KIDNEY AND LIVER KYNURENINE P ATHW A Y
ENZYMES IN CHRONIC RENAL FAILURE
Anna Tankiewicz·, Dariusz Pawlak, Joanna Topczewska-Bruns
and Wlodzimierz Buczko
It has been suggested that kynurenine pathway may be an important pathological
factor during the chronic renal failure (CRF) development. Therefore in the present study,
in rats with end-stage of chronic renal failure, we measured the plasma and tissues
(kidney and liver) concentrations of tryptophan, kynurenine, 3-hydroxykynurenine. We
also evaluated the activity of tryptophan 2,3-dioxygenase in the liver (TDO), indoleamine
2,3-dioxygenase in kidney (IDO) and kynurenine 3-hydroxylase (HK) in the liver and in
The plasma and tissues tryptophan concentrations were decreased, whereas the
concentrations of its metabolites increased when compared to control group. The increase
in the TDO and 3-HK activity was observed, while IDO activity remains unchanged.
In conclusion, the increase in the activity of TDO and HK along with disturbances of
renal excreting function may be responsible for the elevation in the kynurenine and 3-
hydroxykynurenine concentrations in experimental chronic renal failure.
The concentration of cytotoxic compounds, such as kynurenine and 3-
hyroxykynurenine, that are formed from tryptophan (TRP) increases significantly in the
plasma of patients with chronic renal failure (CRF) 1,2. It is speculated that these products
may contribute to the disturbances affecting numerous organs and regulatory systems,
including central nervous system functioning 3, Moreover, peripheral neuropathy,
increased susceptibility to infections, hypertension, lipid disturbances and anemia and are
observed in the uremic patients 4-6.
The catabolism of TRP is differently controlled during health and disease by two
distinct enzymes, namely tryptophan 2,3-dioxygenase (TDO, EC 22.214.171.124) and
• Deparlment of Pharmacodynamics, Medical Academy. Mickiewicza 2C. 15-230 Bialystok. Poland
Phone/Fax: 48 85 7421816 e-mail: aniatanw!.Ooczta.oneLpl
Developments in Tryptophan and Serotonin Metabolism, edited by Allegri et aJ.
Kluwer AcademicIPlenum Publishers, 2003.
A. TANKIEWICZ ET AL.
indoleamine 2,3-dioxygenase (IDO, EC 126.96.36.199) 7,8. These enzymes are differentially
distributed in the body, possess different spectrum for substrates, different molecular
weight and inducers. In spite of this, both of them catalyse the oxidative cleavage of the
tryptophan pyrrole ring to N-formylkynurenine that is further converted to kynurenine
(KYN) 8. Then KYN is transformed to 3-hydroxykynurenine (3-HKYN) by kynurenine
3-hydroxylase (HK, EC 188.8.131.52) 9.
In the present study we evaluated the peripheral TRP metabolism in the end-stages of
the rat experimental chronic renal failure. We determined plasma and tissues levels of
TRP, its metabolites, i.e. KYN and 3-HKYN and TDO, IDO and HK activity.
2. MATERIALS AND METHODS
The study was performed on male Wistar rats weighting 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 the temperature and humidity were controlled.
Rats were anesthetized with pentobarbital (40 mg/kg, i.p.). The resection of renal
tissue was carried out using the method described by Orrnrod and Miller 10. In sham
operated (SO) rats surgical extraction of the renal capsule was performed. The
experimental uremic group chronic renal failures was induced by the removal of the left
kidney, while the right kidney was decorticated in 60 %. After 1 week the additional 20
% of right kidney cortex was removed. The blood and slices of kidney, liver for the
analyses was taken 2 months after the surgical procedure. The study was approved by
Local Ethical Committee.
The animals were anaesthetised with pentobarbital (40 mg/kg i.p.) and the blood was
dra\W by heart puncture and put into a tube containing of 3.13% sodium citrate
(citratelblood = 1/9). The plasma was obtained by a centrifugation of the blood at 3000
rpm for 15 min (temp. 4°C). Urea and creatinine levels were measured by the use of
commercial kits (Corrnay, UK). After bleeding rats tissues (kidney, liver) were prepared,
and slices (500 mg) were homogenized in ice-cold water. Homogenates were and
centrifuged 14000 g for 30 min 4°C. Samples were stored at -80°C until assayed.
TRP concentrations were determined by high-performance liquid chromatography
(HPLC) according to Herve et al. II, KYN concentrations according to Holms 11, whereas
3-HKYN concentrations were determined as described by Heyes 13.
The activity of TDO was assayed according to the method described by Salter et al.
whereas activity ofIDO and HK was assayed according to Saito et al. 15.
The values are expressed as the mean ± SEM. Multiple groups comparisons were
performed by one-way analysis of variance, and significant intergroup's difference were
assessed by Tukey-Kramer Test. A value of p<0.05 were regarded as significant.
The changes in the concentration of classic markers of renal insufficiency, creatinine
and urea, were tested to estimate the effectiveness of the surgical uremia induction. We
found significant an increase in plasma creatinine (p<O.OOI) and urea (p<O.OOI)
concentration in rats with CRF in comparison with the sham-operate rats (Table 1).
EZYMES OF KYNURENINE PATHWAY IN UREMIA
We also observed a significant decrease concentrations of TRP in CRF rats in plasma
(p<O.OO 1), kidney (p<O.OO I) and liver (p<O.05). In contrast all TRP metabolites were
increased both in plasma and tissues. KYN level in CRF rats was significantly increased
in plasma (p<O.05), kidney (p<O.OOl) and liver (p<O.05). We have also noticed
significantly increase of 3-HKYN concentrations in plasma (p<O.OOl), kidney (p<O.OOI)
and liver (p<O.OI) in uremic rats in compare to the sham-operate rats (Table 2).
Activity of tryptophan 2,3-dioxygenase was significantly increased (p<O.OOl) in rats
with CRF in comparison to healthy rats. Whereas kidney activity of indoleamine 2,3-
dioxygenase was not changed in uremic rats (ns). Kidney activity of kynurenine 3-
hydroxylase was significantly increased (p<O.05) in rats with CRF. Also in liver enzyme
activity significantly increased (p<O.OOl) in CRF group in comparison with the sham-
operate rats (Table 3).
Table 1. Plasma creatinine and urea concentration.
23.4 ± 3J
291.5 ± 17.2 •••
3.7 ± 0.3
85.1 ± 10.1 •••
Values are presented as mean ± SEM, significance ot the difference In comparison with the SO group:
Table 2. Concentration ofTRP, KYN and 3-HKYN in plasma, kidney and liver.
Values are presented as mean ± SEM, slgmficance of the difference m companson With the SO group:
• p<0.05, •• p<O.O I, ... p<O.OO I.
Table 3. Activity ofTDO, IDO and HK in kidney and liver.
84.1 ± 43.9
1486.2 ± 122.4
6.9 + 1.7
12.2 ± 1.9
25.3 + 1.9
136.5 ± 10.5
34.6 ± 2.0
54.3 ± 4.1
Values are presented as mean ± SEM. slgmflcance of the difference In comparison With the SO group:
• p<0.05, ••• p<O.OO I.
A. TANKIEWICZ ET AL.
Chronic renal failure leads to numerous metabolic disturbances, which are reflected
in the blood and tissues. In the present study we evaluated the parameters that allowed us
to estimate both, the renal insufficiency severity and the degree of disturbances in the
TRP meta:bolism via kynurenine pathway.
We have showed the decrease in TRP plasma and tissues concentrations in animals
with experimental renal failure, and the increase of its metabolites content, i.e. KYN, 3-
HKYN. We have also observed the increase in TDO and HK activity, but unchanged IDO
activity. The similar results have been reported previously by Saito et al. I .
The decrease in TRP concentration may result from its conversion to KYN in the
reaction catalysed by TDO. As reported Saito et ai. I the increase in TDO activity is
caused by elevated level of circulating inductors, such as glucagon and
glucocorticosteroids. Moreover, the reduction in the concentration of this amino acid may
be caused by the impaired TRP transformation, glomerular hyperfiltration typical for
initial uremia, or its diminished reabsorption in the renal tubules 16. Also reduced TRP
dietary intake should be taken into consideration 17.
In our study the reduction in TRP concentration was linked to the increase in the
TDO activity. Chronic renal diseases lead to the increase concentration of interferon-y
(INF-y). Besides the induction ofIDO activity, INF-y is also able to stimulate nitric oxide
(NO) synthase (NOS), leading to the augmented NO production. NO has been reported to
have the high affinity for the heme iron present in IDO molecules. It may interact with
superoxide ion, an essential IDO cofactor. Additionally, other kynurenine metabolite,
picolinic acid, inhibits IDO by the NOS activity stimulation 18. Thus, the observed
unchanged IDO activity may result from the inhibition of its activity.
In contrast, TRP metabolite - KYN increased in the plasma, kidney and liver when
compared to the sham operated control rats. The increase in the KYN concentration in
uremic animals may be caused by both its enhanced synthesis and reduced clearance.
Saito et al. I have demonstrated that the renal 24-h clearance of KYN was slightly
decreased, whereas the total urinary excretion of KYN increased. Therefore, the
accumulation of KYN in renal insufficiency is probably not mainly related to the
decrease in its excretion, but to the enhanced production of KYN. Moreover, the
enhanced KYN concentration in the kidney may depend on its uptake from the
circulating blood. The same pattern seems to be true in the case ofKYN toxic metabolite
3-HKYN that is converted to xanthurenic and kynurenic acid 19. Hyperkynureninemia
may be also caused by pyridoxal-5-phosphate deficiency, cofactor of enzymes (i.e.
kynureninase and kynurenine aminotransferase) that are responsible for the KYN
metabolism 20. The role of KYN in the kidney disorders has been shown previously. It
could be involved in the pathomechanism ofmesangio-proliferative ~ l o m e r u l o n e p h r i t i s 21
and in the pathological mitosis and apoptosis of the kidney epithelial 2.
Kynurenine 3-hydroxylase converts KYN to 3-HKYN that is cytotoxic substance
with the ability to generate the hydrogen peroxide and hydroxyl radical, leading to the
cells death 7. We observed the increase in HK activity, which reflected the increase in the
3-HKYN concentrations, both in the plasma and the tissues. This enzyme is FAD -
dependent and utilizes NADPH as an electron donor. It is well known that the kidney and
the liver have the highest activity of NAD synthase 9. Therefore, we can speculate that
the observed increase in HK activity resulted from the increased concentration of the
EZYMES OF KYNURENINE PATHWAY IN UREMIA
available substrate. In CRF the deficiency of pyridoxal-5-phosphate, inhibitor of NADPH
dependent enzymes, prevents HK inhibition.
In conclusion, the increase in the activity of TDO and HK may be responsible for the
elevation in the kynurenine and 3-hydroxykynurenine concentrations in experimental
chronic renal failure. Based on our results we can speculate that profoundly changed
kynurenine pathway metabolism in CRF can play an important role in renal failure
K. Saito, S. Fujigaki, M. P. Heyes. K. Shibata, M. Takemura. H. Fujii. H. Wada. A. Noma. M. Seishima,
Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency, Am. J. Physiol.
Renal Pilysiol. 279. F565-F572 (2000).
E.W. Holmes. P. Russell. G .. 1. Kinzler. C.R. Reckard. R.C. Flanigan. K.D. Thompson, E.W. Bermes,
Oxidative tryptophan metabolism in renal allograft recipients: increased kynurenine synthesis is
associated with inflammation and OKT3 therapy. CylOkine 4,205-213 (1992).
W.M. Behan. T. W. Stone. Role of kynurenines in the neurotoxic actions of kail1lc acid, Sr. J. Pharmacal.
129, 1764-1770 (2000).
V. Kapoor, R. Kapoor, 1.P. Chalmers, Altered responsiveness of medullary depressor neurones to 1.-
glutamate and D-serine in SHR rats, Neuroreport, 7,1409-1412 (1996).
Y. Kawashima, T. Sanaka. N. Sugino. M. Takashashi, H. Mizoguchi, Supressive elTect of quinolinic acid
and hippuric acid on bone marrow erythroid grouth and lymphocyte blast fomlation in uremia, Adv.
Exp. Med. Bioi. 223. 69-72 (1987).
S. Berweck, I. Hennig. C. Sternberg. H. Dingerkus. K. Ludat. H. Hampl. Cardiac mortality prevention In
uremic patients. Therapeutic strategies with particular attention to complete correction of renal anemia.
C/in. Nephrol. 53, 80-85 (2000).
TW. Stone. Neuropharmacology of quinolinic and kynurenic acids. Pharmacal. Rev, 45. 309-379 (1993).
M.W. Taylor. G. Feng. Relationship between interferon-y, indoleamine 2.3-dioxygenase, and tryptophan
catabolism. FASEB J. 5.2516-2522 (1991).
J. Breton. N. Avanzi, S. Magagnin, N. Covini. G. Magistrelli, L. Cozzi, A. Isacchi, Functional
characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase. Eur. J.
Biochem. 267,1092-1099 (2000).
10. D. Ormrod. T. Miller, Experimental uremia. Nephron 26. 249-2541 (1980).
II. C. Hevere. P. Beyne. H. Jamault. E. Delacoux. Determination of tryptophan and its kynurenine pathway
metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet
and fluorimetric detection.J. Chronlatogr. 675.157-161 (1996).
12. E. W. Holmes. Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high
liquid chromatography, Anal. Biochem. 172, 518-525 (1988).
13. M.P. Heyes, Quantilication of3-hydroksykynurenine in brain by high-performance liquid chromatography
and electrochemical detection. J. Chromatogr. 428. 340-344 (1988).
14. M. Salter, R. Hazelwood. C. Pogson. R. Iyer. D.1. Madge. The effect of a novel and selective inhibitor of
tryptophan 2.3-dioxygenase on tryptophan and serotonin metabolism in the rat. Biochem Pharmacol
15. K. Saito. M.P. Heyes. Kynurenine pathway enzymes in brain. Properties of enzymes and regulation of
quinolinic acid synthesis. in: Recent Advances in Tryptophan Research. edited by Graziella Allegri
Filippini el £II. (Plenum Press. New York. 1996). pp. 485-492.
16. E.W. Holmes. S.E. Kahan. Tryptophan distribution and metabolism in experimental chronic renal
insufliciency. Exp. Mol. Pa/hol. 46.89-101 (1987).
17. J. Topczewska-Bruns. A. Tankiewicz. D. Pawlak. W. Buczko. Behavioral changes in the course of chronic
renal insufficiency in rats. Pol. J. Plwrmacol. 53,263-269 (2001).
18. A Chiarugi. P Sbarba, D. Paccagnini, S. Donnini. S. Filippi. F. Moroni. Combined inhibition of
indoleamine 2.3-dioxygenase and nitric oxide synthetase modulates neurotoxin release by interferon-y-
activated macrophages. J. Leukocyte Bioi. 68. 260-266 (2000).
19. F. Takeuchi. R. Tsubouchi, S. lzuta. Y. Shibata. Kynurenine metabolism and xanthurenic acid formation
in vitamin B6-deticient rat after tryptophan injection, J. Nutr. Sci. Vitaminol. 35.111-122 (1989).
A. TANKIEWICZ ET AL.
20. A. Martinsons. V. Rudzite. V. Groma. O. Bratslavska. B. Widner, D. Fuchs. Kynurenine and neopterin in
chronic glomerulonephritis. Ad,'. Etp. Med. BioI. 467.72·77 (1999).
21. A. Martinsons, V. Rudzite. E. Jurika. A. Silava. The relationship between kynurenine. catecholoamines.
and arterial hypertension in mesangioproliferative glomerulonephritis. Adv. Exp. Men. Bioi. 398.417·
22. A. Martinsons. V. Rudzite, O. Bratslavska. V. Saulite. The influence of kynurenine. and norepinephrine
on tubular epithelial cells and alveolar fibroblasts. Anv. Exp. Med. Bioi. 467. 347·352 (1999).