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Nephrol Dial Transplant (2003) 18: Editorial Comments

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Nephrol Dial Transplant (2003) 18: 252257

New insights in dialysis membrane biocompatibility: relevance of adsorption properties and heparin binding
Jacques Chanard, Sylvie Lavaud, Christine Randoux and Philippe Rieu
Service de Ne phrologie, Centre Hospitalier et Universitaire et CNRS FRE 2260, Reims, France

Keywords: biocompatibility; dialysis membranes; extracorporeal devices; haemodialysis

Introduction
In the last 10 years, the concept of biocompatibility as applied to haemodialysis membranes has gained a general clinical acceptance in light of the knowledge that the clinical expression of b2-microglobulin (b2m) amyloidosis found in long-term haemodialyzed patients was governed by the characteristics of dialysis membranes. Highly permeable and biocompatible synthetic membranes interfere with the clinical expression of the disease and postpone occurrence of carpal tunnel syndrome and juxta-articular bone cysts [14].
Correspondence and offprint requests to: Prof. Jacques Chanard, Centre Hospitalier et Universitaire, 45 rue Cognacq-Jay, 51100 Reims, France. Email: jchanard@chu-reims.fr
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Biocompatibility is a difcult concept to dene in the absence of well-established clinical correlates. Besides anaphylactoid reactions [5], which are related more frequently to contaminated dialysate or leachable chemicals than to the sustained activation of the contact phase of coagulation in patients treated with angiotensin converting enzyme inhibitors [6,7], the concept of biocompatibility refers to any harmful effects induced by the contact of blood with dialysis membrane [8]. We consider that a membrane is less biocompatible when the sum of adverse humoral and cellular reactions occurring during haemodialysis is higher than for a reference membrane. An extended denition of biocompatibility should also include factors related to the patients clinical conditions, such as diabetes or systemic inammatory disease, and to factors associated with the specic supportive technique: i.e. haemodialysis, haemoltration, haemodialtration, acetate-free dialysis, etc. The aim of this short review is to highlight the key roles played by the adsorptive properties of dialysis

2003 European Renal AssociationEuropean Dialysis and Transplant Association

Nephrol Dial Transplant (2003) 18: Editorial Comments

253 Table 1. Measurement of dialysis membrane electronegativity, plasma kallikrein and bradykinin concentration Membrane Zeta potential (mV) 70"5 60"4 25"2 20"2 10"1 51"4 15"4 Plasma kallikrein (Uul) 60"15 80"20 10"5 -5 -5 -5 -5 Bradykinin generation (fmoluml)

membranes in determining biocompatibility and to extend the advantages of this property to heparin binding.

Membrane components and effect of extracorporeal devices

A wide spectrum of cellulosic and synthetic materials has been used for manufacturing dialysis membranes. Some of the synthetic membranes in wide use are made with one of the following polymers (this list is not exhaustive): polysulfone, polyamide, polyacrylonitrile, and their copolymers, polymethylmetacrylate, polytetrauoroethylene and their various derivatives. It may be pointed out that some of these products were developed primarily for industrial use and later found their way in to medical devices. The use of high-ux dialyzers lead to understanding the role of uraemic toxins [911] and diffusion of endotoxin from contaminated dialysate by backltration, and to the general acceptance of the hypothesis of membrane bioreactor proposed by Henderson et al. [12]. It suggested, in short, that monocyte activation induced by endotoxin stimulated synthesis and secretion of the proinammatory interleukin-1 (IL-1). Furthermore, it has been documented that other proinammatory and chemoattractant cytokines such as TNF-a, IL-6 and IL-12 [13,14] are secreted in excess in haemodialysis patients and, to return to biocompatibility, it was demonstrated that generation of proinammatory cytokines was also induced by the direct contact of leukocytes with cellulosic membranes in the absence of endotoxin [1517]. Studies of plasma levels of cytokines have provided conicting clinical results about their values as prognostic markers in the treatment of acute renal failure. Nevertheless, the use of synthetic highly permeable membranes, which bind endotoxins, cytokines and growth factors in conjunction with high purity dialysates (the lowest possible endotoxin concentration as measured by up-regulation of the endotoxin receptor CD14 at the surface of circulating leukocytes), is recommended by the European Best Practice Guidelines [18]. The binding properties of membranes have made possible the practical application of haemodialtration with one-line products of substitution uid administered intravenously without prior control for biological qualities. Dialysis membranes can be classied according to their permeability to plasma proteins. However, that classication does not take into account their binding capacities, which differ from one membrane to the other and govern, at least partly, the clearance of charged molecules. Membrane biocompatibility, which is mainly associated with the binding characteristics of polymers, is related to the distribution of hydrophobic and hydrophylic domains and electric charges at the surface and in the body of the membrane. A classication can be proposed based on measurements of the Zeta potential (Table 1) [19].

AN69 PANDX PMMA CT CUP PS AN69-ST

32 100 8983 130 65 78 62 150

(26 50041 200) (22 60036 150) (50250) (25100) (2550) (25120) (30450)

Membranes: polyacrylonitrile AN69 (Hospal); PANDX: polyacrylonitrile (Asahi); PMMA: polymethylmetacrylate (Toray); CT, cellulose triacetate (Baxter); CUP, cuprophane (Akzo); PS, polysulfone (Fresenius); AN69-ST, AN69-PEI (Hospal).

Physicochemical mechanisms associated with membrane biocompatibility

In clinical haemodialysis, the concept of biocompatibility was rst coined after the discovery that the dramatic drop of circulating leukocytes noted during a session of haemodialysis performed with a cellulosic membrane did not occur when using the synthetic membranes [20,21]. This effect was the result of complement activation that generated anaphylatoxins, and of the up-regulation of the surface adhesion molecules and the expression of leukocyte antigens, such as CD11b, CD18, complement receptor type 3 and CD45 [2225]. Biomaterial is recognized by the complement system as foreign and triggers its activation. When complement activation is initiated, biologically highly active inammatory mediators are generated, including the anaphylatoxins C3a, C4a and C5a, the opsonin iC3b and the terminal complement complex. C5a exerts a particularly strong proinammatory activity by inducing an up-regulation of granulocytes, monocytes, lymphocytes and platelet adhesion molecules and lysosomal release from polymorphonuclear granulocytes. It is generally accepted that synthetic membranes are less harmful to complement, leukocytes and platelets than cuprophan, because they up-regulate adhesion to a lesser extent and, in addition, some of them have the unique property for binding anaphylatoxins, cytokines and enzymes released by cell activation after membrane contact [17,26,27]. The activation of polymorphonuclears by articial membranes induces a burst of oxygen-free radicals, the so-called oxidative stress and associated with it the inammatory stress that together induce strong biochemical transformations of lipids and proteins (peroxydation) and generate toxic radicals that activate endothelial cells and result in vascular injury and atherosclerosis. It is now well documented that chronic inammation, as indicated by high plasma levels of C-reactive protein, can be generated by either of two mechanisms: blood contamination by bacterial endotoxin originating from contaminated dialysate and diffusing into the blood, and by the simple

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Nephrol Dial Transplant (2003) 18: Editorial Comments

contact of IL-1-generating cells such as monocytes with incompatible membranes [23]. Criteria for measuring dialysis membrane biocompatibility are numerous. Many of them were developed in vitro, and some discrepancies have been reported when they were applied in vivo. We do not know to what extent the disturbances induced by the contact between blood and articial membrane trigger general defence mechanisms, as we are unable to measure all the buffering systems that support our defences against inammatory stress. Nevertheless, checking these pathophysiological mechanisms is mandatory in order to avoid or reduce the reactions associated with subacute inammationaccelerated cardiovascular disease and malnutritionthat characterize patients on chronic haemodialysis.

Haemocompatibility of dialysis membranes

Haemocompatibility is an aspect of dialysis biocompatibility, one that has been particularly well studied in the context of extracorporeal circulation for cardiopulmonary surgery. In the absence of anticoagulants, all membranes induce the contact phase activation and subsequent reactions of blood clotting [2831]. Blood coagulation results from the conversion of brinogen into insoluble brin. The brin is deposited on the membrane surface and forms a mesh of brils. In contact with brin, platelets are activated and aggregate, giving rise to a cooperative interaction that leads to blood clotting. Simultaneously, blood cells are attracted by the thrombus, invade it and, through enzymatic release, contribute to further brin formation and platelet recruitment. These series of reactions and binding of clotting molecules onto the membrane surface contribute to the formation of a biological membrane that spread over the polymer and is conventionally called protein cake. This biological layer contains plasma proteins such as Factor XII, brinogen, vitronektin, high molecular weight kininogens (HMWK) and others whose activation results in further thrombogenesis [3235], strong platelet aggregation and release of procoagulants like platelet Factor 4. We have seen that leukocytes also are involved in this process and by secreting proteases, lactoferrin and myeloperoxidase contribute to the proinammatory reactor of membrane incompatibility [26].

Dialysis membrane adsorption

Exposure of blood to articial membrane surfaces almost immediately leads to the adsorption of plasma proteins. This initial adsorption profoundly inuences all subsequent events, and to a large extent, determines the thrombogenicity, in other words the biocompatibility of the material. It has been proposed that there are two steps of plasma protein membrane adsorption. The rst occurs on the membrane surface as a result of preferential competitive adsorption of high molecular

weight proteins, such as albumin, brinogenubrin, bronectin and globulins. This phenomenon has both a limited capacity and a limited selectivity due to the saturation of the surface with albumin and clotting proteins. The second step of adsorption, which follows surface adsorption, is absorption in the body of the membrane of proteins with low and medium molecular weights. This phenomenon is slower, depends on membrane structure and thickness and is limited by membrane permselectivity. It involves proteins like b2m, cytokines and anaphylatoxins. This process is dynamic, with continuing enzymatic reactions and substrate replacement. However, its overall turnover rate has not been measured in vivo. A technique using radiolabelled proteins and at the end of a session measuring the radioactivity trapped in the dialyzer has yielded some data for the capacity of dialyzers to bind b2m [36]. Adsorption on the AN69 membrane of HMWK, which participates in the contact phase activation of the endogenous blood coagulation cascade has been demonstrated in vitro [3739]. The membranes biological activity was maintained and correlated with the amount of adsorbed protein. The mechanism of HMWK adsorption was dependent on ionic interactions between the membrane and the protein. Neutralization of charged surface anionic sulfonate groups scattered over the polymer by polyethyleneimine (PEI), as in the AN69-ST membrane, signicantly decreased HMWK binding and decreased kinin generation. PEI reduced surface electronegativity without altering the negatively charged sulfonates of the membrane core. The increased biocompatibility of this new membrane was also credited for improving the binding of C3a, C5a [16], Factor D [27] and glycated b2m [40]. The protein layer coating the polymer reduces membrane permeability throughout a dialysis session [41]. This event results from the plasma proteins and adherence of platelets and leukocytes onto haemodialysis membranes. It depends on a number of factors, among them: the nature and the thickness of polymer; the kind of plasma proteins; the interaction between membrane and protein electric charges and local pH; the type of anticoagulant in use; the ow properties of the system. Tailoring the physico-chemical properties of the polymer smooth surface to selectively adsorb a protein layer to include an anticoagulant such as heparin, or an enzyme such as uricase or an antioxidant such as vitamin E has been proposed as a means of improving haemodialysis [42]. The degree to which a particular binding occurs depends to a large extent, on the character of the membranewhether it is porous or non-porous, hydrophobic or hydrophilic, polar or non-polar. The occurrence of so many interactions raises the question of competitive adsorption of proteins and renders the design of membranes with specic sorbent properties more difcult. Table 2 shows changes of protein adsorption induced through the neutralization of surface electronegativity by covering the AN69 surface with PEI.

Nephrol Dial Transplant (2003) 18: Editorial Comments Table 2. Mean value of protein adsorption onto native and modied AN69 membrane Plasma proteins Molecular weight (Da) Isoelectric point Adsorption at pH 7.4 (mgum2) AN69 HMWK Fibrinogen b2m 120 000 340 000 11 800 4.454.65 5.106.3 5.7 1.5 30 36 AN69-ST 0.3 20 34

255

Reducing surface electronegativity by PEI (AN69-ST membrane) signicantly decreased HMWK and brinogen binding, whereas binding of b2m, which occurs mainly in the bulk of the membrane, was unchanged. Adapted from [19]. Data derived from [3942].

Heparin coating of dialysis membrane

Control of mural clotting can be accomplished by the use of anticoagulants. Some naturally occurring anticoagulants, such as antithrombin III, contribute to the mixture of proteins adsorbed onto the membrane surface. They are, however, quantitatively insufcient for preventing clotting. Heparin, a complex carbohydrate, is commonly used in haemodialysis [43]. The presence of sulfate groups in the carbohydrate moiety gives heparin its highly negative charge. Heparin binds to antithrombin III, accelerating the enzymeneutralizing effect of this serine protease inhibitor, and prevents thrombin formation. In the presence of heparin, the interaction of antithrombin with thrombin is virtually instantaneous. The heparinantithrombin complex inhibits the conversion of other coagulation proteins to active serine proteases (Factors XII, XI, IX and X). The binding of unfractionated heparin binding to polymers has been extensively studied in an effort to render dialysis membranes more haemocompatible. The binding of heparin shares chemical characteristics with the binding of other blood components, through ionic and covalent interactions. Surface-coating techniques have been developed, but information about these procedures is covered by manufacturers patents. For efciently binding heparin to cellulosic dialyzers, the membrane must rst be coated with an intermediate polymer. As a bridging element, polyethyleneglycol has been extensively tested [4449]. This approach has improved the haemocompatibility of articial devices used for extracorporeal circulation in cardio-pulmonary surgery, but has not been very successful in haemodialysis. More recently, a techniquestretching PEI over the surface of a polyacrylonitrile polymerhas produced a new membrane, the AN69-ST membrane. In comparison with the parent AN69 membrane, the new AN69-ST membrane is characterized by a less electronegative surface, while the sulfonic charges present in the body of the membrane have not been modied by the apposition of PEI over its surface [19], as indicated previously. Heparin binding has been documented both in vitro and in vivo [50]. Once adsorbed onto the

membrane, heparin keeps its anticoagulant properties. This property presently is used mainly at the bedside, allowing the tapering of heparin doses in haemodialysis, by between half to two-thirds of the regular doses. In some patients at high risk of bleeding and under strict supervision, haemodialysis has been managed without systemic administration of heparin [51]. One may wonder if decreasing heparin doses in the chronically haemodialyzed patient could decrease the risk of heparin-related osteoporosis. Similarly, heparin reduction could decrease hypertriglyceridemia and prevent the increase of plasma very low density lipoprotein and immediate density lipoprotein particles as well as of hyperlipoprotein(a) found in some 80% of chronically haemodialyzed patients, as these anomalies are associated with the heparin-induced decrease of lipoprotein lipase activity [52,53]. These hypotheses remain to be proven. Binding of heparin should prevent further denaturation and hence activation of the adhered proteins and blood cells [54]. This hypothesis, if valid, should perfectly full the requirements of biocompatibility.

Membrane biocompatibility, nutrition and atherosclerosis

The metabolic consequences of haemodialysis performed with a non-biocompatible membrane have been evaluated. In a pioneer study, Berstro m et al. [55], showed that sham haemodialysis in healthy students induced a signicant increase of protein breakdown when cuprophan membrane was used but this harmful effect was blunted when using the polyacrylonitrile AN69 membrane. (In both groups, subjects were exposed to a similar dialysate.) The effect could not be ascribed either to amino acid loss during dialysis, as recently conrmed [56], or to backltration of a contaminated dialysate. It is not presently possible to delineate the biological mediators that support the accelerated vascular disease manifested by the haemodialyzed patient. The contributions of dialysis membrane or technique, or of dialysate quality, to the inammatory status accompanying haemodialysis have been delineated in a recent comprehensive review by Kaysen [57]. Some 5060%, if not more, of mortality in chronically haemodialyzed patients results from cardiovascular disease. Among many effects triggered by haemodialysis, the activation of nucleated cells releasing cytokines and growth factors on one hand and generating oxidative stressassociated peroxidation and glycation of proteins and lipids on the other, may contribute to accelerated atherosclerosis [5860]. Increased acute phase proteins, especially C-reactive protein, presently are the best biological markers of inammation. They reect the diffuse activation of endothelium, which may also be measured by specic markers derived from endothelial cells, such as E-selectin and sICAM-1. Ridker et al. [61] and Koenig et al. [62] provided convincing

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Nephrol Dial Transplant (2003) 18: Editorial Comments 5. Jaber BL, Pereira BJG. Dialysis reactions. Semin Dial 1997; 10: 158165 6. Tielemans C, Madhoun P, Lenaers M, Schandene L, Goldman M, Vanderweghem JL. Anaphylactoid reactions during hemodialysis on AN69 membranes in patients receiving ACE inhibitors. Kidney Int 1990; 38: 982984 7. Verresen L, Waer M, Vanrenterghem Y, Michielsen P. Angiotensin-converting-enzyme inhibitors and anaphylactoid reactions to high-ux membrane dialysis. Lancet 1990; 336: 13601362 8. Matsuda T. Biological responses at non-physiological interfaces and molecular design of biocompatible surfaces. Nephrol Dial Transplant 1989; 4 (Suppl 3): 560566 9. Babb AL, Strand MJ, Uvelli DA, Milutinovic J, Scribner BH. Quantitative description of dialysis treatment: a dialysis index. Kidney Int 1975; 2: S23S29 10. Brunois JP, Melin JP, Lavaud S, Chanard J. Effect of the hemodialysis prescription on morbidity. N Engl J Med 1982; 306: 12371238 11. Clark WR, Gao D. Low-molecular weight proteins in end-stage renal disease: potential toxicity and dialytic removal mechanisms. J Am Soc Nephrol 2002; 13: S45S47 12. Henderson LW, Koch KM, Dinarello CA, Shaldon S. Hemodialysis hypotension: the interkeukin-1 hypothesis. Blood Purif 1983; 1: 38 13. Lonneman G, Haubitz M, Schindler R. Hemodialysis-associated induction of cytokines. Blood Purif 1990; 8: 214222 14. Pereira B. Cytokine production in patients on dialysis. Blood Purif 1995; 13: 135146 15. Laude-Sharp M, Caroff M, Simard L, Pusineri C, Kazatchkine M, Haeffner-Cavaillon N. Induction of IL-1 during hemodialysis: transmembrane passage of intact endotoxines. Kidney Int 1990; 38: 10891094 16. Cheung AK, Parker C, Wilcox L, Janatova J. Activation of complement by hemodialysis membranes: polyacrylonitrile binds more C3a than cuprophan. Kidney Int 1990; 37: 10551059 17. Clark WR, Hamburger RJ, Lysaght J. Effect of membrane composition and structure on solute removal and biocompatibility in hemodialysis. Kidney Int 1999; 56: 20052015 18. European Best Practice Guidelines for Haemodialysis. Nephrol Dial Transplant 2002; 17 (Suppl 7) 19. Renaux JL, Thomas M, Crost T, Loughraieb N, Vantard G. Activation of the kallikrein-kinin system in hemodialysis: role of membrane electronegativity, blood dilution and pH. Kidney Int 1999; 55: 10971103 20. Hakim RM, Breillatt J, Lazarus M, Port FK, Eknoyan G. Complement activation and hypersensitivity reaction to dialysis membranes. N Engl J Med 1984; 311: 877917 21. Hakim R, Fearon D, Lazarus JM. Biocompatibility of dialysis membranes: effects of chronic complement activation. Kidney Int 1984; 26: 194200 22. Dinarello CA. Cytokines: agents provocateurs in hemodialysis? Kidney Int 1992; 41: 683694 23. Herbelin A, Nguyen AT, Zingraff J, Urena P, Descamp-Latscha B. Inuence of uremia and hemodialysis on circulating interleukin-1 and tumor necrosis factor alpha. Kidney Int 1990; 37: 116125 24. Seyfert UT, Helmling E, Hauck W et al. Comparison of blood biocompatibility during haemodialysis with cuprophane and polyacrylonitrile membranes. Nephrol Dial Transplant 1991; 6: 428434 25. Mulvihill J, Crost T, Renaux JL, Cazenave JP. Evaluation of hemodialysis membrane biocompatibility by parallel assessment in an ex vivo model in healthy volunteers. Nephrol Dial Transplant 1997; 12: 19681973 26. Tetta C, David S, Mariano F, Denitti C, Panichi V. Alterations of the cytokine network in hemodialysis. J Nephrol 2001; 14 (Suppl 4): S22S29 27. Pascal M, Schifferli J. Adsorption of complement factor D by polyacrylonitrile dialysis membrane. Kidney Int 1996; 43: 903911 28. Sultan Y, London GM, Goldfarb B et al. Activation of platelets, coagulation and brinolysis in patients on long-term haemodialysis: inuence of cuprophan and polyacrylonitrile membrane. Nephrol Dial Transplant 1990; 5: 362368

evidence that the baseline plasma concentration of C-reactive protein in apparently healthy subjects is predictive of cardiovascular events. Therefore, for these ndings to be applied to the haemodialyzed patient, we must take into account membrane biocompatibility, whatever the mechanisms, which make a membrane biocompatible and haemocompatible. Supporting this recommended approach are clinical data that suggest an antiinammatory effect of low doses heparin used during haemodialysis [63].

Conclusion: adsorption as a specic characteristic of biocompatible dialysis membranes

Criteria of biocompatibility should be taken into consideration when using high purity dialysate. As discussed, several biological pathways, stimulated during haemodialysis, are relevant to any denition of membrane biocompatibility. Enzymes and substrates that characterize these pathways interact together, before and after dialysis membranes are coated by plasma proteins. The relationship between the initial phases of classic complement activation and the contact phase process and brinolysis have been described. Plasmin activation, which governs brinolysis also stimulates the complement cascade either directly or though cleavage of Factor XII. Decreasing membrane binding of complement, Factor XII, platelets and leukocytes with appropriate redistribution of electric charges over the surface and within the body of the membrane appears to be the most efcient way for reducing the inammatory stress of haemodialysis and rendering dialysis membranes more biocompatible. It is possible that in the near future heparin-coated devices that are suitable for chronic haemodialysis and lessen the activation of cellular and humoral mechanisms of haemoincompatibility, will prove to be reliable for regular use. Biocompatibily of haemodialysis membranes nowadays presents the most important challenge to overcome for improving dialysis quality beyond the mechanistic approach of urea removal.
Acknowledgements. We are indebted to J. L. Renaux, PhD, for constructive technical comments.

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