Search

Gut microbiota-derived metabolites play important roles in health and disease. D-amino acids and their L-forms are metabolites of gut microbiota with distinct functions. In this study, we show the pathophysiologic role of D-amino acids in association with gut microbiota in humans and mice with acute kidney injury (AKI). In a mouse kidney ischemia/reperfusion model, the gut microbiota protected against tubular injury. AKI-induced gut dysbiosis contributed to the altered metabolism of D-amino acids. Among the D-amino acids, only D-serine was detectable in the kidney. In injured kidneys, the activity of D-amino acid oxidase was decreased. Conversely, the activity of serine racemase was increased. The oral administration of D-serine mitigated the kidney injury in B6 mice and D-serine-depleted mice. D-serine suppressed hypoxia-induced tubular damage and promoted posthypoxic tubular cell proliferation. Finally, the D-serine levels in circulation were significantly correlated with the decrease in kidney function in AKI patients. These results demonstrate the renoprotective effects of gut-derived D-serine in AKI, shed light on the interactions between the gut microbiota and the kidney in both health and AKI, and highlight D-serine as a potential new therapeutic target and biomarker for AKI.

D-Serine is known to be essential for the activation of the N-methyl-D-aspartate (NMDA) receptor in the excitation of glutamatergic neurons, which have critical roles in long-term potentiation and memory formation. D-Serine is also thought to be involved in NMDA receptor-mediated neurotoxicity. The deletion of serine racemase (SRR), which synthesizes D-serine from L-serine, was recently reported to improve ischemic damage in mouse middle cerebral artery occlusion model. However, the cell type in which this phenomenon originates and the regulatory mechanism for D-/L-serine remain elusive. The D-/L-serine content in ischemic brain increased until 20 hours after recanalization and then leveled off gradually. The results of in vitro experiments using cultured cells suggested that D-serine is derived from neurons, while L-serine seems to be released from astroglia. Immunohistochemistry studies of brain tissue after cerebral ischemia showed that SRR is expressed in neurons, and 3-phosphoglycerate dehydrogenase (3-PGDH), which synthesizes L-serine from 3-phosphoglycerate, is located in astrocytes, supporting the results of the in vitro experiments. A western blot analysis showed that neither SRR nor 3-PGDH was upregulated after cerebral ischemia. Therefore, the increase in D-/L-serine was not related to an increase in SRR or 3-PGDH, but to an increase in the substrates of SRR and 3-PGDH.

The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D-/L-serine from 2.82 ± 0.18 to 1.10 ± 0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D-/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D-/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury.

The N-methyl-D-aspartate receptor (NMDAR) is crucial for pain-related behaviors. D-Serine is synthesized from L-serine by serine racemase (SR) and modulates NMDAR functions by acting as an agonist at the glycine-binding site. We analyzed noxious stimulus-induced ultrasonic vocalization and locomotor activity in the open-field test using SR knockout (SR-KO) mice to examine the role of endogenous D-serine in mammalian behaviors. SR-KO mice emitted less ultrasonic vocalization after noxious stimulation (VAS) than wild-type (WT) mice. The locomotor activity of WT mice decreased with repeated daily exposures to the open field, whereas that of SR-KO mice remained unchanged. VAS was significantly enhanced during arthritis in WT mice, whereas it was not enhanced during arthritis in SR-KO mice. These results indicate that mice lacking the ability to produce D-serine endogenously in the brain differ from normal mice with respect to the chronic pain-induced behavioral changes.

D-Amino acids are stereoisomers of L-amino acids. They are often called unnatural amino acids, but several D-amino acids have been found in mammalian brains. Among them, D-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. D-Amino-acid oxidase (DAO), which degrades neutral and basic D-amino acids, is mainly present in the hindbrain. DAO catabolizes D-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of D-serine and other D-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of D-serine. D-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that D-amino acids and DAO have pivotal functions in the central nervous system.

For elucidation of the regulation mechanisms of intrinsic amounts of D-serine (D-Ser) which modulates the neuro-transmission of N-methyl-D-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and D-amino acid oxidase (DAO) were established, and the amounts of D-Ser in the tissues and physiological fluids were determined. D-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in D-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR(-/-)) mice compared with those of control mice, although the amounts of D-Ser in these tissues were low. The amounts of D-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous D-Ser into the brain tissues, we have determined the D-Ser of SRR(-/-) mice after oral administration of D-Ser for the first time, and a drastic increase in D-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic D-Ser amounts was investigated. In the frontal brain, most of the intrinsic D-Ser was biosynthesized by SRR. On the other hand, half of the D-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic D-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.

The amygdala is implicated in chronic pain-induced emotional changes. Chronic pain induces plastic changes of the N-methyl-d-aspartate receptor (NMDAR) functions in the brain including the amygdala. d-Serine is synthesized endogenously by serine racemase and modulates NMDAR-mediated synaptic transmission as a coagonist of glycine binding site. To clarify the functional roles of endogenous d-serine in chronic pain-induced plasticity of NMDAR mediated synaptic transmission, we investigated the NMDAR-mediated excitatory synaptic current (EPSC) of neurons in the latero-capsular division of the central amygdala (CeLC) using brain slices from serine racemase knockout (SR-KO) mice with chronic pain induced by monoarthritis. The decay time of NMDAR-mediated EPSC was significantly elongated by monoarthritis in wild type (WT) mice, but not in SR-KO mice. The d-serine application-induced increase of NMDAR-mediated EPSC was significantly facilitated by monoarthritis in WT mice, but not in SR-KO mice. These results suggest that endogenous d-serine facilitates chronic pain-induced plastic changes of NMDAR mediated synaptic transmission in CeLC., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. D-serine, an endogenous coagonist of N-methyl-D-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the D-amino acid oxidase (DAO) gene, encoding a D-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and D-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the D-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through D-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing D-serine or controlling DAO activity in ALS should be tested in future studies.

A fully automated two-dimensional HPLC system combining a microbore-ODS column and a narrowbore-enantioselective column was designed and validated, and the amounts of D-serine (D-Ser) and D-alanine (D-Ala) in various tissues and physiological fluids of Long-Evans agouti/SENDAI (LEA/Sen) rats lacking D-amino-acid oxidase (DAO) were determined. Intra- and inter-day precision was less than 4.3% and accuracy ranged between 99.9 and 104%. LEA/Sen rats were reported to lack DAO in their kidneys and expected to be a novel mutant animal lacking DAO, however, the amounts of D-amino acids in the LEA/Sen rats have not been investigated. In the present study, the intrinsic amounts of D-Ser and D-Ala, which are neuromodulators of the N-methyl-D-aspartate (NMDA) receptors, were determined in seven brain tissues, four peripheral tissues, plasma and urine of the LEA/Sen rats and compared to those of the control (Wistar and SD) rats having normal DAO activity. The levels of D-Ser in the tissues and physiological fluids of the LEA/Sen rats were significantly higher than those of the Wistar and SD rats except for the frontal brain regions. Concerning D-Ala, the amounts in the tissues and physiological fluids of the LEA/Sen rats were drastically increased compared to those of the Wistar and SD rats. These results indicate that the intrinsic amounts of D-Ser and D-Ala in the tissues of rats are regulated by DAO, and that LEA/Sen rats would be useful for the study of NMDA receptor-related diseases in which DAO is implicated., (Copyright © 2010 Elsevier B.V. All rights reserved.)

D-serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.

Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of D-serine (D-Ser) and D-alanine (D-Ala), and their amounts in the tissues and physiological fluids of mice with various D-amino-acid oxidase (DAO) activities have been demonstrated. These two D-amino acids are modulators of the N-methyl-D-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the D-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750 mm x 0.53 mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm x 1.5 mm I.D.) was selected for the second dimension. The lower limits of quantitation of D-Ser and D-Ala were 500 amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of D-Ser and D-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these D-amino acids were drastically increased with a lowering of the DAO activity except for the cases of D-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of D-Ser and D-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases.

D-serine is nephrotoxic in rats. Based on circumstantial evidence, it has been suspected that D-amino-acid oxidase is involved in this nephrotoxicity. Since we found that LEA/SENDAI rats lacked D-amino-acid oxidase, we examined whether this enzyme was associated with D-serine-induced nephrotoxicity using the LEA/SENDAI rats and control F344 rats. When d-propargylglycine, which is known to have a nephrotoxic effect through its metabolism by D-amino-acid oxidase, was injected intraperitoneally into the F344 rats, it caused glucosuria and polyuria. However, injection of d-propargylglycine into LEA/SENDAI rats did not cause any glucosuria or polyuria, indicating that D-amino-acid oxidase is definitely not functional in these rats. D-serine was then injected into the F344 and LEA/SENDAI rats. It caused glucosuria and polyuria in the F344 rats but not in the LEA/SENDAI rats. These results indicate clearly that D-amino-acid oxidase is responsible for the D-serine-induced nephrotoxicity.

A positron-emitter (carbon-11) labeled antagonist for the glycine-binding site of NMDA receptors, [(11)C]L-703,717, has a unique in vivo binding characteristic, in which it preferentially binds to cerebellar-specific NMDA receptors consisting of a GluRepsilon3 subunit and eventually accumulates in rodent cerebellum under in vivo conditions, but not under in vitro conditions. In order to understand the in vivo-specific site and subunit localization of this radioligand, we examined the effect of the endogenous glycine site agonists, glycine and D-serine, on in vivo [(11)C]L-703,717 binding. An increase in extracellular glycine concentration by treatment with a glycine transporter 1 (GlyT1)-selective inhibitor, NFPS ethyl ester, significantly decreased the cerebellar localization of [(11)C]L-703,717 in rats. D-serine is known to be concentrated in mammalian forebrain regions. The lack of D-serine detection in the cerebellum may be due to the fact that it has the highest enzymatic activity of D-amino acid oxidase (DAO). It was found that the cerebellar localization of [(11)C]L-703,717 is greatly diminished in mutant mice lacking DAO, in which D-serine content in the cerebellum is drastically increased from a nondetectable level in normal mice. These studies indicate that [(11)C]L-703,717 is susceptible to inhibition by glycine site agonists in its in vivo binding, and suggest that regional differences in inhibitions by endogenous agonists may be a crucial factor in the site- and subunit-specific binding of this glycine-site antagonist., (Copyright 2003 Wiley-Liss, Inc.)

A simple and precise method for the simultaneous determination of free D-aspartic acid, D-serine and D-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-L-cysteine. With the present method, the contents of these three D-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking D-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of D-serine were observed in forebrain (100-400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, D-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, D-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.

We have examined whether D-amino acid oxidase (DAO) regulates free D-serine content using mutant ddY/DAO- mice lacking DAO activity. We find that the content of D-serine in the serum and cerebellum of mutant mice is much higher than that of normal mice, whereas a slight but significant difference in the cerebral D-serine level is observed between the two strains. These results suggest that, although DAO may participate in the catabolism of D-serine in the cerebellum and periphery, there appears to be other mechanisms for catabolism of endogenous D-serine in the brain.

The presence of free D-alanine, D-proline and D-serine was demonstrated in mammalian tissues, using a mutant mouse strain lacking D-amino acid oxidase. In the experiment, free amino acids from the kidney and serum were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) to diastereomers, separated by two-dimensional thin-layer chromatography (TLC), and analysed by reversed-phase high-performance liquid chromatography (HPLC) for the resolution of D- and L-isomers. D/L ratios of alanine, proline and serine were obtained based on the peak areas of HPLC.