TY - JOUR
AU - Sonkodi, Sandor
AB - Abstract Background. This study examined whether administration of l‐carnitine ameliorates gentamicin‐induced renal injury in rats. Methods. Male Sprague–Dawley rats were assigned to one of seven treatment groups: group A (control) rats were given normal saline injections daily for 8 consecutive days; group B, C and D rats were given gentamicin injections, 50 mg/kg body weight/day daily for 8 consecutive days; and group E, F and G rats were given gentamicin injections, 80 mg/kg/day daily for 8 consecutive days. Starting 4 days before these injections, all groups were given additional injections, for 12 consecutive days, of normal saline (groups A, B and E) or l‐carnitine at 40 mg/kg (groups C and F) or 200 mg/kg (groups D and G). Histological scoring of renal cortical pathology was performed after day 12. Results. Among rats injected with gentamicin 50 mg/kg/day, those given either 40 or 200 mg/kg/day of l‐carnitine had higher creatinine clearances at day 12 than the rats not given carnitine. In the rats given 80 mg/kg gentamicin and no carnitine, renal function tended to be lower than in controls. At day 12, the rats given gentamicin 80 mg/kg and l‐carnitine 200 mg/kg/day, compared with rats given gentamicin 80 mg/kg and no carnitine, displayed lower serum urea and probably creatinine concentrations, and higher creatinine clearances, and their serum urea was not different from control (group A) rats. Both doses of gentamicin induced renal cortical histopathology. Changes were milder with gentamicin 50 mg/kg/day, and l‐carnitine, particularly at 200 mg/kg/day, ameliorated the severity of renal pathology induced by both gentamicin doses. In rats given gentamicin 80 mg/kg/day, the animals treated with carnitine 200 mg/kg/day had significantly less severe proximal tubular necrosis and significantly greater mild proximal tubular necrosis compared with rats receiving l‐carnitine 40 mg/kg/day or no carnitine. Conclusions. In rats receiving gentamicin, daily l‐carnitine injections, particularly at 200 mg/kg/day, ameliorate the severity of renal cortical proximal tubular necrosis and maintain greater renal function. acute renal failure, acute tubular necrosis, carnitine, gentamicin, gentamicin nephrotoxicity, kidney disease Introduction Gentamicin nephrotoxicity is one of the most common causes of acute renal failure and promotes both increased morbidity and greater healthcare costs [1]. Although the change in gentamicin dosing from multiple‐daily to once‐daily doses has reduced the risk of nephrotoxicity, the incidence of gentamicin‐induced acute renal failure still remains high [2]. There may be several mechanisms by which gentamicin induces renal injury. These include binding to anionic phospholipids and altering the function and structure of cellular and intracellular membranes [3–6]. Gentamicin may also cause ATP depletion from either mitochondrial damage or direct inhibition of mitochondrial oxidative phosphorylation [7], and gentamicin may cause oxidative injury [8,9]. l‐carnitine (l‐trimethyl‐3‐hydroxy‐ammoniabutanoate) is a zwitter ion that facilitates the transfer of long‐chain (>10 carbon) fatty acids into the mitochondria of skeletal muscle and cardiomyocytes, where they undergo beta‐oxidation [10]. By this mechanism carnitine profoundly influences both skeletal muscle and myocardial fatty acid oxidation, and maintains low pools of fatty acid (acyl)‐coenzyme A compounds, which are potentially toxic [11]. Carnitine may: (i) stabilize certain cellular membranes, most notably in erythrocytes [12,13]; (ii) increase myocardial ATP, presumably through its promotion of fatty acid oxidation [14,15]; and (iii) inhibit free radical production [16–18]. l‐carnitine is also a relatively well tolerated and safe compound. Thus, some of the actions of l‐carnitine may be opposite to the toxic effects of gentamicin. We, therefore, carried out a pilot study to test the hypothesis that treatment with l‐carnitine may prevent or ameliorate gentamicin‐induced acute renal injury. Methods Animals, treatments and specimen collection The experiments were performed in male Sprague–Dawley rats (Charles River) weighing 180–200 g. Animal experimentation was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were maintained in an animal research facility. Rats were provided with rat chow and tap water ad libitum. After allowing 5 days for adaptation to the new environment, rats were assigned in a random order to one of seven groups [n=6 for the control group (A) and n=7 for each other group]. Each group of rats were given two subcutaneous injections in the morning as follows (Table 1): Table 1.  Definition of symbols and schema for study Group  n  Normal  l‐carnitine     Gentamicin         Saline   40 mg/kg   200 mg/kg   50 mg/kg   80 mg/kg   A  6  12a, 8b          B  7  12      8    C  7    12    8    D  7      12  8    E  7  12        8  F  7    12      8  G  7      12    8  Group  n  Normal  l‐carnitine     Gentamicin         Saline   40 mg/kg   200 mg/kg   50 mg/kg   80 mg/kg   A  6  12a, 8b          B  7  12      8    C  7    12    8    D  7      12  8    E  7  12        8  F  7    12      8  G  7      12    8  aNumber of days the daily doses were injected. Injections given for only 8 days were commenced on the 5th day of study; i.e. after the first 4 days of receiving normal saline or l‐carnitine. bA second daily normal saline injection was given for 8 days, starting on day 5. View Large Group A. Injected with normal saline once daily for 12 days, with an additional daily injection of normal saline for the last 8 days. Group B. Injected with 50 mg/kg body weight/day of gentamicin for 8 days and injected with normal saline daily for 12 days, starting 4 days before the gentamicin injections. Group C. Injected with gentamicin 50 mg/kg/day for 8 days and with l‐carnitine 40 mg/kg/day for 12 days, starting 4 days before the gentamicin injections. Group D. Injected with gentamicin 50 mg/kg/day for 8 days and with l‐carnitine 200 mg/kg/day for 12 days, beginning 4 days before the gentamicin injections. Group E. Injected with gentamicin 80 mg/kg/day for 8 days and with normal saline daily for 12 days, starting 4 days before the gentamicin injections. Group F. Injected with gentamicin 80 mg/kg/day for 8 days and with l‐carnitine 40 mg/kg/day for 12 days, starting 4 days before the gentamicin injections. Group G. Injected with gentamicin 80 mg/kg/day for 8 days and with l‐carnitine 200 mg/kg/day for 12 days, beginning 4 days before the gentamicin injections. Thus, the daily normal saline (groups B and E) or carnitine injections (groups C, D, F and G) were commenced 4 days before the onset of the gentamicin injections and were continued for the entire 8 days that the rats received the gentamicin. l‐carnitine was given as Carnitor® (Sigma Tau Laboratories, Gaithersburg, MD, USA) for the 40 mg/kg/day dose or as a powder (Sigma Chemical Co., St Louis, MO, USA) for the 200 mg/kg dose, dissolved in the same volume of water as for the 40 mg/kg/day dose. All injections in each rat were given between 09:00 and 09:30 every day. The carnitine and normal saline injections in groups B–G and the saline injections in group A were of equal volume. Blood was collected into heparinized tubes, from the tail of each rat unless otherwise stated, on four occasions for measurement of serum urea and creatinine, and at the end of days 8 and 12 for gentamicin. Blood was drawn: at the beginning of day 1, before the first carnitine or normal saline injection; at the end of day 4 (i.e. morning of day 5) before the first injection of gentamicin (groups B–G) or normal saline (group A); at the end of day 8 (i.e. morning of day 9) before the fifth injection of gentamicin (groups B–G) or normal saline (group A); and at the end of day 12 [i.e. morning of day 13, 24 h after the last injection of gentamicin (groups B–G) or normal saline (group A)]. The blood drawings were always obtained between 08:00 and 09:00 before that day's injections were given. Twenty‐four hour urine collections were taken for measurement of urine creatinine. The 24 h urine collections were commenced and completed between 08:00 and 09:00 on days 1–2 (i.e. starting on the day of the first injections of normal saline or carnitine), days 4–5, days 8–9 and days 12–13. The urine was collected without preservatives while each rat lived in a metabolic cage. At the end of day 12 (i.e. morning of day 13), 24 h after the last gentamicin (or normal saline) injection, rats were killed. Kidneys were harvested and blood collections were performed. After anaesthesia with ketamine‐xylazine, rats were placed on a thermostatic heating plate to maintain the body temperature at 37°C [19]. The abdominal cavity was opened, and 1 ml of blood was drawn from the abdominal aorta. Serum was separated from this blood sample and used for the measurements described above. One kidney was immediately removed for histological examination. The renal artery of the other kidney was then clamped with a Wollenberger's clamp, and the kidney was dissected from the abdominal cavity, with a fragment (at least 0.8 cm) of the main renal artery attached. This kidney was perfused and processed further for the measurement of renal cortical gentamicin concentrations. The mortality rate of the rats was also assessed. Chemistry measurements Creatinine and urea were measured colorimetrically using kits obtained from the Roche Corporation (Mannheim, Germany) and a Hitachi 917 Autoanalyzer (Roche Diagnostics, Basel, Switzerland). Gentamicin was measured in serum by a polarization fluorescence immunoassay in a TDx analyzer (Abbott Diagnostics, Abbott Park, IL, USA); gentamicin standards were prepared in normal rat sera. Gentamicin content was measured only in the cortex of the kidney because 85% of gentamicin is reported to accumulate in the renal cortex, primarily in the proximal tubules [20,21], as soon as 24 h after a single injection into the rat [22]. Also, the great preponderance of the proximal tubules are located in the cortex. To measure the gentamicin concentrations in renal cortex, the kidney was processed and analysed as reported previously [20,21], with minor modifications. Briefly, the kidney was perfused immediately after extirpation with 10 ml of cold 0.1 M sodium phosphate buffer (pH 7.4) through the renal artery that remained attached to the kidney to wash out the blood remaining in the kidney. The renal cortex was then separated by sharp dissection, weighed and immediately homogenized with 2.5 volumes of cold 0.1 M sodium phosphate buffer (pH 7.5). The homogenate was diluted with the same buffer to 20% (w/v) and diluted further with 19 parts of 0.15% Triton‐X in distilled water. The gentamicin concentration was measured in the Triton‐X diluted homogenate by polarization fluorescence immunoassay with the Tdx analyzer (Abbott Diagnostics). Gentamicin standards were prepared in rat cortex homogenates from normal rats. The protein concentration of the renal cortical homogenate was measured by the Lowry–Folin method, and the renal cortical gentamicin concentrations were expressed per milligram of renal cortical protein. Morphological examination of the kidney One kidney was removed from each rat for histological examination. The kidney was incised perpendicular to its long axis with a razor blade, and a 2 mm thick slice of tissue was removed and transferred immediately into a 4% buffered formol solution kept on ice. After fixation for 24 h at 4°C, the slices were embedded in paraffin and then sectioned. The sections were stained with periodic acid‐Schiff (PAS) and haematoxylin–eosin, and examined by light microscopy by B.I. Histological sections of the kidneys from all rats were qualitatively assessed without knowledge of the treatment group from which each histological section had been obtained. Since the gentamicin‐induced morphological abnormalities in rat kidney are mainly localized in the proximal tubules, and the other structures of the kidney do not exhibit major histological alterations [23,24], only the renal cortex was examined in detail. The photographic field of a 40× magnification objective lens of a Zeiss microscope was used as a sampling frame. The sampling started from one glomerulus chosen arbitrarily. From this glomerulus, the stage was moved in only one direction along the midcortex. Twenty fields were read in two consecutive rows as described previously [25]. All proximal tubular profiles (PTs) present in the 20 fields were evaluated, and the number of proximal tubular profiles present in each field was counted. Care was taken not to count the same profile twice. Four categories of injury were distinguished: 0, no alteration; 1+, isolated cell necrosis (apoptotic bodies and/or disruption of cell membrane and loss of nuclear stain); 2+, several necrotic cells in a tubular profile; and 3+, complete or almost complete necrosis of a tubular profile. The various categories were expressed in percentages, where the total number of tubular profiles examined per kidney was 100%. Some kidney sections were read twice in order to assess and control for the subjectivity of the evaluation. Statistical analyses Comparisons between groups were performed by one‐way ANOVA (analysis of variance) with seven groups. One‐way ANOVA was used so that differences between treatment arms rather than overall effects could be evaluated. If the ANOVA was statistically significant, paired comparisons were performed by the least square means test. Statistical differences were considered significant if the P value was <0.025. If the P value was <0.025, the unadjusted P value was reported. ANOVA was performed on the following measurements obtained at the end of day 12: serum creatinine and urea concentrations, histological scores and renal cortical gentamicin levels. In addition, ANOVA was carried out on the 24 h urinary creatinine clearances obtained from days 12 to 13. For the comparison of the histological scores, group A (saline‐injected controls) was compared with each of the other six groups. Also, groups B, C and D were compared with each other, as were groups E, F and G. Finally, the histopathology in groups B, C and D combined was compared with that in groups E, F and G taken together. Results The serum urea concentrations during the course of the study are shown in Figure 1 and Table 2. In most groups, serum urea rose slightly during the first 4 days. In the control group (group A), the serum urea then essentially plateaued, whereas in the six gentamicin‐treated groups, the serum urea began to rise substantially between days 8 and 12. In the seven groups of rats, the serum urea did not differ significantly at any time measured until the 12th day (8th day of gentamicin injections) at which time differences emerged, as indicated by the ANOVA and least square means tests. In rat groups B, C and D, receiving gentamicin 50 mg/kg/day, the serum urea tended to be greater than in the control (group A) rats at the end of day 12, but the differences were not significant. Similarly, there were no differences within the three 50 mg/kg/day groups at day 12. However, in the rats receiving gentamicin 80 mg/kg/day and either no carnitine (group E) or l‐carnitine 40 mg/kg/day (group F), the serum urea at day 12 was greater than in the control group (group A) (P=0.0001 for each comparison). In contrast, in the rats receiving gentamicin 80 mg/kg and l‐carnitine 200 mg/kg, the serum urea was not significantly different from the control group that received no gentamicin, and it was lower in the former group compared with the rats receiving gentamicin 80 mg/kg/day and no carnitine (P=0.0001). Serum urea also tended to be lower at day 12 in the rats given gentamicin 80 mg/kg and carnitine 200 mg/kg as compared with rats given gentamicin 80 mg/kg and carnitine 40 mg/kg (P=0.023, unadjusted for number of comparisons) (Table 2). A comparison of the changes in serum urea between day 12 and day 1 (i.e. the value at day 12 minus that at day 1) among each of the seven groups of rats showed statistical differences that were identical to those described for the above‐mentioned differences in the mean serum urea value at day 12. Fig. 1.  View largeDownload slide Mean values for serum urea concentrations in rats obtained at the beginning of day 1, and at the end of days 4, 8 and 12 after the onset of daily injections of normal saline injections or l‐carnitine. Open symbols indicate the three groups of rats given gentamicin 50 mg/kg/day. Closed symbols indicate the three groups of rats given gentamicin 80 mg/kg/day. Diamonds represent rats given no carnitine, circles represent rats given l‐carnitine 40 mg/kg/day, and squares represent rats given l‐carnitine 200 mg/kg/day. Crossed open circles indicate control rats (group A) given neither gentamicin nor carnitine. The indicated gentamicin injections were given daily for 8 days, starting on day 5. Fig. 1.  View largeDownload slide Mean values for serum urea concentrations in rats obtained at the beginning of day 1, and at the end of days 4, 8 and 12 after the onset of daily injections of normal saline injections or l‐carnitine. Open symbols indicate the three groups of rats given gentamicin 50 mg/kg/day. Closed symbols indicate the three groups of rats given gentamicin 80 mg/kg/day. Diamonds represent rats given no carnitine, circles represent rats given l‐carnitine 40 mg/kg/day, and squares represent rats given l‐carnitine 200 mg/kg/day. Crossed open circles indicate control rats (group A) given neither gentamicin nor carnitine. The indicated gentamicin injections were given daily for 8 days, starting on day 5. Table 2.  Measures of renal function in rats given saline, gentamicin and/or l‐carnitine   Group   Day 1   Day 4   Day 8   Day 12   Serum urea (mM)  A No Gent; No Carn  5.0±0.4  6.1±0.7  6.2±0.4  6.6±1.1    B 50 mg Gent; No Carn  5.0±0.7  6.0±0.8  6.7±0.8  8.5±1.2    C 50 mg Gent; 40 mg Carn  5.8±0.9  6.2±1.1  6.1±1.0  7.9±1.2    D 50 mg Gent; 200 mg Carn  5.3±0.7  5.6±0.9  6.4±0.8  9.0±1.5    E 80 mg Gent; No Carn  4.8±0.8  5.6±0.7  6.7±0.2  13.5±3.5b,f    F 80 mg Gent; 40 mg Carn  5.3±0.8  6.1±0.9  6.6±0.8  11.6±1.8b,d    G 80 mg Gent; 200 mg Carn  4.8±0.4  5.8±1.1  5.8±0.9  9.2±1.3  Serum creatinine (uM)  A No Gent; No Carn  44.0±1.9  48.3±2.4  49.0±3.0  42.2±1.3    B 50 mg Gent; No Carn  47.3±4.1  47.0±2.2  51.7±3.9  53.1±3.2    C 50 mg Gent; 40 mg Carn  49.1±3.2  47.6±2.2  47.7±2.9  51.0±7.1    D 50 mg Gent; 200 mg Carn  47.4±1.0  46.0±1.4  48.1±1.5  47.9±2.8    E 80 mg Gent; No Carn  46.3±1.6  47.9±3.6  49.0±2.2  65.4±11.1b,d    F 80 mg Gent; 40 mg Carn  48.0±1.4  47.3±1.9  49.3±1.2  59.1±10.5b    G 80 mg Gent; 200 mg Carn  49.6±3.6  47.0±1.8  50.7±3.0  54.1±5.5a  Group     Days 1–2   Days 4–5   Days 8–9   Days 12–13   Creatinine clearance (ml/min)  A No Gent; No Carn  0.23±0.06  0.22±0.03  0.23±0.03  0.28±0.06    B 50 mg Gent; No Carn  0.24±0.05  0.22±0.02  0.17±0.04  0.18±0.02    C 50 mg Gent; 40 mg Carn  0.22±0.03  0.20±0.04  0.31±0.08  0.33±0.15c    D 50 mg Gent; 200 mg Carn  0.24±0.06  0.25±0.07  0.32±0.11  0.34±0.08c    E 80 mg Gent; No Carn  0.29±0.07  0.25±0.03  0.27±0.10  0.17±0.03e    F 80 mg Gent; 40 mg Carn  0.27±0.04  0.25±0.07  0.33±0.10  0.24±0.05    G 80 mg Gent; 200 mg Carn  0.22±0.05  0.25±0.05  0.25±0.08  0.33±0.03    Group   Day 1   Day 4   Day 8   Day 12   Serum urea (mM)  A No Gent; No Carn  5.0±0.4  6.1±0.7  6.2±0.4  6.6±1.1    B 50 mg Gent; No Carn  5.0±0.7  6.0±0.8  6.7±0.8  8.5±1.2    C 50 mg Gent; 40 mg Carn  5.8±0.9  6.2±1.1  6.1±1.0  7.9±1.2    D 50 mg Gent; 200 mg Carn  5.3±0.7  5.6±0.9  6.4±0.8  9.0±1.5    E 80 mg Gent; No Carn  4.8±0.8  5.6±0.7  6.7±0.2  13.5±3.5b,f    F 80 mg Gent; 40 mg Carn  5.3±0.8  6.1±0.9  6.6±0.8  11.6±1.8b,d    G 80 mg Gent; 200 mg Carn  4.8±0.4  5.8±1.1  5.8±0.9  9.2±1.3  Serum creatinine (uM)  A No Gent; No Carn  44.0±1.9  48.3±2.4  49.0±3.0  42.2±1.3    B 50 mg Gent; No Carn  47.3±4.1  47.0±2.2  51.7±3.9  53.1±3.2    C 50 mg Gent; 40 mg Carn  49.1±3.2  47.6±2.2  47.7±2.9  51.0±7.1    D 50 mg Gent; 200 mg Carn  47.4±1.0  46.0±1.4  48.1±1.5  47.9±2.8    E 80 mg Gent; No Carn  46.3±1.6  47.9±3.6  49.0±2.2  65.4±11.1b,d    F 80 mg Gent; 40 mg Carn  48.0±1.4  47.3±1.9  49.3±1.2  59.1±10.5b    G 80 mg Gent; 200 mg Carn  49.6±3.6  47.0±1.8  50.7±3.0  54.1±5.5a  Group     Days 1–2   Days 4–5   Days 8–9   Days 12–13   Creatinine clearance (ml/min)  A No Gent; No Carn  0.23±0.06  0.22±0.03  0.23±0.03  0.28±0.06    B 50 mg Gent; No Carn  0.24±0.05  0.22±0.02  0.17±0.04  0.18±0.02    C 50 mg Gent; 40 mg Carn  0.22±0.03  0.20±0.04  0.31±0.08  0.33±0.15c    D 50 mg Gent; 200 mg Carn  0.24±0.06  0.25±0.07  0.32±0.11  0.34±0.08c    E 80 mg Gent; No Carn  0.29±0.07  0.25±0.03  0.27±0.10  0.17±0.03e    F 80 mg Gent; 40 mg Carn  0.27±0.04  0.25±0.07  0.33±0.10  0.24±0.05    G 80 mg Gent; 200 mg Carn  0.22±0.05  0.25±0.05  0.25±0.08  0.33±0.03  Gent=gentamicin; carn=carnitine. Dosage (mg) refers to mg/kg body weight/day. Significantly different from group A: aP <0.004, bP <0.001; from group B: cP ≤ 0.006; from group G: dP <0.025, eP=0.002, fP <0.001. Values are represented as mean±SD, and statistical analyses were only performed for the data obtained on days 12–13. View Large The serum creatinine also did not differ significantly among the seven groups at any time measured until day 12 (Figure 2 and Table 3). As with the serum urea, the serum creatinine tended to be higher at day 12 in the three groups of rats receiving gentamicin 50 mg/kg as compared with the control rats; these differences were not significant. However, the serum creatinine was greater at day 12 at a borderline level of significance in the rats given gentamicin 50 mg/kg/day and no carnitine (group B) as compared with the control group (P=0.035, unadjusted for number of comparisons) (Table 2). The serum creatinine was significantly greater in each group of rats given gentamicin 80 mg/kg (P=0.0001) as compared with the control rats, who were given no gentamicin [i.e. group A rats vs those given gentamicin 80 mg/kg/day and either no carnitine (P=0.0001), l‐carnitine 40 mg/kg/day (P=0.0001) or l‐carnitine 200 mg/kg/day (P=0.0037)]. Moreover, at day 12 among the rats given gentamicin 80 mg/kg, those that also received l‐carnitine 200 mg/kg displayed a serum creatinine value that was lower than in the rats not given carnitine (P=0.012). A comparison of the changes in serum creatinine concentrations between day 12 and day 1 (i.e. value at day 12 minus that at day 1) among each of the seven groups of rats showed only the following minor statistical discrepancies from the above‐mentioned differences in the mean serum creatinine at day 12: the change in serum creatinine in the rats injected with gentamicin 80 mg/kg/day and l‐carnitine 200 mg/kg/day was not significantly different from the control rats given no gentamicin, but was highly statistically significantly reduced compared with the rats injected with gentamicin 80 mg/kg/day and no carnitine (P=0.0005). Fig. 2.  View largeDownload slide Mean values for serum creatinine concentrations obtained at the beginning of day 1, and at the end of days 4, 8 and 12 after the onset of daily injections of normal saline or l‐carnitine. Symbols are defined in the legend to Figure 1. The indicated gentamicin injections were given daily for 8 days, starting on day 5. Fig. 2.  View largeDownload slide Mean values for serum creatinine concentrations obtained at the beginning of day 1, and at the end of days 4, 8 and 12 after the onset of daily injections of normal saline or l‐carnitine. Symbols are defined in the legend to Figure 1. The indicated gentamicin injections were given daily for 8 days, starting on day 5. Table 3.  Serum and renal gentamicin levels Treatment group  n  Serum gentamicin (mg/100 ml)    Renal cortical gentamicin (ng/mg protein)      Day 9   Day 13   Day 13   B  7  0.19±0.03  0.21±0.01a,c,f,h  911±62b,e,g  C  7  0.17±0.02b,e  0.17±0.01c,f,h  907±49b,e,g  D  7  0.17±0.01a,e  0.18±0.01c,f,h  946±54a,d,g,c  E  7  0.22±0.01  0.46±0.05  1222±79  F  7  0.23±0.02  0.49±0.07  1220±71  G  7  0.20±0.01  0.45±0.03  1312±122  Treatment group  n  Serum gentamicin (mg/100 ml)    Renal cortical gentamicin (ng/mg protein)      Day 9   Day 13   Day 13   B  7  0.19±0.03  0.21±0.01a,c,f,h  911±62b,e,g  C  7  0.17±0.02b,e  0.17±0.01c,f,h  907±49b,e,g  D  7  0.17±0.01a,e  0.18±0.01c,f,h  946±54a,d,g,c  E  7  0.22±0.01  0.46±0.05  1222±79  F  7  0.23±0.02  0.49±0.07  1220±71  G  7  0.20±0.01  0.45±0.03  1312±122  Specimens were collected on the morning of days 9 or 13, at the end of days 8 or 12, respectively. Values are expressed as mean±SEM. Values not measured in group A. Significantly less than values from the same column in group E: aP <0.05, bP ≤ 0.01, cP ≤ 0.001; in group F: dP <0.05, eP <0.01, fP <0.001; and in group G: gP <0.01, hP <0.001. View Large The creatinine clearances at day 12, shown in Figure 3 and Table 2, were not different in the control rats compared with any of the six groups of rats that were injected with gentamicin. However, among the rats given gentamicin 50 mg/kg/day, those that were given no carnitine displayed significantly lower creatinine clearances as compared with those receiving either 40 mg/kg or 200 mg/kg of l‐carnitine (P=0.004 and P=0.006, respectively). Moreover, among the rats injected with gentamicin 80 mg/kg/day, those animals that also received l‐carnitine 200 mg/kg/day had significantly greater creatinine clearances at day 12 than did those not given any carnitine (P=0.002). The former animals also tended to have greater creatinine clearances than the rats given gentamicin 80 mg/kg/day and carnitine 40 mg/kg/day (group F) (P=0.049, unadjusted for the number of comparisons) (Table 2). Also, there was a tendency for the creatinine clearance to be lower in the rats given either gentamicin 50 mg/kg and no carnitine (group B) or gentamicin 80 mg/kg and no carnitine (group E), than animals given no gentamicin (group A) (P=0.054 and P=0.041, respectively, unadjusted for the number of comparisons). Fig. 3.  View largeDownload slide Mean values for creatinine clearances obtained on days 0–1, 4–5, 8–9 and 12–13 after the onset of daily injections of normal saline or l‐carnitine. Symbols are defined in the legend to Figure 1. The indicated gentamicin injections were given daily for 8 days, starting on day 5. Fig. 3.  View largeDownload slide Mean values for creatinine clearances obtained on days 0–1, 4–5, 8–9 and 12–13 after the onset of daily injections of normal saline or l‐carnitine. Symbols are defined in the legend to Figure 1. The indicated gentamicin injections were given daily for 8 days, starting on day 5. A comparison of the changes in creatinine clearances between day 12 and day 1 among each of the seven groups of rats showed the following discrepancies from the above mentioned differences in the mean creatinine clearance values at day 12: The increase in creatinine clearances was also significantly greater in the control rats compared with the rats injected with gentamicin 80 mg/kg/day and no carnitine (P=0.0004). In addition, the rats injected with gentamicin 80 mg/kg/day and l‐carnitine 200 mg/kg/day had a greater increase in creatinine clearances at day 12 than the rats given gentamicin 80 mg/kg/day and l‐carnitine 40 mg/kg (P=0.0025). The results of the histological scoring of the renal cortical tissue are shown in Figure 4A–D. The rats given gentamicin 50 mg/kg/day had, on average, evidence for 1+ proximal tubular necrosis, but not for 2+ or 3+ necrosis. The rats given gentamicin 80 mg/kg/day had 1+, 2+ and 3+ necrosis. In contrast to these six groups, the control (group A) rats not given gentamicin had, on average, no 1+, 2+ or 3+ proximal tubular necrosis. In comparison with the control rats there was significantly more frequent 1+ necrosis in the rats given gentamicin 50 mg/kg/day and no carnitine (group B), and in all three groups of rats given gentamicin 80 mg/kg/day (Figure 4A). Group A rats also had significantly less 2+ necrosis than each of the three groups of rats given gentamicin 80 mg/kg/day, and significantly less 3+ necrosis than the rats given gentamicin 80 mg/kg/day, and either no carnitine (group E) or only 40 mg/kg/day of carnitine (group F) (Figure 4B and C). The rats given gentamicin 80 mg/kg/day and l‐carnitine 200 mg/kg/day (group G) did not have significantly more 3+ necrosis than the control rats (group A) (Figure 4C). Fig. 4.  View largeDownload slide Renal histopathology, determined by a scoring system of 0, 1+, 2+ or 3+ proximal tubular necrosis (see text) in rat renal cortex obtained after day 12 in rats given daily injections of saline (group A), or gentamicin 50 mg/kg/day (groups B, C and D) or 80 mg/kg/day (groups E, F and G), and either normal saline injections (groups B and E) or l‐carnitine injections (40 mg/kg/day, groups C and F; or 200 mg/kg/day, groups D and G). Brackets indicate +1 SD. Asterisks indicate that groups E and F are statistically significantly different from group G (P <0.05). Fig. 4.  View largeDownload slide Renal histopathology, determined by a scoring system of 0, 1+, 2+ or 3+ proximal tubular necrosis (see text) in rat renal cortex obtained after day 12 in rats given daily injections of saline (group A), or gentamicin 50 mg/kg/day (groups B, C and D) or 80 mg/kg/day (groups E, F and G), and either normal saline injections (groups B and E) or l‐carnitine injections (40 mg/kg/day, groups C and F; or 200 mg/kg/day, groups D and G). Brackets indicate +1 SD. Asterisks indicate that groups E and F are statistically significantly different from group G (P <0.05). Among the three groups of rats given gentamicin 50 mg/kg, the animals that also received l‐carnitine 200 mg/kg (group D) had a significantly lower proportion of proximal tubules that displayed 1+ necrosis than the rats given no carnitine (group B) (Figure 4A) (P<0.01). There was no difference in the amount of 1+ necrosis in the rats given 50 mg/kg of gentamicin and either no carnitine (group B) or 40 mg/kg of carnitine (group C) (P=0.066). Among the groups of rats given gentamicin 80 mg/kg, the rats also receiving l‐carnitine 200 mg/kg/day (group G) had a significantly higher percentage of proximal tubular cells with 1+ necrosis as compared with the animals that were injected with either normal saline (group E) or 40 mg/kg of l‐carnitine (group F) (Figure 4A) (P<0.01 for each comparison). Among the rats receiving gentamicin 80 mg/kg/day, there were no differences with regard to the percentage of tubules displaying 2+ necrosis, although there was a tendency for the frequency of 2+ necrosis to decrease as the l‐carnitine dose increased from 0 to 40 to 200 mg/kg/day (Figure 4B). On the other hand, the percentage of proximal tubules with 3+ necrosis was significantly lower in the rats injected with gentamicin 80 mg/kg/day that were given l‐carnitine 200 mg/kg/day (group G) as compared with those given gentamicin 80 mg/kg/day and either carnitine 40 mg/kg/day (group F, P<0.02) or no carnitine (group E, P<0.01) (Figure 4C). The sums of the percentages of proximal tubules showing 0, 1+, 2+ or 3+ necrosis in all six groups of rats that received gentamicin are indicated in Figure 4D. Although each of the three groups of rats that received gentamicin 80 mg/kg had a significantly greater combined percentage of proximal tubules with 1+, 2+ or 3+ necrosis as compared with any of the groups of rats that were given no gentamicin (group A) or 50 mg/kg of gentamicin (groups B, C and D) (P=0.0001 for each comparison), there were no differences within the three groups of rats given gentamicin 80 mg/kg/day. The explanation for this lack of significant difference among the three groups of rats receiving 80 mg/kg of gentamicin is that the rats receiving normal saline or l‐carnitine 40 mg/kg tended to have less 1+ necrosis but more 2+ and 3+ necrosis compared with the animals given l‐carnitine 200 mg/kg/day. Figure 5 shows representative histological appearances of proximal tubules from control rats (group A) (Figure 5a) given no gentamicin, rats given gentamicin 80 mg/kg/day and no carnitine (Figure 5b and c), and rats given gentamicin 80 mg/kg/day and l‐carnitine 200 mg/kg/day (Figure 5d). Normal proximal and distal tubule cells and a normal glomerulus from a group A rat are shown in Figure 5a. Severe 3+ (asterisk) proximal tubular necrosis and also 2+ (hash sign) and 1+ (arrowhead) proximal tubular necrosis are displayed in a rat given gentamicin 80 mg/kg/day (Figure 5b and c). Tubular necrosis is not as severe or extensive in a rat given gentamicin 80 mg/kg/day and l‐carnitine 200 mg/kg/day (Figure 5d). Fig. 5.  View largeDownload slide Representative examples of renal histology from control rats (group A) not given gentamicin or l‐carnitine (A), rats given gentamicin 80 mg/kg/day and no l‐carnitine (B and C), and rats given gentamicin 80 mg/kg/day with l‐carnitine 200 mg/kg/day (D). Periodic acid–schiff stain. (A, C and D) Magnification:×40; (B) magnification:×50. *=3+ tubular necrosis; #=2+ necrosis; arrowhead=1+ necrosis (see Methods section). Fig. 5.  View largeDownload slide Representative examples of renal histology from control rats (group A) not given gentamicin or l‐carnitine (A), rats given gentamicin 80 mg/kg/day and no l‐carnitine (B and C), and rats given gentamicin 80 mg/kg/day with l‐carnitine 200 mg/kg/day (D). Periodic acid–schiff stain. (A, C and D) Magnification:×40; (B) magnification:×50. *=3+ tubular necrosis; #=2+ necrosis; arrowhead=1+ necrosis (see Methods section). The serum gentamicin concentrations at the end of both days 8 and 12, and the renal cortical gentamicin concentrations at the end of day 12 were each significantly greater in the three groups of rats injected with gentamicin 80 mg/kg compared with the three groups of animals given gentamicin 50 mg/kg (Table 3). However, within the three groups of rats given either the high dose or the low dose of gentamicin, there were no differences in the serum or renal cortical gentamicin concentrations. Discussion The results of this study indicate that l‐carnitine can ameliorate gentamicin‐induced renal toxicity in rats. Two different doses of gentamicin were used to create kidney disease. The 50 mg/kg/day dose was utilized because it induces a mild form of renal toxicity, whereas the 80 mg/kg/day dose was employed because it creates a more severe degree of renal injury [23,24,26]. The doses were given for 8 days, because this duration of treatment has been reported to induce renal injury with insufficiency, at least with the larger gentamicin dose. The l‐carnitine dose of 40 mg/kg/day was selected to examine whether an intake that was large but not too dissimilar from that given to humans [27] could protect against gentamicin nephrotoxicity. The l‐carnitine dose of 200 mg/kg/day was used in order to provide some assurance that if no protective effect of l‐carnitine was identified, the lack of effect could not be ascribed to an inadequately small dose. The l‐carnitine injections were initiated 4 days prior to commencing gentamicin treatment, also to provide some assurance that if l‐carnitine was not shown to be protective, it could not be due to a period of time when the kidney tissue was exposed to gentamicin without adequate tissue l‐carnitine levels. Subcutaneous injections of the 80 mg/kg/day gentamicin dose and, to a lesser extent, the 50 mg/kg/day gentamicin dose induced renal injury, as indicated by the renal histopathology and, most apparently with the larger gentamicin intake, a reduction in renal function. The 80 mg/kg/day gentamicin dose, as compared with the dose of 50 mg/kg/day, was clearly associated with a greater loss of renal function, as indicated by generally greater serum creatinine and urea concentrations and lower creatinine clearances, and more severe histopathology, as shown by the histological analyses of the renal cortical tissue obtained at the end of day 12. The present data indicate that the daily injections of l‐carnitine, especially with the larger dose of 200 mg/kg/day, ameliorated the degree of renal injury. Evidence for this was provided in the following findings: in the rats given gentamicin 80 mg/kg/day, those animals injected with 200 mg/kg/day of carnitine, compared with the rats given no carnitine, displayed lower serum urea and probably serum creatinine concentrations and a higher creatinine clearance at day 12. Also, the rats given gentamicin 80 mg/kg/day and l‐carnitine 200 mg/kg/day, compared with the control rats given no gentamicin, showed no differences at day 12 in serum urea levels. Moreover, among the rats injected with gentamicin 50 mg/kg/day, those animals given 40 or 200 mg/kg/day of l‐carnitine had higher creatinine clearances at day 12 than the those not given carnitine. These findings were generally corrobated or strengthened by analyses of the change in the values for these measurements obtained on day 12 vs day 1 (i.e. day 12 values minus day 1 values). Finally, the histological data indicate a protective effect of l‐carnitine. Among the rats given gentamicin 50 mg/kg/day, when no carnitine was administered there was significantly greater 1+ proximal tubular necrosis than was observed in the control (group A) rats. However, l‐carnitine, in doses of both 40 and 200 mg/kg, protected against this histopathological effect of gentamicin (Figure 4A). Similarly, in the rats given the greater dose of gentamicin, 80 mg/kg/day, the larger quantity of carnitine, 200 mg/kg/day, protected against significantly greater 3+ proximal tubular necrosis than was observed in the control (group A) rats (Figure 4C). Among the rats given gentamicin 50 mg/kg/day, there was significantly less 1+ necrosis in the rats given carnitine 200 mg/kg than in those given no carnitine. Among the rats injected with gentamicin 80 mg/kg/day, those animals given l‐carnitine 200 mg/kg/day displayed a significantly greater percentage of 1+ proximal tubular necrosis, but significantly less 3+ proximal tubular necrosis as compared with the rats injected with either no carnitine or l‐carnitine 40 mg/kg/day. A control group of l‐carnitine treatment without gentamicin was not included in this study. This group was excluded because there is no evidence that l‐carnitine has any beneficial or adverse effects on the kidney [10,11]. Indeed, in our study, the rat groups that received carnitine plus gentamicin, in general, had less histological abnormalities and greater renal function than the rats given the same dose of gentamicin but without carnitine, suggesting that l‐carnitine does not adversely affect the kidney. The finding that the creatinine clearance at day 12 was not different in the control (group A) rats as compared with any of the other six groups is somewhat puzzling. It may reflect the fact that the sample size of each group was only six or seven and the variance in creatinine measurements in 24 h urine collections in rats can be substantial. Recently, it has been reported that for humans, serum creatinine, in association with certain other clinical characteristics, may be a more accurate measure of the glomerular filtration rate than creatinine clearance [28]. It may be noteworthy that the only groups of rats that had mean values for creatinine clearance that exceeded the control (group A) rats were the two gentamicin‐treated groups that received the larger dose of carnitine, 200 mg/kg/day, and the animals given the smaller dose of gentamicin, 50 mg/kg/day, with 40 mg/kg/day of carnitine (Figure 3). The two groups of rats given gentamicin without any carnitine had the lowest mean values for creatinine clearance. The mechanisms of action by which l‐carnitine may ameliorate gentamicin‐induced nephrotoxicity in rats are unclear. Research suggests a number of possible mechanisms of gentamicin nephrotoxicity. One set of potential mechanisms involves the binding of gentamicin to the structure and perturbation of the function of biological membranes [3–6]. Gentamicin binds to anionic phospholipids [3,5,29], and may thereby alter the biophysical properties and functions of cell membranes by decreasing the permeability of the glycerol moiety of phosphatidylinositol [4], decreasing membrane fluidity [5,30] and promoting membrane aggregation [6,31]. Membranous structures that can be damaged by gentamicin include plasma membranes [32,33], lysosomes [32], mitochondria [7], microsomes [34] and probably the golgi apparatus [35]. Lysis of lysosomes containing gentamicin may release both potent acid hydrolases and high concentrations of the drug into the cytoplasm, disrupting critical intracellular processes including mitochondrial respiration [7], microsomal protein synthesis [34] and intracellular signalling via the phosphatidylinositol cascade [36], as well as the generation of hydroxyl radicals [37], all of which have been observed in experimental models of gentamicin toxicity. Thus, ATP depletion and also generation of oxidants and free radicals may occur in the kidney with gentamicin toxicity [7,9]. Carnitine plays an essential role in long‐chain fatty acid oxidation [10], and administration of l‐carnitine is associated with a linear dose‐dependent increase in myocardial ATP in a rat model of doxorubicin myocardial injury [14,15]. Indeed, experimental evidence indicates that carnitine can protect against doxorubicin‐induced myocardial injury and mortality [14,15,38]. Carnitine may also maintain membrane stability; this has been shown most clearly in erythrocytes. This effect of carnitine may be mediated either by its actions on membrane phospholipid fatty acids or by interactions between carnitine and cytoskeletal proteins [12]. Although carnitine may itself be an antioxidant, it appears to have a strong inhibitory effect on free radical production [16–18]. l‐carnitine is also reported to have a pronounced protective effect against renal ischaemia‐reperfusion injury [18,39]. Moreover, l‐proprionyl carnitine has been found to reduce cyclosporine‐induced lipid peroxidation [40] and decreased intracellular ATP levels [41] in the rat kidney. Further studies will be necessary to ascertain whether these or other actions of carnitine are responsible for its protective effect against gentomicin‐induced nephrotoxicity. In the present study, there was no evidence that carnitine altered the serum or renal cortical tissue concentrations of gentamicin (Table 2). Thus, it is not likely that l‐carnitine ameliorated gentamicin‐induced renal injury by altering the total body or renal tissue burden of gentamicin. It should be emphasized that in the present study, l‐carnitine was administered for 4 days prior to the onset of the gentamicin injections. Whether this treatment would prevent or ameliorate gentamicin‐induced renal injury in humans is not known. However, for l‐carnitine to become a practical therapy for humans receiving injections of gentamicin, it would first have to be shown that carnitine ameliorates the gentamicin‐induced renal injury even if the l‐carnitine is started concomitantly or after the onset of the gentamicin injections. Correspondence and offprint requests to: Joel D. Kopple, Division of Nephrology and Hypertension, Harbor‐UCLA Medical Center, 1000 W. Carson Street, Torrance, CA 90509, USA.Email: jkopple@rei.edu We thank Dr Nancy Berman for advice on the statistical analysis of the data. This paper was presented in part at the Ninth International Congress on Renal Nutrition and Metabolism, Vienna, Austria, 29 August to 1 September 1998, and the 31st Annual meeting of the American Society of Nephrology, Philadelphia, Pennsylvania, 25–28 October 1998. This research was supported by the Hungarian Ministry of Health (grant No. ETT 658/96), by Sigma‐Tau Inc., and by the GCRC (grant No. M01‐RR00425), National Center for Research Resources. References 1 Kaloyanides GJ, Bosmans J‐L, De Broe ME. Antibiotic‐ and immunosuppression‐related renal failure. In: Schrier RW, Gottschalk CW, eds. Diseases of the Kidney . Vol. II, 1997; 1115–1151 Google Scholar 2 Ferriols‐Lisart R, Alós‐Alminana. Effectiveness and safety of once‐daily aminoglycosides: A meta‐analysis. 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TI - l‐carnitine ameliorates gentamicin‐induced renal injury in rats
JO - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/17.12.2122
DA - 2002-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/l-carnitine-ameliorates-gentamicin-induced-renal-injury-in-rats-jNUYaJLTDW
SP - 2122
EP - 2131
VL - 17
IS - 12
DP - DeepDyve
ER -