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Institut National de la Santé et de la Recherche Médicale U319, Université Paris 7-Denis Diderot, France.
Correspondence to Dr. Thierry Gilbert, Institut National de la Santé et de la Recherche Médicale U319, "Développement normal et pathologique des fonctions épithéliales," Université Paris 7-Denis Diderot, 2 place Jussieu, Tour 33-43, case 7126, 75251 Paris Cedex 05, France. Phone: +33 1 43 25 04 70; Fax: +33 1 43 25 67 89; E-mail: TGilbert{at}Paris7.jussieu.fr
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal organogenesis relies on inductive interactions between the ureteric bud, an epithelial outgrowth of the Wolffian duct, and the metanephrogenic mesenchyme. Dichotomous divisions of the ureteric bud will form the collecting tubules, and epithelium-mesenchyme interactions at its terminal buds will induce the mesenchyme to condense and differentiate into glomerular and tubular structures (reviewed in reference (11). The general features of the events that lead to metanephros formation are known, but the underlying mechanisms—especially those controlling the branching morphogenesis and the formation of a given nephron number—are far from fully understood (12,13,14,15). However, inborn nephron deficits have been reported, and, according to their severity, they may initiate a progressive renal disease or influence the rate of progression of acquired renal disease (16). Little attempt has been made to investigate their origin, although intrauterine growth retardation and vitamin A deficiency might be major causes of inborn nephron deficit (17,18). In rat, in utero exposure to aminoglycoside antibiotics has been shown to induce a permanent nephron deficit (19,20). This particular adverse effect on the developing kidney, leading to oligonephronia, was not predictable from what was known of aminoglycoside nephrotoxicity in the adult. And even if mild, the bilateral nephron deficit observed was sufficient to accelerate the development of glomerular lesions in adulthood (21). Concerning the mechanisms of this oligonephronia, we have demonstrated that an impaired branching capacity of the ureteric bud, but not of its elongation ability, was the initiating event of this drug-induced nephron deficit (22).
No attention has been paid to the effect of ß-lactams on the embryonic kidney. The aim of this study was to determine whether they impair renal organogenesis. We studied two penicillins, ampicillin and amoxicillin, and one third-generation cephalosporin, ceftriaxone. Two experimental approaches were used. The first set of experiments was performed in vitro using whole metanephros organ culture. This culture system combined with specific labeling of glomerular structures has been shown to be well suited for recapitulating the main steps of renal organogenesis and quantifying in vitro nephrogenesis (23,24). Its use was aimed at testing the direct effect of a wide range of antibiotic concentrations on the ability of the kidney rudiment to grow and differentiate in vitro (25). The second experimental design consisted of in utero exposure of pregnant rat to these drugs, at a period overlapping early renal organogenesis. Histology and determination of the nephron mass in pups was performed to determine the potential embryo toxicity of these drugs.
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Materials and Methods |
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Metanephros Organ Culture
Metanephros organ culture was performed as described previously
(23,24).
After collection, metanephroi were freed of exogenous tissue and placed onto a
0.8-µm Millipore AA filter (Millipore, Saint-Quentin-en-Yvelines, France),
floating on a defined serum-free medium and incubated for 6 d in 35-mm Petri
dishes at 37 ± 0.5°C in a humidified incubator (5% CO2).
The following defined medium was used: Dulbecco's modified Eagle's medium/F12
(vol/vol) supplemented with 15 mM Hepes, 45 mM sodium bicarbonate (pH 7.45),
transferrin (6.2 x 10-8 M), selenium (6.8 x
10-9 M), insulin (8.3 x 10-7 M), triiodothyronin
(2 x 10-9 M), and prostaglandin E1 (7 x
10-8 M). Culture medium was changed daily, and no antibiotic or
fungicide was present throughout the control experiment. Under these
conditions, metanephroi can differentiate and nephrogenesis proceeds through
the usual stages (23). We
performed paired experiments using both metanephroi of the same fetus: One was
grown as a control, and the other was grown in the same medium supplemented
with the antibiotic. For each set of experiments, 10 to 15 pairs of
metanephroi were used, collected from at least three different litters.
Antibiotic stock solutions were prepared as follows: Amoxicillin was made at a
concentration of 4 mg/ml in the minimum medium (Dulbecco's modified Eagle's
medium/F12), and ampicillin and ceftriaxone were prepared in
phosphate-buffered saline (PBS) at concentrations of 10 and 30 mg/ml,
respectively. All solutions were stored at -20°C, and serial dilutions
were performed daily in the defined medium. Antibiotics were used at 10, 100,
or 1000 µg/ml.
Determination of Differentiation and Growth States of Cultured
Metanephroi
Differentiation was assessed by counting the glomerular structures present
within the whole explanted metanephroi, as described previously
(24). Briefly, filter-grown
metanephroi were fixed individually in 2% paraformaldehyde in PBS supplemented
with 0.1 mM CaCl2 and 1 mM MgCl2 (PBS/calcium magnesium
[CM]) for 2 h at 4°C. Then, the metanephroi were carefully detached from
the filter and rinsed sequentially in PBS/CM and PBS/CM containing 50 mM
NH4Cl. After permeabilization with saponin, the explants were
treated with vibrio cholera neuraminidase and labeled with fluorescein-coupled
helix pomatia agglutinin and rhodamine-coupled peanut agglutinin. The samples
were then mounted in PBS/glycerol mixed with anti-bleaching agents.
Overall, growth of the cultured metanephroi was determined by protein content measurement performed after glomeruli counting. Labeled metanephroi were rinsed in distilled water and sonicated for 15 s in 0.5 ml of distilled water. Protein content was measured according to Lowry's procedure, modified by Larson using serum bovine albumin as standard (26,27). Data are expressed as micrograms of protein per explant.
To assess the effect of antibiotics on the branching pattern of the ureteric bud, pairs of metanephroi were grown for 2 d with or without the test antibiotic and stained in toto with fluorescein-coupled dolichos biflorus agglutinin. The whole branching morphogenesis of the ureteric bud was visualized, and the number of tips was counted.
Nephron Mass Determination
The total number of nephrons was determined in the entire kidney of
14-d-old pups. Whole kidneys were incubated in 6N hydrochloric acid for 45 to
60 min at 37°C according to the kidney weight. After overnight storage in
water at 4°C, macerated kidneys were placed in a 100-ml gauged flask.
Skillful shaking leads to a suspension of tubular structures and unbroken
glomeruli. Two to four aliquots of 0.5 ml were pipetted and used for
glomerular counting.
Histologic Studies
Cultured metanephroi were fixed with 2.5% glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.4) and washed overnight in the same buffer. They were
then post-fixed in 1% osmium tetroxide, dehydrated through a graded ethanol
series, infiltrated, and flat-embedded in Epon. Serial sections of 1 µm
thickness were cut and stained with toluidine blue. To detect apoptotic cells
in metanephroi grown with or without penicillin, two approaches were used.
First, spatial visualization of apoptosis was performed using a whole-mount
in situ DNA end-labeling technique. Metanephroi were fixed in 2%
paraformaldehyde and submitted to a protocol of indirect Apoptag® in
situ apoptosis detection kit (Oncor, Gaithersburg, MD) modified by Smith
and Cartwright (28). Briefly,
samples were dehydrated through a graded ethanol/Tris buffer saline series,
treated with proteinase K (20 µg/ml for 20 min at 37°C), and incubated
with the terminal deoxynucleotidyl transferase solution containing digoxigenin
nucleotides for 1 h at 37°C. Digoxigenin-labeled DNA ends were recognized
by alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche
Diagnostics, Meylan, France), previously preabsorbed on rat embryonic tissues,
and visualized accordingly without counterstaining. In the second approach, we
determined apoptotic index according to morphologic criteria
(29,30)
following light microscopy examination of semithin sections of Epon-embedded
metanephroi. At a final magnification of x 1000, apoptotic cells can
clearly be distinguished from necrotic cells using the following stereotyped
criteria: occurrence in scattered cells, presence of nuclear condensation,
nondamaged plasma membrane, and cell shrinkage. Two plastic sections taken 25
µm apart were viewed for each group. Examination was focused on the induced
(condensed) metanephric mesenchyme facing the ureteric bud ends and on the
surrounding uninduced (loose) mesenchyme. Six samples were analyzed in control
and penicillin groups, representing more than 700 cells in each group.
Renal morphology of newborn rats was examined on 1-µm-thick sections. A median slice of 1 mm thickness was first cut and treated for Epon embedding. Serial sections were collected for each animal and stained with toluidine blue. The entire cortex and medulla were visible on each section. In 14-d-old pups, the right kidney was removed, cut parallel to the short axis, and immersed in Dubosq Brazil fixative (4% picric acid in acetic acid/ethanol 80%/formaldehyde, 1:10:4). Paraffin/paraplast 3-µm sections were stained with Masson's trichrome or with Jone's reticulin. Sections were examined with a Nikon Optiphot microscope, and photographs were taken on T-Max Kodak films. For morphologic measurements, video images were transferred in a PowerMac station and analyzed using NIH Image software.
Statistical Analyses
Data are reported as means ± SEM. Results obtained from paired
experiments were analyzed using the nonparametric Wilcoxon t test.
2 tests were used to compare the various degrees of severity
observed in vitro after antibiotic exposure. Comparison between
various groups of treated females was performed by variance analysis (ANOVA)
combined with the Fisher test. Significance was determined at P <
0.05.
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Results |
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Quantitative data concerning amoxicillin are reported in Figure 2B. Observation of the whole cultured metanephroi revealed less severe features at low doses than for ampicillin. At the concentration of 10 µg/ml, no effect on both growth and differentiation occurred. A 10-fold increase in the concentration leads to a significant deficit of in vitro nephron formation of about 20% without significant reduction in the protein content. However, further increase of amoxicillin dose in the culture medium had severe toxic effects. The overall growth of the explanted metanephroi was reduced by 62% (20.8 ± 2.3 versus 7.8 ± 1.6 µg protein/explant, n = 10). In vitro differentiation was even more impaired as judged by the reduction of 75% of the number of nephrons. Distribution of the nephron deficit severity for each dose indicated that a higher dose led to a more severe and more frequent nephron deficit.
Ceftriaxone
Metanephros organ cultures that grew in the presence of 10 or 100 µg/ml
of ceftriaxone showed no major histologic alteration, as shown in
Figure 3. Quantification of the
number of nephrons formed in vitro revealed a mild reduction of 14
and 17% for each concentration, respectively
(Figure 2C). No difference in
the protein content was measured compared with controls. Surprisingly, by
using ceftriaxone at the dose of 1000 µg/ml in the medium, a complete
failure of metanephros development occurred in vitro
(Figure 3D). The same results
were observed with 500 µg/ml (data not shown). The nephron deficit severity
for each dose is also reported in Figure
2C.
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In Utero Exposure to ß-Lactams
Effect on Pregnancy Outcome. Several parameters were monitored
following birth. The delivery of most of the females was observed during day
time, allowing us to determine the duration of gestation. Pups were counted
and weighed 4 h after birth. None of these parameters was affected by
ampicillin or amoxicillin treatment (Table
1). In the ceftriaxone group, females delivered 6 h earlier,
leading to slightly lower birth-weight pups.
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Effects on Postnatal Development and Renal Ontogeny. After normalization of each litter to eight pups, newborn rats were kept with their mothers for 2 wk. Their body weight, kidney weight, and the total number of nephrons were then measured. The same postnatal growth was observed among control, ampicillin, and amoxicillin groups (Table 2). The number of nephrons was significantly reduced in pups delivered from the ampicillin group, and to a lower extent in those from the amoxicillin group. However, one pup out of four had a nephron deficit higher than 20% in the ampicillin group (28,656 ± 404, n = 9). By doubling the dose of ampicillin in vivo, we observed the same degree of oligonephronia, but all of the pups were concerned (29,120 ± 720, n = 5). The 14-d-old pups born to ceftriaxone-treated mothers unexpectedly were found to be larger than the controls (Table 2). In addition, the presence of an even larger kidney was noted (+28%), as confirmed by the increased kidney weight to body weight ratio. No sign of obvious morphologic alteration was observed during kidney collection. Determination of the nephron mass did not reveal any reduction of the number of nephrons. Expressed per gram of body weight, ceftriaxone offspring have a lower number of nephrons than controls (Table 2). Because we were quite surprised by the complete block of nephrogenesis induced by ceftriaxone at the highest dose used in vitro, we conducted an additional series of in utero ceftriaxone exposure experiments by treating pregnant rats with 500 mg/kg for 5 d. None of the parameters we measured at birth was affected by this treatment (data not shown). No nephron deficit was detected in 2-wk-old pups, but the kidney was enlarged by 35% compared with controls, leading to a kidney-to-body mass ratio above 1%.
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Histologic Findings after ß-Lactam Exposure
From in vitro studies, semithin sections of ß-lactam-exposed
metanephroi showed no major pathologic findings when used at 10 or 100
µg/ml. In an attempt to investigate the mechanism of the nephron deficit
induced at the dose of 100 µg/ml, first we counted the number of terminal
branches of the ureteric bud after 2 d of culture. No difference was observed
between the pairs of metanephroi, whatever the amount of ß-lactam we used
(data not shown). Second, we examined the localization of cells showing
features of apoptosis in cultured metanephroi with or without 100 µg/ml
ß-lactam. Particular attention has been paid to the nephrogenic zone. As
shown in Figure 4, nick
end-labeled cells were more numerous in this area following penicillin
exposure than in controls. To confirm these findings and to specify which
mesenchymal cells were the most susceptible to apoptosis, a morphologic
approach was performed on plastic sections. In controls, the percentage of
apoptotic cells in the induced and uninduced mesenchyme was 1.1 ± 0.4
and 7.9 ± 1.3, respectively. After ampicillin and amoxicillin exposure,
a marked increase was observed within the condensed mesenchyme surrounding the
ureteric bud ends (5.1 ± 0.7 and 3.4 ± 0.5, respectively;
P < 0.05). The uninduced mesenchyme shows the same percentage of
apoptotic figures compared to controls (7.7 ± 1.3 and 9.3 ± 0.9
for ampicillin and amoxicillin, respectively). At the highest dose, dilation
of the intercellular spaces in ureteric bud extremities was a common feature
in all of the drug-exposed metanephroi (data not shown).
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From in vivo studies, according to the class of ß-lactam we used, two major findings were observed on renal sections from pups born to ß-lactam-treated mothers. In ampicillin or amoxicillin groups, kidneys of newborn rats and 14-d-old pups displayed enlarged tubular segments (Figures 5 and 6). In newborn rats, both the collecting and the proximal tubules were affected, exhibiting slightly or severely dilated tubular lumens (Figure 5). Most of the cysts were predominantly localized in the juxtamedullary cortex, i.e., where the first nephrons have been formed. But they were also observed in the subcapsular zone in connection with ureteric bud ends (Figure 5D) or with Bowman's capsule of glomeruli lying underneath S-shaped bodies (Figure 5E). Enlargement of collecting tubules was noted in the papilla (Figure 5F). Two weeks later, cysts were still present in focal areas in all viewed sections, apparently affecting collecting tubules (Figure 6, B and C). They were more pronounced in animals in utero exposed to ampicillin than to amoxicillin. Concerning the kidneys of pups born to ceftriaxone-treated mothers, a punctuate dark material was readily observed as soon as the histologic blocks were trimmed (Figure 6E). This feature affected most of the medullary areas except the outer stripe. An interstitial edema with some leukocytic inflammation was observed (Figure 6F). Using polarization equipment, microcrystal-like structures were found within these inflammatory cells (data not shown). Occasionally, microcysts and focal areas of tubular necrosis were also observed in the cortex. In all groups, a similar cortical thickness was measured at 2 wk of age.
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Discussion |
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The observation of a permanent nephron deficit induced in utero by amino-penicillin exposure, combined with evidence of cystic tubule dilation, was unexpected. These compounds are widely believed to have no developmental toxicity. Nevertheless, they do possess some nephrotoxicity, since reports have mentioned acute renal failure in pediatric patients receiving supratherapeutic doses of amoxicillin (31,32,33). In our study, therapeutic doses have been used to treat pregnant females. The critical parameter was likely to be the timing of exposure, as already reported (20). Organogenesis is a period when drugs are known to have the greatest potential to cause malformations (6). The mechanism of this oligonephronia was not completely elucidated. Apparently, it did not rely on a defect of ureteric bud branching, as previously shown for an antibiotic of the aminoglycoside family (22). However, the presence of cysts in cortical ducts of the developing collecting system, which is still involved in nephron induction at its tips, may have disturbed nephrogenesis. Alternatively, the increased rate of apoptotic cells among the mesenchyme may have caused the nephron deficit. During kidney development, largescale death is a normal feature in the nephrogenic zone, reaching up to 3% of cells in rat metanephroi (30,34). Here we showed that in vitro exposure to ampicillin or amoxicillin induced more apoptotic cells in condensed mesenchymal cells adjacent to ureteric bud ends. These cells are usually rescued from apoptosis to undergo epithelial morphogenesis upon induction (35). Our findings indicate that a smaller fraction of the mesenchyme will participate in nephrogenesis, which is consistent with previous reports showing that extensive apoptosis reduces the number of nephrons (36,37,38). Another mechanism could be based on the occurrence of hypokalemia, a common feature of penicillin antibiotics (39,40). Despite the fact that such electrolyte imbalance occurring during fetal development remains hypothetical, it has been demonstrated that the early metanephros development is extremely sensitive to potassium concentration variations (41). Decreased amounts of potassium in embryonic kidneys led to abnormal development that was characterized by failure of nephron induction and occasional cystic dilations of the ureteric bud. This has been reported both in murine and human kidneys (41,42). Interestingly, it has been demonstrated recently that a decrease in intracellular potassium is an early event in programmed cell death (43). It is tempting to speculate that a similar phenomenon might be the leading event of the penicillin-induced oligonephronia.
A moderate nephron mass reduction is likely to have no functional consequences at birth due to hyperfiltration of the remaining nephrons. Thus, at first, a mild oligonephronia is unlikely to be detected. However, even if mild, an inborn nephron deficit is susceptible to favor the development of glomerulosclerosis in adulthood, as already reported in rats (20) and mice (44). Some have also suggested that a reduced number of nephrons favor the development of hypertension (45). In this study of in utero ß-lactam exposure, none of these drugs had an effect on fetal growth. This was carefully checked since intrauterine growth retardation is known to induce permanent nephron mass reduction (46,47,48). In the present study, oligonephronia is combined with the development of renal cysts. The presence of enlarged tubular segments may raise some question of the normal differentiation of the juxtamedullary nephrons that have been induced during the peak exposure to the amino penicillin. But most of all, the presence of renal cysts in pups of 2 wk of age, if it persists, is likely to worsen the renal dysfunction in adulthood.
One of the difficulties in assessing potential drug developmental toxicity is to determine the maximal concentration to be used. As mentioned earlier, this was one of the reasons to use metanephros organ culture. Within 1 wk, this model, combined with quantitative approaches of nephron formation, allowed us to test various concentrations of ß-lactams. This confirms its usefulness as a screening test for potentially toxic drugs (25). In humans, intravenous administration of penicillin yields a mean plasma concentration of approximately 50 mg/L (49). We therefore assumed that concentrations of 10 and 100 µg/ml of culture medium were close to physiologic levels, in agreement with the very few data available on concentration measurements of these drugs in the immature kidney (7). For both ampicillin and amoxicillin, data gained from in vivo experiments were consistent with the in vitro data obtained with the dose of 10 µg/ml, and to a lesser extent with the concentration of 100 µg/ml. By contrast, the ceftriaxone data from metanephros organ culture were different from the in vivo results. This discrepancy may be explained by the protein-binding capacity of this cephalosporin. In the plasma, its degree of protein binding can reach 98%. In metanephros organ culture, a weak binding had probably occurred, leading to an increase of free ceftriaxone within the embryonic kidney and to increased potential toxic effects, particularly striking at high concentrations. This highlights a developmental toxicity for ceftriaxone that is unlikely to occur in vivo. Another typical feature of penicillins and cephalosporin-related renal injury is tubulointerstitial nephritis (40,50,51). A clear interstitial inflammation was detected in the medulla of young rats born to ceftriaxone-treated mothers. This ß-lactam is known to have a prolonged elimination half-life compared to amino penicillins, especially within the kidney, which may account for this feature (52). The edema was prominent as confirmed by the increased kidney weight. Whether this interstitial inflammation will be subsequently reversed remains to be demonstrated. However, due to frequent hypersensibility reaction development, reexposure to this drug should be avoided (31,40,51).
Penicillins cross the human placenta by simple diffusion and are well absorbed by the fetal tissues (i.e., the percentage of absorbed drug is considerably greater than in the adult) (8). Consequently, these drugs may persist for prolonged periods within the fetus. During organogenesis, a period of rapid embryonic differentiation, very low concentrations of toxic drugs may have devastating effects (53). And this is favored by a weak capacity for renal elimination and hepatic degradation in the fetus. Penicillins and cephalosporins have long been considered safe in pregnancy due to the absence of known fetal toxicity (2). However, none of these drugs has been classified in the class A of Food and Drug Administration fetal risk drug categories, therefore indicating that their absolute lack of developmental toxicity has not been demonstrated (54,55,56). Even if it is clear that they are not teratogenic, they may induce permanent, although silent at birth, renal defects. It has been proposed that a significant portion of human renal pathologies may have their origins in insults that occurred in utero (17,45).
In conclusion, we acknowledge that it is difficult to resolve the question of a ß-lactam-mediated fetal nephrotoxicity in humans by using animal models or cell/organ cultures. Species differences in handling of the drugs and thresholds of toxicity make the applicability of such studies to humans unclear. However, comparisons of rat and human developmental toxicity databases have yielded frequent overlaps, and laboratory animals may carry weight in predicting human developmental toxicity (57,58). The assessment of potential drug-induced renal maldevelopment in the fetus is urgently needed, but unfortunately, it remains a difficult task (59). Therefore, caution is needed, and the use of ß-lactam antibiotics in obstetrics should be carefully monitored.
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American Society of Nephrology
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) or with ([UNK]) ß-lactam at
three different concentrations. (A) Ampicillin. (B) Amoxicillin. (C)
Ceftriaxone. Below each pair of bars for every concentration of ß-lactam
is reported the distribution of the nephron deficit severity with the
following code: white, <20%; stippled, 20 to 40%; gray, 40 to 60%; black,
>60%. *P < 0.05, **P < 0.01,
and ***P < 0.001, respectively, compared to paired
controls.
