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Cilostazol attenuates gentamicin-induced nephrotoxicity in rats
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Cilostazol attenuates gentamicin-induced nephrotoxicity in rats Ahmed A. Abdelsameeaa , Ahmed M. Mohameda , Mona G. Amerb,* , Shahera M. Attiac a
Clinical Pharmacology Department, Faculty of Medicine, Zagazig University, Egypt Histology & Cell Biology department, Faculty of Medicine, Zagazig University, Egypt c Medical Biochemistry, Faculty of Medicine, Zagazig University, Egypt b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2015 Received in revised form 26 December 2015 Accepted 7 January 2016
Introduction: Gentamycin is a widely used antibiotic. The nephrotoxic adverse effects of the drug may limit its use. Cilostazol, a phosphodiesterase III inhibitor, was reported to protect from renal oxidative stress. This work aimed to investigate the possible protective effect of cilostazol on gentamicin-induced nephrotoxicity and the possible underlying mechanisms. Materials and methods: 40 male albino rats were divided into 4 equal groups: (1) Control; (2) Cilostazol, 10 mg/kg, p.o.; (3) Gentamicin, 80 mg/kg, i.p.; (4) Gentamicin 80 mg/kg, i.p. along with cilostazol 10 mg/ kg, p.o. All drugs were administered once daily for 8days. On 9th day blood samples were collected for the estimation of creatinine, urea and uric acid in serum. Then the rats were sacrificed and kidneys were removed for light and electron microscope studies. Moreover, reduced glutathione (GSH) and malondialdehyde (MDA) levels as well as catalase (CAT) and superoxide dismutase (SOD) activities were determined in renal tissues. Results: Gentamicin elevated the serum levels of creatinine, urea and uric acid as well as the MDA level in the renal tissue, while it decreased CAT, SOD activities and GSH levels as well as produced degenerative changes in glomeruli and tubules associated with increased expression of apoptotic markers and decreased expression of anti-apoptotic markers. Administration of cilostazol decreased urea, creatinine, uric acid and MDA levels while increased CAT and SOD activities and GSH levels as well as ameliorated the histopathological changes in relation to gentamicin group. Conclusion: Cilostazol protected rats from gentamicin-induced nephrotoxicity possibly, in part through its antioxidant and anti-apoptotic activity. ã 2016 Elsevier GmbH. All rights reserved.
Keywords: Gentamicin Nephrotoxicity Cilostazol Ultrastructure Apoptosis
1. Introduction Gentamicin is a widely used aminoglycoside antibiotic either alone or in combination with a cell wall-active drug in management of severe and life-threatening infections caused by Grampositive and Gram-negative aerobes (Choi et al., 2011). The ototoxic and nephrotoxic adverse effects of the drug may limit its use. In some situations, these adverse effects are so severe that the treatment must be discontinued (Ali, 2011). The pathogenesis of gentamicin nephrotoxicity involves multiple pathways, including oxidative stress, inflammation, reduced renal blood flow, and increased nitric oxide (NO) level (Balakumar
Abbreviations: CAT, catalase; DCT, distal convoluted tubule; GSH, Glutathione; H&E, hematoxylin and eosin; MDA, malondialdehyde; PCT, proximal convoluted tubules; SOD, superoxide dismutase. * Corresponding author at: Histology and Cell Biology, Faculty of Medicine, Zagazig University, Zagazig, Egypt. E-mail address: [email protected] (M.G. Amer).
et al., 2010; Christo et al., 2011). Several agents have been used, with various degrees of success, to ameliorate or prevent gentamicin nephrotoxicity (Otunctemur et al., 2013; Rodrigues et al., 2014). Cilostazol, a selective phosphodiesterase III inhibitor, has potent antiplatelet and vasodilator effects. The drug is approved for treatment of intermittent claudication in patients with peripheral vascular diseases (Chen et al., 2015). Cilostazol was generally well tolerated. Adverse events reported are headache, palpitation and tachycardia with mild to moderate intensity and rarely required treatment withdrawal (Kim et al., 2015). Several investigation in different cells and tissues have indicated to inhibitory effect of cilostazol on reactive oxygen species and superoxide generation as well as hydroxyl radicals scavenging action (Kim et al., 2002; Lee et al., 2010). The aim of the present study is to assess the possible protective effect of cilostazol on gentamicin-induced nephrotoxicity and the possible underlying mechanisms.
http://dx.doi.org/10.1016/j.etp.2016.01.002 0940-2993/ ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: A.A. Abdelsameea, et al., Cilostazol attenuates gentamicin-induced nephrotoxicity in rats, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.01.002
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2. Materials and methods 2.1. Materials Gentamicin sulfate (Memphis Pharm., & Chemical Ind., Cairo, Egypt); Cilostazol powder, (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan).
samples were homogenized in ice-cold 50 mM potassium phosphate buffer (pH 7.5), centrifuged at 4 C 12,000 g for 15 min and then the supernatant was collected. MDA in the supernatant can react with freshly prepared thiobarbituric acid (TBA) to form a colored complex which has maximum absorbance at 535 nm (Buege and Aust, 1978). The nmol MDA/g wet tissue was calculated from the plotted standard curve prepared from 1,1,3,3tetraethoxypropane.
2.2. Animals Male albino rats weighing 150–180 gm were used after one week for proper acclimatization to the standard housing conditions (25 2 C temperature and 12 h light/dark cycle) and were supplied with standard rodent chow and tap water ad libitum. All experimental protocols were approved by the Ethics Committee of Zagazig University. 2.3. Experimental design 40 male albino rats were randomly divided into 4 equal groups: Group (1) non-treated control; Group (2) was treated with cilostazol 10 mg/kg, p.o. dissolved in saline (Gokce et al., 2012) and served as cilostazol-treated control. Group (3) was injected with gentamicin 80 mg/kg, i.p. (Reddy et al., 2011) for induction of experimental nephrotoxicity; Group (4) was given cilostazol 10 mg/kg, p.o. 1 h before gentamicin 80 mg/kg, i.p. All drugs were administered daily for 8 consecutive days. On the 9th day blood samples were collected, and serum was separated by centrifugation at 3000 g for 10 min for estimation of creatinine, uric acid and urea levels. A longitudinal section from the left kidney was excised from each animal for histological examination. The renal cortex of the rest of the kidneys was stored at 80 C and subsequently homogenized in ice-cold phosphate buffer (0.05 M, pH 7.4) for biochemical analysis. 2.3.1. Biochemical analysis 2.3.1.1. Serum analysis. Creatinine, uric acid and urea levels were estimated using kits supplied by spinreact (Girona, Spain). 2.3.1.2. Estimation of the reduced glutathione (GSH). The GSH level in the kidney was estimated using the method described by Ellman (1959). Briefly, the renal homogenate was mixed with 10% w/v trichloroacetic acid in ratio of 1:1 and centrifuged at 40 C for 10 min at 5000 rpm. The supernatant obtained (0.5 ml) was mixed with 2 ml of 0.3 M disodium hydrogen phosphate buffer (pH 8.4) and 0.4 ml of distilled water. Then 0.25 ml of 0.001 M freshly prepared DTNB (5,51-dithiobis (2-nitrobenzoic acid)) dissolved in 1% w/v sodium citrate) was added. The reaction mixture was incubated for 10 min and absorbence of yellow colored complex was noted spectrophotometrically at 412 nm. A standard curve was plotted using reduced form of glutathione. 2.3.1.3. Determination of lipid peroxidation. Lipid peroxide was estimated by measurement of malondialdehyde (MDA) levels spectrophotometrically in kidney homogenate whereas kidney
2.3.1.4. Determination of catalase (CAT). It was determined using colorimetric assay kits supplied from Biodiagnostic Company for diagnostic reagents: Dokki, Giza, Egypt. The resulting quinoneimine dye is measured at 520 nm (Aebi, 1984). 2.3.1.5. Determination of superoxide dismutase activity (SOD). Determination of total (Cu, Zn and Mn) SOD activity was determined kinetically (Sun et al., 1988) using kits supplied from Biodiagnostic Company for diagnostic reagents: Dokki, Giza, Egypt. 2.3.2. Histopathological studies At the time of sacrifice, the animals were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg. The kidneys were rapidly removed and opened to be processed. For light microscopy, specimens were fixed in 10% formalin solution and processed to prepare 5 mm thick paraffin sections and stained with hematoxylin and eosin (H&E) (Bancroft and Gamble, 2002). For immunohistochemical study, the deparaffinized sections on charged slides were used for detection of Bax (apoptosis inducers) and BCL2 (antiapoptotic marker). Immunohistochemical reaction was carried using avidin biotin peroxidase system. The primary antibody used was a rabbit polyclonal antibody (Sigma Laboratories, Saint Louis, USA). The primary antibody used for Bax and BCL2 were rabbit polyclonal antibody, sigma laboratories (Cat No 6¼ anti Bax: B3428, anti BCL2 sc-492). Universal kit used avidin biotin peroxidase system produced by NovaCastra Laboratories Ltd., UK. The same method was applied to prepare negative control sections but the primary antibody was not added. Mayer’s hematoxilin was added as counter stain. The cells that displayed brown precipitation were considered positive for Bax and BCL2 expression (Albamonte et al., 2013). Specimens for electron microscope examination were cut into 1 mm3 pieces and fixed in 2.5% glutraldehyde buffered with 0.1 mol/L phosphate buffer at pH 7.4 for 2 h. They were post fixed in 1% osmium tetroxide for 1 h, dehydrated through graded alcohol series, and embedded in epoxy resin. Ultrathin sections (50 nm thick) were collected on copper grids and stained with uranyl acetate and lead citrate (Glauret and Lewis, 1998). 2.3.3. Quantitative morphometric measurements Area percentage of the positive Bax & Bcl2 immune reactive cells lining the renal tubules was estimated by using “Leica Quin 500C” image analyzer computer system (Leica Imaging System Ltd., Cambridge, England). The measuring frame of a standard area
Table 1 Effect of cilostazol, gentamicin alone and in combination on serum urea, creatinine and uric acid in albino rats (n = 10). Groups
Control
Cilostazol
Gentamicin
Gentamicin + cilostazol
Urea (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl)
35 2.33 0.75 0.06 1.22 0.21
38 2.47 0.81 0.09 1.36 0.19
109 11.77* 2.15 0.13* 5.83 0.33*
66.83 5.73# 1.54 0.21# 2.98 0.23#
Data are presented as means SEM; n = number of rats in each group. Statistical analysis was done using one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. *, P < 0.05 versus control and cilostazol groups; #,P < 0.05 versus control, cilostazol, and gentamicin groups.
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was equal to 7286, 78 mm2. Measurements were done within 10 non overlapping fields for each section at 400 magnification.
3
3.2. Immunohistochemical results To determine the mechanism underlying the anti-apoptotic effects of cilostazol; Bax (a marker protein of apoptosis) and BCL2 (anti-apoptotic protein) were detected using immunohistochemistry. The results revealed strong positive immunostaining of Bax in most of cells lining renal tubules and glomeruli of gentamicin treated group (Fig. 2b), faint staining in gentamicin–cilostazol treated group (Fig. 2c) and negative staining in control groups (Fig. 2a). Examination of the kidneys of control group detected positive immunostaining for BCL2 in cells lining renal tubules and glomeruli (Fig. 2d), no reaction was detected in kidney of gentamicin-treated group (Fig. 2e) and strong positive reaction was seen in most of the renal tubules and glomeruli of gentamicin– cilostazol treated group (Fig. 2f). Statistical analysis of morphometric results measuring the area% of positive reaction confirmed the previous results (Table 3). Electron microscopic examination of the renal cortex of control groups revealed renal corpuscles with glomerular capillaries lined by fenestrated endothelium. The podocytes with euchromatic nuclei and foot processes were seen (Fig. 3a). Gentamicin caused several visible and distinct changes in the ultrastructure of the kidney of Group (3). Disrupted blood renal barrier appeared with degenerated endothelium and vacuolated podocytes. Also, thickened basement membrane of the barrier and dense mesangial matrix were noticed (Fig. 3b). The cells lining the PCT appeared with euchromatic nuclei and basal enfolding with elongated mitochondria. The apical surface had a well-developed luminal brush border consisting of numerous closely packed microvilli (Fig. 4a). Gentamicin treatment in Group (3) induced ultrastructure alteration in PCT. The brush border showed areas of complete loss and mitochondria appeared swollen. Disorganized basal enfolding and electron dense shrunk nuclei were observed in most of the tubules (Fig. 4b). Most of the PCT from gentamicin– cilostazol treated group showed normal ultrastructure features. Some showed disrupted basal enfolding and swollen mitochondria (Fig. 4c). The cells of the DCT of control groups showed euchromatic nuclei and basal enfolding with elongated mitochondria (Fig. 4d). In gentamicin-treated group, vacuolation of the cytoplasm is observed in most of the tubules with disorganized basal enfolding, and irregular nuclei with condensed chromatin (Fig. 4e). DCT of gentamicin–cilostazol treated group appeared with irregular nuclei and thickened basement membrane (Fig. 4f).
2.4. Statistical analysis Results were expressed as the means + S.E.M. Statistical significant difference was determined by one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparisons. Probability values (P) less than 0.05 were considered to be statistically significant. 3. Results Serum urea, creatinine and uric acid levels: Gentamicin significantly increased (P < 0.05) urea, creatinine and uric acid levels in relation to control groups. The previous parameters were significantly decreased (P < 0.05) by cilostazol–gentamicin administration in relation to gentamicin-treated group (Table 1). Renal GSH, MDA levels as well as CAT, and SOD activities: Gentamicin significantly (P < 0.05) increased MDA level while, decreased GSH level as well as the activity of CAT&SOD in relation to control groups. Cilostazol–gentamicin treatment significantly decreased (P < 0.05) MDA level in comparison with gentamicintreated group. On the other hand, concomitant treatment of cilostazol with gentamicin significantly increased the GSH level as well as CAT&SOD activities in the renal tissue compared with gentamicin-treated group (Table 2). 3.1. Histology of kidney Histological examination revealed that non-treated and cilostazol-treated control groups showed normal features of renal glomeruli and cortical tubules. Figures for control group were representative for both groups. The normal renal corpuscle consisted of a tuft of capillaries, the glomerulus, surrounded by a double-walled epithelial capsule called Bowman’s capsule. Between the two layers of the capsule is the urinary or Bowman’s space. The convoluted tubules lined by cuboidal cells. Profiles of the proximal convoluted tubules (PCT) made up the bulk of the renal cortex with narrow irregular lumen; however, the distal tubules (DCT) exhibited clear lumen (Fig. 1a). In contrast, gentamicin-treated group showed renal corpuscles formed of either hypertrophied or atrophied glomeruli with widening of their Bowman’s spaces. The endothelial cells lining the glomerular tufts showed swelling and intarcytoplasmic vacuolation (Fig. 1b and c). The epithelial cells lining of the renal tubules showed vacuoles and darkly stained pyknotic nuclei. Interstitial infiltration and marked atrophy of the glomeruli with wide Bowman’s space were also seen (Fig. 1c). Concomitant administration of cilostazol with gentamicin restored the histopathological insult induced by gentamicin, as it showed regular epithelial cells lining the tubules. Glomerular and peritubular capillary congestion were also noticed (Fig. 1d).
4. Discussion The nephrotoxicity of the aminoglycoside antibiotic gentamicin is well documented (Ali et al., 2011). The elevated levels of serum creatinine, urea and uric acid are indicators of renal damage and dysfunction. The ability of the kidney to filter creatinine, a nonprotein waste product of creatinine phosphate metabolism, is reduced during renal dysfunction as a result of diminished glomerular filtration rate. Moreover, the elevated level of urea
Table 2 Effect of cilostazol, gentamicin alone and in combination on SOD and CAT activities as well as GSH and MDA levels in renal tissue in albino rats. Groups
Control
SOD (U/g tissue) CAT (U/mg tissue) MDA (mmol/g tissue) GSH (mmol/g tissue)
29 20 2.53 2.2
3.05 1.43 0.31 0.31
Cilostazol 28 22 3.12 2.3
2.66 2.81 0.41 0.41
Gentamicin 9.35 7.32 9.62 0.66
1.09* 0.87* 1.02* 0.07*
Gentamicin + cilostazol 18 14 4.63 1.5
1.95# 1.87# 0.51# 0.97#
Data are presented as means SEM; n = number of rats in each group. Statistical analysis was done using one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. *, P < 0.05 versus control and cilostazol groups; #, P < 0.05 versus control, cilostazol, and gentamicin groups.
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Fig. 1. H&E stained sections of the renal cortex show (1a): control renal cortex showing renal glomerulus (g), Bowman’s space (s), PCT (P) and distal convoluted tubules (d). (1b and c): renal cortex of gentamycin treated group with either hypertrophied glomeruli (g1) with obliterated Bowman’s space and vacuolated endothelial cells or atrophied glomeruli (g2) with widening of Bowman’s space (s). Renal tubules show epithelial flattening with vacuolar degeneration (t) and pyknotic nuclei (arrows). Interstitial cellular infiltrations (I) is seen. (1d): showing renal cortex of gentamycin + cilostazol treated group with apparently normal glomerulus (g), PCT (P), DCT (d) Glomerular and peritubular congestion (arrow heads) is noticed (arrow heads)400.
and uric acid occurs during renal dysfunction (Perrrone et al., 1992). In the present study, gentamicin administration increased the levels of serum creatinine, urea and uric acid as compared to control group, while no significant difference in the studied parameters between cilostazol (Group 2) and control groups. In parallel with the present findings, Parlakpinar et al. (2005) and Reddy et al. (2011) found that gentamicin administration elevated the levels of serum creatinine and urea. In the clinical picture, aminoglycosides nephrotoxicity manifest as renal failure with a rise in the serum creatinine, disturbed serum electrolytes. Changes in proximal tubular function are manifested as proteinuria, glycosuria and urinary loss of brush border enzymes (Sandhu et al., 2007). Histopathological examination revealed degenerative changes in renal cortex of gentamicin treated rats. Hypertrophied glomeruli with vacuolated endothelial cells together with glomerular atrophy and disrupted blood renal barrier were evident. Also, cells lining renal tubules especially that of PCT appeared affected; apoptotic nuclei, vacuolated cytoplasm and swollen mitochondria were observed. Interstitial inflammatory cellular infiltration was evident. All of these histological alterations were associated with increased immunohistochemical expression of apoptotic marker (Bax) and decreased expression of anti-apoptotic marker (BCL2). These results cope with previous findings obtained by El Mouedden et al. (2000) and confirmed the apoptotic effect of gentamicin. Nephrotoxicity of gentamicin is known to affect both glomerular and tubular cells. Following glomerular filtration, gentamicin accumulates in the epithelial cells of mainly the proximal, but also distal and collecting tubules for an extended period and results in intracellular alterations, causing damage that can range from loss of the brush border to complete tubular necrosis (Quiros et al., 2011). After drug uptake, a number of cellular processes are activated, culminating in apoptosis. This contributes to loss of the renal tubular epithelium and thus kidney dysfunction. Besides tubular injury, persistent contraction of the glomerular mesangial cells, cellular apoptosis, proliferation and necrosis have all been described in the histopathological evidence of aminoglycosides nephrotoxicity (Lopez-Novoa et al., 2011). Other contributing factors to the nephrotoxicity of aminoglycosides are the generation of free radicals and the decrease of renal blood flow through local enhancement of the vasoconstrictors, blocking the vasodilators and promoting leukocyte margination (Zafarghandy and van den Anker, 2013). The results of the present study showed that administration of cilostazol along with gentamicin significantly decreased the levels of serum creatinine, urea or uric acid in relation gentamicintreated group. Also, histological findings were concordant with biochemical results. Renal glomeruli and tubules showed recovered structure. Few tubules were affected. On the ultrastructure observation and with the co-addition of cilostazol, the blood renal barrier with its components and cells lining convoluted tubules showed improvement in their structure in comparison with that treated with gentamicin alone. These results indicate that cilostazol protected rats from gentamicin-induced nephrotoxicity. Indeed, the renoprotective effect of cilostazol was assessed by Ragab et al. (2014). They found that cilostazol was effective to mitigate renal ischemia-reperfusion injury in rats. In the same direction, cyclosporine-induced nephrotoxicity was attenuated by cilostazol administration in rats (Gokce et al., 2012). It is known that oxidative stress plays an essential role in the development of gentamicin nephrotoxicity (Walker et al., 1999). Changes of membrane lipid composition could be induced by free radical-initiated lipid peroxidation with subsequent increase in MDA levels which is one of the products of lipid peroxidation
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Fig. 2. (a–c) show Bax immunostaining with negative reaction in control group (2a), strong positive reaction in most of the lining of renal tubules (arrows) and glomerulus (arrow head) of gentamycin treated group (2b) and faint positive staining (arrows) in some of the tubules of Cilostazol group. (2d–f) show BCL2 immunostaining with strong positive reaction in most of the renal tubules of control group (2d) and negative staining in gentamycin treated group (2e) and positive reaction in most of the tubules (arrows) and glomeruli (arrow heads) of gentamycin + cilostazol treated group (2f) 400.
Table 3 Effect of cilostazol, gentamicin alone and in combination on area percentage of collagen fibers and Bcl2 immunopositivity (n = 10). Groups
Control
Cilostazol
Gentamicin
Gentamicin + cilostazol
Area% of Bax Area% of BCL2
0.01 0.002 25.64 3.7
0.01 0.001 27.21 1.5
23.95 2.09* 2.15 0.13*
2.16 0.451# 16.91 3.5#
Data are presented as means SD.; n = number of rats in each group. Statistical analysis was done using one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. *, P = 0.00 versus control and cilostazol groups; #, P = 0.00 versus control, cilostazol, and gentamicin groups
(Parlakpinar, 2005). GSH has a very important role in protecting against oxygen free radical detrimental effect through providing reducing equivalents for several enzymes act as a scavenger to hydroxyl radicals and singlet oxygen (Ragab et al., 2014). The results of the present study showed that administration of gentamicin increased MDA level while decreased GSH in the renal tissue. Administration of cilostazol with gentamicin decreased MDA level while increased GSH in relation to gentamicin-treated group. The nephrotoxic effect of gentamicin is partially mediated through decreasing GSH level (Abdel-Raheem et al., 2009). The damage in plasma membrane caused by oxidative stress results in loss of osmotic balance and intracellular calcium levels increase.
Cellular swelling is the first manifestation of these reversible changes which can be detected under light microscope (Silan et al., 2007). In addition, swelling of endothelium lining the glomerular tufts and tubular vacuolization is the reflection of these reversible changes in kidneys (Abdel-Raheem et al., 2009). The present findings indicated that, cilostazol prevented lipid peroxidation and opposed the gentamicin-induced redox tissue imbalance, possibly via its free radical scavenging property and/or by increasing the activity of the endogenous antioxidants (SOD and CAT). In harmony with the present findings, Agrawal et al. (2007) reported that cilostazol inhibited lipid peroxidation and reduced oxidative stress through decrease the MDA level and improved glutathione level in blood of diabetic patients. The scavenging of
Fig. 3. electron micrograph of blood renal barrier (3a): control kidney Showing renal corpuscle with glomerular blood capillaries lined by fenestrated endothelium (E) and containing red blood cells (RC) in their lumen. Podocyte (Pd) with euchromatic nucleus (n) and foot processes (arrows) are also seen. (3b) Gentamycin treated group show glomerular blood capillaries with focal thickening of their basement membrane (B) and disrupted endothelial cells (E) and electron dense mesangial matrix (*). Podocytes (pd) are seen with vacuolated cytoplasm and distorted foot processes (arrows). (3c) Gentamycin + cilostazol group show glomerular blood capillaries with uniform endothelial cell (E) and RBCs (RC) in their lumen. Podocyte (Pd) is seen with foot processes (arrows) and euchromatic nucleus (n). Scale bar = 1 mm.
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Fig. 4. (a–c) show ultrastructure features of PCT. (4a): showing cells lining PCT of control group with euchromatic nuclei (n), apical microvilli (mv), basal enfolding and elongated mitochondria (arrows) are resting on basal lamina (BL). (4b) Showing epithelial cells of PCT of gentamycin treated group with small condensed nucleus (n), electron dense nucleus (N) and disrupted basal enfolding with swollen mitochondria (arrows). Notice apical microvilli (mv) lost in some parts (*) and basal lamina (BL). (4c) Showing cells of PCT of cilostazol protected group with euchromatic nuclei (n), apical microvilli (mv). Disrupted basal enfolding and swollen mitochondria (arrows) are noticed. Fig. (4d–f) show ultrastructure features of DCT of studied groups. (4d): showing cells lining the DCT of control group with euchromatic nuclei (n), basal enfolding and elongated mitochondria (arrows) and rounded mitochondria (m). (4e): showing epithelial cells lining distal convoluted tubules of gentamycin treated group. Nuclear margination with heterochromatin clumbs (n), disrupted basal infoldings, slightly thickened (BL) and vaculated (V) cytoplasm are seen. Some of their nuclei are dense and irregular (n), elongated mitochondria (arrow) is seen. 4f (Showing cells lining DCT of gentamycin + cilostazol treated group with euchromatic nuclei (n), elongated mitochondria (arrows) and basal enfolding (arrows). Basal lamina (BL) is thickened. Scale bar = 2 mm.
superoxide radicals is achieved through SOD, which catalyses the dismutation of superoxide to hydrogen peroxide. This reaction has a 10000-fold faster rate than spontaneous dismutation (AbdelRaheem et al., 2009; Lee et al., 2010). The anti-apoptotic effect of cilostazol was investigated in the present work. Immunohistochemical examination of renal tissue of gentamicin-cilostazol treated group revealed a significant decrease in the expression of apoptotic protein marker (Bax) and a significant increase in the expression of anti-apoptotic protein marker (BCL2) when compared with gentamicin-treated group. The anti-apoptotic role of cilostazol was confirmed in endothelial cells via counteract tumor necrosis factor-a (TNFa)-induced cell death (Lim et al., 2009; Park et al., 2011) and by suppressing mitochondria dependent apoptotic signaling with decreased DNA fragmentation and subsequent stimulation of Bcl2 expression and down-regulation of Bax protein and cytochrome c release from mitochondria in different tissues (Jong-Hoon et al., 2009). Apoptosis can be triggered by gentamycin in the kidney by increase in the cytosolic Bax protein content, which activates the so-called mitochondrial pathway of apoptosis (Servais et al., 2006). Our results confirmed that cilostazol pretreatment at a dose of 10 mg/kg for 8 days could actually protect cells from gentamicininduced apoptogenic effect and thereby mitigate its toxicity. The daily oral dose of cilostazol administered to rats in our study comes in accordance with the guidance provided By Food and Drug Administration Center for Drug Evaluation and Research (CDER) and similar to that used by other studies to demonstrate the beneficial role of cilostazol (Sheu et al., 2012; Abdel Kawy, 2015). The authors set a rational use for a selection of this dose and consider that this dose is safe low to moderated efficacious dose although using it for 21 days. 5. Conclusion Cilostazol protected rats from gentamicin-induced nephrotoxicity. The renoprotective effect of cilostazol could be partially
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Experimental and Toxicologic Pathology xxx (2015) xxx–xxx
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Cilostazol attenuates gentamicin-induced nephrotoxicity in rats Ahmed A. Abdelsameeaa , Ahmed M. Mohameda , Mona G. Amerb,* , Shahera M. Attiac a
Clinical Pharmacology Department, Faculty of Medicine, Zagazig University, Egypt Histology & Cell Biology department, Faculty of Medicine, Zagazig University, Egypt c Medical Biochemistry, Faculty of Medicine, Zagazig University, Egypt b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2015 Received in revised form 26 December 2015 Accepted 7 January 2016
Introduction: Gentamycin is a widely used antibiotic. The nephrotoxic adverse effects of the drug may limit its use. Cilostazol, a phosphodiesterase III inhibitor, was reported to protect from renal oxidative stress. This work aimed to investigate the possible protective effect of cilostazol on gentamicin-induced nephrotoxicity and the possible underlying mechanisms. Materials and methods: 40 male albino rats were divided into 4 equal groups: (1) Control; (2) Cilostazol, 10 mg/kg, p.o.; (3) Gentamicin, 80 mg/kg, i.p.; (4) Gentamicin 80 mg/kg, i.p. along with cilostazol 10 mg/ kg, p.o. All drugs were administered once daily for 8days. On 9th day blood samples were collected for the estimation of creatinine, urea and uric acid in serum. Then the rats were sacrificed and kidneys were removed for light and electron microscope studies. Moreover, reduced glutathione (GSH) and malondialdehyde (MDA) levels as well as catalase (CAT) and superoxide dismutase (SOD) activities were determined in renal tissues. Results: Gentamicin elevated the serum levels of creatinine, urea and uric acid as well as the MDA level in the renal tissue, while it decreased CAT, SOD activities and GSH levels as well as produced degenerative changes in glomeruli and tubules associated with increased expression of apoptotic markers and decreased expression of anti-apoptotic markers. Administration of cilostazol decreased urea, creatinine, uric acid and MDA levels while increased CAT and SOD activities and GSH levels as well as ameliorated the histopathological changes in relation to gentamicin group. Conclusion: Cilostazol protected rats from gentamicin-induced nephrotoxicity possibly, in part through its antioxidant and anti-apoptotic activity. ã 2016 Elsevier GmbH. All rights reserved.
Keywords: Gentamicin Nephrotoxicity Cilostazol Ultrastructure Apoptosis
1. Introduction Gentamicin is a widely used aminoglycoside antibiotic either alone or in combination with a cell wall-active drug in management of severe and life-threatening infections caused by Grampositive and Gram-negative aerobes (Choi et al., 2011). The ototoxic and nephrotoxic adverse effects of the drug may limit its use. In some situations, these adverse effects are so severe that the treatment must be discontinued (Ali, 2011). The pathogenesis of gentamicin nephrotoxicity involves multiple pathways, including oxidative stress, inflammation, reduced renal blood flow, and increased nitric oxide (NO) level (Balakumar
Abbreviations: CAT, catalase; DCT, distal convoluted tubule; GSH, Glutathione; H&E, hematoxylin and eosin; MDA, malondialdehyde; PCT, proximal convoluted tubules; SOD, superoxide dismutase. * Corresponding author at: Histology and Cell Biology, Faculty of Medicine, Zagazig University, Zagazig, Egypt. E-mail address: [email protected] (M.G. Amer).
et al., 2010; Christo et al., 2011). Several agents have been used, with various degrees of success, to ameliorate or prevent gentamicin nephrotoxicity (Otunctemur et al., 2013; Rodrigues et al., 2014). Cilostazol, a selective phosphodiesterase III inhibitor, has potent antiplatelet and vasodilator effects. The drug is approved for treatment of intermittent claudication in patients with peripheral vascular diseases (Chen et al., 2015). Cilostazol was generally well tolerated. Adverse events reported are headache, palpitation and tachycardia with mild to moderate intensity and rarely required treatment withdrawal (Kim et al., 2015). Several investigation in different cells and tissues have indicated to inhibitory effect of cilostazol on reactive oxygen species and superoxide generation as well as hydroxyl radicals scavenging action (Kim et al., 2002; Lee et al., 2010). The aim of the present study is to assess the possible protective effect of cilostazol on gentamicin-induced nephrotoxicity and the possible underlying mechanisms.
http://dx.doi.org/10.1016/j.etp.2016.01.002 0940-2993/ ã 2016 Elsevier GmbH. All rights reserved.
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2. Materials and methods 2.1. Materials Gentamicin sulfate (Memphis Pharm., & Chemical Ind., Cairo, Egypt); Cilostazol powder, (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan).
samples were homogenized in ice-cold 50 mM potassium phosphate buffer (pH 7.5), centrifuged at 4 C 12,000 g for 15 min and then the supernatant was collected. MDA in the supernatant can react with freshly prepared thiobarbituric acid (TBA) to form a colored complex which has maximum absorbance at 535 nm (Buege and Aust, 1978). The nmol MDA/g wet tissue was calculated from the plotted standard curve prepared from 1,1,3,3tetraethoxypropane.
2.2. Animals Male albino rats weighing 150–180 gm were used after one week for proper acclimatization to the standard housing conditions (25 2 C temperature and 12 h light/dark cycle) and were supplied with standard rodent chow and tap water ad libitum. All experimental protocols were approved by the Ethics Committee of Zagazig University. 2.3. Experimental design 40 male albino rats were randomly divided into 4 equal groups: Group (1) non-treated control; Group (2) was treated with cilostazol 10 mg/kg, p.o. dissolved in saline (Gokce et al., 2012) and served as cilostazol-treated control. Group (3) was injected with gentamicin 80 mg/kg, i.p. (Reddy et al., 2011) for induction of experimental nephrotoxicity; Group (4) was given cilostazol 10 mg/kg, p.o. 1 h before gentamicin 80 mg/kg, i.p. All drugs were administered daily for 8 consecutive days. On the 9th day blood samples were collected, and serum was separated by centrifugation at 3000 g for 10 min for estimation of creatinine, uric acid and urea levels. A longitudinal section from the left kidney was excised from each animal for histological examination. The renal cortex of the rest of the kidneys was stored at 80 C and subsequently homogenized in ice-cold phosphate buffer (0.05 M, pH 7.4) for biochemical analysis. 2.3.1. Biochemical analysis 2.3.1.1. Serum analysis. Creatinine, uric acid and urea levels were estimated using kits supplied by spinreact (Girona, Spain). 2.3.1.2. Estimation of the reduced glutathione (GSH). The GSH level in the kidney was estimated using the method described by Ellman (1959). Briefly, the renal homogenate was mixed with 10% w/v trichloroacetic acid in ratio of 1:1 and centrifuged at 40 C for 10 min at 5000 rpm. The supernatant obtained (0.5 ml) was mixed with 2 ml of 0.3 M disodium hydrogen phosphate buffer (pH 8.4) and 0.4 ml of distilled water. Then 0.25 ml of 0.001 M freshly prepared DTNB (5,51-dithiobis (2-nitrobenzoic acid)) dissolved in 1% w/v sodium citrate) was added. The reaction mixture was incubated for 10 min and absorbence of yellow colored complex was noted spectrophotometrically at 412 nm. A standard curve was plotted using reduced form of glutathione. 2.3.1.3. Determination of lipid peroxidation. Lipid peroxide was estimated by measurement of malondialdehyde (MDA) levels spectrophotometrically in kidney homogenate whereas kidney
2.3.1.4. Determination of catalase (CAT). It was determined using colorimetric assay kits supplied from Biodiagnostic Company for diagnostic reagents: Dokki, Giza, Egypt. The resulting quinoneimine dye is measured at 520 nm (Aebi, 1984). 2.3.1.5. Determination of superoxide dismutase activity (SOD). Determination of total (Cu, Zn and Mn) SOD activity was determined kinetically (Sun et al., 1988) using kits supplied from Biodiagnostic Company for diagnostic reagents: Dokki, Giza, Egypt. 2.3.2. Histopathological studies At the time of sacrifice, the animals were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 50 mg/kg. The kidneys were rapidly removed and opened to be processed. For light microscopy, specimens were fixed in 10% formalin solution and processed to prepare 5 mm thick paraffin sections and stained with hematoxylin and eosin (H&E) (Bancroft and Gamble, 2002). For immunohistochemical study, the deparaffinized sections on charged slides were used for detection of Bax (apoptosis inducers) and BCL2 (antiapoptotic marker). Immunohistochemical reaction was carried using avidin biotin peroxidase system. The primary antibody used was a rabbit polyclonal antibody (Sigma Laboratories, Saint Louis, USA). The primary antibody used for Bax and BCL2 were rabbit polyclonal antibody, sigma laboratories (Cat No 6¼ anti Bax: B3428, anti BCL2 sc-492). Universal kit used avidin biotin peroxidase system produced by NovaCastra Laboratories Ltd., UK. The same method was applied to prepare negative control sections but the primary antibody was not added. Mayer’s hematoxilin was added as counter stain. The cells that displayed brown precipitation were considered positive for Bax and BCL2 expression (Albamonte et al., 2013). Specimens for electron microscope examination were cut into 1 mm3 pieces and fixed in 2.5% glutraldehyde buffered with 0.1 mol/L phosphate buffer at pH 7.4 for 2 h. They were post fixed in 1% osmium tetroxide for 1 h, dehydrated through graded alcohol series, and embedded in epoxy resin. Ultrathin sections (50 nm thick) were collected on copper grids and stained with uranyl acetate and lead citrate (Glauret and Lewis, 1998). 2.3.3. Quantitative morphometric measurements Area percentage of the positive Bax & Bcl2 immune reactive cells lining the renal tubules was estimated by using “Leica Quin 500C” image analyzer computer system (Leica Imaging System Ltd., Cambridge, England). The measuring frame of a standard area
Table 1 Effect of cilostazol, gentamicin alone and in combination on serum urea, creatinine and uric acid in albino rats (n = 10). Groups
Control
Cilostazol
Gentamicin
Gentamicin + cilostazol
Urea (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl)
35 2.33 0.75 0.06 1.22 0.21
38 2.47 0.81 0.09 1.36 0.19
109 11.77* 2.15 0.13* 5.83 0.33*
66.83 5.73# 1.54 0.21# 2.98 0.23#
Data are presented as means SEM; n = number of rats in each group. Statistical analysis was done using one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. *, P < 0.05 versus control and cilostazol groups; #,P < 0.05 versus control, cilostazol, and gentamicin groups.
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was equal to 7286, 78 mm2. Measurements were done within 10 non overlapping fields for each section at 400 magnification.
3
3.2. Immunohistochemical results To determine the mechanism underlying the anti-apoptotic effects of cilostazol; Bax (a marker protein of apoptosis) and BCL2 (anti-apoptotic protein) were detected using immunohistochemistry. The results revealed strong positive immunostaining of Bax in most of cells lining renal tubules and glomeruli of gentamicin treated group (Fig. 2b), faint staining in gentamicin–cilostazol treated group (Fig. 2c) and negative staining in control groups (Fig. 2a). Examination of the kidneys of control group detected positive immunostaining for BCL2 in cells lining renal tubules and glomeruli (Fig. 2d), no reaction was detected in kidney of gentamicin-treated group (Fig. 2e) and strong positive reaction was seen in most of the renal tubules and glomeruli of gentamicin– cilostazol treated group (Fig. 2f). Statistical analysis of morphometric results measuring the area% of positive reaction confirmed the previous results (Table 3). Electron microscopic examination of the renal cortex of control groups revealed renal corpuscles with glomerular capillaries lined by fenestrated endothelium. The podocytes with euchromatic nuclei and foot processes were seen (Fig. 3a). Gentamicin caused several visible and distinct changes in the ultrastructure of the kidney of Group (3). Disrupted blood renal barrier appeared with degenerated endothelium and vacuolated podocytes. Also, thickened basement membrane of the barrier and dense mesangial matrix were noticed (Fig. 3b). The cells lining the PCT appeared with euchromatic nuclei and basal enfolding with elongated mitochondria. The apical surface had a well-developed luminal brush border consisting of numerous closely packed microvilli (Fig. 4a). Gentamicin treatment in Group (3) induced ultrastructure alteration in PCT. The brush border showed areas of complete loss and mitochondria appeared swollen. Disorganized basal enfolding and electron dense shrunk nuclei were observed in most of the tubules (Fig. 4b). Most of the PCT from gentamicin– cilostazol treated group showed normal ultrastructure features. Some showed disrupted basal enfolding and swollen mitochondria (Fig. 4c). The cells of the DCT of control groups showed euchromatic nuclei and basal enfolding with elongated mitochondria (Fig. 4d). In gentamicin-treated group, vacuolation of the cytoplasm is observed in most of the tubules with disorganized basal enfolding, and irregular nuclei with condensed chromatin (Fig. 4e). DCT of gentamicin–cilostazol treated group appeared with irregular nuclei and thickened basement membrane (Fig. 4f).
2.4. Statistical analysis Results were expressed as the means + S.E.M. Statistical significant difference was determined by one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparisons. Probability values (P) less than 0.05 were considered to be statistically significant. 3. Results Serum urea, creatinine and uric acid levels: Gentamicin significantly increased (P < 0.05) urea, creatinine and uric acid levels in relation to control groups. The previous parameters were significantly decreased (P < 0.05) by cilostazol–gentamicin administration in relation to gentamicin-treated group (Table 1). Renal GSH, MDA levels as well as CAT, and SOD activities: Gentamicin significantly (P < 0.05) increased MDA level while, decreased GSH level as well as the activity of CAT&SOD in relation to control groups. Cilostazol–gentamicin treatment significantly decreased (P < 0.05) MDA level in comparison with gentamicintreated group. On the other hand, concomitant treatment of cilostazol with gentamicin significantly increased the GSH level as well as CAT&SOD activities in the renal tissue compared with gentamicin-treated group (Table 2). 3.1. Histology of kidney Histological examination revealed that non-treated and cilostazol-treated control groups showed normal features of renal glomeruli and cortical tubules. Figures for control group were representative for both groups. The normal renal corpuscle consisted of a tuft of capillaries, the glomerulus, surrounded by a double-walled epithelial capsule called Bowman’s capsule. Between the two layers of the capsule is the urinary or Bowman’s space. The convoluted tubules lined by cuboidal cells. Profiles of the proximal convoluted tubules (PCT) made up the bulk of the renal cortex with narrow irregular lumen; however, the distal tubules (DCT) exhibited clear lumen (Fig. 1a). In contrast, gentamicin-treated group showed renal corpuscles formed of either hypertrophied or atrophied glomeruli with widening of their Bowman’s spaces. The endothelial cells lining the glomerular tufts showed swelling and intarcytoplasmic vacuolation (Fig. 1b and c). The epithelial cells lining of the renal tubules showed vacuoles and darkly stained pyknotic nuclei. Interstitial infiltration and marked atrophy of the glomeruli with wide Bowman’s space were also seen (Fig. 1c). Concomitant administration of cilostazol with gentamicin restored the histopathological insult induced by gentamicin, as it showed regular epithelial cells lining the tubules. Glomerular and peritubular capillary congestion were also noticed (Fig. 1d).
4. Discussion The nephrotoxicity of the aminoglycoside antibiotic gentamicin is well documented (Ali et al., 2011). The elevated levels of serum creatinine, urea and uric acid are indicators of renal damage and dysfunction. The ability of the kidney to filter creatinine, a nonprotein waste product of creatinine phosphate metabolism, is reduced during renal dysfunction as a result of diminished glomerular filtration rate. Moreover, the elevated level of urea
Table 2 Effect of cilostazol, gentamicin alone and in combination on SOD and CAT activities as well as GSH and MDA levels in renal tissue in albino rats. Groups
Control
SOD (U/g tissue) CAT (U/mg tissue) MDA (mmol/g tissue) GSH (mmol/g tissue)
29 20 2.53 2.2
3.05 1.43 0.31 0.31
Cilostazol 28 22 3.12 2.3
2.66 2.81 0.41 0.41
Gentamicin 9.35 7.32 9.62 0.66
1.09* 0.87* 1.02* 0.07*
Gentamicin + cilostazol 18 14 4.63 1.5
1.95# 1.87# 0.51# 0.97#
Data are presented as means SEM; n = number of rats in each group. Statistical analysis was done using one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. *, P < 0.05 versus control and cilostazol groups; #, P < 0.05 versus control, cilostazol, and gentamicin groups.
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Fig. 1. H&E stained sections of the renal cortex show (1a): control renal cortex showing renal glomerulus (g), Bowman’s space (s), PCT (P) and distal convoluted tubules (d). (1b and c): renal cortex of gentamycin treated group with either hypertrophied glomeruli (g1) with obliterated Bowman’s space and vacuolated endothelial cells or atrophied glomeruli (g2) with widening of Bowman’s space (s). Renal tubules show epithelial flattening with vacuolar degeneration (t) and pyknotic nuclei (arrows). Interstitial cellular infiltrations (I) is seen. (1d): showing renal cortex of gentamycin + cilostazol treated group with apparently normal glomerulus (g), PCT (P), DCT (d) Glomerular and peritubular congestion (arrow heads) is noticed (arrow heads)400.
and uric acid occurs during renal dysfunction (Perrrone et al., 1992). In the present study, gentamicin administration increased the levels of serum creatinine, urea and uric acid as compared to control group, while no significant difference in the studied parameters between cilostazol (Group 2) and control groups. In parallel with the present findings, Parlakpinar et al. (2005) and Reddy et al. (2011) found that gentamicin administration elevated the levels of serum creatinine and urea. In the clinical picture, aminoglycosides nephrotoxicity manifest as renal failure with a rise in the serum creatinine, disturbed serum electrolytes. Changes in proximal tubular function are manifested as proteinuria, glycosuria and urinary loss of brush border enzymes (Sandhu et al., 2007). Histopathological examination revealed degenerative changes in renal cortex of gentamicin treated rats. Hypertrophied glomeruli with vacuolated endothelial cells together with glomerular atrophy and disrupted blood renal barrier were evident. Also, cells lining renal tubules especially that of PCT appeared affected; apoptotic nuclei, vacuolated cytoplasm and swollen mitochondria were observed. Interstitial inflammatory cellular infiltration was evident. All of these histological alterations were associated with increased immunohistochemical expression of apoptotic marker (Bax) and decreased expression of anti-apoptotic marker (BCL2). These results cope with previous findings obtained by El Mouedden et al. (2000) and confirmed the apoptotic effect of gentamicin. Nephrotoxicity of gentamicin is known to affect both glomerular and tubular cells. Following glomerular filtration, gentamicin accumulates in the epithelial cells of mainly the proximal, but also distal and collecting tubules for an extended period and results in intracellular alterations, causing damage that can range from loss of the brush border to complete tubular necrosis (Quiros et al., 2011). After drug uptake, a number of cellular processes are activated, culminating in apoptosis. This contributes to loss of the renal tubular epithelium and thus kidney dysfunction. Besides tubular injury, persistent contraction of the glomerular mesangial cells, cellular apoptosis, proliferation and necrosis have all been described in the histopathological evidence of aminoglycosides nephrotoxicity (Lopez-Novoa et al., 2011). Other contributing factors to the nephrotoxicity of aminoglycosides are the generation of free radicals and the decrease of renal blood flow through local enhancement of the vasoconstrictors, blocking the vasodilators and promoting leukocyte margination (Zafarghandy and van den Anker, 2013). The results of the present study showed that administration of cilostazol along with gentamicin significantly decreased the levels of serum creatinine, urea or uric acid in relation gentamicintreated group. Also, histological findings were concordant with biochemical results. Renal glomeruli and tubules showed recovered structure. Few tubules were affected. On the ultrastructure observation and with the co-addition of cilostazol, the blood renal barrier with its components and cells lining convoluted tubules showed improvement in their structure in comparison with that treated with gentamicin alone. These results indicate that cilostazol protected rats from gentamicin-induced nephrotoxicity. Indeed, the renoprotective effect of cilostazol was assessed by Ragab et al. (2014). They found that cilostazol was effective to mitigate renal ischemia-reperfusion injury in rats. In the same direction, cyclosporine-induced nephrotoxicity was attenuated by cilostazol administration in rats (Gokce et al., 2012). It is known that oxidative stress plays an essential role in the development of gentamicin nephrotoxicity (Walker et al., 1999). Changes of membrane lipid composition could be induced by free radical-initiated lipid peroxidation with subsequent increase in MDA levels which is one of the products of lipid peroxidation
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Fig. 2. (a–c) show Bax immunostaining with negative reaction in control group (2a), strong positive reaction in most of the lining of renal tubules (arrows) and glomerulus (arrow head) of gentamycin treated group (2b) and faint positive staining (arrows) in some of the tubules of Cilostazol group. (2d–f) show BCL2 immunostaining with strong positive reaction in most of the renal tubules of control group (2d) and negative staining in gentamycin treated group (2e) and positive reaction in most of the tubules (arrows) and glomeruli (arrow heads) of gentamycin + cilostazol treated group (2f) 400.
Table 3 Effect of cilostazol, gentamicin alone and in combination on area percentage of collagen fibers and Bcl2 immunopositivity (n = 10). Groups
Control
Cilostazol
Gentamicin
Gentamicin + cilostazol
Area% of Bax Area% of BCL2
0.01 0.002 25.64 3.7
0.01 0.001 27.21 1.5
23.95 2.09* 2.15 0.13*
2.16 0.451# 16.91 3.5#
Data are presented as means SD.; n = number of rats in each group. Statistical analysis was done using one-way ANOVA followed by Tukey post-hoc test for multiple comparisons. *, P = 0.00 versus control and cilostazol groups; #, P = 0.00 versus control, cilostazol, and gentamicin groups
(Parlakpinar, 2005). GSH has a very important role in protecting against oxygen free radical detrimental effect through providing reducing equivalents for several enzymes act as a scavenger to hydroxyl radicals and singlet oxygen (Ragab et al., 2014). The results of the present study showed that administration of gentamicin increased MDA level while decreased GSH in the renal tissue. Administration of cilostazol with gentamicin decreased MDA level while increased GSH in relation to gentamicin-treated group. The nephrotoxic effect of gentamicin is partially mediated through decreasing GSH level (Abdel-Raheem et al., 2009). The damage in plasma membrane caused by oxidative stress results in loss of osmotic balance and intracellular calcium levels increase.
Cellular swelling is the first manifestation of these reversible changes which can be detected under light microscope (Silan et al., 2007). In addition, swelling of endothelium lining the glomerular tufts and tubular vacuolization is the reflection of these reversible changes in kidneys (Abdel-Raheem et al., 2009). The present findings indicated that, cilostazol prevented lipid peroxidation and opposed the gentamicin-induced redox tissue imbalance, possibly via its free radical scavenging property and/or by increasing the activity of the endogenous antioxidants (SOD and CAT). In harmony with the present findings, Agrawal et al. (2007) reported that cilostazol inhibited lipid peroxidation and reduced oxidative stress through decrease the MDA level and improved glutathione level in blood of diabetic patients. The scavenging of
Fig. 3. electron micrograph of blood renal barrier (3a): control kidney Showing renal corpuscle with glomerular blood capillaries lined by fenestrated endothelium (E) and containing red blood cells (RC) in their lumen. Podocyte (Pd) with euchromatic nucleus (n) and foot processes (arrows) are also seen. (3b) Gentamycin treated group show glomerular blood capillaries with focal thickening of their basement membrane (B) and disrupted endothelial cells (E) and electron dense mesangial matrix (*). Podocytes (pd) are seen with vacuolated cytoplasm and distorted foot processes (arrows). (3c) Gentamycin + cilostazol group show glomerular blood capillaries with uniform endothelial cell (E) and RBCs (RC) in their lumen. Podocyte (Pd) is seen with foot processes (arrows) and euchromatic nucleus (n). Scale bar = 1 mm.
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Fig. 4. (a–c) show ultrastructure features of PCT. (4a): showing cells lining PCT of control group with euchromatic nuclei (n), apical microvilli (mv), basal enfolding and elongated mitochondria (arrows) are resting on basal lamina (BL). (4b) Showing epithelial cells of PCT of gentamycin treated group with small condensed nucleus (n), electron dense nucleus (N) and disrupted basal enfolding with swollen mitochondria (arrows). Notice apical microvilli (mv) lost in some parts (*) and basal lamina (BL). (4c) Showing cells of PCT of cilostazol protected group with euchromatic nuclei (n), apical microvilli (mv). Disrupted basal enfolding and swollen mitochondria (arrows) are noticed. Fig. (4d–f) show ultrastructure features of DCT of studied groups. (4d): showing cells lining the DCT of control group with euchromatic nuclei (n), basal enfolding and elongated mitochondria (arrows) and rounded mitochondria (m). (4e): showing epithelial cells lining distal convoluted tubules of gentamycin treated group. Nuclear margination with heterochromatin clumbs (n), disrupted basal infoldings, slightly thickened (BL) and vaculated (V) cytoplasm are seen. Some of their nuclei are dense and irregular (n), elongated mitochondria (arrow) is seen. 4f (Showing cells lining DCT of gentamycin + cilostazol treated group with euchromatic nuclei (n), elongated mitochondria (arrows) and basal enfolding (arrows). Basal lamina (BL) is thickened. Scale bar = 2 mm.
superoxide radicals is achieved through SOD, which catalyses the dismutation of superoxide to hydrogen peroxide. This reaction has a 10000-fold faster rate than spontaneous dismutation (AbdelRaheem et al., 2009; Lee et al., 2010). The anti-apoptotic effect of cilostazol was investigated in the present work. Immunohistochemical examination of renal tissue of gentamicin-cilostazol treated group revealed a significant decrease in the expression of apoptotic protein marker (Bax) and a significant increase in the expression of anti-apoptotic protein marker (BCL2) when compared with gentamicin-treated group. The anti-apoptotic role of cilostazol was confirmed in endothelial cells via counteract tumor necrosis factor-a (TNFa)-induced cell death (Lim et al., 2009; Park et al., 2011) and by suppressing mitochondria dependent apoptotic signaling with decreased DNA fragmentation and subsequent stimulation of Bcl2 expression and down-regulation of Bax protein and cytochrome c release from mitochondria in different tissues (Jong-Hoon et al., 2009). Apoptosis can be triggered by gentamycin in the kidney by increase in the cytosolic Bax protein content, which activates the so-called mitochondrial pathway of apoptosis (Servais et al., 2006). Our results confirmed that cilostazol pretreatment at a dose of 10 mg/kg for 8 days could actually protect cells from gentamicininduced apoptogenic effect and thereby mitigate its toxicity. The daily oral dose of cilostazol administered to rats in our study comes in accordance with the guidance provided By Food and Drug Administration Center for Drug Evaluation and Research (CDER) and similar to that used by other studies to demonstrate the beneficial role of cilostazol (Sheu et al., 2012; Abdel Kawy, 2015). The authors set a rational use for a selection of this dose and consider that this dose is safe low to moderated efficacious dose although using it for 21 days. 5. Conclusion Cilostazol protected rats from gentamicin-induced nephrotoxicity. The renoprotective effect of cilostazol could be partially
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