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Grape seed proanthocyanidins ameliorates Cadmium induced renal injury and oxidative stress in experimental rats through the up-regulation of nuclear related factor 2 (Nrf2) and antioxidant responsive elements Nazima Bashir, Vaihundam Manoharan and Selvaraj Milton Prabu *

Department of Zoology, Faculty of Science Annamalai University, Annamalainagar-608002 Tamilnadu, India

* Corresponding author:

Dr. Selvaraj Miltonprabu Assistant Professor Department of Zoology, Faculty of Science, Annamalai University, Annamalai Nagar – 608002 Tamil Nadu, India. Tel: +91 04144 – 238282; Cell : +91 9842325222 Fax: +91 04144 – 238080 Email address: [email protected] [email protected]

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Grape seed proanthocyanidins ameliorates Cadmium induced renal injury and oxidative stress in experimental rats through the up-regulation of nuclear related factor 2 (Nrf2) and antioxidant responsive elements NazimaBashir, Vaihundam Manoharan and Selvaraj Milton Prabu * Faculty of Science, Department of Zoology, Annamalai University, Annamalai Nagar. Abstract Cadmium (Cd) preferentially accumulates in the kidney, the major target for Cd related toxicity. Cd induced reactive oxygen species (ROS) have been considered as a crucial mediator for renal injury. The biologically significant ionic form of cadmium (Cd+), binds to many bio-molecules and these interactions underlie the toxicity mechanisms of Cd. The present study was hypothesized to explore the protective effect of Grape Seed Proanthocyanidins (GSP) on Cd induced renal toxicity and to elucidate the potential mechanism. Male Wistar rats were treated with cadmium (Cd) as cadmium chloride (CdCl2, 5 mg kg−1 bw, orally) and orally pre-administered with GSP (100 mg kg−1 bw) 90 minutes before Cd intoxication for 4 weeks to evaluate cardio damage of Cd and antioxidant potential of GSP. Serum renal function parameters (blood urea nitrogen and creatinine) levels in serum and urine, Renal oxidative stress (lipid peroxidation, protein carbonylation, enzymatic and non-enzymatic antioxidants), inflammatory (NF-kB p65, NO, TNF-α, IL-6), apoptotic (caspase 3, caspase 9, Bax, Bcl-2), membrane bound ATPases and Nrf2 (HO-1, keap1, γ-GCS, and µ-GST) markers were evaluated in Cd treated rats. Pretreatment with GSP revealed a significant improvement in renal oxidative stress markers in kidneys of Cd treated rats. In addition, GSP treatment decreases the amount of iNOS, NF-κB, TNF-α, caspase 3 and Bax and increases the levels Bcl-2 protein expression. Similarly, mRNA and protein analyses substantiated that GSP treatment, notably normalizes the renal expression of Nrf2/Keap1and its downstream regulatory proteins in the Cd treated rats. Histopathological and ultra structural observations also evidenced that GSP

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effectively protects the kidney from Cd induced oxidative damage. These findings suggest that GSP ameliorates renal dysfunction and oxidative stress through the activation of Nrf2 pathway in Cd intoxicated rats. Key words: Cadmium, Grape seed proanthocyanidins, nephrotoxicity, inflammation, apoptosis, Nrf2, ROS, oxidative stress, rats. 1. INTRODUCTION Cadmium (Cd) is a noxious contaminant of continuing great toxicological concern worldwide. Although some forms of life adjust to the presence of this metal, most biological effects of Cd are deleterious, particularly in mammals and other species located at the top of the evolutionary tree (Johri et al. 2010). Cd is used extensively in electroplating, although the nature of the operation does not generally lead to overexposures. Cd is also found in some industrial paints and may represent a hazard when sprayed. Operations involving removal of Cd paints by scraping or blasting may pose a significant hazard. Exposures to Cd are addressed in specific standards for the general industry, shipyard employment, construction industry, and the agricultural industry. Cd enters the organisms by dermal contact, inhalation or ingestion of contaminated drinking water and redistributes itself to the entire organ system of the body, mainly to the red blood cells or high molecular weight proteins in the plasma (EFSA, 2009). The kidney is the primary organ affected by chronic Cd exposure and toxicity. After absorption, Cd is transported in the blood by albumin to the liver, where it binds to metallothionein (MT) forming Cd- MT complex. The synthesis of MT in the kidney is more down and insufficient to tie down all the free Cd, resulting in tubular damage or cell membrane destruction via activation of oxygen species (EFSA, 2009). The Cd-MT complex is then released back into the circulation. This complex of low molecular weight is freely filtered through the glomerulus like free Cd then reabsorbed by transporters such as divalent metal transporter1, megalin and cubilin (Christensen and Nielsen, 2007). Cd accumulates in the kidney as a result of its preferential uptake by receptor-mediated endocytosis of freely filtered and MT bound Cd (Cd-MT) in the

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renal proximal tubule. Cd is predominantly located and induces renal tubular dysfunction and renal cancer in the S1 and S2 segment of kidneys (Llyasova and schwartz2005). Internalized Cd-MT is degraded in endosomes and lysosomes, releasing free Cd2+ into the cytosol, where it can generate reactive oxygen species (ROS) and activate cell death pathways. This further led to the degradation of Na+/K+-ATPase, a membrane bound protein that drives the re - absorption of ions and nutrients through Na dependent transporters in the proximal tubules, via the proteasomal and endo lysosomal proteolytic pathways. This is turn contributes to the ‘Fanconi like syndrome’ in which Na+ dependent transport is diminished and is associated with Cd induced nephrotoxicity (Thevenoid, 2003). As a toxic metal, there is unlikely to be specific transport proteins for Cd. Instead, because Cd has similar chemical and physical properties to essential metals such as iron (Fe), zinc (Zn) and calcium (Ca), it can be transferred and carried up by cells by a procedure referred to as “ionic and molecular mimicry”(Bridges and Zalups, 2005). In order to combat against Cd induced oxidative renal damage, antioxidant phytochemicals are the suitable antagonists because of their high antioxidant nature. Grapes (Vitis vinifera), which are one of the most widely consumed fruits in the world have enormous health benefits. Grapes are rich in proanthocyanidins with 60–70% of the proanthocyanidins being contained in the seeds. Grape seed proanthocyanidins (GSP), also named condensed tannins, are oligomers and polymers of monomeric flavonoids, which belong to a larger group of polyphenolic compounds. In these tannins, the monomeric units are primarily linked through a single 4-6 or 4-8 carbon-carbon bonds (B linkages) or through 4-8 carbon-carbon and 2-7 ether bonds. More specifically, they are polyflavans condensed molecules of those flavonoids with a saturated C ring. The unique polyhydroxy phenolic nature of proanthocyanidins and the resulting electronic configuration allows relatively easy release of protons and, as a consequence, they have significant antioxidant activity, employing many antioxidant systems, and radical scavenging activity (Beninger & Hosfield, 2003). GSP exerts a novel spectrum of biological, therapeutic, and chemopreventive properties, in addition to lipid peroxidation, thrombocyte aggregation and capillary permeability-reducing effects; they also have antibacterial, antiviral and anti-inflammatory characteristics.

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They show these effects by modulating various enzymes, including cyclooxygenase and lipooxygenase (Bagchi et al 2003). Proanthocyanidins are also known as sustained release antioxidants and can remain in the plasma and tissues for up to 7-10 days and exert antioxidant properties, which is mechanistically different from other water soluble antioxidants. The presence of both hydrophobic and hydrophilic residues within the flavan-ol molecule allows these compounds to interact with phospholipid head groups and be absorbed onto the surface of membranes. In a previous study, we demonstrated that GSP inhibits Cd -induced hepatic dysfunctions in rats (Nazimabashir et al 2013). In this study, we aimed to show the effect of GSP on Cd intoxication in kidney of rats, which has not been studied experimentally before. The initial aim of the present study was to investigate the oxidative changes, which play a role in the pathogenesis of Cd by evaluating blood urea nitrogen (BUN) and creatinine levels, total oxidant system (TOS), total antioxidant system, cytokines, ATPases, apoptosis and Nrf2 factor exploring the underlying pathways involved in the renal-protection rendered by GSP.

2.

Material and methods

2.1. Chemicals GSP, containing approximately, 54% dimeric, 13% trimeric procyanidins and 7% tetrameric, proanthocyanidins were obtained from Jianfeng, Inc. (Lot no. G050412; Tianjin, China). Cadmium Chloride (CdCl2) and other fine chemicals were obtained from Pfizer, India. Commercial kits to estimate urea, uric acid and creatinine were from sigma diagnostics (I) Pvt. Ltd. (Baroda, India). All other chemicals and biochemicals were of analytical grade obtained from local firms. 2.2. Animals Healthy male albino Wistar rats (180-200 g) were used for the experiment. Rats were obtained from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, and maintained in an air-conditioned room (25 ± 2º C) with a 12 h light/12 h dark cycle. The study protocol was approved by the Institutional Animal Ethical Committee

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(vide no. 1020, 2013), for the purpose of control and supervision on experimental animals (CPCSEA) at Annamalai University, Annamalai Nagar India. 2.3. Study protocol After 5 days of acclimatization the 24 male rats were randomly divided into four groups of 6 animals each control, GSP, Cd and Cd + GSP groups. The rats were weighed on the first day of the study. Group control, received normal saline; Group Cd, orally received Cd as CdCl2 in 0.5 ml sterile physiological saline at a dose of 5mg/kgbw/ day for 4 weeks; Group GSP received 100 mg/kg/day, dissolved in normal saline by oral gavage for 4 weeks (Renugadevi and MiltonPrabu,2010); and Group Cd + GSP, Rats orally received Cd 5mg/kgbw and GSP at a dose of 100 mg/kgbw, which was dissolved in their respective saline, 90 min after the administration of GSP every day by oral gavage for 4 weeks. GSP dose was selected on the basis of prelimanary studies.The five concentrations, (25,50 and 100) of GSP were used to determine the dose depentdent effect of Cd. It was confirmed that 100mg/kg bw showed better results and hence was used for further biochemical parameters.(unpublished lab report). The rats in the groups were kept in separate cages and had free access to standard rat chow and water (except for the days of dehydration). At the end of the treatment period, the rats were fasted overnight, anesthetized with pentobarbital sodium (35 mg/kg, i.p.) and sacrificed by cervical decapitation. The blood was collected in heparinized tubes and was subsequently centrifuged (1000xg for 15 min) and stored at -80˚C for analysis. Urine samples were obtained from each animal housed in a specially designed metabolic cage, and fecal contamination was avoided. Urine samples were collected in bottles within 24-h cycles. The volume of each sample was recorded and centrifuged at 3000xg for 5 min. Urine samples were collected during the morning between 9.00 and 10.00 h. Kidney tissues from control and experimental groups of rats were excised, rinsed with ice-cold saline and homogenized in 100 mM Tris– HCl buffer (pH 7.4) using Teflon homogenizer and centrifuged at 1200xg for 30 min at 40C. The supernatant was pooled and used for the further biochemical estimations. The protein concentrations were

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assessed using a protein assay kit (Bio-Rad, Hercules, CA, USA) and bovine serum albumin was used as a standard.

3. Biochemical assays 3.1. Estimation of BUN, urea, uric acid, creatinine and creatinine clearance The levels of BUN, urea, uric acid and creatinine in serum and urine were estimated spectrophotometrically using commercial diagnostic kits (Sigma Diagnostics (I) Pvt. Ltd., Baroda, India). Creatinine clearance as an index of glomerular filtration rate was calculated from serum creatinine and a 24 h urine sample creatinine level. 3.2. Determination of lipid peroxidation and protein carbonyl contents. Lipid peroxidation in the kidney was estimated spectrophotometrically by measuring thiobarbituric acid reactive substances (TBARS) and hydroperoxides as described by (Niehiaus and Samuelsson., 1968). In brief, 0.5 ml of the kidney-homogenate was treated with 2 ml of TBA– trichloroacetic acid (TCA) –HCl reagent (0.37% TBA, 0.25 N HCl, 15% TCA, 1 : 1 : 1 ratio) and placed for 15 min in a water bath, then cooled and centrifuged for 10 min (1000 rpm) at room temperature and the clear supernatant was measured at 535 nm against a reference blank. Lipid hydroperoxides were estimated spectrophotometrically by measuring hydroperoxides (HPs) by the method of (Jiang et al. 1992). The homogenate (0.5 ml) was treated with 0.9 ml of Fox reagent (88 mg of BHT, 7.6 mg of xylenol orange and 0.8 mg of ammonium iron sulfate were added to 90 ml of methanol and 10 ml of 250 mmol H2SO4), incubated at 37 °C for 30 min and color development was monitored at 560 nm. As a hallmark of protein oxidation, total protein carbonyl content was determined in the kidney by a spectrophotometric method described by (Levine et al., 1990) and expressed as nmol per mg protein. In brief, the tissue homogenate was centrifuged at 10 000 rpm for 20 min to separate cytosol, and then 0.5 ml of cytosolic fraction and 0.5 ml of TCA were added. Later 0.5 ml of DNPH was added and kept for 1 h

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at room temperature. The pellet was washed thrice with 1 ml of ethanol–ethyl acetate mixture, and the pellet was dissolved in 1 ml of guanidine hydrochloride, and the color developed was read at 360 nm. 3.3. Determination of non-enzymatic antioxidants. Reduced glutathione (GSH) was determined by the method of (Moron et al., 1979). 0.5 ml of supernatant was mixed with 10% TCA in a 1: 1 ratio and then centrifuged for 10 min at 5000 rpm. The clear supernatant was then mixed with 2 ml of phosphate buffer and 0.5 ml of DTNB. After incubation for 10 min, absorbance was measured at 412 nm. Total sulphydryl groups (TSH) in the kidney homogenate were measured after the reaction with dithionitrobis benzoic acid using the method of (Ellman, 1959). 1 ml of supernatant was treated with 0.5 ml of Ellman’s reagent (19.8 mg of 5, 5-dithionitrobis benzoic acid in 100 ml of 0.1% sodium citrate) and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm using a spectrophotometer. Vitamin C concentration was measured by the Omaye et al., (1979) method. To 0.5 ml of homogenate, 1.5 ml of 6% TCA was added and centrifuged (3500g, 20 min). To 0.5 ml of supernatant, 0.5 ml of DNPH reagent (2% DNPH and 4% 9 N H2SO4) was added and incubated for 3 h at room temperature. After incubation, 2.5 ml of 85% H2SO4 was added and the color developed was read at 530 nm after 30 min. Vitamin E was estimated by the method of (Desai, 1984) Vitamin E was extracted from heart tissue by the addition of 1.6 ml ethanol and 2.0 ml petroleum ether and centrifuged. The supernatant was separated and evaporated into the air. To the residue, 0.2 ml of 0.2% 2-dipyridyl and 0.2 ml of 0.5% ferric chloride were added and kept in the dark for 5 min. An intense red colored layer obtained with the addition of 4 ml butanol was read at 520 nm. 3.4. Assay of antioxidant and GSH metabolizing enzymes Superoxide dismutase (SOD) activity was determined by the method of (Kakkar et al., 1984) in which the inhibition of the formation of NADH-phenazinemethosulfate nitroblue tetrazolium formazon was measured spectrophotometrically at 560 nm. MDA was calculated by an assay kit according to the

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manufacturer’s instruction (Biodiagnostic, Egypt). Catalase (CAT) activity was assayed calorimetrically as described by Sinha (1972) using dichromate acetic acid reagent. Glutathione peroxidase (GPx) activity was assayed by the method based on the reaction between glutathione remaining after the action of GPx and 5, 5- dithiobis-2-nitrobenzoic acid to form a complex that absorbs maximally 412 nm (Rostruck et al 1973). Glutathione S-transferase (GST) activity was determined spectrophotometrically by using dichloro-2, 4-dinitrobenzene as the substrate (Habig et al 1974). Glutathione reductase (GR) that utilizes NADPH to convert metabolized Glutathione (GSSG) to the reduced form was assayed by the method of the Horn and Burn 1978. The estimation of Glucose 6-phosphate dehydrogenase (G6PD) was carried out by the method of Beutler 1983, where an increase in the absorbance was measured when the reaction was started by the addition of glucose 6-phosphate. Protein level was determined by using Bovine serum albumin as the standard at 560 nm by (Lowry et al 1951). 3.5. Estimation of TNF-α, NO, IL-6, IL-1B and NF-kB p65 The levels of pro-inflammatory cytokines such as TNF-α, NO, IL-6, IL-1B and NFkBp65 in kidney tissues of control and experimental groups of rats were determined by specific ELISA kits according to the manufacturer's instructions (Biosource, Camarillo, CA). The concentration of proinflammatory cytokines was determined spectrophotometrically at 450 nm. Standard plots were constructed by using standard cytokines and the concentrations for unknown samples were calculated from the standard plot. The nuclear level of p65 may correlate positively with the activation of the NF-κB pathway. The NF-κB/p65 Activ ELISA (Imgenex, San Diego, CA) kit was used to quantify NF-κB free p65 in the nuclear fraction of the kidney tissue homogenate. The analysis was done according to the manufacturer's instructions.

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3.6. Estimation of membrane-bound ATPases Total ATPase activity in kidney homogenate was measured by the method of Evans (1969). The ATPase activity in 0.1 ml of an aliquot of the homogenate was measured in a final volume of 2 ml containing 0.1 ml of 0.1 M Tris– HCl (pH 7.4), 0.1 ml of 0.1 M NaCl, 0.1 ml of 0.1 M MgCl2, 1.5 ml of 0.1 M KCl, 0.1 ml of 1 mM EDTA and 0.1 ml of 0.01 M ATP. The reaction was stopped at 20 min by the addition of 1 ml of 10 % TCA and then centrifuged (1500xg for 10 min) and the inorganic phosphorus (Pi) liberated was estimated in the protein free supernatant. The amount of liberated Pi was estimated according to the method of Fiske and Subbarow (1925). The activity of Na+/K+-dependent ATPase was determined by the method of Bonting (1970) with slight modification. In this assay, 0.2 ml of the kidney tissue homogenate was added to the mixture containing 1 ml of 184 mM Tris–HCl buffer (pH 7.5), 0.2 ml of 50 mM MgSO4, 0.2 ml of 50 mM KCl, 0.2 ml of 600 mM NaCl, 0.2 ml of 1 mM EDTA and 0.2 ml of 10 mM ATP and incubated for 15 min at 37ºC. When present, ouabain was used at a concentration of 1 mM. The reaction was arrested by the addition of 1 ml of ice cold 10 % TCA. Then the amount of Pi liberated was estimated in the protein-free supernatant. Na+, K+ -ATPase activity was taken to be the ouabain sensitive component of the total ATPase activity. The activity of Ca2+–ATPase was assayed according to the method of Hjerten and Pan (1983). In brief, 0.1 ml of tissue homogenate was added with a mixture containing 0.1 ml of 125 mM Tris–HCl buffer (pH 8), 0.1 ml of 50 mM CaCl2 and 0.1 ml of 10 mM ATP. The contents were incubated at 37 ºC for 15 min. The reaction was then arrested by the addition of 0.5 ml of ice cold 10 % TCA and centrifuged. The amount of Pi liberated was estimated in the supernatant. The activity of Mg2+–ATPase was assayed by the method of Ohnishi et al., (1982). The contents were incubated for 15 min at 37 ºC and the reaction was arrested by adding 0.5 ml of 10 %TCA. The Pi liberated was then estimated in the protein-free supernatant. The activities of these ATPase enzymes in tissue homogenate were expressed as log Pi liberated/min/mg protein. 3.7. Western blot analysis for Bax, Bcl-2, Caspase 3, Caspase 9 and Cytochrome C

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Western blotting was performed by the method of Camano et al., (2010) to analyze the expression pattern of Bax, Bcl-2, Cytochrome c, Caspase 9 and Caspase 3. The kidney tissue samples were homogenized in an ice-cold lysis buffer (1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 µg/ml phenymethyl sulfonyl Cd). The homogenate was centrifuged at 12,000 RPM for 10 min at 4 °C, and the supernatant was stored at −80 °C. The protein concentration was determined with the BioRad (Hercules, CA, USA) protein assay reagent. Equivalent amounts of protein were resolved on a 10% SDSpolyacrylamide gel, and then transferred to a PVDF membrane. After blockage of nonspecific binding sites with 5% non fat milk in PBS-T (PBS and 1% Tween 20) for 1 h at room temperature, the membrane was incubated overnight at 4 °c with diluted goat anti-Bcl-2, Bax, cytochrome c, cleaved caspases 3and 9, β-actin monoclonal antibodies (Santa Cruz Inc.,CA, USA). The membranes were then washed thrice with PBS-T, incubated further with a secondary horseradish peroxidase conjugated antibody (Santa Cruz Inc. CA, USA) at room temperature for 1 h, and then washed thrice with PBS-T). Specific binding was detected using diamino benzidine and H2O2 as substrates. Band intensities of Bcl-2, Bax, cytochrome c, cleaved caspase 3, 9 and β-actin were quantified by densitometry analysis using an image analysis system (Image J; National Institute of Health, Bethesda, USA). The results were normalized to the β-actin expression in each group (Mean ± SD) as percent of control. No differences were observed on β-actin expression between experimental groups. 3.8. Western blot analysis for Nrf2, Keap 1, HO-1, γ-GCS, and µ-GST Protein extraction was performed as follows. The rat kidney was homogenized in 1 ml of ice cold hypotonic buffer A [10mM HEPES (pH 7.8), 10mM KCL, 2mM MgCl2, 1 mMDTT, 0.1 mMEDTA, 0.1 mM phenyl methyl sulfonyl Fluoride (PMSF)]. To the homogenate, 80µl of 10% Nonidet P-40 (NP-40) solution was added, and the mixture was then centrifuged for 2min at 14000xg. The supernatant was collected as a cytosolic fraction for HO-1 The precipitated nuclei were washed once with 500µl of buffer A plus 40µl of 10% NP-40, centrifuged, re suspended in 200µl of buffer C [50 Mm HEPES (PH 7-

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8), 50 Mm KCL, 300Mm NaCl, 0.1 mM EDTA, 1mM DTT, 0.1mM PMSF, 20% glycerol] and centrifuged for 5min at 14800xg The supernatant containing nuclear proteins was collected for Nrf2 (Laemmli, 1970). Concentration of the protein was determined according to the procedure described by Lowry using a protein assay kit supplied by Sigma, St. Louis, MO, USA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer containing 2% β-mercaptoethanol was added to the supernatant. Equal amounts of protein (50µg) were electrophoresed and subsequently transferred to nitrocellulose membranes (Schleicher and school lunch, Keene, NH, USA). Nitrocellulose blots were washed twice for 5min each in PBS and blocked with 1%bovine serum albumin in PBS for 1 hour prior to application of the primary antibody. The antibody against Nrf2, µ-GST, γ -GCS, and Keap-1 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody against HO-1, was purchased from Abcam (Cambridge, UK). The primary antibody was diluted (1:1000) in the same buffer containing 0.05% Tween 20. The nitrocellulose membrane was incubated overnight at 40 C with protein antibody. The blots were washed and incubated with horseradish peroxidase conjugated goat anti mouse IgG (Abcam Cambridge, UK). Specific binding was detected using diaminobenzidine and H2O2 as substrates. Band intensities of Nrf2, HO-1, and β-actin were quantified by densitometry analysis using an image analysis system (Image J; National Institute of Health, Bethesda, USA). The results were normalized to the β-actin expression in each group (Mean±SD) as percent of control. No differences were observed on β-actin expression between experimental groups. 3.9. Immunohistochemical Examination of Renal Tissue To examine the protective effects of GSP on apoptosis, autophagy and inflammation in the kidney, cleaved caspase-3 and iNOS expression in the kidney was assessed by Immunohistochemical staining. The iNOS was detected using two distinct antibodies: (1) a rabbit polyclonal antiiNOS

antibody (cat # SC-650) purchased from Santa Cruz Biotechnology (SC) of Santa Cruz, CA; and

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(2) a mouse monoclonal antibody purchased from TL (cat. # N32020). All these antibodies were used with paraffin-embedded tissue. In addition, iNOS was detected in 4-m thick sections cut from frozen renal tissue, incubated with either SC or TL anti-iNOS antibody, and processed in the same manner as for paraffin-embedded tissue. Immunohistochemical analysis of cleaved caspase-3 was performed as previously described by Grande et al. (2010). The procedures were performed according to protocol for cleaved caspase-3 and iNOS immunohistochemistry, with slight modifications. Negative controls included staining tissue sections with the omission of the primary antibody. The sections were graded as 0 (no staining), 1 (staining, 25%), 2 (staining between 25% and 50%), 3 (staining between 50% and 75%) or 4 (staining 75%). 3.10. Histopathology For histopathological examination, the kidney tissues were dissected and the tissue samples were fixed at 20% neutral buffered formalin solution kidneys were gradually dehydrated and embedded in paraffin. Paraffin sections were cut at 5-6 µm thickness and stained with hematoxylin and eosin for light microscopy examination. The sections were viewed and photographed on an Olympus light microscope (Olympus BX51, Tokyo, Japan) with attachment photograph machine (Olympus C-5050, Olympus Optical Co. Ltd., Japan). Histopathological scoring was performed to evaluate the severity of renal tubular damage using a semi quantitative scale. All sections were evaluated for the degree of tubular and glomerular injury, inflammatory cell infiltration, necrosis, edema and calcification. Each kidney slide was examined and assigned for severity of changes using scores on scale of (none - = 0%), mild (+ =

Grape seed proanthocyanidins ameliorates cadmium-induced renal injury and oxidative stress in experimental rats through the up-regulation of nuclear related factor 2 and antioxidant responsive elements.

Cadmium (Cd) preferentially accumulates in the kidney, the major target for Cd-related toxicity. Cd-induced reactive oxygen species (ROS) have been co...
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