J Am Soc Nephrol 12: 1721–1728, 2001 High Adenosine and Deoxyadenosine Concentrations in Mononuclear Cells of Hemodialyzed Patients JEROME SAMPOL,*† BERTRAND DUSSOL,†‡ EMMANUEL FENOUILLET,* CHRISTIAN CAPO,§ JEAN-LOUIS MEGE,§ GILLES HALIMI,* GUY BECHIS,* PHILIPPE BRUNET,†‡ HERVE ROCHAT,†‡ YVON BERLAND,†‡ and REGIS GUIEU*¶ *UMR CNRS 6560, Faculte´deMe´decine Secteur Nord; †Service de Ne´phrologie and ‡Centre d’Investigation Clinique, Hoˆpital Sainte Marguerite; §CNRS UPRESA 6020 Unite´des Rickettsies; and ¶Laboratoire de Biochimie, Hoˆpital de la Timone, Marseille, France. Abstract. Infections are one of the most important complica- clear cells of patients than in healthy volunteers. During HD, tions of hemodialysis (HD). The high concentrations of aden- Ado concentration decreased by 34%, whereas inosine concen- osine (Ado) and of its metabolites during HD may contribute to tration increased by 27%. Before HD, MCADA activity level the dialysis-induced immune deficiency through their known was 2.1-fold lower in patients than in control subjects. After ability to alter lymphocyte function. The influence of HD on HD, MCADA activity increased by nearly 50% but remained Ado metabolism was assessed in mononuclear cells through lower than in control subjects. Ado kinase activity level of the measurement of (1) the concentrations of nucleosides in patients did not differ from that of control subjects and was mononuclear cells and (2) the activities of mononuclear cell unchanged by HD. The influence of Ado on in vitro mononu- Ado deaminase (MCADA) and Ado kinase, two enzymes clear cell proliferation and interferon-␥ production also was involved in Ado concentration regulation. Nine end-stage renal evaluated. Ado inhibited cell proliferation and interferon-␥ failure hemodialyzed patients (five men and four women; mean production in a dose-dependent manner, and these inhibitions age, 69 Ϯ 10 yr) and eight healthy volunteers (four men and were stronger for patients than for healthy volunteers. The high four women; mean age, 53 Ϯ 19 yr) were included in the study. concentrations of Ado and deoxyadenosine in mononuclear Before HD, Ado, deoxyadenosine, and inosine concentrations cells and the low MCADA activity level likely are involved in were respectively 2.9-, 2.5-, and 2.5-fold higher in mononu- the immune defect of patients who are undergoing HD. Despite improvements in dialysis and medical therapies, infec- phosphate. The extracellular metabolism of Ado and d-Ado is tions account for a significant proportion (12 to 15%) of deaths mediated by two mechanisms. First, Ado and d-Ado are taken in the chronic dialysis population (1). The immune defect up quickly and efficiently by red blood cells, via an equilibra- observed in patients with end-stage renal disease could explain tive facilitated diffusion system (10) (Figure 1A). Nucleosides partly the frequency of infections. However, hemodialysis also are taken up efficiently by mononuclear cells via an (HD) actually worsens this immunodeficiency because of re- equilibrative facilitated diffusion system and less so by a current predialytic activation of mononuclear cells induced by sodium-dependent concentration system (11). Because of its blood contact with the dialysis membrane and dialysate, which rapid uptake, the plasma half-life of Ado is very short (a few in turn results in a deactivation state (2). Many humoral and seconds). Second, Ado and d-Ado are deaminated rapidly into cellular factors likely are implicated in this immune defect (3). inosine and deoxyinosine, respectively, by adenosine deami- Among them, adenosine (Ado) and deoxyadenosine (d-Ado), nase (ADA; Figure 1A). both strong immunosuppressive agents (4–6), might be impli- ADA is found in large amounts particularly in mononuclear cated because plasma concentrations of Ado (7) and metabo- cells (mononuclear cell adenosine deaminase [MCADA]) lites (8) are high in patients who are undergoing HD. (12,13), where it plays a major role in Ado concentration Ado is released by endothelial cells and by several tissues, regulation in both extracellular (13) and intracellular spaces more particularly during ischemia (9). Intracellular Ado comes (14,15). It also is implicated in T-cell activation via noncova- from the hydrolysis of nucleotides through a 5'nucleotidase. lent binding to the T-cell antigen CD26 (16). Intracellular Ado d-Ado is formed from the hydrolysis of deoxyadenosine mono- concentration also is regulated strongly by adenosine kinase (AKA) activity (17,18). Indeed, AKA phosphorylates Ado and nucleosides into nucleotides (19). Thus, low MCADA (15) or Received April 21, 2000. Accepted January 26, 2001. AKA activity level (20,21) results in an increased intra- and Correspondence to Dr. Regis Guieu, UMR CNRS 6560, Faculte´deMe´decine extracellular Ado concentration. A normal MCADA activity Secteur Nord, Bd P. Dramard 13015 Marseille, France. Phone: 33-4- 91698843; Fax: 33-4-91657595; E-mail: [email protected] level prevents adenosine accumulation and thus ensures normal 1046-6673/1208-1721 lymphocyte development and function (22). Inherited ADA Journal of the American Society of Nephrology deficiency, which induces high Ado and d-Ado concentrations Copyright © 2001 by the American Society of Nephrology in body fluids, causes severe combined immunodeficiency 1722 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1721–1728, 2001 syndrome (5). The immune system defect occurs because of 30 to 60 yr). Mean hemoglobin level was 15 Ϯ 2.5 g/100 ml; mean the accumulation of toxic purine metabolites, particularly d- hematocrit was 43 Ϯ 2%; lymphocyte count was 1700 Ϯ 200/␮l, and Ado and deoxyadenosine triphosphates (d-ATP), both of which CD3ϩ cell count was 1105 Ϯ 190. There was no significant differ- ϩ inhibit the ribonucleotide reductase activity of T cells (23) ence between CD3 number in patients and in healthy volunteers Ͻ (Figure 1B). Furthermore, lymphocytes are particularly sensi- (ANOVA P 0.05). tive to Ado and d-Ado because of their ability to accumulate d-ATP (24). Reagents ␣ ␤ We showed previously that plasma Ado concentration is Adenosine (crystallized, 99% pure), dipyramidole, , methylene- adenosine-5'-diphosphate, ATP, 9-erythro (2-hydroxy-3-nonyl) ade- increased in patients who are undergoing HD (7). Taking into nine, 6-methylthiopurineriboside (6-MMPR), and dithiothreitol were account the facilitated diffusion system and the concentrative from SIGMA (Saint Quentin Fallavier, France). Deoxycoformycin sodium-dependent transport system of nucleosides across the (dcf) was from Lederle Laboratories (Paris, France). Heparin was cell membrane, we hypothesized that Ado and d-Ado concen- from Sanofi Winthrop (Gentilly, France). Na2-ethylenediaminetet- trations are high in mononuclear cells of patients who are raacetic acid was from SIGMA. Bovine serum albumin was from undergoing HD. Because ADA and AKA are thought to play a Johnson and Johnson Clinical Chemistry (Rochester, NY). Con- major role in the regulation of intracellular nucleoside concen- canavalin A (Con A) was from SIGMA. The reversed phase chroma- tration, we evaluated their activities in mononuclear cells. tography column (Merck LIChrospher C18, and RP8 250 ϫ 4 mm) Finally, we also evaluated the influence of Ado on the T-cell and other reagents were from Merck (Darmstadt, Germany). proliferation and interferon-␥ (IFN-␥) production in patients who are undergoing HD. We chose IFN-␥ rather than interleu- Blood Samples for Ado, d-Ado, and Inosine Assays kin-2 (IL-2) because IFN-␥ is a good marker of human periph- Sample collection has been described elsewhere (28,29). Briefly, eral blood lymphocytic activity (25) and because IL-2 has been blood collected from the brachial vein (8 ml/sample) was drawn into studied carefully during HD in humans (26,27). vacuum tubes containing a stopping solution (0.2 mM dipyridamole, 4.2 mM Na2-ethylenediaminetetraacetic acid, 5 mM 9-erythro (2- hydroxy-3-nonyl) adenine, 79 mM ␣,␤ methylene-adenosine-5'- Materials and Methods ␮ Patients diphosphate, 1 IU/ml heparin sulfate, 200 g/ml dcf, and 0.9% NaCl), which prevents degradation and uptake of Ado. The samples were For patients and control subjects, the ingestion of coffee or tea was transferred to special Vacutainer tubes (Ficoll-based CPT system; suspended 72 h before samples were taken. Patients who had been Becton Dickinson, Le Pont de Claix, France) and centrifuged at treated with papaverine, dipyridamole, or immunosuppressive agents 500 ϫ g for 30 min. Then 1 ml of interphase cells was pipetted off and during the preceding 6 mo were excluded from the study. All partic- washed three times with 3 ml of the stopping solution before assess- ipants gave their consent according to the Helsinki convention. Nine ment of cell viability by trypan blue dye test exclusion. Granulocyte adults (five men and four women) on maintenance HD, who were contamination and mononuclear cell number were measured (Coulter randomly chosen among 128 patients with end-stage renal failure in Beckman, Fullerton, CA). These techniques resulted in cell prepara- the same center, constituted the group of patients who were undergo- tions that were Ͼ98% viable and that contained Ͻ3% of granulocytes. Ϯ Ϯ ing HD. Their mean age SD was 69 11 yr (range, 49 to 85 yr), Lymphocyte proportion was always more than 95%. Cells were re- Ϯ and their mean duration of maintenance HD was 47 34 mo (range, suspended in stopping solution (6.5 Ϯ 1.5 ϫ 106 cell/ml) and frozen 17 to 132 mo). Causes of end-stage renal failure were diabetic (Ϫ80°C). Samples were submitted to four freeze-thaw cycles (Ϫ80°C, ϭ ϭ nephropathy (n 2), chronic glomerulonephritis (n 1), interstitial ϩ37°C) and centrifuged (2500 ϫ g for 10 min) to obtain clear ϭ ϭ nephritis (n 1), polycystic kidney disease (n 1), nephroangio- supernatant cell extracts. Supernatants were deproteinized by addition ϭ ϭ sclerosis (n 3), and cholesterol embolism (n 1). No patient had of 100 ␮l of perchloric acid (6N) and then centrifuged (1500 ϫ g for anti–human immunodeficiency virus or hepatitis B surface antigen or 10 min).