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Increased levels of 3-hydroxykynurenine in different brain regions of rats with chronic renal insufficiency
Brain Research Bulletin, Vol. 58, No. 4, pp. 423–428, 2002 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/02/$–see front matter
PII: S0361-9230(02)00813-4
Increased levels of 3-hydroxykynurenine in different brain regions of rats with chronic renal insufficiency Joanna Topczewska-Bruns,∗ Dariusz Pawlak, Ewa Chabielska, Anna Tankiewicz and Wlodzimierz Buczko Department of Pharmacodynamics, Medical Academy, Bialystok, Poland [Received 27 February 2002; Revised 14 May 2002; Accepted 16 May 2002] occurring in CRI [6,40]. There is also evidence of blood–brain barrier derangement, which—in turn—leads to an alteration in amino acid concentrations in the cerebrospinal fluid (CSF) and brain tissue [19]. Among others, it has been shown that tryptophan (TRP) concentrations in the CNS during CRI changes [38]. TRP is an exogenous amino acid, which could be converted in the brain either to serotonin [27] or to numerous neuroactive kynurenines via the kynurenic pathway. The kynurenic pathway is a main route of TRP metabolism on the periphery (95%) and all stages of this pathway have been shown within the CNS [13]. Most of the kynurenines lead to alterations in cellular metabolism and, as a consequence, to damage and cell death. 3-Hydroxykynureninie (3-HK) especially exhibits strong neurotoxin properties. This compound causes apoptotic neuronal death by generating a reactive oxygen species (ROS) [24]. Another compound of this pathway—kynurenine (KYN) has been shown to induce convulsions when injected intracerebroventriculary into rats’ brain [20]. The end-product of KYN metabolism is quinolinic acid (QUIN), which is a powerful excitant and convulsant substance [35]. Its neurotoxicity is mediated by the activation of the N-methyl-d-aspartate subtype of glutamate receptors (NMDA) in a competitive manner [36]. TRP metabolism via the kynurenic pathway is strongly disturbed in a number of pathological states including cerebral ischemia, infections, Huntington disease or hepatic encephalopathy [3,21,30,32]. Unfortunately, until now the influence of kidney failure on the metabolism of central kynurenines has not been intensively investigated. Therefore, we decided to evaluate some metabolites of the kynurenic pathway including TRP, KYN and 3-HK in different brain regions as well as their plasma concentrations in uremic rats. The accumulation of neuroactive metabolites of the kynurenine pathway within the brain may be of functional and clinical significance.
ABSTRACT: Tryptophan (TRP) metabolism via the kynurenine pathway leads to formations of neuroactive substances like kynurenine (KYN) and 3-hydroxykynurenine (3-HK), which may be involved in the pathogenesis of several human brain diseases. 3-Hydroxykynurenine especially is known to have strong neurotoxic properties. The generation of reactive oxygen species (ROS) leads to neuronal cell death with apoptotic features. Because the chronic renal insufficiency (CRI) results in disturbances in the functioning of the central nervous system (CNS), it is conceivable that the metabolism of some kynurenines may be altered and could play an important role in uremic encephalophaty. The levels of TRP, KYN and 3-HK were measured in the plasma and in different brain regions of uremic rats. The total plasma concentration of TRP as well as in all the studied brain samples was significantly diminished during uremia. Surprisingly, the level of KYN and 3-HK were elevated both in the plasma and different brain regions of CRI animals. KYN concentrations were approximately two times higher in the cerebellum, midbrain and cortex compared to the control group. The changes of 3-HK levels were more pronounced in the striatum and medulla than in other structures. This data suggests that CRI results in deep disturbances on the kynurenine pathway in CNS, which could be responsible for neurological abnormalities seen in uremia. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: Tryptophan, Kynurenine, Uremia, Rat, Central nervous system.
INTRODUCTION Chronic renal insufficiency (CRI) results in profound biochemical disturbances affecting numerous organs and regulatory systems, including the functioning of the central nervous system (CNS). The retention of excessive by-products of protein metabolism in the blood may impair nephron functions and reduce the excretion of urea, creatinine and other products of protein metabolism [4]. The accumulation of these compounds, known as uremic toxins, may lead to several neurological symptoms such as disorientation, memory failure, convulsions and coma [11,14,22]. Unfortunately, the mechanisms involved in the pathologies within the CNS, which occur in renal insufficiency are not fully understood. It has been demonstrated that parathyroid hormone (PTH) as well as certain metabolites of creatinine like methylguanidine or guanidinosuccinic acid are responsible for some neurological abnormalities
MATERIAL AND METHODS Animals Male Wistar rats weighing 260–480 g were housed in cages as appropriate with ad libitum access to chow and water. A 12:12 h light–dark cycle was maintained, and temperature and humidity were controlled. After 1 week of adaptation the rats were randomly divided into two groups: group I served as a control (shamoperated) and surgical induction of chronic renal insufficiency was performed on group II.
∗ Address for correspondence: Joanna Topczewska-Bruns, Department of Pharmacodynamics, Medical Academy, Mickiewicza Str. 2C, 15-230 Bialystok, Poland. Fax: +48-85-7421816; E-mail: [email protected]
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424 The study was approved by the Ethical Committee of the Medical Academy in Bialystok as being in compliance with the guidelines for care and use of animals in physiological sciences recommended by National and International Law and the Guidelines for the Use of Animals in Biomedical Research [10]. Surgical Induction of Chronic Renal Insufficiency Chronic renal insufficiency was induced by a partial resection of the renal tissue according to Ormrod and Miller [26]. Briefly, rats were anesthetized with pentobarbital (40 mg/kg, i.p.). In sham-operated rats (control group—CON) only the surgical extraction of the renal capsule was performed. The end-stage CRI was induced by removal of the left kidney, while the right kidney was decorticated by 60%. After 1 week the additional 20% of the right kidney cortex was removed. Biochemical analyses were carried out 2 months after the last surgical procedure. Blood and Brain Sampling Rats were anesthetized with pentobarbital (40 mg/kg, i.p.). The blood was collected by a heart puncture and transferred to a tube containing 3.13% sodium citrate (citrate/blood = 1:9, v/v). Plasma was obtained by a centrifugation of the blood at 3000×g for 10 min at 4◦ C. Urea and creatinine levels were measured by the use of commercial kits (Cormay, UK). The brains were removed and chilled on ice. Seven regions (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus and cortex) were dissected and thereafter the samples were immediately frozen. Frozen brain tissues were homogenized in 0.15N perichloric acid containing 1.025% EDTA (pH 3.0). The samples were centrifuged at 14,000 rpm for 20 min at 4◦ C. After centrifugation, the supernatant was filtered (0.45-µm Millipore filter) and stored at −80◦ C until assayed. Determination of Tryptophan (TRP) Tryptophan concentrations were determined according to Hever et al. [15]. The reversed-phase HPLC system consisted of a Waters Sherisorb S3 ODS2 150 mm × 2.1 mm column (USA), HP 1050 series pump (Germany) and a Rheodyne injection valve fitted with a sample loop (5 µl). The column effluent was monitored by using a programmable fluorescence detector HP 1046A (Germany). Optimized conditions were determined by recording fluorescence spectra with a stop–flow technique. Excitation and emission wavelengths were set at 254/404 nm for TRP. The output of the detector was connected to a single instrument, LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 0.25 ml/min consisting of 50 mM acetic acid, 0.25 M zinc acetate (pH 4.9), containing 1.2% of acetonitrile. Chromatography was carried out at 25◦ C. Determination of Kynurenine (KYN) Kynurenine concentrations were determined by high-performance liquid chromatography (HPLC) according to Holmes [18]. The chromatographic system Hewlett-Packard (Germany) was composed of a HP 1050 series pump and a Rheodyne injection valve fitted with a sample loop (20 µl). A guard column— LiChrospher 100 RP-18, 5 µm, 4 mm × 4 mm (Germany) was placed before the C18 reversed-phase column LiChrospher 100 RP-18, 5 µm, 125 mm × 4 mm (Germany). Using a HP 1050 series UV detector (Germany) monitored the column effluent (365 nm). The output of the detector was connected to a single instrument LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 1.5 ml/min. It consisted of 0.1 M acetic acid and 0.1 M ammonium acetate (pH 4.65) containing 2% of acetonitrile. Chromatography was carried out at 25◦ C.
TOPCZEWSKA-BRUNS ET AL. Determination of 3-Hydroxykynurenine (3-HK) 3-Hydroxykynurenine was measured using the HPLC technique as described by Heyes and Quearry [16]. The reversed-phase HPLC system consisted of a Waters Sherisorb S3 ODS2 150 mm×2.1 mm column (USA), HP 1050 series pump (Germany) and a Rheodyne injection valve fitted with a sample loop (5 µl). The column effluent was monitored by using a programmable electrochemical detector HP 1049A (Germany). Potential of the working electrode was 0.6 V. The output of the detector was connected to a single instrument LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 0.25 ml/min and consisted of 0.1 M triethylamine, 0.1 M phosphoric acid, 0.3 mM EDTA, 8.2 mM heptane-1-sulfonic acid sodium salt, containing 2% acetonitrile. Chromatography was carried out at 25◦ C. Statistical Analysis The results of the experiment were evaluated by a one-way analysis of variance (ANOVA) followed by the Mann–Whitney U-test. Student’s t-test was used to compare urea and creatinine concentrations in the blood. The data were deemed statistically significant when p < 0.05. RESULTS Renal cortex extraction led to an increase in plasma urea and creatinine concentrations. Urea and creatinine levels were significantly elevated in experimental CRI animals in comparison with the control group (p < 0.001) (Table 1). Plasma Studies The plasma concentration of total TRP was significantly diminished whereas the level of KYN and 3-HK increased compared to the control group (p < 0.001 for all substances) (Table 1). Brain Studies Tryptophan level was significantly decreased in all studied brain regions compared to those this in the control group. The change was strongly expressed in the cerebellum, striatum, hypothalamus and hippocampus (p < 0.001). Nevertheless, in other structures of the CNS, levels also reached statistical significance in the medulla, the cortex (p < 0.01) and the midbrain (p < 0.05) (Fig. 1). The concentration of KYN in CRI rats was significantly elevated in each part of the brain (p < 0.001). The KYN level was approximately two times higher in the cerebellum, midbrain and cortex than in the control group (Fig. 2).
TABLE 1 THE PLASMA CREATININE, UREA, TRYPTOPHAN, KYNURENINE AND 3-HYDROXYKYNURENINE CONCENTRATIONS OF THE CONTROL GROUP AND RATS WITH CRI
CON Creatinine (µM) Urea (mM) Tryptophan (µM) Kynurenine (µM) 3-Hydroxykynurenine (nM)
28.56 3.62 40.6 1.83 59.32
± ± ± ± ±
4.2 0.75 4.85 0.20 8.51
CRI 329.28 90.2 18.27 2.67 158.02
± ± ± ± ±
p 36.7 13.6 2.75 0.31 21.09
<0.001 <0.001 <0.001 <0.001 <0.001
CON—control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group.
KYNURENINES IN UREMIC BRAIN
FIG. 1. Tryptophan (TRP) concentrations in different brain structures in rats with chronic renal insufficiency. CON— control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group. (∗∗∗) p < 0.001; (∗∗) p < 0.01; (∗) p < 0.05.
FIG. 2. Kynurenine (KYN) concentrations in different brain structures in rats with chronic renal insufficiency. CON— control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group. (∗∗∗) p < 0.001.
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FIG. 3. 3-Hydroxykynurenine (3-HK) concentrations in different brain structures in rats with chronic renal insufficiency. CON—control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group. (∗∗∗) p < 0.001; (∗∗) p < 0.01; (∗) p < 0.05.
We have also observed an enhanced level of 3-HK within the brain structure of uremic rats. This was more evident in the striatum and medulla (p < 0.001) than in the hypothalamus, midbrain, hippocampus or cortex (p < 0.01). A smaller increase was found in the cerebellum, but there was still a significant difference between the controls and the experimental animals (p < 0.05) (Fig. 3). DISCUSSION We have demonstrated that during experiments, uremia causes profound disturbances on the kynurenic pathway within the CNS as well as on the periphery. Plasma levels of TRP were significantly diminished whereas concentrations of KYN and 3-HK were elevated. Similar results have been observed previously [28,29]. TRP is an exogenous amino acid and thus, decreased concentrations of TRP may reflect the restriction of food intake, which is a common symptom of CRI [1]. In our previous studies, we have shown that food intake is significantly diminished in uremic rats [28,39]. Moreover, according to Holmes and Kahan [17] the low blood concentrations of TRP may result from decreased intestinal absorption due to an excessive TRP transformation into other indoles competing for bounds to proteins in the bowel epithelium or diminished reabsorption of this amino acid in renal tubules. It has been reported by Saito et al. that the activity of hepatic tryptophan dioxygenase (TDO), the major enzyme responsible for the transformation of TRP to KYN on the periphery, is increased in CRI rats [31]. This could partially explain the elevated level of KYN in serum. KYN is a key compound of the kynurenic pathway leading to the formation of anthranilic acid (AA) by the use of kynureninase (the major route along the periphery) and production of 3-HK catalyzed
by kynurenine hydroxylase (predominantly in the brain). Because the activity of liver kynureninase [31] is decreased in the course of CRI, most of the KYN may be converted to 3-HK resulting in its accumulation in plasma. Impaired kidney functions, which excrete 3-HK, may contribute to an accumulation of this compound in plasma [23,29]. It’s interesting that both KYN and 3-HK can cross blood–brain barrier [8]. This could have a special value when the concentrations of these substances are elevated in plasma. Our experiments have shown significant alterations in TRP, KYN and 3-HK concentrations within different brain regions. We have observed an increase of KYN and 3-HK in CNS and a diminished level of TRP in all brain structures in the course of CRI. Approximately 40% of KYN is synthesized de novo from TRP in the brain; the remainder comes from plasma [9] through the blood–brain barrier by the large neutral amino acid carrier [34]. We have shown that the plasma level of KYN in CRI is significantly elevated. Thus, the accumulation of this compound in the different parts of the brain observed by us could be due to an enhanced influx from the plasma. The cerebrovascular large neutral amino acid carrier is a sodium independent mechanism that facilitates the exchange of 10 or more neutral amino acids between the plasma and the brain. Also TRP is transported by the use of this carrier. It is controversial whether KYN has a higher affinity to large neutral amino acid carriers then TRP. As reported by Speciale et al. [33] TRP and some other amino acid like leucine or phenylalanine are recognized with a higher affinity than KYN. This is opposite to the finding of Green and Curzon who have demonstrated that KYN can inhibit TRP uptake into brain slices [12]. The latter report could partially explain the decreased level of TRP in CNS in rats with CRI. The accumulation of TRP in the brain is also affected by the concentration
KYNURENINES IN UREMIC BRAIN of other amino acids (in order: leucine > tyrosine > threonine > phenyloalanine = histidine > isoleucine = methionine > valine), which are competing for TRP transport [2,41]. According to Jeppson et al., the concentration of some large neutral amino acids like, i.e., phenylalanine or threonine in the blood is significantly increased in uremia [19]. Hence, we can’t exclude that alteration in TRP levels observed in our experiment is a result of the inhibition of its uptake into the brain by competitive amino acids. We have demonstrated that the concentration of 3-HK is significantly higher in all brain regions probably due to increased synthesis as well as an increased influx from the plasma, where the concentration of this compound is highly elevated. Although an elevated level of 3-HK was observed in each studied brain structure, the changes in 3-HK levels are more pronounced in the striatum and medulla than in other structures, particularly the cerebellum. This is in part consistent with the view of Okuda et al. [24] that 3-HK in a low concentration shows brain region selectivity. As they have reported, cortical and striatal neurons are most vulnerable to 3-HK toxicity; hippocampal neurons are less sensitive, while cerebellar cells are totally resistant to damage caused by 3-HK. This probably reflects the differences in cellular uptake of this compound within the CNS structure. The neurons with little sensitivity to 3-HK toxicity (cerebellum) have a poor ability to take up large neutral amino acids by Na+ -dependent transporters. In contrast, the cortex, striatum or even hippocampus has a high affinity to transport 3-HK by a Na+ -dependent process [7]. Because neurotoxicity of a low concentration of 3-HK is dependent on Na+ -dependent cellular uptake, these structures may be more susceptible to damage caused by 3-HK [24]. Nevertheless, the toxicity of higher doses of 3-HK (in the range of 100 µmol) is independent of cellular uptake and, thus, doesn’t show any brain region selectivity [24]. The toxic action of 3-HK is mediated by the generation of ROS like hydrogen peroxide and hydroxyl radicals [25]. A hydroxyl radical is an extremely reactive species responsible for most of the covalent modifications and damages to macromolecules, including DNA, proteins and lipid membranes [5]. Neuronal cell death induced by 3-HK shows typical apoptotic features like perinuclear chromatin condensation and endonuclease-mediated internucleosomal DNA fragmentation [24]. In conclusion, our study demonstrates that chronic renal failure results in serious disorders in the kynurenic pathway on periphery and within all the studied structure of the CNS (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus and cortex). Kynurenines such as KYN and 3-HK may be important mediators of neurological dysfunctions observed both in uremic patients and animals. In our previous studies, we have demonstrated serious behavioral disturbances like decreased locomotor, exploratory and emotional activity of rats suffering from CRI, which closely resemble these observed in uremic patients [39]. If we understand the mechanism causing neurological change in uremia, strategies that can decrease neuroactive kynurenine metabolites or attenuate their effects may offer new therapeutic approaches for CRI-related neurological complications. It is worth noting that some approaches have been undertaken to interfere with and modify the kynurenic pathway in neurodegenerative disorders or hypoxia-ischemic strokes [37]. Thus, it seems to be necessary to further investigate the metabolism of kynurenines in the brain during kidney insufficiency. REFERENCES 1. Aguilera, A.; Selegas, R.; Codoceo, R.; Bajo, A. Uremic anorexia: A consequence of persistently high brain serotonine levels? The tryptophan/serotonin disorder hypothesis. Peri. Dial. Int. 20:810–816; 2000.
427 2. Bender, D. A. Biochemistry of tryptophan in health and disease. Mol. Aspects Med. 6:101–197; 1982. 3. Berqvist, P. B. F.; Heyes, M. P.; Bugge, M.; Bengtsson, F. Brain quinolinic acid in chronic experimental hepatic encephalopathy: Effects of an exogenous ammonium acetate challenge. J. Neurochem. 65:2235–2240; 1995. 4. Burn, D. J.; Bates, D. Neurology and the kidney. J. Neurol. Neurosurg. Psychiatry 65:810–821; 1998. 5. Coyle, J. T.; Puttfarcken, P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689–695; 1993. 6. D’Hoge, R.; Pei, Y.-Q.; Marescau, B.; De Deyn, P. P. Behavioral toxicity of guanidinosuccinic acid in adult and young mice. Toxicol. Lett. 64/65:773–777; 1992. 7. Eastman, C. L.; Guilarte, T. R.; Lever, J. R. Uptake of 3-hydroxykynurenine measured in rat brain slices and in neuronal cell line. Brain Res. 584:110–116; 1992. 8. Fukui, S.; Schwarcz, R.; Rapoport, S. I.; Takadda, Y.; Smith, Q. R. Blood–brain transport of kynurenines: Implications for brain synthesis and metabolism. J. Neurochem. 56:2007–2017; 1991. 9. Gal, E. M.; Sherman, A. D. L-kynurenine: Its synthesis and possible regulatory function in brain. Neurochem. Res. 5:223–239; 1980. 10. Giles, A. R. Guidelines for the use of animals in biomedial research. Thromb. Haemost. 58:1078–1084; 1987. 11. Gin, H. E. Neurobehavioral dysfunction in uremia. Kidney Int. Suppl. 2:217–221; 1975. 12. Green, A. R.; Curzon, G. The effect of tryptophan metabolites on brain 5-hydroxytryptamine metabolism. Biochem. Pharamcol. 19:2061– 2068; 1970. 13. Guidetti, P.; Eastman, C. L.; Schwarcz, R. Metabolism of [5-3 H]kynurenine in the rat brain in vivo: Evidence for the existence of a functional kynurenine pathway. J. Neurochem. 65:2621–2632; 1995. 14. Hegedus, Z. L.; Frank, H. A.; Steinman, T. I.; Nayak, U. Dialysis of plasma soluble lipofucsins in patients with end-stage renal failure. Arch. Int. Physiol. Biochim. Biophys. 99:355–363; 1999. 15. Hever, C.; Beyne, P.; Jamault, H.; Delacoux, E. Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection. J. Chromatogr. 675:157–161; 1996. 16. Heyes, M. P.; Quearry, B. J. Quantification of 3-hydroxykynurenine in brain by high-performance liquid chromatography and electrochemical detection. J. Chromatogr. 428:340–344; 1988. 17. Holmes, E. W.; Kahan, S. E. Tryptophan distribution and metabolism in experimental chronic renal insufficiency. Exp. Mol. Pathol. 46:89– 101; 1987. 18. Holmes, E. W. Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography. Anal. Biochem. 172:518–525; 1988. 19. Jeppson, B.; Freund, H. R.; Gimmon, M. D.; James, J. H.; von Meyenfeldt, M. F.; Fischer, J. E. Blood–brain derangement in uremic nephropathy. Surgery 92:30–35; 1982. 20. Lapin, I. P. Consultant action of intracerebroventriculary administrated L-kynurenine sulphate, quinolinic acid and other derivatives of succinic acid, and effect of amino acid: Structure–activity relationships. Neuropharmacology 21:1227–1233; 1982. 21. Michalak, A.; Chatauret, N.; Butterworth, R. F. Evidence for a serotonin transporter deficit in experimental acute liver failure. Neurochem. Int. 38:163–168; 2001. 22. Moe, S. M.; Sprague, S. M. Uremic encephalophaty. Clin. Nephrol. 42:251–256; 1994. 23. Moller, S. E. Pharmacokinetics of tryptophan, renal handling of kynurenine and the effect of nicotinamide on its appearance in plasma and urine following l-tryptophan loading of healthy subjects. Eur. J. Clin. Pharmacol. 21:137–142; 1981. 24. Okuda, S.; Nishiyama, N.; Saito, H.; Katsuki, H. 3-Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity. J. Neurochem. 70:299–307; 1998. 25. Okuda, S.; Nishiyama, N.; Saito, H.; Katsuki, H. Hydrogen peroxidemediated neuronal cell death by an endogenous mediated neurotoxin, 3-hydroxykynurenine. Proc. Natl. Acad. Sci. U.S.A. 93:12553–12558; 1996. 26. Ormrod, D.; Miller, T. Experimental uremia. Nephron 26:249–254; 1980.
428 27. Park, J. W.; Youn, Y. C.; Kwon, O. S.; Jang, Y. Y.; Han, E. S.; Lee, C. S. Protective effect of serotonin on 6-hydroxydopamineand dopamine-induced oxidative damage of brain mitochondria and synaptosomes and PC12 cells. Neurochem. Int. 40:223–233; 2002. 28. Pawlak, D.; Tankiewicz, A.; Mysliwiec, P.; Buczko, W. Tryptophan metabolism via kynurenine pathway in experimental chronic renal failure. Nephron 90:328–335; 2002. 29. Pawlak, D.; Tankiewicz, A.; Buczko, W. Kynurenine and its metabolites in the rat with experimental renal insufficiency. J. Physiol. Pharmacol. 52:755–766; 2001. 30. Pearson, S. J.; Reynolds, G. P. Increased brain concentration of neurotoxin, 3-hydroxynurenine, in Huntington’s disease. Nuerosci. Lett. 144:199–201; 1992. 31. Saito, K.; Fujigaki, S.; Heyes, M. P.; Shibata, K.; Takemura, M.; Fuji, H.; Wada, H.; Noma, A.; Seishima, M. Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency. Am. J. Physiol. Renal Physiol. 279:F565–F572; 2000. 32. Saito, K.; Nowak, T. S.; Markey, S. P.; Heyes, M. P. Mechanism of delayed increases in kynurenine pathway metabolism in damaged brain regions following transient cerebral ischemia. J. Neurochem. 60:180– 192; 1993.
TOPCZEWSKA-BRUNS ET AL. 33. Speciale, C.; Hares, K.; Schwarcz, R.; Brookes, N. High-affinity uptake of L-kynurenine by a Na+ -independent transporter of neutral amino acids in astrocytes. J. Neurosci. 9:2066–2072; 1989. 34. Speciel, C.; Schwarcz, R. Uptake of kynurenine into rat brain slices. J. Neurochem. 54:156–163; 1990. 35. Stone, T. W.; Connick, J. H. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol. Rev. 45:309–379; 1993. 36. Stone, T. W. Kynurenines in the CNS: From endogenous obscurity to therapeutic importance. Prog. Neurobiol. 64:633–645; 2001. 37. Stone, T. W. Kynurenic acid antagonists and kynurenine pathway inhibitors. Exp. Opin. Invest. Drugs 10:633–645; 2001. 38. Swenseid, M. E.; Wang, M.; Vyhmeister, I.; Chan, W.; Siassi, F.; Tam, C. F.; Kopple, J. D. Amino acid metabolism in the chronically uremic rat. Clin. Nephrol. 3:240–246; 1975. 39. Topczewska-Bruns, J.; Tankiewicz, A.; Pawlak, D.; Buczko, W. Behavioral changes in the course of chronic renal insufficiency in rats. Pol. J. Pharamcol. 53:263–269; 2001. 40. Vanholder, R.; De Smet, R. Pathophysiologic effects of uremic retention solutes. J. Am. Soc. Nephrol. 10:1815–1823; 1999. 41. Yuwiler, A.; Oldendorf, E.; Geller, E.; Braun, L. Effect of albumin binding and amino acid competition on tryptophan uptake into brain. J. Neurochem. 28:1015–1023; 1977.
PII: S0361-9230(02)00813-4
Increased levels of 3-hydroxykynurenine in different brain regions of rats with chronic renal insufficiency Joanna Topczewska-Bruns,∗ Dariusz Pawlak, Ewa Chabielska, Anna Tankiewicz and Wlodzimierz Buczko Department of Pharmacodynamics, Medical Academy, Bialystok, Poland [Received 27 February 2002; Revised 14 May 2002; Accepted 16 May 2002] occurring in CRI [6,40]. There is also evidence of blood–brain barrier derangement, which—in turn—leads to an alteration in amino acid concentrations in the cerebrospinal fluid (CSF) and brain tissue [19]. Among others, it has been shown that tryptophan (TRP) concentrations in the CNS during CRI changes [38]. TRP is an exogenous amino acid, which could be converted in the brain either to serotonin [27] or to numerous neuroactive kynurenines via the kynurenic pathway. The kynurenic pathway is a main route of TRP metabolism on the periphery (95%) and all stages of this pathway have been shown within the CNS [13]. Most of the kynurenines lead to alterations in cellular metabolism and, as a consequence, to damage and cell death. 3-Hydroxykynureninie (3-HK) especially exhibits strong neurotoxin properties. This compound causes apoptotic neuronal death by generating a reactive oxygen species (ROS) [24]. Another compound of this pathway—kynurenine (KYN) has been shown to induce convulsions when injected intracerebroventriculary into rats’ brain [20]. The end-product of KYN metabolism is quinolinic acid (QUIN), which is a powerful excitant and convulsant substance [35]. Its neurotoxicity is mediated by the activation of the N-methyl-d-aspartate subtype of glutamate receptors (NMDA) in a competitive manner [36]. TRP metabolism via the kynurenic pathway is strongly disturbed in a number of pathological states including cerebral ischemia, infections, Huntington disease or hepatic encephalopathy [3,21,30,32]. Unfortunately, until now the influence of kidney failure on the metabolism of central kynurenines has not been intensively investigated. Therefore, we decided to evaluate some metabolites of the kynurenic pathway including TRP, KYN and 3-HK in different brain regions as well as their plasma concentrations in uremic rats. The accumulation of neuroactive metabolites of the kynurenine pathway within the brain may be of functional and clinical significance.
ABSTRACT: Tryptophan (TRP) metabolism via the kynurenine pathway leads to formations of neuroactive substances like kynurenine (KYN) and 3-hydroxykynurenine (3-HK), which may be involved in the pathogenesis of several human brain diseases. 3-Hydroxykynurenine especially is known to have strong neurotoxic properties. The generation of reactive oxygen species (ROS) leads to neuronal cell death with apoptotic features. Because the chronic renal insufficiency (CRI) results in disturbances in the functioning of the central nervous system (CNS), it is conceivable that the metabolism of some kynurenines may be altered and could play an important role in uremic encephalophaty. The levels of TRP, KYN and 3-HK were measured in the plasma and in different brain regions of uremic rats. The total plasma concentration of TRP as well as in all the studied brain samples was significantly diminished during uremia. Surprisingly, the level of KYN and 3-HK were elevated both in the plasma and different brain regions of CRI animals. KYN concentrations were approximately two times higher in the cerebellum, midbrain and cortex compared to the control group. The changes of 3-HK levels were more pronounced in the striatum and medulla than in other structures. This data suggests that CRI results in deep disturbances on the kynurenine pathway in CNS, which could be responsible for neurological abnormalities seen in uremia. © 2002 Elsevier Science Inc. All rights reserved. KEY WORDS: Tryptophan, Kynurenine, Uremia, Rat, Central nervous system.
INTRODUCTION Chronic renal insufficiency (CRI) results in profound biochemical disturbances affecting numerous organs and regulatory systems, including the functioning of the central nervous system (CNS). The retention of excessive by-products of protein metabolism in the blood may impair nephron functions and reduce the excretion of urea, creatinine and other products of protein metabolism [4]. The accumulation of these compounds, known as uremic toxins, may lead to several neurological symptoms such as disorientation, memory failure, convulsions and coma [11,14,22]. Unfortunately, the mechanisms involved in the pathologies within the CNS, which occur in renal insufficiency are not fully understood. It has been demonstrated that parathyroid hormone (PTH) as well as certain metabolites of creatinine like methylguanidine or guanidinosuccinic acid are responsible for some neurological abnormalities
MATERIAL AND METHODS Animals Male Wistar rats weighing 260–480 g were housed in cages as appropriate with ad libitum access to chow and water. A 12:12 h light–dark cycle was maintained, and temperature and humidity were controlled. After 1 week of adaptation the rats were randomly divided into two groups: group I served as a control (shamoperated) and surgical induction of chronic renal insufficiency was performed on group II.
∗ Address for correspondence: Joanna Topczewska-Bruns, Department of Pharmacodynamics, Medical Academy, Mickiewicza Str. 2C, 15-230 Bialystok, Poland. Fax: +48-85-7421816; E-mail: [email protected]
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424 The study was approved by the Ethical Committee of the Medical Academy in Bialystok as being in compliance with the guidelines for care and use of animals in physiological sciences recommended by National and International Law and the Guidelines for the Use of Animals in Biomedical Research [10]. Surgical Induction of Chronic Renal Insufficiency Chronic renal insufficiency was induced by a partial resection of the renal tissue according to Ormrod and Miller [26]. Briefly, rats were anesthetized with pentobarbital (40 mg/kg, i.p.). In sham-operated rats (control group—CON) only the surgical extraction of the renal capsule was performed. The end-stage CRI was induced by removal of the left kidney, while the right kidney was decorticated by 60%. After 1 week the additional 20% of the right kidney cortex was removed. Biochemical analyses were carried out 2 months after the last surgical procedure. Blood and Brain Sampling Rats were anesthetized with pentobarbital (40 mg/kg, i.p.). The blood was collected by a heart puncture and transferred to a tube containing 3.13% sodium citrate (citrate/blood = 1:9, v/v). Plasma was obtained by a centrifugation of the blood at 3000×g for 10 min at 4◦ C. Urea and creatinine levels were measured by the use of commercial kits (Cormay, UK). The brains were removed and chilled on ice. Seven regions (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus and cortex) were dissected and thereafter the samples were immediately frozen. Frozen brain tissues were homogenized in 0.15N perichloric acid containing 1.025% EDTA (pH 3.0). The samples were centrifuged at 14,000 rpm for 20 min at 4◦ C. After centrifugation, the supernatant was filtered (0.45-µm Millipore filter) and stored at −80◦ C until assayed. Determination of Tryptophan (TRP) Tryptophan concentrations were determined according to Hever et al. [15]. The reversed-phase HPLC system consisted of a Waters Sherisorb S3 ODS2 150 mm × 2.1 mm column (USA), HP 1050 series pump (Germany) and a Rheodyne injection valve fitted with a sample loop (5 µl). The column effluent was monitored by using a programmable fluorescence detector HP 1046A (Germany). Optimized conditions were determined by recording fluorescence spectra with a stop–flow technique. Excitation and emission wavelengths were set at 254/404 nm for TRP. The output of the detector was connected to a single instrument, LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 0.25 ml/min consisting of 50 mM acetic acid, 0.25 M zinc acetate (pH 4.9), containing 1.2% of acetonitrile. Chromatography was carried out at 25◦ C. Determination of Kynurenine (KYN) Kynurenine concentrations were determined by high-performance liquid chromatography (HPLC) according to Holmes [18]. The chromatographic system Hewlett-Packard (Germany) was composed of a HP 1050 series pump and a Rheodyne injection valve fitted with a sample loop (20 µl). A guard column— LiChrospher 100 RP-18, 5 µm, 4 mm × 4 mm (Germany) was placed before the C18 reversed-phase column LiChrospher 100 RP-18, 5 µm, 125 mm × 4 mm (Germany). Using a HP 1050 series UV detector (Germany) monitored the column effluent (365 nm). The output of the detector was connected to a single instrument LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 1.5 ml/min. It consisted of 0.1 M acetic acid and 0.1 M ammonium acetate (pH 4.65) containing 2% of acetonitrile. Chromatography was carried out at 25◦ C.
TOPCZEWSKA-BRUNS ET AL. Determination of 3-Hydroxykynurenine (3-HK) 3-Hydroxykynurenine was measured using the HPLC technique as described by Heyes and Quearry [16]. The reversed-phase HPLC system consisted of a Waters Sherisorb S3 ODS2 150 mm×2.1 mm column (USA), HP 1050 series pump (Germany) and a Rheodyne injection valve fitted with a sample loop (5 µl). The column effluent was monitored by using a programmable electrochemical detector HP 1049A (Germany). Potential of the working electrode was 0.6 V. The output of the detector was connected to a single instrument LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 0.25 ml/min and consisted of 0.1 M triethylamine, 0.1 M phosphoric acid, 0.3 mM EDTA, 8.2 mM heptane-1-sulfonic acid sodium salt, containing 2% acetonitrile. Chromatography was carried out at 25◦ C. Statistical Analysis The results of the experiment were evaluated by a one-way analysis of variance (ANOVA) followed by the Mann–Whitney U-test. Student’s t-test was used to compare urea and creatinine concentrations in the blood. The data were deemed statistically significant when p < 0.05. RESULTS Renal cortex extraction led to an increase in plasma urea and creatinine concentrations. Urea and creatinine levels were significantly elevated in experimental CRI animals in comparison with the control group (p < 0.001) (Table 1). Plasma Studies The plasma concentration of total TRP was significantly diminished whereas the level of KYN and 3-HK increased compared to the control group (p < 0.001 for all substances) (Table 1). Brain Studies Tryptophan level was significantly decreased in all studied brain regions compared to those this in the control group. The change was strongly expressed in the cerebellum, striatum, hypothalamus and hippocampus (p < 0.001). Nevertheless, in other structures of the CNS, levels also reached statistical significance in the medulla, the cortex (p < 0.01) and the midbrain (p < 0.05) (Fig. 1). The concentration of KYN in CRI rats was significantly elevated in each part of the brain (p < 0.001). The KYN level was approximately two times higher in the cerebellum, midbrain and cortex than in the control group (Fig. 2).
TABLE 1 THE PLASMA CREATININE, UREA, TRYPTOPHAN, KYNURENINE AND 3-HYDROXYKYNURENINE CONCENTRATIONS OF THE CONTROL GROUP AND RATS WITH CRI
CON Creatinine (µM) Urea (mM) Tryptophan (µM) Kynurenine (µM) 3-Hydroxykynurenine (nM)
28.56 3.62 40.6 1.83 59.32
± ± ± ± ±
4.2 0.75 4.85 0.20 8.51
CRI 329.28 90.2 18.27 2.67 158.02
± ± ± ± ±
p 36.7 13.6 2.75 0.31 21.09
<0.001 <0.001 <0.001 <0.001 <0.001
CON—control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group.
KYNURENINES IN UREMIC BRAIN
FIG. 1. Tryptophan (TRP) concentrations in different brain structures in rats with chronic renal insufficiency. CON— control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group. (∗∗∗) p < 0.001; (∗∗) p < 0.01; (∗) p < 0.05.
FIG. 2. Kynurenine (KYN) concentrations in different brain structures in rats with chronic renal insufficiency. CON— control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group. (∗∗∗) p < 0.001.
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FIG. 3. 3-Hydroxykynurenine (3-HK) concentrations in different brain structures in rats with chronic renal insufficiency. CON—control (sham-operated) group; CRI—chronic renal insufficiency (experimental details are described in ‘MATERIALS AND METHODS’). Values are presented as means ± SEM, significance of the difference in comparison with the control group. (∗∗∗) p < 0.001; (∗∗) p < 0.01; (∗) p < 0.05.
We have also observed an enhanced level of 3-HK within the brain structure of uremic rats. This was more evident in the striatum and medulla (p < 0.001) than in the hypothalamus, midbrain, hippocampus or cortex (p < 0.01). A smaller increase was found in the cerebellum, but there was still a significant difference between the controls and the experimental animals (p < 0.05) (Fig. 3). DISCUSSION We have demonstrated that during experiments, uremia causes profound disturbances on the kynurenic pathway within the CNS as well as on the periphery. Plasma levels of TRP were significantly diminished whereas concentrations of KYN and 3-HK were elevated. Similar results have been observed previously [28,29]. TRP is an exogenous amino acid and thus, decreased concentrations of TRP may reflect the restriction of food intake, which is a common symptom of CRI [1]. In our previous studies, we have shown that food intake is significantly diminished in uremic rats [28,39]. Moreover, according to Holmes and Kahan [17] the low blood concentrations of TRP may result from decreased intestinal absorption due to an excessive TRP transformation into other indoles competing for bounds to proteins in the bowel epithelium or diminished reabsorption of this amino acid in renal tubules. It has been reported by Saito et al. that the activity of hepatic tryptophan dioxygenase (TDO), the major enzyme responsible for the transformation of TRP to KYN on the periphery, is increased in CRI rats [31]. This could partially explain the elevated level of KYN in serum. KYN is a key compound of the kynurenic pathway leading to the formation of anthranilic acid (AA) by the use of kynureninase (the major route along the periphery) and production of 3-HK catalyzed
by kynurenine hydroxylase (predominantly in the brain). Because the activity of liver kynureninase [31] is decreased in the course of CRI, most of the KYN may be converted to 3-HK resulting in its accumulation in plasma. Impaired kidney functions, which excrete 3-HK, may contribute to an accumulation of this compound in plasma [23,29]. It’s interesting that both KYN and 3-HK can cross blood–brain barrier [8]. This could have a special value when the concentrations of these substances are elevated in plasma. Our experiments have shown significant alterations in TRP, KYN and 3-HK concentrations within different brain regions. We have observed an increase of KYN and 3-HK in CNS and a diminished level of TRP in all brain structures in the course of CRI. Approximately 40% of KYN is synthesized de novo from TRP in the brain; the remainder comes from plasma [9] through the blood–brain barrier by the large neutral amino acid carrier [34]. We have shown that the plasma level of KYN in CRI is significantly elevated. Thus, the accumulation of this compound in the different parts of the brain observed by us could be due to an enhanced influx from the plasma. The cerebrovascular large neutral amino acid carrier is a sodium independent mechanism that facilitates the exchange of 10 or more neutral amino acids between the plasma and the brain. Also TRP is transported by the use of this carrier. It is controversial whether KYN has a higher affinity to large neutral amino acid carriers then TRP. As reported by Speciale et al. [33] TRP and some other amino acid like leucine or phenylalanine are recognized with a higher affinity than KYN. This is opposite to the finding of Green and Curzon who have demonstrated that KYN can inhibit TRP uptake into brain slices [12]. The latter report could partially explain the decreased level of TRP in CNS in rats with CRI. The accumulation of TRP in the brain is also affected by the concentration
KYNURENINES IN UREMIC BRAIN of other amino acids (in order: leucine > tyrosine > threonine > phenyloalanine = histidine > isoleucine = methionine > valine), which are competing for TRP transport [2,41]. According to Jeppson et al., the concentration of some large neutral amino acids like, i.e., phenylalanine or threonine in the blood is significantly increased in uremia [19]. Hence, we can’t exclude that alteration in TRP levels observed in our experiment is a result of the inhibition of its uptake into the brain by competitive amino acids. We have demonstrated that the concentration of 3-HK is significantly higher in all brain regions probably due to increased synthesis as well as an increased influx from the plasma, where the concentration of this compound is highly elevated. Although an elevated level of 3-HK was observed in each studied brain structure, the changes in 3-HK levels are more pronounced in the striatum and medulla than in other structures, particularly the cerebellum. This is in part consistent with the view of Okuda et al. [24] that 3-HK in a low concentration shows brain region selectivity. As they have reported, cortical and striatal neurons are most vulnerable to 3-HK toxicity; hippocampal neurons are less sensitive, while cerebellar cells are totally resistant to damage caused by 3-HK. This probably reflects the differences in cellular uptake of this compound within the CNS structure. The neurons with little sensitivity to 3-HK toxicity (cerebellum) have a poor ability to take up large neutral amino acids by Na+ -dependent transporters. In contrast, the cortex, striatum or even hippocampus has a high affinity to transport 3-HK by a Na+ -dependent process [7]. Because neurotoxicity of a low concentration of 3-HK is dependent on Na+ -dependent cellular uptake, these structures may be more susceptible to damage caused by 3-HK [24]. Nevertheless, the toxicity of higher doses of 3-HK (in the range of 100 µmol) is independent of cellular uptake and, thus, doesn’t show any brain region selectivity [24]. The toxic action of 3-HK is mediated by the generation of ROS like hydrogen peroxide and hydroxyl radicals [25]. A hydroxyl radical is an extremely reactive species responsible for most of the covalent modifications and damages to macromolecules, including DNA, proteins and lipid membranes [5]. Neuronal cell death induced by 3-HK shows typical apoptotic features like perinuclear chromatin condensation and endonuclease-mediated internucleosomal DNA fragmentation [24]. In conclusion, our study demonstrates that chronic renal failure results in serious disorders in the kynurenic pathway on periphery and within all the studied structure of the CNS (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus and cortex). Kynurenines such as KYN and 3-HK may be important mediators of neurological dysfunctions observed both in uremic patients and animals. In our previous studies, we have demonstrated serious behavioral disturbances like decreased locomotor, exploratory and emotional activity of rats suffering from CRI, which closely resemble these observed in uremic patients [39]. If we understand the mechanism causing neurological change in uremia, strategies that can decrease neuroactive kynurenine metabolites or attenuate their effects may offer new therapeutic approaches for CRI-related neurological complications. It is worth noting that some approaches have been undertaken to interfere with and modify the kynurenic pathway in neurodegenerative disorders or hypoxia-ischemic strokes [37]. Thus, it seems to be necessary to further investigate the metabolism of kynurenines in the brain during kidney insufficiency. REFERENCES 1. Aguilera, A.; Selegas, R.; Codoceo, R.; Bajo, A. Uremic anorexia: A consequence of persistently high brain serotonine levels? The tryptophan/serotonin disorder hypothesis. Peri. Dial. Int. 20:810–816; 2000.
427 2. Bender, D. A. Biochemistry of tryptophan in health and disease. Mol. Aspects Med. 6:101–197; 1982. 3. Berqvist, P. B. F.; Heyes, M. P.; Bugge, M.; Bengtsson, F. Brain quinolinic acid in chronic experimental hepatic encephalopathy: Effects of an exogenous ammonium acetate challenge. J. Neurochem. 65:2235–2240; 1995. 4. Burn, D. J.; Bates, D. Neurology and the kidney. J. Neurol. Neurosurg. Psychiatry 65:810–821; 1998. 5. Coyle, J. T.; Puttfarcken, P. Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689–695; 1993. 6. D’Hoge, R.; Pei, Y.-Q.; Marescau, B.; De Deyn, P. P. Behavioral toxicity of guanidinosuccinic acid in adult and young mice. Toxicol. Lett. 64/65:773–777; 1992. 7. Eastman, C. L.; Guilarte, T. R.; Lever, J. R. Uptake of 3-hydroxykynurenine measured in rat brain slices and in neuronal cell line. Brain Res. 584:110–116; 1992. 8. Fukui, S.; Schwarcz, R.; Rapoport, S. I.; Takadda, Y.; Smith, Q. R. Blood–brain transport of kynurenines: Implications for brain synthesis and metabolism. J. Neurochem. 56:2007–2017; 1991. 9. Gal, E. M.; Sherman, A. D. L-kynurenine: Its synthesis and possible regulatory function in brain. Neurochem. Res. 5:223–239; 1980. 10. Giles, A. R. Guidelines for the use of animals in biomedial research. Thromb. Haemost. 58:1078–1084; 1987. 11. Gin, H. E. Neurobehavioral dysfunction in uremia. Kidney Int. Suppl. 2:217–221; 1975. 12. Green, A. R.; Curzon, G. The effect of tryptophan metabolites on brain 5-hydroxytryptamine metabolism. Biochem. Pharamcol. 19:2061– 2068; 1970. 13. Guidetti, P.; Eastman, C. L.; Schwarcz, R. Metabolism of [5-3 H]kynurenine in the rat brain in vivo: Evidence for the existence of a functional kynurenine pathway. J. Neurochem. 65:2621–2632; 1995. 14. Hegedus, Z. L.; Frank, H. A.; Steinman, T. I.; Nayak, U. Dialysis of plasma soluble lipofucsins in patients with end-stage renal failure. Arch. Int. Physiol. Biochim. Biophys. 99:355–363; 1999. 15. Hever, C.; Beyne, P.; Jamault, H.; Delacoux, E. Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection. J. Chromatogr. 675:157–161; 1996. 16. Heyes, M. P.; Quearry, B. J. Quantification of 3-hydroxykynurenine in brain by high-performance liquid chromatography and electrochemical detection. J. Chromatogr. 428:340–344; 1988. 17. Holmes, E. W.; Kahan, S. E. Tryptophan distribution and metabolism in experimental chronic renal insufficiency. Exp. Mol. Pathol. 46:89– 101; 1987. 18. Holmes, E. W. Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography. Anal. Biochem. 172:518–525; 1988. 19. Jeppson, B.; Freund, H. R.; Gimmon, M. D.; James, J. H.; von Meyenfeldt, M. F.; Fischer, J. E. Blood–brain derangement in uremic nephropathy. Surgery 92:30–35; 1982. 20. Lapin, I. P. Consultant action of intracerebroventriculary administrated L-kynurenine sulphate, quinolinic acid and other derivatives of succinic acid, and effect of amino acid: Structure–activity relationships. Neuropharmacology 21:1227–1233; 1982. 21. Michalak, A.; Chatauret, N.; Butterworth, R. F. Evidence for a serotonin transporter deficit in experimental acute liver failure. Neurochem. Int. 38:163–168; 2001. 22. Moe, S. M.; Sprague, S. M. Uremic encephalophaty. Clin. Nephrol. 42:251–256; 1994. 23. Moller, S. E. Pharmacokinetics of tryptophan, renal handling of kynurenine and the effect of nicotinamide on its appearance in plasma and urine following l-tryptophan loading of healthy subjects. Eur. J. Clin. Pharmacol. 21:137–142; 1981. 24. Okuda, S.; Nishiyama, N.; Saito, H.; Katsuki, H. 3-Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity. J. Neurochem. 70:299–307; 1998. 25. Okuda, S.; Nishiyama, N.; Saito, H.; Katsuki, H. Hydrogen peroxidemediated neuronal cell death by an endogenous mediated neurotoxin, 3-hydroxykynurenine. Proc. Natl. Acad. Sci. U.S.A. 93:12553–12558; 1996. 26. Ormrod, D.; Miller, T. Experimental uremia. Nephron 26:249–254; 1980.
428 27. Park, J. W.; Youn, Y. C.; Kwon, O. S.; Jang, Y. Y.; Han, E. S.; Lee, C. S. Protective effect of serotonin on 6-hydroxydopamineand dopamine-induced oxidative damage of brain mitochondria and synaptosomes and PC12 cells. Neurochem. Int. 40:223–233; 2002. 28. Pawlak, D.; Tankiewicz, A.; Mysliwiec, P.; Buczko, W. Tryptophan metabolism via kynurenine pathway in experimental chronic renal failure. Nephron 90:328–335; 2002. 29. Pawlak, D.; Tankiewicz, A.; Buczko, W. Kynurenine and its metabolites in the rat with experimental renal insufficiency. J. Physiol. Pharmacol. 52:755–766; 2001. 30. Pearson, S. J.; Reynolds, G. P. Increased brain concentration of neurotoxin, 3-hydroxynurenine, in Huntington’s disease. Nuerosci. Lett. 144:199–201; 1992. 31. Saito, K.; Fujigaki, S.; Heyes, M. P.; Shibata, K.; Takemura, M.; Fuji, H.; Wada, H.; Noma, A.; Seishima, M. Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency. Am. J. Physiol. Renal Physiol. 279:F565–F572; 2000. 32. Saito, K.; Nowak, T. S.; Markey, S. P.; Heyes, M. P. Mechanism of delayed increases in kynurenine pathway metabolism in damaged brain regions following transient cerebral ischemia. J. Neurochem. 60:180– 192; 1993.
TOPCZEWSKA-BRUNS ET AL. 33. Speciale, C.; Hares, K.; Schwarcz, R.; Brookes, N. High-affinity uptake of L-kynurenine by a Na+ -independent transporter of neutral amino acids in astrocytes. J. Neurosci. 9:2066–2072; 1989. 34. Speciel, C.; Schwarcz, R. Uptake of kynurenine into rat brain slices. J. Neurochem. 54:156–163; 1990. 35. Stone, T. W.; Connick, J. H. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol. Rev. 45:309–379; 1993. 36. Stone, T. W. Kynurenines in the CNS: From endogenous obscurity to therapeutic importance. Prog. Neurobiol. 64:633–645; 2001. 37. Stone, T. W. Kynurenic acid antagonists and kynurenine pathway inhibitors. Exp. Opin. Invest. Drugs 10:633–645; 2001. 38. Swenseid, M. E.; Wang, M.; Vyhmeister, I.; Chan, W.; Siassi, F.; Tam, C. F.; Kopple, J. D. Amino acid metabolism in the chronically uremic rat. Clin. Nephrol. 3:240–246; 1975. 39. Topczewska-Bruns, J.; Tankiewicz, A.; Pawlak, D.; Buczko, W. Behavioral changes in the course of chronic renal insufficiency in rats. Pol. J. Pharamcol. 53:263–269; 2001. 40. Vanholder, R.; De Smet, R. Pathophysiologic effects of uremic retention solutes. J. Am. Soc. Nephrol. 10:1815–1823; 1999. 41. Yuwiler, A.; Oldendorf, E.; Geller, E.; Braun, L. Effect of albumin binding and amino acid competition on tryptophan uptake into brain. J. Neurochem. 28:1015–1023; 1977.