Original Article Levofloxacin and Gentamicin Induced Nephrotoxicity Pak Armed Forces Med J 2015; 65(4): 439-43 DETERMINATION OF THE EFFECTS OF LEVOFLOXACIN ON GENTAMICIN INDUCED NEPHROTOXICITY IN RABBITS: A COMPARATIVE STUDY Uzma Naeem, Shahid Jamal, Akbar Waheed Army Medical College, National University of Sciences and Technology Islamabad, Pakistan ABSTRACT Objective: To determine the effects of levofloxacin on gentamicin induced nephrotoxicity in rabbits. Study Design: Comparative experimental study. ORIGINAL ARTICLES Place and Duration of Study: The animal house of Army Medical College, Rawalpindi, and the pathology department of Army Medical College, Rawalpindi, from July 2009 to January 2010. Material and Methods: The effects of levofloxacin on gentamicin-induced nephrotoxicity were evaluated in rabbits. Twenty four rabbits were used in this study which were randomly divided into four groups (n= 6 in each group). Six animals were injected for 15 days with saline (NaCl; 0.9%), six with gentamicin alone at doses of 20 mg/kg of body weight/12 h (intramuscularly), six with combination of gentamicin (20 mg/kg/12 h) with low therapeutic doses of levofloxacin (30 mg/kg/24 h) and the last six were treated with gentamicin and high therapeutic doses of levofloxacin (50 mg/kg/24 h). Levofloxacin was given by intraperitoneal route. Results: Gentamicin induced nephrotoxicity was evaluated by histopathological and serum analysis. The extent of nephrotoxicity was significantly increased when gentamicin was given in combination with levofloxacin both in low and high doses. Conclusion: Levofloxacin enhances gentamicin induced nephrotoxicity and extent of this nephrotoxicity increased with increasing dose of levofloxacin. Keywords: Gentamicin, Histopathology, Levofloxacin, Nephrotoxicity, Serum analysis. INTRODUCTION Kidney is the target organ for numerous environmental toxins and therapeutic agents like antibiotics 1. One of the most important side affects of antibiotics is acute or chronic renal failure. Wide range of toxicity has been reported with commonly rates of 5-14% for nephrotoxicity 2. Although aminoglycosides are being used for the treatment of several gram negative bacterial infections, but consumption of large quantities may cause nephrotoxicity. Gentamicin is probably one of the most commonly used aminoglycosides for the treatment of gram negative infections 3. Like all other aminoglycosides, gentamicin is essentially eliminated by glomerular filtration. Gentamicin gets accumulated in proximal tubules and leads to the production of free radicals in these Correspondence: Dr Uzma Naeed, House No. 04, Main Street, Salah ud din Avenue, Link Airport Road, Rawalpindi, Pakistan Email: uzmanaem88@gmail.com Received: 12 Jul 2010; revised received 29 Apr 2015; accepted 30 Apr 2015 tubules 4. However the pathological mechanisms involved in gentamicin induced nephrotoxicity include induction of oxidative stress, apoptosis, necrosis, up regulation of transforming growth factor B (TGF-B), elevation of endothelin1 and increase in monocyte/macrophage infiltration 5. Levofloxacin is a synthetic broad spectrum antimicrobial belonging to the class of fluoroquinolones. Levofloxacin has an extended spectrum of activity compared with oldergeneration fluoroquinolones (ciprofloxacin, ofloxacin), with improved activity against gram-positive bacteria and excellent activity against gram-negative bacteria and atypical organisms 6. It is primarily excreted by the kidneys. The effects on kidney that have been reported rarely with levofloxacin include mild interstitial nephritis, occult blood in urine, decreased urine function and crystallurea 7. Levofloxacin has proven efficacy against Pseudomonas aeruginosa infections and is also in use in combination with gentamicin for treatment of pseudomonal infections 7. As levofloxacin and gentamicin are used synergistically, it is imperative that effects of 439
levofloxacin on gentamicin induced nephrotoxicity should be evaluated. MATERIAL AND METHODS This study was a comparative experimental study which was carried out in the animal house of Army Medical College and analysis was done in the pathology department of Army Medical College Rawalpindi. Duration of study was six months. Gentamicin preparations commercialized for clinical applications were used for the current study 8. Injection gentamicin (containing 80 mg of gentamicin sulfate in 2ml solution) prepared by Reckette and Colman Pharmaceuticals, Karachi, Pakistan were purchased from local market. Pure salt of levofloxacin was generously donated by Amson Pharmaceuticals, Rawalpindi, Pakistan. From this salt solutions were prepared for intraperitoneal injections. All 36 animals were divided randomly into four groups, with six rabbits in each. Gentamicin was administered intramuscularly 9, whereas levofloxacin was administered by intraperitoneal route 16. Gentamicin was given at twelve hour interval at 8 a.m and at 8 p.m 8 while levofloxacin was given at 24 hour interval 17 for a period of fourteen days according to the following regimen: Group 1 (G-1) served as control and received 0.5 ml of isotonic saline twice daily 8. Group 2 (G-2) received gentamicin sulfate Table: Renal functions of the rabbits on serum analysis. TESTS G-1 G1 G-2 G-2 G-3 G-3 G-4 G-4 BUN 7.2 15.92 27.32 29.38 mmol/l 7.3 6.82 5.88 7.33 ±0.82 ±0.7 ±0.56 ± 0.52 ±0.3 ± 0.81 ±0.84 ± 10.18 p<0.001 p<0.001 p<0.001 S. Creatinine 92.5 139.83 161.5 186.5 µmol/l 89.67 87 87.83 85.83 ±6 ±5.65 ±6.72 ± 13.20 ±4.51 ± 15.07 ±6.10 ± 29.09 p<0.01 p<0.01 p<0.001 S. Sodium 137.5 138.17 135 122.5 mmol/l 138 136.5 136.33 139.33 ±1.86 ± 1.07 ±1.75 ± 3.98 ±2.06 ± 1.68 ±1.09 ± 3.71 p= NS p = NS p<0.04 S. Potassium 6 5.63 6.1 8.67 mmol/l 6.17 4.88 6.03 4.85 ±0.17 ± 0.15 ±0.3 ± 0.31 ±0.17 ± 0.23 ±0.2 ± 0.35 p = NS p = NS p<0.04 S. Calcium 3.1 2.86 2.8 2.8 mmol/l 3.18 3.13 3.22 3.15 ±0.05 ± 0.07 ±0.07 ± 0.21 ±0.1 ± 0.5 ±0.05 ± 0.11 p = NS p = NS p = NS 40mg/kg/day, nephrotoxic dose 10. Group 3 (G-3) received combination of gentamicin 40 mg/kg/day and levofloxacin 30 mg/kg/day 11. Group 4 (G-4) received gentamicin 40 mg/kg/day and levofloxacin 50 mg/kg/day 12. Blood was collected twice during the study period from the marginal ear veins. First samples were taken at the start of study and second after 15 days. The collected blood was allowed to clot at room temperature and then centrifuged at 3000 rpm for 15 minutes. Serum was separated with the help of an automatic micropipette and stored in a clean and dry serum storage vial at -20 0 C for estimation of 440
Levofloxacin and Gentamicin Induced Nephrotoxicity urea, creatinine and electrolytes potassium and calcium) later on. (sodium, Pak Armed Forces Med J 2015; 65(4): 439-43 RESULTS After 15 days of treatment, a significant difference was observed between the groups. Gentamicin given for 15 days at 40 mg/kg/day All the animals were sacrificed 24 hours after the last dose of the drug8. The abdomen was opened by a longitudinal incision immediately after death. Both the kidneys were dissected out and removed after cutting the ureters and renal vessels. Capsules were stripped off gently. Kidney specimens were sliced sagitally and placed in 10% formaline for 24 hours. The kidney tissue then processed for paraffin embedding. Approximately 3-5µm thick sections were taken with a rotary microtome. Sections were mounted on glass slides and stained with Hematoxylin and Eosin stain (H. & E. stain) for routine histopathological study under light microscope with special attention to proximal tubules. Figure-1: Microscopic structures of rabbit renal cortex of animals treated with gentamicin showing tubular necrosis (X400). Serum creatinine and BUN levels were measured by automated enzymatic assays. Serum creatinine was measured by Jaffe reaction13 and BUN levels by urease kinetic method14. Serum sodium and potassium were measured by ion selective electrode method and serum calcium by CPC method. Histopathological abnormalities were scored on plastic sections at X400 magnification. Each slide was coded. Sections were taken from three different regions of renal cortex for each rabbit. The lesions in renal cortex were scored: 0= No cell necrosis, 1= Mild, only single cell necrosis in sparse tubules, 2= Moderate, more than one cell involved in sparse tubules, 3= Marked, tubules exhibiting total necrosis in almost every power field, 4= Massive total necrosis15. Figure-2: left; Microscopic structures of proximal tubules of the rabbits treated with combination of gentamicin and levofloxacin (low doses) showing necrosis, inflammatory infiltrate and flattening and desquamation of tubular cells (X400). Right; Microscopic structures of proximal tubules of rabbit kidneys treated with gentamicin and high doses of levofloxacin showing inflammatory cells and lost tubular as well as cellular pattern (X400). The standard error of means was calculated on computer using Microsoft Office Excel 2007. In order to find the significance of the difference between two observations Student s t test was used. The difference between two observations was considered significant if the p value was less than 0.05. The results of histopathology were analyzed by using the Chi Square Test. The difference between the two observations was considered significant if the p value was less than 0.05. induced a significant change in serum creatinine levels and BUN compared with control animals, their p values were 0.001 and 0.01 respectively. Concomitant administration of levofloxacin with gentamicin both in low and high doses significantly raised serum creatinine and BUN levels, although both parameters were still significantly higher in the rabbits receiving combination of gentamicin and high doses of levofloxacin (p < 0.001 for both parameters). 441
Serum sodium, potassium and calcium levels were statistically almost unchanged in animals treated with gentamicin alone and with gentamicin-levofloxacin (low doses) combination on day 15. But a significant change in serum sodium (p < 0.04) and serum potassium levels (p < 0.01) was observed in animals treated with combination of gentamicin and high doses of levofloxacin as compared with control. Data is shown in the table. After 15 days of treatment with gentamicin alone, renal proximal tubular cells showed patchy necrosis, grade 2 necrosis (figure-1). While treatment with gentamicin plus low doses of levofloxacin resulted in slightly increased focal necrosis of proximal tubules but overall assessment was also that of grade 2 (figure 2 left). In this group infiltration of interstitium with different inflammatory cells including some eosinophils (showing type ш hypersensitivity reaction) was also observed, particularly in the surroundings of necrotic tubules. As the dose of levofloxacin was increased element of necrosis and inflammatory component both were augmented, but the tubular necrosis was still in grade 2 with more marked inflammatory infiltration of interstitium (figure-2 right). By applying chi square test p value was not significant. DISCUSSION This study strongly suggests that levofloxacin enhances the gentamicin-induced nephrotoxicity. This assumption is based on various criteria such as the rise in serum creatinine and BUN levels and more severe histopathological lesions in the renal cortex of the animals treated with gentamicinlevofloxacin combination (although p value is insignificant) compared with the animals treated with gentamicin alone. Levofloxacin is one of the safest and best tolerated fluoroquinolone over a wide range of daily doses 16. It is not primarily nephrotoxic, but it has nephrotoxic potential especially in old age and in immuno compromised patients 17. Our present work was based upon the hypothesis that fleroxacin, a fluoroquinolone can attenuate gentamicin induced nephrotoxicity in rats 8. Protection against aminoglycoside induced nephrotoxicity has been demonstrated for many antibiotics or chemical compounds. Actually, several antimicrobials including ceftriaxone, ticarcillin, carbenicillin, latamoxef and daptomycin 18,19, decrease gentamicin induced nephrotoxicity. This protection occurs with or without a reduction in the uptake of gentamicin, suggesting different mechanisms of protection. Even though such combinations of gentamicin and levofloxacin are not additive or synergistic in vitro 8, they are often used clinically, like the combination of gentamicin and antipseudomonal fluoroquinolones (ciprofloxacin and levofloxacin) in the treatment of Pseudomonas aeruginosa infections 20. Besides gentamicin persists within the renal parenchyma for many months after their use, and since they are often used as step down therapy following intravenous therapy, the chances of exposing the kidneys to these combinations are high. CONCLUSION It can be concluded from present work that caution must be exercised when using gentamicin with levofloxacin due to enhancement of nephrotoxic potential. It is also concluded that nephrotoxic potential enhances with increasing the dose of levofloxacin. CONFLICT OF INTEREST This study has no conflict of interest to declare by any author. Acknowledgement: The authors acknowledge the assistance of Mr Mukarram Shah, Lab Tech Histopathology, for preparing histopathology slides. REFERENCES 1. Solhauq MJ, Bolqer PM, Jose PA. The developing kidney and environmental toxins. Pediatrics 2004; 113(4): 1084-91. 2. Baykal A, Sarigul F, Suleymanlar G, Moreira PI, Perry G, Smith MA, et al. Ciprofloxacin does not exert nephrotoxicity in rats. Am J Infec Dis 2005; 1(3) : 145-48. 3. Martinez-Salqado C, Lopez-Hernandez FJ, Lopez-Novoa JM. Glomerular nephrotoxicity of aminoglycosides. Toxicol Appl Pharmacol 2007; 223(1): 86-98. 4. Abdel-Raheem IT, Abdel-Ghany AA, Mohammad GA. Protective effects of quercetin against gentamicin induced nephrotoxicity in rats. Boil Pharm Bull 2009; 32(1): 61-7. 5. Balakumar P, Rohilla A, Thangathirupathi A. gentamicin-induced nephrotoxicity: do we have a promising therapeutic approach to blunt it. Pharmacol Res 2010; 62(3): 179-86. 6. Nelson JM, Chiller TM, Powers JH, Angulo FJ. Fluoroquinoloneresistant Campylobactera species and the withdrawal of 442
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