Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats

Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats

Toxicology 207 (2005) 169–177 Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats H. Parlakpina...

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Toxicology 207 (2005) 169–177

Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats H. Parlakpinara,∗ , S. Tasdemira , A. Polatb , A. Bay-Karabulutc , N. Vardid , M. Ucard , A. Aceta a

Department of Pharmacology, Faculty of Medicine, Inonu University, 44280 Malatya, Turkey b Department of Physiology, Faculty of Medicine, Inonu University, 44280 Malatya, Turkey c Department of Biochemistry, Faculty of Medicine, Inonu University, 44280 Malatya, Turkey d Department of Histology, Faculty of Medicine, Inonu University, 44280 Malatya, Turkey Received 22 July 2004; received in revised form 14 August 2004; accepted 14 August 2004 Available online 23 November 2004

Abstract The toxicity of gentamicin (GEN) in the kidney seems to relate to the generation of reactive oxygen species (ROS). Caffeic acid phenethyl ester (CAPE) has been demonstrated to have antioxidant, free radical scavenger and anti-inflammatory effects. It has been proposed that antioxidant maintain the concentration of reduced glutathione (GSH) may restore the cellular defense mechanisms and block lipid peroxidation thus protect against the toxicity of wide variety of nephrotoxic chemicals. We investigated the effects of CAPE on GEN-induced changes in renal malondialdehyde (MDA), a lipid peroxidation product, nitric oxide (NO) generation, superoxide dismutase (SOD), catalase (CAT) activities, GSH content, blood urea nitrogen (BUN) and serum creatinine (Cr) levels. Morphological changes in the kidney were also examined. A total of 32 rats were equally divided into four groups which were: (1) control, (2) injected with intraperitoneally (i.p.) GEN, (3) injected with i.p. GEN + CAPE and (4) injected with i.p. CAPE. GEN administration to control rats increased renal MDA and NO generation but decreased SOD and CAT activities, and GSH content. CAPE administration with GEN injections caused significantly decreased MDA, NO generation and increased SOD, CAT activities and GSH content when compared with GEN alone. Serum level of BUN and Cr significantly increased as a result of nephrotoxicity. CAPE also, significantly decreased serum BUN and Cr levels. Morphological changes in the kidney due to GEN, including tubular necrosis, were evaluated qualitatively. In addition, CAPE reduced the degree of kidney tissue damage induced by GEN. Both biochemical findings and histopathological evidence showed that administration of CAPE reduced the GEN-induced kidney damage.

Corresponding author. Tel.: +90 422 341 0660 1309; fax: +90 422 341 0036. E-mail address: [email protected] (H. Parlakpinar).

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.08.024


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Our results indicated that CAPE acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of GEN both at the biochemical and histological level. Thus, CAPE could be effectively combined with GEN treatment. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Caffeic acid phenethyl ester; Gentamicin; Reactive oxygen radicals; Renal toxicity; Rat

1. Introduction Aminoglycoside antibiotics are commonly used for the treatment of severe gram negative bacterial infections. Despite their beneficial effects, aminoglycosides have considerable nephrotoxic side effects (Parlakpinar et al., 2003). The most widely used drug in this category is gentamicin (GEN) (Reiter et al., 2002). A major complication of GEN treatment is nephrotoxicity, accounting for 10–20% of all cases of acute renal failure (ARF) according to experimental results (Erdem et al., 2000). Also, 30% of the patients treated with GEN for more 7 days show some signs of nephrotoxicity that markedly limits its use (Pedraza-Chaverri et al., 2000). So, GEN nephrotoxicity promotes both increased morbidity and health-care costs. Although the change in GEN dosing from multiple-daily to once-daily dose has reduced the risk of nephrotoxicity, the incidence of GEN-induced ARF still remains high (Kopple et al., 2002). Neprotoxicity induced by GEN is a complex phenomenon characterised by an increase in blood urea nitrogen (BUN) and serum creatinine (Cr) concentration, and severe proximal renal tubular necrosis followed by deterioration and renal failure (Smetana et al., 1988; Al-Majed et al., 2002). Although the pathogenesis is still not well understood, the toxicity of GEN in the kidney seems to relate to the generation of destructive reactive oxygen species (ROS) in these cells (Reiter et al., 2002; Al-Majed et al., 2002). ROS have been implicated in a wide range of biological functions, but they can express both beneficial and highly toxic effects on cellular homeostasis (Mates, 2000). A large body of in vivo and in vitro evidence indicates that ROS are important mediators of GEN-induced nephrotoxicity (Pedraza-Chaverri et al., 2000; Kopple et al., 2002; AlMajed et al., 2002; Abdel-Naim et al., 1999). ROS have been proposed as a causative agent of cell death in many different pathological states as well as, in glomerular disease (Smetana et al., 1988), in renal ischemia and

reperfusion injury (Longoni et al., 2002), and in various models of toxic renal failure (Piotrowski et al., 1996). Several studies have demonstrated that various agents including melatonin (Ozbek et al., 2000), vitamin E, superoxide dismutase (SOD) (Pedraza-Chaverri et al., 2000), lipoic acid (Al-Majed et al., 2002; Sandhya and Varalakshmi, 1997), zinc (Kumar et al., 2000), ginkgo biloba extract (Maldonado et al., 2003), diallyl disulfide (Pedraza-Chaverr´y et al., 2003) and etc. can prevent GEN-induced renal damage. To date there is no study the protective effect of caffeic acid phenethyl ester (CAPE), a known potent antioxidant, free radical scavenger and anti-inflammatory (Martins et al., 2002) on aminoglycoside antibiotics including GEN. CAPE is an active component of honeybee propolis extracts and has been used as a folk medicine for many years. At a concentration of 10 ␮mol, it completely blocks production of ROS in human neutrophils and in the xanthine/xanthine oxidase system (Ozyurt et al., 2001). Therefore, this experimental study was designed to investigate the possible protective effects of CAPE on nephrotoxicity induced by GEN in a rat model, and to clarify the association between renal malondialdehyde (MDA), nitric oxide (NO) production, SOD, catalase (CAT) activities, glutathione (GSH) content, Cr, BUN levels and GEN-induced nephrotoxicity.

2. Materials and methods 2.1. Experimental conditions Female Wistar rats (aged 8–12 weeks) weighing 200–250 g were placed in a temperature (21 ± 2 ◦ C) and humidity (60 ± 5%) controlled room in which a 12 h:12 h light: dark cycle was maintained. Thirty-two rats were randomly assigned to four groups equally: (1) control group; injected intraperitoneal (i.p.) diluted 1% ethanol with saline (vehicle) for 12 days, (2) GENtreated group; firstly injected i.p. with vehicle for 2

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days and afterwards i.p. with 100 mg/kg GEN (Genta 80 mg, I.E. ULAGAY, Istanbul) for 8 days. At the end of the GEN treatment, continued only vehicle treatment for 2 days, (3) GEN + CAPE-treated group; injected i.p. with 10 ␮mol/kg CAPE for 2 days before GEN treatment and daily during GEN treatment and after the GEN treatment for 2 days, (4) CAPE-treated group; injected i.p. diluted 1% ethanol with CAPE for 12 days. At 24 h after the last injection, a time chosen according to our previous aminoglycoside-related study (Parlakpinar et al., 2003), rats in all groups were killed and the kidneys quickly removed, decapsulated and divided equally into two longitudinal sections. One of these was placed in formaldehyde solution for routine histopathologic examination by light microscopy. The other half was placed in liquid nitrogen and stored at −85 ◦ C until assayed for MDA, NO production, SOD, CAT activities, and GSH content. Trunk blood was extracted to determine the serum levels of BUN and Cr. For these studies, CAPE (Sigma–Aldrich Chemie Gmbh, Steinheim, Germany, 3 mg kg−1 of the salt) was dissolved in ethanol and diluted in saline to give a final concentration of 1% ethanol. Because of the very variable CAPE dosage schemes reported in literature, we administrated CAPE at the dose of 10 ␮mol/kg which concentration was reported that (Ozyurt et al., 2001) completely blocks production of ROS. All experiments in this study were performed in accordance with the guidelines for Animal Research from the National Institutes of Health and were approved by the Committee on Animal Research at Inonu University, Malatya, Turkey. 2.2. Biochemical determination A total of 200 mg of kidney tissue was homogenized in ice-cold 150 mM KCl for determination of MDA. The MDA content of homogenates was determined spectrophotometrically by measuring the presence of thiobarbituric acid reactive substances (TBARS) (Lim et al., 2002). Results are expressed as nmol/g tissue. Since tissue nitrite (NO2 − ) and nitrate (NO3 − ) levels can be used to estimate NO production (Gurlek et al., 2004), we measured the concentration of these stable NO oxidative metabolites (Fadillioglu et al., 2003) in homogenate according to the methods described elsewhere. Quantification of NO2 − and NO3 − was based on the Griess reaction, in which a chromophore


with a strong absorbance at 545 nm is formed by reaction of NO2 − with a mixture of naphthlethylenediamine and sulfanilamide. Results are expressed as ␮mol/g tissue. SOD enzyme activity determination was based on the production of H2 O2 from xanthine by xanthine oxidase and reduction of nitroblue tetrazolium as previously described (Goering et al., 2002). The product was evaluated spectrophotometrically at 560 nm. Results are expressed as U/g protein. CAT enzyme activity was determined according to Aebi’s method (Aebi, 1974). The principle of the assay is based on the determination of the rate constant (s−1 k) or the H2 O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) k/g protein. GSH was determined by the spectrophotometric method based on the use of Ellman’s reagent (Gupta et al., 1999). Results are expressed as ␮mol/g tissue. Serum levels of Cr and BUN were determined using the Olympus Autoanalyzer (Olympus Instruments, Tokyo, Japan). 2.3. Histological analysis For light microscopic evaluation, portions of each kidney were fixed in 10% neutral phosphate-buffered formalin solution. Following dehydration in an ascending series of ethanol (70, 80, 96, 100%), tissue samples were cleared in xylene and embedded in paraffin. Tissue sections of 6 ␮m were stained with hematoxylin–eosin (H–E). Five coded slides from each group were examined by an observer blinded to the treatments. Light microscopy was used to evaluate tubular necrosis which was graded as follows: • Mild (+): only single cell necrosis and slight degenerative changes. • Moderate (++): tubular necrosis at different foci throughout the cortex. • Severe (+++): extensive and marked tubular necrosis throughout the cortex. 2.4. Data analysis Kidney MDA, NO, SOD, CAT, GSH, serum BUN and Cr levels were analyzed by one-way ANOVA. Posthoc comparisons were done using Tukey’s tests. Differences were considered significant at P < 0.05. Results are expressed as mean ± S.E.M.


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3. Results

Table 2 Histological damage in rats (n = number of animals)

3.1. Effect of CAPE on GEN-induced changes in kidney tissue enzymes, lipid peroxides, NO production and GSH content


Grade of tubular necrosis −




Control (n = 8) GEN (n = 8) GEN + CAPE (n = 8) CAPE (n = 8)

8 0 0 8

0 0 5 0

0 2 3 0

0 6 0 0

No animals died during or after the injections, as shown in Table 1. According to the control group; GEN-induced ARF manifested by a significantly increased kidney MDA and NO levels [61.9 ± 3.7 versus 89.1 ± 4.0; 104.9 ± 7.7 versus 159.8 ± 13.8, respectively] while SOD, CAT activities and GSH content significantly decreased [1.69 ± 0.05 versus 1.04 ± 0.01; 242.9 ± 19.1 versus 77.1 ± 8.5 and 12.8 ± 0.5 versus 8.6 ± 0.6, respectively]. CAPE administration with GEN injections caused significantly decreased in MDA, NO generation [89.1 ± 4.0 versus 63.4 ± 3.9; 159.8 ± 13.8 versus 49.3 ± 2.8, respectively] and increased in SOD, CAT activities and GSH content [1.04 ± 0.01 versus 2.04 ± 0.08; 77.1 ± 8.5 versus 236.0 ± 15.6; and 8.6 ± 0.6 versus 12.9 ± 0.9, respectively] in kidney when compared with GEN alone. There were no statistically differences between control and only CAPE-treated groups.

Light microscopy was used to evaluate tubular necrosis and was graded as follows: mild (+): only single cell necrosis and slight degenerative changes; moderate (++): tubular necrosis at different foci throughout the cortex; severe (+++): extensive and marked tubular necrosis throughout the cortex.

tically differences between control and only CAPEtreated groups. 3.3. Effect of CAPE on GEN-induced morphological changes in kidney tissue The histological results are shown in Table 2. The morphological changes in kidney were graded and results were scored as described in Section 2. The kidneys of the control group showed normal kidney parenchyma (Fig. 1). GEN-treated rats showed more extensive and marked tubular necrosis (+++) (Fig. 2). In the GEN + CAPE-treated rats sparse tubular changes were observed (Fig. 3). CAPE apparently reduced kidney tissue damage. On the other hand, there were not any microscopical differences between the control and only CAPE-treated groups.

3.2. Effect of CAPE on GEN-induced changes in serum parameters As shown in Table 1 serum levels of BUN and Cr are significantly higher in the GEN-treated animals when compared with control group [15.57 ± 0.48 versus 27.71 ± 1.08; 0.34 ± 0.02 versus 0.77 ± 0.04, respectively]. Pretreatment of the animals with CAPE significantly reduced the high level of serum BUN and Cr [27.71 ± 1.08 versus 15.0 ± 0.37; 0.77 ± 0.04 versus 0.45 ± 0.03, respectively]. There were no statis-

4. Discussion Since the clinical use of aminoglycosides may be limited by the development of nephrotoxicity, it is im-

Table 1 The effects of gentamicin (GEN) administration to rats with or without caffeic acid phenethyl ester (CAPE) Parameters





MDA (nmol/g tissue) NO (␮mol/g tissue) SOD (U/g protein) CAT (k/g protein) GSH (␮mol/g tissue) BUN (mg/dL) Cr (mg/dL)

61.9 ± 3.7 104.9 ± 7.7 1.69 ± 0.05 242.9 ± 19.1 12.8 ± 0.5 15.57 ± 0.48 0.34 ± 0.02

89.1 ± 4.0a 159.8 ± 13.8a 1.04 ± 0.01a 77.1 ± 8.5a 8.6 ± 0.6a 27.71 ± 1.08a 0.77 ± 0.04a

63.4 ± 3.9b 49.3 ± 2.8b 2.04 ± 0.08b 236.0 ± 15.6b 12.9 ± 0.9b 15.0 ± 0.37b 0.45 ± 0.03b

60.3 ± 2.3 95.8 ± 5.6 1.55 ± 0.01 254.7 ± 9.4 12.7 ± 0.4 14.37 ± 0.55 0.38 ± 0.02

a b

P < 0.05 vs. control group. P < 0.05 vs. GEN

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Fig. 1. Tubules appear normal in the control group. H–E X 66.

portant to be aware of those risk factors associated with a greater incidence of renal damage. The onset of decreasing renal function caused by aminoglycosides usually occurs after 1 week’s treatment (Walker and Duggin, 1988; Solgaard et al., 2000). We are, however,

still short of having brought the safety of aminoglycosides to that of the main other wide-spectrum antibiotics. GEN-induced nephrotoxicity may not only be due to other mechanisms (Hagiwara et al., 1988) but also result from free radical damage. It has been also

Fig. 2. Marked tubular necrosis is observed in the GEN-treated group. Desquamated and degenerated epithelial cells are visible in the lumens of necrotic tubules (*). H–E X 66.


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Fig. 3. Tubules show slight histological changes in the GEN + CAPE-treated group. The tubules revealed normal except the flattening of the epithelia of some tubules (*). H–E X 66.

reported that ROS play major role in the pathophysiology of aminoglycosides-induced nephrotoxicity (Baliga et al., 1998; Rao et al., 1999). Due to the treatment with some antioxidants protects the GEN-induced renal injury, in the present study we focused the effect of CAPE on the renal damage and oxidative injury induced by GEN. We have evaluated the following endpoints of renal damage: (1) renal hemodynamics, (2) determination of antioxidant enzyme activities, lipid peroxidation, NO generation and GSH content, (3) kidney histopathology. Several dosage schemes have been reported for GEN administration. We administrated GEN at the dose of 100 mg/kg/day for 8 days i.p., which is the dosage scheme reported to cause marked nephrotoxicity (Erdem et al., 2000). There are some experimental data suggesting that nephrotoxic drugs may also change levels of MDA, glutathione peroxidase (GSHPx), CAT, SOD, GSH, BUN and Cr (Ozbek et al., 2000), which are commonly used to monitor the development and extent of renal tubular damage due to oxidative stress. CAPE is known to have antioxidative (Sud’ina et al., 1993; Bhimani et al., 1993; Ozen et al., 2004), anti-inflammatory (Sud’ina et al., 1993), reperfusion injury preventive (Koltuksuz et al., 1999), antiprolifer-

ative (Chen et al., 1996), immunostimulatory (Lin et al., 1999), antibacterial (Setzer et al., 1999), antiviral (King et al., 1999), anticancer (Chen et al., 1996; Lee et al., 2000), antiatherosclerotic (Nardini et al., 1995), and neuroprotective (Kim and Kim, 2000), these properties are associated with either their as antioxidants and enzyme inhibitors or their binding activity specific receptors. Related to the above beneficial properties of CAPE, this experimental study was designed to investigate the effects of CAPE on GEN-induced renal changes using both biochemical determinations and the morphology of the kidney using light microscopy. Results of this study confirmed that GEN at a dose of 100 mg/kg/day produces nephrotoxicity as evident by the reduction in GFR which is shown by increase in serum Cr. Recently, it has been reported that for humans, serum Cr in association with certain other clinical characteristics may be a more accurate measure of the GFR than Cr clearance (Kopple et al., 2002). This impairment in glomerular function was accompanied by an increase in BUN. Serum Cr concentration is more significant than the BUN level in the earlier phases of kidney disease. On the other hand, BUN begins to rise only after a marked renal parenchymal injury occurs (Erdem et al., 2000). In the present study, increase in serum Cr and BUN levels induced by GEN was signif-

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icantly blocked by CAPE. The success of CAPE in reducing Cr and BUN concentrations could be attributed to its antioxidant properties because it has been found that ROS may be involved in the impairment of GFR (Pedraza-Chaverri et al., 2000). It has been proposed that antioxidants maintain the concentration of reduced GSH and may restore the cellular defense mechanisms and block lipid peroxidation, thus protecting against the toxicity of wide variety of nephrotoxic chemicals (Babu et al., 1995). Depletion of renal GSH, which is one the primary reasons for the resulting lipid peroxidation, may cause increases in MDA levels. As with the current findings, these results indicate that the generation of free radicals and subsequent lipid peroxidation may play a role in GEN toxicity. In the current study, GEN given alone significantly decreased MDA and NO levels while SOD, CAT activities and GSH content were reduced in the kidney tissue. Similar results were also observed by Ozbek et al. (2000). GEN nephropathy was associated with low activity of GSH-Px, CAT, SOD and GSH content in the renal cortex. This decreased renal antioxidant enzymatic defense could aggravate the oxidative damage in these rats. The exaggerated production of ROS in GEN-induced nephrotoxicity could induce inactivation of antioxidant enzymes. Administration of CAPE injection caused significantly decreased MDA levels in the kidney when compared with GEN alone. MDA, measured as TBARS, in the renal cortex resulting a decrease in the polyunsaturated fatty acid content, which serve as substrate for free radical attack (Al-Majed et al., 2002). Additionally, CAPE treatment prevented the decrease in SOD and CAT activities may be used as a marker of tubular damage. In accordance with the our results, more recently reported that treatment with free-radical scavenger CAPE attenuated the increase in plasma BUN and kidney NO levels, and showed histopathological protection against cisplatininduced acute renal failure. Also CAPE reduced levels of CAT, SOD and GSH-Px (Ozen et al., 2004). However, recently Gurel et al. (2004) reported that in rat renal ischemia-reperfusion injury CAPE could not a significant increase or decrease in the activity of CAT but significantly increased SOD activities. CAPE may be beneficial in breaking the vicious circle by increasing the activities of SOD, CAT and GSH contents. Our results strongly indicate that CAPE is important in pro-


tecting the kidney from GEN-induced injury, through improvement in oxidant status and a possible antioxidant activity. These findings correlated well with the renal histological examination which revealed that more extensive and marked tubular necrosis in the GEN-treated kidney. Similar changes were also reported by Kumar et al. (2000) and others (Al-Majed et al., 2002) demonstrating structural changes in renal tissue of GEN-treated animals and its protection by various agents. Administration of CAPE reversed kidney damage with especially a marked reduction in tubular damage (Table 2) induced by GEN. In accordance with the previous reports, the results of the present study demonstrate that GEN-treated rats show accelerated lipid peroxidation in the renal tissue as reflected by an increase in MDA and reduced antioxidant enzyme activities. Considering the reduced oxidative damage due to CAPE treatment, all investigators attributed protective actions of CAPE to its antioxidative, free radical scavenger and anti-inflamatory activity. This data is consistent with our results that CAPE administration significantly afforded protection against nephrotoxicity induced by GEN treatment. The success of CAPE in GEN toxicity implies the involvement of free radicals in the renal damage, although other destructive processes may also be involved. According to our findings, which was in parallel with histopathological evidence administration of CAPE abolished certain effects of GEN such as the decreased levels of antioxidant enzymes or the increased levels of MDA, NO, Cr and BUN. We propose that CAPE acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of GEN both at the biochemical and histological level. However, further studies are essential to elucidate the exact mechanisms of protection and the effect of CAPE.

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