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Servicio de Nefrología and Unidad de Investigación, Hospital Universitario Reina Sofía, Cordoba, Spain
Correspondence to Dr. Alejandro Martín-Malo, Servicio de Nefrología, Hospital Universitario "Reina Sofía," Avda Menéndez Pidal s/n, 14004-Cordoba, Spain. Phone: +34 95 72 17229; Fax: +34 95 72 02542; E-mail: amartinma{at}senefro.org
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A beneficial effect of biocompatible membranes on hemodialysis patient survival has been recently reported (1,5,6). Studies based on a large number of patients show that the mortality rate of subjects dialyzed with unsubstituted cellulose membranes was higher than with synthetic and modified cellulose membranes (1). Interestingly, infection was one of the major causes of mortality associated with bioincompatible membranes (6,7). Impaired cellular host defense has been proposed to be one of the main mechanisms for increased susceptibility to infections in the dialysis population (8). However, the definite mechanism responsible for this host defense alteration is not well understood. Optimal host defense requires a fine balance between recruitment and death of immunocompetent cells; an alteration in the regulation of cell death by apoptosis may negatively affect the mechanism of host defense (9). We have shown that in vitro, the cell contact of bioincompatible membranes results in apoptosis (10).
Mononuclear cells play a major role in host defense and are one of the main
factors responsible for the first step in the control of infection. Apoptosis
of mononuclear cells may normally occur with aging or may be induced by
inflammatory mediators such as cytokines
(11,12).
Hemodialysis therapy is challenged by the functional abnormalities derived
from the contact of cells with membranes. During hemodialysis, mononuclear
cells are stimulated resulting in interleukin-1 and TNF-
production and
increased expression of adhesion molecules
(13,14,15,16).
Stimulation of mononuclear cells is likely caused by the interaction of
cell-surface proteins with the dialysis membrane. In addition, stimuli related
to the hemodialysis procedure may participate in cell activation
(17). Hemodialysis-induced
cell activation may result from cell contact with hemodialysis membrane plus
other stimuli generated during the hemodialysis procedure, such as complement
factors derived from complement activation or the exposure to dialysate-borne
bacterial product contamination
(17,18).
Thus, it seems that during hemodialysis with nonbiocompatible membranes, cell
activation may be caused by more than one stimulus
(10,14).
It is known that mononuclear cell apoptosis frequently occurs when cells are activated by two different stimuli or when they are subjected to an unphysiologic stimulation (10,19). These are conditions that may be present during hemodialysis when cells are exposed to membranes with a low degree of biocompatibility (10,20). In addition, recent reports have suggested that uremia may directly cause mononuclear cell apoptosis (12,19). The aim of the present study was to evaluate the influence of both uremia itself and different hemodialysis membranes on mononuclear cell apoptosis in vivo and in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mononuclear Cell Preparation
Circulating human mononuclear cells were isolated from 10 ml of heparinized
whole blood. In all patients, the blood samples were drawn immediately before
the first HD session of the week (44 h since the last HD session), and
patients were asked to fast the night before dialysis. In healthy volunteers,
blood was obtained the morning after 8 h of fasting. Buffy coat cells were
separated by differential centrifugation gradient (Ficoll/Hypaque; Pharmacia
LKB, Uppsala, Sweden). Mononuclear cells were washed and seeded in 12-well
culture plates with complete culture medium as described below. Monocytes were
isolated from adherence to plates. A purity of >75% of cells was
demonstrated by staining with anti-CD14 mononuclear antibody (mAb M5E2;
PharMingen, San Diego, CA). Contamination with CD3+ and CD19+ (Leu-4 and
Leu-12; Becton-Dickinson, Mountain View, CA) lymphocytes was <8%. Because
monocytes were not absolutely pure after enrichment, representative control
experiments were performed in cells isolated by flow cytometry and sorted
using a monoclonal antibody against the CD14 molecule (AB383; R&D Systems,
Abingdon, Oxon, United Kingdom). All maneuvers were done under strict sterile
conditions.
Cell Culture
Cells were cultured in RPMI 1640 cell culture medium supplemented with
L-glutamine (2 mM), Hepes (20 mM), sodium pyruvate (1 mM), streptomycin (50
mcg/ml), penicillin (100 UI/ml), and 10% fetal calf serum (FCS) at 37°C in
5% CO2/95% air atmosphere. Fetal calf serum was heated during 1 h
at 56°C to eliminate the complement-activating fractions. Cells were
cultured in 96-well microtiter plates (Falcon; Becton Dickinson, Lincoln Park,
NJ) at 2 x 105 cells per well for 48 h.
Cell Apoptosis
Cell apoptosis was measured by Annexin V staining. One of the cell membrane
changes during the early and intermediate stages of cell apoptosis is the
translocation of phosphatidylserine from the inner side of the cell membrane
to outside. Annexin binds only to cells in which phosphatidylserine has been
translocated to the outside membrane. To evaluate apoptosis, cells were washed
in phosphate-buffered saline and density was adjusted to 5 x
105/ml. Then, cells were resuspended in binding buffer (10 mM
Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; filtered through a
0.2-µm filter); 5 µl Annexin V FITC (Bender MedSystems, Vienna, Austria)
was added to 195 µl of cell suspension. After 10 min of incubation in the
dark, cells were washed and resuspended in 190 µl of binding buffer and 10
µl of propidium iodide stock solution (20 µg/ml). The degree of
apoptosis was assessed by flow cytometry. Live cells were considered those
cells that were negative for both dyes, dead cells were positive for both
fluorochromes, while apoptotic cells were positive only for Annexin V FITC and
negative for propidium iodide. Background fluorescence was determined by
FITC-conjugated mouse immunoglobulins.
In Vitro Study
To evaluate the independent effect of the dialysis membrane on the
induction of apoptosis, and exclude other potential confounding factors, THP-1
cells and whole blood from healthy donors were circulated through a
mini-dialyzer.
Cell Culture
THP-1 cells, a human mononuclear cell line (American Type Culture
Collection, Manassas, VA), were cultured at 37°C in RPMI 1640 supplemented
with L-glutamine (2 mM), Hepes (20 mM), sodium pyruvate (1 mM), streptomycin
(50 µg/ml), penicillin (100 UI/ml), and 10% fetal bovine serum. Fetal
bovine serum was preheated at 56°C for 60 min to inactivate
complement.
Mini-Dialyzers
Mini-dialyzers were 1:50 scale mini-modules made of a plastic case and
hollow fibers resembling the geometry of clinical commercial hollow fiber
dialyzers. Fibers were made of cuprophan, hemophan, cellulose acetate, AN69,
and high-flux polysulfone. Minidialyzers were kindly provided by Hospal (Lyon,
France). Lines were obtained from Excorim Medical Lines (Lund, Sweden). An
Ismatec MC-360 peristaltic roll pump (Glattbrugg, Zurich, Switzerland) was
used to set a flow between 2 to 4 ml/min, simulating a clinical blood flow of
100 to 200 ml/min. Before use, mini-dialyzers and lines were rinsed with 250
ml of 0.9% saline solution. Dialysis circuit was designed as a closed-loop
circuit containing 20 ml of RPMI 1640 and THP-1 cells adjusted at a density of
1 x 106 cells/ml. Dialysate circuit was not used, allowing
cells to circulate only in the "blood" circuit without any
exchange of water and solutes through the capillary wall. The procedure was
performed under strict sterile conditions at 37°C into a CO2
incubator. Samples were obtained before and after 120 min from the start of
the procedure, and were placed in plastic wells in the incubator for 48 h.
Then, cells were fixed and stained with Annexin V FITC to determine the
percent of apoptosis, as mentioned above. Four experiments were performed with
each membrane.
To evaluate the effect of plasma proteins and other cells on the mononuclear cell apoptosis induced by the dialysis membrane, additional in vitro experiments were carried out using fresh whole blood obtained from four healthy subjects. A 48-ml blood sample from a single individual was divided into four aliquots of 12 ml each. One aliquot was used as control and the other three aliquots were circulated during 120 min through mini-dialyzers of cuprophan, hemophan, and high-flux polysulfone, respectively.
Reagents
RPMI 1640, L-glutamine, hepes, Sodium pyruvate, streptomycin, penicillin,
and fetal bovine serum were purchased from BioWhittaker (Walkersville, MD).
Propidium iodide and DNase-free RNase A were purchased from Sigma Chemical Co.
(St. Louis, MO). FITC-labeled Annexin V was purchased from Boehringer Mannheim
(Mannheim, Germany).
Statistical Analyses
Results are expressed as mean ± SD. Nonparametric data were compared
by Kruskal-Wallis test. Comparison between two means was analyzed by
Mann-Whitney test for unpaired data and Wilcoxon signed rank test for paired
data. Spearman rank correlation test was used for correlation analysis.
Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mean percentage of cell apoptosis was significantly elevated in Non-D patients (11.5 ± 5.5%) compared with control subjects (2.1 ± 0.7%, P < 0.001) and CAPD patients (7.0 ± 5.8%, P < 0.05). In patients with chronic renal failure on regular HD with cuprophan, the percentage of apoptosis (21.1 ± 12.6%) was significantly higher than in control subjects (P < 0.01) and Non-D (P < 0.01) and CAPD (P < 0.01) patients. Cell apoptosis in CAPD patients was higher than in control subjects, but differences did not reach statistical significance (Figure 1). In Non-D subjects, the renal creatinine clearance and blood sample for apoptosis were obtained on the same day. The results show that in these patients, apoptosis was inversely correlated with the renal creatinine clearance (r = -0.62, P = 0.003) (Figure 2).
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Effect of Dialysis Membrane on Apoptosis
The effect of the HD membrane on mononuclear cell apoptosis was evaluated
in uremic patients dialyzed with different types of dialyzers. These patients
were not significantly different with regard to age, gender, etiology of
chronic renal failure, body mass index, time on dialysis, duration of
dialysis, dose of dialysis, and erythropoietin therapy.
Figure 3 shows the results obtained with the five types of dialyzers evaluated. The percentage of apoptosis was high in hemophan (29.3 ± 13.3%) and cuprophan dialyzers (21.1 ± 12.6%) and relatively low in AN69 (14.9 ± 9.3%) and polysulfone (15.6 ± 8.2%) membranes. The values observed with hemophan were significantly increased compared with controls (P < 0.01), Non-D (P < 0.001), CAPD (P < 0.01), and with the rest of the dialysis membranes (P < 0.05). The percentage of apoptosis with cuprophan membranes was greater than with the other three membranes, but the difference was not statistically significant. The percentages of cell apoptosis with cellulose acetate, polysulfone, and AN69 were significantly increased compared with controls (P < 0.01) and CAPD (P < 0.05), but similar to Non-D.
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In seven patients chronically hemodialyzed with hemophan membranes, the change to polysulfone dialysis for an 8-wk period resulted in a marked decrease of apoptosis (from 25.7 ± 7.1 to 9.8 ± 5.0%, P < 0.01) (Figure 4).
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In Vitro Experiments
To evaluate an independent effect of dialysis membrane on cell apoptosis,
we performed additional in vitro experiments. The same five dialyzers
tested in vivo were evaluated in vitro. Studies were
performed using a human mononuclear cell line THP-1.
Figure 5a shows the percentage
of apoptosis before and after the circulation of the cells for 120 min through
the five dialyzers. At baseline, the percentage of apoptosis was similar for
all membranes. A significant increase of the apoptosis was observed in the
five membranes after 120 min of cell circulation through the dialyzer:
cuprophan (baseline 8.0 ± 1.8% versus 120 min 35.7 ±
3.3%, P < 0.001); hemophan (9.2 ± 3.5% versus 41.5
± 3.0%, P < 0.001); cellulose acetate (8.2 ± 1.0%
versus 16.5 ± 3.7%, P < 0.01); AN69 (6.3 ±
2.0% versus 14.3 ± 2.0%, P < 0.01), and
polysulfone membranes (7.5 ± 1.0% versus 13.8 ± 2.7%,
P < 0.05). There was a statistically significant difference in the
mean values obtained at 120 min among the five membranes evaluated. The
percentage of apoptosis was significantly increased in hemophan when compared
to the other dialyzers (P < 0.05). There was also a significant
increase in the apoptosis observed with cuprophan in comparison with cellulose
acetate, AN69, and polysulfone dialyzers (P < 0.05). However, at
120 min, there was not a significant difference among the other three
membranes tested.
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Additional experiments in which whole blood from healthy donors was circulated through the mini-dialyzers for 2 h showed that the mononuclear cell apoptosis was significantly increased in hemophan (35.6 ± 6.5%) and cuprophan (30.5 ± 4.8%) versus polysulfone membrane (8.8 ± 1.0%) and control (5.6 ± 2.4%) (Figure 5b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the HD procedure, the blood is exposed to the dialysis membrane resulting in mononuclear cell activation (10). Events associated with conventional HD, such as complement activation, upregulated expression of cell surface integrins, and release of proinflammatory cytokines, are related to cell membrane interaction (13,23,24,25,26,27). Uncontrolled monocyte activation may cause adverse effects; apoptosis may be a mechanism by which these chronically activated cells are deleted from the system. Previous studies by our group demonstrated that cell apoptosis might occur as the direct result of membrane-induced cell activation (10). The results obtained in the present study are in agreement with our recently reported in vitro data, which show that the interaction between mononuclear cell and cuprophan membranes resulted in cell aggregation and apoptosis. This effect was enhanced by preactivation of protein kinase C (10); and, in a different study, it was demonstrated that cuprophan-induced apoptosis was inhibited by pertussis toxin-susceptible G proteins (20). The results of these experiments suggest that the cell contact with cuprophan membrane can be transduced through cell surface proteins into specific intracellular apoptotic signals.
Independently of HD, the present study shows that uremia per se induces apoptosis. This is in agreement with a previous study performed by Heidenreich et al. (28). The normal immune response requires a fine balance between proliferation and cell deletion by apoptosis. We reason that both uremia and HD may affect the immune response by increasing the cell apoptosis.
There is no clear clinical evidence demonstrating a close relationship
between the chronic activation of monocytes and the long-term complications of
the HD patients
(1,6,29,30,31).
It has been proposed that some of the clinical adverse effects in long-term HD
patients are due to the release of cytokines such as interleukin-1ß,
interleukin-6, and TNF- during the HD procedure
(14,17,25,32,33).
However, these cytokines have been found to be elevated in uremic nondialyzed
patients, indicating that monocyte stimulation may be induced by uremia
itself, and therefore it is not related to the dialysis procedure only
(34,35).
The fact that there is a negative correlation between renal creatinine
clearance and apoptosis suggest that uremia itself is in part the responsible
for the increase in apoptosis observed in the HD patients. It is likely that
in these uremic patients, cell stimulation induced by the HD membrane was able
to provoke a further increase in the apoptosis.
The second aim of this study was to evaluate the role of membranes on the induction of mononuclear cell apoptosis. The percentage of apoptosis was higher with hemophan than AN69 and polysulfone. In dialyzed patients with cuprophan, the percentage of apoptosis was greater than with AN69 and polysulfone, although this difference was not statistically significant (P = 0.07). It is important to note that the period of time required for observing a decrease in apoptosis after switching from hemophan to polysulfone was only 8 wk.
Heidenreich et al. (28) reported an acute decrease in the percentage of apoptosis in end-stage renal patients after the HD procedure independently of the membrane used. Therefore, it seems that the correction of uremia was able to reduce apoptosis. It is likely that the induction of apoptosis is related to two different stimuli: uremia and type of membrane. To separate both effects, additional experiments were designed using a human mononuclear cell line and healthy whole blood circulating through mini-modules with the same membranes that were used in vivo. interestingly, the results from the in vitro experiments were quite similar to the in vivo observations: a high degree of apoptosis with hemophan and cuprophan, and relatively low apoptosis with polysulfone. These results were reproduced in vitro using both THP-1 cells and whole blood. The properties of polysulfone membranes made by various manufacturers may differ from each other; therefore, the results of the present study may not be extended to all polysulfone dialyzers.
In summary, our results show that both uremia and HD induce an increase in cell death by apoptosis. By contrast, CAPD treatment decreases apoptosis. The percentage of apoptosis observed in HD patients depends on the type of membrane used, being significantly increased with hemophan dialyzers. The present study supports the hypothesis that both the uremia itself and the properties of HD membrane have an effect on monocyte apoptosis. In conclusion, cell apoptosis seems to be closely related to the severity of uremia and type of dialysis therapy. A more complete understanding of the role that apoptosis plays in the pathophysiology of end-stage chronic renal failure may lead to new strategies for renal replacement therapy. Differences in the degree of apoptosis may help to explain why HD with a biocompatible membrane can reduce the morbidity and mortality of uremic patients with chronic infection episodes.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, interleukin-6 and
ß2-microglobulin. Nephrol Dial Transplant6
[Suppl 2]: 18-23,1991
. Kidney Int 37:116
-125, 1990[Medline]
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