1 Department of Internal Medicine and 2 Department of Radiology, Klinikum Innenstadt, University of Munich, Munich, Germany
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Abstract |
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Methods. In this retrospective investigation, 89 patients with end-stage renal disease maintained on regular haemodialysis for at least 10 years and treated with one type of dialysis membrane exclusively were selected for analysis. They were divided into three groups: low-flux, bioincompatible cellulose (I), low-flux, intermediately biocompatible polysulphone or PMMA (II), or high-flux, highly biocompatible polysulphone or AN69 (III). In addition, the patients were analysed according to the microbiological quality of the dialysis fluid, which had been tested regularly and was classified either as standard or as intermittently contaminated. The clinical manifestations indicative of AB-amyloidosis, namely, carpal tunnel syndrome, arthropathy and bone cysts, were diagnosed after recruitment.
Results. Clinical symptoms were most pronounced in group I, intermediate in group II, and lowest in group III. Patients treated with intermittently contaminated dialysis fluid showed a higher prevalence of AB-amyloidosis than patients with less contaminated dialysis fluid. Logistic regression analysis demonstrated that the flux characteristics of the dialyser and the microbiological quality of the dialysis fluid as well as the biocompatibility of the dialyser were independent determinants of AB-amyloidosis.
Conclusion. It would be prudent clinical practice to employ high-flux biocompatible membranes in conjunction with ultrapure dialysis fluid for the treatment of end-stage renal disease patients who need to remain on long-term haemodialysis.
Keywords: AB-amyloidosis; biocompatibility; dialysis fluid; haemodialysis membrane
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Introduction |
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The aims of our investigations were to address two questions in long-term dialysis patients (defined as more than 10 years on regular haemodialysis):
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Subjects and methods |
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Causes of end-stage renal disease were determined based on history, urine protein analysis, radiological signs and histological examination of biopsy specimens. Chronic glomerulonephritis was diagnosed in 35 patients, chronic tubulointerstitial nephritis in 33, polycystic kidney disease in 19 patients, and diabetic nephropathy in two patients. Coexisting diseases known to be associated with other forms of amyloidosis were excluded, none of the patients had haemosiderosis or excessive hyperparathyroidism. Haemodialysis was performed three times a week for 45 h, with nominal dialysate flow rates of 500 ml/min, blood flow rates of 200350 ml/min, and individual adjustment of the fluid removal rates according to clinical needs. Commercial bicarbonate dialysis fluid was used in all patients. The delivered Kt/V urea was at least 1.2, all patients were anuric. Three groups of membranes were analysed. Group I, low-flux (Kuf<15 ml/mm Hg/h) cellulosic membranes (Cuprophane) with low biocompatibility (intensive potential to generate complement compounds and to activate cells); group II, low-flux medium biocompatible synthetic membranes (polysulphone 400 or polymethylmethacrylate); and group III, high-flux highly biocompatible synthetic membranes (polysulphone 600 or acrylonitrile). These dialysis membranes had comparable surface areas ranging between 1.25 and 1.33 m2. All patients gave their informed consent to participate in the prospective systematic evaluation of AB-amyloidosis manifestations. The investigations were approved by the local Ethical Committee.
Retrospective collection of demographic and clinical data
In all patients age, sex, body weight (post-dialysis dry weight), underlying renal disease, time on haemodialysis, type of membrane, and results of the microbiological tests of the dialysis fluid were taken from the medical records and dialysis protocols.
Microbiological parameters
Dialysis fluid samples (1 ml) were processed in duplicate in plate count agar by the pour plate method and incubated for 48 h at 37°C and at 20°C for up to 5 days. The membrane filtration technique was used to test Pseudomonas organisms, enterococci, staphylococci, and coliform bacteria in 100-ml water samples. The microbiological quality of dialysis fluid was defined as standard dialysis fluid (<200 c.f.u./ml) according to the guidelines issued by national dialysis societies or to directives from national pharmacopoieae in Europe and Canada; or defined as non-complying bacterial sample or contaminated dialysis fluid (>200 c.f.u./ml). The median of colony forming units was 65 c.f.u./ml in the standard dialysis fluid and 550 c.f.u./ml in the non-complying samples.
In all 89 patients a total of 14.8% (598 of 4046) of non-complying bacterial samples was recorded. The majority of these non-complying samples (416) were found in 18 patients. Each of these patients, recruited from different dialysis centres, had at least one non-complying bacterial test per year (eight patients had one non-complying sample per year, six patients had two non-complying samples per year, and four patients had three non-complying samples per year). Patients with contaminated dialysis fluid were spread among groups I to III. Eight patients received high-flux haemodialysis, six patients were treated with regenerated cellulosic membranes, and in four patients low-flux synthetic membranes were utilized. These 18 patients form the subgroup treated with so-called intermittently contaminated dialysis fluid. By contrast to the clearly defined duration of the exposure of the patients to the membrane, the chronicity of exposure to different microbiological qualities of the dialysis fluid is less well known. Based on the time intervals between two non-complying bacterial tests, the time of exposure to contaminated dialysis fluid ranged from 3 to 7 years (mean 6 years).
Diagnosis of clinical AB-amyloidosis manifestations
CTS was diagnosed either by a history of surgical intervention with the histological detection of amyloid by positive Congo red staining, or by typical complaints and electroneurographic criteria. Electroneurography was carried out in all patients participating in the evaluation. The AB-amyloidosis arthropathy was diagnosed by generally accepted ultrasonographic and radiological signs. The ultrasonographic criteria for arthropathy included a maximum thickness of the rotor cuffs of more than 8 mm and the presence of echogenic pads between muscle groups. Ultrasonography was performed in 83 of 89 patients. Radiological criteria encompassed soft-tissue fullness and/or subchondral juxta-articular erosions at the capsular, ligamental, and synovial zones of insertions with well-preserved joint space. The criteria used for bone cysts indicating amyloid deposits were (i) a diameter of at least 10 mm in the humerus or the hip, and of 5 mm in other bones; (ii) location outside the weight-bearing area of the joint or outside areas prone to synovial effusions; (iii) normal joint space adjacent to the subchondral defect. In all patients X-ray evaluation of the shoulders, hands, hips and knees was performed. The diagnosis of the clinical manifestations indicative of AB-amyloidosis was made by local neurologists, orthopaedic surgeons or radiologists according to accepted criteria. The ultrasound images and the X-rays were re-evaluated by the same independent radiologist of the university hospital. None of these physicians was aware of the dialysis status of the patients.
Laboratory data
Plasma levels of intact parathyroid hormone (by immunoradiometric assay), serum levels of aluminium (by atomic adsorption spectrometry), ß2-microglobulin levels, and C- reactive protein concentrations were measured in all participants after recruitment.
Statistical analysis
All values were expressed as mean±SD. Statistical significance (defined as P<0.05) was calculated by the MannWhitney U test, Pearson 2 test, and logistic regression using the SPSS software (SPSS 8.0, SPSS Inc. Chicago Il, USA). For each analysis, the categorical variables presence of clinical manifestations of AB-amyloidosis, type of membrane, and microbiological quality of dialysis fluid (contamination exceeding the standards or not) were contrasted using the deviation method (each category of the predictor variable except the reference category was compared to the overall effect). All variables were entered in the model by the block entry method. For each variable in the equation we calculated coefficient (B), standard error of B, estimated odds ratio R, and 95% confidence interval for exp B.
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Results |
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Carpal-tunnel syndrome indicative of AB-amyloidosis
Forty-two long-term dialysis patients had carpal-tunnel syndrome; 21 were in group I, 14 in group II, and seven patients in group III. Surgical therapy for CTS was performed in 15 patients of group I (bilateral CTS operations in six patients), in nine patients of group II (bilateral CTS operations in two patients), and in five patients of group III. In all 37 surgical specimens amyloid deposits were detected microscopically by Congo red staining. Surgical specimens from all patients (29) were examined further and showed immunoperoxidase staining of ß2 microglobulin. In 13 patients the diagnosis of CTS was based on clinical complaints and positive electroneurographical criteria. None of the affected patients suffered from any disease known to be associated with an increased prevalence of CTS.
Arthropathy indicative of AB-amyloidosis
Shoulder pain and stiffness, occurring bilaterally in the majority of patients, were observed in 35 of 89 patients (19 patients in group I, 10 patients in group II, six patients in group III). Thickening of the biceps tendons and the rotor cuffs as well as echogenic structures were found in all 35 patients. Significant thickening of the femoral neck capsules could be demonstrated in 25 patients (14 patients in group I, seven patients in group II, four patients in group III). Synovial biopsies and synovial fluid aspirates were obtained from 18 patients and showed amyloid deposits (Congo red) in eight patients (four patients in group I, two in group II, two in group III). Radiological evidence of significant subchondral bone cysts were present in the wrists of 40 patients (20 patients in group I, 13 in group II, seven in group III), in the hips of 26 patients (15 patients in group I, seven in group II, four in group III), and in the shoulders of 14 patients (seven patients in group I, five in group II, two in group III). These cystic bone radiolucencies were multiple in each region and occurred in at least two regions.
Dialytic factors and clinical manifestations indicative of AB-amyloidosis
The prevalence of clinical manifestations indicative of AB-amyloidosis differed substantially between the three groups (Table 3). The percentage of patients suffering from CTS, arthropathy or bone cysts was significantly higher in the cellulosic membrane group (group I) compared to the low-flux synthetic or high-flux synthetic group (groups II and III). Intermittent bacterial contamination of the dialysis fluid resulted in a higher percentage of patients with AB-amyloidosis (Table 4
). Logistic regression analyses demonstrated an increased risk of clinical manifestations of AB-amyloidosis in patients with impermeable bioincompatible membranes or with contaminated dialysis fluid (Table 5
).
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Discussion |
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The molecular pathogenesis of AB-amyloidosis remains unknown [15]. Given the restriction of this amyloid syndrome to advanced renal failure, the increased body burden of ß2-microglobulin in long-term dialysis patients appears to be one of the basic prerequisites for amyloidogenesis. Recent studies suggested a pathogenetic role of a recently identified modification of ß2-microglobulin in amyloid fibrils, i.e. advanced glycation end-products (AGEs) which are formed by non-enzymatic glycation and oxidation reactions in uraemic patients (carbonyl stress and oxidative stress of uraemia). Pentosidine, imidazolone, and carboxymethyllysine are clinically important with respect to the development of AB-amyloidosis.
The bioincompatibility of the dialytic procedure may influence the pathogenesis of AB-amyloidosis through multiple, mutually non exclusive mechanisms. The lower prevalence of AB-amyloidosis in patients on high-flux biocompatible dialysers seems to be related to a number of specific membrane characteristics which differ from low-flux cellulosic or low-flux synthetic membranes. Firstly, a number of studies have indicated that high-flux biocompatible membranes preserve residual renal function better than low-flux cellulosic membranes [16]. During the first 1224 months on dialysis serum ß2 microglobulin levels are strongly determined by residual renal function. Thus differences in renal excretion of ß2 microglobulin may slow the progressive accumulation of ß2 microglobulin.
Secondly, highly permeable membranes remove generated ß2 microglobulin by adsorption (especially the AN 69 membrane) and convection (especially polysulphone membranes), whereas all low-flux membranes are virtually impermeable to the precursor protein. Pre-dialysis serum ß2-microglobulin levels are significantly lower (by 30%) in patients receiving high-flux dialysis membranes compared with patients treated with low-flux membranes.
Thirdly, the potential of cellulosic membranes such as cuprophane to activate complement and circulating cells has been considered a potential stimulus of ß2-microglobulin synthesis and/or release. However, the results of both in-vitro and in-vivo studies are conflicting [6]. Overall, the increased release of ß2-microglobulin appears of marginal significance when compared with daily ß2-microglobulin synthesis [17].
Fourthly, a more recent but still speculative explanation for the membrane effect is a potential influence of the membrane type on AGE levels. There is evidence that both low-flux and high-flux membranes can reduce elevated pre-dialysis pentosidine concentrations, and that high-flux membranes permit some elimination of AGE-modified proteins [18,19]. Of greater importance is that the repeated exposure of patient blood to bioincompatible membranes may intensify the systemic inflammatory response syndrome and aggravate the oxidative stress of uraemia. In fact the use of high-flux polysulphone may be associated with lower pentosidine levels [20].
Finally, another explanation for the beneficial effects of high-flux biocompatible membranes and of ultrapure dialysis fluid, as demonstrated in previous studies, on the development of AB-amyloidosis, relates to repeated stimulation of cytokine production during haemodialysis. Numerous in-vitro experiments have demonstrated different possible mechanisms involved in haemodialysis-related cytokine induction: direct activation of mononuclear cells by the dialysis membrane, complement activation, and the passage of cytokine-inducing bacterial fragments from contaminated dialysate through the dialyser membrane into the blood. Bacterial contamination of commercial bicarbonate dialysate is common, and exceeds often the recommended standards (lower than 200 c.f.u./ml) or even the currently allowed upper limits (lower than 2000 c.f.u./ml). Evidence has accumulated over the last decade that low-molecular-size pyrogens derived from bacterial contamination of the dialysate are able to penetrate low-flux as well as high-flux dialysers by back diffusion or back filtration [2126]. However, the adsorptive capacity of the specific dialysis membrane itself as well as the formation of a pyrogen-adsorbing protein layer may reduce the permeability to small-molecular-weight bacterial products. A number of in-vitro studies have shown that the adsorptive capacity is higher for synthetic high-flux membranes than for cellulosic low-flux membranes [21,27]. The increased adsorptive capacity of synthetic membranes for pyrogens (particularly high-flux polysulphone and polyamide) has been used as ultrafilters to reduce the pyrogen content of contaminated dialysate and to produce ultrapure dialysis fluid.
The clinical importance of the dialysis-related cytokine induction for the development of AB amyloidosis can be derived not only from the lower prevalence of AB amyloidosis in patients treated with membranes reducing endotoxins derived from contaminated dialysis fluid but also from observations, that the use of ultrapure dialysis fluid reduces cytokine levels, lowers pre-dialysis ß2-microglobulin concentrations and delays the appearance of clinical signs of the carpal-tunnel syndrome even in patients treated with a low-flux bioincompatible membrane [13].
The long-term use of low-flux synthetic membranes resulted in our study in an intermediary prevalence compared to high-flux synthetic membranes or low-flux cellulosic membranes. These data obtained by logistic regression analysis clearly indicate that the main mechanism important for amyloidogenesis is the capacity of a membrane to remove ß2-microglobulin or AGE-modified proteins by adsorption and/or diffusion and by a lower endotoxin reduction factor rather than the biocompatibility of the membrane.
The optimal treatment option to prevent AB-amyloidosis is undoubtedly a successful renal transplantation. However, the time when every dialysis patient will receive a renal transplant in the first years of dialysis therapy is far away. Therefore, better methods of extracorporeal detoxification inducing less retention of ß2-microglobulin and inflammation are mandatory to postpone the manifestations of this complication. We recommend that current treatment should employ high-flux synthetic membranes along with ultrapure dialysis fluid in patients receiving at least 5 years dialytic or filtrative renal replacement therapy or starting dialysis at ages older than 55 years.
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Acknowledgments |
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Notes |
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References |
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