BW SA with CD significantly ameliorated the reduction in the kidney-body weight ratio in comparison to the CD-only nephropathy rats.

CD-intoxicated rats had a significant decline in their level of endogenous enzymes such as catalase (CAT), non-sulfhydryl content (NP-SH), and total proteins (TP) as compared to normal rats: 55.19%, 76.85%, and 32.90% respectively. However, SA (10 and 20 mg/kg BW) pretreatment significantly augmented these levels in a dose-dependent manner: 29.30% and 69.81% for CAT (6.60 ± 0.34–8.54 ± 0.30 and 11.21 ± 0.39 IU), 143.73% and 217.73% for NP-SH (2.07 ± 0.14–5.05 ± 0.18 and 6.59 ± 0.34 nmol/mg protein), 7.13% and 25.48% for TP (84.82 ± 1.34–90.87 ± 1.35 and 106.43 ± 1.83 g/l). This clearly shows that SA at 10 and 20 mg/kg BW has the ability to restore the endogenous antioxidant defense system, composed of enzymes such as CAT and NP-SH as well as TP, to normal levels (Fig. 2B–D). 3.5. Effect of SA on myeloperoxidase (MPO) and nitric oxide level As illustrated in Fig. 3A, MPO levels were significantly induced, up to 82.65% (19.49 ± 0.56–35.61 ± 1.39, P < 0.01) in the kidneys of CD-intoxicated rats as compared to control. SA (10 and 20 mg/kg) pretreatment significantly inhibited this increase in MPO level about 21.28% (35.61 ± 1.39–28.07 ± 0.50) and 42.81% (35.61 ± 1.39 to 20.39 ± 0.66), respectively. These results demonstrate the antiinflammatory activity of SA through inhibition of neutrophil infiltration. We further determined the anti-inflammatory potential of SA by examining the level of nitric oxide in the kidney tissue of CD-intoxicated rats. As illustrated in Fig. 3B, CD induces a 65.88% increase in the nitric oxide level (33.07 ± 1.14–54.85 ± 1.63) in intoxicated rats, as compared to control. Pretreatment of SA (10 and 20 mg/kg) inhibits this increase in nitric oxide in the kidney in a dose-dependent manner that is about 20.05 and 33.58% (54.85 ± 1.63–43.85 ± 1.40 and 36.43 ± 0.83, respectively, P < 0.05). This further indicates that SA has potent anti-inflammatory as well as anti-nitrosative effects. 3.6. Effect of SA on cytokine (TNF-˛ and IL-6)

3.2. Effect of SA on kidney function tests Oral CD administration produced an elevation in serum Ca++ (1.14 ± 0.06–23.43 ± 0.92, P < 0.05), LDH (65.89 ± 2.03–230.42 ± 7.24, P < 0.05), (0.45 ± 0.06–1.87 ± 0.08, P < 0.05), uric creatinine acid (1.14 ± 0.06–2.46 ± 0.15, P < 0.001), and urea (38.68 ± 1.18–152.72 ± 2.25, P < 0.05) as compared to the control. Pretreatment of SA to CD-intoxicated nephrotoxic rats significantly prevented the elevation of serum biomarkers in a dose-dependent manner: Ca++ (23.43 ± 0.92–17.27 ± 1.79 and 9.16 ± 0.76, P < 0.05);

As illustrated in Fig. 3, CD-intoxicated rats display a significant 4.17-fold elevation of TNF-␣ levels (27.28 ± 1.56–113.86 ± 1.76 pg/mg protein, P < 0.05) and 3.17fold induction of IL-6 levels (29.92 ± 2.27–94.93 ± 3.73 pg/mg protein) (Fig. 3C–D.) when compared to normal control rats. SA pretreatment (10 and 20 mg/kg) significantly decreased the TNF-␣ levels by 0.64 and 0.51-fold (73.13 ± 2.52 and 57.82 ± 3.22 pg/mg protein) and IL-6 levels by 0.84 and 0.74-fold (79.38 ± 3.77 and 70.04 ± 3.16 pg/mg protein), respectively, when compared with CD-intoxicated rats (P < 0.01). These results show that SA down-

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Table 1 Effect of sinapic acid on kidney function markers in cadmium intoxicated rats. (n = 5)

Creatinine (mg/dl) ± SEM

Urea (mg/dl) ± SEM

LDH ± SEM

Uric acid (mg/dl) ± SEM

Calcium ± SEM

Normal Control CD SA 10 mg/kg + CD SA 20 mg/kg + CD

0.45 ± 0.02* 1.87 ± 0.08# 0.95 ± 0.04*# 0.72 ± 0.07*#

38.68 ± 1.18 152.72 ± 2.25 107.94 ± 1.23*# 85.82 ± 2.83*#

65.89 ± 2.03 230.42 ± 7.24 138.35 ± 5.37*# 104.77 ± 1.09*#

1.14 ± 0.06 2.46 ± 0.15 1.54 ± 0.07*# 1.11 ± 0.04*#

5.50 ± 0.14 23.43 ± 0.92 17.27 ± 1.79*# 9.16 ± 0.76*#

‘*’ Denotes significant differences compared with the control group (P < 0.05); ‘#’ denotes significant differences compared with the CD group (P < 0.05). (n = 5 animals for each group).

Fig. 2. Effect of SA (10 and 20 mg/kg) on CD-intoxicated rats with respect to kidney lipid peroxidation MDA (A), catalase activity (B), NP-SH (C), and total protein (D). Results are means ± SEM of five animals per group. ‘*’ Denotes significant differences compared with the control group (P < 0.05); ‘#’ denotes significant differences compared with the CD group (P < 0.05).

regulates cytokine levels, producing potent anti-inflammatory activity. 3.7. Effect of SA on NF-B in CD-inflicted renal damage CD significantly augments the nuclear translocation of the p65 subunit of NF-␬B (Fig. 4A) and NF-␬B-DNA binding activity (Fig. 4B) in intoxicated rats as compared to normal control rats. SA pretreatment at 10 and 20 mg/kg significantly diminished both nuclear

NF-␬B (p65) protein expression and DNA-binding activity when compared to CD-intoxicated rats. CD-intoxicated rats exhibited significantly higher renal protein expression of Bax and caspase 3 activities, with significant inhibition of the elevation of Bcl-2 protein expression when compared to vehicle control rats (P < 0.05) (Fig. 4C–D). Additionally, the anti-inflammatory potential of SA was examined via immunoblot of iNOS in the kidney tissue of CDintoxicated rats, and similar results were found as in nitric oxide (Fig. 4E).

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Fig. 3. Effect of SA on pro-inflammatory cytokine levels in the kidney tissues of CDintoxicated rats. (A) MPO, (B) Nitric oxide, (C) Tumor necrosis factor-␣ (TNF-␣), (D) Interleukin-6 (IL-6); results are mean ± SEM of five animals per group. ‘*’ denotes significant differences compared with the control group (P < 0.05); ‘#’ denotes significant differences compared with the CD group (p < 0.05).

3.8. Effect of SA on histopathology of CD-inflicted renal damage Histopathological examination of the CD-intoxicated kidney reveals focal interstitial nephritis composed of neutrophils, myocytes, and plasma cells, with desquamation of renal tubular epithelium and vacuolization. Pretreatment of SA restored the normal architecture of renal tubules, indicating significant mitigation of inflammation as well as necrosis of cells. Overall, our results indicate that SA has potential as a protective agent from CD-induced nephrotoxicity (Fig. 5). 4. Discussion Several reports have described the toxic and carcinogenic effects of the heavy metal CD on human health owing to its extensive availability in the air (cigarette smoke, factory wastes), water (from pipeline and batteries), and plants (Chargui et al., 2011). CD is a known toxic industrial pollutant. It is poorly excreted and accumulates in various organs, such as the lungs, kidney, testis, liver, and bones, and is responsible for serious organ damage (Anetor, 2012). The production of ROS is considered the key mechanism of metalinduced carcinogenesis, since heavy metals such as CD are capable of changing the redox equilibrium in cells (Lee et al., 2012). Chronic administration of CD damages the proximal tubules in the kidney via apoptosis and necrosis, leading to a characteristic nephropathy (Agirdir et al., 2002; Rehm and Waalkes, 1990). Aktoz et al. and

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Fouad and Jresat used 1 and 1.2 mg/kg CD for 30 days in their experiments, respectively; therefore, we used 1.2 mg/kg CD for 30 days in order to induce proximal tubules and glomeruli dysfunction in the kidneys (Aktoz et al., 2012; Fouad and Jresat, 2011). CD intoxication also increased the weight of the kidneys. The CD-induced increase in the kidney-body weight ratio has been associated with inflammatory processes triggered by this metal (Donpunha et al., 2011). Treatment of the CD-intoxicated rats with SA returned the kidneybody weight ratio toward normal. A pilot study for dose selection was carried out for SA, and concentrations from 10 to 20 mg/kg were found to be effective in ameliorating the kidney injuries of CD-intoxicated rats. CD-induced glomerular and tubular necrosis occurs in response to increases in urea, uric acid, and creatinine in the serum (Hooper et al., 1998). Increased serum levels of NP-SH and LDH indicate CD toxicity in rat kidneys. These elevations are in agreement with previous results (Erboga et al., 2016; Renugadevi and Prabu, 2010). SA treatment (10 and 20 mg/kg) significantly downregulated the elevated levels of urea, uric acid, creatinine, and calcium in the serum of CD-intoxicated rats. The current study substantiate the findings of Morales et al. and Erboga et al., in which quercetin and thymoquinone were found to be protective against CD-induced nephrotoxicity via their antioxidant properties (Erboga et al., 2016; Morales et al., 2006a). In this study, we observed that CD intoxication induces renal dysfunction by elevating lipid peroxidation and upsetting the native antioxidant defense system. Results suggest that SA (10 and 20 mg/kg) treatment could prevent CD-induced modifications of antioxidant-related parameters in experimental animals. The mitigating effect of SA is due its potent antioxi´ dant nature (Ansari et al., 2016; Niciforovi c´ and Abramoviˇc, 2014). Administration of SA (10 and 20 mg/kg) significantly modulated the lipid peroxidation and antioxidant status in CD-intoxicated rats, which suggests a reduction in ROS and/or replenishment of cellular antioxidant defenses. Numerous previous reports have suggested that biophenols and flavonoids may act as antioxidants, free-radical scavengers, and radio protectors (Ansari et al., 2016; Chen, 2016). The reduction of CAT and intracellular NP-SH by CD is a prerequisite for ROS generation and disruption of intracellular organelles (Oladipo et al., 2016; Valko et al., 2005). SA (10 and 20 mg/kg) treatment inhibits the depletion of antioxidant enzymes such as CAT and NP-SH. A significant reduction in the level of non-enzymatic antioxidants as seen in the CD-intoxication state could lead to increased susceptibility of the renal tissue to free-radical damage. The metal-chelating and antioxidant capability of SA to inhibit ROS generation further reduces oxidative stress induced by CD intoxication, which could inhibit the further depletion of antioxidative enzymes in kidney. Our results also confirm a reduction of the levels of enzymatic antioxidants such as CAT and NP-SH in the kidneys of CD-intoxicated rats (Sener et al., 2005). Most of the antioxidant enzymes become inactive after CD exposure due to the direct binding of CD to thiol (SH) groups (Quig, 1998). It has been reported that SA metabolites can also inhibit peroxynitrate-mediated oxidation, which further supports our hypothesis of the antioxidant potential of SA (Ansari et al., 2016). CD-induced toxicity is mediated through the generation of ROS and NOS in the early phase of CD-induced nephrotoxicity (Zhong et al., 2015). CD encourages the generation of NO, and in presence of oxygen NO forms intermediates that displace CD from metallothionein. Free CD has been observed to induce DNA damage and apoptosis (Misra et al., 1996). In the present study, CD-intoxicated rats displayed increased level of NO, whereas SA (10 and 20 mg/kg) treatment prevent the increase in renal NO in rats. Therefore, we conclude that SA has a potent ability to curb CD-induced nitrosative stress. NO is a biomarker for inflammation; thus has ability to curb the CD-induced inflammatory response.

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Fig. 4. Effect of SA on inflammatory and apoptotic markers in kidney tissues of CD-intoxicated rats. Immunoblot analysis of apoptotic markers: (A) NF-␬B [p65], (B) nuclear NF-␬B [p65], (C) Bax, (D) caspase 3, (E) Bcl2, I␬B␣, and (F) iNOS. Results are mean ± SEM of five animals per group. ‘*’ Denotes significant differences compared with the control group (P < 0.05); ‘#’ denotes significant differences compared with the CD group (P < 0.05).

Fig. 5. Effect of SA (10 and 20 mg/kg/day) on histopathological features of CDintoxicated kidney (H&E, 40 × ). (A) Kidney tissue of a normal control rat. Note the normal appearance of the glomerular capillaries (arrow), with both proximal convoluted tubules (PC) and distal convoluted tubules (DC) showing a normal epithelium. (B) Kidney tissue of CD-intoxicated rats; the glomerular capillaries are widened, irregular, and attached to the Bowman’s capsule (arrow). Some glomerular capillaries show nodular sclerosis (arrowhead). The tubular epithelium is also affected (asterisk). (C) Kidney tissue after treatment with 10 mg SA; the histological features are relatively improved compared to untreated CD-intoxicated rats. The glomerular capillaries retain their normal size and appearance (arrow), but the mesangial cell number is still relatively higher than that of normal rats (arrowhead) and the tubular epithelium is diminished (asterisk). (D) Kidney tissue from a CD-intoxicated rat treated with 20 mg SA; the histological features greatly improved; there is a nearly normal structure of glomerular capillaries (arrow) and tubular epithelium (asterisk).

MPO is a chemokine released by activated neutrophils and macrophages that has potent pro-oxidative and pro-inflammatory properties (Kataranovski et al., 1999). Treatment with SA (10 and 20 mg/kg) significantly reduced the enhanced MPO level in CD-intoxicated rats in a dose-dependent manner. CD-induced nephrotoxicity is often associated with inflammatory cytokines such as TNF-␣ and IL-6 and the infiltration of neutrophils (Kayama et al., 1995). An increase in renal TNF-␣ and IL-6 levels in CDintoxicated rats is consistent with previous reports (Kataranovski et al., 1998; Kataranovski et al., 1999). SA treatment (10 and 20 mg/kg) significantly and dose-dependently prevented an upregulation in cytokine response, specifically TNF-␣ and IL-6, in CD-intoxicated rats. There are several reports describing SA downregulation of TNF-␣ and IL-6 levels (Ansari et al., 2016; Shin et al., 2013; Yun et al., 2008). Apoptosis is a unique form of cell death that occurs in tissues under certain set of physiological conditions and differs from necrosis (Walker et al., 1987). Apoptosis is one of the main features of CD-induced nephrotoxicity. CD intoxication has been shown to induce apoptosis in proximal tubular cells, leading to kidney dysfunction (Xie and Shaikh, 2006; Yuan et al., 2014). In mitochondrial pathways, the expression of caspase 3 is activated by caspase 9 (Chen et al., 2011). Caspase 3 and Bax function as pro-apoptotic proteins, while Bcl-2 acts as an anti-apoptotic protein. Bcl-2 binding to the mitochondrial outer membrane inhibits cytochrome c activation (Kalkan et al., 2012). The CD-intoxicated rats show an increase in caspase 3; Bax protein expression, however, decline in Bcl-2 expression. These results further suggest that CD intoxica-

Please cite this article in press as: Ansari, M.A., et al., Sinapic acid ameliorate cadmium-induced nephrotoxicity: In vivo possible involvement of oxidative stress, apoptosis, and inflammation via NF-␬B downregulation. Environ. Toxicol. Pharmacol. (2017), http://dx.doi.org/10.1016/j.etap.2017.02.014

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tion induces apoptosis as well as inflammation, resulting in tubular necrosis in kidney tissues. SA pretreatment in CD-induced nephrotoxic rats significantly protected and prevented injuries associated with renal tubular apoptosis and necrosis. Several studies have shown that NF-␬B activation promotes CD-induced apoptosis in the renal tubules of rats (Xie and Shaikh, 2006; Yuan et al., 2014). NF-␬B may directly activate the apoptotic protein caspase 3 or downregulate anti-apoptotic proteins, such as Bcl-2 (Xie and Shaikh, 2006). In the present study, we observed an upregulation in NF-␬B p65 protein expression in CD-intoxicated rats, which is consistent with previous reports. The increase in NF-␬B p65 and NF-␬BDNA-binding significantly and dose-dependently regressed with SA treatment at 10 and 20 mg/kg. These findings are consistent with previous findings on antioxidants that are able to ameliorate proximal tubular injuries produced by apoptosis (Erboga et al., 2016; Luo et al., 2016; Nazima et al., 2015). 5. Conclusion These outcomes indicated that SA pretreatment mitigates renal impairment and structural injuries via the downregulation of oxidative/nitrosative stress, inflammation, and apoptosis in the kidney Furthermore, Cd-induced ROS appears to be involved in NF␬B–mediated apoptosis in renal tubules. SA may be an appropriate target molecule for renal protection from Cd-induced nephrotoxicity. Conflict of interest The authors declare no conflict of interests. Acknowledgments The authors would like to express their appreciation to the Deanship of Scientific Research at King Saud University for the provision of funding for publication through the College of Pharmacy Research Center. References Adefegha, S.A., Omojokun, O.S., Oboh, G., 2015. Modulatory effect of protocatechuic acid on cadmium induced nephrotoxicity and hepatoxicity in rats in vivo. SpringerPlus 4, 619. Agirdir, B.V., Bilgen, I., Dinc, O., Ozcaglar, H.U., Fisenk, F., Turhan, M., Oner, G., 2002. Effect of zinc ion on cadmium-induced auditory changes. Biol. Trace Elem. Res. 88, 153–163. Aktoz, T., Kanter, M., Uz, Y.H., Aktas¸, C., Erbo˘ga, M., Atakan, I˙ .H., 2012. Protective effect of quercetin against renal toxicity induced by cadmium in rats. Balkan Med. J. 2012. Andreasen, M.F., Landbo, A.-K., Christensen, L.P., Hansen, Å., Meyer, A.S., 2001. Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins. J. Agric. Food Chem. 49, 4090–4096. Anetor, J.I., 2012. Rising environmental cadmium levels in developing countries: threat to genome stability and health. Niger. J. Physiol. Sci. 27, 103–115. Ansari, M.A., Raish, M., Ahmad, A., Ahmad, S.F., Mudassar, S., Mohsin, K., Shakeel, F., Korashy, H.M., Bakheet, S.A., 2016. Sinapic acid mitigates gentamicin-induced nephrotoxicity and associated oxidative/nitrosative stress, apoptosis, and inflammation in rats. Life Sci. 165, 1–8. Chargui, A., Zekri, S., Jacquillet, G., Rubera, I., Ilie, M., Belaid, A., Duranton, C., Tauc, M., Hofman, P., Poujeol, P., El May, M.V., Mograbi, B., 2011. Cadmium-induced autophagy in rat kidney: an early biomarker of subtoxic exposure. Toxicol. Sci. 121, 31–42. Chen, Y.C., Chen, C.H., Hsu, Y.H., Chen, T.H., Sue, Y.M., Cheng, C.Y., Chen, T.W., 2011. Leptin reduces gentamicin-induced apoptosis in rat renal tubular cells via the PI3K-Akt signaling pathway. Eur. J. Pharmacol. 658, 213–218. Chen, C., 2016. Sinapic acid and its derivatives as medicine in oxidative stress-induced diseases and aging. Oxid. Med. Cell. Longev. 2016, 3571614. Donpunha, W., Kukongviriyapan, U., Sompamit, K., Pakdeechote, P., Kukongviriyapan, V., Pannangpetch, P., 2011. Protective effect of ascorbic acid on cadmium-induced hypertension and vascular dysfunction in mice. Biometals 24, 105–115.

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Please cite this article in press as: Ansari, M.A., et al., Sinapic acid ameliorate cadmium-induced nephrotoxicity: In vivo possible involvement of oxidative stress, apoptosis, and inflammation via NF-␬B downregulation. Environ. Toxicol. Pharmacol. (2017), http://dx.doi.org/10.1016/j.etap.2017.02.014