Life Sciences, Vol. 66, No. 26, pp. 2603-261 I Zoo0 Copyright 0 2000 Elsevier Science Inc. Printed in the USA. All rights reserved

0024-320S/OOI.%see front matter

PII s0024-3205(08)80594-4

CARVEDJLOL : A BETA BLOCKER WlTH ANTIOXIDANT PROPERTY PROTJXTS AGAINST GENTAMK!WmUCED NEPEROTOXIClTY IN RATS

K.Vijay Kumar, Anwar A. Shifow, M.U.R.Naidu, l K.S.Ratnakar

Central Research Laboratory, Departments of Clinical Pharmacology & Therapeutics & *Pathology, Nizam’s Institute of Medical Sciences, Hyderabad, India

(Received in foal form February 2,200O)

SnlllM~

Gentamicin is an antiiiotic effective against gram negative infections, whose clinical use is limited by its nephrotoxicity. Since the pathogenesis of gentamicin-induced nephwtoxicity involves oxygen free radicals, the antioxidant carvedilol may protect against gentamicin-induced renal toxicity. We therefore tested this hypothesis using a rat model of gentamicin nephrotoxicity. Carvedilol (2 mg/kg) was administered intra- peritoneally 3 days before and 8 days concurrently with gentamicin (80 mg/kg BW). Estimations of urine creatinine, glucose, blood urea, serum creatinine, plasma and kidney tissue malondialdehyde (MDA) were carried out, after the last dose of gentamicin. Kidneys were also examined for morphological changes. Gentamicin caused marked nephrotoxicity as evidenced by increase in blood urea, serum creatinine and decreased in creatinine clearance. Blood urea and serum creatinine was increased by 883% and 480% respectively with gentamicin compared to control. Carvedilol protected the rats from gentamicin induced nephrotoxicity. Rise in blood urea, serum creatinine and decrease in creatinine clearance was significantly prevented by carvedilol. There was 190% and 377% rise in plasma and kidney tissue MDA with gentamicin. Carvedilol prevented the gentamicin induced rise in both plasma and kidney tissue MDA. Kidney from gentamicin treated rats, histologically showed necrosis and desquamation of tubular epithelial cells in renal cortex, whereas it was very much comparable to control with carvedilol. In conclusion, carvedilol with its antioxidant property protected the rats from gentamicin-induced nephrotoxicity.

Key ~o’ords: carvedilol, antioxidant, gentamicin-induced nephrotoxicity

Most clinical studies have shown that an aminoglycoside antibiotic gentamicin with potent activity against gram-negative infections induced nephrotoxicity that limits its therapeutic utility (1,2). Nepbrotoxicity induced with gentamicin is a complex phenomenon characterized by proximal tubular injury followed by deterioration and renal failure (3). The specificity of gentamicin renal toxicity is related to its accumulation in the renal convoluted tubules and also its effect on biological membranes. Gentamicin-induced nephrotoxicity also results in the loss of brush border integrity of proximal convoluted tubule (4). Expression of heat shock proteins such as HSP47 in tubular epithelial cells and interstitial cells was also noted in gentamicin treated animals (5). A role of platelet-activating factor (PAF) has also been suggested in gentamicin-induced nepbrotoxicity (6). Numerous studies, both in vitro and in vivo have demonstrated that reactive oxygen metabolites including superoxide anion, hydrogen peroxide, hydroxyl radical are implicated in gentamicin-induced nephrotoxicity (7-10). Free radicals have also been implicated in glomerular diseases ( 11,12) and neutrophil mediated glomerular

Corresponding author: Dr.M.U.R.Naidu, Department of Clinical Pharmacology L Therapeutics, Nizam’s Institute of Medical Sciences, Punjagutta, Hyderabad 500082, India. Fax: 009140 33 10076.

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diseases (13,14). Ramasammy and coworkers have demonstrated that there is an increase in renal cortical lipid peroxidation in gentamicin treated rats (7,15) and in vitro enhances generation of hydrogen peroxide by renal cortical mitochondria (8). Hydrogen peroxide in particular is toxic to both glomerular and the tubulointerstitial corn- either directly or tbrough generation of the hydroxyl radical under the catalytic effect of iron (16,17). The normal cells also generate large amounts of hydrogen peroxide, however they are easily scavenged by the antioxidant enzymes (18). Our recent studies were also suggested that nephrotoxicity induced by cyclosporine was mediated by oxygen free radicals and lipid peroxidation (19,20).

Many different chemical agents were used to prevent gentamicin-induced nephrotoxicity. Administration of Zinc has been shown to prevent gentamicin-induced nephrotoxicity in rats by enhancing the metallothionine synthesis (9). Lipoic acid had also been shown to protect gentamicin nephrotoxicity (2 1). Rats treated with platelet activating factor (PAF) antagonist BN- 5202 1 prevented the gentamicin-induced nephrotoxicity (6). Carvedilol (CAR) is an antihypertensive drug, with non- selective beta-adrenergic and non-selective alpha-adrenergic blocking activity. In addition to antihypertensive action, carvedilol has potent antioxidant activity (22). The antioxidant activity of carvedilol has been characterized in great detail in a variety of in vitro test systems and animal models. It inhibits superoxide anion release from activated neutrophils and inhibits the lipid peroxidation in myocardial cell membrane in vitro (23). Carvedilol prevents the loss of cardiac myocytes that occur in heart failure as a result of oxidative stress (24) and prevents lipid peroxidation in cardiac tissue (25). In cultured endothelial cells carvedilol has been shown to protect against oxygen radical induced cell injury (26). Based on these evidences we therefore utilised a rat model of gentamicin nephrotoxicity to determine carvedilol nephroprotective activity.

Materials & Methods

Male wistar rats were maintained under 1Zhour light02 hour dark cycle at 22°C and were fed standard laboratory chow obtained Tom National Facility for Supply of Animals, National Institute of Nutrition, Hyderabad, India. The composition of diet contains wheat flour 22.5%, roasted bengal gram flour 60%, skim milk powder 5%, casein 4%, refined oil 4%, salt mixture with starch 4%, vitamin & choline mixture with starch 0.5%. Experiments were performed in four groups of adult male wistar rats (weighing 200-250 g) injected intraperitoneally with: (1) ethanol (0.4%/kg BW) for 11 days for control; (2) gentamicin (80 mg/kg BW) for 8 days; (3) carvedilol(2 mg/kg BW in 0.4% ethanol/kg BW) for 11 days; (4) carvedilol(2mg&g BW) 3 days before and 8 days concurrently with gentamicin. At dose 80 mg/kg body wt intraperitoneally for eight days gentamicin is known to cause significant nephrotoxicity (27). The water intake by each animal was 15 f 2.0 ml/day.

Biochemical assays: At the end of study, rats were kept in an individual metabolic cage for 24 hour urine collection. Blood was collected by ocular puncture method. After 24 hours of last dose of gentamicin treatment, animals were sacrificed by cervical dislocation under ether anesthesia. For plasma, blood was collected in a heparinized vial and serum in plain vial and were separated at 4°C in a refrigerator centrifuge for 15 minutes at 3500 rpm. Blood urea was measured by L-glutamate dehydrogenase (GLDH>kinetic method and creatinine clearance was calculated after estimating urine and serum creatinine by alkaline picrate method (28) using Beckman Spectrophotometer. Glucose was determined in urine by glucose ox&se-peroxidase (GOD-POD) method using a commercially available kit from Boehringer Manheim, Germany.

Lipid Peroxidation products: The degree of lipid peroxidation in the renal cortex was determined by measuring thiobarbituric acid reactive substances (TBARS) as described previously (29). Portions of renal cortical tissue were disseLted, weighed, minced and homogenized in PBS (pH 7.4) containing 1 mM EDTA by use of a Potter-Elvehjem smooth glass homogenizer. The homogenate were centrifuged at 3500 rpm and supematant were collected and used for the estimation of protein and MDA. The 10% of the

Vol. 66, No. 26,200O Carvedilol on Gentamicin-induced Nephrotoxicity 2605

homogenate ( 1 to 1.5 mg protein) was precipitated with 20% trichloroacetic acid, vortex and centrifuged at 3500 rpm for 30 minutes. To the protein free supematant (1 .Oml), 0.33% thiobarbituric acid (0.25ml) was added and boiled for 1 hour at 95“C, the TBA reactive products were extracted in butanol (l.Oml) and the intensity of the pink colour was read at 520 run. Freshly diluted tetramethoxypropane (Sigma Chemical Co., USA) which yields malondialdehyde (MDA) was used as a standard. The protein content of the homogenate was measured by the method of Lowry (30) and the data were expressed as nmoles of MDA-equivalents/mg of protein. Similar procedure was followed to estimate MDA in plasma, where 0.5 ml of plasma was used instead of homogenate.

Histopathological Examination: The kidneys from all the four treated groups were fixed in 10% formalin in phosphate buffer (pH 7.0) for 24 hours at room temperature (37°C) for light microscopy.. The sections were cut at 5 urn and stained with hematoxylin and eosin. The slides were coded and were examined by a histopathologist who was ignorant about the treatment groups. Sections were examined and assigned scores as reported by Houghton et al (31), which is as follow: 0 = Normal, 1 = areas of focal granulovacoular epithelial cell degeneration and granular debris in tubular lumens with or without evidence of tubular epithelial cell desquamation in small foci (1% of total tubule population involved in desquamation); 2 = tubular epithelial necrosis and desquamation easily seen but involving less than half of the cortical tubules; 3 = more than half of proximal tubules showing desquamation and necrosis but involved tubules are easily found; 4 = complete or almost complete tubular necrosis.

Statistical Analysis: Reported values are mean f SD (n=6). The significance of differences among four groups was assessed using one way analysis of variance (ANOVA) and for multiple comparison TUKEY’S HSD test was used. The significance was set at the p < 0.05.

Data reported in Table 1 showed that the body weight of gentamicin and gentamicin plus carvedilol treated groups was significantly lower than in control. There was a significant decrease in 24-hour urine volume in gentamicin treated rats than in control. In carvedilol plus gentamicin treated rats 24-hr urine volume was significantly increased compared to gentamicin and control group. Blood urea and serum creatinine levels were markedly increased with gentamicin. In control, serum creatinine was 0.5& 0.04 mg/dl, with gentamicin it increased to 2.4 f 0.22 mg/dl, while it was 0.52 f 0.09 mg/dl and 1.76 a. 12 mg/dl in CAR and CAR + gentamicin group. Blood urea concentration was also markedly elevated with gentamicin, CAR treatment prevented gentamicin-induced increase in blood urea. In

TABLE 1

Effect of gentamicin and carvedilol on body weight and renal parameters in rats

Parameter Control Carvedilol Gentamicin Carvedilol + Gentamicin

B W &change) +4 +2 -8 -7 UV(mY24 hrs) 20.00 f 5.0 22.20 f 4.2 7.00 f 2.8*$ 33.00 f lo.o*+$ Ccr (ml/hr) 0.50 f 0.04 0.49 f 0.08 0.05 * 0.04’9 0.32 f O.lO*+$

Ur. Glucose(mg/dl) 0.46 f 0.20 0.42 f 0.26 56.00 f 19.00*$ 7.65 f 1.60” $

Data are mean f SD of 6 rats in each group; BW, body weight. UV, urine volume, Ccr, creatinine clearance. *p < 0.05 vs control group; +p < 0.05 vs gentamicin group %p < 0.05 vs carvedilol control group

2606 Carvedilol on Gentamicin-induced Nepbrotoxicity Vol. 66, No. 26,200O

400, 37

0 0 GEN CAR GEN+CM

(4 (W Fig. 1.

Effect of carvedilol on gentamicin-induced changes of (a) blood urea; (b) serum creatinine. GEN indicates gentamicin; CAR, carvedilol. *p < 0.05 vs control group; +p < 0.05 vs gentamicin group. $p < 0.05 vs carvedilol control group

(b) (4 Fig. 2.

Effect of carvedilol on gentamicin-induced enhancement of (a) Plasma; (b) Kidney tissue TBARS “p < 0.05 vs control group; +p < 0.05 vs gentamicin group; $p < 0.05 vs carvedilol control group

Vol. 66, No. 26,200O Carvedilol on Gentamicin-induced Nephrotoxicity 2607

control, gentamicin, CAR and CAR + gentamicin treati groups the mean blood urea was 31 i 4.2, 274 f 40 mgldl, 30 f 3.8,115 l 18 mg/dl respectively (fig 1). Rats treated with gentamicin showed decrease in creatinine cleamnce(Ccr) (0.050 JZ 0.004 ml/h&g) compared to control (0.50 * 0.04). CAR did not alter Ccr perae, but sign&antly prevented gentamicin-mduced fill in Ccr (0.32 f 0.10 ml/hr/kg). Urinary excretion of glucose significantly increased in gentamicin treated group, CAR treatment prevented gentamicin-induced glycosuria.

Malondialdehyde (MDA) a product of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS) significantly increased in plasma and kidney tissue of gentamicin treated rata. &administration of CAR plus gentamicin produced a significant decrease in kidney tissue and plasma MDA (fig. 2). The histological changes of all the groups were graded and the results are expressed in table 2. Gentamicin treated rats showed necrosis and desquamation of the tubular epithelial cells in the renal cortex (fig. 3). In addition to the necrosis of proximal tubules, formation of hyaline casts, dilation of distal tubules was also observed in gentamicin treated rats (score 3). CAR administration per se did not produced any changes, whereas the extent of tubular injury was markedly reduced in CAR plus gentamicin group (score 1).

TABLE 2

Grading of h&topathological examha tion of rat lddney treated with pntamlcin and arvcdilol

Group Grade

Control Carvedilo Gentamicin Gentamicin +

0+ 0+ 4+ 1+

The clinical usefulness of gentamicin is limited due to nephrotoxicity, manifested as acute tubular necrosis and impairment in renal function. Results of this study contirmed that gentamicin at a dose of 80 m&g BW produces significant nephrotoxicity as evidenced by increase in blood urea, serum creatinine, decrease in creatinine clearance and histopathological changes in the kidney tissues. Similar results was also observed by Walker and Shah (32). Gentamicin produced significant decrease in body weight. Ali & coworkers observed dose dependent decrease in body weight in gentamicin (20, 40, SOm&kg BW) treated rats (33). There was a significant increase in urinary glucose in gentamicin treated rats. This increase was also reported by others in rats (27) and dogs (34). Carvedilol protected the animals against gentamicin-induced nephrotoxicity. There was minimal impairment in renal tinction with gentamicin in carvedilol treated animals. Pretreatment of carvedilol showed marked functional and histological protection against gentamicin nephrotoxicity. Increase in blood urea and serum creatinine and reduction in cm&nine clearance induced by gentamicin was significantly blocked by carvedilol. Several studies have demonstrated that gentamicin induced nephrotoxicity can be prevented by the various agents. Use of polyaspartic acid prevented functional and histological changes of gentamicin nephrotoxicity (35). Administration of pyridoxal-5’ phosphate (36), diphenyl-phenybnediamine (37) and sodium (38) ameliorate gentamicin nephrotoxicity. Wong et al showed that rats supplemented with magnesium had better &&nine clearance and histology than gentamicin (40). Piperacillin has protective role in gentamicin nephrotoxicity by interfering renal tubular uptake of gentamicin (41).

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Fig. 3. Histopathological examination of rat kidney. (A) Control, (B) gentamicin, (C) gentamicin +

Carvedilol (HE x 60).

Vol. 66, No. 26,200O Carvedilol on Gentamicin-induced Nephrotoxicity 2609

The results of our present experiment confirms the earlier studies (15,32) that gentamicin treated rats show accelerated lipid peroxidation in the renal tissue as reflected by increase in MDA, an end product of lipid peroxidation. Increase in lipid peroxidation in renal cortex resulted in decrease in polyunsaturated fatty acid content, which serve as substrate for free radical attack (42,43). Pretreatment of carvedilol significantly prevented the gentamicin-induced lipid peroxidation in plasma and kidney tissue. This is most probably due to less damage by oxygen free radicals with carvedilol. The involvement of oxygen tiee radicals in tissue injuries is well-established (3,44,45). A relationship between nepbrotoxicity and oxidative stress has been confimed in many experimental models. Many investigators described the role of reactive oxygen species including hydroxyl radical in gentamicin- induced nephrotoxicity (8,32). A role of lipid peroxidation in gentamicin-induced lipid peroxidation has been described by evaluating the effect of diphenylene diamine and vitamin-E (37,46). The nephrotoxic symptoms due to adriamycin were significantly reduced with superoxide dismutase and alpha-tocopherol (47,48). Administration of large doses of vitamin-E, prevented the gentamicin- induced proximal tubular cell lipid peroxidation in vitro (49) and in vivo (50). It also increases the activities of superoxide dismutase, glutathione peroxidase in renal cortex of remnant kidney and lipid peroxidation, glomeruiosclerosis were reduced (5 1).

The effects of carvedilol on renal hemodynamics and renal function have been studied in animals (52) and humans (53). It reduces proteinuria, serum creatinine and blood urea nitrogen and glomerulosclerosis in rat model of chronic renal failure (54). Carvedilol has renoprotective effect in patients (55). Results of the present study indicate tbat carvedilol protects the kidneys from gentamicin-induced nephrotoxicity through its antioxidant property. It prevents electron adduct formation in both aqueous (5-5-dimethylpyrroline-l-oxide, DMPO) and lipid (2- methylnitrosopropane, MNP) environments containing either superoxide or hydroxyl-radical generating system (22). Carvedilol prevents lipid peroxidation in brain and heart membranes both in vitro and in vivo (56). The metabolite of carvedilol (SB 209905 and SB 211475) are extremely potent antioxidant being 50 to 100 fold more potent than carvedilol and 1000 to 10,000 fold more potent than vitamin-E (57,58). Carvedilol protects endothelial cells and vascular smooth cells from oxygen radical mediated injury and inhibits the formation of oxidized LDL (59). It preserves the endogenous antioxidant system (vitamin E and glutathitine) which is normally consumed when tissues or organs are exposed to oxidative stress (60). Recent study showed that carvedilol protects against glomerulosclerosis in rat remnant kidney without general changes in antioxidant enzyme status (61). Pretreatment of superoxide diimutase (8000 W/kg) showed significant protection against gentamicin nepbrotoxicity (62). In our recent study, we observed that melatonin, an antioxidant significantly protected the gentamicin-induced nephrotoxicity in rats (63).

Histopathology of the renal tissues of the rats treated with gentamicin showed necrosis and desquamation of the tubular epithelial cells in renal cortex. Similar changes were also reported by Nakakuki (64). There was a significant glycosuria in gentamicin treated rats indicating proximal tubular injury(34). Many investigators have demonstrated the structural changes in renal tissue of gentamicin treated animals and its protection by various agents. Use of hydroxyl radical scavenger Dimethylthiourea (DMTU), protected the histological changes observed in gentamicin treated rats (32). Results of the present study showed marked histological protection by CAR. Sandhya et al (21) reported that gentamicin treated rats showed the presence of homogenous materials in the form of droplets of masses in the proximal convoluted tubules and tbe interstitium showed inflammatory filtrate and the changes were markedly reduced with lipoic acid treatment. In summary, gentamicin- induced nepbrotoxicity is related to lipid peroxidation. Pretreatment of carvedilol provided protection against gentamicin nephrotoxicity by inhibiting the free radical mediated process.

Acknowledgements

This work was supported by the Indian Council for Cultural Relations, Govt. of India to Anwar Ahmed Shifow.

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