ANTIMICROBIAL AGENTS AND CHEMOTHERAPY

,
0066-4804/99/$04.00ϩ0
May 1999, p. 1020–1026 Vol. 43, No. 5
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Effectiveness and Toxicity of Gentamicin in an Experimental Model
of Pyelonephritis: Effect of the Time of Administration
MICHEL LEBRUN,
1
LOUIS GRENIER,
1
PIERRETTE GOURDE,
1
MICHEL G. BERGERON,
1
GASTON LABRECQUE,
2
AND DENIS BEAUCHAMP
1
*
Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Que ´bec,
1
and
Faculte ´ de Pharmacie, Universite ´ Laval,
2
Sainte-Foy, Que ´bec,
Canada G1V 4G2
Received 27 July 1998/Returned for modification 26 December 1998/Accepted 21 February 1999
Temporal variations in the renal toxicity of aminoglycosides have been reported for experimental animals as
well as for humans. In fact, maximal renal toxicity of aminoglycosides was observed when the drug was given
during the rest period, while a lower toxicity was observed when the drug was injected during the activity
period. The aim of the present study was to evaluate temporal variations in the effectiveness and renal toxicity
of gentamicin in an experimental model of pyelonephritis in rats. The experiments were carried out with female
Sprague-Dawley rats (185 to 250 g). They had free access to food and water throughout the study and were
maintained on a 14-h light–10-h dark cycle. Animals were divided into four groups corresponding to the
respective time of induction of pyelonephritis and treatment: 0700, 1300, 1900, and 0100 h. Pyelonephritis was
induced by a direct inoculation of Escherichia coli (10
7
to 10
8
CFU) in the left kidney. Animals were treated for
3 and 7 days with a single daily dose of gentamicin (20 and 40 mg/kg of body weight, respectively) or saline
(NaCl, 0.9%) at either 0700, 1300, 1900, or 0100 h. Animals treated at 0100 h for 3 days with gentamicin (20
mg/kg) showed a significantly lower number of bacteria in their kidneys than did all other groups (P < 0.01).
After 7 days of treatment, the efficacy, evaluated by the log CFU per gram of tissue and by the percentage of
sterilized kidneys, was also higher when gentamicin was administered at 0100 h. The ␤-galactosidase and the
N-acetyl-␤-D-glucosaminidase activities were significantly higher in urine of rats given gentamicin at 1300 h
than in urine of rats treated at another time of day (P < 0.05). Gentamicin injected at 1300 h induced a
significantly greater increase of [
3
H]thymidine incorporation into DNA of renal cortex (P < 0.01), a signifi-
cantly greater inhibition of sphingomyelinase activity (P < 0.05), and significantly more histopathological
lesions than the same dose injected at another time of the day. Creatinine and blood urea nitrogen levels in
serum were significantly higher (P < 0.05) and the creatinine clearance was significantly lower (P < 0.05) when
gentamicin was injected at 1300 h than when it was injected at another time of day. Our data suggest temporal
variations in both the toxicity and the effectiveness of gentamicin, the drug being more effective and less toxic
when injected during the activity period of the animals.
Although antibiotics have been used in therapeutics for
more than 60 years, no rational and standardized approaches
have been defined for the treatment of urinary tract infections
(12, 27, 47). In Canada and the United States, patients consult
physicians more than 6 million times a year for this type of
infection, which is one of the most commonly observed in
clinical practice (48). Approximately 250,000 patients suffer
from acute pyelonephritis, and hospitalization is often needed
(12, 41).
Aminoglycosides are widely used in the treatment of severe
gram-negative bacterial infections. Unfortunately, the clinical
use of aminoglycosides is limited by their potential ototoxicity
and nephrotoxicity (25). Nevertheless, their concentration-de-
pendent bactericidal action, their antibacterial synergism with
␤-lactam antibiotics, their postantibiotic effect, their low cost,
and the better understanding of risk factors associated with the
use of these agents are responsible for the maintenance of
their clinical use.
In the last 10 years, new approaches to the management of
renal pathologies have been evaluated, but the treatment of
pyelonephritis has not changed significantly (11, 12). However,
several approaches to reducing the incidence of renal toxicity
associated with aminoglycoside treatment have been studied.
For instance, animal studies showed that the coadministration
of agents such as poly-L-aspartic acid (8), daptomycin (9),
ceftriaxone (10), and fleroxacin (7) with aminoglycosides sig-
nificantly reduced the nephrotoxicity of aminoglycosides. How-
ever, the clinical application of these approaches remains un-
certain.
The once-daily administration of aminoglycosides is actually
the most attractive alternative for reducing the renal toxicity of
these agents in patients. Animal studies indicated that extend-
ing the frequency of injection was as effective as and less toxic
than more frequent administration of aminoglycosides (18,
21). Several meta-analyses of randomized human clinical trials
compared the clinical efficacy, the bacteriological cure, and the
incidence of nephrotoxicity of the same total daily dose of
aminoglycosides administered once daily with the effects of
multiple doses injected during the day (1–3, 20, 22, 23, 36).
These analyses suggested that the once-daily dosing with ami-
noglycosides is at least as effective and safe as the multiple-
dose regimen of aminoglycosides.
Unfortunately, these studies did not take into account the
time of injection of these antibiotics throughout the day. In
fact, we previously reported for rats temporal variations in the
renal toxicity of aminoglycosides (30, 33), and these data were
* Corresponding author. Mailing address: Centre de Recherche en
Infectiologie, RC 709, Centre de Recherche du Centre Hospitalier
Universitaire de Que ´bec, Pavillon CHUL, 2705 Boul. Laurier, Ste-Foy,
Que ´bec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-
2715. E-mail: denis.beauchamp@crchul.ulaval.ca.
1020

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in agreement with those obtained by other investigators (15,
37, 39, 40, 51, 52). Peak renal toxicity was observed when
aminoglycosides were injected in the middle of the rest period
of the experimental animals, while lower toxicity was found
when they were treated in the middle of the activity period.
These data are also in agreement with those of Prins et al. (44),
recently obtained for patients. In fact, they reported that neph-
rotoxicity occurred more frequently when aminoglycosides
were administered to patients between midnight and 0730 h
than when administered at any other hour of the day.
The temporal variations in the effectiveness of aminoglyco-
sides have never been investigated for animals. Thus, the ob-
jective of this study was to investigate the circadian variations
in the toxicity and the efficacy of gentamicin in an experimental
model of pyelonephritis in rats.
MATERIALS AND METHODS
Animals and treatment. Female Sprague-Dawley rats (Charles River Breeding
Laboratories, Inc., Montre ´al, Que ´bec, Canada) aged 58 to 82 days and weighing
185 to 250 g were used in this study. They had free access to food and water
throughout the experiment. They were maintained on a 14-h-light (rest period)
and a 10-h-dark (active period) schedule with the light on at 0600 h, for 1 week
before induction of pyelonephritis. Animals were divided into four groups on the
basis of the time of day that infection was induced and treatment was given.
Pyelonephritis model. The bacterial strain used to induce pyelonephritis was
Escherichia coli Yale EY-9 (furnished by V. Andreoli, Yale University). Before
the induction of pyelonephritis, animals were anesthetized with sodium pento-
barbital (Nembutal sodium [Abbott Laboratories]) at 50 mg/kg of body weight,
intraperitoneally (i.p.). The side of the animals was shaved and asepticized, and
a small incision was made at the level of the kidney. The left kidney was exposed,
and 0.05 ml of an inoculum containing 10
7
to 10
8
bacteria was injected through
the upper and lower poles of the kidney. This technique, described previously by
Kaye (26), produces a constant and severe pyelonephritis in the left kidney with
extensive inflammation and abscess formation induced by the direct inoculation
of E. coli and a less severe pyelonephritis in the right kidney due to the reflux of
infected urine. Pyelonephritis was induced at either 0700, 1300, 1900, or 0100 h,
and the treatment was initiated exactly 24 h after the time of inoculation. The
MIC of gentamicin against the E. coli strain was 0.5 ␮g/ml.
Effectiveness study. Infected animals were treated with a single daily injection
of gentamicin (20 and 40 mg/kg, i.p.) for 3 and 7 days at the same time of day at
which the infection was previously induced: 0700, 1300, 1900, or 0100 h. Saline
(NaCl [0.9%]) was injected into control animals in a volume of 0.2 ml at the same
hour of the day as that for the aminoglycoside. Twenty-four hours after the last
injection, a minimum of 10 rats per group were anesthetized as described above,
blood was sampled by cardiac puncture, and animals were killed by decapitation.
At the time of sacrifice, a midline abdominal incision was made and both kidneys
were aseptically removed, decapsulated, weighed, and homogenized in 3 ml of
sterile saline at 4°C. Appropriate dilutions of homogenized kidneys were made,
and 10-␮l samples were placed in triplicate on MacConkey agar. The number of
CFU of E. coli in the kidneys was determined after an incubation of 18 h at 37°C
(the CFU per milliliter of homogenate were transformed into CFU per gram of
tissue). The bacterial enumeration was done at the dilution that allowed us to
detect between 30 and 300 CFU/g of kidney. The limit of detection was 30 CFU/
g of kidney. Kidneys were considered sterile when no CFU were detected on the
agar. The efficacy of gentamicin was assessed by comparing the number of CFU
measured in the infected and treated rats with that of animals injected with saline
at the same time. The efficacy of gentamicin was also evaluated by comparing the
percentages of sterile kidneys in the infected and treated animals at the four
times of day.
Nephrotoxicity study. Groups of rats were infected as described above at 0700,
1300, 1900, and 0100 h. Exactly 24 h after the induction of the infection, groups
of at least six infected rats were treated with a single daily i.p. injection of
gentamicin (40 mg/kg) or saline for 7 days at the same time at which the infection
was induced. During treatment, animals were placed individually in metabolic
cages to collect urine over a 24-h period for the measurement of specific enzyme
activities as markers of toxicity. Urine was collected under mineral oil immedi-
ately after the first gentamicin injection (day 1) and 24 h before sacrifice (day 7),
and the volume was noted. All samples were centrifuged (1,340 ϫ g) for 15 min,
and the enzymuria was assessed immediately after centrifugation. At the time of
sacrifice, a midline abdominal incision was made and the right kidney of each
animal was rapidly removed and bisected. The blood was centrifuged, and the
serum collected was quickly frozen at Ϫ20°C for the determination of creatinine
and blood urea nitrogen (BUN) levels. The cortex of the kidneys was dissected,
and a piece of tissue was quickly frozen in dry ice for further determination of
sphingomyelinase activity and the [
3
H]thymidine/DNA ratio. Another part of the
cortical tissue was cut into small blocks (approximately 1 mm
3
) in a drop of 0.5%
glutaraldehyde–0.1 M phosphate buffer (pH 7.4) and left overnight in the same
fixative at 4°C.
Enzymuria. The activities of ␤-galactosidase, N-acetyl-␤-D-glucosaminidase,
and ␥-glutamyltransferase were measured as an index of tubular damages. The
activities of ␤-galactosidase and N-acetyl-␤-D-glucosaminidase, lysosomal en-
zymes, were measured by the method of Maruhn (35). The activity of ␥-glutamyl
transpeptidase, an enzyme of the brush border membrane, was evaluated by the
methodology of Persijn and van der Slik (42).
Biochemical analysis. The cellular regeneration in the renal cortex was eval-
uated by measuring the radioactivity of [
3
H]thymidine incorporated into DNA as
described by Laurent et al. (29) on purified DNA obtained from the cortical
tissue of the right kidney. Sphingomyelinase (EC 3.1.4.12) activity was assayed in
the renal cortex as described previously (28). Serum creatinine and BUN levels
were evaluated with a Hitachi 737 apparatus.
Histology. Cubes previously fixed overnight were washed with phosphate
buffer (0.1 M, pH 7.4), fixed in 1% osmium tetroxide for 60 min at room
temperature, dehydrated in ascending grades of alcohol, and embedded in
Araldite 502 epoxy resin. Thick sections (1 ␮m) were cut with an Ultracut E
ultramicrotome (Leica Canada, Inc., Que ´bec, Que ´bec, Canada), stained with hot
toluidine blue, and examined with a blind code to identify gross lesions. Micro-
scopic renal lesions were scored on plastic sections at a magnification of ϫ400.
Each slide was coded so that identification of the groups was not possible for the
observer. Slices came from three different pieces of renal cortex for each rat, and
five rats per group were used. The following lesions in the renal cortex were
recorded: isolated cell necrosis, tubular necrosis (proximal tubule with more than
50% necrotic cells), tubular desquamation (proximal tubule with 100% necrotic
cells), the number of proximal tubules with metachromatic material in their
lumens, and the number of interstitial cells (no specific identification of cell type
was made). The total number of proximal tubules was also measured on each
slice. The number of isolated necrotic cells, the number of necrotic proximal
tubules, the number of desquamated proximal tubules, and the number of prox-
imal tubules with metachromatic material in their lumens were recorded as the
percentages of the total number of proximal tubules on each respective slice and
assigned a score as follows: 0 to 9%, 1; 10 to 19%, 2; 20 to 29%, 3; etc. The score
for the interstitial cells was obtained by dividing the total number of interstitial
cells (excluding endothelial capillary cells) by the total number of proximal
tubules on each respective slice. The lesion scores were summed up to produce
a single toxicity score for each animal.
Statistics. All statistical analyses were performed with the Stat-View SE ϩ
Graphics 1988 program (Abacus Concepts, Inc., Berkeley, Calif.). Analysis of
variance by a least-squares method was used to determine the statistical signif-
icance of the difference between groups. A p value smaller than 0.05 (P Ͻ 0.05)
was considered significant. Group comparisons were performed by Fisher’s pro-
tected least significant difference test.
RESULTS
Effectiveness study. Figure 1 shows the effectiveness of gen-
tamicin (20 and 40 mg/kg) given once daily to infected rats for
3 days. In infected animals treated with gentamicin (20 mg/kg),
the log CFU per gram was significantly lower than that for the
infected controls only when gentamicin was administered at
0100 h (P Ͻ 0.05). When gentamicin was injected at a dose of
40 mg/kg, a significant reduction (P Ͻ0.05) in the log CFU per
gram of tissue compared with that for control infected rats was
observed at all injection times, but the reduction in the bacte-
rial counts was slightly greater (not significant) when gentami-
cin was injected at 0100 h than at other times of day.
Figure 2 shows the effectiveness of a once-daily gentamicin
(40 mg/kg) injection of infected rats for 7 days. Gentamicin
produced a significant reduction (P Ͻ0.05) of the log CFU per
gram of tissue in all treated groups (Fig. 2, dotted line) in
comparison to that of their time-matched controls (solid line).
Figure 2 shows also that gentamicin injected at 0100 h steril-
ized 40% of infected kidneys, while the percentage of sterile
kidneys was 25, 0, and 33% in animals treated at 0700, 1300,
and 1900 h, respectively.
Nephrotoxicity study. Figure 3 shows excretion of ␤-galac-
tosidase, N-acetyl-␤-D-glucosaminidase, and ␥-glutamyltrans-
ferase in the 24-h urine of infected rats treated for 7 days with
gentamicin (40 mg/kg/day) at 0700, 1300, 1900, and 0100 h.
The excretion of each enzyme was significantly higher in
urine of animals injected at 1300 than in urine of animals
injected at any other time of day. Similar effects were observed
VOL. 43, 1999 GENTAMICIN IN PYELONEPHRITIS 1021

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on the first day of treatment, but the level was much greater on
day 7.
The inhibition of sphingomyelinase activity, the cellular re-
generation, and the histopathological scores measured in the
renal cortex of rats treated for 7 days are presented in Fig. 4.
The inhibition of sphingomyelinase activity was significantly
more severe when gentamicin was injected at 1300, 1900, and
0100 h than when animals were injected with gentamicin at
0700 h (P Ͻ 0.01). In comparison to their time-matched con-
trols, the animals treated at 1300 h were the only ones to show
a significant inhibition of the sphingomyelinase activity (P Ͻ
0.01). The cellular regeneration was significantly higher in the
renal cortex of animals treated with gentamicin at 1300 than in
their time-matched controls (P Ͻ 0.01) and in other treated
groups (P Ͻ 0.01). The histopathological nephrotoxicity scores
were also significantly higher for animals treated with genta-
micin at 1300 h than for their time-matched controls (P Ͻ0.01)
and for the other groups treated at the other times of day (P Ͻ
0.01).
Figure 5 shows plastic sections of the left renal cortex of
infected nontreated animals (controls) (A) and the renal cor-
tex of infected animals treated with gentamicin for 7 days at
0100 (B) or 1300 (C) h. The renal cortex of infected control
animals shows typical signs of pyelonephritis such as the pres-
ence of intensive peritubular infiltration with inflammatory
cells and the loss of the structural integrity of the tissue. The
peritubular cell infiltration was less severe in gentamicin-
treated infected rats. In infected animals treated with genta-
micin at 1300 h, the renal cortex still shows peritubular cell
infiltration as well as signs of gentamicin toxicity such as des-
quamated and necrotic proximal tubular cells (Fig. 5C) while
these changes were not observed for animals treated with gen-
tamicin at 0100 h (Fig. 5B). However, large lysosomes can be
observed in proximal tubular cells in the latter group (Fig. 5B).
FIG. 1. Log CFU per gram of kidney (Ϯstandard deviations [SD]) of animals infected with E. coli and treated with either saline (infected) or a single daily injection
of gentamicin (infected and treated) at doses of 20 and 40 mg/kg for 3 days at either 0700, 1300, 1900, or 0100 h. Treatment was initiated 24 h after the induction of
infection. ء, significantly different from time-matched infected and treated animals (P Ͻ 0.05). Open boxes, light period; closed boxes, dark period.
FIG. 2. Log CFU per gram of kidney (Ϯ standard deviations [SD]) and percentages of sterile kidneys (treated animals only) of animals infected with E. coli and
treated with either saline (infected) or a single daily injection of gentamicin (infected and treated) at doses of 40 mg/kg for 7 days at either 0700, 1300, 1900, or 0100 h.
ء, significantly different from time-matched infected and treated animals (P Ͻ 0.05). Open box, light period; closed box, dark period.
1022 LEBRUN ET AL. ANTIMICROB. AGENTS CHEMOTHER.

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These observations are consistent with the data for the his-
topathological quantification of the cellular toxicity induced by
gentamicin presented in Fig. 4.
Renal function. Figure 6 shows creatinine and BUN levels in
FIG. 3. Twenty-four-hour urinary excretion of ␤-galactosidase (A), N-acetyl-
␤-D-glucosaminidase (B), and ␥-glutamyl transpeptidase (C) in infected rats
treated for 7 days with saline or gentamicin. Gentamicin was given as a single
daily dose of 40 mg/kg/day i.p. at either 0700, 1300, 1900, or 0100 h. The
enzymuria was expressed as a percentage of control animals Ϯ standard devia-
tion (SD). §, significantly different from the respective time-matched controls
(P Ͻ 0.01); ء, statistical differences (P Ͻ 0.05) between the groups under the
extremities of the line. Open boxes, light period; closed boxes, dark period.
FIG. 4. Inhibition of sphingomyelinase activity (A), cellular regeneration
(B), and total histopathologic nephrotoxicity scores (C) in renal cortices of
infected rats treated for 7 days with gentamicin. Gentamicin was given once daily
at either 0700, 1300, 1900, or 0100 h at a dose of 40 mg/kg/day. The data are
presented as the means Ϯ standard deviations (SD) and expressed as the per-
centages of the values measured for control animals. §, significantly different
from the respective time-matched controls (P Ͻ 0.01); ء, statistical differences
(P Ͻ 0.01) between the groups under the extremities of the line. Open boxes,
light period; closed boxes, dark period.
VOL. 43, 1999 GENTAMICIN IN PYELONEPHRITIS 1023

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serum as well as the creatinine clearance of infected rats
treated with a single daily injection of gentamicin at a dose of
40 mg/kg for 7 days. Serum BUN and creatinine levels were
significantly higher in animals treated with gentamicin at
1300 h than in their time-matched controls (P Ͻ 0.05) and in
rats treated at 0700 and 0100 h (P Ͻ 0.05). The creatinine
clearance was significantly lower in animals treated with gen-
tamicin at 1300 than in their time-matched controls (P Ͻ 0.05)
and in rats treated at 0700 and 0100 h (P Ͻ0.05). Furthermore,
the creatinine clearance was significantly lower in animals
treated with gentamicin at 1900 than in animals treated at
0700 h (P Ͻ 0.05).
DISCUSSION
The objective of our study was to evaluate the effects of the
time of administration on the effectiveness and the renal tox-
icity of gentamicin in an experimental model of pyelonephritis
in rats. Our data show that both the effectiveness and the
toxicity of gentamicin varied over the 24-h period and that the
efficacy was best at the time when the toxicity of the drug was
the lowest. Data indicated also that the presence of infection
does not modify the circadian rhythm of aminoglycoside tox-
icity.
Temporal variations in the renal toxicity of aminoglycosides
have been reported for experimental animals since 1982.
Briefly, Nakano and Ogawa (37) showed for the first time that
the survival rate of mice treated once daily with gentamicin was
higher when the drug was injected in the middle of the activity
period (0200 h) and lower when gentamicin was injected in the
middle of the rest period (1400 h) of the animals. Similar
results were reported by Cambar and his colleagues (13, 39)
with high doses of gentamicin, dibekacin, and netilmicin. Tem-
poral variations in the nephrotoxicity of aminoglycosides were
also observed with lower doses of gentamicin (19, 40, 51),
amikacin (14, 16), and isepamicin (52). In all these studies, the
high acute doses of aminoglycosides produced greater toxicity
when they were injected in the middle of the rest period, while
toxicity was less when the antibiotics were injected in the mid-
dle of the activity period of the experimental animals. Studies
done in our laboratory showed similar results, as we injected
daily small doses of aminoglycosides over 7 to 10 days. In fact,
our data showed that tobramycin (30, 33) and isepamicin (49)
induced higher toxicity when injected at 1400 h than when the
same dose was injected at 0200 h. Recently, Prins et al. showed
for the first time that the incidence of nephrotoxicity was sig-
nificantly higher in patients receiving aminoglycosides during
their sleeping period (midnight to 0730 h) than in patients
receiving drugs at other times of day (44).
The mechanisms responsible for the temporal variation in
renal toxicity of aminoglycosides are still unknown. Some au-
thors suggested that circadian rhythms in the urinary excretion
of water, electrolyte, and renal perfusion (45); in membrane
structure and fluidity (17); and in the number and size of
autophagic vacuoles in the cytoplasm of tubular cells (43)
might be responsible for the temporal variations in the renal
toxicity of aminoglycosides. Temporal variations in the phar-
macokinetics of aminoglycosides in serum could also be in-
volved in the time-dependent changes in aminoglycoside neph-
rotoxicity. Such changes were found in the levels of these
antibiotics in plasma in animals (24, 30, 38, 46, 52) and in
humans (34, 38, 50). Time-dependent changes were also found
in the cortical accumulation (30, 40, 51, 52) and in the kinetics
of the intracortical accumulation rate (31) of different amino-
glycosides.
Other experiments in our laboratory showed that the 24-h
variation in the serum corticosterone levels or changes in the
subcellular distribution of aminoglycosides could not explain
the chrononephrotoxicity of aminoglycosides (6). However,
our data showed that a time-restricted feeding schedule in-
duced a shift in the maximal and minimal levels of gentamicin
nephrotoxicity in animals injected at different times of the day
(5). Furthermore, fasting abolished the 24-h variations in the
nephrotoxicity of gentamicin (4). Fasting was also associated
with higher levels of tobramycin in the serum and cortex (32).
We thus concluded that the time of dosing of gentamicin rel-
FIG. 5. Plastic sections of the right renal cortex of infected control animals
(A) and infected animals treated for 7 days with gentamicin at either 0100 h (B)
or 1300 h (C). G, glomerulus; PT, proximal tubule; DT, distal tubule; NPT,
necrotic proximal tubule; DPT, desquamated proximal tubule. Arrowheads,
metachromatic material in tubular lumen. Magnification, ca. ϫ300.
1024 LEBRUN ET AL. ANTIMICROB. AGENTS CHEMOTHER.

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ative to the time of feeding seems to be a more important
modulator of aminoglycoside nephrotoxicity than is the light-
dark cycle.
In this study, we showed again that gentamicin induced
higher toxicity when injected in the middle of the rest period
(1300 h) and that toxicity was lower when the drug was injected
in the middle of the activity period (0100 h). Moreover, no
temporal variations in the cortical accumulation of gentamicin
were observed in the present study (data not shown), as re-
cently demonstrated with tobramycin (33). As these data were
observed for animals suffering from pyelonephritis, it can thus
be concluded that the presence of infection did not modify the
temporal variations in the renal toxicity of gentamicin.
Our results also demonstrate a circadian rhythm in the ther-
apeutic efficacy of gentamicin in the treatment of experimental
E. coli pyelonephritis. The effectiveness of gentamicin was
evaluated by two methods: (i) the log CFU of bacteria in the
renal tissue in the treated animals compared to those in their
time-matched infected controls and (ii) the percentage of ster-
ile kidneys, observed at the end of therapy. In fact, the per-
centage of sterile kidneys was higher when the drug was in-
jected at 0100 h than when it was injected at 0700, 1300, and
1900 h. Thus, the effectiveness of gentamicin was highest dur-
ing the activity period in animals treated with gentamicin at
doses of 40 mg/kg for 7 days.
Several factors might explain the temporal variations in the
effectiveness of gentamicin in this experimental model. First,
the higher water and food intake, resulting in a higher urine
output, might have contributed to increasing the efficacy and
decreasing the toxicity of gentamicin in infected animals when
injected during the activity period. In fact, it might have con-
tributed also to increasing the clearance of bacteria from the
kidney and increasing the efficacy of gentamicin during this
period of the day. However, since no temporal variations were
observed in the cortical accumulation of gentamicin in the
present study, these drug levels could not contribute to the
temporal variations in the effectiveness of the drug. It is inter-
esting to note that Hosokawa et al. (24) also showed temporal
variations in the effectiveness of a single dose of amikacin in an
experimental model of i.p. infection with Pseudomonas aerugi-
nosa. In infected mice, the 50% effective dose of amikacin was
lower in the midlight phase (1300 h) at time of lower clearance
than in the middark phase (0100 h), when clearance was
higher.
Based on our studies and on the evidence available in the
current literature, it is quite clear that the renal toxicity of
aminoglycosides can be reduced by giving the drug as a single
daily injection when patients are active. Even if extrapolation
of experimental data to humans is always uncertain, it is rea-
sonable to believe that the administration of gentamicin in the
beginning or the middle of the day in humans may reduce renal
toxicity and increase the efficacy of these antibiotics. In fact,
some chronopharmacologic studies have already been applied
to patients with very conclusive results (34, 38, 50). In humans,
the clearance of aminoglycosides was lower during the night
(in the rest period) and maximal during the activity period
(day). These results perfectly correlate with those obtained for
experimental animals. The prospective study of Prins et al. (44)
clearly showed that the incidence of toxicity in 179 patients was
significantly higher when the drug was given as a once-daily
injection during the sleeping period of patients. Further inves-
tigations must be done to investigate the effect of the time of
administration on aminoglycoside effectiveness in patients, but
physicians should already be careful when administering ami-
noglycosides to patients at night. The clinical applications of
these results may be eventually extended to pathologies such as
FIG. 6. BUN level (A), serum creatinine level (B), and creatinine clearance
(C) of infected rats treated for 7 days with saline or gentamicin. Gentamicin was
given as a single daily dose of 40 mg/kg at either 0700, 1300, 1900, or 0100 h. The
data are presented as the means Ϯstandard deviations (SD) and expressed as the
percentages of the values measured for control animals. §, significantly different
from the respective time-matched controls (P Ͻ 0.05); ء, statistical differences
(P Ͻ 0.05) between the groups under the extremities of the line. Open boxes,
light period; closed boxes, dark period.
VOL. 43, 1999 GENTAMICIN IN PYELONEPHRITIS 1025

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pyelonephritis and pneumonia that could be treated with once-
a-day aminoglycosides.
ACKNOWLEDGMENTS
This study was supported by the Kidney Foundation of Canada. M.
LeBrun is the recipient of a studentship from the Fonds pour la
Formation de Chercheurs et l’Aide a ` la Recherche (F.C.A.R.).
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1026 LEBRUN ET AL. ANTIMICROB. AGENTS CHEMOTHER.

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