Division of Nephrology, Department of Medicine, University of Vienna, Vienna, Austria
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Abstract |
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Methods. Serum/plasma proteins were separated by non-reducing SDSPAGE and transferred to a nitro-cellulose membrane. FLCs were detected by specific antibodies and an enhanced chemiluminescence detection system. The FLC concentrations were calculated. We studied 15 healthy subjects, 10 patients with chronic renal failure, 71 patients undergoing haemodialysis treatment and 33 patients treated with haemodiafiltration. Different membranes were compared: low- and high-flux polysulfone membranes, low- and high-flux cellulose triacetate membranes, high-flux polymethylmethacrylate and polyacrylonitrile membranes.
Results. Chronic renal failure patients showed elevated FLC concentrations as compared with controls. In haemodialysis or haemodiafiltration patients these values were even higher. This was mainly due to an increased concentration of FLC of the -type. The treatment modality per se did not influence the FLC concentrations. Only haemodialysis or haemodiafiltration with the polymethylmethacrylate membrane lead to a significant reduction in FLC concentrations; however, these did not reach control levels. We did not observe differences in FLC levels between patients with different underlying diseases, nor did we find a correlation between age or the duration of the dialysis treatment and FLC concentrations. We found a positive correlation between FLC concentrations at the beginning of dialysis treatment and the amount of IgLCs removed during treatment. However, the average FLC level after treatment did not reach control values.
Conclusions. Currently available haemodialysis or haemodiafiltration treatments are unable to normalize the elevated serum/plasma levels of FLCs in end-stage renal disease patients.
Keywords: chronic renal failure; haemodialysis; immunoglobulin light chains; kappa chains; lambda chains
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Introduction |
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Wagasugi et al. [3] reported in a study published in 1991 that haemodialysis (HD) treatment was not able to lower FLC levels in serum/plasma and that patients receiving HD have even higher FLC serum/plasma levels than pre-HD patients with chronic renal failure (CRF). Since then, there has been no report on the removal of FLCs by HD. We have therefore investigated in the present study the effect of HD and haemodiafiltration (HDF) with different synthetic membranes to answer the question of whether currently available HD or HDF treatments can normalize FLC levels in serum/plasma in end-stage renal disease patients. We used a novel assay based on the immunodetection of FLCs separated from intact immunoglobulins by electrophoresis.
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Subjects and methods |
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Determination of FLC concentrations in sera/plasma
Phosphate-buffered saline pH 7.4 (PBS; 30 µl) or 30 µl light chain solution of known concentration (internal standard) was added to 30 µl serum. For FLCs we used purified human Bence Jones kappa (P 016; Nordic Immunology, Tilburg, the Netherlands) and for
FLCs purified human Bence Jones lambda (P 017; Nordic Immunology). The protein concentration of the added FLCs was determined by the bicinchoninic (BCA) protein assay (Pierce, Rockford, IL). To avoid interference in FLC dimer (
50 kDa) detection due to the presence of albumin (
68 kDa), albumin was largely removed from the serum by BlueSepharose treatment prior to analysis. Blue Sepharose 6 Fast Flow (Amersham Pharmacia Biotech AB, Uppsala; Sweden) was washed twice with 2 vol. PBS, 140 µl of the BlueSepharose suspension was added, and the samples were incubated at 37°C for 30 min on a shaker. The BlueSepharose was then spun down and 10 µl of non-reducing SDSPAGE sample buffer was added to 10 µl supernatant. After 1530 min incubation at 37°C the serum proteins were separated by non-reducing SDSPAGE (12.5% homogenous gel, Phast System; Pharmacia LKB Biotechnology, Uppsala, Sweden) and electroblotted (Phast System) onto a nitro-cellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Buckinghamsire, UK). Four samples with and four samples without light chain addition were run on one gel. The quantity of added light chains was in the same range (up to twofold) as the FLCs in the serum. This approximate concentration was determined by running all sera on a non-reducing SDSPAGE gel; a sample with known light chain concentration (100 µg/ml) was run on each gel as an external standard.
The transferred FLCs were detected with antibodies specific for and
FLCs (K1255 and L7646, respectively; Sigma, St Louis, MO, USA), horseradish peroxidase (HRP)-labelled goat-anti-rabbit antibodies (NIP824; Amersham Pharmacia Biotech) and the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech) using Hyperfilm ECL (Amersham Pharmacia Biotech). The intensity of each of the
and
FLC bands in the monomeric and dimeric form was determined by scanning with the Ultro Scan XL (Pharmacia LKB) and the image master 1D program (Amersham Pharmacia Biotech). Using the known amount of added light chains, the concentration of FLCs in the serum was calculated.
Preparation of whole Igs
Serum from a healthy donor was passed through a 5-ml protein G column (Amersham Pharmacia Biotech). Whole Igs were eluted by lowering the pH (0.2 M GlycineHCl, pH 2.8) and neutralized immediately after elution by a 1/5 vol. of 1 M TrisHCl, pH 8.0. Igs were separated further from small amounts of co-eluting proteins of lower molecular weight by size-exclusion fast-protein liquid chromatography (FPLC, Superdex 200 prep grade, high load 16/50; Amersham Pharmacia Biotech).
Statistical analysis
All values are expressed as means±SEM (standard error of the mean). The FLC levels were compared by analysis of variance (ANOVA). For paired samples we used the Wilcoxon test. P values <0.05 were considered statistically significant.
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Results |
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We found a positive correlation between the light chain concentration at the beginning of HD/HDF treatment and the amount of light chains removed during dialysis. Whereas we observed an increase in light chain levels during HD/HDF treatment in patients with low pre-dialysis concentrations of light chains, the dialysis treatment lead to a significant reduction in light chain levels only in patients with very high pre-dialysis concentrations (Figure 2).
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Discussion |
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FLCs isolated from HD and continuous ambulatory peritoneal dialysis patients inhibit PMNL chemotaxis and the stimulation of glucose uptake [1]. On the other hand, FLCs stimulate basal levels of glucose uptake and of the oxidative metabolism of PMNL [4]. Furthermore, the presence of FLCs increases the percentage of viable PMNL by inhibiting spontaneous apoptotic cell death [5]. Therefore, FLCs may not only contribute to the diminished immune function but also to the state of baseline pre-activation of the immune system in uraemia.
Kappa- and -type FLCs exist as monomers and dimers with molecular weights of approximately 25 and 50 kDa, respectively. Significantly increased levels of FLCs in sera from patients with severely reduced kidney function has been reported by Solling [6] and by Wagasugi et al. [3]. Whereas there is a negative correlation between FLC serum concentration and glomerular filtration rate [6], there is no significant increase in the concentration of intact Igs in uraemia [3].
The focus of the present work is the measurement of FLC levels in serum/plasma of patients undergoing regular HD or HDF before and 2 h after the start of treatment. We used a method based on the separation of serum proteins by non-reducing SDSPAGE. This allowed us to measure FLCs in their monomeric and dimeric forms independently from Igs, which exist in a 1000-fold excess in normal human serum. However, one must be aware that because FLCs have a tendency to polymerize that is dependent on the protein concentration [7], the measured monomer to dimer ratio does not necessarily reflect precisely the actual values in the sample. Other FLC tests use antibodies that react only with free IgLCs [6,8]. However, only a small subpopulation of Igs, denatured in a manner such that unexposed epitopes on the LCs are turned outward, would be enough to interfere with FLC determination. As shown in Figure 1, whole Igs stayed intact during our assay procedure and therefore did not interfere with the determination of FLC levels.
Table 2A shows that the FLC concentration in HD patients is even higher than in CRF patients. This is mainly due to the higher
chain concentration. This was also found by Wakasugi et al. [3] who demonstrated a predominance of
chains in HD patients. Despite some differences in the actual concentrations, partly due to the different pool of donors, the results regarding CRF patients published by Solling [6] correspond to ours in the following points: (i) in the sera of CRF patients, the FLC concentrations of both
- and
-type are higher than in the sera of healthy controls; and (ii) in CRF patients the
/
ratio is higher than in the controls. The
/
ratio of light chains as part of the intact Igs is 1.86 [9]. The lower ratio of FLCs in normal sera is explained by the faster renal elimination of free
- compared with
-light chains as a result of the difference in polymerization behaviour [10]. Impaired renal elimination, therefore, causes a relative increase in
light chains.
The increase in FLC concentration in HD patients does not correlate with the duration of HD therapy, as demonstrated by our group (data not shown) and by Wakasugi et al. [3]. An increased FLC concentration was found in all groups of renal failure patients with different underlying diseases. We did not find any correlation between age and FLC concentration. Solling [6] measured FLC concentrations in different age groups and found a gradual increase from 1- to 1020-year-old people. For adults, the FLC concentration did not change any more with increasing age. As we investigated only sera from adults our results are in agreement with those of Solling.
Table 2B shows the effect of HD or HDF treatment with different membranes on FLC concentrations. Only treatment with the PMMA membrane significantly decreased FLC concentrations, especially of the
-dimer, which has been found at very high levels in the serum/plasma of patients undergoing dialysis treatment. The HDF treatment with other membranes did not lead to a significant decrease in FLC levels. Therefore, we conclude that the removal of FLCs by the PMMA membrane is mainly due to adsorption. This is in agreement with other reports concerning the adsorptive properties of the PMMA membrane: >85% of factor D [11] and >90% of ß2-microglobulin removal [12] occurs by adsorption using PMMA. On the other hand, we did not find a significant FLC reduction using the polyacrylonitrile membrane, a dialyser also known for high protein adsorption.
The average FLC concentration 2 h after the start of treatment did not reach control levels. Furthermore, we found that the amount of FLCs removed by dialysis treatment decreases with decreasing serum/plasma FLC levels and that dialysis treatment is most efficient for very high FLC concentrations (Figure 2). A similar situation has been reported for ß2-microglobulin [13].
Our observation suggests the existence of two different counteracting mechanisms influencing light chain levels during HD/HDF. The dialysis treatment per se seems to lead to an increased rate of light chain appearance in the serum/plasma by a yet unknown mechanism. This effect is most pronounced at low light chain concentrations. Only at very high light chain levels does the second mechanism, light chain removal by dialysis and/or adsorption, become dominant and lead to concentrations which are still far higher than normal.
Therefore, we conclude from our results that currently available HD or HDF treatments cannot normalize elevated serum/plasma levels of FLCs in end-stage renal disease patients.
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Notes |
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References |
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