1H NMR Pattern Recognition and 31P NMR Studies with d-Serine in

Chem. Res. Toxicol. 2003, 16, 1207-1216

1H

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NMR Pattern Recognition and 31P NMR Studies with D-Serine in Rat Urine and Kidney, Time- and Dose-Related Metabolic Effects Rebecca E. Williams,* Matthew Jacobsen, and Edward A. Lock Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom Received April 17, 2003

Proton NMR spectroscopy of urine has previously been used to gain insight into the site and mechanism of toxic injury to the kidney. D-Serine injures the rat kidney, causing selective necrosis of the proximal straight tubules. Damage is accompanied by proteinuria, glucosuria, and amino aciduria, the latter preceding the onset of necrosis. This study has employed 1H NMR spectroscopy of urine and 1H NMR and 31P NMR spectroscopy of kidney extracts to examine the nephrotoxic action of D-serine. Urine was collected 0-8 h (all doses) and 8-24, 24-48, 48-72, 72-96, and 96-120 h (500 mg/kg only) postdosing from Alderley Park rats given D-serine (62.5, 125, 250, and 500 mg/kg ip). 1H NMR spectra were monitored for markers of tubular damage. Additionally, ATP and ADP were quantitated in kidney perchloric acid extracts, prepared after 0.5, 1, 2, 4, and 8 h (500 mg/kg) to assess energy status; serine was also measured in these samples. At 500 mg/kg, glucosuria, amino aciduria, and reduced citrate, R-ketoglutarate, and succinate were observed in urine at 0-8 h. Furthermore, serine and pyruvate levels were elevated at this time. After 8-24 h, similar changes were observed; however, they were more severe reflecting the development of the lesion prior to recovery. These perturbations were dose-related, in particular, for serine and pyruvate, with no alterations seen at 62.5 mg/kg. Kidney serine concentration rapidly increased, where it was maximal after 30 min and cleared by 8 h. A decline in ATP, to approximately 60-70% of control, was observed within the kidney at 2-4 h postdosing, when necrosis first becomes evident suggesting that mitochondrial function might be impaired in the early stages of D-serine-induced nephrotoxicity. The use of NMR spectroscopy has given a comprehensive overview of the effects of D-serine in vivo. Information on the excretion of serine and its effect on renal energy metabolism provides insight into the possible mechanism of renal tubule injury.

Introduction A number of groups have previously reported the use of proton NMR spectroscopy and pattern recognition studies of urine to give insight into the site and mechanism of toxic injury to the kidney (1-4). In this study, we have investigated the application of 1H NMR of urine and 1H NMR and 31P NMR of kidney extracts at various postdosing time points and doses to examine the nephrotoxic action of the naturally occurring amino acid D-serine, in the rat. We have taken an integrated metabonomics approach alongside classical routine screening of toxicology endpoints such as clinical chemistry measurements and renal histopathology to analyze the onset, progression, and recovery from toxicity caused by D-serine to the pars recta of the proximal tubule. D-Serine is present in the plasma, in particular of humans (5) and in the brain (6). It can be formed endogenously as a result of spontaneous racemization of aged proteins (7) or following de novo synthesis in the brain (8). Furthermore, in addition to naturally formed D-amino acids, our diet also contains those formed due to food processing as exposure of proteins to certain * To whom correspondence should be addressed. Tel: 01625514653/01625 510341. Fax: 01625-516962. E-mail: becky.williams@ astrazeneca.com.

conditions induces racemization of L-amino acids to (9). The presence of D-amino acids in the body may be both beneficial and deleterious. For example, recent studies have shown that D-serine is an endogenous ligand for the glycine binding site on the N-methyl-Daspartate receptor (10-12). However, at high doses in the rat, D-serine produces selective damage to the pars recta region of the proximal tubule (13, 14). The onset of injury is rapid with focal lesions being observed by electron microscopy within 1 h in the tubular epithelial cells of male rats given 400 mg/kg D-serine ip (15). The injury is accompanied by marked proteinuria, glucosuria, and amino aciduria (16, 17) with the amino aciduria becoming evident prior to the onset of cellular necrosis (16). The mechanism whereby D-serine, but not L-serine, produces renal injury in the rat is currently not fully understood. D-Serine is however reabsorbed in the pars recta region of the rat proximal tubule (18, 19) where it is metabolized by D-AAO,1 a peroxisomal enzyme that is highly expressed in the epithelial cells (20). D-AAO catalyzes the oxidative deamination of D-amino acids D-isomers

1 Abbreviations: D-AAO, D-amino acid oxidase; HCBD, hexachloro1,3-butadiene; HgCl2, mercury(II) chloride; MDP, methylene diphosphonic acid; NAG, N-acetyl glucosaminidase; PCA, principal component analysis; TCA, tricarboxylic acid; TSP, 3-(trimethylsilyl)propionate2,2,3,3-d4.

10.1021/tx030019q CCC: $25.00 © 2003 American Chemical Society Published on Web 09/03/2003

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producing the corresponding R-keto acid plus ammonia and hydrogen peroxide (20). Studies with [14C]-D-serine have shown that radiolabel specifically accumulates in the pars recta region of the kidney whereas [14C]-L-serine has a more widespread tissue distribution (21). These studies indicate that localization, concentration, and metabolism of D-serine in renal tubule cells in the pars recta may account for the selective toxicity. Administration of D-propargylglycine to mice also results in selective necrosis to the pars recta region of the proximal tubule with proteinuria, glucosuria, and amino aciduria (22). Although mice are not affected by D-serine, it is of interest that the injury caused is very similar to that observed in rats with D-serine. The injury is not observed in mice that lack D-AAO demonstrating that metabolism of D-propargylglycine by D-AAO is important in its mechanism of selective toxicity (22). In this study, we have shown that the combination of 1 H NMR and 31P NMR spectral data on the kidney with 1H NMR pattern recognition data for urine plus conventional clinical chemistry and histopathology gave a comprehensive overview of the effects of D-serine in vivo. We have also provided information on the concentration of the amino acid in the target organ and its clearance from the body as well as some insight into a possible mechanism of toxicity.

Materials and Methods Animals and Treatments. Male (190-220 g; 7-8 weeks) Alderley Park (Wistar-derived) rats were obtained from the animal breeding unit, Alderley Park, Cheshire, U.K. Following an acclimatization period of 7 days, rats were either housed individually in metabolism cages for the collection of urine or housed two per cage according to the experimental protocol. Controlled humidity (30-70%), temperature (22 ( 3 °C), and a 12 h light/dark cycle were maintained. The animals’ bodyweight and clinical observations were recorded at the start of the study and immediately prior to termination. Measurements were repeated every 24 h in situations where animals were housed in metabolism cages in excess of 24 h. All animals had free access to food (RM-1 diet; Special Diet Services) and water throughout the study. Animal procedures were performed in accordance with licenses issued under the animals (Scientific Procedures) Act, 1986. Three separate studies were conducted. 1H NMR Spectroscopic Time Course Study in Urine. Fifteen animals were housed individually in metabolism cages, and a 24 h urine sample was collected over dry ice immediately prior to dosing (-24-0 h). The animals were then dosed with either D- or L-serine (500 mg/kg; 4 mL/kg ip; n ) 5) or vehicle (deionized water; 4 mL/kg ip; n ) 5). Urine was then collected over dry ice at 0-8, 8-24, 24-48, 48-72, 72-96, and 96-120 h postdosing. An interim blood sample was also taken after 72 h. At 120 h postdosing, the rats were killed by exposure to halothane followed by exsanguination. Terminal blood samples were taken from the heart into heparinized tubes, and the plasma was separated by centrifugation (3000g, 4 °C, 10 min). Plasma was submitted for clinical chemistry analysis, and the right kidney was taken for pathological examination. 1H NMR and 31P NMR Spectroscopic Dose-Response Study in Urine and Kidney. Twenty-five animals were housed individually in metabolism cages, and a 24 h urine sample was collected over dry ice immediately prior to dosing (-24-0 h). The animals were then dosed with D-serine at 0, 62.5, 125, 250, or 500 mg/kg in deionized water (4 mL/kg ip; n ) 5 per group). Urine was collected over dry ice 0-8 h postdosing. After 8 h, the rats were deeply anaesthetized by injection of pentobarbitone sodium (Rhone Merieux Ltd.; 300 mg/kg ip), and the left kidney was exposed and frozen in situ using tongs prechilled

Williams et al. in liquid nitrogen. The renal blood vessel was then clamped, and the frozen kidney was removed and immersed in liquid nitrogen. Terminal blood samples, taken from the inferior vena cava into heparinized tubes, were submitted for clinical chemistry analysis of the plasma, and the right kidney was taken for pathological examination. 1H NMR and 31P NMR Spectroscopic Time Course Study in the Kidney. Thirty-two animals were housed in pairs and dosed with either D-serine (500 mg/kg; 4 mL/kg ip; n ) 16) or vehicle (deionized water, 4 mL/kg ip; n ) 16). After 0.5, 1, 2, or 4 h, the rats (four control and four treated at each time point) were deeply anaesthetized by injection of pentobarbitone sodium (Rhone Merieux Ltd.; 300 mg/kg ip), and the kidney was frozen in situ as described in the section above. Terminal blood samples, taken from the inferior vena cava into heparinized tubes, were submitted for clinical chemistry analysis of the plasma, and the right kidney was taken for pathological examination.

Analytical Procedures Clinical Chemistry and Histopathology. Urine volume was measured, and then, the urine was analyzed for NAG activity, glucose, and total protein content. Creatinine and urea concentrations were analyzed in plasma. The measurements were made using a Konelab 60i instrument (Labmedics) using standard assay kits supplied by Labmedics (NAG assay kit supplied by PPR Diagnostics Ltd.). The right kidney was weighed, and a transverse section including the papilla was fixed in 10% neutral buffered formalin, embedded in paraffin wax, and 5 µm thick sections were cut and stained with haematoxylin and eosin for histopathological assessment. 1 H NMR Spectroscopy Analysis of Urine. Aliquots of urine (500 µL) were mixed with 250 µL of phosphate buffer (0.2 M; pH 7.4; prepared in D2O) containing TSP (0.5 mg TSP/mL buffer) (4) and centrifuged at 14 000g for 10 min at 4 °C. The supernatants (600 µL) were placed into a 5 mm NMR tube (Wilmad). 1H NMR spectra were acquired using a Bruker DPX400 spectrometer operating at 400.13 MHz for 1H. 1H and 31P NMR Spectroscopic Analysis on Kidney Extracts. Frozen kidneys were ground under liquid nitrogen, and the resulting powder was weighed. Perchloric acid (6% v/v; 5 mL; 0 °C) was added to the powder, and the sample was vortexed and then held on ice for 10 min. The sample was homogenized using a polytron (Kinematica AG) and centrifuged (2000g for 20 min at 4 °C), and the supernatant was decanted into another tube and kept ice-cold. The pellet was resuspended in perchloric acid (3 mL) prior to a second centrifugation step. The supernatants were then combined and neutralized using potassium hydroxide (3 M). The neutralized extract was centrifuged (2000g for 20 min at 4 °C), and the resulting supernatant was freeze-dried prior to taking up in D2O containing TSP (1 mM), MDP (2 mM), and EDTA (20 mM). The extract was neutralized to pH 7.0 using sodium hydroxide (1 M) and hydrochloric acid (1 M) and placed into a 5 mm NMR tube (Wilmad). 1H and 31P NMR spectra were acquired using a Bruker DPX400 spectrometer operating at 400.13 MHz for 1H and 161.98 MHz for 31P. NMR Spectroscopy of Urine and Tissue Extracts. 1H and 31P NMR spectra were acquired using a 5 mm QNP probe. For 1H NMR spectra, the standard “noesypr1d” pulse sequence (Bruker Analytik GmbH, Germany) was utilized for data acquisition; this pulse

NMR Studies on D-Serine-Induced Nephrotoxicity

sequence efficiently suppresses the large signal that arises from water. Sixty-four free induction decays were accumulated into 64k data points with a spectral width of 8012.82 Hz, an acquisition time of 4.09 s, and an interpulse delay of 2 s. A line broadening of 0.3 Hz was applied to the data, and all spectra were phase and baseline corrected. For quantitation of metabolites, calibration curves were prepared with reference to the internal standard TSP. It is not possible to distinguish between the D-isomer and the L-isomer of serine using this technique; therefore, results will be expressed as “serine”. 31P NMR spectra were acquired using a 30° pulse with WALTZ decoupling and an interpulse interval of 0.5 s. Free induction decays (1500) were accumulated into 32k data points with a spectral width of 8116.88 Hz. A line broadening of 3.0 Hz was applied to the data. For quantitation of ATP, a calibration curve was constructed from the β-ATP resonance with reference to the internal standard MDP (set to 17.5 ppm). Automated Data Reduction and PCA of 1H NMR Spectra. Using the AMIX software package (version 2.7.5, Bruker Analytische), each NMR spectrum was segmented into regions of 0.04 ppm giving rise to 256 segments (from 0.2 to 10.0 ppm) and the integral value for each segment was calculated (4, 23). In the segmentation routine, the regions of the spectrum giving rise to water and urea peaks (4.5-6.05 ppm) were removed, as were those associated with serine (3.80-3.88 and 3.924.04 ppm). No major metabolites associated with nephrotoxicity are found exclusively in the spectral region that contains serine; hence, the removal of this region is not considered to adversely affect the analysis. The segmented data were exported from AMIX into Microsoft Excel (version 7.0a), where the integral values were scaled to the total of the summed integrals of each spectrum in order to partially compensate for differences in urinary dilution between samples. The scaled data were then imported into SIMCA (version 8.0; Umetrics, Sweden) for PCA. For this analysis, the data set was mean centered. In addition to the analysis carried out on the individual data, a metabolic trajectory was also constructed for the time course study. Prior to analysis with SIMCA, the segmented data for each experimental group at each time point were averaged resulting in a single data set per group. PCA was carried out on these data to generate the metabolic trajectory. Statistical Analysis. The data are expressed as mean ( SD. Significant differences between the experimental groups were determined for each measurement using a one way ANOVA followed by the Bonferroni test. A corrected P value < 0.05 was considered statistically significant. The nonresponders defined in the doseresponse study were excluded from the statistical analysis.

Results 1H NMR Spectroscopic and PR Analysis of Urines Time Course Study over 5 Days. A typical time course of 1H NMR spectra of urine following dosing with D-serine (500 mg/kg ip) is shown in Figure 1. Major perturbations in metabolite levels can be clearly seen throughout the study. Metabolites that are important in the characterization of D-serine-induced nephrotoxicity were identified using PCA and are summarized in Table 1. These

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Figure 1. Time courses1H NMR spectra. Typical 1H NMR spectra (0.7-4.7 ppm) of urine obtained prior to dosing and at specified time intervals following exposure to D-serine (500 mg/kg). Table 1. Time CoursesUrinary Metabolite Alterations: Metabolites that Are Important in the Characterization of D-Serine-Induced Nephrotoxicity (Identified using PCA) metabolite

time point of increase (h)

metabolite

time point of decrease (h)

serine pyruvate glutamate leucine glucose valine lactate alanine acetate

0-24 0-48 0-48 0-72 0-96 0-120 0-120 0-120 48-120

creatinine allantoin hippurate dimethylglycine R-ketoglutarate malate fumarate citrate succinate

0-48 0-48 0-48 0-72 0-96 0-96 0-96 0-120 8-72

changes were not seen in control animals or those treated with L-serine. Over the first 8 h postdosing, an elevation in lactate, pyruvate, alanine, glucose, glutamate, leucine, and valine was observed. Decreases in creatinine, allantoin, hippurate, dimethylglycine, R-ketoglutarate, citrate, malate, and fumarate were also seen at this time. Hippurate, malate, fumarate, and allantoin levels, which


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Figure 2. Time coursesmetabolic trajectory. A scatter plot of the first two principal component scores (t(1) vs t(2)) showing the trajectories calculated from the mean integral values of NMR spectra acquired from each experimental group at the specified time intervals. The regions of the spectrum containing resonances arising from serine were removed prior to this analysis.

are not shown in Figure 1 due to their chemical shift or signal overlap, fell by approximately 4-5-fold at this time as compared to control values. Furthermore, a large resonance arising from serine was present in these spectra. At 8-24 h postdosing, the alterations that were observed during the first 8 h became more prominent with the addition of a reduction in succinate excretion. Contrary to these observations, the observed elevations of serine and pyruvate were less marked at this time as compared to the first 8 h postdosing. D-Serine-treated animals excreted approximately 23 mg of serine during the first 8 h postdosing and only 7 mg over the following 16 h, which, in total, represents about 23% of the administered dose. Serine was not detected in urine samples collected after 24 h and was not detected in the urine of control animals or those treated with L-serine. The metabolic trajectory shown in Figure 2 clearly shows the progression of D-serine-induced nephrotoxicity leading to maximal alterations in metabolite levels by 24 h. After 24 h, the trajectory starts to return toward control values as the urinary metabolic profile gradually recovers. Despite the general recovery in metabolite levels, the D-serine trajectory continues to separate from control and L-serine trajectories due primarily to an elevation in acetate at later time points. Acetate excretion starts to increase at 48 h postdosing, and by 96-120 h, acetate excretion is marked (Figure 1). By this stage, the urinary metabolite profile is otherwise normal apart from a slight depletion in citrate and a slight elevation in lactate and alanine that persist. Furthermore, at later time points, a small increase in acetate is observed in control animals, which causes the control trajectory to move away from the earlier time points (Figure 2); the increase is not as marked as observed in treated animals. Clinical Chemistry and HistopathologysTime Course Study over 5 Days. Administration of D-serine resulted in a loss of bodyweight at 48, 72, 96, and 120 h postdosing (-1.9 ( 2.6, -4.6 ( 3.3, -8.6 ( 4.6, and -7.2 ( 3.8% of the predose bodyweight, respectively) as compared to control and L-serine-treated animals. Clinical chemistry analysis revealed an elevation in urinary protein, glucose, NAG, and volume (Figure 3A-D) in animals treated with D-serine, with a maximal elevation in urinary glucose occurring at 8-24 h postdosing. By 96-120 h postdosing, urinary protein, glucose, and volume had returned to control levels. A significant elevation in plasma creatinine and urea was also ob-

served at 72 and 120 h postdosing; the increase was most marked at 72 h (Figure 3E,F). These changes were not observed in animals treated with L-serine. Furthermore, the kidneys obtained from D-serine-treated animals at 120 h postdosing were pale and swollen and were significantly larger than the kidneys obtained from control and L-serine-treated animals (kidney weight (g): control, 1.97 ( 0.17; L-serine, 2.13 ( 0.16; D-serine, 4.08* ( 0.28; *P < 0.05 as compared to control, one way ANOVA). At this time, tubular basophilia, indicative of tubular regeneration, and dilation of the straight portion of the proximal tubule were observed (Figure 4B). 1H NMR Spectroscopy and PR Analysis of Urine and 1H and 31P NMR Spectroscopy of Kidneys Dose-Response Study. In the dose-response study, nonresponders to D-serine were observed in the 125 and 250 mg/kg dose groups. These animals exhibited normal clinical chemistry and histopathology as discussed later in this section. There were no metabolite perturbations in the 1H NMR spectra of urine obtained from animals treated with D-serine at 62.5 mg/kg or from the nonresponders that were treated with D-serine at 125 and 250 mg/kg. Examination of the data using PCA demonstrated that these spectra clustered with the control data in a scatter plot of the first two principal components (Figure 5). The urine collected from the two animals that responded at 125 mg/kg separated away from the control cluster. The metabolites that were perturbed were consistent with those identified during the time course study except that the alterations were less prominent. The spectra acquired from the four animals that responded at 250 mg/kg and those animals treated at 500 mg/kg were further separated from the control data demonstrating a more pronounced perturbation in the urinary metabolic profile at the higher doses. It was not possible however to separate the 250 and 500 mg/kg data sets using PCA, as the major metabolite alterations were similar at both doses, as seen for lactate in Figure 6. Serine and pyruvate excretion were measured in the urine (Figure 6), and a clear dose-response relationship was demonstrated for both metabolites. Neither serine nor pyruvate was detected in the urine of those animals treated at 62.5 mg/ kg or those animals that did not respond at 125 and 250 mg/kg. The inability to detect serine in the urine of these animals may be due to the limited sensitivity of the technique.



Figure 4. Histopathology. Haematoxylin- and eosin-stained sections (×40 magnification) from (A) control and (B) D-serine (500 mg/kg ip)-treated rat kidney showing (A) control straight proximal tubules and (B) tubular basophilia (arrowhead) with multifocal tubular dilation (arrow) (120 h postdosing).

Figure 3. Time coursesclinical chemistry. (A-D) Urinary protein, glucose, NAG, and volume measured at specified time intervals following a single dose of D-serine (500 mg/kg). (E,F) Plasma creatinine and urea measured 72 and 120 h after a single dose of D-serine (500 mg/kg). (*P < 0.05 as compared to control; one way ANOVA). 1

H and 31P NMR spectroscopy of kidney extracts were unable to detect any metabolite changes following dosing with D-serine at 62.5, 125, or 250 mg/kg. The data obtained from animals treated at 500 mg/kg are reported in the following section together with the data obtained at the earlier time points. Clinical Chemistry and HistopathologysDoseResponse Study. Histopathological examination did not reveal any treatment-related changes in the kidney of rats 8 h after administration of a low dose of D-serine (62.5 mg/kg). Following exposure to D-serine at 125 mg/ kg, slight eosinophilia and necrosis of the straight proximal tubules were present in two animals; three of the animals at this dose were histologically normal and were termed “nonresponders”. At 250 mg/kg, four of the animals had eosinophilia and necrosis of the epithelium

Figure 5. Dose-responsesPCA. A scatter plot of the first two principal component scores (t(1) vs t(2)) calculated from 1H NMR spectra acquired from urine collected 0-8 h after dosing with D-serine (62.5, 125, 250, and 500 mg/kg). The regions of the spectrum containing resonances arising from serine were removed prior to this analysis.

of the straight proximal tubules; the severity of the tubular necrosis was increased as compared to the necrosis seen at 125 mg/kg. Furthermore, minimal tubular dilation of scattered cortical tubules was observed in two animals. At this dose, one animal was histologically normal and was termed a nonresponder. All rats dosed at 500 mg/kg had eosinophilia and necrosis of the straight proximal tubular epithelium (Figure 7A), the severity of which was increased as compared to the 250 mg/kg group. Tubular dilation of scattered cortical tu-


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postdosing, which remained increased until 8 h in the case of glucose. A depletion of ATP was seen at 2 and 4 h postdosing (Figure 8B,C), which returned to control levels by 8 h (Figure 8B). Clinical Chemistry and HistopathologysTime Course Study over 8 h. Plasma creatinine was statistically significantly increased 4 h after D-serine administration (61.5* ( 4.0 µmol/L) and 8 h (94.6* ( 7.6 µmol/ L) postdosing as compared to control (37.8 ( 3.9 µmol/ L). Plasma urea was also significantly increased (control, 5.8 ( 0.4; 4 h, 6.9* ( 0.6; 8 h, 8.0* ( 1.0 mmol/L; *P < 0.05, one way ANOVA). No histological changes were present in rats treated with D-serine (500 mg/kg) at 30 min postdosing. After 1 h, pronounced eosinophilia of the epithelium of the straight proximal tubules was observed in three out of four animals; this extended multifocally to involve the epithelium of the medullary rays. By 2 h postdosing, all rats had eosinophilia of the straight proximal tubules, which again extended into the epithelium of occasional medullary rays. In addition, all rats had rare scattered necrotic cells within the straight proximal tubules. After 4 h, necrosis of the straight proximal tubular epithelium was present in all treated animals. The severity of the tubular necrosis was increased as compared with the animals terminated at 2 h postdosing. Tubular epithelial eosinophilia was decreased as compared to the earlier time points, reflecting the transition from eosinophilia to necrosis among affected tubules. As described previously, all rats dosed at 500 mg/kg had eosinophilia and necrosis of the straight proximal tubular epithelium by 8 h, the severity of which was increased as compared to the 4 h group.

Discussion

Figure 6. Dose-responsesserine, pyruvate, and lactate excretion. (A-C) Amount of lactate, serine, and pyruvate in urine samples collected 0-8 h after dosing with D-serine (62.5, 125, 250, and 500 mg/kg) (*P < 0.05 as compared to control; one way ANOVA). R, responders; N, nonresponders as observed with histopathology.

bules was also observed in all five animals, and eosinophilic casts were observed within medullary tubules in two animals. Clinical chemistry analysis revealed a dose-related elevation in urinary protein, glucose, NAG, and volume (Figure 7B-E) and plasma creatinine and urea (Figure 7F,G). There were no changes in clinical chemistry parameters following exposure to D-serine at 62.5 mg/kg or in the animals termed as nonresponders at 125 and 250 mg/kg in agreement with the histopathological findings. 1 H NMR and 31P NMR Spectroscopic Analysis of KidneysTime Course Study over 8 h. 1H NMR spectroscopy of kidney extracts revealed that serine was present in large amounts in the kidney by 30 min postdosing (Figure 8A) after which serine levels decreased and had returned to control levels by 4 h postdosing. A significant elevation in lactate (Figure 8B) and glucose (data not shown) was observed at 4 h

Time Course of Onset and Recovery from DSerine Nephrotoxicity. The alterations in urine and plasma clinical chemistry parameters are consistent with damage to the proximal renal tubules (24, 25) and demonstrate the progression of and recovery from Dserine-induced nephrotoxicity. Histological examination of the kidney confirmed selective damage to the pars recta of the proximal tubules as reported by others (15, 26). The onset of renal injury is rapid with glucosuria, proteinuria, and enzymuria being detected during the first 8 h after dosing. The injury then increased in severity and persisted until at least 48 h postdosing before it gradually started to return toward normal. That the kidney is in recovery by the end of the study is supported by the finding of renal tubular basophilia 120 h postdosing, which is indicative of regeneration, following necrosis (27). During the first 24 h postdosing, a marked alteration in the urinary metabolic profile (as detected by 1H NMR spectroscopy) was observed as compared to the profile obtained prior to dosing. Glucosuria and amino aciduria (including alanine, valine, glutamate, and leucine) were observed as has previously been reported following D-serine-induced injury using conventional analytical techniques (16, 17). In addition, a perturbation in lactate and TCA cycle intermediates citrate, succinate, malate, and fumarate was observed. Depletion of urinary citrate, a normal component of rat urine, has been attributed to either an impairment of the TCA cycle or renal tubular acidosis (28). Lactate, which is normally reabsorbed in



Figure 7. Dose-responseshistopathology and clinical chemistry. (A) Haematoxylin- and eosin-stained kidney section (×40 magnification) obtained from a D-serine-treated rat (500 mg/kg) at 8 h postdosing showing acute tubular necrosis of the straight proximal tubules (arrows denote necrotic tubules; for study control, see Figure 4A). (B-E) Urinary protein, glucose, NAG, and volume measured in samples collected 0-8 h postdosing. (F,G) Plasma creatinine and urea measured in samples collected at 8 h postdosing (*P < 0.05 as compared to control; one way ANOVA). R, responders; N, nonresponders as observed with histopathology.

the proximal tubule, can be elevated due to impaired reabsorption or to increased plasma lactate or renal lactate production as a result of a disturbance in energy metabolism (28). Our observations suggest that energy metabolism is perturbed at an early stage in the development of D-serine-induced injury. Comparison of the urinary 1H NMR metabolic profile observed following administration of D-serine is very similar to that seen

following HCBD and HgCl2-induced damage to the pars recta of the proximal tubule (1, 2). Both compounds cause an early elevation of amino acids, lactic acid, and glucose together with depletion in succinate, R-ketoglutarate, citrate, and hippurate (1, 2, 4). Elevated pyruvate is not seen with either compound suggesting that the increase in pyruvate is specific to D-serine-induced toxicity and is not related to an increased production of lactate.


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Figure 9. Metabolism of D-serine.

Figure 8. Kidney extractss1H and 31P NMR analysis. Amount of (A) serine (expressed as µmol/g wet weight) and (B) lactate and ATP (expressed as µmol/g wet weight) present in kidney extracts acquired at specified times postdosing (500 mg/kg) (*P < 0.05 as compared to control; ∼P < 0.05 as compared to 4 and 8 h; one way ANOVA). (C) 31P NMR spectra of kidney extracts acquired from a control animal and a treated animal at 4 h postdosing (500 mg/kg).

During the 24-120 h postdosing, the alterations observed during the first 24 h become gradually less pronounced in agreement with the documented recovery from D-serine-induced nephrotoxicity (16). The onset of the recovery process is clearly evident in the metabolic trajectory as the course of the trajectory turns back toward control after 24 h postdosing. Alanine and lactate are still elevated at day 5 while most metabolites have returned to control levels. As both of these metabolic components are reabsorbed by transporters in the proximal tubule (29), their continued elevation is likely to reflect the fact that the epithelial cells have not fully recovered by this time, which may limit their transport ability. Of particular interest is the rise in acetate excretion 48 h postdosing that becomes very pronounced by 96-120 h. Examinations of urinary 1H NMR profiles from rats treated with S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and 1,1,2-trichloro-3,3,3-trifluoro-1-propene (TCTFP) demonstrate a similar rise in urinary acetate at 48 h, the latest time point examined, becoming marked as glucosuria returned toward control. Both DCVHC and TCTFP also cause selective damage to the proximal tubules (3). Furthermore, a rise in acetate was observed in the urine of rats exposed to HCBD and HgCl2; however, the rise occurred at an earlier stage, before

48 h, than was seen with DCVHC, TCTFP, and D-serine occurring as glucosuria first became evident and returning to control levels during recovery from the injury (1, 2). Increased urinary acetate has been observed during recovery from ischemic injury to the proximal tubule during renal transplantation (29, 30) suggesting that elevated acetate may reflect cellular recovery in this region. Acetate plays a role in luminal acidification in the pars recta of the proximal tubule (31); therefore, it is possible that increased acetate may occur as a result of its involvement in acid-base balance in the recovering epithelial cells. Dose-Response Relationships for the Onset of D-Serine Nephrotoxicity. Clinical chemistry analysis demonstrated a clear dose-response relationship following D-serine-induced injury that was further supported by the histopathological findings. Previous papers have stated that the lesion is highly reproducible (15); therefore, it is of interest that some nonresponders were observed in this study. It is possible that miss-dosing of D-serine could have occurred as serine was not detected in the urine of the nonresponders. This study examined the effect of D-serine over the first 8 h only; however, the time course demonstrated that the most marked urinary perturbations were observed at 8-24 h postdosing. Examination of nonresponders over an extended time course would be of interest to determine whether the animals might have responded over a longer time scale. PCA of urine 1H NMR spectra demonstrated the development of toxicity with increasing dose. The inability to distinguish between the 250 and the 500 mg/ kg data sets however suggests that above 250 mg/kg the metabolite perturbations that characterize D-serineinduced nephrotoxicity do not become further pronounced. This was demonstrated by quantitation of urinary lactate, which was excreted in comparable amounts at both 250 and 500 mg/kg. In contrast, pyruvate was excreted in a dose-dependent manner at all doses suggesting that the elevation in pyruvate might occur as a result of the metabolism of serine. D-Serine can undergo metabolism either by D-AAO producing hydroxypyruvate, ammonia, and hydrogen peroxide or by serine racemase, which converts D-serine to L-serine, which is then deaminated by serine dehydratase to produce pyruvate (Figure 9). Pyruvate was not detected in the urine following administration of L-serine (500 mg/ kg); however, it has been demonstrated that L-serine is


readily accumulated within tissues due to its incorporation into proteins rather than undergoing metabolism (21). In this study, approximately 17% of the administered dose of D-serine was excreted in the urine over the first 8 h, which agrees well with previously published excretion rates for D-serine (32). In contrast, serine is not detectable in the 0-8 h urine samples from rats treated with L-serine (see time course study) indicating a marked difference in the handling of D- vs L-serine. If the observed increase in pyruvate is derived from D-serine, then this can only account for about 1-2% of the original dose suggesting that metabolism of D-serine via L-serine is not the major metabolic pathway. Hydroxypyruvate was not detected in the urine; however, its 1H NMR resonances lie in the region of the spectrum containing glucose resonances that were all pronounced during the first 24 h postdosing due to marked glucosuria. Furthermore, hydroxypyruvate is readily metabolized to glycolate and oxalate and the latter is not detectable by 1H NMR spectroscopy. Whether the metabolism of serine by D-AAO is a key stage in the toxic mechanism is certainly of interest. Correlation between the D-AAO activity and the extent of toxicity has been excluded as the renal activity of D-AAO is greater in the dog than in the rat and lower in the guinea pig than in the rat, despite the absence of toxicity in both the dog and the guinea pig (33). However, the excretion rate of D-serine was seen to be lower in rats as compared to humans and dogs suggesting that Dserine reabsorption is more extensive in rats enabling more substrate to be available for D-AAO (5). It has also been demonstrated that compounds protective against D-serine-induced nephrotoxicity have common structural characteristics (34) suggesting that protection is afforded due to competition with D-serine at specific transport sites supporting the concept that concentration of Dserine in renal tubule cells is a requirement for the induction of toxic injury. Furthermore, it has been suggested that the production of reactive oxygen species as a consequence of D-serine metabolism by D-AAO might lead to its selective toxicity (34-37). It has been reported that glutathione prevents D-serine-induced amino aciduria suggesting that oxidative damage may be involved (34, 36, 37). In urine, the breakdown products of lipid peroxides, including acetone, acetaldehyde, and malondialdehyde, have been identified as noninvasive markers of oxidative damage (38). The spectra in this study were examined for any change in urinary acetone; however, in control samples and those obtained from treated animals after 72 h, the acetone peak was barely detectable. Interestingly, in the treated samples at 0-8 h postdosing, acetone was present suggesting that acetone is produced during the onset and development of necrosis. Time Course of Early Biochemical Perturbations in the Kidney. 1H NMR analysis on the kidney revealed that serine was present at maximal levels within the first 30 min postdosing and then rapidly declined due to either excretion or metabolism. In the proton spectra, no resonance attributable to metabolites of D-serine could be determined; however, this could be due to sensitivity of the technique rather than an absence of the metabolites. The first signs of injury were seen after 1 h, suggesting that the rapid accumulation and possible metabolism of D-serine are key triggers in the onset of necrosis as the


damage progresses rapidly despite the clearance of serine from the tissue at later times. A disturbance in energy metabolism was detected by 31P NMR spectroscopy as early as 2 h postdosing, prior to an elevation in lactate, which presumably reflects the occurrence of cellular necrosis and cell death. It is possible that energy metabolism is perturbed at an earlier time than 2 h as in this study the extracts were prepared from the entire kidney thus any small effect within the pars recta region of the proximal tubule would be masked. As only rare scattered necrosis is observed at 2 h postdosing, it is considered likely that the disturbance in energy metabolism does not just reflect necrotic cells but could be a factor in the mechanism of toxicity. In summary, the integration of clinical chemistry and histopathology findings with metabolic data gives a holistic approach to the study of time and dose-response relationships during the development and recovery from D-serine-induced renal injury. Additional information on the excretion of serine by the kidney and its effect on renal energy metabolism provides some insight into the possible mechanism whereby renal tubule injury may occur.

Acknowledgment. We thank Andy Gyte for his technical assistance with this work.

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1H NMR Pattern Recognition and 31P NMR Studies with d-Serine in

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