Post-dilution haemodiafiltration and low-flux haemodialysis have dissimilar effects on platelets: a side study of CONTRAST

Nephrology Dialysis Transplantation, Nov 2009

Background. Cardiovascular disease (CVD) is the leading cause of death in patients with end-stage renal disease (ESRD). Platelet (PLT) dysfunction, which is a well-known phenomenon in advanced chronic renal failure, corresponds positively with CVD in these patients. The accumulation of retained uraemic toxins might play an important role in this respect. During haemodialysis (HD), both an increase in the expression of the platelet (PLT) cell surface molecule P-selectin (CD62p) and the release of intra-granular substances, such as platelet factor 4 (PF4) and ß-thromboglobulin (BTG), have been described. As the removal of uraemic toxins is superior during haemodiafiltration (HDF), this form of treatment may have quite another impact on PLTs than HD. Methods. Nineteen chronic HD patients who were treated with low-flux HD for at least 2 months were included in the Dutch CONvective TRAnsport STudy (CONTRAST). After randomization, 10 patients continued low-flux HD and 9 patients switched to post-dilution HDF. The present study describes various parameters of PLT activation and degranulation at baseline (during HD) and after 3 months (during HDF) in the latter group of patients. At both time points, multiple blood samples were drawn. During the first 30 min of treatment, differences over the extracorporeal circuit (ECC) were calculated by taking samples from both afferent (arterial) and efferent (venous) lines. Correlations between various parameters were calculated in the total group of patients after 3 months. Results. Immediately after the start of HD, PLT counts dropped over the ECC. During HDF, PLT counts decreased even more and reached a nadir at t30. CD62p expression increased early during HD and returned to baseline thereafter. During HDF, these changes were more pronounced and more protracted. With respect to degranulation, rather dissimilar results were obtained. During HD, both PF4 and BTG increased over time, whereas during HDF, PF4 increased but BTG did not change. Haemoconcentration and transmembrane pressure (TMP) within the dialyser were, respectively, ∼10 and 3× higher during HDF than during HD. There was a striking correlation between the changes in haemoconcentration and the changes in both PLT counts and CD62p over the ECC. Summary and Conclusions. PLT activation, as measured by the expression of CD62p, was more pronounced and more protracted during HDF than during HD. During HDF, PLTs were trapped abundantly within the ECC, not only after first passage, but also thereafter. The degranulation product BTG increased during HD, but did not change during HDF. These observations may well be explained by the greater haemoconcentration and/or higher TMP during HDF on the one hand, and superior convective transport at the other. Whether the potential harmful effects of enhanced PLT activation are counterbalanced by the beneficial effects of an increased convective transport of degranulation products remains to be established.

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Post-dilution haemodiafiltration and low-flux haemodialysis have dissimilar effects on platelets: a side study of CONTRAST

Mareille Gritters-van den Oever 1 2 Muriel P. C. Grooteman 1 3 Piet C. M. Bartels 0 Peter J. Blankestijn 3 6 Michiel L. Bots 3 5 Marinus A. van den Dorpel 3 4 Marianne Schoorl 0 Margreet Schoorl 0 Piet M. ter Wee 1 3 Menso J. Nube 1 2 3 0 Department of Clinical Chemistry, Haematology and Immunology, Medical Centre Alkmaar , Alkmaar 1 Department of Nephrology, VU University Medical Centre , Amsterdam 2 Department of Nephrology, Medical Centre Alkmaar , Alkmaar 3 Executive Committee of CONTRAST 4 Department of Internal Medicine, Maasstad Hospital , Rotterdam, The Netherlands 5 Julius Centre for Health Sciences and Primary Care, University Medical Centre Utrecht , Utrecht 6 Department of Nephrology, University Medical Centre Utrecht Background. Cardiovascular disease (CVD) is the leading cause of death in patients with end-stage renal disease (ESRD). Platelet (PLT) dysfunction, which is a wellknown phenomenon in advanced chronic renal failure, corresponds positively with CVD in these patients. The accumulation of retained uraemic toxins might play an important role in this respect. During haemodialysis (HD), both an increase in the expression of the platelet (PLT) cell surface molecule P-selectin (CD62p) and the release of intra-granular substances, such as platelet factor 4 (PF4) and -thromboglobulin (BTG), have been described. As the removal of uraemic toxins is superior during haemodiafiltration (HDF), this form of treatment may have quite another impact on PLTs than HD. Methods. Nineteen chronic HD patients who were treated with low-flux HD for at least 2 months were included in the Dutch CONvective TRAnsport STudy (CONTRAST). After randomization, 10 patients continued low-flux HD and 9 patients switched to post-dilution HDF. The present study describes various parameters of PLT activation and degranulation at baseline (during HD) and after 3 months (during HDF) in the latter group of patients. At both time points, multiple blood samples were drawn. During the first 30 min of treatment, differences over the extracorporeal circuit (ECC) were calculated by taking samples from both afferent (arterial) and efferent (venous) lines. Correlations between various parameters were calculated in the total group of patients after 3 months. Results. Immediately after the start of HD, PLT counts dropped over the ECC. During HDF, PLT counts decreased even more and reached a nadir at t30. CD62p expression increased early during HD and returned to baseline thereafter. During HDF, these changes were more pronounced and more protracted. With respect to degranulation, rather dissimilar results were obtained. During HD, both PF4 and BTG increased over time, whereas during HDF, PF4 inC The Author 2009. Published by Oxford University Press [on behalf of ERA-EDTA]. All rights reserved. For Permissions, please e-mail: - creased but BTG did not change. Haemoconcentration and transmembrane pressure (TMP) within the dialyser were, respectively, 10 and 3 higher during HDF than during HD. There was a striking correlation between the changes in haemoconcentration and the changes in both PLT counts and CD62p over the ECC. Summary and Conclusions. PLT activation, as measured by the expression of CD62p, was more pronounced and more protracted during HDF than during HD. During HDF, PLTs were trapped abundantly within the ECC, not only after first passage, but also thereafter. The degranulation product BTG increased during HD, but did not change during HDF. These observations may well be explained by the greater haemoconcentration and/or higher TMP during HDF on the one hand, and superior convective transport at the other. Whether the potential harmful effects of enhanced PLT activation are counterbalanced by the beneficial effects of an increased convective transport of degranulation products remains to be established. Introduction During haemodialysis (HD), various side effects occur, including the activation of protein systems in the blood and the stimulation of circulating elements. The sum of these undesirable side effects has been termed bio-incompatibility [1]. With respect to platelets (PLT), both an increase in the expression of cell surface molecules and the release of intra-granular substances, such as platelet factor 4 (PF4), platelet-derived growth factor (PDGF) and -thromboglobulin (BTG), have been well documented [2]. During HD, activated PLTs adhere to circulating elements, including other PLTs, neutrophils, lymphocytes [3], monocytes and erythrocytes [4]. As a result of these cellcell interactions, a mixture of micro-aggregates is formed, with specific pathophysiological effects. Flow cytometric analysis showed production of reactive oxygen species after the intra-dialytic formation of plateletneutrophil aggregates [5]. In the clinical situation, PLT reactivity corresponded positively with cardiovascular morbidity and mortality, both in non-renal patients [6] and in patients with end-stage renal disease (ESRD) [7]. Moreover, in HD patients, PLT activation correlated with bleeding and thrombotic diathesis [8] and vascular access failure [9]. The cause of PLT dysfunction in patients with ESRD is multi-factorial. Both uraemic and HD-related factors might play a role. Not only the type of dialyser [4] but also other components of the extracorporeal circuit (ECC) [10] and the type of anticoagulant [11] influence PLTs. As PLT abnormalities occur early in the course of renal failure [12], the uraemic milieu and particularly the retention of uraemic toxins probably also have a profound influence on these elements [13]. Therefore, dialysis techniques with a high convective transport, such as haemodiafiltration (HDF), may reduce the degree of PLT activation and improve PLT reactivity. However, as a high convection volume can only be reached at the cost of substantial haemoconcentration, at least during post-dilution HDF, and an increased transmembrane pressure (TMP) within the dialyser, it is conceivable that undesirable side effects occur. The present analysis is a single-centre side study of CONTRAST, the Dutch multicentre randomized controlled trial comparing the effects of post-dilution HDF with low-flux HD on all-cause mortality and cardiovascular morbidity and mortality [14]. At baseline and 3 months after randomization, PLT numbers, the expression of the cell surface marker CD62p and the degranulation products PF4 and BTG were measured. In order to establish the role of the ECC in PLT activation and degranulation, blood was sampled from both afferent (arterial) and efferent (venous) lines at various time points during treatment. Subjects and methods In our centre, 19 stable ESRD patients [11 males and 8 females, median age 63 years (2882)] undergoing low-flux HD treatment for at least 2 months [median 25 months (783)] participated in CONTRAST. Of these patients, 10 were randomized to treatment with low-flux HD and 9 to post-dilution HDF. In the latter group, PLT parameters were measured prospectively before (during HD) and after the switch to HDF at 3 months, thus excluding patient and non-HD(F)-treatment-related factors as much as possible. Correlations between various parameters were calculated cross-sectionally by combining the data of the total group of patients (n = 19) 3 months after randomization. The aetiology of renal insufficiency was hypertensive nephrosclerosis in eight patients, diabetic nephropathy in five and adult dominant polycystic kidney disease in three patients. The other three patients suffered from IgA nephropathy, membranous nephropathy and tubulo-interstitial nephritis. Individuals who used platelet inhibitors or other medication interfering with PLT function were excluded from this study. EPO dose [median 10 000 (2000 30 000 IU)] per week remained unchanged over the study period. Written informed consent was obtained in all cases. The protocol was approved by the local Medical Ethical Committee and the Executive Committee of CONTRAST. All parameters were measured over time both prior to randomization (M0) and after 3 months (M3). Samples were taken from the fistula (t0) and from the efferent line, i.e. after the dialyser and before the addition of substitution fluid in HDF, after 1 (t1), 5 (t5), 30 (t30), 60 (t60) and 150 (t150) min. Furthermore, samples were drawn from the afferent lines, i.e. before the roller pump, after 5 min (t5aff) and 30 min (t30aff). The calculated differences over the ECC (delta, = efferent value afferent value) at t1, t5 and t30 were designated as t1 (first passage), t5 and t30, respectively. Platelet numbers were corrected for changes in plasma volume based on haematocrit (Ht) measurements: [corrected valuetx = valuetx (Htt0/Httx)]. All other results were corrected for changes in plasma volume, based on a different, more complex, formula: corrected valuetx = valuetx [Htt0/(1 Htt0)][(1 Httx)/Httx]. Haemodialysis procedure and materials Only first-use low-flux polysulfone (PS) dialysers [F8 HPS, Fresenius Medical Care, Bad Homburg, Germany; ultrafiltration (UF) factor 18 ml/h mmHg, surface area 1.8 m2, steam sterilized] were utilized on Fresenius 4008 and 5008 series dialysis machines. Bicarbonate dialysate was used with a dialysate flow of 500 ml/min, whereas dialysate temperature was kept at 36C. The production of ultrapure water was similar to HDF (see below). All dialysers were pre-rinsed with 1000 ml 0.9% NaCl. Individual doses of dalteparin were based on bodyweight (50 IU/kg) and given as a bolus injection at the beginning of the dialysis session (mean 4750 1419 IU). According to the CONTRAST protocol, treatment times were fixed at baseline and spKt/V urea was 1.2 per treatment. Haemodiafiltration procedure and materials Only first-use high-flux PS dialysers [FX80 Helixone R , Fresenius Medical Care, Bad Homburg, Germany; ultrafiltration (UF) factor (Kuf) 59 ml/h mmHg, surface area 1.8 m2, steam sterilized] were used. A sterile and non-pyrogen substitution solution was prepared online from a dialysate concentrate and ultrapure dialysate using the ONLINEplustm system (Fresenius Medical Care), which contains two ultrafilters (DIASAFE R plus) and is integrated in the Fresenius 4008 and 5008 series dialysis machines. Ultrapure quality was defined as bacterial counts <0.1 CFU/ml and endotoxin levels <0.025 EU/ml. The microbiological water quality of the dialysis solutions was regularly monitored according to the guidelines of the Dutch Federation of Nephrology. Bicarbonate was provided from powder cartridges (biBAG, Fresenius Medical Care). The dialysate flow was kept at 500 ml/min, with a dialysate temperature of 36C. According to the CONTRAST protocol, the blood flow was 250400 ml/min, treatment times were fixed and spKt/V urea was 1.2 per treatment. HDF was provided in the post-dilution modus with a target convection volume of 6 l/h. All dialysers were pre-rinsed with 1000 ml 0.9% NaCl. The dalteparin protocol was similar to HD, although in the first months after randomization two patients were switched to higher doses (mean 5250 1845 IU, n = 9) following an episode of coagulation in the dialyser. Transmembrane pressure (TMP) and haemoconcentration As the TMP is not displayed on the dialysis machines, an estimation was made based on dialyser membrane characteristics (Kuf) and ultrafiltration (UF) rate (Quf) according to the formula: TMP = Quf/Kuf. In addition, the UF pressures, which are shown on the display of the dialysis machines, were noted throughout the HD and HDF sessions and served as a surrogate marker of TMP. Both filtration fraction (FF) and haemoconcentration (% increase in hematocrit) in the ECC were calculated based on Ht levels in the afferent and efferent lines at t5 and t30. Platelet counts. Blood samples were collected in K2EDTA (ethylene diamine tetraacetic acid) tubes (Becton Dickinson, Plymouth, UK) and PLT numbers were determined using a Sysmex XE2100 Haematology analyser (Sysmex Corporation, Kobe, Japan). Platelet surface markers. The platelet surface markers CD62p (P-selectin; clone CLB Thromb 6, Beckman Coulter, Mijdrecht, The Netherlands) and CD41 (clone P2, Beckman Coulter, Mijdrecht, The Netherlands) were detected by direct labelling and flow cytometry. Blood was drawn into K2EDTA tubes and within 2 h after collection incubated with a glycoprotein-specific fluorochrome-labelled monoclonal antibody. A flow cytometer (Epics XL, Beckman Coulter, Mijdrecht, The Netherlands) was used to determine the percentage of platelets with CD62p surface expression. CD41 served as a platelet-specific label. Platelet factor 4 and beta-thromboglobulin. Blood samples were drawn into CTAD tubes (Vacutainer R CTAD, Becton Dickinson, Plymouth, UK), cooled on ice and centrifuged for 20 min at 2500 g. Plasma samples were stored at 70C until measurement. PF4 and BTG were determined using commercially available sandwich ELISA kits (Asserachrom PF4 R and Asserachrom b-TG kit R , Diagnostica Stago, Asnie`res, France). All analyses were performed with the SPSS 15.0 software system. A general linear model (GLM) for repeated measures was applied to study various aspects of platelet activation during a single session both at baseline (HD) and 3 months after randomization (HDF). The GLM was applied as it allows the simultaneous assessment of the effects of both time and treatment modality on outcome measures. Furthermore, GLM was used to analyse the effect of treatment modality and sampling point [efferent versus afferent, delta () over the ECC] on the same outcome parameters. In the case of significance, post hoc paired t-tests were applied. Finally, correlations between haemoconcentration, PLT numbers and CD62p were calculated and expressed as Pearsons coefficients. Data were expressed as mean standard error. Differences were considered significant at P < 0.05. Platelet counts Baseline (M0) versus 3 months (M3). PLT counts at M3 t0 were not different from M0 t0 (data not shown). Intra-dialytic changes over time (t0 t150). As shown in Figure 1A, during HD PLT counts changed only slightly over time: from 196 12 109/l at t0 to 180 11 109/l at t30 and 188 13 109/l at 150 (P = 0.010). In the HDF group, after an initial decline from 195 13 109/l at t0 to 162 11 109/l at t30 (efferent line), the number of these cells increased substantially to 233 17 109/l at t150 (P < 0.001). The difference between the two modalities was highly significant (P < 0.001). In the HDF group, both at t5 and at t30 a marked increment was observed in the afferent line (Figure 1B). Changes over the ECC (t1, t5 and t30). As shown in Figure 1C, during HD PLT counts declined only at t1, i.e. after first passage (t1 31 11 109/l, P = 0.021). During HDF, however, PLTs declined significantly over the circuit not only at t1 (t1: 45 16 109/l), but also at t5 (t5: 53 13 109/l) and t30 (t30: 77 12 109/l). The difference between HD and HDF was highly significant, both at t5 and at t30 (P = 0.009). Baseline (M0) versus 3 months (M3). CD62p expression at M3 t0 was not different from M0 t0 (data not shown). Intra-dialytic changes over time (t0 t150). During HD, CD62p expression increased markedly: from 29 6% at t0 to 48 8% at t30 and declined thereafter to 30 4% at t150 (P < 0.001, Figure 2A). During HDF, however, CD62p expression increased and remained elevated until the end of treatment (t150: 55 8%, P < 0.001). The difference between HD and HDF was highly significant (P = 0.002). Changes over the ECC (t1, t5 and t30). During both HD and HDF, the expression of CD62p increased significantly over the ECC at t1, t5 and t30 (Figure 2B). The difference between HD and HDF was significant (P = 0.039) at t30. Platelet factor 4 Baseline (M0) versus 3 months (M3). PF4 levels at M3 t0 were not different from M0 t0 (data not shown). Intra-dialytic changes over time (t0 t150). During both HD and HDF, an increase in PF4 levels was observed over time (P < 0.001, Figure 3A), reaching maximum values at t5 (HD: 107 10 IU/ml, HDF: 84 3 IU/ml). At t150, in both HD and HDF, PF4 levels were still elevated if compared to t0. No differences were observed between the two treatment modalities. Changes over the ECC (t1, t5 and t30). During HD, PF4 increased across the ECC (t1: P = 0.005, t30: P = 0.046, Figure 3B), whereas PF4 concentrations decreased over the ECC during HDF (t5: P = 0.021). The difference between HD and HDF was highly significant (P = 0.001). Beta-thromboglobulin Baseline (M0) versus 3 months (M3). BTG levels at M3 t0 were not different from M0 t0 (data not shown). Intra-dialytic changes over time (t0 t150). During HD, a significant increase was observed from 214 46 IU/ml at t0 to 361 51 IU/ml at t30, with a decline thereafter to 281 33 IU/ml at t150 (P = 0.013, Figure 4A). During HDF, changes were not observed. The difference between HD and HDF was significant (P = 0.03). Changes over the ECC (t1, t5 and t30). During HD, BTG increased moderately over the ECC (t30: P = 0.06, Figure 4B). During HDF, marked changes were not observed. No differences were observed between the two treatment modalities. Transmembrane pressure and haemoconcentration Based on the formula (TMP = Quf/Kuf), the estimated TMPs in HDF were 100 mmHg and <50 mmHg in HD. UF pressures as measured and displayed by the machine ranged from 120 to 260 mmHg during HDF, whereas in HD these pressures were generally <100 mmHg and mostly <50 mmHg. The correlation between the estimated TMP and the UF pressures was highly significant (r2 = 0.8, P = 0.01). Hence, it appears that TMP during HDF is 3 higher than during HD. The filtration fraction, as calculated from Ht levels in the afferent and efferent lines at t5 and t30 based on the formula [1 (Htaff/Hteff)], was 0.03 0.01 in HD and 0.29 0.01 Fig. 1. (A) PLT numbers (109/l, mean SE) in the efferent line during HD and HDF. Changes over time were observed during both treatments: (HD) P = 0.010, + (HDF) P < 0.001. PLT counts evolved differently over time during treatment with both techniques (P < 0.001). (B) PLT numbers (109/l, mean SE) in the both afferent and efferent lines during HDF: P = 0.038, P = 0.002. NB. P-values for changes over the ECC are depicted in C. (C) Changes in PLT numbers (109/l, mean SE) over the ECC (delta: efferent value afferent value) during HD and HDF: P < 0.05. Difference between HD and HDF: P = 0.009. Fig. 3. (A) PF4 concentrations (IU/ml, mean SE) in the efferent line during HD and HDF. Changes over time were observed during both treatments: (HD)/+ (HDF): P < 0.001. (B) Changes in PF4 concentrations (IU/ml, mean SE) over the ECC (delta: efferent value afferent value) during HD and HDF: P < 0.05. Difference between HD and HDF: P = 0.001. Fig. 4. (A) BTG (IU/ml, mean SE) concentrations in the efferent line during HD and HDF. Changes over time were observed during HD: P = 0.013. Difference between HD and HDF: P = 0.030. (B) Changes in BTG (IU/ml, mean SE) concentrations over the ECC (delta: efferent value afferent value) during HD and HDF. in HDF, and the resulting haemoconcentrations [(Hteff Htaff/Htaff) 100%] 4 1% and 41 1%, respectively. Correlations between the haemoconcentration (%) within the ECC and platelet numbers and activation markers (CD62p) The combined data from both HD and HDF group at M3 (n = 19) at t30 showed a robust correlation between the haemoconcentrationand likewise the filtration fractionand the changes in both CD62p expression (t30, r2 0.46, P = 0.002, Figure 5) and PLT numbers over the ECC (t30, r2 0.68, P < 0.001). Discussion PLT activation and degranulation are well-known side effects of HD, which may aggravate the PLT dysfunction that is already observed in the preceding stages of CRF [8,15]. Hence, in ESRD, both bio-incompatible effects of repetitive HD and accumulation of uraemic toxins may have a damaging impact on PLTs. Activated PLTs release inflammatory and mitogenic substances that influence endothelial cell function, chemotaxis and adhesion, and promote the transmigration of monocytes to sites of damaged endothelium, as in early atherosclerotic plaques [16]. In cell cultures, it was demonstrated that the degranulation product PF4 binds directly to oxidized LDL, thereby increasing its binding to vascular cells and macrophages [17], thus enhancing the formation of foam cells and contributing to the process of atherosclerosis. Hence, it has been suggested that HD-induced PLT activation contributes to the vascular damage and accelerated atherosclerosis, which is observed in patients with ESRD [6]. In the present study, the activation and degranulation of PLTs were estimated prospectively in patients who were first treated with low-flux HD and thereafter with Fig. 5. Correlations between the haemoconcentration (% increase in haematocrit over the ECC) and changes in platelet numbers (10E9/l, r2 0.68) and CD62p expression (% PLT, r2 0.46) over the ECC at t30 (delta: efferent value afferent value). Combined data from both HD and HDF groups at M3 (n = 19). post-dilution HDF. As the material of the dialysers was similar in both treatment arms (polysulfone), at first sight the main difference between the two modalities is the amount of convective transport. Theoretically, PLT dysfunction could improve by HDF, as uraemic retention products play an important role in this respect [18]. However, as demonstrated in this study, haemoconcentration increased by 40%, whereas TMP was 3 higher than in HD. In fact, both these effects may have an adverse influence on PLTs that traverse the dialyser in HDF patients. During HD, PLT counts dropped over the ECC, but only directly after the start of treatment. During HDF, however, PLT counts reached their nadir at t30, indicating that PLTs are trapped within the ECC not only after first passage, but also thereafter. In the HDF group, both at t5 and at t30 PLT numbers were higher in the afferent line, indicating the release of PLTs from the bone marrow or storage pools. Furthermore, a substantial rebound was noticed at t150. Together, these data suggest that fresh PLTs are continuously released during HDF, which is quite different from the situation during HD. As samples taken before treatment at baseline and after 3 months were similar, these data indicate that PLT numbers return to baseline within days. During low-flux HD, CD62p expression increased over the ECC immediately after the start and returned to baseline at t150. During HDF, however, the alterations were more pronounced and more protracted. Moreover, whereas the changes over the ECC during HD at t1, t5 and t30 remained relatively stable, these values gradually increased during HDF. Samples taken before treatment at baseline and after 3 months showed no differences, neither in HD nor in HDF. Hence, the combined data on PLT numbers and CD62p expression suggest that activated PLTs disappear from the circulation before the next treatment and are replaced by an influx of fresh PLTs from storage pools. Although we did not assess TMP directly by a four-point pressure measurement, a highly significant correlation was found between the calculations of the formula TMP = Quf/Kuf and the UF-pressures, as assessed by a two-point (4008) or threepoint (5008) pressure measurement, which are displayed on the machines. The estimated TMPs were 3 higher in HDF than in HD, whereas in HDF, with a mean FF of 0.29, the haemoconcentration over the dialyser was 40%. Therefore, in our opinion, the above-mentioned differences between HD and HDF may very well be attributable to a dissimilar haemoconcentration and/or TMP, leading to different flow and shear conditions within the dialyser during treatment [19]. The former conclusion is obviously supported by the striking correlation between the haemoconcentration observed and the deltas of both PLT counts and CD62 expression in the total group of patients at M3. With respect to PLT degranulation, PF4 levels reached peak values shortly after the start of treatment and decreased gradually thereafter, during both HD and HDF. Comparable findings were published before, both in low-flux [2] and in high-flux HD [11]. As PF4 increased particularly in the afferent line, and the changes over the ECC were only modest (HD) or even absent (HDF), these findings imply that the rise in PF4 occurred mainly outside the ECC. Moreover, these data suggest loss of PF4 from the blood compartment through the dialyser to the dialysate compartment during HDF. Previously, we described a sharp increment in PF4 after the isolated administration of low-molecular-weight heparin (LMWH) 10 min before the actual start of HD [10]. From these data, it was concluded that this effect is mainly caused by LMWH-induced detachment of PF4 from the endothelium and only to a limited extent by release from PLTs. Clearly, this view is confirmed and strengthened by the present data. Hence, other PLT granule products, which are not released by LMWH from the endothelium, may be more appropriate for the estimation of HD-induced PLT degranulation. Indeed, recently we demonstrated that BTG, which is also stored in the -granules of PLTs, is almost exclusively released within the ECC [20]. Likewise, in the present study, BTG levels increased markedly over the ECC during HD, which is in line with other studies in low-flux HD [2,21]. During HDF, however, BTG levels did not change, neither in the ECC, nor over time. As the molecular weights (MWs) of PF4 and BTG are 27 kD and 36 kD, respectively, it seems plausible that these substances are removed by convective transport, which is obviously not the case in low-flux HD. Currently, it is unclear whether, and to what extent, BTG is removed by convective transport during high-flux HD. In two studies comparing regenerated cellulose (RC) and cellulose triacetate (CTA) with polysulfon (PS) membranes, BTG levels increased significantly during HD with RC and CTA, but not with PS [22,23]. However, in these two studies, the UF characteristics of the dialysers were not specified. Although PLTs in HD patients appear hypo-responsive to stimulation [24], exhaustion due to the repetitive stimulation of the treatment [6] is unlikely in the present analysis. In both modalities, a substantial PF4 increase was observed in the venous line directly after first passage, which clearly could not originate from the endothelium but only from PLTs traversing the ECC. Obviously, our study has some limitations. First, this analysis was a side study of CONTRAST, comparing PLT activation during online post-dilution HDF to regular low-flux HD. Since a high-flux filter was applied in HDF, the difference between the two modalities comprised not only convective transport related matters (i.e. haemoconcentration and higher TMPs), but also membrane flux characteristics. Hence, it is not readily apparent from our data whether the dissimilar patterns of PLT activation and degranulation result from differences in the permeability characteristics of the membranes used and/or the dissimilar flow and shear conditions within the dialysers. As the CONTRAST study protocol precluded the inclusion of other treatment modalities, such as pre-dilution HDF or high-flux HD, we can only speculate on the effects of membrane permeability. According to the formula TMP = QUF/KUF, the larger pore size (higher KUF) of high-flux membranes would result in lower TMPs, whereas haemoconcentration would be greater due to internal filtration. In this respect, it should be mentioned that PLT activation during high-flux HD [4,10] was not different from lowflux HD, suggesting that a pore size alone has no major effect on this process. Another shortcoming in this study concerns the degree of anticoagulation. As anti-Xa activity is not monitored routinely in our centre, the dosage of dalteparin was based on body weight and adjusted in the case of clotting of the ECC. Considering the molecular weight of dalteparin, which is 5000 D, it is likely that part of it is removed during HDF [25]. Moreover, recently it appeared that procoagulatory activity was higher during pre-dilution HDF than during high-flux HD, despite comparable anti-Xa activity [26]. Therefore, it cannot be excluded that in the present study, during HDF some unnoticed clotting occurred within the dialyser, which altered local flow conditions and contributed to PLT activation. Finally, our data only refer to post-dilution HDF, which is quite different from pre-dilution HDF, in particular with respect to the degree of haemoconcentration and possibly coagulation. On the other hand, as all patients were treated according to standard treatment protocols of the Dutch Federation of Nephrology and the HDF protocol of CONTRAST is consistent with current guidelines, our study is representative for todays clinical practice. To conclude, PLT activation, as measured by the expression of CD62p, is more pronounced and more protracted during post-dilution HDF than during low-flux HD. During HDF, PLTs are trapped in large quantities within the ECC, not only after first passage, but also thereafter. Moreover, as PLT counts in the afferent line increased over time and a marked rebound was observed during the course of treatment, it is likely that fresh PLTs are continuously released from the resident pool. As CD62p expression did not return to baseline, it seems plausible that these fresh PLTs are activated during the entire course of treatment. During HD, these phenomena are either absent or much less prominent. Whereas mainly quantitative differences were observed with respect to PLT counts and CD62p expression, the levels of degranulation products were markedly lower during post-dilution HDF as compared to low-flux HD. As the MWs of both PF4 and BTG are clearly below the cut-off value of the high-flux dialysers used, it is conceivable that these substances are cleared from the circulation by convective transport. The different kinetics of BTG and PF4 may be explained by the fact that BTG is exclusively released within the ECC and PF4 originates mainly from the endothelium. Hence, in the case of PF4, only a fraction of the total body burden crosses the ECC per unit of time, whereas in the case of BTG all production is directly subjected to convective transport. Due to the fact that neither PF4, nor BTG, traverses low-flux membranes, the amount of both products increased during HD. The observations during HDF may well be explained by the greater haemoconcentration and/or higher TMP on the one hand, and superior convective transport at the other. Whether the potential harmful effects of enhanced PLT activation are counterbalanced by the possible beneficial effects of increased convective transport and hence removal of degranulation products is currently unknown. Acknowledgements. We wish to thank the patients of the Medical Centre Alkmaar Dialysis Department for their participation in this study and the nursing staff for their indispensable support. We are grateful to the Dutch Kidney Foundation, Fresenius Medical Care and Gambro for their financial support to CONTRAST. The present study was financially supported by unrestricted grants from Baxter and Fresenius Medical Care. Conflict of interest statement. The sponsors were neither involved in the analysis of the results, nor in the writing of the manuscript. Received for publication: 7.1.09; Accepted in revised form: 3.6.09 Qunying Guo2, Juan Jes us Carrero1, Xueqing Yu2, Peter Barany1, Abdul Rashid Qureshi1, Monica Eriksson1, Bj orn Anderstam1, Michal Chmielewski1, Olof Heimb urger1, Peter Stenvinkel1, Bengt Lindholm1 and Jonas Axelsson1 Background. Vascular endothelial growth factor (VEGF) was recently shown to predict survival in prevalent haemodialysis patients. Soluble VEGF receptors (sVEGFR)-1 and -2 are circulating endogenous modulators of VEGF activity. We thus studied the relationship between sVEGFR-1 and -2 and survival in a cohort of prevalent haemodialysis (HD) patients. Methods. Components of the VEGF system were measured (ELISAs) in 185 prevalent HD patients and levels related to clinical characteristics, biochemical markers and survival. The patients were followed up prospectively for a median 31 (2037) months. Results. While ischaemic heart disease was independently associated with a lower sVEGFR-2 (OR = 2.75, P = 0.02), sVEGFR-1 was positively associated with IL-6 ( = 0.22, P = 0.003) and white blood cell count ( = 0.22, P = 0.002). In survival analysis, the patients with a high sVEGFR-1 level had a higher all-cause mortality (KaplanMeier ChiSquare = 5.6, P = 0.02) and a higher adjusted mortality risk (Cox HR = 1.93, P = 0.009) than those with low levels. Conclusion. In the first clinical study of sVEGFR-1 and -2 in CKD, we found novel associations between the sVEGFRs and cardiac disease. This may be of clinical importance, as a high sVEGFR-1 was an independent risk factor for allcause mortality.


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Mareille Gritters-van den Oever, Muriel P. C. Grooteman, Piet C. M. Bartels, Peter J. Blankestijn, Michiel L. Bots, Marinus A. van den Dorpel, Marianne Schoorl, Margreet Schoorl, Piet M. ter Wee, Menso J. Nubé. Post-dilution haemodiafiltration and low-flux haemodialysis have dissimilar effects on platelets: a side study of CONTRAST, Nephrology Dialysis Transplantation, 2009, 3461-3468, DOI: 10.1093/ndt/gfp308