accompanied by increase activity of liver tryptophan 2,3-dioxygenase, the rate- limiting enzyme of kynurenine pathway in rats, while indoleamine 2,3- ...
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2003, 54, 2, 175189 www.jpp.krakow.pl
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 Bia³ystok, Bia³ystok, 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
conclude
activity
that
kynurenine
kynurenine renal
metabolites,
uremic syndromes.
Key
of
chronic
3-hydroxylase
failure
which
may
is
be
was
associated involved
significantly
with
in
the
the
increased.
accumulation
pathogenesis
of
of
We
L-
certain
w o r d s : 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
176
L-TRYPTOPHAN [-44.1± 4.3%] TDO [+427.7±37.2%]
IDO [+9.6±1.0%]
KAT [-62.1±5.7%]
KYNURENIC ACID
KZ [-49.8±4.0%]
L-KYNURENINE
ANTRANILIC ACID
[+72.5±4.6%]
[+245.6±31.8%]
[+579.1±68.5%] HK [+53.5±4.0%]
3-HYDROXYKYNURENINE [+261.0±24.9%] KAT [-62.1±5.7%]
HAO [-52.8±6.6%]
XANTHURENIC ACID
QUINOLINIC ACID
[+274.6±34.4%]
[+200.7±24.5%]
Fig. 1. Scheme of kynurenine pathway. TDO - tryptophan 2,3-dioxygenase, IDO - indoleamine 2,3dioxygenase,
KAT
hydroxylase,
HAO
-
kynurenine
aminotransferase,
KZ
-
kynureninase,
3-hydroxyanthranilate-3,4-dioxygenase.
The
total
HK
-
kynurenine
conentration
of
3-
TRP
metabolites and activity of kynurenic pathway enzymes was prsented (bracketedes). Details are given in the text.
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). 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).
177
There is accumulating evidence suggesting that disturbances in kynurenine pathway of TRP degradation in uremia might have clinical relevance. It has been demonstrated excitotoxins
that by
in
its
central
action
nervous
as
system
endogenous
QA
may
agonist
of
favor
the
effects
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 dihydrogen phosphate, tri-sodium citrate di-hydrate, tri-chloric acid were obtained from Merck, Germany;
zinc
kynurenic
acid,
acetate,
potassium
phosphate,
3-hydroxykynurenine,
tri-ethylamine,
anthranilic
acid,
L-tryptophan,
xanthurenic
acid,
L-kynurenine,
quinolinic
amid,
methylene blue, catalase, ascorbic acid, sucrose, met-hemoglobin, tri-chloroacetic acid, pyridoxal phosphate,
α-ketoglutarate,
glucose-6-phosphate
Tris-HCl buffer, magnesium chloride (MgCl2), 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
178
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 shamoperated 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
µl of substrate solution (100 mM µM methylene blue, 10 µg catalase, 50 mM ascorbic acid
of tissue homogenate supernatant and 50
potassium phosphate buffer (pH 6.5), 50
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) trichloroacetic 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.
179
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
µl of substrate solution µM pyridoxal phosphate, 20 mM α-
of tissue homogenate supernatant and 50
containing 200 mM potassium phosphate buffer (pH 8.0), 200
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 µM pyridoxal phosphate and 3.0 mM KYN. The reaction was
mM Tris-HCl buffer (pH 8.0), 100
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 containing
100
mM
µl
of tissue homogenate supernatant and 50
potassium
phosphate
buffer
(pH
7.5),
4
mM
µl
of substrate solution
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
containing 100 mM MES buffer (pH 6.5), 10
µM
µl
of substrate solution
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).
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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). 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. 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) 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).
Table 1. The effect of experimental chronic renal failure of various severity (CRF 1-3) 1. The effect of experimental chronic renal failure of various severity (CRF 1-3) on on biochemical parameters.
Table
biochemical parameters.
creatinine [mg/dl] urea [mg/dl] albumin [g/dl]
CON
CRF 1
CRF 2
CRF 3
0.35±0.04 22.8±0.6 3.8±0.2
0.72±0.08 ** 73.2±6.6 * 3.5±0.5
1.1±0.09 *** 128.4±18.0 ** 3.2±0.4
3.3±0.2 *** 501.6±39.0 *** 2.7±0.4 *
Values are presented as means ± SEM, n = 8-10. Statistical significance vs control group: *p
