Adsorption of unactivated complement proteins by hemodialysis membranes

Hemodialysis using polyacrylonitrile (PAN) membranes has been reported to be associated with depletion of complement in the plasma, yet the increase in plasma C3a antigen concentrations and the degree of leukopenia are modest. These observations suggest that PAN membranes may have a large propensity to adsorb native complement proteins; as a consequence, complement depletion can occur without activation. In the present study, we observed that incubation of human serum in the presence of PAN membrane resulted in a 50% loss of serum hemolytic activity of C3. When radiolabeled purified components were offered, PAN membranes were found to adsorb C3 and C5 in a dose-dependent manner. Adsorption of these proteins by PAN was more than 20 times greater than adsorption by cuprophan, cellulose acetate or Hemophan (Akzo, formerly Enka, Wuppertal, FRG) at all concentrations examined. These results suggest that depletion of complement when serum is exposed to hemodialysis membranes may result from adsorption of complement components onto the membrane surfaces and does not necessarily indicate complement activation.     

Activation of the alternative pathway of complement by cellulosic hemodialysis membranes

Compared to cellulose acetate, hemodialysis with cuprophan membranes is associated with greater activation of the alternative pathway of complement. Previous studies have shown that this difference is not due to a greater number of potential covalent binding sites for activated C3 on cuprophan. To investigate further the factors that influence complement activation by hemodialysis membranes, proteins were eluted from serum-treated cuprophan and cellulose acetate membranes with hydroxylamine at alkaline pH and analyzed by SDS-PAGE and Western blot. Approximately 23 times more total protein was removed from cellulose acetate. Virtually all the C3 in the cellulose acetate eluate was in the form of inactive fragments C3c and C3dg. In contrast, the functionally active form of C3 (C3b) was a prominent constituent of the cuprophan eluate. The binding of factor B (precursor of the catalytic subunit of the C3 convertase) and factor H (regulatory protein of C3 activation) to serum-treated membranes was also analyzed. By Scatchard's method, the affinity constant at equilibrium for factor B binding (KB) to the two types of membranes was not significantly different; however, there were approximately four times more factor B binding sites on the cuprophan than on the cellulose acetate. For cuprophan, the number of factor B binding sites was 1.6 times greater than the number of factor H binding sites. These studies demonstrate that a portion of the C3b molecules that bind to cuprophan are protected from degradation, and suggest that the complement activating capacity of hemodialysis membranes is determined by biochemical properties that modulate both the binding of serum proteins to the membrane and the interactions of the endogenous regulatory proteins with membrane-associated C3b.     

Modulation of the immune response to HBV vaccination by hemodialysis membranes

Introduction  

Seroconversion response to Hepatitis B virus (HBV) vaccination is limited in uremic patients because of impaired humoral and cellular immune activity. Recent studies show that high-flux (HF) hemodialysis (HD) membranes can improve T cell functions and decrease the proinflammatory cytokine activation more effectively than low-flux (LF) membranes. In regard to HF membranes may have immune modulator effects; we compared the antibody responses to hepatitis B vaccination between HF HD and LF HD membranes.     

Adherence of neutrophils to hemodialysis membranes: role of complement receptors.

Complement activation occurs during hemodialysis using cellulosic dialysis membranes with the consequent deposition of C3 activation and degradation products on the membrane surface. To determine if these complement fragments are functionally active, we examined their capacity to mediate leukocyte adherence to cuprophan membranes. Immunoblotting of proteins eluted from plasma-treated cuprophan membranes confirmed the presence of both C3b and iC3b. Incubation of cuprophan membranes with heparinized whole blood resulted in adherence of leukocytes but not erythrocytes. Neutrophils were the primary cell type bound, with monocytes comprising less than 5% of the adherent cells. Studies using indium-labeled neutrophils demonstrated that the binding was plasma dependent and increased with time up to two hours. Neutrophil binding was inhibited by preincubation of the plasma-treated cuprophan membrane with anti-C3 or preincubation of neutrophils with an antibody directed against the alpha chain of complement receptor type 3 (CR3). These observations indicate that iC3b deposited on cuprophan membrane surface as a result of complement activation mediates neutrophil adherence via interaction with CR3. They also support the hypothesis that, in addition to the anaphylatoxins released into the fluid phase, complement activation products that remained membrane bound during hemodialysis also stimulate pathophysiological responses.     

The effects of complement activation during cardiopulmonary bypass. Attenuation by hypothermia, heparin, and hemodilution.

Complement activation was examined prospectively in 100 cardiopulmonary bypass (CPB) patients. Plasma C3a desArg (C3a) increased (cannulation: 234 +/- 33 ng/mL; 20 minutes on CPB: 622 +/- 51; 2 hours after CPB: 1143 +/- 109, p less than 0.0001). C3a at 2 hours was higher in the 13 patients requiring mechanical ventilation for longer than 1 day (1023 +/- 274) than in the 67 without respiratory complication (568 +/- 45, p less than 0.004). Five more patients were studied for neutrophil activation to confirm that a biologic effect of complement activation occurs during CPB; in these five patients C3a increased to 317% of baseline after 10 minutes on CPB with a corresponding rise in neutrophil cell surface receptors for the complement opsonin C3b (as measured by indirect immunofluorescence) to 168% (p less than 0.05). Both increases were sustained at 30 minutes. Temperature, dilution, and heparin were studied as variables relevant to CPB. Exposure of normal neutrophils to C5a in vitro caused an increase in C3b receptors which was dependent on temperature (0 specific fluorescence at 0 C, 30 at 25 C, 180 at 30 C, and 275 at 37 C). Generation of C3a and C5a in normal serum by zymosan was also temperature-dependent (ng/mL C5a generated: 0.7 at 25 C, 200 at 30 C, and 897 at 37 C; ng/mL C3a generated: 546 at 25 C, 10,872 at 30 C, and 65,667 at 37 C). Serum dilution to 33% decreased ng/mL C5a generated in the same system from 200 to 76 with no effect on C3a. Addition of heparin to 20 U/mL decreased ng/mL C3a generated from 10,872 to 913 and C5a from 200 to 8. Thus, hypothermia, dilution, and heparin protect CPB patients from complement activation by reducing both generation of C3a/C5a and the subsequent cellular response of neutrophil activation.     

Analysis of the complement C3 fragments associated with hemodialysis membranes

During hemodialysis with cuprophan membranes, bioactive peptides are generated because the alternative pathway of complement is activated. When cellulose acetate membranes are employed, complement activation is attenuated. The molecular basis for this improved biocompatibility is unknown. It has been postulated, however, that the complement activating potential of dialysis membranes is influenced by the availability of free hydroxyl groups which would provide an acceptor site for activated C3. To investigate this hypothesis, the forms of C3 associated with cellulose acetate and cuprophan membranes have been analyzed. By Western blot, the predominant form of C3 present on both types of membranes is C3c, a degradation product of C3 that lacks the thiolester necessary for covalent bonding. Minimal amounts of C3d (the region of C3 which contains the thiolester) were observed on both membranes; however, by ELISA, there was no difference in the amount bound to cellulose acetate compared to cuprophan. Further, membrane-associated C3d could be removed by urea, suggesting that it was not bound covalently. These studies indicate that the complement activating potential of dialysis membranes is not determined primarily by the availability of potential covalent binding sites for activated C3b.     

Activation of complement by hemodialysis membranes: polyacrylonitrile binds more C3a than cuprophan

Conventionally, complement activation by hemodialysis membranes has been determined by measuring fluid phase C3a. Based on such measurements, polyacrylonitrile (PAN) membranes have been classified as weak activators compared to cuprophan. Previous studies have demonstrated, however, that PAN adsorb fluid phase C3a. Based on that observation, we hypothesized that complement activation by PAN might be artifactually underestimated if relatively large amounts of C3a remained membrane bound. In the present study, a method that allows the simultaneous quantification of both fluid phase and membrane bound C3a was used to assess complement activation by PAN and cuprophan. Pieces of membrane were incubated with C3-depleted serum that had been repleted with radiolabeled C3. Subsequently, the supernates and membranes were subjected to SDS-PAGE, and complement activation was quantified by determining the radioactivity of the C3a bands in the gel. The results showed that while the serum exposed to cuprophan membranes contained almost five times more C3a than that exposed to PAN, approximately 80 times more C3a was bound to the PAN membranes. Consequently, the total amount of C3a generated in the presence of PAN was higher than that generated in the presence of cuprophan. We conclude that assessment of complement activation by hemodialysis membranes using fluid phase C3a measurements alone may be misleading.     

Polymorphonuclear leukocyte function during hemodialysis: relationship to complement activation

Phagocytosis, H2O2 production, and C3bi receptor (CR3) expression by polymorphonuclear leukocytes (PMN) obtained from patients before, during, and after a hemodialysis treatment were evaluated by flow microfluorometry. The results were compared to changes in plasma levels of C3ades Arg and C5ades Arg. Prior to hemodialysis C3ades Arg and C5ades Arg levels, CR3 expression and phagocytosis were not different from normal controls. However, both basal and phagocytosis-induced H2O2 production were increased. C3ades Arg and C5ades Arg were increased after 15 min of dialysis; this was accompanied by transient but significant reductions in PMN count and phagocytosis and increased CR3 expression. No changes in basal or stimulated H2O2 production were observed. We conclude that PMN of hemodialysis patients are primed for an enhanced respiratory burst before dialysis is initiated. Dialysis-induced complement activation after the initiation of dialysis does not further stimulate H2O2 production or enhance the response to phagocytosis. However, complement activation may cause leukopenia and CR3 expression.     

Apoptosis of human polymorphonuclear neutrophils accelerated by dialysis membranes via the activation of the complement system.

BACKGROUND: Haemodialysis (HD) with bioincompatible cellulosic membranes like Cuprophan (CU) is considered to influence negatively the clinical outcome of acute and chronic renal failure. In this effect, apart from the disturbance of phagocytosis or oxygen species production by leukocytes, increased apoptosis also has been implicated recently. The objective of this study was to study the effect of HD membranes on apoptosis induction in polymorphonuclear neutrophils (PMN). METHODS: PMN from healthy donors and uraemic patients were isolated and apoptosis was induced by co-incubation with CU, Hemophan or polyamide hollow fibres in the presence of serum from healthy or uraemic humans. Apoptosis was quantified by flow cytometry using Annexin V-FITC and propidium iodide staining and was confirmed by the detection of DNA fragmentation on gel electrophoresis. The deposition of immunoglobulins (Ig) and complement factors on hollow fibres was detected by direct immunofluorescence. RESULTS: Heat inactivation or the depletion of complement components or Ig significantly reduced apoptosis, indicating its dependence on classical complement activation. The detection of IgG on hollow CU fibres and the restored acceleration of apoptosis by the appropriate replenishment of Ig-deficient sera additionally confirmed these findings. Inhibition experiments revealed that caspases were necessary mainly, but not exclusively, for apoptosis to occur after complement activation. Uraemia led to increased PMN apoptosis in the presence of bioincompatible, but not biocompatible, membranes. CONCLUSIONS: Our results suggest that the acceleration of PMN apoptosis in the presence of CU is mediated via an antibody-dependent activation of the classical complement pathway mobilizing both caspase-dependent and -independent pathways.     

Biocompatibility of dialysis membranes: effects of chronic complement activation

The ability of three dialysis membranes (cuprophane, cellulose acetate, and polymethylmethacrylate) to activate complement was studied prospectively in ten chronic dialysis patients using new and reused membranes. Patients were dialyzed for 1 month with each type of membrane. New cuprophane membranes caused the most intense activation, while polymethylmethacrylate (PMMA) surfaces caused the least degree of complement activation. Reuse decreases the capacity of the cuprophane membrane to activate complement but does not significantly alter the capacity of cellulose acetate membranes. The extent of complement activation paralleled the ability of these membranes to induce neutropenia. Recurrent dialysis with new cuprophane and cellulose acetate membranes leads to a decrease in pre-dialysis and "rebound leukocytosis" neutrophil count, as well as a more intense activation of complement and an enhanced endogenous clearance of products of complement activation. The clinical sequelae of recurrent complement activation are discussed.     

Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation

BACKGROUND: Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. METHODS: A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. RESULTS: Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs-C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs-C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. CONCLUSIONS: Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.     

Effect of regional citrate anticoagulation on leukopenia, complement activation, and expression of leukocyte surface molecules during hemodialysis with unmodified cellulose membranes

BACKGROUND: Dialysis with complement-activating membranes is associated with leukopenia, which is related to an increased expression of adhesion molecules on leukocytes. Citrate chelates calcium and has been claimed to attenuate leukopenia. METHODS: In this study, the effects of citrate anticoagulation on leukocyte and granulocyte counts, complement activation, and the expression of CD11b, CD11c, and CD45 on the surface of granulocytes were evaluated during hemodialysis with unmodified cellulose membranes. Standard heparin was compared to citrate in three different schedules: citrate was infused to obtain a concentration of either 7 or 10 mmol/l blood. CaCl(2) was administered into the dialyzer outlet at 8. 25 mmol Ca(2+)/h (citrate 10 mmol/l) or at 11 mmol Ca(2+)/h (citrate 7 and 10 mmol/l) to reconstitute the calcium levels in the blood returning to the patient. RESULTS: The use of citrate at a high concentration (10 mmol/l) was associated with a blunted upregulation of CD11b, both at the inlet and at the outlet bloodline; for CD11c a reduced upregulation was observed on granulocytes harvested from the inlet bloodline. No effects of citrate were observed on leukopenia, granulocytopenia, or complement activation. A positive correlation between the decrease in systemic ionized Ca(2+) concentration and the increase in CD11b and CD11c expression was found. CONCLUSION: Citrate/CaCl(2) administration affects leukocyte adhesion molecule expression in a dose-dependent way; however, no significant effect could be demonstrated on leukopenia and complement activation.     

Symptoms and activation of granulocytes and complement with two dialysis membranes

Complement (C) activation, neutropenia, and mild pulmonary dysfunction attend hemodialysis (HD) with cellophane [for example, cuprophan (Cu)] membranes. While usually asymptomatic, these phenomena may cause distress in patients with cardiopulmonary disease, and "start-up" symptoms of HD might be mediated by C-stimulated granulocytes (PMNs). Cellulose acetate (CA) hemodialysis membranes have been devised and claimed more blood compatible than Cu. In a blinded series of HD patients, pruritus, fatigue, and sense of well-being were each scored statistically more favorably by the patients during HD with CA than during HD with Cu (P less than 0.05). Postulating that less C activation might underlie the benefit, we showed that neutropenia was less severe with CA (nadir 77.6% of initial count, +/- 4 SEM) than with Cu (38.3% +/- 2.9; P less than 0.01). In vitro, incubation of CA membranes with plasma led to less C3 conversion (20% vs. 40%), less PMN aggregating activity (5.9 ZAP units vs. 36.3) and less decrement in CH50 (6.5% vs. 22%) than like incubations of Cu. C activation was also less potent in vivo: During HD plasma C3a rose from a mean 401 ng/ml to a peak 6,325 in patients on Cu dialyzers, but from 426 to only 3,637 in patients on CA devices (P less than 0.05). Time-course studies suggested CA was initially as potent an activator as Cu but rapidly lost ability to activate C, possibly because of saturation of C3b binding sites. As an index of PMN activation, we also assayed plasma lactoferrin and found levels significantly higher during Cu than CA dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Standard heparin versus low-molecular-weight heparin. A medium-term comparison in hemodialysis

BACKGROUND: To compare standard heparin (SH) and low molecular weight heparin (LMWH) in terms of anticoagulation, platelet activation and lipid metabolism, we selected 54 patients who had been on 4-hour hemodialysis three times weekly for at least 12 months, without bleeding disorders or dyslipidemic diseases. 28 were on hemodialysis with Polysulfone low-flux, 26 were on hemodiafiltration with Polysulfone high-flux. All patients underwent EPO. METHODS: During the first 18 months, we administered SH 1,500 IU on starting dialysis and 1,500 +/- 500 IU in continuous intradialytic infusion per session. In the following 18 months, we administered LMWH 64.6 IU/kg on starting dialysis in a single arterious bolus. We assessed aPTT, anti-factor Xa activity, TAT and FPA, beta-TG and PF4. Blood samples were taken monthly at times 0, 30, 60, 180 and 240 min, as well as 1, 4 and 20 h after dialysis end. Predialysis cholesterol, HDL, LDL, triglycerides and lipoprotein(a) were checked monthly. RESULTS: During both LMWH and SH sessions no clotting or major bleeding complications were observed. APTT with LMWH was lower than that found with SH (p < 0.001); aFXa using LMWH was higher than when using SH (p < 0.001); TAT and FPA were lower in LMWH sessions (p < 0.01) than in SH sessions. We also detected lower beta-TG (p < 0.05) and PF4 levels (p < 0.05) using LMWH than using SH. As regards lipids, we only observed a significant decrease in triglycerides after 18 months of LMWH treatment. CONCLUSIONS: Routine use of LMWH during hemodialysis affords a safe and effective alternative to SH, and causes reduced platelet activation.     

Effect of dialyzer reprocessing with Renalin on serum beta-2-microglobulin and complement activation in hemodialysis patients.

The peracetic acid-based sterilant Renalin is increasingly being used for reprocessing hemodialyzers. In order to evaluate the effects of reprocessing on beta 2-microglobulin (beta 2M) kinetics and complement activation in chronic hemodialysis patients, we compared 4 dialyzer membranes on 1st, 2nd and 4th use of the membrane. Dialysis with new cuprammonium rayon dialyzers (0.8 m2) for 4 h resulted in a nonsignificant increase in serum beta 2M concentrations of 10.7% (corrected for changes in extracellular volume) and significant generation of the complement component C3a des Arg. On reuse, minimal changes in serum beta 2M levels were noted and complement activation was absent. Dialysis with new cellulose acetate (CA, 1.5 m2), polyacrylonitrile (AN69 HF, 1.6 m2) or polymethylmethacrylate (PMMA, 1.6 m2) membranes resulted in significant decreases in serum beta 2M levels (19.5, 31.7 and 50.8%, respectively). Reprocessing had negligible effects on the removal of beta 2M by CA and AN69, but by the 4th use halved the effectiveness of PMMA. Reprocessing reduced the significant generation of C3a des Arg observed with new CA and PMMA membranes. We conclude that, except for PMMA, Renalin reprocessing has minor effects on the ability of the membranes to remove beta 2M and improves the biocompatibility of all membranes studied.     

Citrate anticoagulation control by ionized calcium levels does not prevent hemostasis and complement activation during hemodialysis

The purpose of this study was to determine whether or not regional citrate anticoagulation (RCA) controlled by ionized calcium (iCa(2+)) would overcome thrombogenicity, prevent hemostasis, and complement activation during hemodialysis (HD). RCA was performed in 10 patients during 10 HD sessions using a polysulfone membrane in an effort to keep iCa(2+) at dialyzer outlet at < or =0.4 mmol/L. Compared to baseline, plasma levels of thrombin-antithrombin III complexes rose significantly at 240 min, and tissue factor and complement C5a component levels at 30 and 240 min of the procedure. Thrombocyte count declined significantly at 30 and 240 min, while activated clotting time (ACT) did not increase significantly, and platelet factor 4 as well as von Willebrand factor levels did not alter significantly. While ACT correlated significantly with some thrombogenicity markers, iCa(2+) did not correlate with ACT, changes in hemostasis, or C5a. We conclude the usually recommended iCa(2+) levels in the HD extracorporeal circuit did not guarantee the complete overcoming of thrombogenicity, prevention of hemostasis, and complement activation.     

血液透析应用低分子肝素诱发Ⅱ型血小板减少症一例

血液透析常用的抗凝剂低分子肝素诱发的Ⅱ型血小板减少症（HIT）较为罕见，现报告1例。 患者，女，68岁，因多尿、多饮10余年，间断颜面水肿5年，恶心、呕吐3d入院。既往无药物过敏史。实验室检查：血小板148×10^9／L，BUN26．25mmol／L，Ser503．9μmol／L，诊断为糖尿病肾病，慢性肾衰竭。入院后行右股静脉双腔管置管血液透析，动静脉内瘘成形术。