Melamine-Induced Renal Toxicity Is Mediated by the Gut Microbiota Xiaojiao Zheng et al. Sci Transl Med 5, 172ra22 (2013); DOI: 10.1126/scitranslmed.3005114

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An Unwitting Microbial Culprit in Melamine Toxicity?

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A tragic incident in China in 2008 involving the deliberate and illicit supplementation of milk with melamine resulted in the deaths of children from renal failure and highlighted the toxicity of this compound. In a new study, Zheng et al. study the toxicity of melamine in rats and report that microbial metabolism of melamine is crucial for forming the key metabolite that causes kidney damage. They discover that a species of Klebsiella can form cyanuric acid from melamine, which then forms complex precipitates that lead to kidney stone formation. The gut microbiota in general play an important role in human health and are known to affect the metabolism and toxicity of a number of drugs. The new findings suggest that individual variation in the gut microbial composition of children exposed to melamine may have been important in the observed patterns of mortality in exposed individuals.

RESEARCH ARTICLE KIDNEY DISEASE

Melamine-Induced Renal Toxicity Is Mediated by the Gut Microbiota Xiaojiao Zheng,1,2* Aihua Zhao,1,2*† Guoxiang Xie,3 Yi Chi,2 Linjing Zhao,2 Houkai Li,3 Congrong Wang,1 Yuqian Bao,1 Weiping Jia,1 Mike Luther,4 Mingming Su,4 Jeremy K. Nicholson,5 Wei Jia1,2,3†

Melamine poisoning has become widely publicized after a recent occurrence of renal injury in infants and children exposed to melamine-tainted milk in China. This renal damage is believed to result from kidney stones formed from melamine and uric acid or from melamine and its cocrystallizing chemical derivative, cyanuric acid. However, the composition of the stones and the mechanism by which the stones are formed in the renal tubules are unknown. We report that cyanuric acid can be produced in the gut by microbial transformation of melamine and serves as an integral component of the kidney stones responsible for melamine-induced renal toxicity in rats. Melamine-induced toxicity in rats was attenuated, and melamine excretion increased after antibiotic suppression of gut microbial activity. We further demonstrated that melamine is converted to cyanuric acid in vitro by bacteria cultured from normal rat feces; Klebsiella was subsequently identified in fecal samples by 16S ribosomal DNA sequencing. In culture, Klebsiella terrigena was shown to convert melamine to cyanuric acid directly. Rats colonized by K. terrigena showed exacerbated melamine-induced nephrotoxicity. Cyanuric acid was detected in the kidneys of rats administered melamine alone, and the concentration after Klebsiella colonization was increased. These findings suggest that the observed toxicity of melamine may be conditional on the exact composition and metabolic activities of the gut microbiota.

INTRODUCTION Melamine has received intense media attention as the cause of renal failure and death in both animals and children (1–3) because of its addition to pet food and infant formula as a way of boosting the apparent protein content. The low acute toxicity of melamine alone with an oral median lethal dose (LD50) of 3161 mg/kg in rats (4) and a solubility in water of 3.24 g/liter at room temperature (5) has raised questions regarding the mechanism of melamine-induced toxicity. Previous reports (6–9) suggest that severe renal toxicity in animals is associated with the combination of melamine and its structural analog, cyanuric acid, which can readily self-assemble into supermolecular aggregates through a hydrogen-bonding mechanism, leading to insoluble melamine-cyanurate cocrystals (10, 11). Recent studies have identified melamine-cyanurate crystals in the kidneys of rats, fish, and pigs that consume melamine alone (12, 13); however, the mechanism remains unknown. In contrast to the findings in animals, however, the calculi in infants suffering from melamine-induced renal toxicity were identified as being mostly melamine and uric acid in composition (4, 14–16). On the basis of these findings, we set out to explore the mechanism of melamine-induced renal toxicity. Previous studies in our laboratory have shown that melamine-induced toxicity in Wistar rats is dose-dependent, with

marked toxicity and metabolic changes observed at a high dose of melamine (600 mg/kg) similar to that seen in rats treated with a low dose of melamine combined with cyanuric acid [melamine (50 mg/kg) and cyanuric acid (50 mg/kg)] (6). We also found substantial fluctuation in urinary metabolite levels such as lowered phenylacetylglycine and elevated trimethylamine-N-oxide and 3-phenylpropionate, which are related to gut microbial-mammalian cometabolism in rats receiving a high dose of melamine compared to those in the control group (6). Certain bacterial strains, such as Klebsiella terrigena (strain DRS-1), can produce cyanuric acid from melamine through a series of deamination steps (17–19). This aerobic strain may be part of the mammalian gut microbiota (20), which prompted us to hypothesize that melamine is partially converted to cyanuric acid through intestinal microbial transformation and that the high renal toxicity in mammals exposed to melamine is a result of melamine-cyanurate or melamine-cyanurate-urate coprecipitation in renal tubules. Here, we perform pharmacological and metabolomic studies to investigate the role of the gut microbiota, and in particular Klebsiella species, in the conversion of melamine to cyanuric acid.

RESULTS 1 Center for Translational Medicine, and Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China. 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China. 3Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC 28081, USA. 4David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA. 5Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London SW7 2AZ, UK. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected] (W.J.); [email protected] (A.Z.)

Gut microbiota suppression in rats attenuates melamine-induced toxicity We performed animal studies to assess the impact of suppressed or nonsuppressed microbiota activity on melamine-induced toxicity in Wistar rats. Microbial suppression was achieved by oral treatment with a broad-spectrum antibiotic agent, imipenem/cilastatin sodium, at a daily dose of 50 mg/kg for 4 days before melamine exposure. Melamine (SigmaAldrich, ≥99% purity, without cyanuric acid contamination) was administered orally to rats at a daily dose of 600 mg/kg for 15 days. The

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(Published 13 February 2013; revised 3 April 2013)

RESEARCH ARTICLE benzaldehyde (26–28). These metabolites returned to control levels after microbiota suppression. The results suggest that the gut microbiota play an important role in melamine-induced metabolic changes. Gut microbiota suppression increases urinary excretion of melamine in rats We also observed that with the suppression of the gut microbiota, urinary excretion of melamine was substantially increased, presumably due to the reduced microbial conversion of melamine to cyanuric acid in vivo. We quantitatively analyzed urinary melamine from the rats in the Mel group and the AB + Mel group at 15 days after melamine intervention. Only 11.7 ± 1.8% of the melamine dose (600 mg/kg) was excreted in urine of the rats in the Mel group, whereas 23.8 ± 8.4% of the melamine dose was detected in urine of the rats in the AB + Mel group, representing a twofold increase (P < 0.01) (Fig. 2A). In addition, as melamine administration was increased to 100, 300, and 600 mg/kg in a parallel study performed in our laboratory, renal toxicity increased and the urinary excretion of melamine decreased significantly at a ratio of about 6:2:1 relative to the melamine dose (P < 0.001) (Fig. 2B). The urinary excretion of both melamine and cyanuric acid was the lowest at the low dose of melamine combined with cyanuric acid (11.4% of melamine dose shown in Fig. 2B, and 4.5% of cyanuric acid dose shown in Fig. 2C). The histological findings of renal toxicity in this group were similar to those observed in the Mel group rats. The excretion rates of melamine and cyanuric acid are shown in tables S3 and S4. These results suggest that a substantial portion of the “inert” melamine is somehow converted to other chemical forms in vivo and that gut microbiota suppression inhibits such a conversion. Gut microbiota can convert melamine to cyanuric acid We further performed in vitro studies to confirm the existence of intestinal microbes in experimental rats that can convert melamine to cyanuric acid. Fecal specimens collected from Wistar rats were incubated in nutrient broth medium supplemented with melamine (1000 mg/ml) under aerobic conditions. The concentration of melamine in the culture continuously decreased to 635.12 mg/ml after 36 hours of cultivation (Fig. 3A). The concentration of cyanuric acid in the medium after 24 hours of cultivation was 0.19 mg/ml (Fig. 3A). In controls without fecal specimens, we were not able to detect cyanuric acid in the melaminesupplemented medium. These results confirmed the microbial conversion of melamine to cyanuric acid in vitro by bacteria derived from rat feces. We suspected that melamine is degraded by intestinal microbes through a mechanism of nitrogen consumption by environmental aerobic bacteria as previously reported (17, 19, 29). This type of deamination process is highly efficient if melamine is used as the sole nitrogen source, but melamine degradation may be suppressed if there are other nitrogen sources present. To test this hypothesis, we monitored melamine utilization efficiency in high–nitrogen content tryptic soy broth (30) with the same concentration of melamine supplementation. We found that melamine degradation in nitrogen-rich tryptic soy broth was markedly hindered and that cyanuric acid was not detectable throughout 36 hours of cultivation. Other intermediate products of melamine metabolism, including ammeline, ammelide, and biuret, which are a result of successive deaminations of triazines, were detected in the fecal samples supplemented with melamine (Fig. 3A). Ammelide reached a peak concentration (0.47 ± 0.12 mg/ml) after 8 hours of cultivation, and cyanuric

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experimental rats were divided into four groups: control group, antibiotic group (AB group), melamine group (Mel group), and antibiotic + melamine group (AB + Mel group). The antibiotic used, imipenem/ cilastatin, is non-absorbable and is effective on both Gram-positive and Gram-negative bacteria in the gastrointestinal tract without systemic effects (5). To rule out the possibility of exogenous contamination of melamine and cyanuric acid, we analyzed the water, feed, and antibiotic solutions used for the animal experiment using ultra performance liquid chromatography/triple quadrupole tandem mass spectrometry (UPLC/TQMS). Melamine and cyanuric acid were not detected in these materials. Kidney histology (Fig. 1, A to F) showed severe damage from melamine administration (600 mg/kg per day), including crystal formation (Fig. 1D), obvious dilatation of renal tubules (Fig. 1E), and renal interstitial hemorrhage (Fig. 1F). These effects were ameliorated in the animals with gut microbial suppression, as shown by less hemorrhage in the renal interstitium (Fig. 1C). No histological abnormalities were observed in the kidney tissues of control (Fig. 1A) or AB rats (Fig. 1B), suggesting that the short-term administration of antibiotic did not produce histological damage in kidney tissues. At necropsy, the kidneys of melamine-treated rats appeared to have a rough surface (fig. S3), and the relative kidney weight of these rats was higher (P < 0.05) than those of the rats in the control group (Fig. 1G). In contrast, the relative kidney weights of the rats in AB and AB + Mel groups were not significantly different from that of the rats in the control group (P > 0.05). Serum urea nitrogen (Fig. 1H) and creatinine (Fig. 1I) concentrations were slightly higher in the Mel group only (P = 0.08). These results show that microbial inhibition can ameliorate melamine-induced toxicity. Metabolomic approaches have potential for deciphering the global phenotypic changes resulting from functional alterations of both host metabolism and the gut microbiome (21–23). To explain the metabolic fluctuations induced by melamine with or without microbiota suppression at different time points, we constructed urinary metabolic trajectories (Fig. 1J) using a multivariate statistical method [partial least squares discriminant analysis (PLS-DA)] (24, 25). The trajectory represents the time course of metabolic changes in each group measured at different time points. The four trajectories started at the same spatial position in the upper right quadrant, indicating that the metabolic profiles of the four groups were similar on day 0. As shown in Fig. 1J, the trajectory of the Mel group (red) drastically shifted away from that of the control group (gray) after 1 day of melamine treatment and continued to drift away throughout the experiment, demonstrating that the metabolic profile of the Mel group was largely different because of the melamine intervention. Moreover, the AB + Mel group (pink) showed a different time-dependent trajectory, moving closer to that of the control group, suggesting that the melamine-induced metabolic alterations were attenuated. A metabolic heat map (Fig. 1K) illustrated fluctuation of altered urinary metabolites after microbiota suppression compared to unsuppressed microbiota. The metabolites (table S1) were selected in accordance with the criteria of nonparametric univariate statistics (Kruskal-Wallis, P < 0.05) and multivariate statistics [variable importance in the projection value (VIP) > 1 and the absolute value of correlation coefficients (Pcorr) > 0.5]. Differentially expressed metabolites in the Mel group were attenuated in the AB + Mel group, as indicated by the changes in heat map intensities. Among the identified differential metabolites in the Mel group, several have been previously reported as gut microbiota–related metabolites (Fig. 1L), including phenylpyruvic acid, phenylacetylglycine, indoleacetic acid, trimethylamine oxide, 5-phenylvaleric acid, and

Fig. 1. E v a l u a t i o n o f melamine-induced renal toxicity with or without gut microbiota suppression. (A to F) Representative photographs of histological examination of kidneys in (A) control rats, (B) antibiotictreated rats (AB group), (C) rats treated with antibiotics and melamine (AB + Mel group), and (D to F) rats treated with melamine alone (Mel group). The red arrow indicates crystals, the white arrows indicate hemorrhage, and the asterisks indicate tubular dilatation. Scale bars, 50 mm. (G) Relative kidney weights (expressed as percentage of the total body weight). Mean values ± SD are plotted. (H and I) Serum urea nitrogen and creatinine concentrations. Mean values ± SD are plotted. (J) Time-dependent trajectories of urinary metabolomic profiles. Control, AB, Mel, and AB + Mel groups are shown across the time course from days 0 to 19. Each arrow represents mean values of the scores from the first (x axis) and the second (y axis) principal components at a certain time point of the corresponding group. The dots represent the pre-dose time points, and the diamonds represent the endpoint at 15 days after melamine or vehicle treatment (day 19). (K) Heat map of identified differential metabolites with a urinary metabolomic profile. Each cell in the heat map represents the fold change of a particular metabolite, which is the ratio of the intensity of each sample in the treatment group to the mean value of the control group [(a) Mel group versus control group; (b) AB + Mel group versus control group; and (c) AB + Mel group versus AB group]. Red indicates increased concentrations, and purple indicates decreased concentrations. (L) The box plot of each metabolite represents the ratio of the mean value of peak intensity in the treatment group to that for the control group (Mel versus control group, AB + Mel group versus control group, and AB + Mel group

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versus AB group from left to right, respectively). The values on the y axis in the plot of trimethylamine oxide, 5-phenylvaleric acid, and benzaldehyde are shown as log plots. n = 6 to 7 per group. *P < 0.05, one-way analysis of variance (ANOVA).

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well as one Rhodococcus isolate were previously reported to have a high capacity for triazine conversion (29, 31). Klebsiella seemed to be a strong candidate because the K. terrigena strain has been shown to convert melamine to cyanuric acid (19). Two other strains, Klebsiella pneumonia and Klebsiella planticola DSZ, were also reported to have a capacity for degrading triazines (29, 32). We performed 16S ribosomal DNA (rDNA) sequencing analysis of cultivated rat feces and identified the presFig. 2. The urinary excretion of melamine and cyanuric acid. (A) Urinary excretion of melamine in rats from ence of seven species of Klebsiella genus, the high-dose Mel and AB + Mel groups. Mean values ± SD are plotted. (B) Urinary excretion of melamine including oxytoca, terrigena, pneumoniae, administered at different doses: low-dose melamine (100 mg/kg), mid-dose melamine (300 mg/kg), high- planticola, singaporensis, ornithinolytica, and dose melamine (600 mg/kg), and low-dose melamine combined with cyanuric acid [melamine (50 mg/kg) variicola (Fig. 3B). The species of Klebsiella and cyanuric acid (50 mg/kg)]. Mean values ± SD are plotted. (C) Urinary excretion of cyanuric acid in the genus as well as other bacterial species were low-dose cyanuric acid (100 mg/kg) group and low-dose melamine and cyanuric acid group [melamine (50 mg/kg) and cyanuric acid (50 mg/kg)]. Mean values ± SD are plotted. n = 6 to 7 per group. **P < 0.01, identified in the cultivated rat feces using the BLASTN program available on the ***P < 0.001, one-way ANOVA. National Center for Biotechnology Information (NCBI) nucleotide sequence database Web site (http://blast.ncbi.nlm.nih. gov) based on 98 to 97% similarity with the hits (Fig. 3B). Among the seven species, three (K. terrigena, K. pneumonia, and K. planticola) were previously reported to have the capability of converting melamine to cyanuric acid (19, 29, 32). We further cultivated K. terrigena in medium containing melamine (1000 mg/ml). Cyanuric acid was immediately detected within 1 hour of cultivation (Fig. 3A), indicating the strong capacity of K. terrigena for melamine biotransformation. After 10 hours of cultivation, the concentration of melamine decreased by about 14% of its original level in the Klebsiella culture, and cyanuric acid reached a peak concentration of 0.57 ± 0.03 mg/ml, remaining stable for the entire experimental period. Other melamine derivatives, including ammeline, ammelide, biuret, and urea, were all detected within 36 hours of cultivation (Fig. 3A). Fig. 3. The conversion of melamine to cyanuric acid. (A) Concentrations of melamine, ammeline, Together, these findings show that ammelide, cyanuric acid, biuret, and urea in fecal cultures containing different microbes and in cultures aerobic bacteria of the Klebsiella gecontaining K. terrigena alone at different time points. Mean values ± SD are plotted. n = 3. (B) Several nus exist in rodents and that microbial genera of gut microbes with high abundance in the mammalian gut are listed including Bacteroides, transformation of melamine to cyanuClostridium, Lactobacillus, Bifidobacterium, Escherichia, and Klebsiella. Seven Klebsiella species were idenric acid can occur in the mammalian tified and are listed with similarity hits (shown as percentages) after each species name. gastrointestinal tract. Intestinal aerobic microbes of the Klebsiella genus, in paracid and its downstream product biuret were detected after 24 hours ticular the K. terrigena strain, may be key players in the conversion of cultivation. of melamine. The Klebsiella species are responsible for deamination of melamine We next sought to determine which aerobic bacteria were responsible for the biotransformation of melamine. Three Pseudomonas isolates as

Colonization of rat guts with Klebsiella exacerbates melamine-induced toxicity To confirm the impact of Klebsiella species on melamine-induced toxicity in mammals, we colonized the guts of Wistar rats with K. terrigena.

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K. terrigena bacterial cells were harvested from cultures and were diluted to a final concentration of 109 colony-forming units/ml. Colonization of rat guts was achieved by oral administration of 500 ml of bacterial suspension for four consecutive days. The rats with or without Klebsiella colonization were then orally administered melamine (600 mg/kg per day) for 15 days (Mel group and K + Mel group, respectively). Two control groups (an untreated control group and a group receiving Klebsiella but no melamine) were also used in parallel in the study. At necropsy, the kidneys of rats receiving melamine with or without Klebsiella colonization were edematous, and the relative kidney weight was significantly higher than that of the rats in the two control groups (P < 0.05) (Fig. 4A). The kidneys of rats in the K + Mel group

seemed to have more histological lesions and were pale in appearance compared to those from the other three groups (fig. S4). Kidney tissue slides were also prepared by wet-mount procedure and short-term fixation with formalin followed by hematoxylin and eosin (H&E) staining. Microscopically, wet-mount sections from frozen kidneys revealed the presence of golden green crystalline deposits in the kidneys of all rats in the K + Mel group, whereas the same type of kidney stones was observed only in three rats of the Mel group (table S5). The kidney stones in the Mel and K + Mel groups appeared to be birefringent crystals based on polarized light optical microphotography (Fig. 4, D to F), which is consistent with previous reports (8, 33–35). Marked tubular lesions were observed in the H&E-stained sections from the K + Mel group, including crystal formation (Fig. 4J), hemorrhage, dilatation of renal tubules, and inflammatory cell infiltration (Fig. 4, K and L). In the Mel group, inflammatory cell infiltrates were observed (Fig. 4I), but no histological abnormalities were observed in the kidneys of control (Fig. 4G) and Klebsiella alone (Fig. 4H) groups. In addition, biochemical analysis showed that serum urea nitrogen (Fig. 4B) and creatinine (Fig. 4C) concentrations were significantly (P < 0.05) increased in the rats of Mel and K + Mel groups and that these two markers were substantially higher in the K + Mel group than in the Mel alone group. These results confirm aggravated renal toxicity in the rats with Klebsiella colonization.

Fig. 4. Evaluation of melamine-induced renal toxicity in rats with or without Klebsiella colonization. (A) Relative kidney weights (expressed as percentage of the total body weight). Mean values ± SD are plotted. (B and C) Serum urea nitrogen and creatinine concentrations. Mean values ± SD are plotted. (D to F) Representative photographs of wet-mount analysis of rat kidney tissue analyzed by polarized light microscopy. The crystals observed in the kidneys from rats administered melamine without Klebsiella colonization (Mel group) and rats administered melamine with Klebsiella colonization (K + Mel group) were scattered in the kidneys and were characteristically birefringent. (G to L) Representative photographs of the histological sections of kidneys in control group (G), Klebsiella group (H), Mel group (I), and K + Mel group (J to L). The red arrow (J) indicates crystals, the white arrows (K) indicate hemorrhage, the asterisks indicate tubular dilatation, and the black arrows indicate inflammatory cell infiltrates. Scale bars, 50 mm. n = 6 to 8 per group. *P < 0.05, **P < 0.01, one-way ANOVA. www.ScienceTranslationalMedicine.org

Colonization of rat guts with Klebsiella increases cyanuric acid concentrations To determine the composition of kidney stones in rats, we extracted and dissolved the kidney tissues using a mixture of acetonitrile/water/diethylamine at a ratio of 5:4:1 and analyzed the contents by UPLC/TQMS (table S6). Melamine was detected in the rats from the Mel and K + Mel groups at concentrations of 16.58 ± 6.79 nmol/g and 89.68 ± 19.14 nmol/g, respectively (Fig. 5A). In these two groups, cyanuric acid was also detected at concentrations of 0.45 ± 0.14 nmol/g and 2.32 ± 0.73 nmol/g, respectively (Fig. 5B). There was a fivefold increase (P < 0.01) in the concentration of cyanuric acid in the kidneys of rats with Klebsiella colonization. In addition to melamine and cyanuric acid, uric acid was also detected in the kidneys from all groups. The concentrations of uric acid in the kidneys of Mel and K + Mel groups were much higher than those in the control and Klebsiella groups (Fig. 5C). We also performed in vitro chemical precipitation assays by mixing the 13 February 2013

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acid being produced by the gut microbial conversion of melamine, which leads to the development of melamine-cyanurate crystals in the kidneys. The crystal sedimentation of melamine-cyanurate facilitates further conglomeration and crystallization of melamine-urate in renal tubules, thereby causing acute or chronic kidney failure. These data from animal studies are relevant to melamine-induced nephrotoxicity in humans. K. terrigena was previously Fig. 5. Assessment of crystals in the rat kidney. (A) Melamine concentrations in the kidneys of rats given detected in the stool specimens of 0.9% of melamine without Klebsiella colonization (Mel group) and rats given melamine with Klebsiella colonization 5377 healthy subjects (40). A recent study (K + Mel group). (B) Cyanuric acid concentrations in the kidneys of rats from the Mel and K + Mel groups. of children in rural China who consumed (C) Uric acid concentrations in the kidneys of rats in the control, Klebsiella, Mel, and K + Mel groups. Mean melamine-contaminated dairy products values ± SD are plotted. n = 6 to 8 per group. **P < 0.01, one-way ANOVA. reported that the overall prevalence of urinary tract abnormalities among 7933 excompounds together in the following pairs: melamine–uric acid, posed children was 0.61% (3). These clinical data suggest that the melamine–cyanuric acid, uric acid–cyanuric acid, and melamine– incidence of melamine-induced toxicity in humans is similar to the inuric acid–cyanuric acid. We observed that crystals readily formed cidence of K. terrigena colonization in humans, suggesting that the (within ~24 hours) when cyanuric acid was present, but melamine– population that is susceptible to melamine adulteration may correuric acid did not precipitate, even after 96 hours. late with high levels of gut Klebsiella, although this correlation has not yet been formally established. A direct linkage to melamine-induced renal toxicity would require genotyping of the gut microbiota present in the fecal samples of infants suffering from adulterated milk powder– DISCUSSION induced nephrotoxicity. The genotyping would reveal whether infants It is well established that the mammalian gut microbiota interact ex- with nephrotoxicity had a higher abundance of the Klebsiella species. tensively with the host through metabolic exchange and cometabolism However, this type of study would be very challenging because 4 years of substrates. Although the mechanisms are poorly understood, they has passed since the major incident of melamine poisoning in Chinese have been suggested to play a role in the etiology of many human dis- infants, which occurred in late 2008. Our study demonstrates that gut eases as well as adverse drug effects (36). Recent studies have shown microbial activities can affect the metabolism and toxicity of food conthat melamine-cyanurate crystals can form in the kidneys of rats, fish, taminants and pollutants and, therefore, should be taken into consideraor pigs that consume only melamine (8, 12, 13). Although some reports tion in measuring the impact of human environmental exposure events. have speculated that the source of cyanuric acid was contaminated food (37), no cyanuric acid was detected in water, feed, or antibiotic solutions used in our animal studies. Therefore, our results suggest that the cya- MATERIALS AND METHODS nuric acid detected in the kidneys of rats was derived from the gut microbial conversion of melamine. Because the concentration of cyanuric Animal study 1: Melamine-induced toxicity in rats with gut acid was much lower than that of melamine in the kidneys of melamine- microbiota suppression dosed rats, we suspect that cyanuric acid may serve as a nidus for crystal The protocols of gut microbiota suppression and induction of melaformation by melamine-cyanurate and melamine-urate, which constitute mine toxicity were based on our previous reports (6, 26). After 1 week the chemical composition of kidney stones reported in renal tubules of of acclimation, a total of 26 rats were randomly divided into four groups: control group (n = 6), which received the same volume of mammals exposed to melamine. The concentrations of uric acid in the kidneys of melamine-dosed water as the other three groups from days 1 to 4 and then 1% CMC-Na animals were much higher than those in the animals of control groups, (vehicle of melamine) from days 5 to 19; antibiotic group (AB group) suggesting that uric acid is an important component of melamine- (n = 6), which received antibiotic solution at an oral dose of 50 mg/kg induced kidney stones. Melamine and cyanuric acid are able to form per day from days 1 to 4 and then 1% CMC-Na from days 5 to 19; self-assembling complexes through organized intramolecular networks antibiotic + melamine group (AB + Mel group) (n = 7), which received of hydrogen bonds and pi-pi aromatic ring stacking (38). Uric acid has antibiotic solution at a daily dose of 50 mg/kg from days 1 to 4 and imide groups known to interact with melamine in self-associating then melamine (600 mg/kg) from days 5 to 19 [this was equivalent to complexes (39). Minimum solubility for the complex of melamine- the high-dose melamine group in our previous study (6)]; melamine cyanurate was observed at a pH of 4.5 to 7, whereas melamine-urate group (Mel group) (n = 7), which received water from days 1 to 4 and exhibited tighter binding under acidic conditions (pH 4) (38). At neu- then melamine at a daily dose of 600 mg/kg from days 5 to 19. Animals tral pH, melamine tends to bind to cyanuric acid with an affinity that is were sacrificed on the 21st day. 29-fold greater than that of melamine-urate (38). At the pH of urine, which is about 6.0, melamine should combine with cyanuric acid more Biochemical assays easily than uric acid. Together, these results suggest that melamine- Blood was collected from the ocular orbit of rats before sacrifice. The induced renal toxicity occurs as a result of a small amount of cyanuric blood was clotted at room temperature for 30 min, and serum was www.ScienceTranslationalMedicine.org

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Wet-mount analysis At necropsy, the left and right kidneys from each rat were weighed and then sectioned longitudinally. One half of each kidney was placed in 10% neutral buffered formalin for 12 hours. The other half of kidney was flash-frozen. The left kidney was used for wet-mount preparation, and the right kidney was used for crystal analysis. The wet-mount analysis was conducted by pressing about 2-mmthick sections of fresh tissue between two microscope slides and then observing the tissue under bright field and polarized light microscopy (Leica DM LP, Hitachi Co.). On the basis of a method described in a previous report (35), the presence of crystals was scored from 0 to 5 as shown in table S5. Histological analysis Half of one kidney was fixed in formalin and embedded in paraffin wax. Histological sections (3 mm thick) of the paraffin-embedded tissue were stained with H&E and prepared for light microscopy using Leica DMRE Microsystems equipment with a SPOT Flex Microscope Digital Camera (Diagnostic Instruments Inc.). Metabolomic studies The metabolomic study was performed following our previously published protocol (6). Details regarding sample preparation, instrumental analysis, and compound annotation can be obtained in the Supplementary Materials. Quantitative analysis of urinary melamine and cyanuric acid excretion Melamine and cyanuric acid in each 300 ml of urine sample were prepared by adding 300 ml of mix solvent (water/diethylamine at a ratio of 9:1). The preparation procedures used were the same as those described for the metabolomic study. Melamine and cyanuric acid were analyzed by UPLC/quadrupole time-of-flight MS (UPLC/QTOFMS) with optimized chromatography and MS conditions (table S2). Melamine and cyanuric acid stock standard solutions were prepared at a concentration of 1 mg/ml for quantitation. To eliminate the sample matrix effect, we diluted the stock solutions with control urine samples to concentration series of 0.1, 0.5, 1, 5, 10, 25, 50, 100, or 500 mg/ml, and then we prepared each standard solution using the same procedures as the sample preparation before analysis. The calibration curve was constructed by plotting the peak area of each standard versus concentration. Melamine and cyanuric acid excretion percentages were the portions of the administered amounts that appeared in the urine. Biotransformation of melamine to cyanuric acid Feces samples were collected from 6-week-old male Wistar rats. Nutrient broth was used as culture medium in this study. Fresh feces pellets (about 5 g) were placed into 100 ml of sterile medium. Fecal microbes were propagated in a shaking incubator (Labnet 311DS) at a speed of 120 rpm at 37°C for 36 hours until the medium was obviously turbid. Each 500 ml of the propagated fecal culture was then transferred to 4.5 ml of sterile nutrient broth supplemented with melamine (1 mg/ml) for cultivation. During the cultivation, samples were taken out at 0, 1, 2, 3, 4, 6, 8,

12, 24, and 36 hours and centrifuged at 12000g for 20 min. The collected supernatants were stored at −80°C until analysis. For analysis, an aliquot of 500 ml supernatant was mixed with 500 ml of mix solution (methanol/water/diethylamine at a ratio of 5:4:1) and then centrifuged at 12000g for 10 min. The supernatant was filtered through a syringe filter (0.22 mm) for UPLC/QTOFMS analysis. The standard solutions of melamine, cyanuric acid, ammeline, ammelide, biuret, and urea for quantitation were dissolved in nutrient broth and prepared with the same procedures as those for sample preparation before analysis. The calibration curves were constructed with the same procedure described above. 16S rDNA sequence analysis of Klebsiella in gut microbiota The Klebsiella sp. strain was previously reported to have the capability of converting melamine to cyanuric acid (19). To identify whether Klebsiella strains exist in mammalian gut microbiota, we collected fresh feces from 6-week-old Wistar male rats. The feces was propagated in nutrient broth for 24 hours at a rotation speed of 120 rpm to facilitate bacterial growth. The procedures of DNA extraction, primer design and synthesis, polymerase chain reaction, and sequence analysis can be obtained in the Supplementary Materials. K. terrigena [American Type Culture Collection (ATCC) 700372] was used as a positive control, and Escherichia coli was used as a negative control. Melamine biotransformation to cyanuric acid by a Klebsiella sp. strain K. terrigena was used to confirm the Klebsiella strain–mediated biotransformation because this strain has been shown to be one of the most important of the genus (19). Freeze-dried K. terrigena powder was propagated following instructions from ATCC in tryptic soy broth and a shaking incubator at 26°C for 36 hours. Each 500 ml of K. terrigena liquid culture was inoculated into 4.5 ml of tryptic soy broth supplemented with melamine (1 mg/ml) or without melamine (negative control). The standard solutions of melamine, cyanuric acid, ammeline, ammelide, biuret, and urea for quantitation were dissolved in tryptic soy broth. The calibration curve was constructed with the same procedure described above. Animal study 2: Melamine-induced toxicity in rats colonized with Klebsiella Klebsiella colonization was performed according to a previously described method (41). The preparation of Klebsiella bacterial cell suspension can be obtained in the Supplementary Materials. After 1 week of accommodation, a total of 28 rats were randomly divided into four groups: control group (n = 6), which received 0.5 ml of saline as the other three groups from days 1 to 4 and 1% CMC-Na (vehicle of melamine) from days 5 to 19; Klebsiella group (n = 6), which received 0.5 ml of Klebsiella bacterial cell suspension from days 1 to 4 and 1% CMC-Na from days 5 to 19; melamine group (Mel group) (n = 8), which received 0.5 ml of saline from days 1 to 4 and then administered with melamine at a daily dose of 600 mg/kg from days 5 to 19; Klebsiella and melamine group (K + Mel group) (n = 8), which was dosed with 0.5 ml of Klebsiella bacterial cell suspension from days 1 to 4 and then with melamine (600 mg/kg) from days 5 to 19 (an equivalent dose of the highdose melamine group). Animals were sacrificed on the 21st day. Crystal composition assessment by UPLC/TQMS The extraction method was conducted based on a previously described method with minor improvements (42). Half of one kidney, including

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separated by centrifugation at 3000g for 10 min for biochemical assessment. Serum biochemical markers, including urea nitrogen and creatinine, were measured with a Hitachi 7600 Automatic Analyzer (Hitachi Co.).

the capsule, cortex, and medulla, was weighted into a 15-ml tube. Each 100-mg tissue was homogenized with 1 ml of acetonitrile/water/ diethylamine (5:4:1) for 5 min with a BioSpec Mini-Beadbeater and centrifuged at 3000g for 20 min. A 2-ml aliquot of extraction was transferred into a new 15-ml tube, 4 ml of acetonitrile was added, and the sample was vortexed briefly. The tube was then centrifuged for 20 min at 4000g, and a 2-ml aliquot of supernatant was filtered through a 0.8-mm syringe filter. The supernatant was then evaporated dry under nitrogen with a nitrogen evaporator (Organomation Associates Inc.). The dried extract was reconstituted in 400 ml of water/acetonitrile at a ratio of 4:1 for UPLC/TQMS analysis. The settings of UPLC/TQMS can be obtained in the Supplementary Materials and table S6. Standard solutions were diluted with water/acetonitrile (4:1) in seven concentrations. To eliminate the sample matrix effect, we added a 10-ml standard solution to 390 ml of control sample. These standard solutions were used to generate calibration curves for quantitation. Sample assessment was performed with a Waters Acquity UPLC system coupled to an AB Sciex Triple Quad 5500 mass spectrometer with Analyst 1.5.1 software. The optimized instrumental parameters are listed in the Supplementary Materials. Statistical analysis All data are shown as means ± SD. The statistical significance was evaluated by one-way ANOVA with Bonferroni posttest. Metabolomic data were analyzed with Simca-P+ 12.0 software package (Umetrics). PLS-DA was used to determine metabolomic patterns (fig. S1), and a perturbation trajectory was then constructed by plotting score parameters (Fig. 1J). The differential metabolites selected should meet the following three requirements: nonparametric Kruskal-Wallis test (P < 0.05), multivariate statistical analysis–orthogonal projections to latent structures discriminant analysis (OPLS-DA) (Pcorr > 0.5), and OPLS-DA VIP > 1. The fold change was the ratio of mean intensity of each metabolite in the treatment group to that in the control group. Heat maps were created in MATLAB 7.1 (The MathWorks Inc.). The DNA sequences were analyzed with the BLASTN program from the NCBI Web site.

SUPPLEMENTARY MATERIALS www.sciencetranslationalmedicine.org/cgi/content/full/5/172/172ra22/DC1 Materials and Methods Fig. S1. PLS-DA scores plot derived from urinary metabolites. Fig. S2. Daily physiological records. Fig. S3. The gross appearance of kidneys of rats in control, AB, AB + Mel, and Mel groups. Fig. S4. The gross appearance of kidneys of rats in control, Klebsiella, Mel, and K + Mel groups. Table S1. List of urinary differential metabolites relevant to melamine-induced toxicity. Table S2. Optimized UPLC/QTOFMS conditions for melamine and cyanuric acid quantitation. Table S3. The absolute excretion rate of melamine. Table S4. The absolute excretion rate of cyanuric acid. Table S5. The crystal intensities in rat kidneys. Table S6. Optimized UPLC/TQMS conditions for melamine, cyanuric acid, and uric acid quantitation.

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RESEARCH ARTICLE

RESEARCH ARTICLE 39. W. Tolleson, Renal toxicity of pet foods contaminated with melamine and related compounds, paper presented at the 235th National Meeting of the American Chemical Society, New Orleans, LA, 2008. 40. R. Podschun, Isolation of Klebsiella terrigena from human feces: Biochemical reactions, capsule types, and antibiotic sensitivity. Zentralbl. Bakteriol. 275, 73–78 (1991). 41. S. Ohkawara, H. Furuya, K. Nagashima, N. Asanuma, T. Hino, Oral administration of butyrivibrio fibrisolvens, a butyrate-producing bacterium, decreases the formation of aberrant Crypt Foci in the colon and rectum of mice. J. Nutr. 135, 2878–2883 (2005). 42. M. S. Filigenzi, B. Puschner, L. S. Aston, R. H. Poppenga, Diagnostic determination of melamine and related compounds in kidney tissue by liquid chromatography/tandem mass spectrometry. J. Agric. Food Chem. 56, 7593–7599 (2008). Funding: This study was supported by the National Basic Research Program of China (2007CB914700) and the National Natural Science Foundation of China Program (81170760). Author contributions: Wei Jia and A.Z. designed the study. X.Z., A.Z., Y.C., L.Z., C.W., and Y.B. performed the animal, in vitro bacterial, and metabolomic studies. X.Z. and H.L. performed the gene expression analysis. X.Z. and M.S. analyzed the data. G.X., H.L., Weiping Jia, and M.L. helped prepare the manuscript. X.Z., A.Z., and Wei Jia wrote the manuscript. Wei Jia and J.K.N. contributed to the overall metabolomic design. Competing interests: The authors declare that they have no competing interests. Data and materials availability: 16S rDNA sequence data have been deposited in GenBank under accession numbers KC287217 (cultivated rat gut microbiome) and KC287218 (K. terrigena DRS-1). Submitted 9 October 2012 Accepted 25 January 2013 Published 13 February 2013 10.1126/scitranslmed.3005114 Citation: X. Zheng, A. Zhao, G. Xie, Y. Chi, L. Zhao, H. Li, C. Wang, Y. Bao, W. Jia, M. Luther, M. Su, J. K. Nicholson, W. Jia, Melamine-induced renal toxicity is mediated by the gut microbiota. Sci. Transl. Med. 5, 172ra22 (2013).

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E r r at u m Kidney Disease

A Correction to the Research Article Titled: “Melamine-Induced Renal Toxicity Is Mediated by the Gut Microbiota” by X. Zheng, A. Zhao, G. Xie, Y. Chi, L. Zhao, H. Li, C. Wang, Y. Bao, W. Jia, M. Luther, M. Su, J. K. Nicholson, W. Jia

The sentence in the abstract that reads “Melamine-induced toxicity in rats was attenuated, and melamine excretion decreased after antibiotic suppression of gut microbial activity” is incorrect. The correct sentence should read “Melamine-induced toxicity in rats was attenuated and melamine excretion increased after antibiotic suppression of gut microbial activity.” The corrected online version of the abstract is at http://stm.sciencemag.org/content/5/172/172ra22.abstract, the corrected online version of the full text is at http://stm.sciencemag.org/content/5/172/172ra22.full, and the corrected PDF version of the article is at http://stm.sciencemag.org/content/5/172/172ra22.full.pdf. 10.1126/scitranslmed.3006179 Citation: A correction to the Research Article titled: “Melamine-induced renal toxicity is mediated by the gut microbiota” by X. Zheng, A. Zhao, G. Xie, Y. Chi, L. Zhao, H. Li, C. Wang, Y. Bao, W. Jia, M. Luther, M. Su, J. K. Nicholson, W. Jia. Sci. Transl. Med. 5, 179er3 (2013).

www.ScienceTranslationalMedicine.org    3 April 2013    Vol 5 Issue 179 179er3        1