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Research Paper
Journal of Pharmacy And Pharmacology
S-allylcysteine prevents cisplatin-induced nephrotoxicity and oxidative stress Tania Gómez-Sierraa, Eduardo Molina-Jijónb, Edilia Tapiac, Rogelio Hernández-Pandod, Wylly Ramsés García-Niñoa, Perla D. Maldonadoe, José Luis Reyesb, Diana Barrera-Oviedof, Ismael Torresg and José Pedraza-Chaverria a Department of Biology, Faculty of Chemistry, fDepartment of Pharmacology, gAnimal Care Unit, Faculty of Medicine, National Autonomous University of Mexico (UNAM), bDepartament of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies, National Polytechnic Institute (Cinvestav-IPN), cLaboratory of Renal Pathophysiology, Department of Nephrology, National Institute of Cardiology, d Experimental Pathology Section, National Institute of Medical Sciences and Nutrition ‘Salvador Zubirán’, eLaboratory of Vascular Pathology, National Institute Neurology and Neurosurgery ‘Manuel Velasco Suárez’, Mexico City, DF, Mexico
Keywords cisplatin; nephrotoxicity; nuclear factor-erythroid 2-related factor-2; oxidative stress; S-allylcysteine Correspondence José Pedraza-Chaverri, Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico (UNAM), Laboratory 209, Building F, University City, 04510, DF, México. E-mail: [email protected] Received February 3, 2014 Accepted March 23, 2014 doi: 10.1111/jphp.12263
Abstract Objectives Cisplatin (CP) is an antineoplastic agent that induces nephrotoxicity and oxidative stress. S-allylcysteine (SAC) is a garlic-derived antioxidant. This study aims to explore whether SAC protects against CP-induced nephrotoxicity in rats. Methods In the first stage, the SAC protective dose was determined by measuring renal damage and the oxidative stress markers malondialdehyde, oxidized proteins and glutathione in rats injected with CP. In the second stage, the effect of a single dose of SAC on the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), protein kinase C beta 2 (PKCβ2) and nicotinamide adenine dinucleotide phosphate oxidase subunits (p47phox and gp91phox) was studied. In addition, the effect of SAC on oxidative stress markers and on the activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in isolated proximal and distal tubules were evaluated. Key findings SAC (25 mg/kg) prevented the CP-induced renal damage and attenuated CP-induced decrease in Nrf2 levels and increase in PKCβ2, p47phox and gp91phox expression in renal cortex and oxidative stress and decrease in the activity of CAT, GPx and GR in proximal and distal tubules. Conclusions These data suggest that SAC provides renoprotection by attenuating CP-induced oxidative stress and decrease in the activity of CAT, GPx and GR.
Introduction Cisplatin, cis-diamminedichloroplatinum II (CP), is an antineoplastic drug used for the treatment of many cancers such as bladder, lung, stomach, testis, ovary and breast. Food and Drug Administration approved its clinical use in 1978; however, its application is limited because the 25–35% of patients treated with CP develop kidney damage.[1–4] CP-induced nephrotoxicity has been associated with the increased production of reactive oxygen species (ROS)[3,5]; inhibition of antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR), glutathioneS-transferase (GST), catalase (CAT) and glutathione pero-
xidase (GPx)[6,7]; the high expression of copper transporter 1 and organic cation transporter 2 in kidney[2,3]; and the induction of the pathway that involves the γ-glutamyl and S-cysteine conjugate β-lyase which diminishes glutathione (GSH) content and produces reactive thiols.[5,8,9] Also, CP induces apoptosis and necrosis in renal tubular cells by activating multiple cell death and inflammation pathways such as p53, c-Jun N-terminal kinases, p38 α, tumour necrosis factor alpha (TNF-α), nuclear factor kappa B (NF-κB) and induction of caspases 2, 3, 8 and 9.[2,3,5,10] Garlic (Allium sativum) has been used by several cultures to treat and prevent cardiovascular, neurodegenerative
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1271–1281
1271
SAC prevents cisplatin-induced renal damage
Tania Gómez-Sierra et al.
and inflammatory diseases.[11–14] The beneficial effects of garlic are associated with the content of sulfur compounds including S-allylcysteine sulfoxide, γ-glutamylS-allylcysteine, S-methylcysteine sulfoxide, trans-1propenylcysteine sulfoxide, S-2 carboxypropopylglutathione and S-allylcysteine (SAC). The latter has been reported to exert antioxidant activity.[11–13,15] However, in garlic, SAC is present in low amounts but during the ageing process of garlic, SAC concentration increases due to enzymatic and natural chemical reactions.[16,17] Due to its antioxidant properties, SAC has been used in several in-vivo and in-vitro models of oxidative stress to decrease lipoperoxidation; to scavenge ROS such as superoxide anion (O2●−), hydrogen peroxide (H2O2), hydroxyl (●OH), peroxynitrite (ONOO−), hypochlorous acid (HOCl), singlet oxygen (1O2) and peroxyl radicals (ROO●); also, to increase GSH levels and the activity of antioxidant enzymes, such as SOD, CAT, GR and GPx,[18–21] to inhibit the activation of NF-κB implicated in the regulation of transcription of genes involved in apoptosis[22,23] and to promote the activation of nuclear factor (NF)-erythroid 2-related factor-2 (Nrf2) in liver and brain.[17,24] Due to the effects described above, the capacity of SAC to exert protection in different models of renal damage and its intake through the diet or commercially available garlic products, this antioxidant was used in this experimental model. So the purpose of this study was to evaluate whether SAC provides a protective effect against CP-induced nephrotoxicity in rats.
ice bath; acetic acid (2.5 ml) was added (final pH 5.6). The white precipitate was filtered and dried by suction. An analytical sample was obtained by crystallization from absolute ethanol.[19] Commercial kits for the determination of blood urea nitrogen (BUN) and plasma creatinine were from Spinreact (Girona, Spain) and micro BCA protein assay reagent from Pierce (Rockford, IL, USA). CP (cisdiamminedichloroplatinum (II)), L-cysteine hydrochloride monohydrate, allyl bromide, bovine serum albumin, 1-chloro-2,4-dinitrobenzene, oxidized glutathione, reduced GSH, dimethyl sulfoxide, nicotinamide adenine dinucleotide phosphate (NADPH), streptomycin sulfate, guanidineHCl, 1-methyl-2-phenylindole, dinitrophenylhydrazine, tetramethoxypropane (TMPO), glucose, alanine, Percoll, GST, collagenase from Clostridium histolyticum type 1, GR, phenylmethylsulfonyl fluoride (PMSF), rabbit anti-Nrf2 and anti-protein kinase C beta 2 (PKCβ2) antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA); goat anti-p47phox and goat anti-gp91phox antibodies were purchased from Santa Cruz Biotech, Inc. (Santa Cruz, CA, USA). ECLTM prime Western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). Absolute ethanol was from JT Baker (Xalostoc, Edo México, México). All other chemicals and reagents used were of analytical grade and commercially available.
Materials and Methods
The aim of this first set of experiments was to determine the SAC protective dose; for this purpose, a dose–response curve was performed to determine the effect of SAC on CP nephrotoxicity. The animals were divided in 11 groups and treated as follows: Control group: vehicle (saline solution 0.9%) intraperitoneal for 4 days. CP group: a single dose of CP (7.5 mg/kg b.w.) intraperitoneal. CP + SAC group: On day 1, two doses of SAC were administered in an interval of 2 h. On day 2, SAC was given 1 h before and after the CP dose. On days 3 and 4, a single dose of SAC was carried out. SAC dose assayed were: 6.25, 12.5, 15, 20, 25, 50, 100, 150 and 200 mg/kg b.w.; intraperitoneal SAC was dissolved in 0.9% saline solution at 25°C and the doses were prepared fresh every day of injection. On fifth day, the animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) to obtain the blood through the aorta using a syringe and an 18 gauge needle with heparin. Blood samples were centrifuged for 10 min at 3000g to separate the plasma which was stored at −40°C until analysis. Both kidneys were removed, one for histological studies and the other to measure oxidative stress markers. The group treated only with different doses of SAC
Animals All experiments were conducted using male adult Wistar rats with initial body weight of 200–225 g. Animals were housed in a controlled environment with a 12 h light/dark cycle and had free access to food and water. Animals were used according to the Guidelines of the Official Mexican Standard care and use of laboratory animals (NOM-062ZOO-1999) and protocol were approved by the local ethics committee (FQ/CICUAL/062/13).
Reagents SAC was synthesized into a round-bottomed flask, equipped with a mechanical stirrer, was placed L-cysteine hydrochloride monohydrate (30 mmol), and absolute ethanol (90 ml) was added. After 5 min under stirring, 11 mmol of sodium was added in several portions during 30 min to the suspension under vigorous stirring. Next, allyl bromide (31 mmol) was added. After, the mixture was stirred for 1 h, and then cold water (30 ml) was added to obtain a colourless solution. The ethanol was distilled off under reduced pressure and the solution was cooled in an 1272
Experimental Design First stage
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1271–1281
Tania Gómez-Sierra et al.
SAC prevents cisplatin-induced renal damage
was not included because the sole aim of the first stage was to find the protective dose of SAC against CP-induced nephrotoxicity.
370 nm,[29] and GSH content was measured by fluorescence at a 390 and 478 nm excitation and emission wavelengths, respectively.[30]
Second stage
Activity of antioxidants enzymes
The SAC dose of 25 mg/kg was used to determine the renal expression of Nrf2, PKCβ2, p47phox and gp91phox and to isolate proximal and distal tubules for measurement of oxidative stress markers and the activity of antioxidant enzymes. SAC administration protocol was similar to the used in the first stage; SAC group 25 mg/kg was included. On fourth day of the study, 24 h urine was collected, and on fifth day, samples were obtained as described above. Both kidneys were removed for Western blot analysis and to isolate proximal and distal fractions.
CAT activity was assayed by measuring H2O2 concentration at 240 nm.[31] The activity of GPx and GR was determined through the disappearance of NADPH and monitored at 340 nm.[32,33]
Preparation of kidney homogenates To measure oxidative stress markers, tissue samples were homogenized with a polytron (PT model 2000 Brinkmann; Kinematica, Westbury, NY, USA) in cold phosphate buffer 50 mM, pH 7.0. The homogenates were centrifuged at 19 000g at 4°C for 30 min, supernatant was collected and the amounts of total proteins were measured by the Lowry method.[25]
Determination of renal function BUN and plasma and urine creatinine were measured with commercial kits according to the manufacturers’ instructions. Creatinine clearance was calculated with the standard formula using the creatinine concentrations obtained in plasma and urine. Proteinuria was measured by the Bradford method.[26]
Histological studies Tissue samples were fixed in 10% formalin dissolved in phosphate-buffered saline buffer pH 7.4, gradually dehydrated and embedded in paraffin. Sections of 4 μ width were stained with hematoxylin and eosin.[27] The quantitative histological damage was measured by using a Leica Microsystems Imaging Solutions Ltd. Software (Cambridge, UK). Damaged tubules were quantified. The histological profile of 20 proximal tubules randomly selected per rat (four rats per group) was measured. Then, the percentage of damaged cells was calculated. The magnification used for automated morphometry was 200×.
Markers of oxidative stress Malondialdehyde (MDA) was measured by a colorimetric method, using a standard curve of TMPO.[28] The content of protein-hydrazone was measured by spectrophotometry at
Extraction of total fractions of proteins for western blot Protein extraction was performed as previously described.[34] Briefly, kidneys were excised and decapsulated; the cortex was dissected out. Pieces of renal cortex were placed in ice-cold Krebs-bicarbonate solution (KB, mM): 110 NaCl, 25 NaHCO3, 3 KCl, 1.2 CaCl2, 0.7 MgSO4, 2 KH2PO4, 10 sodium acetate, 5.5 glucose, 5 alanine and 0.5 g/L bovine serum albumin, pH 7.4. Pieces of renal cortex were placed in ice-cold KB, washed three times and resuspended in 10 ml of KB containing 0.2 g/100 ml collagenase. Samples were gassed with 95% O2/5% CO2 in a shaking water bath at 37°C for 30 min. After digestion, approximately 10 ml of ice-cold KB with a protease inhibitor cocktail (Complete, Roche, Mannheim, Germany) and PMSF (20 μg/ml) were added, and suspension was gently shook to disperse tissue fragments. Collagen fibres were removed by filtration, and tissue suspension was gently centrifuged (18g for 30 s). This washing procedure was repeated three times. Thereafter, pellet was resuspended in RIPA buffer (mM): 40 Tris–HCl, 150 NaCl, 2 EDTA, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate and 0.2% SDS, pH 7.6. Samples were incubated for 30 min at 4°C. Thereafter, samples were sonicated three times for 30 s each at low intensity in an ultrasonic processor (Vibra cell; Sonics & Materials Inc., Danbury, CT, USA). After that, samples were centrifuged at 14 000g, at 4°C, for 40 min, and supernatants were collected. Total protein quantification was performed using the Micro BCA Protein Assay Reagent Kit (Pierce).
Western blot analysis Western blot analysis was performed as previously described.[34] PVDF membranes were incubated overnight at 4°C with the appropriate primary antibodies against Nrf2 (1 : 1000), PKCβ2 (1 : 1000), p47phox (1 : 500) and gp91phox (1 : 500); peroxidase-conjugated anti-rabbit antibodies (1 : 5000) were used. Immunoblots were developed using the ECL prime Western blotting detection reagent (Amersham, GE Healthcare, Buckinghamshire, UK). Chemiluminescence was detected in an EC3 Imaging
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1271–1281
1273
SAC prevents cisplatin-induced renal damage
Tania Gómez-Sierra et al.
(a)
Isolation of proximal and distal tubules Renal cortex was dissected out and washed with Krebs Ringer Bicarbonate solution (KRB). Subsequently, tissue was resuspended with a collagenase solution and was centrifuged at 1300g at 4°C; four washes were performed with KRB. Finally, tissue samples were resuspended in Percoll solution and centrifuged at 20 000g at 4°C for 40 min. Proximal and distal tubules were obtained[35] and total protein was measured by the Lowry method.[25]
2.5
Plasma creatinine (mg/dl)
System (UVP Biolmaging Systems, Cambridge, UK). Protein band density was quantified by transmittance densitometry (Image J Software; National Institutes of Health, Bethesda, MD, USA).
2.0
*
* * *
1.5 #
#
0.5
+ + + + + + + + + + – 6.2512.5 15 20 25 50 100 150 200
150
*
* *
*
Statistical analysis
*
#
1.0
0.0 CP (7.5 mg/kg) – SAC (mg/kg) – (b)
*
*
* *
*
All the values were expressed as mean ± standard error of the mean. Statistical analysis was performed for each determination using Prism 5.0 software (GraphPad, La Jolla, CA, USA) comparing the means through a one-way analysis of variance test followed by Bonferroni multiple comparisons differences; a P value
Research Paper
Journal of Pharmacy And Pharmacology
S-allylcysteine prevents cisplatin-induced nephrotoxicity and oxidative stress Tania Gómez-Sierraa, Eduardo Molina-Jijónb, Edilia Tapiac, Rogelio Hernández-Pandod, Wylly Ramsés García-Niñoa, Perla D. Maldonadoe, José Luis Reyesb, Diana Barrera-Oviedof, Ismael Torresg and José Pedraza-Chaverria a Department of Biology, Faculty of Chemistry, fDepartment of Pharmacology, gAnimal Care Unit, Faculty of Medicine, National Autonomous University of Mexico (UNAM), bDepartament of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies, National Polytechnic Institute (Cinvestav-IPN), cLaboratory of Renal Pathophysiology, Department of Nephrology, National Institute of Cardiology, d Experimental Pathology Section, National Institute of Medical Sciences and Nutrition ‘Salvador Zubirán’, eLaboratory of Vascular Pathology, National Institute Neurology and Neurosurgery ‘Manuel Velasco Suárez’, Mexico City, DF, Mexico
Keywords cisplatin; nephrotoxicity; nuclear factor-erythroid 2-related factor-2; oxidative stress; S-allylcysteine Correspondence José Pedraza-Chaverri, Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico (UNAM), Laboratory 209, Building F, University City, 04510, DF, México. E-mail: [email protected] Received February 3, 2014 Accepted March 23, 2014 doi: 10.1111/jphp.12263
Abstract Objectives Cisplatin (CP) is an antineoplastic agent that induces nephrotoxicity and oxidative stress. S-allylcysteine (SAC) is a garlic-derived antioxidant. This study aims to explore whether SAC protects against CP-induced nephrotoxicity in rats. Methods In the first stage, the SAC protective dose was determined by measuring renal damage and the oxidative stress markers malondialdehyde, oxidized proteins and glutathione in rats injected with CP. In the second stage, the effect of a single dose of SAC on the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), protein kinase C beta 2 (PKCβ2) and nicotinamide adenine dinucleotide phosphate oxidase subunits (p47phox and gp91phox) was studied. In addition, the effect of SAC on oxidative stress markers and on the activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in isolated proximal and distal tubules were evaluated. Key findings SAC (25 mg/kg) prevented the CP-induced renal damage and attenuated CP-induced decrease in Nrf2 levels and increase in PKCβ2, p47phox and gp91phox expression in renal cortex and oxidative stress and decrease in the activity of CAT, GPx and GR in proximal and distal tubules. Conclusions These data suggest that SAC provides renoprotection by attenuating CP-induced oxidative stress and decrease in the activity of CAT, GPx and GR.
Introduction Cisplatin, cis-diamminedichloroplatinum II (CP), is an antineoplastic drug used for the treatment of many cancers such as bladder, lung, stomach, testis, ovary and breast. Food and Drug Administration approved its clinical use in 1978; however, its application is limited because the 25–35% of patients treated with CP develop kidney damage.[1–4] CP-induced nephrotoxicity has been associated with the increased production of reactive oxygen species (ROS)[3,5]; inhibition of antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR), glutathioneS-transferase (GST), catalase (CAT) and glutathione pero-
xidase (GPx)[6,7]; the high expression of copper transporter 1 and organic cation transporter 2 in kidney[2,3]; and the induction of the pathway that involves the γ-glutamyl and S-cysteine conjugate β-lyase which diminishes glutathione (GSH) content and produces reactive thiols.[5,8,9] Also, CP induces apoptosis and necrosis in renal tubular cells by activating multiple cell death and inflammation pathways such as p53, c-Jun N-terminal kinases, p38 α, tumour necrosis factor alpha (TNF-α), nuclear factor kappa B (NF-κB) and induction of caspases 2, 3, 8 and 9.[2,3,5,10] Garlic (Allium sativum) has been used by several cultures to treat and prevent cardiovascular, neurodegenerative
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1271–1281
1271
SAC prevents cisplatin-induced renal damage
Tania Gómez-Sierra et al.
and inflammatory diseases.[11–14] The beneficial effects of garlic are associated with the content of sulfur compounds including S-allylcysteine sulfoxide, γ-glutamylS-allylcysteine, S-methylcysteine sulfoxide, trans-1propenylcysteine sulfoxide, S-2 carboxypropopylglutathione and S-allylcysteine (SAC). The latter has been reported to exert antioxidant activity.[11–13,15] However, in garlic, SAC is present in low amounts but during the ageing process of garlic, SAC concentration increases due to enzymatic and natural chemical reactions.[16,17] Due to its antioxidant properties, SAC has been used in several in-vivo and in-vitro models of oxidative stress to decrease lipoperoxidation; to scavenge ROS such as superoxide anion (O2●−), hydrogen peroxide (H2O2), hydroxyl (●OH), peroxynitrite (ONOO−), hypochlorous acid (HOCl), singlet oxygen (1O2) and peroxyl radicals (ROO●); also, to increase GSH levels and the activity of antioxidant enzymes, such as SOD, CAT, GR and GPx,[18–21] to inhibit the activation of NF-κB implicated in the regulation of transcription of genes involved in apoptosis[22,23] and to promote the activation of nuclear factor (NF)-erythroid 2-related factor-2 (Nrf2) in liver and brain.[17,24] Due to the effects described above, the capacity of SAC to exert protection in different models of renal damage and its intake through the diet or commercially available garlic products, this antioxidant was used in this experimental model. So the purpose of this study was to evaluate whether SAC provides a protective effect against CP-induced nephrotoxicity in rats.
ice bath; acetic acid (2.5 ml) was added (final pH 5.6). The white precipitate was filtered and dried by suction. An analytical sample was obtained by crystallization from absolute ethanol.[19] Commercial kits for the determination of blood urea nitrogen (BUN) and plasma creatinine were from Spinreact (Girona, Spain) and micro BCA protein assay reagent from Pierce (Rockford, IL, USA). CP (cisdiamminedichloroplatinum (II)), L-cysteine hydrochloride monohydrate, allyl bromide, bovine serum albumin, 1-chloro-2,4-dinitrobenzene, oxidized glutathione, reduced GSH, dimethyl sulfoxide, nicotinamide adenine dinucleotide phosphate (NADPH), streptomycin sulfate, guanidineHCl, 1-methyl-2-phenylindole, dinitrophenylhydrazine, tetramethoxypropane (TMPO), glucose, alanine, Percoll, GST, collagenase from Clostridium histolyticum type 1, GR, phenylmethylsulfonyl fluoride (PMSF), rabbit anti-Nrf2 and anti-protein kinase C beta 2 (PKCβ2) antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA); goat anti-p47phox and goat anti-gp91phox antibodies were purchased from Santa Cruz Biotech, Inc. (Santa Cruz, CA, USA). ECLTM prime Western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). Absolute ethanol was from JT Baker (Xalostoc, Edo México, México). All other chemicals and reagents used were of analytical grade and commercially available.
Materials and Methods
The aim of this first set of experiments was to determine the SAC protective dose; for this purpose, a dose–response curve was performed to determine the effect of SAC on CP nephrotoxicity. The animals were divided in 11 groups and treated as follows: Control group: vehicle (saline solution 0.9%) intraperitoneal for 4 days. CP group: a single dose of CP (7.5 mg/kg b.w.) intraperitoneal. CP + SAC group: On day 1, two doses of SAC were administered in an interval of 2 h. On day 2, SAC was given 1 h before and after the CP dose. On days 3 and 4, a single dose of SAC was carried out. SAC dose assayed were: 6.25, 12.5, 15, 20, 25, 50, 100, 150 and 200 mg/kg b.w.; intraperitoneal SAC was dissolved in 0.9% saline solution at 25°C and the doses were prepared fresh every day of injection. On fifth day, the animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) to obtain the blood through the aorta using a syringe and an 18 gauge needle with heparin. Blood samples were centrifuged for 10 min at 3000g to separate the plasma which was stored at −40°C until analysis. Both kidneys were removed, one for histological studies and the other to measure oxidative stress markers. The group treated only with different doses of SAC
Animals All experiments were conducted using male adult Wistar rats with initial body weight of 200–225 g. Animals were housed in a controlled environment with a 12 h light/dark cycle and had free access to food and water. Animals were used according to the Guidelines of the Official Mexican Standard care and use of laboratory animals (NOM-062ZOO-1999) and protocol were approved by the local ethics committee (FQ/CICUAL/062/13).
Reagents SAC was synthesized into a round-bottomed flask, equipped with a mechanical stirrer, was placed L-cysteine hydrochloride monohydrate (30 mmol), and absolute ethanol (90 ml) was added. After 5 min under stirring, 11 mmol of sodium was added in several portions during 30 min to the suspension under vigorous stirring. Next, allyl bromide (31 mmol) was added. After, the mixture was stirred for 1 h, and then cold water (30 ml) was added to obtain a colourless solution. The ethanol was distilled off under reduced pressure and the solution was cooled in an 1272
Experimental Design First stage
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1271–1281
Tania Gómez-Sierra et al.
SAC prevents cisplatin-induced renal damage
was not included because the sole aim of the first stage was to find the protective dose of SAC against CP-induced nephrotoxicity.
370 nm,[29] and GSH content was measured by fluorescence at a 390 and 478 nm excitation and emission wavelengths, respectively.[30]
Second stage
Activity of antioxidants enzymes
The SAC dose of 25 mg/kg was used to determine the renal expression of Nrf2, PKCβ2, p47phox and gp91phox and to isolate proximal and distal tubules for measurement of oxidative stress markers and the activity of antioxidant enzymes. SAC administration protocol was similar to the used in the first stage; SAC group 25 mg/kg was included. On fourth day of the study, 24 h urine was collected, and on fifth day, samples were obtained as described above. Both kidneys were removed for Western blot analysis and to isolate proximal and distal fractions.
CAT activity was assayed by measuring H2O2 concentration at 240 nm.[31] The activity of GPx and GR was determined through the disappearance of NADPH and monitored at 340 nm.[32,33]
Preparation of kidney homogenates To measure oxidative stress markers, tissue samples were homogenized with a polytron (PT model 2000 Brinkmann; Kinematica, Westbury, NY, USA) in cold phosphate buffer 50 mM, pH 7.0. The homogenates were centrifuged at 19 000g at 4°C for 30 min, supernatant was collected and the amounts of total proteins were measured by the Lowry method.[25]
Determination of renal function BUN and plasma and urine creatinine were measured with commercial kits according to the manufacturers’ instructions. Creatinine clearance was calculated with the standard formula using the creatinine concentrations obtained in plasma and urine. Proteinuria was measured by the Bradford method.[26]
Histological studies Tissue samples were fixed in 10% formalin dissolved in phosphate-buffered saline buffer pH 7.4, gradually dehydrated and embedded in paraffin. Sections of 4 μ width were stained with hematoxylin and eosin.[27] The quantitative histological damage was measured by using a Leica Microsystems Imaging Solutions Ltd. Software (Cambridge, UK). Damaged tubules were quantified. The histological profile of 20 proximal tubules randomly selected per rat (four rats per group) was measured. Then, the percentage of damaged cells was calculated. The magnification used for automated morphometry was 200×.
Markers of oxidative stress Malondialdehyde (MDA) was measured by a colorimetric method, using a standard curve of TMPO.[28] The content of protein-hydrazone was measured by spectrophotometry at
Extraction of total fractions of proteins for western blot Protein extraction was performed as previously described.[34] Briefly, kidneys were excised and decapsulated; the cortex was dissected out. Pieces of renal cortex were placed in ice-cold Krebs-bicarbonate solution (KB, mM): 110 NaCl, 25 NaHCO3, 3 KCl, 1.2 CaCl2, 0.7 MgSO4, 2 KH2PO4, 10 sodium acetate, 5.5 glucose, 5 alanine and 0.5 g/L bovine serum albumin, pH 7.4. Pieces of renal cortex were placed in ice-cold KB, washed three times and resuspended in 10 ml of KB containing 0.2 g/100 ml collagenase. Samples were gassed with 95% O2/5% CO2 in a shaking water bath at 37°C for 30 min. After digestion, approximately 10 ml of ice-cold KB with a protease inhibitor cocktail (Complete, Roche, Mannheim, Germany) and PMSF (20 μg/ml) were added, and suspension was gently shook to disperse tissue fragments. Collagen fibres were removed by filtration, and tissue suspension was gently centrifuged (18g for 30 s). This washing procedure was repeated three times. Thereafter, pellet was resuspended in RIPA buffer (mM): 40 Tris–HCl, 150 NaCl, 2 EDTA, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate and 0.2% SDS, pH 7.6. Samples were incubated for 30 min at 4°C. Thereafter, samples were sonicated three times for 30 s each at low intensity in an ultrasonic processor (Vibra cell; Sonics & Materials Inc., Danbury, CT, USA). After that, samples were centrifuged at 14 000g, at 4°C, for 40 min, and supernatants were collected. Total protein quantification was performed using the Micro BCA Protein Assay Reagent Kit (Pierce).
Western blot analysis Western blot analysis was performed as previously described.[34] PVDF membranes were incubated overnight at 4°C with the appropriate primary antibodies against Nrf2 (1 : 1000), PKCβ2 (1 : 1000), p47phox (1 : 500) and gp91phox (1 : 500); peroxidase-conjugated anti-rabbit antibodies (1 : 5000) were used. Immunoblots were developed using the ECL prime Western blotting detection reagent (Amersham, GE Healthcare, Buckinghamshire, UK). Chemiluminescence was detected in an EC3 Imaging
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1271–1281
1273
SAC prevents cisplatin-induced renal damage
Tania Gómez-Sierra et al.
(a)
Isolation of proximal and distal tubules Renal cortex was dissected out and washed with Krebs Ringer Bicarbonate solution (KRB). Subsequently, tissue was resuspended with a collagenase solution and was centrifuged at 1300g at 4°C; four washes were performed with KRB. Finally, tissue samples were resuspended in Percoll solution and centrifuged at 20 000g at 4°C for 40 min. Proximal and distal tubules were obtained[35] and total protein was measured by the Lowry method.[25]
2.5
Plasma creatinine (mg/dl)
System (UVP Biolmaging Systems, Cambridge, UK). Protein band density was quantified by transmittance densitometry (Image J Software; National Institutes of Health, Bethesda, MD, USA).
2.0
*
* * *
1.5 #
#
0.5
+ + + + + + + + + + – 6.2512.5 15 20 25 50 100 150 200
150
*
* *
*
Statistical analysis
*
#
1.0
0.0 CP (7.5 mg/kg) – SAC (mg/kg) – (b)
*
*
* *
*
All the values were expressed as mean ± standard error of the mean. Statistical analysis was performed for each determination using Prism 5.0 software (GraphPad, La Jolla, CA, USA) comparing the means through a one-way analysis of variance test followed by Bonferroni multiple comparisons differences; a P value
