Microinflammation induces endothelial damage in hemodialysis patients: the role of convective transport

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1 originl rticle & 7 Interntionl Society of Nephrology Microinflmmtion induces endothelil dmge in hemodilysis ptients: the role of convective trnsport R Rmirez 1, J Crrcedo 1, A Merino 1, S Noguers 1, MA Alvrez-Lr 1, M Rodríguez 1, A Mrtin-Mlo 1, C Tett 2 nd P Aljm 1 1 Unidd de Investigción, Servicio de Nefrologí. Hospitl Universitrio Rein Sofí, Córdo, Spin nd 2 Fresenius Medicl Cre, Bd Homurg, Germny Crdiovsculr complictions re mjor cuse of mortlity in hemodilysis ptients. On-line hemofiltrtion comines convective clernce for removing lrge solutes with diffusion to remove smll solutes nd is ssocited with significnt reduction of inflmmtion nd improved ptient survivl. We compred on-line hemofiltrtion to high-flux hemodilysis () in ptients in sequentil mnner. At seline, 15 stle ptients on s compred with five control sujects showed significnt increses in CD14 þ CD16 þ cells, endothelil microprticles, nd endothelil progenitor cells (EPCs). After 4 months of on-line hemofiltrtion, the numer of CD14 þ CD16 þ cells, microprticles, nd EPCs decresed. After returning to for 4 months, ll mesured prmeters returned to their respective seline vlues. The numer of CD14 þ CD16 þ cells correlted with oth endothelil microprticles nd EPCs. We conclude tht on-line hemofiltrtion ttenutes endothelil dysfunction possily y decresing microinflmmtion. This effect my e directly cused y modultory effect of on-line hemofiltrtion on proinflmmtory cells or y complex interction tht encompsses wider removl of uremic toxins. Kidney Interntionl (7) 72, ; doi:1.138/sj.ki.5225; pulished online 11 April 7 KEYWORDS: circulting endothelil progenitor cells; endothelil microprticles; inflmmtion; on-line hemodifiltrtion; vsculr dmge; vsculr reprtion Correspondence: R Rmirez, Unidd de Investigción, Hospitl Universitrio Rein Sofí Avd Menendez Pidl S/N, Córdo 14. Spin. E-mil: mnuelr.rmirez.ssp@juntdendluci.es Received 31 Octoer 6; revised 15 Jnury 7; ccepted 14 Ferury 7; pulished online 11 April 7 End-stge chronic kidney disese (CKD) is ssocited with high prevlence of crdiovsculr complictions. 1 These CKD ptients suffer from chronic inflmmtory process, which cuses endothelil injury, 2 criticl event in the pthogenesis of therosclerosis. 3,4 Furthermore, in these ptients, uremic toxins nd inflmmtion re more potent predictors of rteriosclerosis thn the clssic risk fctors such s hypertension, dyslipoproteinemi, oesity, nd smoking; 5,6 mrkers of inflmmtion such s C-rective protein nd cytokines re independent predictors of ll cuse nd crdiovsculr mortlity. 7 9 In the lst yers, reserch in the iology of the endothelium hs provided new trgets to exmine endothelil dmge nd repir in humn pthology. Upon noxious insult, endothelil cells my undergo vesicultion ensuing in the relese of microprticles endothelil microprticles (EMP) into the loodstrem. EMP express endothelilspecific surfce mrkers reflective of prent cell sttus (e.g., ctivted nd poptotic). 1 On the other hnd, the moiliztion of one mrrow-derived endothelil progenitor cells (EPC) is criticl for the preservtion of the integrity of endothelium. 11 Endothelil repir requires the presence of EPC, which originte from the one mrrow s demonstrted y the expression of CD34 ( surfce mrker common to hemtopoietic stem cells nd mture endothelil cells) nd vsculr endothelil cell growth-fctor receptor 2 (VEGFR2 or kinse domin-relted (KDR) receptor). 12 Severl studies hve shown evidence of endothelil dmge nd dysfunction in hemodilysis ptients. Incresed levels of poptotic EMP hve een reported in plsm from CKD-5 ptients. In ddition, severl uthors hve reported tht in CKD-4 5 ptients there is decrese in EPC; 13 however, these findings re not uniform s other groups hve reported norml 14 or even incresed numer of circulting EPC. 15 The potentil for reversl of vsculr disese in CKD-5 ptients is unknown. The qulity nd technology of hemodilysis hve improved in reltion to iocomptiility of the mterils used nd the ility to remove lrger size uremic toxins molecules. By comining ultrfiltrtion (convective clernces for removing lrger solutes) with diffusion 18 Kidney Interntionl (7) 72,

2 (for removl of smll solutes), on-line hemodilfiltrtion (OL-HDF) offers highly effective dilysis modlity expnding the spectrum of removed uremic toxins from smll to middle-sized moleculr solutes. Recently, OL-HDF hs een ssocited with significnt reduction of inflmmtion nd improved survivl. 16,17 The im of this study ws to exmine whether meliortion of microinflmmtion using OL-HDF hs n effect on EMP nd ECP s iomrkers of endothelil injury nd repir, respectively. RESULTS Ptients Tle 1 illustrtes the chrcteristics nd lortory dt of ptients included in the study t seline. These prmeters remined unchnged during the study. Evlution of CD14 nd CD16 mononucler expression As compred with norml sujects, t seline (fter 2 months on high-flux hemodilysis ()), CKD-5 ptients exhiited high percentge of peripherl lood mononucler cells (PBMC) positive for CD14 nd CD16 s compred with helthy controls ( % vs %; P ¼.1). After 4 months on OL-HDF, these sme ptients showed mrked decrese in the percent of CD14 þ CD16 þ cells ( %, P ¼.1). A decrese in CD14 þ CD16 þ cells fter OL-HDF ws oserved in ll 15 ptients. During the following period, (ck to ), the percent of CD14 þ CD16 þ cells incresed to % (Po.1 vs OL-HDF) (Tle 2). Evlution of CD31 þ /Annexin V þ EMP nd EPC As oserved in representtive histogrm from the flowcytometer (Figure 1), smples from ptients t seline showed n incresed numer of EPC nnexin-v þ / CD31 þ s compred with helthy sujects. In ddition, in PBMC from ptients t seline the percentge of CD31, CD14, nd VEGFR2 EPC is incresed s compred with controls (Figure 2). Ptients on (seline) presented high numer of EMP events ( events/ml vs controls, P ¼.1, Figure 3). After 4 months on OL-HDF, the numer of events significntly decresed in ll ut one ptient ( events/ml, P ¼.45 vs seline). During the following 4 months of, the numer of events incresed in ll ptients to events/ml, level superimposle to tht of seline. Figure 3 shows high percentge of EPC in ptients t seline in comprison with controls ( %, P ¼.1). After 4 months on OL-HDF, the numer of events significntly decresed to % (P ¼.1 vs seline). In the following 4 months of, the numer of EPC returned to level superimposle to tht of seline ( , P ¼.1 vs OL-HDF, P ¼.9 vs seline). Tle 2 Mononucler expression of CD14 nd CD16 in ptients during the study periods seline OL-HDF CD * CD14+CD * All dt re expressed s mens7s.d. from 15 ptients studied. *Po.1 vs seline nd. Tle 1 Chrcteristics nd lortory vlues of the included ptients t seline Prmeters Bsl Helthy sujects SSC height R.1 Anexin. V Numer of ptients 15 Gender, mle/femle 11/4 Age (yer; men, minimum mximum) 55 (22 77) Dilysis vintge (months; men, minimum mximum) 49 (14 122) Durtion of dilysis tretment (minutes; men, 226 (18 272) minimum mximum) ekt/v (men, minimum mximum) 1.39 ( ) Leucocytes (men, minimum mximum) ( ) Hemogloin (g/dl; men, minimum mximum) (9.2 14) Ferritin (mcg/ml, men, minimum mximum) ( ) Alumin (gr/dl; men, minimum mximum) 4.6 ( ) CRP (mg/l; medin, minimum mximum) 2 (.7 9.1) Plsm TNF (pg/ml; men, minimum mximum) 41.9 ( ) Plsm IL-6 (pg/ml; men, minimum mximum) 18.9 ( ) 2 -microgloulin (men, minimum mximum) 27.2 ( ) PO 4 (mg/dl; men, minimum mximum) 5.24 ( ) C (mg/dl; men, minimum mximum) 9.4 ( ) C PO 4 (mg 2 /dl 2 ; men, minimum mximum) ( ) EPO dosge (mg) (1 8) CRP, C-rective protein; EPO, erythropoietin; IL-6, interleukin-6; TNF, tumor necrosis fctor. c seline SSC height FSC height R.1 d Anexin. V FSC height Figure 1 Phenotype of endothelil microprticles (EMP). Representtive grphs of cytofluorometry nlysis of EMP in plteletfree plsm from helthy sujects ( nd ) nd ptients in t seline (c nd d). ( nd c) EMP re represented on forwrd sctter/side-sctter dot-plot histogrm. EMP re defined s events, with size of.1 1 mm, nd re gted in R1 window. ( nd d) Sizeselected events re plotted s function of their doule fluorescence for CD31-FITC nd specific Annexin-PE inding. Kidney Interntionl (7) 72,

3 Helthy sujects c seline Counts Counts R1 YEGFR2PE R1 d CD14TC YEGFR2PE CD14TC Figure 2 Phenotype of endothelil progenitor cells (EPC). Representtive grphs of cytofluorometry nlysis of EPC in lood from helthy sujects ( nd ) nd ptients in t seline (c nd d). The phenotype of EPC s indicted y triple-positive for CD31, CD14, nd VEGFR2. ( nd c) VEGFR2 re represented on histogrm of fluorescence, nd re gted in R1 window. ( nd d) Positive events for VEGFR2 (R1 gted) re plotted s function of their fluorescence for CD14 nd CD31. EMP (events/μl) EPC (%) seline seline * HDF-OL * HDF-OL Figure 3 Evlution of EMP nd EPC during study periods. () EMP t seline ptients on ( events/ml), fter 4 months on OL-HDF ( events/ml), nd fter 4 months in ( events/ml). *P ¼.45, OL-HDF vs seline, P ¼.48 vs OL-HDF). The open circles represent the vlues for EMP in helthy sujects. () EPC t seline ptients on ( %), fter 4 months on OL-HDF ( %), nd fter 4 months in ( %). *Po.1, OL-HDF vs seline, Po.1 vs OL-HDF). The open circles represent the vlues for EMP in helthy sujects. Correltion etween microinflmmtion (CD14 þ CD16 þ cells) nd endothelil dmge (EMP nd EPC) Interestingly, the numer of CD14 þ CD16 þ cells correlted significntly with oth EMP (r ¼.63, P ¼.3; Figure 4) nd EPC (r ¼.57, P ¼.4; Figure 4) in lood smples from nd OL-HDF. However, there ws no correltion etween EPC nd EMP (dt not shown). To evlute the effects of one dilysis session of ech modlity on endothelil dmge, EPC nd EMP were evluted efore nd fter tretment session t the eginning of ech of the two periods. Although EMP incresed significntly t the end of (t oth, seline nd fter the lst 4 months on ), no significnt chnges occurred fter OL-HDF. A significnt reduction of EPC ws oserved in oth nd in OL-HDF (Figure 5). These results could not e ttriuted to dilutionl effect, s net fluid lnce ws not different in oth procedures. DISCUSSION Our results clerly show tht significntly high numer of circulting CD31 þ /Annexin V þ EMP were oserved in ptients on HD-HF s compred with helthy controls. The EMP (events/μl) r =.63 P =.3 EPC (%) r =.57 P = CD14 + CD16 + cells Figure 4 Correltion etween CD14 þ CD16 þ cells nd EMP or EPC. () CD14 þ CD16 þ cells percent were correlted with the numer of EMP of ptients in in seline (closed circles) nd OL-HDF (open circles). () CD14 þ CD16 þ cells percent were correlted with the numer of EPC of ptients in in seline (closed circles) nd OL-HDF (open circles). EMP (events/μl) 2 * ** OL-HDF EPC (%) * # ** OL-HDF Figure 5 Evlution of EMP nd EPC fter tretment session. EMP () nd EPC () t seline ptients on, fter 4 months on OL-HDF, nd fter 4 months in. The lck rs represents vlues efore tretment session HD, nd the empty rs re vlues fter HD session. () EMP incresed fter hemodilysis session in oth group of ptients in ut not in OL-HDF. *P ¼.2, **P ¼.7. () A significnt reduction in EPC ws oserved in oth HD-HF nd in OL-HDF. *P ¼.21, **P ¼.1, # P ¼ Kidney Interntionl (7) 72,

4 elevted numer of EMP ws ssocited with n lso remrkly high count of EPC. Both circulting EMP nd EPC were reduced fter 4 months of OL-HDF. This study confirms our previously pulished dt on the reduction of proinflmmtory CD14 þ CD16 þ monocytederived dendritic cells in ptients on OL-HDF. 17 This study shows tht the rise of CD14 þ CD16 þ cells is ssocited with proportionl increses in oth ECM nd EPC. An emerging interest hs een drwn from recent humn studies underlying the ssocition of EMP to endothelil dysfunction nd rteril dysfunction in CKD. 18,19 By the liner regression nlysis, we documented significnt correltion etween proinflmmtory CD14 þ CD16 þ cells, nd oth the numer of EMP s well s the numer of EPC. It is tempting to speculte tht CD14 þ CD16 þ monocytederived dendritic cells my hve dul effect triggering endothelil ctivtion nd injury leding to the relese of EMP nd the stimultion of the one mrrow to increse the production of circulting EPC. We were unle to clim direct cuse-nd-effect reltionship etween microinflmmtion, formtion of EMP, nd incresed numers of circulting EPC. Other fctors such s uremic toxins, or other ctivted cells (e.g., pltelets, leukocytes) my come into ply. Jcoi et l. 2 hve implicted the priming of polymorphonucler neutrophils s the min mechnism of the initition of endothelil injury. It is of interest tht no increse in EMP ws oserved in postdilysis lood smples fter OL-HDF. In ll cses, HF- HD ws ssocited with remrkle increse of EMP. In oth nd OL-HDF, EPC decresed significntly in the postdilysis lood smples in respect to the predilysis ones. The unique relevnce of convection on the oserved chnges ws due to the fct tht oth dilysis modlities, HD- HF nd OL-HDF were in fct equl for the memrne used nd wter qulity. To insure uniform procedures nd qulity stndrds, we performed these studies in one single center. Our study shows, for the first time, tht reduction of the monocyte supopultion responsile for cytokine production y OL-HDF my decrese the dmge of the endothelium. Our study hs numer of limittions. First, the lck of rndomiztion of the ptients to different sequences of tretments might in principle hve left the potentil for residul confounding y unrecognized fctors or cointerventions over time. However, s in our previous puliction, lso in the present studies we oserved tht the percent of CD14 þ /CD16 þ cells ws not chnged fter 4 months of (first nd third period), thus ttenuting mjor impct y time nd/or sesonl fctors. Second, lthough EPM nd EPC re considered s mrkers of endothelil dmge nd repir, respectively, they cnnot e considered s surrogte mesures for crdiovsculr outcome. Furthermore, there re controversies on the identifiction, definition, nd significnce of EPC in the pthophysiology of different diseses. According with Ingrm et l., 12 our EPC were sorted y three monoclonl ntiodies nti-cd14, -CD31, nd -VEGFR2. The ltter hs een shown to e expressed on peripherl lood cells tht proliferte in vivo nd tht contriute to re-endotheliliztion. Recent studies lso suggest tht circulting detched endothelil cells nd stimulted EPC from the one mrrow my reflect endothelil injury. 21,22 In this study, we did not ssess the plsm levels of VEGF, stimulus for EPC relese from one mrrow. Therefore, we cnnot exclude tht VEGF levels my hve some responsiility of the oserved chnges. Also erythropoietin (EPO) is strong stimulus for EPC relese. However, there ws no significnt chnge in the routine EPO dose in ny of the ptients throughout the study period. Third, the effect of OL-HDF ws only 15%, wheres the difference etween ptients nd helthy controls ws fr more sustntil (% higher levels in ptients thn in helthy sujects). Even the most effective dilysis technique (OL- HDF) ws fr from producing iologiclly relevnt effect on endothelil dysfunction. Nevertheless, one must rememer tht the ptient popultion ws selected to identify ptients who hd no signs or symptoms s well s lortory mrkers of n ongoing overt inflmmtion (s shown y stly low C-rective protein nd EPO dose during the 8-month follow up. In cross-sectionl study, Chn et l. 15 showed the normliztion of EPC counts nd function in ptients on nocturnl dily hemodilysis for t lest 1 yer. Finlly, the uremic milieu my lso ply role. Fure et l. 19 evluted the effect of specific uremic fctors on EMP relese in vitro, nd showed tht some uremic toxins such s p-cresol nd indoxyl sulfte re le to induce the vesicultion of cultured endothelil cells. Bmmens et l. 23 showed tht OL-HDF significntly reduces the levels of p-cresol in respect to, thus possily suggesting n effect of high convective trnsfer in the removl of wider spectrum of uremic toxins thn tht oserved in predominntly diffusive therpies such s. The eneficil effect of high convective trnsport (OL-HDF) my e directly cused y modultion of proinflmmtory CD14 þ CD16 þ monocyte-derived dendritic cells or y more complex interction tht lso encompsses removl of wider spectrum of uremic toxins. Whtever the mechnism/s might e t work, OL-HDF is ssocited with less formtion of EMP nd relese of EPC in the circultion. In conclusion, our studies provide the evidence tht microinflmmtion is ssocited to endothelil dmge nd tht meliortion of the microinflmmtory sttus using high convective trnsport ppers to reduce endothelil dmge. Further studies will need to wrrnt whether long-term OL- HDF ttenutes endothelil dysfunction nd microinflmmtion in ptients with overt systemic inflmmtion nd whether this cn e ssocited to the improved survivl. MATERIALS AND METHODS Study design A totl of 15 stle HD ptients were included. All ptients hd een treted for more thn 6 months in the stellite dilysis fcility of Kidney Interntionl (7) 72,

5 Rein Sofi University Hospitl of Cordo nd remined in the sme fcility throughout the study. The sme group of ptients ws sequentilly treted with two different modlities of HD during 4 months ech: nd OL-HDF. Both procedures re regulrly used in ptients on mintennce hemodilysis; however, OL-HDF offers more convective trnsport, which results in removl of wider spectrum of uremic toxins. All ptients were on during t lest 2 months (seline), then, ptients were switched to OL-HDF for n dditionl period of 4 months nd finlly ptients were chnged ck to for 4 months. Five pprently helthy sujects (ge: 4976 yers; gender: mle 3/femle 2) were studied. Ptient nd seline dt At seline ll ptients were on using polysulfone memrnes (1.8 m 2, HF8, Fresenius Medicl Cre, Bd Homurg, Germny) using 8 dilysis monitors (Fresenius Medicl Cre). nd OL-HDF were performed with ultrpure icronteuffered dilyste three times week. The verge volume of on-line reinfusion fluid per session ws 19.7 l (rnge: ). Anlysis of the dilysis system lwys reveled sence of cteri (o1 cfu/ml (colony-forming units/milliliter)) or cteriologicl contminnt products (endotoxin levels elow.25 endotoxin units) during the whole period of the study. The dilysis chrcteristics were similr in the two modlities, the only difference eing higher convective trnsport in the OL-HDF thn. The therpeutic regime consisted in EPO, clcium-contining phosphte inders, ntihypertensive drugs, nd intrvenous iron. All ptients were on EPO from the eginning of the study. EPO dosge ws djusted to mintin the hemogloin trget of g/dl. Weekly EPO dose ws similr in oth modlities of HD long the study. Ptients with symptoms or signs of inflmmtion, infection, nd cncer were excluded. None of the ptients were on nti-inflmmtory or immunosuppressive drugs during the 3 months efore inclusion nd for the whole period of the study. None of them hd dietes. Informed consent ws otined from ll ptients. All routine lortory mesurements were done using certificte ssy methods. The effect of ech experimentl period ws ssessed, y otining the lood smples just efore the first dilysis session of the following study period. To nlyze the effects of one dilysis session of ech dilysis modlities ( nd OL-HDF), lood smples were otined just efore nd fter the corresponding first weekly dilysis session. Experimentl procedures Humn mononucler cell culture. PBMC nd plsm were otined from 2 ml of citrte-uffered lood drwn immeditely efore dilysis session. To nlyze the effect of single dilysis procedure, lood smples were otined efore nd fter completion of the dilysis session. Buffy cot cells were seprted y differentil centrifugtion grdient (Ficoll/Hypque; Phrmci LKB, Uppsl, Sweden). Therefter, cells were wshed nd seeded in 12-well t 371C in complete culture medium contining RPMI- 16 supplemented with L-glutmine (2 mm), Hepes (2 mm), sodium pyruvte (1 mm), streptomycin (5 ng/ml), penicillin (1 UI/ml), nd 1% humn utologous serum (Bio-Whittker, Wlkersville, MD, USA). Serum ws heted (561C, 6 min) to inctivte complement. Cytometric ed rry immunossy. The mount of cytokines in plsm ws evluted using Cytometric ed rry immunossy. The cytometric ed rry technique ws sed on the inding of microprticles leled with different fluorescence intensities to ntiodies. The mximum wvelength emission ws out 65 nm (fluorescence-3 chnnel). The prticles were ound y covlent ond to n ntiody ginst one of the cytokines tumor necrosis fctor- or interleukin-6 (Phrmingen, Sn Diego, CA, USA). This method llowed the simultneous determintion of different cytokines in the sme smple. The cytokines were cptured directly in the immunossy using different ntiodies ound to phycoerythrin (PE), which emitted t 585 nm (fluorescence-2 chnnel). The PE-conjugted detector ntiody completed the sndwich, nd the intensity mesured in fluorescence-2 chnnel ws proportionl to the concentrtion of cytokines in the smple, which ws quntified from clirtion curve. An importnt chrcteristic of the ssy system ws tht the clirtors, the ntiody ed regent, nd the second ntiody regent, ntiody PE were mde from mixtures of cytokines. Stndrd curves ( 5 pg/ml) were derived from set of clirtors nd the sme set ws used for ll ssys. Then, 5 ml of the smple or cytokine stndrd ws dded to the mixture of 5 ml ech of ntiody PE detector nd ntiody - ed regent. The mixture (15 ml) ws incuted for 16 min in the drk t room temperture, wshed, nd the test smples were cquired using the flow cytometer. Evlution of CD14 nd CD16 mononucler expression. To quntify proinflmmtory cells (CD14 þ CD16 þ ) smple of 1 5 /ml PBMC ws incuted during 3 min, t 41C with the monoclonl ntiodies (M) M5E2 ginst the molecule CD14 nd 3G8 ginst the molecule CD16 (Becton Dickinson, Sn José, CA, USA). Cytofluorometric nlysis ws performed with FACSCliur cytometer (Becton Dickinson). Determintion. of CD31 þ /Annexin V þ EMP. Apoptotic ECM were isolted s descried erlier. 1 Plsm derived from 2-ml citrte-uffered lood ws seprted from whole lood nd immeditely centrifuged t 13 g for 5 min to generte plteletpoor plsm. Pltelet-poor plsm (5 ml) ws incuted with 4 ml of monoclonl ntiody ginst CD31 (MHCD314-PE Cltg L, Burlingme, CA, USA) followed y incution with fluorescein isothiocynte-conjugted Annexin V kits ccording to the mnufcturer s instructions (Bender MedSystem, Vienn, Austri). Fluorescence-ctivted cell sorter nlysis ws performed in FACSCliur cytometer. The negtive control (zero vlue) ws the vlue otined using the isotype ntiodies. Ech result (one single vlue) ws the verge of three independent determintions of the sme smple. Chrcteriztion of EPC. The EPCs were defined s cells triplepositive for CD31, CD14, nd VEGFR2. 12 Previous studies hve demonstrted tht these cells re ctively involved in endothelil repir. 12. Immeditely fter lood extrction, PBMC were seprted y Ficoll, wshed with phosphte-uffered sline, nd resuspended in EBM-2 medium supplemented with ntiiotics nd 2% of fetl ovine serum. Therefter cells were incuted for 3 min in the drk t 41C with sturting concentrtions of the following monoclonl ntiodies (ma): nti-vegfr2 (ntihumn VEGFR2 KDR-PE; R&D System, Minnepolis, MN USA), nti-cd31 (MHCD311FITC, Cltg L), nd nti-cd14 (MHCD16TRI- COLOR) nd corresponding isotype controls. Then, cells were wshed with phosphte-uffered sline, nd the test smples were nlyzed using the flow cytometer. Quntittive nlysis ws performed on FACSCliur cytometer (Becton Dickinson) mesuring events per smple. Dt were nlyzed using CELLQuest softwre (Becton Dickinson) y side sctter-fluorescence 112 Kidney Interntionl (7) 72,

6 dot plot nlysis. The numer of EPCs ws defined s events triplepositive for CD31, CD14, nd VEGFR2. The negtive control (zero vlue) ws the vlue otined using the isotype ntiodies. Ech result (one single vlue) ws the verge of three independent determintions of the sme smple. Sttisticl nlysis. Comprison etween mens from different periods ws performed y repeted mesures nlysis of vrince followed y Bonferroni test. The comprison of two mens ws done y Student s t-test for pired dt. Non-prmetric dt were nlyzed y Wilcoxon or Mnn Whitney test for pired nd unpired comprison, respectively. The Spermn rnk correltion coefficient ws used to nlyze correltion. Differences were considered significnt when Po.5. ACKNOWLEDGMENTS This work ws supported y grnts from Fondo de Investigciones Científics de l Seguridd Socil (FIS PI3/946, PI5/896, PI6/724, PI6/747, RETICs Red Renl RD6/16/7), Junt de Andlucí (27/5, TCRM 6/6), nd Fundción Nefrológic. We re grteful to ll professionls from Stellite Dilysis Unit of Rein Sofi Hospitl for the effort to complete this work nd to MJ Jimenez for technicl ssistnce. J Crrcedo nd M Rodríguez were supported y contrct Instituto de Slud Crlos III/Fundción Progreso y Slud (Progrm de Estilizción e Incentivción de l Investigción 6). REFERENCES 1. Foley RN, Prfrey PS, Srnk MJ. Clinicl epidemiology of crdiovsculr disese in chronic renl disese. Am J Kidney Dis 1998; 32(Suppl 3): S112 S Pwlk K, Numnik B, Brzosko S et l. Oxidtive stress link etween endothelil injury, cogultion ctivtion, nd therosclerosis in hemodilysis ptients. Am J Nephrol 4; 24: Dvignon J, Gnz P. Role of endothelil dysfunction in therosclerosis. Circultion 4; 19(Suppl 1): III27 III Lndmesser U, Hornig B, Drexler H. Endothelil function: criticl determinnt in therosclerosis? Circultion 4; 19(Suppl 1): II27 II Vnholder R, De Smet R, Glorieux G et l. Europen Uremic Toxin Work Group (EUTox). Review on uremic toxins: clssifiction, concentrtion, nd interindividul vriility. Kidney Int 3; 63: Ronco C, Tett C. Dilysis ptients nd crdiovsculr prolems: cn technology solve the complex eqution? Expert Rev Med Devices 5; 2: Kysen GA, Duin JA, Muller HG et l. The cute-phse response vries with time nd predicts serum lumin levels in hemodilysis ptients. The HEMO Study Group. Kidney Int ; 58: Pnichi V, Mggiore U, Tccol D et l. Interleukin-6 is stronger predictor of totl nd crdiovsculr mortlity thn C-rective protein in hemodilysis ptients. Nephrol Dil Trnsplnt 4; 19: Zoccli C. Trditionl nd emerging crdiovsculr nd renl risk fctors: n epidemiologic perspective. Kidney Int 6; 7: Jimenez JJ, Jy W, Muro LM et l. Endothelil microprticles (EMP) s vsculr disese mrkers. Adv Clin Chem 5; 39: Msud H, Klk C, Ashr T. Endothelil progenitor cells for regenertion. Hum Cell ; 13: Ingrm DA, Cplice NM, Yoder MC. Unresolved questions, chnging definitions, nd novel prdigms for defining endothelil progenitor cells. Blood 5; 16: Herrig K, Pistrosch F, Oelschlegel U et l. Incresed totl numer ut impired migrtory ctivity nd dhesion of endothelil progenitor cells in ptients on long-term hemodilysis. Am J Kidney Dis 4; 44: Choi JH, Kim KL, Huh W et l. Decresed numer nd impired ngiogenic function of endothelil progenitor cells in ptients with chronic renl filure. Arterioscler Throm Vsc Biol 4; 24: Chn CT, Li SH, Verm S. Nocturnl hemodilysis is ssocited with restortion of impired endothelil progenitor cell iology in end-stge renl disese. Am J Physiol Renl Physiol 5; 289: F679 F Cnud B, Brgg-Greshm JL, Mrshll MR et l. Mortlity risk for ptients receiving hemodifiltrtion versus hemodilysis: Europen results from the DOPPS. Kidney Int 6; 69: Crrcedo J, Merino A, Noguers S et l. On-line hemodifiltrtion reduces the proinflmmtory CD14+CD16+ monocyte-derived dendritic cells: prospective, crossover study. J Am Soc Nephrol 6; 17: Amile N, Guerin AP, Leroyer A et l. Circulting endothelil microprticles re ssocited with vsculr dysfunction in ptients with end-stge renl filure. J Am Soc Nephrol 5; 16: Fure V, Dou L, Stier F et l. Elevtion of circulting microprticles in ptients with chronic renl filure. J Throm Hemost 6; 4: Jcoi J, Sel S, Cohen HI et l. Priming of polymorphonucler leukocytes: culprit in the initition of endothelil cell injury. Am J Physiol Hert Circ Physiol 6; 29: Herrig K, Pistrosch F, Oelschlegel U et l. Incresed totl numer ut impired migrtory ctivity nd dhesion of endothelil progenitor cells in ptients on long-term hemodilysis. Am J Kidney Dis 4; 44: Shintni S, Murohr T, Iked H et l. Moiliztion of endothelil progenitor cells in ptients with cute myocrdil infrction. Circultion 1; 13: Bmmens B, Evenepoel P, Vereke K et l. Removl of the protein-ound solute p-cresol y convective trnsport: rndomized crossover study. Am J Kidney Dis 4; 44: Kidney Interntionl (7) 72,