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. Author manuscript; available in PMC: 2009 Aug 20.
Published in final edited form as: Toxicol Sci. 2007 Jul 17;99(2):637–648. doi: 10.1093/toxsci/kfm184

The Utility of a Rodent Model in Detecting Pediatric Drug-Induced Nephrotoxicity

Parvaneh Espandiari *,1, Jun Zhang *, Barry A Rosenzweig *, Vishal S Vaidya †, Jinchun Sun ‡, Laura Schnackenberg ‡, Eugene H Herman *, Alan Knapton *, Joseph V Bonventre †, Richard D Beger ‡, Karol L Thompson *, Joseph Hanig *
PMCID: PMC2729403  NIHMSID: NIHMS129798  PMID: 17636248

Abstract

A multi-age rat model was used to identify potential age-related differences in renal injury following exposure to gentamicin (GM). In this study, 10-, 25-, 40-, and 80-day-old Sprague-Dawley rats were dosed with GM at 0, 50, or 100 mg kg-1 body weight per day (mkd) sc for 6 or 14 days. Urine samples were collected up to 72 h after initial dosing. The maximum tolerated dose was lower in 10-day-old rats than for other ages (none survived 11 days of treatment). Eighty-day-old rats given the highest dose showed a diminished rate of growth and an increase in serum creatinine, blood urea nitrogen (BUN), urinary kidney injury molecule-1 (Kim-1), and renal pathology. Ten- and 40-day-old rats given 100 mkd of GM for 6- or 14 days also had increased levels of serum BUN and Cr and renal pathology, whereas only mild renal alterations were found in 25-day-old rats. After 6 days of treatment with 100 mkd GM, significant increases in Havcr-1 (Kim-1) gene expression were detected only in 10- and 80-day-old rats. In urine samples, nuclear magnetic resonance and ultra performance liquid chromatography/mass spectrometry analysis detected changes related to GM efficacy (e.g., hippurate) and increases in metabolites related to antioxidant activity, which was greatest in the 80-day-old rats. The magnitude of the genomic, metabonomic, and serum chemistry changes appeared to correlate with the degree of nephropathy. These findings indicate that an experimental animal model that includes several developmental stages can detect age-related differences in drug-induced organ toxicities and may be a useful predictor of pediatric drug safety in preclinical studies.

Keywords: gentamicin, age-related nephrotoxicity, biomarkers, Kim-1

The spectrum and intensity of drug activity can be influenced by the level of organ development. In some instances responses are of sufficient magnitude to provoke an adverse drug reaction (ADR) (Makri et al., 2004; Stephenson, 2005). An ADR in pediatric patients may be different from that occurring in adults receiving the same drug. The incidence of ADRs in pediatric patients could be related to the nonlinear maturation of certain pathways responsible for drug absorption, distribution, metabolism, and excretion (ADME) (Faustman et al., 2000; Pirmohamed et al., 1998). The biological conditions present at a young age that could alter drug ADME include: differences in levels of drug metabolizing enzymes, stomach pH, gastrointestinal emptying time, levels of serum albumin, and in the nonadult body H2O:fat ratio (Cresteil et al., 1985; Heyman, 1998). In addition, drug activities can also be affected by developmental differences in the ratio of metabolites as well as lower levels of both biliary activity and renal excretion (Bates and Balistreri, 2004). One of the limiting factors in understanding ADRs in pediatric populations is the lack of appropriate animal models that can be used to predict the possible consequences of exposure to drugs during the early years of human development.

Gentamicin (GM), an aminoglycoside antibiotic, prescribed for the treatment of life-threatening infections (gram-negative and -positive bacilli) is associated with a 10-15% incidence of nephrotoxicity (acute renal failure [ARF] and increases in serum creatinine [Cr]) (Ali, 1995; Mingeot-Leclercq and Tulkens, 1999). GM-induced nephrotoxicity is attributed to an accumulation of a small percentage (∼5%) of the administered dose in renal cortical tubular epithelial cells (Nagai and Takano, 2004; Wiland and Szechcinski, 2003). The exact pathgenic mechanism responsible for GM nephrotoxicity is not completely delineated. Several hypotheses have been suggested including the generation of reactive oxygen species, renal cortical phospholipidosis, and inhibition of Na+--K+- ATPase (Ali, 1995).

There is a continuing need to develop specific and sensitive biomarkers for the early detection of renal toxicity. At present, the most common means of detecting renal injury is to monitor levels of blood urea nitrogen (BUN) and serum Cr. Detectable changes in the concentration of these two substances are observed only after a considerable amount of renal function is lost. This study was initiated to develop and characterize an animal model that could mimic the different stages of human development and to use this model to evaluate potential age-related differences in renal sensitivity to a nephrotoxic agent (GM).

MATERIALS AND METHODS

Animals

Sprague-Dawley rats (Harlan, Indianapolis, IN) that were 10, 25, 40, or 80 days old were used in this study. The acclimation period was different for each age group to allow dosing at the youngest age feasible; 7 days for 33- and 73-day-old rats and 2 days for 23-day-old rats. In order to obtain 10-day-old rats, pregnant females (gestation day 15) were allowed to deliver. After birth, both female and male pups were housed with their dams and were treated beginning at 10 days of age. Male rats were used in all age groups (n = 4) except for the 10-day old where both female and male pups were included (n = 12-15). In this age group, data from both genders were combined when analysis showed no female or male treatment group differences. The animals were housed in plastic cages and maintained in a controlled environment (22°C with a 12-h light-dark cycle). Rats had access to Purina rodent laboratory chow (Purina Mills, St Louis, MO) and water ad libitum.

Chemicals

GM was purchased from Sigma Chemical Co. (St Louis, MO). The drug was dissolved in normal saline at concentrations (0, 10, and 20 mg ml-1) before use. High-performance liquid chromatography (HPLC) grade acetonitrile and water were purchased from Burdick & Jackson (Muskegon, MI). Formic acid and leucine enkephalin were from Sigma Aldrich (St Louis, MO). NMR (nuclear magnetic resonance) solvents trimethylsilyl-2,2,-3,3-tetradeuteropropionic acid (TMSP) and deuterium oxide were obtained from Cambridge Isotope Laboratories (Andover, MA).

Experimental Protocol

Animals were placed in metabolism cages (25-day-old, two per cage and 40- and 80-day old, one per cage) 12 h before and up to 72 h after the first of three injections. Animals in the metabolism cages had free access to food and water. Rats were dosed once daily (early morning at the same time) with saline or GM at 50 or 100 mg kg-1 day-1 (mkd) (for 6 or 14 consecutive days). The dose volume of the sc injections of GM was 5 ml kg-1 body weight. For metabonomic analysis, urine samples were collected from all age groups at 0, 8, 24, 48, and 72 h after the initial dosing except for 10-day-old rats because maternal and pup urine could not be separated. Twenty-four hours after the last injection, all groups were anesthetized with isoflurane and terminal blood samples collected from the abdominal vena cava. The animals were then euthanized by exsanguination. At necropsy, liver, spleen, heart, intestine, and kidney were removed, weighed, and processed for pathology and other studies. All procedures performed during the course of the study were approved by the Center for Drug Evaluation and Research Institutional Animal Care and Use Committee and were in accord with the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Pathology

A portion of each tissue collected was fixed in neutral buffered formalin, embedded in paraffin (sectioned at 5 μm), and stained with hematoxylin-eosin. GM-induced renal lesions were evaluated by light microscopy according to the severity of tubular cell alterations (necrosis, degeneration, regeneration), tubular dilatation, tubular protein casts, glomerular vacuolization, and interstitium mononuclear cell infiltration.

Clinical Chemistry Analysis

The VetScan analyzer was used (Abaxis, Inc., Union City, CA) for all clinical chemistry analysis. For the 10-day-old group, blood from three pups was pooled to obtain a sufficient volume for the different assays.

Kim-1

Kim-1 protein in urine was measured by Microsphere-based Luminex xMAP technology. This technique is an adaptation of the recently developed and validated sandwich ELISA assay from Harvard Medical School (Vaidya et al., 2005, 2006). For measurements, 30 μl of urine from respective control and treated groups was analyzed in duplicate.

NMR Analysis

Samples were prepared by combining 400 μl of urine, 200 μl of sodium phosphate buffer (pH 7.4), and 60 μl of a mixture of 10mM TMSP (sodium 3-trimethyl-silyl-[2,2,3,3,-d4]propionate) and 100mM imidazole. Proton (1H) NMR spectra were acquired on a Bruker Advance spectrometer operating at 600.133 MHz. The NMR spectrometer was equipped with a triple resonance cryoprobe. Water suppression was achieved through application of the Bruker “noesypresat” pulse sequence, which irradiates the water resonance during a delay time (d1 = 2 s) and a mixing time (d8 = 100 ms). For each sample, 32 scans were collected into 65,536 data points. A spectral width of 9615.39 Hz was utilized with an acquisition time of 3.41 s.

Liquid Chromatography

For ultra performance liquid chromatography (UPLC)-mass spectrometry (MS) analysis, 100 μl aliquots of each urine sample was centrifuged at 15,866 g for 12 min at room temperature, and the supernatant liquid was transferred to autosampler vials. Metabolites were separated using a Waters Acquity UPLC system by injecting 5 μl samples of urine onto a Waters BEH C18 (2.1 mm × 10 cm, 1.7 μm) column held at 40°C. The metabolites were eluted from the column with a linear gradient of 0-30% B over 0-6 min, 30-50% B over 6-9 min, and 50-95% B over 9-11 min; the composition was constant for 1 min and then returned to 100% A at 12.1 min at a flow rate 0.15 ml min-1 (A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile). After each injection, a strong/weak wash cycle was employed on the autosampler to minimize the carryover between analyses. The weak needle wash solvent was 0.1% formic acid, and the strong needle wash solvent was 50:50 acetonitrile/water.

Mass Spectrometry

Mass spectrometric data were obtained with a Waters LCT premier single time-of-flight mass spectrometer equipped with an electrospray ion source. LCT premier was operated in W optics mode with 11,000 resolution using dynamic range extension. The source temperature was set up to 120°C with a cone gas flow of 50 l h-1, a desolvation temperature of 200°C, and a desolvation gas flow of 550 l h-1. The capillary voltage was 3.2 kV for positive mode and 2.6 kV for negative mode with a cone voltage of 40 V. A scan time of 0.5 s with an inter-scan delay of 0.05 s was used for all the analyses. Leucine enkephalin at a concentration of 250 pg μl-1 (in 50:50 acetonitrile:0.1% formic acid) was used as a lock-mass in positive mode ([M + H] = 556.2771) and 29 ng μl-1 for negative mode ([M - H] = 554.2615). The lock spray frequency was 5 s, and the lock mass data averaged over 10 scans for collection. A full scan mode from m/z 50 to 850 from 0 to 12 min was used for data collection, in both positive and negative mode. Compounds detected by MS were confirmed by comparison with authentic standards.

Renal Gene Expression Sample Processing and Analysis

For RNA isolation, kidneys were resected and quickly dissected into 0.5 cm sections while submersed in RNALater (Ambion, Austin, TX). Samples were stored in RNALater at 4°C for a minimum of 24 h and a maximum of 72 h. Kidney RNA was isolated using the Qiagen Maxi Kit Protocol after homogenization of the whole organ tissue using a VirTis Tempest rotor-stator homogenizer. RNA quality was assessed on an Agilent 2100 Bioanalyzer. RNA samples were quantitated on a NanoDrop ND-1000 spectrophotometer, aliquoted, and frozen at - 70°C. Quantitative analysis of transcript levels in total RNA was determined using real time quantitative reverse transcription (qRT)-PCR with kits from Applied Biosystems (Foster City, CA) (ABI). cDNA was generated using random hexamer primers and ABI TaqMan Reverse Transcription Reagents kits. PCR was performed using ABI SYBR Green PCR Core Reagents kits on the ABI Prism 7900HT sequence detection system as described in User Bulletin #2 (updated 10/2001). Five fourfold serial dilutions were used to prepare relative standard curves for each of the targets and an endogenous reference. Four replicate wells were performed on each sample, with an average technical variation of less than 4.3% across the entire dataset. Delta Ct values for target genes were imputed from standard curves from four technical replicates per sample and normalized to the control gene average. The endogenous reference gene used in these experiments was 60s ribosomal protein L7 (Rpl7), based on its low signal variance in microarray data from control and GM-treated kidney samples. Relative fold changes in renal gene expression were calculated for individual animals by dividing the amount of normalized target mRNA by the mean of three control animals in a given age group and time point. The statistical significance of differences in relative gene expression levels between time-matched control and treatment groups was calculated using a Student’s t-test comparison of fold change values between age-matched GM-treated and control groups.

Forward (For) and reverse (Rev) primer sequences were designed for the selected targets using ABI Primer Express software. HPLC-purified oligonucleotide primers were obtained from Bioserve Biotechnologies (Laurel, MD) for the following targets: Havcr1(Kim-1): For-GTGAGTGGACCAGGCACACA, Rev-AATCCCTTGATCCATTGTTTTC; Tnfrsf12a: For-CCACCCACTCGGATGGACT, Rev-CCCAGGGCTAAAACTCAGGG; Lcn2: For-ACAACGTCACTTCCATCCTCG, Rev-TGATCCAGTAGCGACAGCCC; Spp1: For-TGAACAGTATCCCGATGCCA, Rev-CTCTTCATGCGGGAGGTGA; Clu: For-GCTTCATTCCCTCCAGTCCA, Rev-TGTTCCAGCAGGGATGAGGT; Rpl7 (60s ribosomal protein L7): For-CAGTACGCGAAGGACATAGGC, Rev-CATTCAGGTCGCTTAGTCCAACT.

Statistical Analysis

All data from the serum, body, and organ weights were expressed as means ± SE and analyzed using ANOVA. Data from female and male 10-day-old rats were combined for cases in which there were no differences between genders.

Data Analysis

NMR data

Spectra were processed using ACD/Labs 1D NMR Manager (Toronto, Canada). The raw free-induction decays were zero filled to 131,072 points, multiplied by a 0.3 Hz exponential function and Fourier transformed. The transformed spectra were then phased using the simple method and baseline corrected using the “SpAveraging” method with a box half width of 61 points and a noise factor of 3. All spectra were autoreferenced to the TMSP peak at 0.0 ppm. The spectra were overlaid in the processing window and grouped. Regions containing the resonances for water, urea, and other solvent peaks were removed prior to integration. The intelligent bucketing module was employed for integration with the bucket width set to 0.04 ppm and the looseness set to 50% for bin size optimization. The table of integrals was exported as a text file for statistical analysis. All statistical analyses of NMR data were done using Statistica version 6.0 (Statsoft, Tulsa, OK). Principal component analysis (PCA) based on covariance of the data was applied to the bucketed intensities. Metabolite identification within the individual spectra was accomplished using the Chenomx NMR Suite (Chenomx, Calgary, Canada), which has a database of >250 compounds.

MS data

After data acquisition using UPLC-MS, the raw data were analyzed using the Micromass MarkerLynx Application version 4.0 (Waters Corporation, Milford, MA). MarkerLynx was employed for peak finding and peak alignment. The original data were processed using the following parameters: initial retention time 0 min, final retention time 12 min, mass tolerance 0.02 Da, retention time tolerance 0.1 min, 20 masses in a 0.2-min retention window. The raw data were then transformed into a single matrix containing aligned peaks with the same mass/retention time pair along with peak normalized intensities and sample name. The resulting two-dimensional data were analyzed by PCA and evaluated for markers that related to age, efficacy, and renal toxicity.

RESULTS

Mortality

GM-induced lethality was noted only in the 10-day-old pups. In this age group, all pups died after 10 or 11 days of 50 or 100 mkd GM treatments (data not shown).

Growth, Liver, Spleen, and Heart

The effects of GM on body weight and other major organ weights are summarized in Table 1. The rate of growth (final body weight to the initial body weight expressed as percent of control) was significantly decreased only in 80-day-old rats treated with 100 mkd GM for 14 days. As a means of assessing GM-induced organ toxicity, the ratio of kidney, liver, heart, and spleen weights to the final body weights of the animals were determined (expressed as percent of control). Kidney weight was significantly increased in 40- and 80-day-old rats treated with 100 mkd of GM for 14 days. A similar change was noted in the kidney of 10-day-old rats given a 100 mkd dose of GM for 6 days. Liver weights significantly decreased in 40-day-old rats following 6- or 14-day treatment with either 50 or 100 mkd GM. A similar decline in liver weight was observed in 80-day-old rats but only in those animals treated with 100 mkd GM for 6 days. No changes in heart or spleen weight were found in any of the age or treatment groups (data not shown).

TABLE 1. Significant Changes in Body, Kidney, and Liver Weights in Rats of Different Ages following Treatment with GM.

Days old GM dose/duration Final body weight/initial body weight % control (g) Kidney weight/Final Body weight % control (g) Liver weight/final body weight % control (g)
80 50 mkd/6 NS NS 82 ± 2.1*
100 mkd/14 83 ± 6.5* 170 ± 34* NS
40 50 mkd/6 NS NS 91 ± 3.0*
50 mkd/14 NS NS 87 ± 6.5*
100 mkd/14 NS 128 ± 4.8* 81 ± 2.5*
10 100 mkd/6 NS 187 ± 15* NS
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The results are mean ± SE, n = 4 for 25-, 40-, and 80-day-old rats. N = 12-15 for 10-day-old pups. NS 1/4 not significant.

*

Significantly different from control ( p < 0.05). Body, kidney, and liver weights from 25-day-old rats were not significantly affected by GM treatment.

Clinical Chemistry

Dose levels of 100 mkd GM caused significant increases in serum BUN and Cr in 40- and 80-day-old rats after 14 days of treatment and in 10-day-old pups after 6 days of treatment (Figs. 1 and 2). The 80-day-old rats that were given the highest dose of GM for 14 days had significant declines in certain clinical chemistry parameters. The average values of the following serum markers in the control and treated groups, respectively, were alkaline phosphatase (266 ± 41 ul-1 and 163 ± 31 ul-1), albumin (4.1 ± 0.2 g dl-1 and. 3.47 ± 0.1 g dl-1), and total protein (5.27 ± 0.1 mMol l-1 and. 1.4 ± 0.1 mMol l-1). In contrast, serum cholesterol levels were significantly increased (p < 0.05, n = 4) after 6 days of treatment with GM (71 ± 2.3 mg dl-1 at 50 mkd and 73 ± 9.2 mg dl-1 at 100 mkd compared to 55 ± 3.3 mg dl-1 in controls). No significant changes in clinical chemistry concentrations were seen in the other age groups at any GM doses.

FIG. 1.

FIG. 1

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Changes in BUN levels following treatment with 50 or 100 mg kg-1 day-1 GM in 10- (for 6 days of treatment), 25-, 40-, or 80-day-old (for 6 and 14 days of treatment) Sprague-Dawley rats. *Significantly higher than control group (p < 0.05).

FIG. 2.

FIG. 2

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Changes in serum Cr levels following treatment with 50 or 100 mg kg-1 day-1 GM in 10- (for 6 days of treatment), 25-, 40-, or 80-day-old (for 6 and 14 days of treatment) Sprague-Dawley rats. *Significantly higher than control group (p < 0.05).

Kidney Injury Molecule-1

Urine samples from 25-, 40-, and 80-day-old animals were collected in metabolism cages for up to 72 h after GM injection in order to detect early biomarkers of nephrotoxicity. For 25-day-old rats, samples were obtained by pooling the urine from two rats per cage (n = 4 pooled samples). The levels of Kim-1 increased significantly only in 80-day-old rats treated with 100 mkd GM at 48 and 72 h (386 ± 48 pg ml-1 vs. 140 ± 23 pg ml-1 at 48 and 271 ± 32 pg ml-1 vs. 138 ± 16 pg ml-1 at 72 h). No significant changes were seen at either GM dose in the other age groups.

Gene Expression Markers of Nephrotoxicity

Treatment with nephrotoxic drugs or ischemic injury induces the upregulation of a number of gene transcripts in a dose- or time-dependent manner in kidney tissue, including Havcr1 (named kidney injury molecule-1 [Kim-1] in rats and hepatitis A virus cellular receptor 1 in other species), clusterin (Clu), lipocalin2 (Lcn2), osteopontin (Spp1), and tweak receptor (Tnfrsf12a) (Amin et al., 2004; Davis and Kramer, 2006; Thompson et al., 2004). In this study, the age-dependent effects of GM on these five putative gene expression markers of nephrotoxicity were assayed after 6 or 14 days of dosing with GM. The transcript levels of the five genes in kidney tissue were measured in qRT-PCR assays relative to an endogenous control gene and compared to age- and time-matched controls. After 6 days of dosing with 100 mkd GM, only the 10-day-old age group showed significant elevations in the levels of all five nephrotoxicity marker genes (p < 0.01) (Fig. 3A). In the 80-day-old group, levels of Havcr1, Tnfrsf12a, and Spp1 transcripts were significantly increased (p < 0.05) at this dose and time point. In 25- and 40-day-old rats, significant treatment-related increases were observed for only one of the five markers (Tnfrsf12a and Spp1, respectively). Six days of treatment with 50 mkd GM also significantly increased all five indicator genes in 10-day-old rats (data not shown) but was not assayed in the older groups.

FIG. 3.

FIG. 3

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Effect of GM on changes in gene expression indicators of nephrotoxicity as a function of age, dose, and treatment time. Transcript levels for Havcr1 (H), Tnfrsf12a (T), Lcn2 (L), Spp1 (S), and Clu (C) are shown for individual animals within each age group of 10-, 25-, 40-, or 80-day-old Sprague-Dawley rats relative to the average level in controls. Target gene transcript levels in each sample are normalized to an endogenous control gene transcript (Rpl7). (A) Treatment groups received 100 mkd GM (open box, n = 4) or vehicle control (circle, n = 3) for 6 days. (B) Treatment groups received either 50 mkd (shaded box, n = 3) or 100 mkd (open symbol, n = 4) GM or vehicle control (circle, n = 3) for 14 days. Statistical significance in a Student’s t-test comparison of age- and time-matched treated and control values is indicated (*p < 0.01; ‡p < 0.05). Solid gray lines demarcate 1.5-fold change levels.

After 14 days of dosing with 50 mkd GM, Havcr1, Tnfrsf12a, and Clu were significantly upregulated in the 25-, 40-, and 80-day-old age groups (Fig. 3B). No samples from 10-day-old rats were available for this time point due to premature deaths. Lcn2 and Spp1 transcript levels were significantly elevated in 40- and 80-day-old rats, but not in 25-day-old rats, at this dose level. After 14 days of dosing with 100 mkd GM, a considerable amount of renal damage (Fig. 8B) and significant upregulation of all five expression biomarkers of nephrotoxicity (Fig 3B) are observed in all three age groups (p < 0.01), except for Lcn2 expression in 25-day-old rats. The observed effect of treatment on the level and significance of change in genes associated with nephrotoxicity suggests the following order of sensitivity to GM-induced renal damage: 10-day-old ⪢ 80-day-old > 40-day-old > 25-day-old.

FIG. 8.

FIG. 8

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Photomicrographs showing GM-induced renal lesions. H&E stain, × 400 (A-J). Epithelial cell degeneration (arrow) and few necrotic cells (arrow head) were seen in 10-day-old female (A) or male rats (B) treated with 100 mkd GM for 6 days. Epithelial cell necrosis (arrow) was observed in 25-day-old rats (C) treated with 100 mkd GM for 14 days but not for 6 days (data not shown). In 40-day-old rats treated with 100 mg kg-1 day-1 of GM, tubular cell necrosis is (arrow) more severe at 14 days (E) than at 6 days (D). In 80-day-old rats treated with GM for 6 days, tubular cell necrosis (arrow) is more severe at a dose of 100 mg kg-1 day-1 (G) than at a dose of 50 mg kg-1 day-1 (F). Severe tubular necrosis (arrow) in the kidneys of 80-day-old-rats treated with 50 mg kg-1 GM for 14 days (H). Severe tubular necrosis (arrow) (I) and glomerular vacuolization (white arrow) in the kidneys of 80-day-old-rats treated with 100 mg kg-1 GM for 14 days (J).

Metabonomics Analysis

NMR results

Figure 4A shows the three-dimensional PCA (3D PCA) of the NMR data of urine samples from three age groups (25-, 40-, and 80-day-old rats), at both GM doses (50 and 100 mkd), and all time points (0, 8, 24, 48, and 72 h). The PCA plot shows that the samples cluster primarily by age group in different regions of the plot. Closer inspection revealed that within each age cluster, there are sub-clusters for the doses and the various time points. There is a clear time-dependent response to GM with the trajectory moving away from the predose (0 h) time points.

FIG. 4.

FIG. 4

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The scores plot for 3D PCA based on covariances of the NMR data from 25-, 40-, and 80-day-old Sprague-Dawley rats at all time points and doses (A). The scores plot for 3D PCA based on covariances of the UPLC/MS negative data from 25-, 40-, and 80-day-old Sprague-Dawley rats at all time points (B).

At both GM doses, urinary hippurate levels were significantly decreased in all age groups at 48 and 72 h after dosing. Figure 5A shows the decrease of hippurate as detected by NMR on day 3 (72 h time point) of the 6-day study. Since GM is a known antibiotic, the decrease in hippurate is a drug efficacy effect as it decreases the total gut microflora content (Lenz et al., 2005). Urine glucose concentrations were significantly increased in all age groups 72 h after dosing at the 50 and 100 mg kg-1 level when compared to age-matched controls except for the low dose 25-day-old animals at 48 h after dosing (Fig. 5B). Glucosuria has been shown previously to be a potential marker of renal toxicity (Lenz et al., 2004, 2005; Williams et al., 2003).

FIG. 5.

FIG. 5

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Effects of treatment with 50 and 100 mg kg-1 day-1 GM on urinary hippurate (A) and urinary glucose (B) determined by NMR in 25-, 40-, and 80-day-old (after 72 h of treatment) Sprague-Dawley rats.

UPLC/MS Analysis of Urine

The urinary metabonomic data obtained via UPLC/MS revealed marked metabolic alterations related to animal age and GM treatment at all time points, in both positive and negative modes. Figure 4B shows the 3D PCA plot of the negative mode UPLC/MS data from all the urine samples in the study. The three different age groups separate into distinct clusters similar to the pattern seen with the NMR data. Additionally, a clear separation of the treated and control groups at each age group at 24, 48, and 72 h time points was observed in PCA analyses of UPLC/MS data (data not shown). The spectra from the dosed animals began to deviate from controls at the 24 h time point and were further separated at 48 and 72 h. Furthermore, UPLC/MS analysis also showed a decrease in the concentration of hippuric acid following GM administration as was also noted by the NMR analysis. Many other unidentified UPLC/MS peaks were detected that were decreased significantly by day 3 in all three age groups and could be GM efficacy markers. Other metabolites detected and identified by UPLC/MS will be reported in a separate publication. Figure 6A illustrates the trend view for 6-hydroxymelatonin from UPLC/MS analysis in negative mode ionization in 25-, 40-, and 80-day rats dosed with 100 mkd GM. 6-Hydroxymelatonin appeared to increase at 48 h in both 40- and 80-day-old groups after treatment with 100 mkd GM, but not in the 25-day-old group. Figure 6B shows a typical positive ion trend plot for an ion with m/z 227.0983 and tR 6.73 min from urine analysis in 25-, 40-, and 80-day-old rats dosed with 100 mkd GM. This unknown ion at tR 0f 6.73 min and m/z 227.0983 sharply increases on day 3 in 80-day-old rats treated with 100 mkd GM and could be a potential marker of GM-induced renal toxicity. The identifications of many of the detected ions are still being elucidated. Further studies are needed to confirm the identity of the ion with m/z 227.0983 as well as to confirm it as a biomarker of GM toxicity.

FIG. 6.

FIG. 6

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(A). Negative mode UPLC/MS trends plot for 6-hydroxymelatonin in individual 25-, 40-, and 80-day-old Sprague-Dawley rats dosed with 100 mg kg-1 day-1 GM (A). Positive mode UPLC/MS trends plot of unknown ion with tR 6.73 min and m/z 227.0983 in individual 25-, 40-, and 80-day old rats dosed with 100 mg kg-1 day-1 GM (B). Percent on y-axis represents the ion’s relative intensity.

Age-Related Differences in Normal Kidney Histopathology

Light microscopic examination revealed significant age differences in normal kidney histology between saline-treated controls. Kidneys from 10-day-old pups showed a very thin cortex compared with the medulla and papilla (Figs. 7A and 7B). The glomerulus was seen as a densely arranged group of cells, without distinguishable tufts of anastomosing capillaries. Cortical epithelial cells located in the proximal convoluted tubules (PCT) appeared low cuboidal in shape and small in size, without recognizable brush borders on the surface (Figs. 7C and 7D). In 25- (Figs. 7E and 7F) and 40-day-old rats (Figs. 7G and 7H), the cortical area was increased; however, the glomerular capillaries and brush borders on the surface of PCT cells were barely discernable (Figs. 7G and 7H). At 80 days, the rat kidney appeared fully developed (thick cortex, tufts of anastomosing glomerular capillaries, and PCT cells with surface brush borders) (Figs. 7I and 7J).

FIG. 7.

FIG. 7

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Micrographs showing age-related changes of the kidney in saline-treated rats. Hematoxylin and eosin (H&E) stain × 50 for A, C, E, G, I, and × 100 for B, D, F, H, and J. Ten-day-old female rats (A and B) and 10-day-old male rats (C and D) have thin cortex and undeveloped glomerulus and tubules. Twenty-five-day-old male rats (E and F) and 40-day-old rats (G and H) show an increase in width of the cortex enlargement of the glomerulus and barely recognized brush borders on the surface of the PCT. Eighty-day-old male rats (I and J) show mature cortex of normal width, developed tufts of glomerular capillaries, and brush borders.

Age-Related Differences in GM-Induced Renal Alterations

Dosing with 100 mkd GM caused a slight pale discoloration in the kidneys of 40- and 80-day-old rats after 14 days and in 10-day-old rats after 6 days of treatment. Renal lesions induced by both GM doses in 10-day-old pups consisted mainly of PCT epithelial cell degeneration (cytoplasmic vacuoles) and occasional necrosis (Figs. 8A and 8B). PCT epithelial cell necrosis was found frequently in 25-day-old rats treated with 100 mkd GM for 14 days (Fig. 8C). PCT epithelial cell necrosis was found to be more severe in 40-day-old rats that were treated with GM for 14 days compared to 6 days treatment (Figs. 8D and 8E). Similarly, 80-day-old animals dosed for 6 days with 100 mkd GM had more severe PCT epithelial necrosis than those dosed in the same time frame with 50 mkd GM (Figs. 8F and 8G). In general, the most severe lesions induced by either dose of GM (massive necrosis and regeneration of PCT epithelial cells, tubular dilatation and casts, glomerular vacuolization, and interstitial lymphocytic infiltration) were seen in the 80-day-old animals treated for 14 days (Figs. 8H-J).

DISCUSSION

ADRs in the pediatric population are a major health issue and are associated with increased morbidity and/or mortality (Impicciatore et al., 2001). The FDA Guidance for Industry: Non-Clinical Safety Evaluation of Pediatric Drugs (2006) has stressed the importance of developing animal models for the pediatric population. The guidance cites several reports on the differential toxic effects of therapeutic drugs in pediatric patients. For example, acetaminophen is less toxic in children (American Academy of Pediatrics, 2001) while valproic acid, chloramphenicol, and lamotrigine are more toxic in the pediatric population (Dreifuss, 1987; Guberman et al., 1999; Kapusnik-Uner et al., 1996). As mentioned in the guidance, the developmental characteristics of animals are similar to humans; however, it is important to consider the comparability of the stages of development in animal models to that of the intended population. With regard to the kidney, the glomerular nephrogenesis in the prenatal human is comparable with that found in 8- to 14-day-old rats, the level of glomerular filtration and tubular secretion occurring in 45-to 180-day-old infants is similar with that observed in 15- to 21-day-old rats, and the completion of nephrogenesis of 35-week prenatal human is comparable with that reported in 4-6 weeks rat (Travis, 1991; Zoetis, 2003).

A significant proportion (20%) of currently prescribed medication for adults can cause kidney injury leading to ARF. As many as 2.5% of these cases are caused by giving antibiotics to hospitalized patients (Kleinknecht and Pallot, 1998; Nolan and Anderson, 1998). The incidence of ARF resulting from administration of drugs in newborns is higher than in adults (Simeoni et al., 1996). Data regarding the incidence of drug-induced nephrotoxicity in premature newborns are not available (Fanos and Cataldi, 2002). GM treatment has been reported to cause a 20% reduction of the number of nephrons and a delay in maturity of the glomerular barrier in the rat (Beauchamp et al., 1992; Smaoui et al., 1993).

In this study, we demonstrated that rats at different stages of development elicit a differential sensitivity to GM. It is apparent that the age is an important factor that needs to be considered in preventing drug-induced nephrotoxicity. For the present study, 10-day-old pups were more sensitive to the toxic effects of GM than other age groups. The 10-day-old rats treated with 50 or 100 mkd of GM for 6 days developed similar levels of mild tubular cell degeneration. High levels of BUN, Cr, and increased expression of Havcr1 were detectable in samples from the groups of pups given 100 mkd GM. Further, treatment with either dose of GM for 10-11 days was lethal to all pups. The thinness of the cortex, the undeveloped glomerular capillaries, and the limited brush borders of tubular epithelial cells could affect the renal distribution and ultimately the toxicity of GM. The nephrotoxic effects of GM are thought to be due to selective accumulation of drug in the PCT epithelial cells (an increased binding affinity on the luminal surface and increased membrane permeability) (Kiyomiya et al., 2000). It was not determined, in the present study, whether there was significant accumulation of GM in the PCT epithelial cells after 6 days of treatment in the pups.

In 25-day-old rats given GM for 6 days, no significant changes were observed in the pathology of the kidneys at either dose or in levels of the majority of renal injury biomarkers at the high dose. After 14 days treatment, the low dose of GM caused minimal effects on renal pathology but a significant increase in Havcr1 expression. Only 14 days of treatment with the high dose of GM induced a mild dose-related renal alterations and significant elevations in the levels of four gene expression markers of nephrotoxicity (Havcr1, Tnfrsf12a, Spp1, and Clu) in the 25-day-old rats.

As rats matured (40- and 80-days-old), the histological appearance and function of the kidney became more adult like (broad cortex, tufts of anastomosing capillaries in the glomerulus, abundant brush borders on the surface of tubular cells). It is thought that at this age, glomerular filtration and selective tubular reabsorption of GM was normal. From evidence provided by traditional and novel biomarkers of renal injury (pathology and gene expression markers at 6 and 14 days, serum BUN and Cr at 14 days), 40-day-old rats were more sensitive to GM-induced nephrotoxicity than 25-day-old rats but less sensitive than 80-day-old rats.

To determine whether the sensitivity of various nephrotoxic biomarkers is similar in different age groups, we evaluated a variety of traditional as well as potential new nephrotoxic biomarkers. Based on the serum level of BUN and Cr data in this study, nephrotoxicity was more severe in 80- and 10-day- old compared to 25- and 40-day-old rats. Detectable changes in the serum levels of BUN and Cr are reported when significant kidney function is lost (Star, 1998). Consequently, if these two substances were used as sole biomarkers, some potential nephrotoxic drugs might not be detected. A more recently identified protein Kim-1 (a type 1 transmembrane protein, with an immunoglobulin and mucindomain) is markedly up-regulated following renal injury in both rats (postischemic) and human (acute tubular necrosis) (Ichimura et al., 1998; Vaidya et al., 2005). Several studies have demonstrated that Kim-1 may be a more sensitive early detector of drug-induced nephrotoxicity (Vaidya and Bonventre, 2006). In the present study, the concentration of Kim-1 in the urine started to increase significantly after 48 h only in GM-treated 80-day-old rats. Based on results from Kim-1 (urinary and mRNA), the 80-day-old rats were more sensitive to GM compared to 40- and 25-day-old groups. With the apparatus available, we were not able to collect urine samples for 10-day-old rats. Although it could not be directly assessed in this study, Kim-1 could be a useful biomarker for drug-induced nephrotoxicity in the youngest animals because we observed a large induction in Havcr1 gene expression in response to GM-induced injury in 10-day-old rats and a good correlation between the induction of Havcr1 mRNA in the kidney and increased levels of Kim-1 in urine after nephrotoxicant treatment in other studies.

PCA analysis of the NMR and UPLC/MS metabonomics data from different ages, time points, and GM doses indicated a distinctly separate and unique pattern for each of the different age groups. These patterns in the 3D PCA plots represent urinary metabolic changes due to aging, GM efficacy, and early biomarkers of GM renal toxicity. Urine levels of glucose and 6-hydroxymelatonin correlated with GM toxicity and levels of hippurate correlated with the known antibiotic action of GM. Many more metabolites detected by NMR and UPLC/MS correlated with age, efficacy, and toxicity, and further analysis of the NMR and UPLC/MS markers will be published separately. The metabonomics study was able to detect GM toxicity much earlier than traditional clinical chemistry end-points. As such, metabonomics was only applied to analyze urine samples from the first 3 days of the 6-day study where there was less toxicity than observed in the 14-day study. In this study, the urinary metabonomic bioindicators are all early markers that appear between days 1 and 3. The genomic markers were measured at later time points (days 6 and 14), so a direct comparison of relative sensitivity between -omic markers could not be made. As mentioned in the results section, the identity of 6-hydroxymelatonin in urine indicated that GM-induced antioxidants appear as early as 3 days after initiating GM treatment. 6-Hydroxymelatonin is an oxidized product of melatonin, which is a powerful antioxidant (Ma et al., 2006). Melatonin has been demonstrated to be a direct scavenger of nitric oxide, which has been implicated in the tissue damage observed following ischemic and toxin-induced ARF. There is also some evidence that melatonin increases tissue arginase activity, which by consequence inhibits nitric oxide syntheses activity (Aydogdu et al., 2006). Melatonin supplementation has been shown to protect the kidney from GM and mercuric chloride-induced renal toxicities (Sener et al., 2002). Therefore, the increased levels of the melatonin metabolite, 6-hydroxymelatonin may represent the body’s first defense against tissue damage caused by a toxic dose of GM. This early toxicity marker may, therefore, permit intervention prior to irreversible renal damage. Glucose and the unknown ion with tR 6.73 min and m/z 227.0983 were also noted as possible early markers of GM-induced toxicity. Glucose has been previously shown to be a general marker of renal toxicity (Lenz et al., 2004, 2005; Williams et al., 2003). Further studies are required in order to identify and qualify the unknown ion m/z 227.0983 as a marker of GM toxicity.

In conclusion, 10- and 80-day-old rats were found to be more susceptible to GM nephrotoxicity than 25- and 40-day-old rats. It is apparent that a multi-age animal model could be appropriate to more fully characterize the toxicity of potential agents across different stages of development. This is of considerable significance since, at present, many drugs that are used in the pediatric population are not sufficiently evaluated to determine the appropriate dosing level for efficacy and safety.

ACKNOWLEDGMENTS

Authors thank Dr Terry Miller for assistance during this project and Dr Ricky Holland for work on evaluating urinary metabolites by LC/MS that were not reported in this manuscript.

FUNDING

This work was supported in part by Scientist Development Grant 0535492T from the American Heart Association to V.S.V. and NIH grant DK 074099 to J.V.B.

Footnotes

The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration.

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