Contribution of quinolinic acid in the development of anemiain renal insufficie - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：DariuszPawlak, MariuszKoda, SebastianPawlak, SlawomirWolczynski,  WlodzimierzBuczko作者单位：Departments of Pharmacodynamics, Clinical Pathology, Cardiosurgery, and GynaecologicalEndocrinology, Medical Academy, 15-230 Bialystok, Poland

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      【摘要】
   Quinolinic acid (QA) is a potentendogenous excitotoxin; elevation of its concentration in an organismhas been implicated in the pathogenesis of various disorders. Thepurpose of this study was the assessment of QA impact on the process oferythropoiesis. Marked increase of QA concentration was observed inplasma and peripheral tissues of uremic rats. These changes wereproportional to the amount of the removed renal tissue and positivelycorrelated with the concentration of creatinine but negativelycorrelated with hematological parameters, i.e., hematocrit and Hb redblood cells count. The changes were accompanied by a slightdecrease in the concentration of endogenic erythropoietin (EPO) in the plasma of animals with uremia. Chronic treatment with QA diminished theincrease in EPO concentration after introduction of cobalt in rats.These changes were associated with the decrease in all hematologicalparameters after QA administration. The in vitro study in theconditions of hypoxia showed that QA inhibited the EPO release fromHepG2 cells to the culture base. Additionally, in HepG2 cells QA had adose-dependent inhibitory effect on hypoxia- and cobalt-induced EPOgene expression without any cell toxicity. In conclusion, theerythropoiesis in chronic renal failure could be attributed to theinfluence of QA on EPO synthesis. Thus we propose that QA can be auremic toxin responsible for anemia in animals or patients with renal failure.
      【关键词】 Contribution quinolinic development anemiain insufficiency' P( j5 N9 k; g- X
   renal failure

INTRODUCTION% I6 _# ?9 D$ o  o. D9 U

ERYTHROPOIESIS DEPENDS ON the proliferative capacity of erythroid progenitorcells in the bone marrow and their stimulation, mainly byerythropoietin (EPO) ( 2 ). In chronic renal failure (CRF),the most important trigger of anemia is disturbances in erythropoiesiscaused by reduced renal production of EPO as well as resistance of bonemarrow cells to this hormone ( 3 ).
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The literature data and our observations have indicated that in CRFpatients, an increased degradation of tryptophan occurs, accompanied bya significant increase in the concentration of its plasma metabolites( 18, 19 ). Quinolinic acid (QA) is the product oftryptophan oxidation that increases after enzymatic changes in thekynurenine pathway (Fig. 1 )( 21 ). QA is an endogenous, specific N -methyl- D -aspartate (NMDA) receptor agonist,which on activation may direct disturbances in cellular metabolicprocesses promoting apoptosis ( 22 ). In in vitroexperiments, it has been demonstrated that QA possesses a suppressiveeffect on erythroid colony and lymphocyte blast formation( 12 ). QA is excreted in the urine of healthy subjects, andit could be accumulated in the blood of uremic patients( 17 ). Therefore, the increased blood concentration in CRFmay account for uremic symptoms, such as anemia.

Fig. 1. Scheme of kynurenine pathway.  K2 B( C' i" [) k( r0 w& F
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The present study was undertaken to investigate plasma QAconcentrations in rats with chronic renal insufficiency and its influence on EPO production. We provide experimental evidence supporting the hypothesis that the inadequate EPO production in uremicpatients might be at least partially attributed to the inhibitoryeffect of QA on EPO production. We also used the human hepatoma HepG2cell line, which is a well-characterized in vitro system, to study themechanisms of EPO production ( 8 ). We have demonstratedthat these cells release EPO in the culture medium in response tohypoxia or transition metals (cobalt) and that this regulation has beenshown to occur at the EPO mRNA level. In addition, we examined therelationship between the QA plasma concentration and the essentialhematological and biochemical parameters of healthy rats and animalswith experimental renal failure.5 [/ h$ [; F# n. y! A" d# O
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MATERIALS AND METHODS0 J4 l' R! }% a: E( v

Chemicals. The chemicals, which were of analytic reagent grade, werepotassium dihydrogen phosphate, phosphoric acid, methanol, hydrochloric acid, and potassium chloride (Merck, Darmstadt, Germany); cobalt chloride hexahydrate, sodium citrate, potassium phosphate,Dulbecco's modified Eagle's medium, penicillin, streptomycin,heat-inactivated fetal bovine serum,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT), isopropanol, Tris(hydroxymethyl)aminomethane hydrochloride, and magnesium chloride (Sigma, St. Louis, MO); QA(ICN); and pentobarbital sodium (Biowed, Pulawy, Poland).( ~% u) g) E- b6 Y/ U, j% L9 H+ f

Animals. Inbred adult (2 mo old) male albino rats (Wistar strain) of initial~180-200 g body wt were used in the experiment. The animals werehoused in conventional conditions, at 22 ± 1°C, with a relative humidity of 50 ± 10% and a 12:12-h light-dark cycle. They were allowed free access to drinking water (redistilled water) and rat chow(LSM, total protein 15.9%, dry diet, Fodder Manufactures, Motycz, Poland).
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Ethics. Procedures involving the animals and their care conformed to theinstitutional guidelines, in compliance with national and internationallaws and Guidelines for the Use of Animals in Biomedical Research( 7 ).
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Surgical induction of experimental chronic renal insufficiency inrats. Rats were anesthetized with pentobarbital sodium (40 mg/kg ip). Theresection of renal tissue was carried out by using the method describedby Ormrod and Miller ( 16 ). In sham-operated rats, surgicalextraction of the renal capsule was performed. The other experimentalgroups were as follows. "Moderate" renal insufficiency (CRF1) wasinduced by the removal of the left kidney, while the right kidney wasdecorticated in 60%. The rats with "severe" renal failure(CRF2) were subjected to the same surgical procedure as was CRF1, andafter 1 wk the additional 20% of the right kidney cortex was removed.The "severe" group of animals was divided into two subgroups, CRF2and CRF3. The blood and tissues for the biochemical analyses were takena month after the surgical procedure on CRF2, with the exception ofCRF3, in which the biological samples were collected 2 mo after thelast surgical intervention.

Effects of QA on erythropoiesis in rats. The erythropoietic effects of sustained intraperitoneal administrationof cobalt chloride in a dose of 10 mg/kg daily for 20 days in rats werecompared with that in the animals that received the QA in doses of 10 or 100 mg/kg ip for 20 days 6 h before each cobalt injection.Blood was removed for determination of hematological and biochemicalparameters at days 0-5, 10, 15,and 20 after treatment.

Blood and tissues sampling. The animals were anesthetized with pentobarbital sodium (40 mg/kg ip),and the blood was drawn by heart puncture and put into a tubecontaining 3.13% sodium citrate (citrate/blood = 1:9). The plasmawas obtained by blood centrifugation at 3,000 rpm for 15 min (4°C).After bleeding, rat tissues (kidney, liver, lung, intestine, heart,spleen, and muscle) were prepared and slices (500 mg) were homogenizedin ice-cold water. Homogenates were additionally sonificated andcentrifuged at 14,000 g for 30 min at 4°C. Samples werestored at 80°C until assayed.% z( J( z$ S4 B3 i5 ~. r* D+ M

Determination of QA. QA was measured by using the HPLC technique as described byWerner-Felmayer et al. ( 26 ). The chromatographic system(Hewlett-Packard) was composed of an HP 1050 series pump with aRheodyne injection valve fitted with a sample loop (20 µl). Partisil10 SAX 250 × 4.6 mm (Phase Separations) column was eluted with 50 mM potassium phosphate (pH 2.0) containing 12% methanol at a flow rateof 2 ml/min. The amount of 2 ml plasma or supernatant of tissuehomogenates was concentrated on Sep-Pack cartridges (Waters Accell PlusQMA), washed in 2 ml of water, and eluted with 0.2 ml 4 MH 3 PO 4 (92% recovery of spiked QA). Using an HP1050 series UV detector, the column effluent was monitored (272 nm).The output of the detector was connected to a single instrument LC-2DChemStation. Chromatography was carried out at 24°C.
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Determination of other biochemical parameters. The following parameters were measured with commercially availablekits: creatinine (CRT; Creatinine 30, Cormay), urea (UR; Urea 30, Cormay), and EPO (EPO-Trac I 125 RIA Kit, DiaSorin). Thebiochemical and hematological parameters were measured by the standardmethods using an automatic Konelab 4.0.5 and Technikon H1 analyzers.Reticulocytes were stained with methylene blue, and their values[corrected reticulocytes count (CRC)] were adjusted to the degree ofanemia ( 13 ).& m6 ?" W1 h$ J2 W* i

Cell cultures. The HepG2 cell line was obtained from the American Type CultureCollection (HB 8065; tissue: hepatoblastoma, liver; sex: male; agestage: 15 yr; and ethnicity: caucasian). These cells were cultured inDulbecco's modified Eagle's medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% heat-inactivated fetalbovine serum in a humidified atmosphere (5% CO 2 -95% air) at 37°C (Heraeus incubator). Starting from the day before the experiment, 5 × 10 5 cells/cm 2 ofconfluent cultures were fed with a serum-free medium (24-well polystyrene dishes). At the beginning of the 24-h experimental period,HepG2 cells received fresh medium containing QA (1, 10, 100, and 1,000 µM). In preliminary experiments, we observed that the addition of QAto cultures had no effect on the EPO levels (normoxic conditions)compared with a control group. EPO production by HepG2 was induced byincubation of the cultures with a low (1%) oxygen tension atmosphereor 100 µM of cobalt chloride for 24 h. At the end of theincubation period, supernatants were harvested, clarified bycentrifugation, and stored frozen at 80°C until assayed.9 }$ ^7 g7 r" p$ D' D) ~

Cytotoxicity of QA toward HepG2 cells. QA cytotoxicity (1, 10, 100, and 1,000 µM) was carried out accordingto Mosmann ( 15 ). Exposition time to QA was 24 h. MTT was dissolved in PBS at 5 mg/ml and filtered to sterilize and remove asmall amount of insoluble residue present in some batches of MTT. Atthe times indicated below, stock MTT solution (100 µl/1 ml medium)was added to all wells of the assay and plates were incubated at 37°Cfor 4 h. Acid-isopropanol (1 ml of 0.04 M HCl in isopropanol) wasadded to all wells and mixed thoroughly to dissolve the dark bluecrystals. After a few minutes at room temperature, the plates were readon a Multiskan EX Labsystems micro-ELISA reader to ensure that allcrystals were dissolved, using a test wavelength of 570 nm and areference wavelength of 630 nm. Plates were normally read within 1 h of adding the isopropanol.# }1 n/ N6 R& F5 Q8 [! `: }

Quantification of EPO mRNA. We have examined the relative levels of EPO mRNA by using asemiquantitative RT-PCR procedure. The total RNA was extracted fromHepG2 cells by using TRIzol reagent (Life Technologies, Grand Island,NY) according to the manufacturer's instructions. RNA was quantifiedspectrophotometrically at 260 nm. RNA was then stored in RNase-freewater at 80°C. cDNA synthesis was performed in 50 mMTris · HCl (pH 8.3), 75 mM KCl, 3 mMMgCl 2, 10 mM DTT, 1 mM 2-deoxynucleotide 5'-triphosphatemix, 2.5 µM oligo(dT) 15, 20 U RNasin ribonucleaseinhibitor, and 200 U M-MLV RT (Promega, Madison, WI) with 1 µg oftotal RNA in a final volume of 20 µl. The mixture was incubated at42°C for 15 min and then heated to 95°C for 5 min. PCR wasperformed in 10 mM Tris · HCl (pH 8.8), 1.5 mM MgCl 2, 50 mM KCl, 0.1% Triton X-100, 50 µM2-deoxynucleotide 5'-triphosphate mix, 200 nM of each primer, 1 unit of DyNAzyme II DNA polymerase (Finnzymes), and 5 µl ofcDNA mixture in a final volume of 25 µl. The expression of thehousekeeping gene, -actin, was considered as a semiquantitativecontrol. Sequences of specific primers for PCR were used: sense5'-ATCACGACGGGCTGTGCTGAACAC-3', positions 335-358, GenBankaccession no. X02157, and antisense 5'-GGGAGATGGCTTCCTTCTGGGCTC-3', positions 623-600, GenBankaccession no. X02157 for EPO ( 23 ); and sense5'-CCAGATCATGTTTGAGACCT-3', positions 913-932, GenBank accessionno. BC009275, and antisense 5'-GCACAGCTTCTCCTTAATGT-3', positions1204-1185, GenBank accession no. BC009275 for -actin. PCR wascarried out under the following conditions: 30 s of denaturationat 94°C, 30 s of annealing at 58°C, and 30 s of extensionat 72°C for 30 cycles, with an additional 5 min of extension for thelast cycle on an MJ Research Thermocycler (model PTC-200, Watertown,MA). Amplification products were run on a 2% agarose gel. Ethidiumbromide-stained gels were visualized under UV illumination andphotographed, and for each sample the intensity of the signal wasmeasured with One Dscan/Zero Dscan v2.02 and v1.0 software(Scanalytics). Ratios of the corresponding peak areas, EPO/ -actin,were calculated for each sample and used for quantitative calculationsand comparisons.( m' C+ Q5 ?, M; y0 [2 p" b; [

Statistical analysis. The values are expressed as means ± SE; n representsthe number of experiments. Multiple group comparisons were performed byone-way analysis of variance, and significant intergroup differences were assessed by a Tukey-Kramer test. Values of P regarded as significant. Correlations between plasmaconcentrations of studied QA and other biochemical or hematologicalparameters were analyzed by using a Spearman test.

RESULTS

Effect of experimental renal insufficiency in rats on biochemicaland hematological parameters. The parameters of renal insufficiency are summarized in Table 1. Rats with moderate, severe 1, andsevere 2 renal failure had significantly increased blood CRTand UR compared with the control group. At the same time, these changeswere associated with the decrease in hematological parameters, i.e.,red blood cells count (RBC), Hb, Hct, and CRC. However, meancorpuscular volume, mean corpuscular Hb, mean corpuscular Hbconcentration, and parameters of iron metabolism, such as serum ironconcentration, transferrin, and total iron-binding capacity, were notchanged.
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Table 1. Effect of varying degrees of experimental renal insufficiency onbiochemical and hematological parameters
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In rats with CRF compared with control animals, the more extensiverange of EPO plasma concentrations was demonstrated. However, the meanvalues of CRF of studied groups and the control group were notsignificantly different ( P 0.05). The administration of cobalt resulted in a significant increase in EPO plasmaconcentration, this effect was noticeably weaker in animals withpartial ablation of the kidney (severe 1 and 2)., N( ^" X! \0 E
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Effect of renal insufficiency on QA plasma and tissue concentrationin rats. The essential increase in plasma QA concentrations in moderate (CRF1)and severe 1 and 2 groups (CRF1 and CRF2, respectively) of renalinsufficiency was observed (Fig. 2 ). At the same time, increases in QAconcentrations in the tissues (kidney, liver, lung, intestine, andspleen) were observed mainly in CRF2 rats. Additionally, therelationship between the increase in the plasma QA concentration andthe stage of renal insufficiency was demonstrated. The multipleregression analysis (Fig. 3 ) showed thatthere was a linear correlation between plasma concentration of CRT andQA ( r = 0.848, P r = 0.808, P was asignificant negative correlation between QA plasma concentrations andhematological parameters such as RBC ( r = 0.841, P Hb ( r = 0.704, P r = 0.843, P ( r = 0.427, P 4 ).

Fig. 2. Quinolinic acid (QA) concentrations in plasma andperipheral tissues of control and partially nephrectomized rats.CRF1-3, moderate, severe 1, and severe 2 chronic renal failuregroups, respectively. * P P P3 ?5 I( T/ c: `' z
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Fig. 3. Correlations between plasma concentrations of creatinine( A ) or urea ( B ) and plasma concentration of QA.
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Fig. 4. Statistical correlations between the plasma concentration of QA andhematological parameters of rats with chronic renal failure. Depictedare Hb, Hb ( A ); Hct ( B ); RBC, red blood cellscount ( C ); and CRC, corrected reticulocytes count( D ).) E6 U& e. T2 E  J7 h
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Effect of chronic QA administration in cobalt chloride-stimulatederythropoiesis in rats. After 1 h, administration of QA in 10 and 100 mg/kg ip dosesproduced plasma concentrations of 11.2 ± 5.3 and 67.9 ± 16.4 µM, respectively (Fig. 5 ). Thelevel of QA in rats then decreased and at 4 h was 0.5 ± 0.1 and 6.3 ± 4.5 µM, respectively. During the next 2 h, theplasma concentration of QA raised to 0.9 ± 0.1 and 2.2 ± 0.8 µM, respectively.- C, G. W: T+ G# C$ w
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Fig. 5. Kinetics of QA in plasma after intraperitonealadministration in doses of 10 and 100 mg/kg.1 `+ F# ?( G9 S, k( a

The exposure of rats to cobalt chloride on day 1 resulted ina significant increase in plasma EPO titers, reached the highest levelafter day 2, and then gradually declined (Fig. 6 A ). However, significantincreases in RBC (Fig. 6 B ), Hb (Fig. 6 C ), and Hct (Fig. 6 D ) were observed after 15 days of intraperitonealadministration of cobalt chloride. Chronic treatment with QA in a doseof 100 mg/kg ip inhibited the increase in EPO concentration aftercobalt chloride administration. Additionally, these changes wereassociated with the decrease in all hematological parameters, i.e.,RBC, Hb, and Hct ( P

Fig. 6. Effect of chronic QA administration in cobalt chloride-stimulatederythropoiesis in rats. Depicted are EPO-plasma erythropoietin (EPO)concentration ( A ), RBC ( B ), Hb concentration( C ), and Hct ( D ). * P P P1 d7 |0 w" _$ m# n  A! w
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Effect of QA on cell viability. As shown in Fig. 7 A, theviability of HepG2 cells was decreased only at a higher concentrationof QA (1,000 µM). QA in concentrations of 1, 10, and 100 µM had noeffect on this parameter.2 R5 `: F# Z/ F

Fig. 7. Effect of QA on the HepG2 cells viability ( A ),EPO medium concentration ( B ), and EPO mRNA induction byhypoxia or cobalt chloride ( C ). * P P P, j( t/ w5 j- O& b- {, S2 P9 @
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Effect of QA on medium concentration of EPO. HepG2 cells, when grown in 1% O 2 for 24 h, producedapproximately five- to sixfold more EPO compared with the cells grown in 21% O 2 (Fig. 7 B ). QA (100 and 1,000 µM)inhibited hypoxia-induced EPO production by 32.1 ± 2.9 and49.6 ± 2.2% in a dose-dependent manner. However, 24-h expositionto 10 µM of QA had no effect on the EPO production, but 48-h HepG2exposition significantly decreased the EPO medium concentration by30.6 ± 4.1%. In normoxic HepG2 cultures, cobalt chloridesignificantly stimulated EPO production during a 24-h incubationperiod, but this effect was weaker than under hypoxic conditions. Theaddition of QA in concentrations of 100 or 1,000 µM inhibited EPOproduction by 24.9 ± 1.6 and 37.7 ± 2.4%, respectively.7 z* V5 P# V- G0 R  q6 [' D

Effect of QA on EPO mRNA induction. In HepG2 cells, hypoxia or cobalt chloride induced EPO gene expression(Fig. 7 C ). We observed a strong, dose-dependent reduction inEPO gene expression by QA in hypoxic conditions at concentrations ranging from 1 to 100 µM, without any cell toxicity. We observed 35.2 ± 5.4% inhibition of mRNA synthesis with 1 µM of QA, and this concentration is achievable in the plasma of uremic rats. However,this dose of QA had no effect on the induction of EPO mRNA by cobaltchloride. The inhibition of EPO gene expression in these conditions wasobserved in higher concentrations of QA (100 and 1,000 µM).5 T: ^" v! ?- U* b8 g" v2 G

DISCUSSION

The present study was undertaken to investigate QA concentrationsin rats with chronic renal insufficiency and their influence on the EPOproduction in both in vivo and in vitro experimental conditions. Weprovide experimental evidence supporting the hypothesis that theinadequate EPO production in uremic patients might be partiallyattributable to the inhibitory effect of QA on the EPO production.2 D* ^9 A2 B" W% v8 z
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We used a well-established model of CRF in rats to define mechanismsthat may be involved in anemia development, which is observed in kidneyfailure ( 16 ). The course of experimental renal failure wasmonitored by means of the plasma concentration of CRT and UR, and bothof them increased proportionally to the extent of renal tissueresection. In addition, we evaluated basal hematological parameters(Hb, Hct, RBC, and CRC) as the markers of the renal insufficiencyprogression. We found that they were significantly decreased dependingon the stage of the experimental renal insufficiency.

In CRF, a great number of the endogenous metabolites that are normallyexcreted in urine accumulate in the blood ( 24 ). Among these are the products of kynurenate sodium (KYN) degradation ( 18, 19 ). We observed a significant increase in the QAconcentrations in plasma and peripheral tissues of uremic animals.These changes were proportional to the amount of the removed renaltissue and correlated with the concentration of CRT, a marker ofdecreased glomerular filtration rate and impaired kidney excretoryfunction. In the CRF2 group, QA plasma concentration was ~10-foldhigher than in the control animals. The changes in QA concentration are in accordance with other reports that demonstrated similar patterns inthe plasma QA of human patients with CRF. As reported by Saito et al.( 20 ), the increase in serum QA concentrations during uremia is due to the decline in the activity ofaminocarboxymuconate-semialdehyde decarboxylase (the enzyme responsiblefor the degradation of QA to the glutarate pathway). Although serumconcentration of QA in CRF was elevated, its renal clearance valueswere slightly decreased by 20% compared with control subjects. Becauseurinary excretion of QA also increased, the authors presume thatincreased solute concentration is not related to a decrease in renalexcretion but to an increase in production and decrease in degradation( 20 ).
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Our laboratory's earlier study demonstrated that hemodialysis is oneof the therapeutic approaches that significantly reduces QA plasmaconcentration; despite this, QA was still elevated in uremic patientscompared with the healthy volunteers ( 17 ). Saito et al. ( 20 ) found that serum KYN and QAconcentrations after hemodialysis were significantly decreased by ~30and 75%, respectively, compared with prehemodialysis values. The risein dialysis frequency decreases QA concentration ( 17 ) andsimultaneously increases erythropoiesis ( 11 ).
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In our study, the plasma level of QA negatively correlated withhematological parameters.
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The predominant reason for insufficient erythropoiesis in renaldiseases is the impossibility of increasing EPO production in responseto the initial anemia ( 3 ). The mean values of EPO in CRFand control rats were not significantly different. The cobaltadministration in rats resulted in a significant increase in EPO plasmaconcentration in the control group, and this effect was significantlyweaker in animals with partial ablation of the kidney. Although therate of EPO production is clearly related to the degree of anemia andin turn to the supply of oxygen to the tissues, this relationship isquite broad, suggesting that a number of other factors play a role.Among potential agonists are cobalt ( 4 ), androgens, andinsulin-derived growth factors ( 1 ). It is known thatcobalt may lead to the depression of respiration, oxidativephosphorylation, and reduced oxygen uptake in kidneys. This metal hasbeen found to mimic the hypoxia-induced expression of the EPO gene( 4 ). Antagonists include inflammatory cytokines, such astumor necrosis factor, IL-1, and transforming growth factor- ( 6 ). It would seem likely that toxic metabolites retainedin CRF may also impair activation of the EPO gene expression, butrelevant observations are not available.) c# j8 f' g- k7 t) ?

In the next step of our study, we used an animal model to definemechanisms that may be involved in the observed changes( 16 ). We investigated several parameters of erythropoiesisin healthy rats in response to 20 days of daily exposure to cobaltchloride. The plasma concentration of EPO was significantly increased,and peak concentration was seen after 1-4 days from the start ofthe experiment. The increase in EPO production results in enhanced redcells formation in bone marrow, which causes the elevation in RBC, Hct,and Hb. This effect was observed after 15 days of cobalt chlorideexposure. The chronic administration of QA clearly inhibited the EPOlevel after cobalt chloride treatment. Additionally, these changes wereassociated with the decrease in all hematological parameters, i.e.,RBC, Hb, and Hct. The concentrations of QA obtained after the lowerdose (10 mg/kg) reached the level observed in patients with chronicrenal insufficiency ( 17 ). QA in a dose of 100 mg/kg causedan increase in the plasma concentration that persisted for 3 h andwas much higher that those observed in uremic patients. QA in a dose of10 mg/kg caused a decrease in the EPO plasma concentration and theinhibition of erythropoiesis, both induced by cobalt chloride. Asmentioned above, the resulting concentration is typical for patientswith chronic renal insufficiency. Thus we assume that also in vivo QAinhibits erythropoiesis.
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We also used the human hepatoma HepG2 cell line, which is awell-characterized in vitro system, to study mechanisms regulating EPOproduction ( 8 ). In this experiment, HepG2 cells were grown in 1% O 2 or in the presence of cobalt chloride. Theincreased EPO production during 24 h in cells grown in 1%O 2 was much stronger compared with cells grown in 21%O 2. These results demonstrated that QA dose dependentlyinhibited the production of EPO stimulated by hypoxia or transitionmetal (cobalt), without any cell toxicity. The lack of changes in -actin mRNA and -fetoprotein levels (data not shown) indicatedthat observed activity of QA on EPO production is specific. Thespecific effects of QA on EPO synthesis in HepG2 cells suggest that QAmay also be an important regulator of EPO production in vivo. Kynurenicacid (NMDA receptor antagonist), which acts as an antagonist of QA( 21 ), did not eliminate the QA effect on EPO production byHepG2 cells (data not shown), thus the inhibitory activity of QA on EPOsynthesis seems to be not connected with the NMDA receptor. Inaddition, the changes observed in EPO synthesis resulted from theinhibition of the EPO mRNA level. The fact that in these studies theconcentrations of QA used to inhibit hypoxia-induced expression of theEPO gene in HepG2 cells were within the range of QA in the plasma ofhumans ( 17 ) or rats ( 19 ) with chronic renalinsufficiency suggests that QA could play a role in the anemia observedin uremia. In our in vitro HepG2 study, we observed that QA in theconcentrations of 1 and 10 µM inhibits EPO gene expression, but wedid not detect any changes in the EPO concentration in the medium. Thefirst cell reaction after stimulus (in our case QA) is the change in the EPO gene expression, which is followed by the activation of intracellular processes and modulation of EPO synthesis and release. The decrease in the EPO mRNA synthesis does not always reflect the EPOconcentration (in the same interval time). Twenty-four-hour HepG2exposition to 10 µM of QA (the QA concentrations observed in patientswith chronic renal insufficiency) inhibits the EPO gene expression butwithout the decrease in EPO concentration in the medium. In contrast,48-h HepG2 exposition to 10 µM of QA significantly decreased the EPOconcentration in the medium.

Tissue hypoxia, whether due to altered O 2 tension,O 2 -carrying capacity, or O 2 affinity of theblood, is the primary stimulus for EPO production ( 14 ).The oxygen sensor is likely to be a heme protein, perhaps a cytochromeb-like flavo-heme NADPH oxidase that signals by activated oxygencompounds. H 2 O 2 generated by NADPH oxidase in aO 2 -dependent manner is a possible candidate for anintracellular messenger ( 5 ). It is a freely diffusible signaling molecule between the sensor and the transcriptional activator-hypoxia inducible factor (HIF). Hypoxia- or cobalt- inducedexpression of the EPO gene depends on the activation of an enhancerelement by HIF. HIF is a heterodimeric nonheme iron protein composed of - and -subunits ( 25 ). HIF1- is continually synthesized but rapidly degraded in normoxia.H 2 O 2 can react with iron in HIF1- togenerate OH radicals. Hypoxia induces stabilization of the HIF-1 subunit, allowing the formation of the complex HIF1- -aryl hydrocarbon nuclear translocator protein. The dimerizationinduces a conformational change that allows it to bind DNA( 9 ). Furthermore, there is evidence for a possible role ofthe nitric oxide-cGMP system in hypoxic regulation of EPO production( 10 ). QA may lead to the formation of radical species(including nitric oxide) that can induce degradation of HIF1- and,finally, negatively regulate EPO gene expression. Moreover,decarboxylation and conversion of QA to nicotinate mononucleotide byphosphoribosyltransferase is a step in biosynthesis ofNAD  . It is also possible that the concentration of NAD caninfluence the redox situation.( k9 C; @2 V: x* n4 e

In conclusion, this study provides the evidence for the accumulation ofQA in the plasma and peripheral tissues in the course of CRF. Theerythropoiesis in CRF could be attributed to the influence of QA on EPOsynthesis. Thus we proposed that QA could be an uremic toxinresponsible for anemia in CRF./ v$ \% r  L0 C; D2 p5 r% U7 L- U
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ACKNOWLEDGEMENTS" f. D- {+ R* f, C. \( @
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The authors thank Krzysztof Zolbach for technical assistance.+ L, l0 T* H; R( V
      【参考文献】/ A$ h7 W# m/ }
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