Nephrology Dialysis Transplantation 2007 22(Supplement 5):v13-v19; doi:10.1093/ndt/gfm295
© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. for Permissions, please email: journals.permissions@oxfordjournals.org
Clinical significance of protein adsorbable membranes—Long-term clinical effects and analysis using a proteomic technique
Ikuo Aoike
Internal Medicine, Shinrakuen Hospital, Niigata, Japan
Correspondance and offprint requests to: Ikuo Aoike, MD, Koyo Medical Clinic, 3-9-25, Kameda-koyo, Konan-ku, Niigata 950-0121, Japan. Email: aoike190{at}ecatv.home.ne.jp
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Abstract
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Polymethylmethacrylate (PMMA) membranes were first developed
in Japan in 1977. Over its 30-year history, use of PMMA has
given rise to a new generation of dialysers and continues to
evolve. In long-term observation of patients treated with PMMA
membranes (BK series), it has been found that serum ß2-microglobulin
is maintained at a significantly lower level than in haemodialysis
(HD) patients undergoing treatment with conventional cellulosic
membranes. Regarding long-term side effects, radiolucent bone
cysts (RBC) and carpal tunnel syndrome (CTS) have developed
in relatively few patients undergoing HD with PMMA membranes.
The profiles of various proteins in the blood of HD patients differ from those in normal subjects. PMMA membranes remove a wide variety of solutes not only via permeation, but also adsorption. Numerous reports have noted that pruritus can be ameliorated with the use of slightly anionic PMMA membranes (BG series). We have been performing proteomic analysis of plasma from HD patients with pruritus. We have confirmed that slightly anionic PMMA membranes can adsorb components with a molecular weight of 160 000 Da, which have stimulatory effect on mast cells from pruritus.
Keywords: haemodialysis; polymethylmethacrylate (PMMA) membrane; protein adsorption; proteomics; pruritus
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Introduction
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Polymethylmethacrylate (PMMA) membranes were developed in Japan
in 1977 and have contributed to haemodialysis (HD) therapy for
30 years. We observed patients treated with PMMA membranes (BK
series) over 5 years and investigated their effects. In patients
treated with PMMA membranes, serum ß2-microglobulin,
total joint pain score, and the rates of occurrence of carpal
tunnel syndrome (CTS) and radiolucent bone cysts (RBC) were
maintained at significantly lower levels than with HD using
conventional cellulosic membranes. We believe that though the
blood of HD patients has a protein profile differing from that
in normal subjects, virtually no dialysers can remove solutes
with a molecular weight (MW) higher than that of albumin, since
they are designed to retain albumin and remove solutes solely
by permeation, mainly via diffusion with a small contribution
from convection. However, PMMA membranes can remove a wide variety
of solutes via not only permeation but adsorption as well, which
may yield some effects to HD therapy.
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Long-term clinical effects
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The Department of Internal Medicine (II), Niigata University
School of Medicine and 30 affiliated hospitals have been conducting
a long-term clinical joint study on HD with PMMA membranes (BK
series) since 1988[
1,
2]. The aim of this study is to clarify
the effects of the HD membrane in preventing or postponing dialysis-related
amyloidosis. This study was composed of two studies, one was
prospective and another was retrospective. In the former, the
subjects were classified into three groups basically upon their
HD duration: 46 patients; <0.17 years, 57 patients; >5
years/with joint pains, and 32 patients; >1 year/without
joint pains. They were followed on their serum ß2-microglobulin
and changes in their total joint pain score. In the latter,
more than 200 patients were grouped in two, one group mainly
using conventional membrane and another mainly using PMMA BK
series. They were compared with each other on the rates of occurrence
of CTS and RBC.
As shown in Figure 1, serum ß2-microglobulin rose gradually from the introduction of HD and reached a plateau at 3 years. On the other hand, in the conventional cellulosic membrane group, serum ß2-microglobulin rose steadily and reached levels higher than those observed with PMMA use. With ongoing use of the PMMA membranes, plasma ß2-microglobulin was maintained at a significantly lower level than that observed with HD using conventional cellulosic membranes. In the case of patients whose HD duration exceeded 5 years and who had joint pain, the serum ß2-microglobulin concentration dropped immediately after the change from cellulosic to the PMMA membranes. In such cases, the total joint pain scores of those receiving HD using the PMMA membranes were significantly decreased and remained low throughout the 5 years period (Figure 2). As shown in Figures 3 and 4, in patients on long-term HD using the PMMA membranes, the rates of incidence of CTS and RBC were lower and their onsets were delayed, as compared with patients receiving HD using conventional cellulosic membranes. Switching from conventional cellulosic to PMMA membranes reduces the risk of complications such as CTS and RBC.
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Fig. 1. Changes in pre-HD serum ß2-microglobulin in the long-term clinical study. Two lines on the left show changes of two patients groups whose HD duration were <0.17 years. The right line shows the change of patients group whose HD duration was >5 years.
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Fig. 2. Changes in the total joint pain score of HD patients after switching from cellulosic to PMMA membranes (BK series). Patients’ HD duration was >5 years and patients were classified into three subgroups: 5–10 years, 10–15 years and 15–20 years. The grey bars show the data of patients who could be followed for 8 years after 5 years follow-up.
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Fig. 3. Comparison of the CTS incidence between the PMMA membrane (BK series) and conventional membrane groups. More than 200 patients were grouped, one group mainly using conventional membrane and another mainly using PMMA BK series.
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Fig. 4. Comparison of the RBC incidence between PMMA membrane (BK series) and conventional membrane groups. More than 200 patients were grouped, one group mainly using conventional membrane and another mainly using PMMA BK series.
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Adsorptive removal characteristics of dialysers
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The PMMA membrane is a unique membrane capable of removing proteins
not only via permeation, but also adsorption [
3]. As shown in
Figure 5, as compared with polysulfone membranes, PMMA membranes
remove a wider variety of solutes, greater amounts of solutes,
and adsorbed proteins over the entire range of MWs. The pore
size distribution was designed to improve removal by adjusting
adsorption and convection.
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Fig. 5. Protein adsorption characteristics, (a) Amounts of adsorbed total proteins, (b) Profile of adsorbed proteins (electrophoresis pattern). As compared with polysulfone membranes, PMMA membranes remove a wider variety of solutes, greater amounts of solutes, and adsorb proteins over the entire range of MWs.
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Figure 6 shows the protein adsorption characteristics of various
membranes and proteins. The PMMA membrane adsorbs a wide variety
of molecules. The rate of adsorption of ß2-microglobulin
is especially high. The removal of ß2-microglobulin
by adsorption is one of the typical characteristics of PMMA
membranes. The weakly anionic PMMA membrane (BG series) has
superior adsorption characteristics for basic proteins.
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Fig. 6. Characteristics of protein adsorption by various membranes. The PMMA membrane adsorbs a wide variety of molecules. The rate of adsorption of ß2-microglobulin is especially high. The removal of ß2-microglobulin by adsorption is one of the typical characteristics of PMMA membranes. The weakly anionic PMMA membrane (BG series) has superior adsorption characteristics for basic proteins. The number in the brackets denotes the iso-electric point of each protein.
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Recent studies have also confirmed its ability to remove several
other types of solutes with larger MWs. As an example, we found
that PMMA membranes can remove free immunoglobulin light chains.
These chains have a MW of 28 000 Da and usually exist as dimers.
Free immunoglobulin light chains cannot be removed by membranes
designed for the removal of solutes by permeation, such as high-flux-type
polysulfone membranes (
Figure 7).
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Fig. 7. Removal of free light chains (FLCs) by PMMA membrane. These chains have a MW of 28 000 Da and usually exist as dimers. Free immunoglobulin light chains cannot be removed by membranes designed for the removal of solutes by permeation, such as high-flux-type polysulfone membranes.
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Proteomic approach for patients suffering from uraemic pruritus
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According to an epidemiological survey conducted in Japan, 38.7%
of all dialysis patients had experienced itching. In a survey
on itching in dialysis patients treated in 41 facilities in
Niigata Prefecture in 2000,
73% responded that they had experienced
itching once or more times, and
75% of these respondents complained
of itchiness every day. In 69 HD patients with marked itching,
the dialysis membrane was switched from other membranes to the
BG series, which are weakly anionic PMMA membranes. The symptom
of itching evaluated on the visual analog scale, VAS, had reportedly
decreased significantly at 8 weeks after the membrane change
(
Figure 8). Several facilities have also reported decreases
in itching after switching from polysulfone membranes, cellulose
triacetate membranes or others to BG membranes, as shown in
Table 1 [
4].
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Fig. 8. Improvement of pruritus with a ‘Filtryzer-BG’. In 69 HD patients with marked itching, the dialysis membrane was switched from other membranes to the BG series, which are weakly anionic PMMA membranes. The symptom of itching evaluated on the VAS, had reportedly decreased significantly at 8 weeks after the membrane change.
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For this reason, our group has been examining uraemic plasma
using proteomic analysis [
5]. We compared the blood of normal
subjects with that of patients using protein analysis techniques.
We are attempting to apply two proteomic approaches in this
study. One is a purification approach, the other a novel pre-filtration
approach.
Purification approach
A subgroup of stable patients with severe pruritus was selected as subjects, and their plasma samples were separated by gel filtration. The degranulation activities of mast cells in these fractions were estimated based on the rate of histamine release from rat mast cells. Figure 9 shows the rat mast cell histamine-releasing activities of fractions with different MWs. High histamine-releasing activity was observed only in the IgG region. This activity was barely detectable in plasma from healthy volunteers. To determine whether this active component was IgG, fractions with peak activity were put together and separated using a protein G-coupled IgG purification column into flow-through and adsorbed fractions. The protein G-adsorbed fractions, i.e. the fractions which contained IgG, showed no activity. This activity was, however, restored in the non-IgG fractions.
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Fig. 9. Rat mast cell histamine-releasing activities of different MW fractions. High histamine-releasing activity was observed only in the IgG region. This activity was, however, restored in the non-IgG fractions as a result by separation with a protein G-coupled IgG purification column.
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Figure 10 shows activities before and after co-incubation of
cut hollow fibers and patient-derived fractions for 3 h at 37°C.
To date, we have confirmed that slightly anionic PMMA membranes
can adsorb components with a MW of up to 160 000 Da from the
plasma of HD patients with pruritus. In the next stage, we analysed
protein profiles based on 2D electrophoresis and compared the
profiles of a dialysis patient and a normal subject (
Figure 11).
It is noteworthy that we confirmed the existence of immunoglobulin
-chains in the plasma from the HD patient. The
-chain cannot
bind to an immunoglobulin removal column.
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Fig. 10. Activities before and after co-incubation of cut hollow fibers and patient-derived fractions for 3 h at 37°C. The two left bars show that there was no change by incubation only. The two right bars compare the difference in residual activity after co-incubation with polysulfone membrane and BG membrane.
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Pre-filtration approach
The protein profile of plasma is well known to be dominated
by a handful of abundant proteins such as albumin and immunoglobulins.
These proteins interfere with the identification of less-abundant
proteins. Thus, in order to separate and concentrate the low-molecular
weight plasma proteins, we treated the plasma with a Toray protein
separator. More than 99.99% of human serum albumin was removed,
and the low-molecular weight proteins were concentrated nearly
300-fold.
Figure 12 shows the peptide patterns of a dialysis
patient and a normal subject. Pre-treatment with membrane fractionation
using the separator clearly showed a large peptide pattern difference
between the HD patient and the normal subject. According to
protein separator-assisted analysis, the expressions of several
proteins, such as complement factor D, myoglobin, macrophage
colony stimulating factor and osteopontin, were increased in
plasma from a patient with uraemic pruritus. We have not yet
been able to pinpoint the causative protein. Nevertheless, it
is anticipated that this method will be effectively applied
in the future.
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Fig. 12. Peptide patterns of a dialysis patient and a normal subject, (a) No pre-treatment (plasma), (b) Pre-treatment with a protein separator. These peptides from serum proteins were obtained by digestion with trypsin, and they were injected into reverse phase chromatography. Pre-treatment with membrane fractionation using the separator clearly showed a large peptide pattern difference between the HD patient and the normal subject.
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Discussion
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Recently, polysulfone membranes designed to retain albumin and
remove solutes solely by permeation, mainly via diffusion but
with a small contribution from convection, have come into common
use. However, our findings suggest that PMMA membranes contribute
to the amelioration of adverse long-term clinical outcomes,
such as RBC, CTS and bone joint pain (
Figures 2–4
). The
profiles of various proteins in the blood of HD patients differ
from those in normal subjects and may be related to various
morbid states. As shown in
Figure 5, PMMA membranes can, via
adsorption and permeation, remove a variety of solutes more
than polysulfone membranes. Removal of ß2-microglobulin
by adsorption is one of the typical characteristics of PMMA
membranes. Removal of some other solutes of higher MW has also
recently been confirmed. For example, we have confirmed that
slightly anionic PMMA membranes can adsorb components whose
MW is 160 000 Da, from the plasma of HD patients with pruritus
(
Figures 9 and
10).
This article is mainly a review of previous reports. Though the adsorbability of PMMA membranes have some effects on the morbid states, further studies with proteomic analysis, epidemiological observations or development of new membranes for aiming at those effects are needed.
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Conclusions
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The profiles of various proteins in the blood of HD patients
differ from those in normal subjects and may be related to various
morbid states. PMMA membranes can remove a variety of solutes
by adsorption in addition to permeation, with some effects on
HD therapy. We anticipate the development of new PMMA membranes
in the future.
Conflict of interest statement. None declared.
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Appendix
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This review report is based mainly on results obtained by the
following Joint-Research Groups:
| Niigata University |
Prof. F. Gejyo |
| Niigata Prefectural |
Dr M. Shimada |
| Central Hospital |
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| Niigata Rinko Hospital |
Dr M. Okada |
| Japanese Red Cross Akita |
Dr T. Yamagishi |
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Mr M. Kumagai |
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Mr A. Birukawa |
| Tojinkai Hospital |
Dr T. Ono |
| Toray Industries, Inc. |
Mr I. Itagaki |
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Mr H. Sugaya |
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Ms S. Yamada |
Niigata University and 30 Affiliated Hospitals
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(Long-term Clinical Effects) (1.2)
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References
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- Aoike I, Gejyo F, Arakawa M. Niigata research programme for ß2-M removal membrane. Learning from the Japanese registry: how will we prevent long-term complications? Nephrol Dial Transplant (1995) 10([Suppl. 7]):7–15.[Medline]
- Aoike I. Long –term clinical experience with PMMA membrane. Contrib Nephrol (1999) 125:205–212.[ISI][Medline]
- Birk HW, Kistner A, Wizemann V, Schütterle. Protein adsorption by artificial membrane materials under filtration. Artificial Organs (1995) 19:411–415.[ISI][Medline]
- Kato A, Takita T, Furuhashi M, et al. Polymethylmethacrylate efficacy in reduction of renal itching in hemodialysis patients: crossover study and role of tumor necrosis factor-
. Artificial Organs (2001) 25:441–447.[CrossRef][ISI][Medline] - Aoike I, Suzuki M, Yamada S, et al. Proteomic analysis of mast cell stimulatory components in uraemic pruritus patient plasma with a novel pretreatment device. ASN Abstracts (2005) TH-PO756.
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Abstract
Introduction