1 Department of Haemodialysis and Nephrology, Medical Centre Alkmaar, Wilhelminalaan, Alkmaar and 2 Department of Nephrology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands
Introduction
Recently, in haemodialysis (HD) concern has been raised about the long-term consequences of non-sterile dialysate and the concomitant use of dialysers with large pore size, usually high-flux devices [1,2]. According to current opinion, monocytes are activated by the repeated transfer of bacteria-derived substances from the dialysate compartment to the circulation of the patient, resulting in the secretion of a variety of pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF). Due to the recurrent character of the stimulus, a chronic inflammatory state is induced, leading to a number of long-term HD-related complications, such as infections, malnutrition, dialysis-related amyloidosis (DRA), accelerated atherosclerosis and increased mortality [3].
However, the cause and nature of HD-induced monocyte activation is a controversial issue. Various provoking factors have been recognized, including complement activation [4], coagulation [5], mechanical stress and shear forces [6] and direct contact between blood cells and the membrane of the dialyser [7]. In addition, dialysate factors, such as the presence of acetate [8] and the backtransfer of contaminated dialysate may induce cell activation as well. In the following section we will discuss the arguments supporting backtransport of endotoxins (ETX), which are mainly indirect. Thereafter, we will review the available evidence with reference to backtransport of contaminated dialysate in clinical practice, as measured by antibodies against ETX, the appearance of limulus amoebocyte lysate (LAL) activity in the blood during HD, and recent clinical intra- and peri-dialytical data relating to this topic. Finally, we shall review a number of relevant clinical follow-up studies with respect to the occurrence of long-term complications during HD with different types of dialysers.
Monocyte activation in experimental closed-loop circuits
A large body of evidence on the influence of contaminated dialysate on monocyte activation has been obtained from closed-loop recirculation experiments, sometimes performed under highly unphysiological conditions (for overview see [9]). In fact, most of these studies showed that bacterial contamination of the dialysate has a major influence on the activation of monocytes in the blood compartment, irrespective of the type of dialyser used [10]. However, on careful examination a tenable interpretation of the results seems hardly possible. In many studies normal saline, tissue culture medium or 10% plasma was circulated in the blood compartment for several hours [1113]. In some experiments acetate containing dialysate was used, which may enhance monocyte activation [8]. Generally, supra-physiological concentrations of a bacterial filtrate were added to the dialysate compartment. Lastly, blood samples were stored in a culture medium for 1824 h. Thereafter, monocyte activation, as measured by the induction of the cytokines IL-1ß, TNF, and the IL-1 receptor antagonist (IL-1Ra) was estimated in the culture supernatant and/or cell lysates [11].
However, when the blood compartment of the test system was filled with diluted or whole blood, conflicting results were obtained. Both the absence [14,15] and the presence [16,17] of cytokine induction were reported. Notably, in the latter study the challenge dose of ETX was 200 ng/ml, which is about 5001000x as high as current recommended maximum levels. Apart from the various ETX doses employed, both the deposition of a protein layer covering the membrane [18] and the presence of the ETX-neutralizing bactericidal/permeability increasing protein (BPI) [19], which is released from granulocytes during HD, may have influenced these discrepant findings [20]. Remarkably, contradictory results were published in two separate papers from the same group [14,16]. On the basis of the most recent report, the authors concluded that the reverse transfer of bacterial contaminants is unlikely to be of clinical significance [14]. Comparable data were reported by Tielemans et al. who assessed the effects of sterile and non-sterile dialysate during closed-loop HD with cuprophane (CU) and polyacrylonitrile (PAN) membranes [15]. Again, the blood circuit was filled with heparinized whole blood. From these experiments it appeared that monocyte activation depended exclusively on the type of membrane and not on the bacterial quality of the dialysate. Finally, Evans and Holmes showed that the transfer of cytokine inducing substances (CIS) across cellulose triacetate (CTA) and PAN membranes was critically dependent on the nature and concentration of the bacterial challenge employed [13]. If the ETX concentrations approached a worst-case clinical situation, transfer of CIS was not observed.
In our opinion, results obtained in vitro from closed-loop circuits cannot invariably be transferred to the practice of clinical HD for several reasons. First, in the clinical situation monocytes that have been activated during a single passage through the dialyser may leave the circulation and enter the marginating pool or get sequestered in the lungs [21]. Second, the induction of pro-inflammatory cytokines can be attenuated in vivo by neutralizing factors, including receptor antagonists, soluble receptors and BPI in the case of lipopolysaccharide (LPS)-induced stimulation [22,23]. Furthermore, evidence has been obtained that transcription of cytokines does not lead necessarily to translation and production of these peptides, as blockade at various levels and even intracellular degradation may occur [24].
Clinical evidence of transfer across haemodialysis membranes
Antibodies against endotoxins
Antibodies against ETX were demonstrated during both low-flux CU and high-flux polymethylmethacrylate (PMMA) dialysis, the latter yielding markedly higher levels, suggesting that this high permeable polymer is a risk factor for the transfer of ETX [25]. However, dialysate ETX levels appeared extremely high (50100 ng/ml) in this study. Interestingly, differences were not observed between CU and a healthy control group.
LAL activity in the blood
With respect to the transfer of ETX, as indicated by the appearance of LAL positive material in the blood, only scarce data have been published in clinical HD. Vanholder et al. described the appearance of LAL reactivity in the circulation after HD with high-flux polysulphone (PS) devices, which was not observed after HD with low-flux PS dialysers [26]. However, the pre-treatment levels of LAL-reactivity in the blood differed markedly between the study groups. Moreover, no data were given on the clinical condition and cardiovascular state of this patient group. This information is critically important, as moderate levels of ETX have been described in apparently healthy persons [27]. Interestingly, in another analysis the presence of LAL-positive material in the blood was found before the initiation of HD with high-flux PAN membranes, whereas substantially lower LAL-reactivity was found during dialysis with low-flux CU devices [28]. Again, in both studies dialysate contamination levels were exceptionally high (bacterial contamination 103104 CFU/ml [26] and ETX levels of 1.442.3 ng/ml [28]). It is of note that ETX levels up to 209 pg/ml, which is about 30 times as high as the upper ETX values measured in the study of Vanholder et al. [26], have been described in apparently healthy persons [27]. In the latter analysis, the presence of endotoxaemia emerged as one of the strongest risk predictors of atherosclerosis and cardiovascular disease (CVD). Previously, it has been shown that endotoxaemia may originate from chronic infections, smoking, gut barrier dysfunction and chronic heart failure [29,30]. It has been well documented that most HD patients exhibit multi-organ disease. In addition, in this patient group atherosclerosis significantly contributes to the high incidence of CVD, whereas mortality from CVD is 1020 times greater than that in the general population [31]. Therefore, it is highly doubtful whether the endotoxaemia in these persons depends mainly on the backtransport of contaminated dialysate or on other predisposing conditions that are commonly observed in chronic HD (CHD) patients.
Intra- and peri-dialytical studies
In order to investigate the influence of dialysate contamination on monocyte activation in the clinical situation, three prospective studies were conducted in our centre recently. The first included 13 CHD patients who were randomly assigned to bicarbonate HD with medium-flux CU, high-flux PS, high-flux CTA and high-flux CTA with an ETX filter [32]. Apart from IL-1ß, TNF, IL-1Ra, the soluble TNF receptors p55 and p75 (sTNF-p55 and sTNF-p75), mRNA coding for IL-1ß was measured. In this study, soluble TNF receptors showed the highest values during HD with low-flux CU. Neither mRNA coding for IL-1ß nor the cytokines mentioned showed any difference between the four modalities, indicating that the plasma levels of these cytokines and cytokine antagonists are independent from the degree of dialysate contamination. Blood samples showed LAL-positivity both before and after CU dialysis, but not during the other modalities. Interestingly, the concentration in the blood was significantly higher than in the dialysate, suggesting that some cross-reacting material, such as ß-glucan [33], had been released from the dialyser. Next, a prospective study was initiated, examining the acute phase reaction (APR) both at the beginning and end of HD, and after 24 h [34]. Eight patients were prospectively randomized to bicarbonate HD with medium-flux CU, medium-flux CU with an ETX filter (CUf) and high-flux PS. Apart from the cytokine IL-6, the acute phase proteins C-reactive protein (CRP) and secretory phospholipase A2 (sPLA2) were measured. From this study it appeared that, whereas PS induced no changes at all, both CU and CUf induced a marked increase in IL-6 at the end of HD, which correlated with an increase in both the CRP and sPLA2 levels after 24 h. Therefore, it was concluded that the type of dialyser rather than the bacterial quality of the dialysate is responsible for the HD-induced APR after 24 h, at least at the conditions used in our centre. Comparable data have been published by Schindler et al. [35] in a long-term analysis on the APR in CHD patients. According to this report, the HD-induced inflammatory reaction was markedly affected by type of membrane, CU eliciting higher CRP levels than both high-flux polyamide and low-flux polycarbonate dialysers. As the mean degree of bacterial contamination was only 20.5±5.8 CFU/ml in this study, these results suggest that the type of membrane plays a pivotal role in the HD-induced inflammatory response. Likewise, Lonnemann [36] showed that plasma CRP levels were similar in two groups of HD patients who were treated for at least one year with either ultrafiltered dialysate (no detectable bacterial growth) or standard dialysate (CFU/ml: 95, range 141000). In this study only low-flux PS devices were used. Recently, we achieved a study comparing monocyte activation in eluates of medium-flux CU and high-flux PS dialysers with plasma samples, taken at the end of HD [37]. The elution technique itself has been described elsewhere [38]. In this study, not only the cell surface markers CD11b, CD62L, CD25, HLA-DR, CD14 and CD64 were measured, but also the cytokine IL-1ß, both in the supernatant of the eluates and in the plasma. From this analysis it appeared that monocytes, eluted from the membrane of the dialyser after HD, are more intensively activated than peripheral blood monocytes at the end of HD. However, neither the type of membrane nor the bacterial quality of the dialysate correlated with the phenotypic changes observed. Therefore, it was concluded that other factors, such as mechanical stress, may play a pivotal role in the activation of dialyser-adherent monocytes. As for IL-1ß, release of this cytokine could be demonstrated in 10 out of 16 eluates. The difference between CU and PS was significant, CU yielding more IL-1ß than PS. Although a modality with filtered dialysate was not included in this study, there was no correlation whatsoever between the amount of IL-1ß in the supernatants and the bacterial quality of the dialysate.
Follow-up studies: clinical parameters
As mentioned before, a substantial amount of evidence indicates that monocyte activation may occur in vitro as a result of backtransport of bacterial products from the dialysate into the blood compartment, depending on the specific conditions of the test system. What about the clinical situation? Is there any evidence that dialysate-induced monocyte activation is a relevant feature of routine HD and plays an important role in the development of long-term HD-associated complications, such as an increased incidence of infections, DRA, malnutrition and mortality? If so, one would expect that high-flux devices, with a high risk of backfiltration [39], are specifically associated with the development of these complications. On theoretical grounds, liquid bicarbonate dialysate should be avoided, as this buffer has a strong tendency for bacterial colonization and hence a high potential for the repetitive induction of monocyte activation in CHD patients [40]. However, it should be kept in mind that dialysis membranes differ considerably in their adsorbing capacity for proteins and bacterial fragments [41]. In fact, ultrafiltration through pyrogen-adsorbing high-flux membranes, such as PS and polyamide, improved the bacteriological quality of contaminated dialysate [42].
A number of clinical follow-up studies have compared the incidence of long-term HD-related complications between different types of dialysers (Table 1 ). In most of these studies various types of dialysers were compared using standard liquid bicarbonate as buffer. In only one analysis standard dialysate was replaced by ultrafiltered dialysate during follow-up [43].
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Conclusions
In vitro monocyte activation has been demonstrated clearly in closed-loop experiments, depending on both the composition of the fluids that were pumped through the blood and dialysate compartment, and the amount and type of bacterial filtrate that was administered to the test system. However, if more clinically relevant conditions were simulated, cytokine induction could not be demonstrated. Although monocyte activation has been observed in vivo, little evidence supports the view that backtransport of contaminated dialysate plays a key role in the development of long-term HD-associated complications, such as infections, malnutrition, DRA and cardiovascular mortality. In fact, HD with various types of highly permeable membranes, including synthetic and modified cellulosic devices which are associated with a high risk on backfiltration, seems less correlated with long term HD-related complications than treatment with low-flux CU. As for CU, unanswered questions are the relative roles of both bioincompatibility and/or backtransport of contaminated dialysate in promoting the development of long term HD-related complications. Intra- and peri-dialytical investigations showed monocyte activation that was independent from the bacterial quality of the dialysate. The exact cause for monocyte activation in clinical HD is most probably multifactorial, as both bioincompatibility, direct contact with the dialyser, mechanical stress and shear forces, may stimulate peripheral blood monocytes during HD. Only a well designed prospective clinical trial can reveal whether or not the use of ultrapure dialysate can postpone and/or attenuate the occurrence of long term HD-related complications, if compared to standard non-filtered dialysate.
Editor's note
See also Controversy by Bommer (pp. 19921994).
Notes
Correspondence and offprint requests to: M. J. Nubé, Department of Haemodialysis and Nephrology, Medical Centre Alkmaar, Wilhelminalaan 12, 1815 JD Alkmaar, The Netherlands.
References