Comparison of the Nephrotoxicity of Netilmicin and Gentamicin in Rats RALPH L
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ANTIMICRoBIAL AGENTs AND CHUMOTHKRAY, Oct. 1977, P. 474-478 Vol. 12, No. 4 Copyright 0 1977 American Society for Microbiology Printed in U.S.A. Comparison of the Nephrotoxicity of Netilmicin and Gentamicin in Rats RALPH L. BOWMAN,1 FREDRICK J. SILVERBLATT,' AND GEORGE J. KALOYANIDES2* Veterans Administration Hospital,' and Nephrology and Infectious Disease Divisions, University of California at Los Angeles San Ferando Valley Medical Program,' Sepulveda, California 91343 Received for publication 17 March 1977 The nephrotoxicity of netilmicin relative to that of gentamicin was examined in Sprague-Dawley rats. Balance studies were performed on rats injected with netilmicin or gentamicin (50 mg/kg per day for 14 days, 100 mg/kg per day for 8 days, and 150 mg/kg per day for 8 days). Control rats were injected with saline. Both drugs caused a dose-related decrease in urine osmolality and increases in urine volume, water intake, and serum creatinine; however, the magnitude of these changes was significantly less in netilmicin- than in gentamicin-injected rats. Light microscopy of renal tissue revealed less proximal tubular cell necro- sis in netilmicin- than in gentamicin-injected rats. There was no significant difference between the renal cortical concentrations ofthe two drugs. Both drugs stimulated uptake ofp-aminohippurate in rat renal cortical slices to the same degree. The data indicate that netilmicin is less nephrotoxic than gentamicin in rats, that the difference in nephrotoxicity cannot be explained by a difference in drug concentration in the renal cortex, and that the ability ofaminoglycosides to stimulate the organic acid transport system of proximal tubular cells does not correlate with their nephrotoxic potential. Renal failure is a well recognized but poorly were begun. After 3 days ofcontrol measurements of understood complication of gentamicin ther- body weight, water intake, urine volume, urine os- apy. Recognition ofthis problem has stimulated molality, solute excretion, and protein excretion, the rats were injected with one of three agents. In the development of semisynthetic analogs of group I, rats received a daily subcutaneous injection gentamicin that possess equivalent antimicro- of gentamicin sulfate or netilmicin sulfate (40 mg of bial efficacy along with a higher ratio ofthera- base per ml) at a dose of50 mg ofbase per kg ofbody peutic effect to toxicity. Netilmicin (Schering weight per day for 14 days. Control rats for each 20569, Schering Laboratories, Bloomfield, N. group were injected with an equivalent volume of J.) is one such semisynthetic analog ofgentami- 0.9% saline (1.25 ml/kg of body weight per day). In cin (10). Recent reports indicate that the anti- group II, rats were injected with drugs at the same microbial spectrum of netilmicin is not only concentration but at a dose of 100 mg/kg of body similar to that of gentamicin but that this weight per day for 8 days. In group Im, the dose was increased to 150 mg/kg of body weight per day for 8 agent exhibits antimicrobial activity against days. many aminoglycoside-resistant strains of At the end of the study, the rats were sacrificed gram-negative bacteria (7, 8). Ofequal interest and the combined weight of both kidneys from each is the recent report of Luft et al. (6) that states rat was determined. Blood was collected from the that netilmicin is less nephrotoxic than genta- aorta and analyzed for urea nitrogen (BUN) and micin in rats. serum creatinine with a Technicon autoanalyzer. In the present study, we examined the effects Urine was collected under oil, the daily volume was ofthree dose schedules ofnetilmicin and genta- measured, and a portion was analyzed for solute micin on renal function and morphology in rats concentration with a Precision osmometer. Urine to protein was precipitated with 11% trichloroacetic further establish the relative nephrotoxicity acid and quantitated by the Lowry method (4). of these two agents. In a separate group of experiments, rats were injected with gentamicin or netilmicin at the same MATERIALS AND METHODS doses and for the same durations as described above. Balance studies were performed in three groups of At the end ofthe experiment, the kidneys were fixed female Sprague-Dawley rats initially weighing 180 by in vivo perfusion ofthe renal arteries (3) with 3% to 210 g. The rats were placed in individual meta- glutaraldehyde buffered to pH 7.4 with 0.1 M sodium bolic cages and permitted free access to tap water phosphate. Immediately thereafter, the kidneys and standard rat chow. A minimum of 10 days was were removed and placed in 3% glutaraldehyde for 4 allowed for acclimatization before measurements h and then placed in a solution of 0.1 M cacodylate 474 VOL. 12, 1977 NETILMICIN AND GENTAMICIN NEPHROTOXICITY 475 with 7% sucrose (pH. 7.4). Subsequently, 1-mm injected with gentamicin at 100 and 150 mg/kg, blocks of cortex were embedded in Epon, and 1-,um- BUN and serum creatinine increased sharply thick sections were cut and stained with toluidine and significantly (P < 0.01) above the levels blue. The sections were coded and examined by blind study under light microscopy for evidence of observed in netilmicin- and saline-injected rats. tubular necrosis and lysosomal changes. In contrast, at each dose schedule, no difference In a fourth group of experiments, rats were in- in BUN was observed between netilmicin- and jected with gentamicin or netilmicin at a dose of 100 saline-injected rats (P > 0.1). Although serum mg/kg of body weight per day for 2, 4, and 8 days. creatinine increased slightly in the netilmicin Twenty-four hours after the last injection, the rats group as a function of drug dose, the increase were sacrificed, and the drug concentration in the was impressively less than that observed in renal cortex and medulla was measured by a micro- gentamicin-injected rats. At 150 mg/kg, serum biological assay technique (5). Bacillus globigii was used as the marker organism. The renal cortex and creatinine increased to 0.84 ± 0.02 mg/100 ml in medulla from each rat were dissected, weighed, ho- netilmicin-injected rats as compared with 4.19 mogenized, and diluted as necessary with phosphate + 0.45 mg/100 ml in gentamicin-injected rats buffer (pH 8). Antibiotic standards were prepared (P < 0.01). with the same buffer. Kidney weight was significantly greater in In a fifth group of experiments, the effect of gen- gentamicin- and netilmicin-injected rats than tamicin and netilmicin on p-aminohippurate (PAH) in saline-injected rats (P < 0.01). At 100 and uptake by renal-cortical slices was examined. Rats 150 mg/kg, kidney weight of gentamicin-in- were injected with drugs at a dose of 100 mg/kg per jected rats exceeded that of rats injected with day for 2 days. Twenty-four hours after the last injection, the kidneys were removed and sliced with netilmicin (P < 0.01). Expressing kidney a Stadie-Riggs microtome. Cortical slices were incu- weight as a function of body weight did not bated for 120 min in a medium containing 10-4 M reveal any correlation between kidney weight, PAH, after which the concentration ratio of cortical drug dosage, BUN, or serum creatinine. The slice to medium PAH was determined as previously increase in kidney weight most likely indicates described (1). interstitial edema reflecting drug-induced, tu- The data in the text and figures are expressed as bular injury. the mean + standard error. Analysis of variance Light microscopic examination of renal tis- was used to compare the effects of drug treatment sue revealed dose-related injury characterized within and between groups. by increased lysosomal bodies, disruption of RESULTS brush borders, cellular swelling, and frank ne- crosis of proximal tubular epithelium. The Table 1 summarizes the balance data for the changes observed in renal tissue of netilmicin- control period (day 0) and the last day of treat- injected rats were less severe than those found ment for the three-dose schedules of each drug. in renal tissue of gentamicin-injected rats. The first sign ofgentamicin or netilmicin neph- The concentration of gentamicin or netilmi- rotoxicity was a decrease in urine osmolality cin in the renal cortex and medulla was mea- followed by a rise in urine volume and water sured after administration of 100 mg/kg per day intake with no consistent changes in solute for 2, 4, and 8 days. In all experiments, the excretion. At 50 mg/kg per day, the changes in cortical concentrations of the drugs exceeded these variables were similar in gentamicin- and that of the medulla (P < 0.01). After 2 days of netilmicin-injected rats. At 100 and 150 mg/kg injections, the cortical concentrations of genta- per day, they became more pronounced. How- micin and netilmicin were similar (1,029 + 114 ever, compared with gentamicin-injected rats, and 1,067 + 124 umg/g of cortex, respectively; P netilmicin-injected rats exhibited significantly > 0.4) and increased to the same extent (2,230 greater weight gain (P < 0.01), less depression ± 275 and 2,142 + 222 ,ug/g of cortex, respec- ofurine osmolality (P < 0.01), and no change in tively; P > 0.4) after 4 days of injections. After solute excretion during both higher dose sched- 8 days of injections, netilmicin measured 1,705 ules. Urinary protein excretion increased simi- ± 151 ,ug/g of cortex, whereas gentamicin de- larly in response to gentamicin or netilmicin. creased to 832 + 108 ,ug/g of cortex, P < 0.01. Figure 1 summarizes the changes in BUN, The decrease in the cortical concentration of serum creatinine, and kidney weight in experi- gentamicin most likely reflects the extensive mental groups I, II, and III. At 50 mg/kg, no proximal tubular cell necrosis observed in this difference in BUN was evident among the three group of rats.