diphenyl diselenide protects against glycerol-induced renal damage in rats

Copyright © 2009 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29: 612–618

Research Article

Received: 3 February 2009, Revised: 16 April 2009, Accepted: 22 April 2009, Published online in Wiley Interscience: 29 May 2009

(www.interscience.wiley.com) DOI 10.1002/jat.1449

Diphenyl diselenide protects against glycerol-induced renal damage in ratsRicardo Brandão, Carmine I. Acker, Marlon R. Leite, Nilda B. V. Barbosa and Cristina W. Nogueira*

ABSTRACT: In this study we evaluated the eff ect of diphenyl diselenide (PhSe)2 on glycerol-induced acute renal failure in rats. Rats were pre-treated by gavage every day with (PhSe)2 (7.14 mg kg-1) for 7 days. On the eighth day, rats received an intra-muscular injection of glycerol (8 mL kg-1). Twenty-four hours afterwards, rats were euthanized and the levels of urea and creatinine were measured in plasma. Catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), d-aminolevulinate dehydratase (d-ALA-D) and Na+, K+-ATPase activities and ascorbic acid levels were evaluated in renal homog-enates. Histopathological evaluations were also performed. The results demonstrated that (PhSe)2 was able to protect against the increase in urea and creatinine levels and histological alterations in kidney induced by glycerol. (PhSe)2 protected against the inhibition in d-ALA-D, CAT and GPx activities and the reduction in ascorbic acid levels induced by glycerol in kidneys of rats. In conclusion, the present results indicate that (PhSe)2 was eff ective in protecting against acute renal failure induced by glycerol. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: glycerol; selenium; acute renal failure; oxidative stress; sulfhydryl enzyme

* Correspondence to: C. W. Nogueira, Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: [email protected]

Introduction

Glycerol is a well-known material for the induction of acute renal failure (ARF) in vivo. ARF is a syndrome characterized by an acute loss of renal function. Despite the reversibility of the loss of renal function in most patients who survive, the mortality rate of ARF remains high (over 50%) (Liano and Pascual, 1996). ARF can be associated with ischemic or hypoxic injury or exposure to neph-rotoxic substances. The pathogenesis of glycerol-induced acute renal failure can involve decreased renal blood fl ow as well as myoglobin release from damaged muscle and increased reactive oxygen species (ROS) (Zager et al., 1995; Chander et al., 2003). ROS, including the superoxide anion, hydroxyl radical, single oxygen and hydrogen peroxide, are oxygen-containing mole-cules with unpaired electrons or abstract electrons from other molecules. These ROS can lead to functional damage in lipid, proteins and DNA, which can eventually result in cell death (Halliwell and Gutteridge, 1990).

The exposure to oxidant agents can cause inhibition of sulfhy-dryl enzymes such as d-aminolevulinate dehydratase (d-ALA-D) (Emanuelli et al., 1996) and Na+, K+-ATPase (Carfagna et al., 1996). d-ALA-D enzyme is essential for all aerobic organisms and takes part in the route of formation of tetrapyrrolic molecules (heme and chlorophyll). The main importance of these compounds is their function as prosthetic groups of proteins, such as hemoglo-bin, myoglobin, cytrochromes, catalase and peroxidase (Jaff e, 1995). Na+, K+-ATPase plays a central role in whole body osmo-regulation (Alam and Frankel, 2006). It is an enzyme embedded in the cell membrane and responsible for the active transport of sodium and potassium ions (Doucet, 1988).

The oxidative eff ects of ROS are controlled by non-enzymatic antioxidant defenses, such as ascorbic acid (Perottoni et al., 2004) and glutathione (GSH) (Zalups, 2000), and also by enzymatic anti-oxidant defenses, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Benov et al., 1990).

Selenium is a structural component of several enzymes with physiological antioxidant properties, including glutathione per-oxidase and thioredoxin reductase (Nogueira et al., 2004). Diphenyl diselenide, a simple organoselenium compound, has been reported in view of its important pharmacological proper-ties (Nogueira et al., 2003, 2004; Borges et al., 2005, 2006; Barbosa et al., 2006; Savegnago et al., 2006, 2007) among them its antioxi-dant activity (Rossato et al., 2002; Meotti et al., 2004). Although we have reported in previous papers the benefi cial eff ects of (PheSe)2 by reducing oxidative stress in diff erent organs includ-ing the kidney (Borges et al., 2006; Brandão et al., 2006), the eff ect of this potential pharmacological agent was never evaluated in an ARF model. Based on these reports, the present study was designed to determine the possible protective eff ect of (PhSe)2 on ARF induced by glycerol in rats.

Materials and Methods

Chemicals

Glycerol, d-aminolevulinic acid (d-ALA), 1-chloro-2,4-dinitroben-zene (CDNB) and p-dimethylaminobenzaldehyde were pur-chased from Sigma (St Louis, MO, USA). Diphenyl diselenide (PhSe)2 was synthesized according to Paulmier (1986). Analysis of the 1H NMR and 13C NMR spectra showed analytical and spectro-scopic data in full agreement with its assigned structure. The chemical purity of (PhSe)2 (99.9%) was determined by GC/HPLC. All other chemicals were of analytical grade and obtained from standard commercial suppliers.

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Diphenyl diselenide protects against glycerol-induced renal damage

J. Appl. Toxicol. 2009; 29: 612–618 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jat

Animals

Male adult Wistar rats (200–250 g) from our own breeding colony were used. The animals were kept in a separate animal room, on a 12 h light/dark cycle, at a room temperature of 22 ± 2 °C and with free access to food and water. The animals were used accord-ing to the guidelines of the Committee on Care and Use of Experimental Animal Resources, the Federal University of Santa Maria, Brazil.

Experimental Procedure

A group of six rats was tested in each experimental group. Rats were orally administered by gavage every day with (PhSe)2 (7.14 mg kg−1, dissolved in canola oil) or vehicle (canola oil) for 7 days. On the eighth day, the rats received an intramuscular injec-tion of glycerol (8 mL kg−1, dissolved in saline at 50%) or vehicle as a divided dose into the hind limbs (Yehuda, 1993). The dose of (PhSe)2, which did not induce toxicity in rodents, was chosen based on previous study (Savegnago et al., 2007).

The protocol of rat treatment is given below:

• group 1 – canola oil + saline;• group 2 – (PhSe)2 + saline;• group 3 – canola oil + glycerol;• group 4 – (PhSe)2 + glycerol.

Twenty-four hours after glycerol exposure, the blood samples were collected directly from the ventricle of the heart in anesthe-tized animals. Subsequently, rats were euthanized by decapita-tion, left kidney was removed and rapidly homogenized in 50 mM Tris–HCl, pH 7.4 (1 : 10, w/v) and centrifuged at 2500 g for 10 min. The low-speed supernatant (S1) was separated and used for bio-chemical assays.

Renal Markers of Damage

Renal function was analyzed using a commercial Kit (Labtest, Diagnostica S.A., Minas Gerais, Brazil) by spectophotometrically assay of urea (Mackay and Mackay, 1927) and creatinine (Jaff e, 1886) levels in plasma.

Catalase (CAT) Activity

Renal CAT activity was determined in S1 by the decomposition of H2O2 at 240 nm according to Aebi (1984). The enzymatic activity was expressed in units mg−1 protein (1 unit decomposes 1 µmol of H2O2 per min at pH 7 at 25 °C).

Glutathione Peroxidase Activity

Renal GPx activity was assayed spectrophotometrically by the method of Wendel (1981), through the glutathione (GSH)–NADPH–glutathione reductase system, by the dismutation of H2O2 at 340 nm. S1 was added to the GSH–NADPH–glutathione reductase system and the enzymatic reaction was initiated by adding H2O2. In this assay, the enzyme activity is indirectly mea-sured by means of NADPH decay. H2O2 is decomposed, generat-ing GSSG from GSH. GSSG is regenerated back to GSH by glutathione reductase present in the assay media at the expenses of NADPH. The enzymatic activity was expressed as nmol NADPH mg protein−1 min−1.

Glutathione S-transferase Activity

Renal GST activity was assayed in S1 through the conjugation of GSH with CDNB at 340 nm as described by Habig et al. (1974). The results are expressed as µmol mg protein−1 min−1.

Ascorbic Acid Levels

Renal ascorbic acid determination was performed as described by Jacques-Silva et al. (2001). S1 was precipitated in 10 vols of a cold 5% trichloroacetic acid solution. An aliquot of S1 sample (0.3 mL), in a fi nal volume of 1 mL of the solution, was incubated at 37 °C for 3 h, then 0.5 mL H2SO4 65% (v/v) was added to the medium. The reaction product was determined using color reagent containing 4.5 mg mL−1 dinitrophenyl hydrazine and CuSO4 (0.075 mg mL−1). The absorbance was read at 520 nm and the data expressed as µg ascorbic acid (AA) g−1 tissue.

δ-Aminolevulinate Dehydratase Activity

Renal d-ALA-D activity was assayed according to Sassa (1982) by measuring the rate of product (porphobilinogen) formation except that 100 mM potassium phosphate buff er, pH 6.8 and 2.4 mM of d-aminolevulinic acid (d-ALA) were used. Incubations of S1 were carried out for 60 min at 37 °C. The reaction product was determined using modifi ed Ehrlich’s reagent at 555 nm, with a molar absorption coeffi cient of 6.1 × 104 per M−1 cm−1 for the Ehrlichporphobilinogen salt. The results are expressed as nmol porphobilinogen (PBG) mg protein−1 h−1.

Na+, K+-ATPase Activity

The reaction mixture for Na+, K+-ATPase activity assay contained 6 mM MgCl2, 100 mM NaCl, 20 mM KCl, 1 mM EDTA and 40 mM Tris–HCl, pH 7.4, in a fi nal volume of 500 µL and an aliquot of S1. The reaction was initiated by the addition of ATP to a fi nal con-centration of 3.0 mM. Controls were carried out under the same conditions with the addition of 1 mM ouabain. Na+, K+-ATPase activity was calculated by the diff erence between the two assays. Released inorganic phosphate (Pi) was measured by the method of Fiske and Subbarow (1925) at 650 nm. The results are expressed as nmol PI mg protein−1 min−1.

Histopathology

Kidneys were fi xed in 10% formalin. For light microscopy exami-nation, tissues were embedded in paraffi n, sectioned at 4 µm and stained with hematoxylin and eosin (Nath et al., 2000). Rats from all groups were examined by histopathology of the param-eters: cellular vacuolization, loss of cellular architecture in the renal tubules and vascular congestion (n = 3 per each group).

Protein Determination

Protein was measured in S1 according to Bradford (1976), using bovine serum albumin as standard.

Statistical Analysis

Data are expressed as means ± SD. Statistical analysis was per-formed to compare treated groups with respective control groups using a two-way analysis of variance (ANOVA), followed

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by the Duncan’s multiple range test when appropriate. Main eff ects are presented only when the higher second-order interac-tion was non-signifi cant. Values of P < 0.05 were considered sta-tistically signifi cant.

Results

Renal Markers of Damage

Two-way ANOVA of plasmatic urea data revealed a signifi cant glycerol × (PhSe)2 interaction (P < 0.012). Post-hoc comparisons demonstrated that urea levels were increased (~2.3 times) in rats exposed to glycerol when compared with the control group. Pre-treatment with (PhSe)2 was eff ective in protecting against the increase in urea levels in rats exposed to glycerol (Table 1).

A signifi cant interaction between glycerol and (PhSe)2 in cre-atinine levels was observed (P < 0.001). Post-hoc comparisons demonstrated that creatinine levels were increased (~3.5 times) in rats exposed to glycerol when compared with the control group. (PhSe)2 was able in protecting against this increase (Table 1).

CAT Activity

A signifi cant main eff ect of glycerol (P < 0.001) and (PhSe)2 (P < 0.001) in renal CAT activity was observed. Post-hoc comparisons demonstrated that rats exposed to glycerol presented a reduc-tion (~28%) in CAT activity when compared with the control group. Rats treated with (PhSe)2 presented an increase (~22%) in CAT activity when compared with the control group. (PhSe)2 pre-treatment protected against the reduction in CAT activity induced by glycerol in kidney of rats [Fig. 1(A)].

GPx Activity

Two-way ANOVA of renal GPx activity data yielded a signifi cant glycerol × (PhSe)2 interaction (P < 0.016). Post-hoc comparisons demonstrated that GPx activity was inhibited (~54%) in rats exposed to glycerol when compared with the control group. (PhSe)2 was partially eff ective in protecting against the inhibition of GPx activity induced by glycerol in kidney of rats [Fig. 1(B)].

GST Activity

A signifi cant main eff ect of (PhSe)2 (P < 0.001) in renal GST activity was found. Post-hoc comparisons revealed that rats exposed to

(PhSe)2 presented an increase (~64%) in GST activity when com-pared to the control group. Glycerol exposure did not modify GST activity increased by (PhSe)2 administration [Fig. 1(c)].

Ascorbic Acid Levels

Two-way ANOVA of renal ascorbic acid data yielded a signifi cant glycerol × (PhSe)2 interaction (P < 0.002). Post-hoc comparisons showed that ascorbic acid levels were reduced (~34%) in rats exposed to glycerol when compared to the control group. Rats treated with (PhSe)2 presented an increase (~27%) in ascorbic acid levels when compared with the control group. Pre-treatment with (PhSe)2 protected against the reduction in ascorbic acid levels induced by glycerol in kidney of rats (Fig. 2).

δ-ALA-D Activity

A signifi cant main eff ect of glycerol (P < 0.001) and (PhSe)2 (P < 0.001) in renal d-ALA-D activity was observed. Post-hoc compari-sons demonstrated that d-ALA-D activity was inhibited (~3.1 times) in rats exposed to glycerol when compared with the control group. (PhSe)2 was partially eff ective in protecting against the inhibition of d-ALA-D activity induced by glycerol in kidney of rats (Table 2).

Na+, K+-ATPase Activity

Na+, K+-ATPase activity remained unaltered in rats exposed to glycerol or (PhSe)2 (Table 2).

Histopathology

Histological evaluation revealed a normal aspect of kidney struc-tures in control and (PhSe)2 groups [Fig. 3(A–D)]. Rats exposed to glycerol presented intense loss of cellular architecture [Fig. 3(E, F)]. Rats that received glycerol presented severe proximal and distal tubular damage and marked vascular congestion between tubules. In addition, glycerol-exposed rats presented cellular vacuolization [Fig. 3(E, F)].

Evaluation of kidneys from rats exposed to glycerol and (PhSe)2 showed tubules with histological characteristics more preserved than those from the glycerol group. Renal tubules displayed moderate damage [Fig. 3(G, H)].

Table 1. Eff ect of (PhSe)2 on plasmatic urea and creatinine levels of rats exposed to glycerol

Urea (mg dl−1)

Creatinine (mg dl−1)

Control 47.7 ± 6.9 0.37 ± 0.14(PhSe)2 36.0 ± 6.2 0.35 ± 0.06Glicerol 110.2 ± 56.3* 1.28 ± 0.63*(PhSe)2 + Glicerol 44.2 ± 8.9# 0.43 ± 0.14#

Data are reported as means ± SD from six rats in each group. *P < 0.05 as compared with the control group (two-way ANOVA/Duncan). #P < 0.05 as compared with the glycerol group (two-way ANOVA/Duncan).

Table 2. Eff ect of (PhSe)2 on renal d-ALA-D and Na+, K+-ATPase activities of rats exposed to glycerol

d-ALA-D activity (nmol PBG mg protein−1 h−1)

Na+, K+-ATPase activity (nmol PI mg

protein−1 min−1)

Control 7.29 ± 1.19 13.48 ± 5.43(PhSe)2 7.89 ± 1.31 10.48 ± 3.44Glicerol 2.35 ± 0.80*,# 12.66 ± 6.51(PhSe)2 + Glicerol 4.24 ± 1.05 *,# 13.11 ± 3.70

Data are reported as means ± SD from six rats in each group. *P < 0.05 as compared with the control group (two-way ANOVA/Duncan). #P < 0.05 as compared with the glycerol group (two-way ANOVA/Duncan).

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Figure 1. Eff ect of (PhSe)2 on CAT (A), GPx (B) and GST (C) activities in kidneys of rats exposed to glycerol. Data are reported as means ± SD of six animals per group. *P < 0.05 as compared with the control group (two-way ANOVA/Duncan). #P < 0.05 as compared with the glycerol group (two-way ANOVA/Duncan).

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Figure 2. Eff ects of (PhSe)2 on renal ascorbic acid levels of rats exposed to glycerol. Data are reported as means ± SD of six animals per group. *P < 0.05 as compared with the control group (two-way ANOVA/Duncan). #P < 0.05 as compared with the glycerol group (two-way ANOVA/Duncan).

A

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Figure 3. Microscopic analysis of rat kidney structures showing details in the left side. (A, B) Control group shows rats with normal kidney structures, with normal aspect of the glomerulus and proximal and distal tubules; (C, D) (PhSe)2 group shows rats with normal aspect of kidneys similar to the control group; (E, F) glycerol group shows kidneys with intense proximal and distal tubular damage (loss of cellular architecture) and marked vascular congestion between tubules; (G, H) glycerol and (PhSe)2 group shows tubules with histological structures more preserved than those in the glycerol group, with moderate proximal and distal tubular damage. Glomerulus (G), proximal tubule (white arrow), distal tubules (black arrow), vascular conges-tion (*) (Figures A, C, E and G: H&E, 100×; Figures B, D, F and H: H&E, 400×).

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DiscussionIn the present study the protective eff ect of (PhSe)2 in an ARF model induced by glycerol in rats is reported. Studies have dem-onstrated that glycerol is a good inductor of renal damage, causing ARF (Chander et al., 2003; Soares et al., 2007). It is known that intramuscular injection of glycerol causes muscle damage and myoglobin release. Iron released from heme pigment, myo-globin, can promote ROS production, lipid peroxidation and acute tubular necrosis (Singh et al., 2003; Chander et al., 2003). In this way, antioxidant molecules are usually tested against renal damage induced by glycerol (Chander and Chopra, 2006).

(PhSe)2 has been reported because of its ability to protect against toxicity caused by various chemicals. The mechanisms by which (PhSe)2 acts have been primarily attributed to the reduc-tion of oxidative stress induced by chemicals (Santos et al., 2005; Borges et al., 2006). Accordingly, renal damage induced by a model of hyperglycemia in rats was ameliorated by (PhSe)2 administration and this eff ect was attributed, at least in part, to its antioxidant action (Barbosa et al., 2006).

In the current study, the renal damage induced by glycerol was evidenced by an increase in urea and creatinine levels, which are renal markers of damage. Marked alterations in histological anal-ysis from rats exposed to glycerol further confi rmed the renal damage. These fi ndings were similar to those of previous studies performed using a similar model (Aydogdu et al., 2004; Singh et al., 2004). The pre-treatment with (PhSe)2 was able to protect against the increase in the levels of urea and creatinine as well as the development of histological alterations, demonstrating the protective eff ect of (PhSe)2 against the renal damage induced by glycerol.

The results reported in this paper show that glycerol exposure alters oxidative–antioxidative balance in rat kidneys. In accor-dance, the oxidative damage induced by glycerol has been reported in the literature (Aydogdu et al., 2006). CAT, GPx and ascorbic acid have been reported to have antioxidant functions (Benov et al., 1990; Fang et al., 2002). In this study, an inhibition of CAT and GPx activities with a reduction in the levels of ascorbic acid in the rat kidneys exposed to glycerol was demonstrated. However, (PhSe)2 protected against alterations of CAT, GPx and ascorbic acid induced by glycerol in rat kidneys, suggesting that this protective eff ect may involve its antioxidant action.

Interestingly, CAT and GST activities and ascorbic acid levels were increased in kidneys of rats treated with (PhSe)2 alone. The increase in GST activity could be attributed to the fact that (PhSe)2 exposure causes an increase in NPSH (mainly GSH) levels in diff erent tissues (De Bem et al., 2007; Luchese et al., 2007) including kidneys (Barbosa et al., 2006). GSH is a substrate to GST action and is required to recycle ascorbic acid from dehydro-ascorbic acid (oxidized form of ascorbic acid; Li et al., 2001). Therefore, the possible increase in GSH levels induced by (PhSe)2 can be attributed to the increase in GST activity and ascorbic acid levels observed in rats treated with (PhSe)2. Since CAT (Benov et al., 1990), GST (Casalino et al., 2004) and ascorbic acid (Fang et al., 2002) are components of the antioxidant defense system of the cells, the increase caused by (PhSe)2 in these parameters could be one of the mechanisms by which (PhSe)2 acts as a protective agent in kidneys of rats.

The inhibition of d-ALA-D activity further supports the idea that glycerol has pro-oxidative eff ect in kidneys of rats. d-ALA-D is a sulfhydryl-containing enzyme that is very sensitive to situa-tions in which oxidative stress plays a role (Rocha et al., 1995;

Santos et al., 2005; Farina et al., 2003). In ischemic kidney Na+, K+-ATPase activity is markedly depressed as reported by Matsuzaki et al. (2007). In contrast, in this study Na+, K+-ATPase activity was not altered in kidney of rats exposed to glycerol. These diff erent results lead us to suppose that the mechanisms involved in ARF induced by glycerol are diff erent from those implicated in isch-emia/reperfusion. On one hand the results on sulfhydryl contain-ing enzymes may indicate that d-ALA-D is more sensitive to glycerol than Na+, K+-ATPase in kidney of rats. Accordingly, d-ALA-D has been reported to be more sensitive to oxidative stress than Na+, K+-ATPase in the brain of rats (Prigol et al., 2007). On the other hand, one of the mechanisms by which glycerol inhibits d-ALA-D activity could be related to Zn2+ displacement. Mammalian d-ALA-D is a metalloenzyme that requires Zn2+ for maximal catalytic activity (Jaff e, 2000).

In conclusion, this study demonstrated that the pre-treatment with (PhSe)2 was eff ective in protecting against ARF induced by glycerol in rats. The antioxidant action of (PhSe)2 seems to be related to the protective eff ect of this compound on ARF induced by glycerol. These fi ndings are of great importance, since ARF is a very common disease and new therapies are needed to prevent this syndrome.

Acknowledgements

The fi nancial support by FAPERGS, CAPES and CNPq is gratefully acknowledged.

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