Ex vivo evaluation of the blood compatibility of mixed matrix haemodialysis membranes.

The patients with end stage kidney disease need haemodialysis therapies, using an artificial kidney. Nevertheless, the current therapies cannot remove a broad range of uremic toxins compared to the natural kidney. Adsorption therapies, using sorbent-based columns, can improve the clearance of uremic toxins, but the sorbent particles often require polymeric coatings to improve their haemocompatibility leading to mass transfer limitations and to lowering of their performance. Earlier, we have developed a dual layer Mixed Matrix fiber Membrane (MMM) based on polyethersulfone/polyvinylpyrrolidone (PES/PVP) polymer blends. There, the sorbent activated carbon particles are embedded in the outer membrane layer for achieving higher removal whereas the inner blood contacting selective membrane layer should achieve optimal blood compatibility. In this work, we evaluate in detail the haemocompatibility of the MMM following the norm ISO 10993-4. We study two generations of MMM having different dimensions and transport characteristics; one with low flux and no albumin leakage and another with high flux but some albumin leakage. The results are compared to those of home-made PES/PVP single layer hollow fiber and to various control fibers already applied in the clinic. Our results show that the low flux MMM successfully avoids contact of blood with the activated carbon and has good haemocompatibility, comparable to membranes currently used in the clinic. STATEMENT OF SIGNIFICANCE: Haemodialysis is a life-sustaining extracorporeal treatment for renal disease, however a broad range of uremic toxins cannot still be removed. In our previous works we showed that a double layer Mixed Matrix Membrane (MMM) composed of polyethersulfone/polyvinylpyrrolidone and activated carbon can achieve higher removal of uremic toxics compared to commercial haemodialysers. In this work we evaluate the haemocompatibility profile of the MMM in order to facilitate its clinical implementation. The lumen particle-free layer of the MMM successfully avoids the contact of blood with the poorly blood-compatible activated carbon. Moreover, thanks to the high amount of polyvinylpyrrolidone and to the smoothness of the lumen layer, the MMM has very good haemocompatibility, comparable to membranes currently used in the clinic.

In vitro biocompatibility evaluation of a heparinizable material (PUPA), based on polyurethane and poly(amido-amine) components.

The biocompatibility of a new heparinizable material based on polyurethane and poly(amido-amine) (PUPA) was evaluated both in the heparinized and non-heparinized forms. The quantity of heparin present on the material was measured using radiolabelled heparin and biological tests. Heparin release in plasma from heparinized PUPA was investigated using in vitro methods. The behaviour of PUPA towards cellular and plasmatic blood components was studied. The influence of sterilization on the cytocompatibility response of both heparinized and non-heparinized PUPA was investigated; gamma-rays were found to be a suitable method of sterilization as no toxic response was noticed.

Kinin generation by hemodialysis membranes as a possible cause of anaphylactoid reactions.

1. Severe anaphylactoid reactions have been reported in some patients treated with angiotensin converting enzyme (ACE) inhibitors during hemodialysis with a polyacrylonitrile (PAN) membrane. Generation of bradykinin via contact activation at the negatively charged membrane surface and reduced bradykinin breakdown due to ACE inhibition have been suggested as possible causes. This hypothesis was evaluated in the present study. 2. PAN or cellulose dialyzer membranes were incubated with plasma at different concentrations of ACE inhibitor. The rate and extent of kinin accumulation was dependent on the ACE inhibitor concentration. 3. Bradykinin levels were determined in "historical" plasma samples drawn from patients treated with ACE inhibitor at the onset of anaphylactoid reactions during hemodialysis with PAN dialyzers. The kinin levels were significantly higher (2.4 +/- 0.05 pmol/ml) than in samples from a group of control patients (0.29 +/- 0.02 pmol/ml). 4. Plasma kinin levels were measured in patients who developed anaphylactoid reactions during dialysis with a PAN membrane though not being treated with ACE inhibitor. At the onset of the reaction, kinin levels increased to 2.1 pmol/ml in the line entering the dialyzer and to 10.5 pmol/ml in the line leaving the dialyzer compared to not more than 0.12 pmol/ml upon dialysis with other membranes. 5. These in vitro and in vivo results demonstrate that contact of blood with a polyacrylonitrile membrane leads to the generation of kinins which accumulate if ACE is inhibited. It is very likely that kinin accumulation in the circulation is the cause of anaphylactoid reactions during hemodialysis with PAN membranes in patients treated with ACE inhibitors and, in some cases, in patients not receiving ACE inhibitor medication.

Cellulose-ester as membrane materials for hemodialysis.

The majority of dialysis membranes are fabricated from regenerated unmodified cellulose. This standard type of cellulosic membrane is frequently under attack because of its alleged lack of biocompatibility. Recent developments, however, have proven that a chemical modification of the reactive surface groups of regenerated cellulose, the hydroxyl-groups, limits the complement-activating potential of these materials and thus improves its blood-compatibility. We extended the idea of modifying cellulose for improved blood-compatibility to a series of different cellulose esters. Special focus was directed towards the question whether a variation of the type of substituent and degree of substitution could influence the blood-compatibility pattern of these materials: the analysis of blood-compatibility profiles showed a direct dependency on the type of substituent and the degree of substitution (DS). As an example, it was found that the DS, necessary for a complete reduction of complement activation, decreases with increasing chain lengths of aliphatic substituents. Optimal degrees of substitution are characteristic of the type of substituents and enable us to tailor materials specifically for optimized blood compatibility.

Bradykinin is a mediator of anaphylactoid reactions during hemodialysis with AN69 membranes.

Anaphylactoid reactions (AR) are the most feared complications of hemodialysis. Recently, a high incidence of AR has been reported during dialysis with AN69 membranes in patients treated with ACE inhibitors. Plasma levels of C3a, histamine and bradykinin were measured in 12 patients at the onset of AR during dialysis with AN69. We also investigated bradykinin generation in 10 symptom-free patients dialyzed with four different membranes. None of the 12 patients studied during AR displayed excessive complement activation or histamine release. In contrast, high bradykinin plasma levels (2392 +/- 53 fmol/ml; mean +/- SEM) were observed in all nine patients of whom bradykinin was measured. One patient developed two consecutive episodes of hypersensitivity on AN69 membranes even without taking ACE inhibitors. Bradykinin levels were high in both episodes (5280 and 10467.7 fmol/ml). Furthermore, this patient showed no symptoms and normal bradykinin levels (123.4 fmol/ml) when dialyzed with other membranes. The role of the membrane type in the AR is further substantiated by the observation that AN69 also provoked a significantly higher bradykinin generation (327.6 +/- 18 fmol/ml; mean +/- SEM) during symptom-free sessions compared to other membranes like CuprophanR (5.1 +/- 7.3), HemophanR (17.2 +/- 6.3) and PolysulfoneR (39.7 +/- 6.6). Our findings strongly suggest that bradykinin is the principal mediator of AR during hemodialysis with AN69 membranes. To our knowledge it is the first time that data support the hypothesis of a more general role of bradykinin in shock-like symptoms. Furthermore, bradykinin generation must be regarded as a new marker of biocompatibility of extracorporeal treatments.

Pyrogen retention by highly permeable synthetic membranes during in vitro dialysis.

Pyrogen permeability of the new highly permeable synthetic membrane polyethersulfone (DIAPES) was compared to polysulfone in vitro dialysis experiments with heparinized human donor blood in the blood compartment. After sterile dialysis for 5 min, dialysate was contaminated with a culture filtrate of Pseudomonas aeruginosa using high and moderate challenge doses (Limulus assay reactivity 20,000 EU/ml and 50 EU/ml, respectively). Whole blood samples were separated from the blood compartment during the sterile (5 min) and contaminated (60 min) phases of dialysis and incubated for 6 h at 37 degrees C. Blood samples were lysed, and interleukin-1beta and tumor necrosis factor alpha were measured by specific ELISAs. Moderate dialysate contamination (50 EU/ml) did not induce detectable cytokine production in whole blood. High challenge dose (20,000 EU/ml) induced whole blood interleukin-1beta and tumor necrosis factor alpha production in the blood compartment, which was higher with DIAPES than with polysulfone after 30 min. After 60 min, membrane-dependent differences were no longer detectable. Pyrogen concentrations in the dialysate decreased with time indicating adsorption of cytokine-inducing substances to the dialyzer membrane. Pyrogens adsorbed to dialyzer membranes were resuspended during recirculation of sterile phosphate-buffered saline in the dialysate compartment and retained cytokine-inducing activity as seen from whole blood incubation experiments. DIAPES and polysulfone adsorbed pyrogens in the presence of whole blood. Pyrogen adsorption to the membrane polymer and/or to the protein coat on the membrane prevented the passage of pyrogens in the presence of moderately contaminated dialysate. High grade dialysate contamination caused breakthrough of pyrogens into the blood with DIAPES and polysulfone. In order to reduce the risk of a dialysis-dependent inflammatory response, dialysate of high bacteriological quality (ultrapure dialysate) should be mandatory.

Reversible inhibition of the proton pump bacteriorhodopsin by modification of tyrosine 64.

Five mol of lysine per mol of bacteriorhodopsin were modified with methylacetimidate. This treatment did not inactivate bacteriorhodopsin but prevented all lysines from subsequent reaction with diazotized sulfanilic acid. This reaction predominantly modified tyrosine 64 and light-induced proton translocation was abolished. Reduction of the mono(p-azobenzene sulfonic acid) tyrosine 64 to the corresponding 3-amino derivative with sodium dithionite led to complete reactivation of the proton translocation activity of bacteriorhodopsin. The relative location of tyrosines 26 and 64 and the COOH terminus on the two surfaces of the purple membrane was determined by incorporation into phospholipid vesicles, subsequent modification, and proteolytic treatment. The results obtained support the models proposed by Engelmann et al. (Engelman, D. M., Henderson, R. McLauchlan, A. D., and Wallace, B. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2023-2027) and by Ovchinnikov et al. (Ovchinnikov, Yu. A., Abdulaev, N. G., Feigina M. Yu., Kiselev A. V., and Lobanov, N. A. (1979) FEBS Lett. 100, 219-224). Tyrosine 64 is located on the extracellular side of the membrane, whereas tyrosine 26 and the COOH terminus are located on the cytoplasmic side. Because specific nitration of tyrosine 26 also leads to inactivation of bacteriorhodopsin (Lemke, H. D., and Oesterhelt, D. (1981) Eur. J. Biochem. 115, 595-604), the results obtained demonstrate that amino acid residues located on both surfaces of the purple membrane are involved in proton translocation.

No generation of bradykinin with a new polyacrylonitrile membrane (SPAN) in haemodialysis patients treated with ACE inhibitors.

BACKGROUND: Anaphylactoid reactions occurring in uraemic patients haemodialysed with polyacrylonitrile haemodialysis (HD) membranes and being treated with ACE inhibitors have been attributed to an excessive generation of bradykinin. METHODS: Here we tested in a prospective trial a new type of polyacrylonitrile membrane (SPAN) with respect to bradykinin generation in nine HD patients receiving either captopril or enalapril. Each patient had three consecutive HD sessions with each of the three tested membranes, high-flux SPAN, high-flux polysulphone (F60) and low-flux Hemophan (GFS Plus 16). RESULTS: No clinical signs of anaphylactoid reactions were observed in any of these patients but the number of patients was relatively small and the duration of exposure to different membranes relatively short. At 5 min after the start of HD session, plasma bradykinin levels were significantly higher in the venous than in the arterial line for all three HD membranes: SPAN, 18.5 +/- 11.9 versus 12.4 +/- 5.3 fmol/ml (P < 0.05); F60, 19.0 +/- 13.8 versus 11.5 +/- 6.5 fmol/ml (P < 0.01); and GFS Plus 16, 39.1 +/- 22.9 versus 15.8 +/- 12.4 fmol/ml (P < 0.005), mean +/- SD respectively. Higher venous line levels were still observed at the 15 and 60 min time points for F60 and GFS Plus 16, but not for SPAN. However, these levels were still insignificant compared to levels measured during episodes of anaphylactic shock from the literature. Plasma histamine and C5a anaphylatoxin levels did not show any increase during HD with SPAN. CONCLUSION: The SPAN membrane did not induce significant bradykinin release in dialysis patients on ACE-inhibitor therapy. It may therefore be used for high-flux dialysis in such patients.

Anaphylactoid reactions during hemodialysis in sheep are ACE inhibitor dose-dependent and mediated by bradykinin.

Anaphylactoid reactions (AR) have been attributed to the generation of bradykinin (BK) when AN69 membranes are used together with angiotensin converting enzyme (ACE) inhibitors during hemodialysis. However, conclusive evidence for the involvement of the BK as the mediator of these AR is still lacking. This study examined the degree of contact activation in an animal model caused by three PAN membranes--AN69, PAN DX, and SPAN- and the effects of different doses of the ACE inhibitor enalapril (ENA) and the BK B2-receptor antagonist icatibant on AR during hemodialysis. Six sheep were dialyzed for one hour with or without ENA pre-treatment using the different membranes in random order. Severe AR were observed only during hemodialysis with AN69 dialyzers together with ENA pre-treatment; the severity of AR increased with the ENA dose. Mild hypotension was noted during hemodialysis with AN69 without ACE inhibition and with PAN DX and 20 mg ENA. Compared to pre-dialysis values, maximum generation of BK after blood passage through the dialyzer was found at five minutes: 73-fold (AN69 without ENA), 161-fold (AN69 with 10 mg ENA), 97-fold (AN69 with 20 mg ENA), 108-fold (AN69 with 30 mg ENA), 154-fold (AN69 with 30 mg ENA and 0.1 mg/kg icatibant), 18-fold (PAN DX without ENA), and 42-fold (PAN DX with 20 mg ENA). Elevated BK levels in arterial blood were detected during hemodialysis with AN69 membranes even without ACE inhibition (2.5-fold); pre-treatment with 20 mg ENA further increased arterial BK concentrations (4-fold). Furthermore, a marked decline of prekallikrein and high molecular weight kininogen concentrations was noted for both AN69 and PAN DX membranes. Anaphylactoid reactions during hemodialysis were completely prevented by icatibant even after pre-treatment with ENA and in the presence of high BK concentrations. Concentrations of prekallikrein, high molecular weight kininogen, and BK remained unchanged and no AR were observed during hemodialysis with SPAN and pre-treatment with 20 mg ENA. Our findings confirm that AR during hemodialysis with the negatively charged AN69 membrane are mediated by BK, since they can be prevented by the BK B2-receptor antagonist icatibant.