Article
Orchiectomy attenuates oxidative stress induced by aluminum in rats
Toxicology and Industrial Health 1–12 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714566876 tih.sagepub.com
Marı´a del Carmen Contini, Ne´stor Millen, Marcela Gonza´lez, Adriana Benmelej, Ana Fabro and Stella Mahieu Abstract The aim of this work was to study whether the increase in antioxidant defenses associated with orchiectomy may account for the reduced susceptibility to aluminum (Al) in male kidney and also to examine whether the reduced antioxidant defenses are associated with androgen levels in orchiectomized (ORX) rats treated with testosterone propionate (TP). Rats were divided into nine groups, namely, intact males (without treatment, treated with sodium lactate, and treated with Al), sham males, ORX males (without treatment, treated with sodium lactate, treated with TP, treated with Al, and treated with TP and Al). Al groups were chronically treated with aluminum lactate for 12 weeks (0.575 mg Al/100 g of body weight, intraperitoneally, three times per week). We reported that ORX rats treated with Al had significantly less lipid peroxidation and an increased level of reduced glutathione (GSH) and GSH/oxidized glutathione ratio in the kidney when compared with intact and TP-treated ORX rats. The activity of superoxide dismutase, catalase, and glutathione peroxidase in ORX rats was much greater than in intact or TP-administered ORX rats. Castration reduced the glomerular alterations caused by Al as well as the number of necrotic tubular cells and nuclear abnormalities. However, we observed a slight alteration in brush border, dilation of proximal tubules, mononuclear infiltrates, and interstitial fibrosis. Castrated males treated with TP showed that this intervention cancels the protective effect of the ORX. This finding suggests that androgens contribute to the development of renal alterations and proteinuria in rats treated with Al. Our results showed that ORX rats are protected against the induction of oxidative stress by Al, but the morphological damage to the kidney tissue induced by the cation was only reduced. Male intact rats treated with Al had more severe glomerulosclerosis, tubular damage, and proteinuria than ORX rats. Keywords Aluminum, oxidative stress, renal, orchiectomy, histological damage
Introduction Aluminum (Al) is considered a nonessential metal; however, its increased biological availability has been linked with both acute and chronic disease in humans. Al accumulates in mammalian tissues such as brain, bone, liver, and kidney. Several therapeutic maneuvers also lead to increased Al exposure. Ingestion of Al-containing food, water, and pharmaceutical products is the primary route of exposure to Al in most humans (Mahieu and Contini, 2010; Wang et al., 2010). Al, a non-redox-active metal, is known to be prooxidant and has recently been implicated in the generation of an oxidative milieu by aiding in the
production of reactive oxygen species (ROS) such and hydrogen peroxide (H2O2) (Exley, as O 2 2004; Kumar and Gill, 2009; Singh et al., 2005). Earlier studies from our laboratory in male rats have shown that the accumulation of Al in renal tissue affects cellular metabolism, promotes oxidative Facultad de Bioquı´mica y Ciencias Biolo´gicas (FBCB), Universidad Nacional del Litoral (UNL), Santa Fe, Argentina Corresponding author: Marı´a del Carmen Contini, Facultad de Bioquı´mica y Ciencias Biolo´gicas (FBCB), Universidad Nacional del Litoral (UNL), Ciudad Universitaria, 3000 Santa Fe, Argentina. Email: [email protected]
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Toxicology and Industrial Health
stress, and induces alterations in renal tubular transport (Mahieu and Calvo, 1998; Mahieu et al., 2001, 2003). We found a decreased urinary concentrating ability in situations where the endogenous or exogenous plasma antidiuretic hormone level was increased (Mahieu et al., 2006). The kidney has a very active oxidative metabolism that results in the production of ROS. As the ratio between oxidant and antioxidant enzyme activities determines the oxidant status of renal cells and tissues, the failure of a compensatory rise in antioxidant enzyme activities or an increase in oxidant enzyme activities may both contribute to renal oxidant stress. (Haugen and Nath, 1999; Ichikawa et al., 1994). In previous studies in male rats, we showed that Al treatment negatively affects the balance between the production of free radical and antioxidant defense in kidney tissue, leading to increased lipid susceptibility to peroxidation (Mahieu et al., 2005, 2009). Male sex hormones have been linked with the progression of renal injury (Park et al., 2004; Song et al., 2006), while female sex hormones have been postulated to be renal protective (Gross et al., 2004; Ji et al., 2007; Sullivan et al., 2007; Yanes et al., 2008). Recently, we demonstrated that females are protected against the prooxidant effect of Al and that modifications of the hormonal status of female rats could affect this response. Al treatment in female rats modifies the homeostasis of glutathione (GSH) and the antioxidant capacity of rat kidneys. The alteration of GSH homeostasis and of oxidative status was not associated with an increased lipid peroxidation (Contini et al., 2008, 2011). A chronic cytotoxic renal effect of Al in mammalian kidney was reported using light and electron microscopy. These results indicated atrophy and degeneration of some tubules, interstitial fibrosis, and some glomerulus that were undergoing partial sclerosis (Kutlubay et al., 2007; Sargazi et al., 2001; Somova et al., 1997). As it has been documented, testosterone has oxidative effects; we have conducted two experiments to determine whether male rats with orchiectomy were protected against the prooxidant effect of chronic treatment with Al. In the first experiment, we assessed the effect of Al on the oxidative stress parameters of orchiectomized (ORX) male rats. A second study was therefore performed in order to assess the effect of testosterone on the susceptibility of the kidney to lipid peroxidation in male rats, using testosterone-supplemented ORX animals. We also examined the relationship between oxidative stress and renal histology changes.
Materials and methods Animals and treatments Male Wistar rats at 12 weeks of age were used (n ¼ 54). One group of the animals was gonadectomized under pentobarbital sodium anesthesia (50 mg/kg body weight (b.w.), intraperitoneally (i.p.)) for bilateral removal of the testes under aseptic surgical conditions. Orchiectomy was performed by a trans-scrotal approach. The efficacy of the orchiectomy was confirmed by the assessment of the seminal vesicles and prostate gland weight and by serum testosterone levels at the end of the experiment. After 2 weeks, ORX rats were divided into two groups. One was implanted with 30-mm capsules filled with testosterone propionate (TP; 4-androsten-17b-ol3-one 17 propionate; Sigma-Aldrich Chemical, St Louis, Missouri, USA; 1.57 mm internal diameter (ID) Silastic tubing) subcutaneously at the base of the neck (ORX þ T) (Gonzales et al., 2004). All capsules were checked for leakage and pre-equilibrated in sterile saline for 24 h before implantation. The encapsulated TP diffuses through the walls of the Silastic tubing (Damassa et al., 1977). A second group of rats did not receive an implant and were used as testosteronedeficient controls (ORX), and Al treated (ORX þ Al). Capsules were changed after 45 days. Rats were randomly divided into nine groups according to treatment with sodium lactate (Na), aluminum lactate (Al), sham (SH), and orchiectomy with and without testosterone as follows: intact males without treatment (C), treated with sodium lactate (C-Na), orchiectomized without treatment (ORX), sham without treatment (SH), ORX treated with sodium lactate (ORX-Na), ORX treated with testosterone (ORX-T), intact males treated with Al (Al), ORX treated with Al (ORX-Al), and ORX treated with testosterone and Al (ORX-T-Al). C-Na and ORX-Na rats did not present differences compared with C and ORX rats, respectively, without any treatment in all the experiments described in this manuscript. For practical reasons, we decided to report only C and ORX. In the same way, SH rats did not present significant differences with C rats in the evaluated parameters. Moreover, we decided to report only C. The Al dose, frequency, and route of administration used were described previously. Al-treated rats received an intraperitoneal injection of sterile solution of aluminum lactate (Merck, White House Station, New Jersey, USA) containing 2.7 g/l of Al and 26.7
Downloaded from tih.sagepub.com at SETON HALL UNIV on April 2, 2015
Contini et al.
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g/l of anion lactate, pH 7, during 12 weeks (0.575 mg Al/100 g of b.w., three times per week). Aluminum lactate was dissolved in deionized water and the solution was filtered through a 0.2-m microfilter (Nalgene, Rochester, New York, USA). Al concentration was measured in the solution (Ballanti et al., 1989; Mahieu et al., 2003).This treatment avoided the death of the animals and produced high Al concentrations in serum, intestine, liver, kidney, and bone. C-Na and ORX-Na were injected with a sterile solution of sodium lactate (Merck) containing 26.7 g/l of anion lactate, pH 7 (5.68 mg of lactate/100 g of b.w., i.p.) during the same period and frequency. The rats were housed under conditions of constant temperature (22–24 C) and humidity (45–50%) in room with a fixed 12-h artificial light/12-h artificial dark cycle. Animals were allowed free access to tap water and a standard diet. Al exposure from food and tap water resulted negligible with respect to the administered Al dose (tap water: 1.6 mol/l; food: 30. 5 mol/g wet weight). The experimental protocol was approved by the Human and Animal Research Committee of the School of Biochemistry, University of Litoral, Santa Fe, Argentina. (Res. C.S. 388/06. PI 20-124).
Biochemical studies in urine After 11 weeks of treatment, the rats (n ¼ 6, each group) were housed in metabolic cages to determine urine volume and food and water intake. Animals received demineralized water and standard diet. After allowing to acclimatize for 1 day, daily urine volume was measured during the last 2 days. Creatinine, protein excretion, and osmolality were analyzed in urine collected during the last 24 h.
Biochemical studies in blood and renal tissues After 12 weeks of treatment, the rats (n ¼ 6, each group) were weighed and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg b.w., i.p.). Blood samples were collected by cardiac puncture. Both kidneys were removed, decapsulated, and dissected. One part was processed for histology, and the rest were frozen and stored in liquid nitrogen. These tissues were used to measure the oxidative state of renal tissue, -glutamyl transferase (GGT) activity in renal plasma membrane and Al content. Then, the seminal vesicles and the prostatic gland were isolated and weighed. Testosterone, creatinine, and Al concentrations were measured in serum
samples. Creatinine clearance was obtained by conventional formulae for each animal. Different sets of renal cortex samples were homogenized with a Polytron homogenizer (Kinematica Inc, USA) in different buffers as follows: (1) buffered sucrose medium (0.25 M sucrose, 10 mM Tris-hydrochloric acid (HCl), pH 7.0) for isolation of renal plasma membrane for GGT activity measurement; (2) buffered sucrose medium (0.25 M sucrose, 10 mM Tris-HCl, pH 7.0) for superoxide dismutase (SOD) activity measurement; (3) 50 mM phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid, for glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT) activity measurements; (4) cold 1, 15% potassium chloride for thiobarbituric acid reactive substances (TBARS) measurement; and (5) a mixture of acetonitrile/water (62.5:37.5 v/v) for reduced GSH and oxidized glutathione (GSSG) measurement. Measurement of oxidative stress markers. SOD activity was assayed according to the method of Misra and Fridovich (1972). GPx activity was determined according to the method of Lawrence and Burk (1976). GR activity was assayed according to the method described by Goldberg and Sparner (1987) after modification and CAT activity was determined following the method of Aebi (1985). TBARS was assayed according to the method of Ohkawa et al. (1979). Determination of renal GSH and GSSG was carried out in homogenates with a mixture of acetonitrile/ water (62.5:37.5 v/v). The homogenates were centrifuged at 18,000 r/min for 10 min. The supernatant was measured by capillary electrophoresis (CE) immediately (Maeso et al., 2005). The separation was performed on a CE P/ACE 5010 (Beckman) with ultraviolet detection at 200 + 10 nm. It was equipped with an uncoated capillary with 40-cm effective length and 50-m ID and using 0.200 M borate as buffer, pH was made up to 8.0 with sodium hydroxide. Protein concentration in renal homogenates was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. GGT activity in whole plasma membrane was measured kinetically at 405 nm employing a commercial kit (Wiener Lab., Rosario, Argentina) using -glutamyl p-nitroanilide as substrate. Histopathological examination. Kidney tissues were fixed in 10% buffered formalin (pH 7.2) and dehydrated
Downloaded from tih.sagepub.com at SETON HALL UNIV on April 2, 2015
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Toxicology and Industrial Health
through a series of ethanol solutions, embedded in paraffin, and routinely processed for histological analysis. Sections of 5 m thickness were cut and stained with hematoxylin–eosin (for general morphological examination), periodic acid-Schiff (PAS; for assessment of basement membrane changes and brush border tubules), and Masson’s trichome stain (for demonstration of collagen deposition). All specimens were examined by two independent observers who were blinded with respect to the identity of the tissue under scrutiny. Stained sections were examined using an Olympus light microscope (400). One hundred glomeruli and 300 tubules were observed in each nonoverlapping fields of each group. According to the score established by Ji et al. (2005), the degree of glomerular sclerosis (GS) was graded on a scale of 0–4: 0 (normal glomeruli); 1sclerotic area up to 1–25% (minimal sclerosis); 2 sclerotic area 26–50% (moderate sclerosis); 3 sclerotic area 51–75% (moderate–severe sclerosis); and 4 sclerotic area 76–100% (severe sclerosis). The glomerular sclerotic index (GSI) was calculated using the following formula: GSI ¼ ð1 n1 Þ þ ð2 n2 Þ þ ð3 n3 Þ þ ð4 n4 Þ= n0 þ n1 þ n2 þ n3 þ n4 , where nx is the number of glomeruli in each grade of GS. The degree of tubulointerstitial injury was evaluated by a semiquantitative scoring system and was graded on a scale of þ to þþþþ (–: normal; þ: affected parameters
Orchiectomy attenuates oxidative stress induced by aluminum in rats
Toxicology and Industrial Health 1–12 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714566876 tih.sagepub.com
Marı´a del Carmen Contini, Ne´stor Millen, Marcela Gonza´lez, Adriana Benmelej, Ana Fabro and Stella Mahieu Abstract The aim of this work was to study whether the increase in antioxidant defenses associated with orchiectomy may account for the reduced susceptibility to aluminum (Al) in male kidney and also to examine whether the reduced antioxidant defenses are associated with androgen levels in orchiectomized (ORX) rats treated with testosterone propionate (TP). Rats were divided into nine groups, namely, intact males (without treatment, treated with sodium lactate, and treated with Al), sham males, ORX males (without treatment, treated with sodium lactate, treated with TP, treated with Al, and treated with TP and Al). Al groups were chronically treated with aluminum lactate for 12 weeks (0.575 mg Al/100 g of body weight, intraperitoneally, three times per week). We reported that ORX rats treated with Al had significantly less lipid peroxidation and an increased level of reduced glutathione (GSH) and GSH/oxidized glutathione ratio in the kidney when compared with intact and TP-treated ORX rats. The activity of superoxide dismutase, catalase, and glutathione peroxidase in ORX rats was much greater than in intact or TP-administered ORX rats. Castration reduced the glomerular alterations caused by Al as well as the number of necrotic tubular cells and nuclear abnormalities. However, we observed a slight alteration in brush border, dilation of proximal tubules, mononuclear infiltrates, and interstitial fibrosis. Castrated males treated with TP showed that this intervention cancels the protective effect of the ORX. This finding suggests that androgens contribute to the development of renal alterations and proteinuria in rats treated with Al. Our results showed that ORX rats are protected against the induction of oxidative stress by Al, but the morphological damage to the kidney tissue induced by the cation was only reduced. Male intact rats treated with Al had more severe glomerulosclerosis, tubular damage, and proteinuria than ORX rats. Keywords Aluminum, oxidative stress, renal, orchiectomy, histological damage
Introduction Aluminum (Al) is considered a nonessential metal; however, its increased biological availability has been linked with both acute and chronic disease in humans. Al accumulates in mammalian tissues such as brain, bone, liver, and kidney. Several therapeutic maneuvers also lead to increased Al exposure. Ingestion of Al-containing food, water, and pharmaceutical products is the primary route of exposure to Al in most humans (Mahieu and Contini, 2010; Wang et al., 2010). Al, a non-redox-active metal, is known to be prooxidant and has recently been implicated in the generation of an oxidative milieu by aiding in the
production of reactive oxygen species (ROS) such and hydrogen peroxide (H2O2) (Exley, as O 2 2004; Kumar and Gill, 2009; Singh et al., 2005). Earlier studies from our laboratory in male rats have shown that the accumulation of Al in renal tissue affects cellular metabolism, promotes oxidative Facultad de Bioquı´mica y Ciencias Biolo´gicas (FBCB), Universidad Nacional del Litoral (UNL), Santa Fe, Argentina Corresponding author: Marı´a del Carmen Contini, Facultad de Bioquı´mica y Ciencias Biolo´gicas (FBCB), Universidad Nacional del Litoral (UNL), Ciudad Universitaria, 3000 Santa Fe, Argentina. Email: [email protected]
Downloaded from tih.sagepub.com at SETON HALL UNIV on April 2, 2015
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Toxicology and Industrial Health
stress, and induces alterations in renal tubular transport (Mahieu and Calvo, 1998; Mahieu et al., 2001, 2003). We found a decreased urinary concentrating ability in situations where the endogenous or exogenous plasma antidiuretic hormone level was increased (Mahieu et al., 2006). The kidney has a very active oxidative metabolism that results in the production of ROS. As the ratio between oxidant and antioxidant enzyme activities determines the oxidant status of renal cells and tissues, the failure of a compensatory rise in antioxidant enzyme activities or an increase in oxidant enzyme activities may both contribute to renal oxidant stress. (Haugen and Nath, 1999; Ichikawa et al., 1994). In previous studies in male rats, we showed that Al treatment negatively affects the balance between the production of free radical and antioxidant defense in kidney tissue, leading to increased lipid susceptibility to peroxidation (Mahieu et al., 2005, 2009). Male sex hormones have been linked with the progression of renal injury (Park et al., 2004; Song et al., 2006), while female sex hormones have been postulated to be renal protective (Gross et al., 2004; Ji et al., 2007; Sullivan et al., 2007; Yanes et al., 2008). Recently, we demonstrated that females are protected against the prooxidant effect of Al and that modifications of the hormonal status of female rats could affect this response. Al treatment in female rats modifies the homeostasis of glutathione (GSH) and the antioxidant capacity of rat kidneys. The alteration of GSH homeostasis and of oxidative status was not associated with an increased lipid peroxidation (Contini et al., 2008, 2011). A chronic cytotoxic renal effect of Al in mammalian kidney was reported using light and electron microscopy. These results indicated atrophy and degeneration of some tubules, interstitial fibrosis, and some glomerulus that were undergoing partial sclerosis (Kutlubay et al., 2007; Sargazi et al., 2001; Somova et al., 1997). As it has been documented, testosterone has oxidative effects; we have conducted two experiments to determine whether male rats with orchiectomy were protected against the prooxidant effect of chronic treatment with Al. In the first experiment, we assessed the effect of Al on the oxidative stress parameters of orchiectomized (ORX) male rats. A second study was therefore performed in order to assess the effect of testosterone on the susceptibility of the kidney to lipid peroxidation in male rats, using testosterone-supplemented ORX animals. We also examined the relationship between oxidative stress and renal histology changes.
Materials and methods Animals and treatments Male Wistar rats at 12 weeks of age were used (n ¼ 54). One group of the animals was gonadectomized under pentobarbital sodium anesthesia (50 mg/kg body weight (b.w.), intraperitoneally (i.p.)) for bilateral removal of the testes under aseptic surgical conditions. Orchiectomy was performed by a trans-scrotal approach. The efficacy of the orchiectomy was confirmed by the assessment of the seminal vesicles and prostate gland weight and by serum testosterone levels at the end of the experiment. After 2 weeks, ORX rats were divided into two groups. One was implanted with 30-mm capsules filled with testosterone propionate (TP; 4-androsten-17b-ol3-one 17 propionate; Sigma-Aldrich Chemical, St Louis, Missouri, USA; 1.57 mm internal diameter (ID) Silastic tubing) subcutaneously at the base of the neck (ORX þ T) (Gonzales et al., 2004). All capsules were checked for leakage and pre-equilibrated in sterile saline for 24 h before implantation. The encapsulated TP diffuses through the walls of the Silastic tubing (Damassa et al., 1977). A second group of rats did not receive an implant and were used as testosteronedeficient controls (ORX), and Al treated (ORX þ Al). Capsules were changed after 45 days. Rats were randomly divided into nine groups according to treatment with sodium lactate (Na), aluminum lactate (Al), sham (SH), and orchiectomy with and without testosterone as follows: intact males without treatment (C), treated with sodium lactate (C-Na), orchiectomized without treatment (ORX), sham without treatment (SH), ORX treated with sodium lactate (ORX-Na), ORX treated with testosterone (ORX-T), intact males treated with Al (Al), ORX treated with Al (ORX-Al), and ORX treated with testosterone and Al (ORX-T-Al). C-Na and ORX-Na rats did not present differences compared with C and ORX rats, respectively, without any treatment in all the experiments described in this manuscript. For practical reasons, we decided to report only C and ORX. In the same way, SH rats did not present significant differences with C rats in the evaluated parameters. Moreover, we decided to report only C. The Al dose, frequency, and route of administration used were described previously. Al-treated rats received an intraperitoneal injection of sterile solution of aluminum lactate (Merck, White House Station, New Jersey, USA) containing 2.7 g/l of Al and 26.7
Downloaded from tih.sagepub.com at SETON HALL UNIV on April 2, 2015
Contini et al.
3
g/l of anion lactate, pH 7, during 12 weeks (0.575 mg Al/100 g of b.w., three times per week). Aluminum lactate was dissolved in deionized water and the solution was filtered through a 0.2-m microfilter (Nalgene, Rochester, New York, USA). Al concentration was measured in the solution (Ballanti et al., 1989; Mahieu et al., 2003).This treatment avoided the death of the animals and produced high Al concentrations in serum, intestine, liver, kidney, and bone. C-Na and ORX-Na were injected with a sterile solution of sodium lactate (Merck) containing 26.7 g/l of anion lactate, pH 7 (5.68 mg of lactate/100 g of b.w., i.p.) during the same period and frequency. The rats were housed under conditions of constant temperature (22–24 C) and humidity (45–50%) in room with a fixed 12-h artificial light/12-h artificial dark cycle. Animals were allowed free access to tap water and a standard diet. Al exposure from food and tap water resulted negligible with respect to the administered Al dose (tap water: 1.6 mol/l; food: 30. 5 mol/g wet weight). The experimental protocol was approved by the Human and Animal Research Committee of the School of Biochemistry, University of Litoral, Santa Fe, Argentina. (Res. C.S. 388/06. PI 20-124).
Biochemical studies in urine After 11 weeks of treatment, the rats (n ¼ 6, each group) were housed in metabolic cages to determine urine volume and food and water intake. Animals received demineralized water and standard diet. After allowing to acclimatize for 1 day, daily urine volume was measured during the last 2 days. Creatinine, protein excretion, and osmolality were analyzed in urine collected during the last 24 h.
Biochemical studies in blood and renal tissues After 12 weeks of treatment, the rats (n ¼ 6, each group) were weighed and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg b.w., i.p.). Blood samples were collected by cardiac puncture. Both kidneys were removed, decapsulated, and dissected. One part was processed for histology, and the rest were frozen and stored in liquid nitrogen. These tissues were used to measure the oxidative state of renal tissue, -glutamyl transferase (GGT) activity in renal plasma membrane and Al content. Then, the seminal vesicles and the prostatic gland were isolated and weighed. Testosterone, creatinine, and Al concentrations were measured in serum
samples. Creatinine clearance was obtained by conventional formulae for each animal. Different sets of renal cortex samples were homogenized with a Polytron homogenizer (Kinematica Inc, USA) in different buffers as follows: (1) buffered sucrose medium (0.25 M sucrose, 10 mM Tris-hydrochloric acid (HCl), pH 7.0) for isolation of renal plasma membrane for GGT activity measurement; (2) buffered sucrose medium (0.25 M sucrose, 10 mM Tris-HCl, pH 7.0) for superoxide dismutase (SOD) activity measurement; (3) 50 mM phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid, for glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT) activity measurements; (4) cold 1, 15% potassium chloride for thiobarbituric acid reactive substances (TBARS) measurement; and (5) a mixture of acetonitrile/water (62.5:37.5 v/v) for reduced GSH and oxidized glutathione (GSSG) measurement. Measurement of oxidative stress markers. SOD activity was assayed according to the method of Misra and Fridovich (1972). GPx activity was determined according to the method of Lawrence and Burk (1976). GR activity was assayed according to the method described by Goldberg and Sparner (1987) after modification and CAT activity was determined following the method of Aebi (1985). TBARS was assayed according to the method of Ohkawa et al. (1979). Determination of renal GSH and GSSG was carried out in homogenates with a mixture of acetonitrile/ water (62.5:37.5 v/v). The homogenates were centrifuged at 18,000 r/min for 10 min. The supernatant was measured by capillary electrophoresis (CE) immediately (Maeso et al., 2005). The separation was performed on a CE P/ACE 5010 (Beckman) with ultraviolet detection at 200 + 10 nm. It was equipped with an uncoated capillary with 40-cm effective length and 50-m ID and using 0.200 M borate as buffer, pH was made up to 8.0 with sodium hydroxide. Protein concentration in renal homogenates was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. GGT activity in whole plasma membrane was measured kinetically at 405 nm employing a commercial kit (Wiener Lab., Rosario, Argentina) using -glutamyl p-nitroanilide as substrate. Histopathological examination. Kidney tissues were fixed in 10% buffered formalin (pH 7.2) and dehydrated
Downloaded from tih.sagepub.com at SETON HALL UNIV on April 2, 2015
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Toxicology and Industrial Health
through a series of ethanol solutions, embedded in paraffin, and routinely processed for histological analysis. Sections of 5 m thickness were cut and stained with hematoxylin–eosin (for general morphological examination), periodic acid-Schiff (PAS; for assessment of basement membrane changes and brush border tubules), and Masson’s trichome stain (for demonstration of collagen deposition). All specimens were examined by two independent observers who were blinded with respect to the identity of the tissue under scrutiny. Stained sections were examined using an Olympus light microscope (400). One hundred glomeruli and 300 tubules were observed in each nonoverlapping fields of each group. According to the score established by Ji et al. (2005), the degree of glomerular sclerosis (GS) was graded on a scale of 0–4: 0 (normal glomeruli); 1sclerotic area up to 1–25% (minimal sclerosis); 2 sclerotic area 26–50% (moderate sclerosis); 3 sclerotic area 51–75% (moderate–severe sclerosis); and 4 sclerotic area 76–100% (severe sclerosis). The glomerular sclerotic index (GSI) was calculated using the following formula: GSI ¼ ð1 n1 Þ þ ð2 n2 Þ þ ð3 n3 Þ þ ð4 n4 Þ= n0 þ n1 þ n2 þ n3 þ n4 , where nx is the number of glomeruli in each grade of GS. The degree of tubulointerstitial injury was evaluated by a semiquantitative scoring system and was graded on a scale of þ to þþþþ (–: normal; þ: affected parameters
