Original article

D. Pawlak, A. Tankiewicz, T. Matys, W. Buczko


PERIPHERAL DISTRIBUTION OF KYNURENINE METABOLITES AND ACTIVITY OF KYNURENINE PATHWAY ENZYMES IN RENAL FAILURE


Department of Pharmacodynamics, Medical Academy of Bialystok, Bialystok, Poland


  We investigated L-kynurenine distribution and metabolism in rats with experimental chronic renal failure of various severity, induced by unilateral nephrectomy and partial removal of contralateral kidney cortex. In animals with renal insufficiency the plasma concentration and the content of L-tryptophan in homogenates of kidney, liver, lung, intestine and spleen were significantly decreased. These changes were accompanied by increase activity of liver tryptophan 2,3-dioxygenase, the rate-limiting enzyme of kynurenine pathway in rats, while indoleamine 2,3-dioxygenase activity was unchanged. Conversely, the plasma concentration and tissue content of L-kynurenine, 3-hydroxykynurenine, and anthranilic, kynurenic, xanthurenic and quinolinic acids in the kidney, liver, lung, intestine, spleen and muscles were increased. The accumulation of L-kynurenine and the products of its degradation was proportional to the severity of renal failure and correlated with the concentration of renal insufficiency marker, creatinine. Kynurenine aminotransferase, kynureninase and 3-hydroxyanthranilate-3,4-dioxygenase activity was diminished or unchanged, while the activity of kynurenine 3-hydroxylase was significantly increased. We conclude that chronic renal failure is associated with the accumulation of L-kynurenine metabolites, which may be involved in the pathogenesis of certain uremic syndromes.

Key words: L-kynurenine metabolites, experimental uremia, rats



INTRODUCTION

The main product of L-tryptophan (TRP) kynurenine pathway degradation in peripheral tissues is L-kynurenine (KYN), which is further converted to a series of metabolites, such as 3-hydroxykynurenine (3-HKYN), and anthranilic (AA), kynurenic (KYNA), xanthurenic (XA) and quinolinic (QA) acids (Fig.1). The first step of TRP catabolism is catalyzed by two distinct enzymes, tryptophan 2,3-dioxygenase (TDO, EC 1.13.11.11) and indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.42), which vary in distribution, substrates affinity, and inducing factors (1). Both TDO and IDO lead to oxidative cleavage of tryptophan pyrrole ring resulting in formation of N-formylkynurenine, which is subsequently converted to KYN (2,3). Depending on the content and activity of enzymes in individual organs, KYN can be further metabolized via three distinct pathways - to KYNA by kynurenine aminotransferase (KAT, EC 2.6.1.7), to 3-HKYN by kynurenine 3-hydroxylase (HK, EC 1.14.13) and to AA by kynureninase (KZ, EC 3.7.1.3) (4).

Fig. 1. Scheme of kynurenine pathway. TDO - tryptophan 2,3-dioxygenase, IDO - indoleamine 2,3-dioxygenase, KAT - kynurenine aminotransferase, KZ - kynureninase, HK - kynurenine 3-hydroxylase, HAO - 3-hydroxyanthranilate-3,4-dioxygenase. The total conentration of TRP metabolites and activity of kynurenic pathway enzymes was prsented (bracketedes). Details are given in the text.

The main route of elimination of KYN and its metabolites is renal excretion (5). In addition, kidney is able to uptake KYN and 3-HKYN from the blood, which are metabolized and excreted in the form of KYNA and XA, respectively (6). Thus, the impairment of kidney function is likely to be associated with the retention of KYN and its metabolites. Indeed, abnormalities in TRP metabolism, such as a decrease in serum TRP concentration with increased levels of KYN have been reported in humans and rats with chronic renal insufficiency (7-9).

There is accumulating evidence suggesting that disturbances in kynurenine pathway of TRP degradation in uremia might have clinical relevance. It has been demonstrated that in central nervous system QA may favor the effects of excitotoxins by its action as endogenous agonist of N-methyl-D-aspartate (NMDA) receptor (4) and cause neuronal death by generation of reactive oxygen species (10). Niwa et al. (11) showed that QA is able to penetrate into brain and evoke seizures, convulsions and muscle cramps. Apart from their actions in the central nervous system, KYN metabolites exert a number of disadvantageous peripheral effects. For example, QA has been shown to inhibit gluconeogenesis (4), erythropoiesis (12) and lymphocyte blast formation (13); therefore, QA accumulation might be related to cellular metabolism disturbances, anemia and immunosuppression observed in uremia. Garacia et al. (14) have proposed that also XA, due to its hydrophilic properties and binding to erythrocyte membrane, could be involved in the pathogenesis of anemia. In contrast, KYNA appears to be beneficial both in central nervous system by blocking NMDA receptor and, in peripheral tissues, by its action on mitochondria, resulting in improvement of respiratory parameters and cellular alkalosis (15).

The above data suggests that exploration of KYN metabolism could help to explain the pathogenesis of certain uremic symptoms. However, products of KYN degradation have been evaluated so far only in blood, cerebrospinal fluid and brain (9,16). In the present study we aimed to evaluate distribution of KYN and its metabolites in plasma and in peripheral tissues (kidney, liver, lung, intestine, spleen and muscles) as well as to assess the activity of kynurenine pathway enzymes in rats with chronic renal failure.


MATERIAL AND METHODS

Chemicals

All the chemicals use in the study were of analytical grade. Ammonium acetate, acetic acid, acetonitrile, phosphoric acid, ethylene-di-nitrilo-tetra-acetic acid di-sodium salt di-hydrate (EDTA), heptane-1-sulfonic acid sodium salt, di-potassium hydrogen phosphate, potassium di-hydrogen phosphate, tri-sodium citrate di-hydrate, tri-chloric acid were obtained from Merck, Germany; zinc acetate, potassium phosphate, tri-ethylamine, L-tryptophan, L-kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid, xanthurenic acid, quinolinic amid, methylene blue, catalase, ascorbic acid, sucrose, met-hemoglobin, tri-chloroacetic acid, pyridoxal phosphate, alpha-ketoglutarate, Tris-HCl buffer, magnesium chloride (MgCl2), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, (NADP), 2-morpholinoethansulfonic amid (MES), ferric sulfate (Fe2(SO4)3), were purchased from Sigma, USA. Sodium pentobarbital and thrombin were from Biovet, Poland.

Animals

The study was performed on male Wistar rats weighing 180-240 g. The animals were housed in group cages as appropriate, in a 12:12 hour light-dark cycle and controlled temperature (20°C) and humidity conditions. Standard rat chow (LSM - total protein 15.9%) and tap water were available ad libitum.

Experimental model of uremia

Chronic renal failure (CRF) was induced in pentobarbital - anaesthetized (40 mg/kg, i.p.) rats by a partial resection of the renal tissue according to Ormrod and Miller (17). Three different levels of the CRF were induced, further referred to as CRF 1, 2 and 3. Induction of CRF1 (moderate CRF) was performed by a total removal of the left kidney and 60% of the right kidney cortex; then the animals were left for one month to allow the development of renal insufficiency. Two weeks after the surgery, a group of the rats subjected to the above procedure were re-operated and additional 20% of the right kidney cortex was removed; then the animals were allowed to develop chronic renal insufficiency for one month (CRF2) or two months (CRF3) after the second surgery. In sham-operated rats (control group) only the surgical extraction of the renal capsule was performed.

Blood and tissues sampling

The animals were anaesthetized with pentobarbital (40 mg/kg i.p.), the blood was drawn by heart puncture and collected into a tube containing 3.13% sodium citrate (citrate/blood ratio = 1:9). The plasma was obtained by centrifugation of the blood at 5000 x g for 15 min at 4°C and was stored at -80°C until assayed. After exsanguination, kidney, liver, spleen, lungs, intestine and muscle samples were removed and cut on ice into slices weighing 100-200 mg. Samples were homogenized in ice-cold homogenization buffer (140 mM potassium chloride/20 mM potassium phosphate, pH 7.0; 0.5ml per 100 mg of tissue). Homogenates were sonicated, centrifuged at 12000 × g for 30 min at 4°C and the supernatant was collected. For kynurenine 3-hydroxylase activity measurement, the tissues were homogenized in 10 volumes of ice-cold 0.32 M sucrose. Homogenates were centrifuged at 12000 × g for 30 min. at 4°C and the pellet was washed three times with 0.32 M sucrose by centrifugation. The pellet was finally resuspended in ice-cold 140 mM potassium chloride/20 mM potassium phosphate buffer (pH 7.0) and sonicated.

The activity of enzymes was expressed as pmol of product formed per hour per gram of tissue. Tissues for HPLC analysis were homogenized in 20% tri-chloroacetic acid (50 mg/0.25ml acid) in ice-cold coat and centrifuged at 14000 × g for 60 min.

Assay of indoleamine 2,3-dioxygenase (IDO) activity

The activity of IDO was quantified by conversion of TRP to KYN (18). The reaction mixture consisted of 50 µl of tissue homogenate supernatant and 50 µl of substrate solution (100 mM potassium phosphate buffer (pH 6.5), 50 µM methylene blue, 10 µg catalase, 50 mM ascorbic acid and 3 mM TRP). The samples were incubated at 37°C while shaking at 100 strokes/min. The enzymatic reaction was terminated after 60 min by the addition of 0.1 ml of 20% (w/v) tri-chloroacetic acid, and the concentration of KYN was measured.

Assay of tryptophan 2,3-dioxygenase (TDO) activity

The activity of TDO was measured according to the method described by Salter et al. (19). The tissue homogenate supernatant was incubated for 60 min at 37°C while shaking at 100 strokes/min in 200 mM potassium phosphate buffer (pH 7.0), 0.136 mg/ml methemoglobin and 3 mM TRP. Reaction was stopped by addition of 0.1 ml of 20% (w/v) tri-chloroacetic acid and the concentration of KYN was measured.

Assay of kynurenine aminotransferase (KAT) activity

The activity of KAT was measured by the conversion of KYN to KYNA (18). The reaction mixture consisted of 50 µl of tissue homogenate supernatant and 50 µl of substrate solution containing 200 mM potassium phosphate buffer (pH 8.0), 200 mM pyridoxal phosphate, 20 mM alpha-ketoglutarate and 3 mM KYN. The reaction was terminated after 60 min by the addition of 0.1 ml of 20% (w/v) tri-chloroacetic acid, and the concentration of KYNA was quantified.

Assay of kynureninase (KZ) activity

The activity of KZ was measured by the conversion of KYN to AA (18). The reaction mixture consisted of 50 µl of tissue homogenate supernatant, and 50 µl of substrate solution containing 200 mM Tris-HCl buffer (pH 8.0), 100 mM pyridoxal phosphate and 3.0 mM KYN. The reaction was terminated after 30 min by the addition of 0.1 ml of 20% (w/v) tri-chloroacetic acid, and the concentration of AA was quantified.

Assay of kynurenine 3-hydroxylase (HK) activity

The activity of HK was measured by the conversion of KYN to 3-HKYN (18). The reaction mixture consisted of 50 µl of tissue homogenate supernatant and 50 µl of substrate solution containing 100 mM potassium phosphate buffer (pH 7.5), 4 mM MgCl2, 3 mM glucose-6-phosphate, 0.4U of glucose-6-phosphate dehydrogenase, 0.8 mM NADP, and 3.0 mM KYN. After 5 min the reaction was terminated by the addition of 0.1 ml of 20% (w/v) tri-chloroacetic acid, and the concentration of 3-HKYN was quantified.

Assay of 3-hydroxyanthranilate-3,4-dioxygenase (HAO) activity

The activity of HAO was measured by the conversion of 3-HAO to QA (18). The reaction mixture consisted of 50 µl of tissue homogenate supernatant and 50 µl of substrate solution containing 100 mM MES buffer (pH 6.5), 10 µM ascorbate, 6 mM Fe2(SO4)3, and 3 mM 3-HAA. After 60 min of incubation the reaction was terminated by fast cooling of the mixture to 4°C and the concentration of QA was quantified.

Determination of tryptophan and its metabolites concentrations

The concentrations of TRP and its metabolites were determined by high-performance liquid chromatography (HPLC), using fluorescence (TRP, KYNA and AA), electrochemical (3-HKYN) or UV (QA) detection as previously described (7,8).

Statistical analysis

The values are expressed as the mean ± standard error mean (SEM); n - represents the number of experiments. Multiple groups comparisons were performed by one-way analysis of variance (ANOVA), and differences between groups were estimated with Student t or Tukey-Kramer test. P value less than 0.05 was considered statistically significant.

Ethics

The study was approved by the Local Ethical Committee as being in accordance with the institutional guidelines for the care and use of research animals, which comply with national, and international laws and Guidelines for the Use of Animals in Biomedical Research (20).


RESULTS

To estimate the effectiveness of surgical uremia induction, we measured the concentration of widely used renal insufficiency markers, creatinine and urea. We found that in the animals in which the mass of the renal cortex was diminished, the level of both creatinine and urea was significantly increased in comparison to control animals and that the changes were proportional to the supposed severity of renal failure, thus confirming the efficacy of the surgical CRF induction (Tab. 1).

Table 1. The effect of experimental chronic renal failure of various severity (CRF 1-3) on biochemical parameters.
Values are presented as means ± SEM, n = 8-10. Statistical significance vs control group: *p<0.05, **p<0.01, ***p<0.001.

The plasma concentration of TRP in the uremic animals (Tab.2) was significantly lower than in control rats and this decrease was dependent on the severity of uremia. The content of this amino acid in animals with CRF2 and CRF3 was also significantly decreased in all tested tissues except for muscles; the most pronounced changes were observed in kidneys and the intestine.

Table 2. Plasma and tissues concentrations of L-kynurenine metabolites.
Values are presented as means ± SEM, n = 8-10. Statistical significance vs control group: *p<0.05, **p<0.01, ***p<0.001. TRP - tryptophan, KYN - kynurenine, KAT - kynurenic acid, AA - anthranilic acid, 3-HKYN - 3-hydroxykynurenine, XA - xanthurenic acid, QA - quinolinic acid

Analysis of total TRP degradation through the kynurenine pathway (plasma and tissues) demonstrated that in animals with CRF3 the concentration of this aminoacid was decreased by 44.1±4.3% in comparison with control rats (Fig. 1). These changes were accompanied by significant increase in the activity of tryptophan 2,3-dioxigenase (TDO) in the liver (427.7±37.2%), while the activity of indoleamine 2,3-dioxygenase, which is present in extrahepatic tissues, remained unchanged (Tab.3)

Table 3. Activity of kynurenine pathway enzymes in chronic renal failures.
Values are presented as means ± SEM, n = 8-10. Statistical significance vs control group: *p<0.05, **p<0.01, ***p<0.001. TDO - tryptophan 2,3-dioxygenase, IDO - indoleamine 2,3-dioxygenase, KAT - kynurenine aminotransferase, KZ - kynureninase, HK - kynurenine 3-hydroxylase, HAO - 3-hydroxyanthranilate-3,4-dioxygenase.

In contrast to TRP, the concentration of KYN in the plasma and examined tissues was increased (Tab. 2). We did not observe any correlation between the increase in the plasma KYN concentration and the stage of the renal insufficiency. The total content of KYN in animals with CRF3 was increased by 72.5±4.6% (Fig. 1).

The plasma and tissues concentration of KYNA in CRF2 and CRF3 was also significantly increased in proportion to the severity of renal failure (Tab. 2). Total body content of KYNA in rats with CRF3 was increased by 245.6±31.8% (Fig.1), while activity of kynurenine aminotransferase (KAT) - the enzyme that produces KYNA from KYN - decreased by 62.1±5.7%. To examine if this decrease in KAT activity could be due to the increase in its product concentration, we performed in vitro experiments in which we added KYNA to homogenate of kidney obtained from intact rat. Indeed, in the presence of KYNA (0.1 and 1 µM), the activity of KAT was inhibited from 4483.8±145.8 nmol/h/g to 3184.3±210.9 and 2872.5±207.4 nmol/h/g, respectively (Tab. 4).

Similarly as with KYNA, rats with CRF had significantly increased plasma and tissues concentrations of AA, which was dependent on the severity of renal insufficiency (Tab. 2). The increase of total body content of AA in animals with CRF3 reached 579.1±68.5% of the control value (Fig. 1). The activity of enzyme that converts KYN to AA - kynureninase (KZ) - was inhibited by 49.8±4.0% (Tab. 3). Incubation of intact rat liver homogenate with 1 µM AA inhibited KZ activity from 1128.9±102.5 to 635.1±94.0 nmol/h/g (Tab. 4).

Table 4. The influence of products of L-kynurenine degradation on enzymes activity.
Values are presented as mean ± SEM, n = 6-10. Statistical significance vs control group: **p<0.01, ***p<0.001. KAT - kynurenine aminotransferase, KZ - kynureninase, HAO - 3-hydroxyanthranilate-3,4-dioxygenase

Increased concentration of KYN in animals with CRF was also accompanied by an increase in the plasma and tissue concentration of 3-HKYN in proportion to the severity of renal insufficiency (Tab. 2). The most pronounced changes in 3-HKYN concentration were observed in kidneys and the liver. Total body content of 3-HKYN in animals with CRF3 was increased by 261.0±24.9% (Fig. 1), which was accompanied by increased activity of the enzyme responsible for the conversion of KYN to 3-HKYN - kunurenine-3-hydroxylase (HK) - by 53.5±4.0% (Tab. 3).

We also examined the metabolism of 3-HKYN metabolites, i.e. XA and QA. Plasma and tissues concentrations of both these substances in uremic rats were increased in proportion to the stage of the renal failure (Tab. 2). Similarly like in the case of 3-HKYN, XA concentration correlated with plasma creatinine level. The increase in the total body content of XA and QA in animals with the most severe renal insufficiency (CRF3) reached 274.6±34.4% and 200.7±24.5% of the control values, respectively (Fig. 1). Changes in the activity of KAT, the enzyme catalyzing the conversion of 3-HKYN to XA (as well as KYN to KYNA) has been described above. The activity of 3-hydroxyanthranilate-3,4-dioxygenase (HAO), which converts 3-HKYN to QA, was decreased in CRF3 by 52.8±6.6% (Tab. 3). However, in contrast to the other enzymes of kynurenine pathway assessed in this study, the presence of QA did not influence HAO activity in intact rat liver homogenate (Tab. 4).


DISCUSSION

In the present study, we used a well established model of CRF in rats to assess changes in kynurenine pathway of TRP metabolism associated with renal function impairment. We observed significant decrease in the concentration of TRP in uremic animals that was accompanied by an accumulation of KYN and a series of its metabolites, i.e., KYNA, AA, 3-HKYN, XA and QA in plasma and peripheral tissues. These changes were proportional to the severity of the renal function impairment and correlated with the concentration of creatinine, a marker of decreased glomelural filtration rate and kidney excretory function.

The decrease in total TRP content in rats with CRF3 reached approximately 40%. This result is in line with other reports, which demonstrated similar changes in the concentration of this amino acid in the blood of human patients with uremia (21,22). Several putative mechanisms of this aminoacidopathy have been proposed, including diminished intake of TRP with food (23), transformation of TRP in bowel epithelium to other indoles and diminished reabsorption of this compound in renal tubules (24). However, there is accumulating evidence that the reduction of plasma TRP level and simultaneous increase in the tissue KYN concentration in CRF could be also a result of induction of enzymes involved in TRP degradation.

Our studies demonstrate that CRF is associated with increased activity of TDO in the liver (over 4-fold increase in the animals with CRF3 in comparison with controls), while IDO activity in extrahepatic tissues remains unchanged. The studies on the role of both these enzymes in TRP metabolism provide conflicting results. Initially it was supposed that in pathological conditions the main enzyme responsible for the conversion of TRP to KYN is IDO, while the role of TDO was restricted to physiological situations. However, studies performed by Saito et al. (9) indicate that chronic renal insufficiency leads to increased activity of TDO, while IDO seems to play only a minor role in these conditions. According to this study, the key factor in TDO induction could be glucagone, which level increases in course of uremia and which acts as an endogenous activator of TDO. Another putative mechanism of this induction of TDO activity could be increased concentration of glucocorticosteroids, which activate this enzyme by promoting mRNA synthesis. On the other hand, glucocorticosteroids inhibit the synthesis of certain cytokines, mainly Il-1 and Il-2, which are important inducers of IDO (25).

As mentioned above, TDO and IDO significantly vary in distribution and substrate affinity. Studies of Taylor and Feng (26), as well as Thomas et al. (27) demonstrate the requirement of TDO for oxygen (O2). In contrast, there is a requirement of IDO for superoxide (O2·-), which is derived mainly from neutrophils and monocytes. Since uremia leads to dysfunction of these cells in terms of chemotaxis, phagocytosis and O2·- synthesis (28), the lack of changes in IDO activity in our studies could be due to diminished bioavailability of superoxide. Thus, it cannot be excluded that the surgery itself could lead to enhanced synthesis of cytokines, which in turn could activate IDO and nitric oxide synthase (29). As shown by Thomas and Stocker (30) nitric oxide (NO) can inhibit IDO activity in two distinct manners. First, NO easily binds to hem iron in IDO moiety, preventing it from being oxidized to the active form. Second, NO can react with superoxide to form peroxynitrite (ONOO-); this reaction leads to depletion of O2·- and accumulation of ONOO-, which is devoid of the ability to induce IDO.

The increased catabolism of TRP in rats with chronic renal failure was accompanied by accumulation of its metabolite, KYN, in plasma and examined tissues. Total body content of KYN increased by 72%. Saito et al. (9) propose that the accumulation of KYN could be related to enhanced synthesis and/or decreased degradation of this compound and not to diminished excretion. However, the increase of KYN concentration can possibly exacerbate kidney damage, inducing pathological mitosis and enhanced apoptosis in kidney epithelial cells, impairing epithelial and endothelial cells function and promoting leading to glomerular membrano-proliferative nephropathy (31-33).

It is known, that KYN can be metabolized via three pathways (Fig. 1) - kynurenine aminotransferase (KAT) converts KYN to kynurenine acid (KYNA), kynureninase to anthranilic acid (AA) and kynurenine 3-hydroxylase to 3-hydroxykynurenie (HK).

In our study we observed a marked accumulation of these metabolites. KYNA concentration was increased in plasma and all examined tissues, with the highest level present in the kidney. Total body content of KYNA in rats with CRF3 increased by 246% in comparison to the control animals. One possible explanation of these changes could be enhanced conversion of KYN to KYNA due to increased activity of KAT. However, we instead observed a decrease of KAT activity in the kidney that was proportional to the severity of renal insufficiency, while in other examined organs the activity of this enzyme remained unchanged. In the in vitro assay (Table 4) we observed inhibition of KAT activity in kidney homogenate by KYNA, which provides evidence that the inhibition of KAT could be due to accumulation of its product. Thus, it is most likely that the increase in KYNA concentration was not caused by enhanced biosynthesis of this compound, but probably by impaired excretion.

Similarly as in the case of KYNA, the concentration of AA was increased in the plasma and tissues and the total body content of this compound in the animals with CRF3 was increased by nearly 590%. The activity of enzyme catalyzing the conversion of KYN to AA - kynureninase (KZ) was decreased in proportion to the severity of renal failure. In the rats with CRF3 total activity of KZ was diminished by 62% and the most pronounced inhibition was observed in kidney and liver. To examine the mechanism of this inhibition we incubated liver homogenate in the presence of AA and we demonstrated that KZ activity can be diminished by its product in a concentration-dependent manner. Another reason of KZ inhibition could also be the deficiency of its cofactor - vitamin B6 (32), as well as the presence of other toxins that inhibit this enzyme.

Our experiments also provide evidence for accumulation of 3-HKYN, which concentration in the plasma and examined tissues was markedly increased and the total body content of this compound in the animals with the most sever uremia was over 2.5-fold higher than in control rats. However, opposite to KYNA and AA, the mechanism of 3-HKYN accumulation seems to be associated not only with impaired excretion, but also with increased synthesis of this compound by HK, which activity in kidneys and the liver was significantly increased. Induction of HK activity correlated with concentrations of creatinine and urea and in the animals with the most pronounced renal failure the total activity of this enzyme increased by 53%. The mechanism of HK induction remains to be elucidated. One possibility could be deficiency of vitamin B6 in course of uremia (32). Since vitamin B6 inhibits most of NADPH-dependent enzymes, including HK (34), lack of this inhibitory action would lead to enhanced activity of the enzyme. This hypothesis is supported by the observation that HK activity was mainly induced in kidneys and the liver, which have high activity of NAD synthetase (35,36). Abundance of NAD provides optimal conditions for HK, which requires reduced form of NAD - NADPH - as electron donor.

We also examined further conversion of 3-HKYN to XA (catalyzed by KAT) and QA (two-step reaction catalyzed by KZ and HAO). As expected, concentration of XA in the plasma and examined tissues was significantly increased, with the most pronounced changes in the kidney. The increase of the total body content of XA in rats with CRF3 reached 275%. The high concentration of XA in the kidney can be explained by the high concentration of 3-HKYN, being the substrate for XA biosynthesis. Similarly, we observed accumulation of another product of 3-HKYN metabolism, QA, in the plasma and all examined tissues, excluding skeletal muscles. The total body content of QA in animals with the most severe stage of uremia increased 2-fold.

Degradation of 3-HKYN involves the same enzymes that catalyze the degradation of KYN, i.e. KAT (converting 3-HKYN to XA) and KZ, which catalyzes the first step of conversion of 3-HKYN to QA (synthesis of 3-hydroxyanthranilic acid, 3-HAA). As mentioned above, activity of both this enzyme was decreased. The second step of QA biosynthesis, is catalyzed by HAO, which converts 3-HAA to 2-amino-3-carboxymuconic semialdehyde; the latter forms QA in spontaneous cyclization reaction (4,9). In our experiments, the activity of HAO was decreased activity in proportion to the severity of renal failure. The most pronounced inhibition of HAO activity was observed in the liver, lungs and the intestine and the total activity of HAO in CRF3 decreased by 53%. In contrast to inhibition of KAT and KZ by their products, incubation of the liver homogenate with QA did not influence the activity of HAO. Thus, inhibition of HAO cannot be associated with a direct action of QA on the enzyme; however, it is likely that in vivo QA could potentially contribute to HAO inhibition in indirect manner, for example generating active forms of oxygen. It has been demonstrated that QA acts as an endogenous agonist of NMDA receptor, which activation results in increased intracellular calcium concentration and activation of proteolytic enzymes that degrade proteins of intracellular cytoskeleton and extracellular matrix (37). This is accompanied by activation of phospholipase A2 and cyclooxygenase and, as a consequence, the production of active oxygen forms which can damage cell membrane and intracellular organelles. Dang et al. demonstrated (38) that HAO requires the presence of molecular oxygen, while reactive oxygen species exert inhibitory action on this enzyme. Moreover, impairment of the function of mitochondria provides additional pool of reactive oxygen species and enhances inhibition of the enzyme. As suggested by Chiarugi and Moroni (39), HAO can be also inhibited by certain proteins that are present on the inner surface of mitochondrial membranes. Another mechanism of HAO inhibition in vivo could be associated with accumulation of uremic toxins, which can directly inhibit the enzyme or change its structure.

As reported by Saito et al. (9) the increase in serum QA concentrations in uremia could also be due to diminished activity of aminocarboxymuconate-semialdehyde decarboxylase (enzyme responsible for the degradation of QA via the glutarate pathway). Although in their study serum concentration of QA in CRF was elevated, renal clearance of this compound was only slightly decreased and the urinary excretion of QA was increased. The authors conclude that increased concentration of QA cannot be related to a decrease in renal excretion but to increased synthesis and decreased (9).

In conclusion, this study provides evidence for accumulation of KYN in plasma and peripheral tissues in course of chronic renal failure. Increased concentration of KYN is associated with accumulation of its metabolites. Since activity of most of the enzymes involved in degradation of KYN is decreased, accumulation of its metabolites is most likely due to impaired renal excretion of these compounds. Increased concentration of KYN degradation products might be an important factor in the pathogenesis of certain disturbances associated with renal insufficiency.


REFERENCES
  1. Saito K, Crowley JS, Markey SP, Heyes MP. A mechanism for increased quinolinic acid formation following acute systemic immune stimulation. J Biol Chem 1993; 268: 15496-15503.
  2. Ren S, Liu H, Licad E, Correia MA. Expression of rat liver tryptophan 2,3-dioxygenase in Escherichia coli: structural and functional characterization of purified enzyme. Arch Biochem Biophys 1996; 333: 96-102.
  3. Takikawa O, Yoshida R, Kido R, Hayaishi O. Tryptophan degradation in mice initiated by indoleamine 2,3-dioxygenase. J Biol Chem 1986; 261: 3648-3653.
  4. Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev 1993; 45: 309-379.
  5. Holmes EW. Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high liquid chromatography. Anal Biochem 1988; 172: 518-525.
  6. Takeuchi F, Tsubouchi R, Izuta S, Shibata Y. Kynurenine metabolism and xanthurenic acid formation in vitamin B6-deficient rat after tryptophan injection. J Nutr Sci Vitaminol 1989; 35: 111-122.
  7. Pawlak D, Tankiewicz A, Buczko W. Kynurenine and its metabolites in the rat with experimental renal insufficiency. J Physiol Pharmacol 2001; 52: 755-766.
  8. Pawlak D, Tankiewicz A, Myśliwiec P, Buczko W. Tryptophan metabolism via the kynurenine pathway in experimental chronic renal failure. Nephron 2002; 90: 328-335.
  9. Saito K, Fujigaki S, Heyes MP et al. Mechanism of increases in KYNurenine and quinolinic acid in renal insufficiency. Am J Physiol 2000; 279: F565-F572.
  10. Chiarugi A, Meli E, Moroni F. Similarities and differences in the neuronal death processes activated by 3OH-kynurenine and quinolinic acid. J Neurochem 2001; 77: 1310-1318.
  11. Niwa T, Yoshizumi H, Emoto Y, Miyazaki T, Hashimoto N, Takeda N, Tatematsu A, Maed K. Accumulation of quinolinic acid in uremic serum and its removal by hemodialysis. Clin Chem 1991; 37: 159-161.
  12. Pawlak D, Koda M, Pawlak S, Wolczynski S, Buczko W. Contribution of quinolinic acid in the development of anemia in renal insufficiency. Am J Physiol 2003; 284: 693-700.
  13. Kawashima Y, Sanaka T, Sugino N, Takahashi M, Mizoguchi H. Suppressive effect of quinolinic acid and hippuric acid on bone marrow erythroid growth and lymphocyte blast formation in uremia. Adv Exp Med Biol 1987; 223: 69-72.
  14. Gracia GE, Wirtz RA, Barr JR, Woolfitt A, Rosenberg R. Xanthurenic acid induces gametogenesis in plasmodium, the malaria parasite. J Biol Chem 1998; 273: 12003-12005.
  15. Baran H, Staniek K, Kepplinger B, Gille L, Stolze K, Nohl H. Kynurenic acid influence the respiratory parameters of rat heart mitochondria. Pharmacology 2001; 62: 119-123.
  16. Bell MJ, Kochanek PM, Heyes MP et al. Quinolinic acid in the cerebrospinal fluid of children after traumatic brain injury. Crit Care Med 1999; 27: 493-497.
  17. Ormrod D, Miller T. Experimental uremia. Nephron 1980; 26: 249-254.
  18. Saito K, Heyes MP. Kynurenine pathway enzymes in brain. Properties of enzymes and regulation of quinolinic acid synthesis. Recent Advanced in Tryptophan Research, Filipini GA et al (eds), New York, Plenum Press, 1996; 75: 485-492.
  19. Salter M, Hazelwood R, Pogson ChI, Iyer R, Madge DJ. The effect of a novel and selective inhibitor of tryptophan 2,3-dioxygenase on tryptophan and serotonin metabolism in the rat. Biochem Pharmacol 1995; 49: 1435-1442.
  20. Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost 1987; 58:1078-1084.
  21. Holmes EW, Russell PM, Kinzler GJ, Bermes EW. Inflammation-associated changes in the cellular availability of tryptophan and kynurenine in renal transplant recipients. Clin Chim Acta 1994; 227: 1-15.
  22. Holmes EW, Russell P, Kinzler GJ et al. Oxidative tryptophan metabolism in renal allograft recipients: increased kynurenine synthesis is associated with inflammation and OKT3 therapy. Cytokine 1992; 4: 205-213.
  23. Topczewska-Bruns J, Tankiewicz A, Pawlak D, Buczko W. Behavioral changes in the course of chronic renal insufficiency in rats. Pol J Pharmacol 2001; 53, 263-269.
  24. Niwa T. Organic acid and the uremic syndrome: protein metabolite hypothesis in the progression of chronic renal failure. Semin Nephrol 1996; 16: 167-182.
  25. Orlowski T. Przeszczepianie nerek. Warszawa, Wydawnictwo Lekarskie PZWL, 1995.
  26. Taylor MW, Feng G. Relationship between interferon-g, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J 1991; 5: 2516-2522.
  27. Thomas SR, Mohr D, Stocker R. Nitric oxide inhibits indoleamine 2,3-dioxygenase activity in interferon-g primed mononuclear phagocytes. J Biol Chem 1994; 269: 14457-14464.
  28. Cendoroglo M, Jaber BL, Balakrishnan VS, Perianayagam M, King AJ, Pereira BJG. Neutrophil apoptosis and dysfunction in uremia. J Am Soc Nephrol 1999; 10: 93-100.
  29. Kudoh A, Katagai H, Takazawa T, Matsuki A. Plasma proinflammatory cytokine response to surgical stress in elderly patients. Cytokine 2001; 15: 270-273.
  30. Thomas SR, Stocker R. Redox reactions related to indoleamine 2,3-dioxygenase and tryptophan metabolism along the kynurenine pathway. Redox Rep 1999; 4: 199-220.
  31. Martinsons A, Rudzite V, Bratslavska O, Saulite V. The influence of kynurenine and norepinephrine on tubular epithelial cells and alveolar fibroblasts. Adv Exp Med Biol 1999; 467: 347-352.
  32. Martinsons A, Rudzite V, Groma V, Bratslavska O, Widner B, Fuchs D. Kynurenine and neopterin in chronic glomerulonephritis. Adv Exp Med Biol 1999; 467: 72-77.
  33. Martinsons A, Rudzite V, Jurika E, Silava A. The relationship between kynurenine, catecholoamines, and arterial hypertension in mesangioproliferative glomerulonephritis. Adv Exp Med Biol 1996; 398: 417-419.
  34. Breton J, Avanzi N, Magagnin S et al. Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase. Eur J Biochem 2000; 267: 1092-1099.
  35. Ericson JB, Flanagan EM, Russo S, Reinhard JFJ. A radiometric assay for kynurenine 3-hydroxylase based on the release of 3H2O during hydroxylation of L-[3,5-3H]kynurenine. Anal Biochem 1992; 205: 257-262.
  36. Kawai J, Okuno E, Kido R. Organ distribution of rat kynureninase and changes of its activity during development. Enzyme 1988; 39: 181-189.
  37. Kristian T, Siesjo BK. Calcium in ischemic cell death. Stroke 1998; 29: 705-718.
  38. Dang Y, Xia C, Brown O. Effects of oxygen on 3-hydroxyanthranilate oxidase of the kynurenine pathway. Free Radic Biol Med 1998; 25: 1033-1043.
  39. Chiarugi A, Moroni F. Regulation of quinolinic acid synthesis by mitochondria and o-methoxybenzoylalanine. Adv Exp Med Biol 1999; 467: 233-239.

R e c e i v e d : February 1, 2003
A c c e p t e d : April 24, 2003
Dr Dariusz Pawlak, Department of Pharmacodynamics, Medical Academy, Mickiewicza Str. 2 C, 15-230 Białystok, Poland. Tel/ fax: +48/85/7485601.
e-mail: dariuszpawlak@poczta.onet.pl