1 Department of Internal Medicine, Section of Applied and Clinical Biochemistry, University of Perugia, 2 G. Fornaini Institute of Biological Chemistry, University of Urbino, 3 Department of Biology, University of Rome Tor Vergata, 4 Nephrology and Dialysis Unit, R. Silvestrini Hospital, Perugia and 5 Department of Nephrology, San Bortolo Hospital, Vicenza, Italy
Correspondence and offprint requests to: Francesco Galli, PhD, Department of Internal Medicine, Section of Applied and Clinical Biochemistry, University of Perugia, Via del Giochetto, 06126 Perugia, Italy. Email: f.galli{at}unipg.it The authors wish it to be known that, in their opinion, the first two authors contributed equally to this work
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Abstract |
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Methods. Apoptosis and SHi were evaluated in vitro in PBMLs obtained from 40 ESRD patients (HD, n = 30 and PD, n = 10) and 10 healthy controls. A subgroup of HD patients was also studied before and after 1 month of treatment with a vitamin E-coated dialyser (CL-E). Cell thiols and viability were also assessed in the monocyte-like cell line U937 and PBMLs after incubation in the presence of uraemic plasma with or without supplementation of the antioxidants vitamin E (70 µM) or N-acetyl-cysteine (NAC) (0.5 mM).
Results. After 24 h in culture, the PBMLs of HD patients, but not those of CAPD patients, showed an apoptotic rate twice that of healthy controls and a 40% decrease of SHi levels (P < 0.01 in both). A negative correlation between the apoptotic rate and SHi was observed in both patients and controls (r = 0.648, P < 0.001). Plasma and ultrafiltrate samples from HD patients contained solutes (mainly in the lowmiddle molecular weight range) able to trigger apoptosis and oxidative stress in U937 cells. The treatment of HD patients with CL-E, as well as the in vitro supplementation of U937 cells with vitamin E or NAC during the exposure to uraemic plasma, decreased the rate of apoptosis and partially restored SHi.
Conclusions. This study showed an association between an increased apoptotic rate and decreased SHi in PBML of HD patients, but not of CAPD patients. These changes are partially due to different pro-apoptogens that accumulate in the plasma and are at least partially prevented by exogenous antioxidants able to restore SHi, such as vitamin E or thiol suppliers.
Keywords: apoptosis; haemodialysis; mononuclear leukocytes; oxidant stress; thiols; vitamin E
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Introduction |
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In the last few years, the presence of an accelerated rate of cell death by apoptosis of both peripheral blood mononuclear cells (PBMLs) and polymorphonuclear cells (PMNs) has been described as a further hallmark of the challenge that uraemia and dialysis provide to immune cells, possibly contributing to sustain leukopenia, phagocyte dysfunctions and increased susceptibility to infections [14]. Interestingly, a different apoptotic rate was observed in cells from conservative pre-dialysis, HD and continuous ambulatory peritoneal dialysis (CAPD or PD) patients [1,4]. This abnormal apoptosis of HD patients is still poorly understood as regards its underlying mechanism(s) and possible therapies.
Several lines of evidence suggest that oxidant (or oxidative) stress may represent a universal trigger for apoptosis, in that it can drive early apoptotic signals originating from different inducing stimuli into common signalling pathways involving specific disturbances of intracellular thiol (SHi) homeostasis (reviewed in Coppola and Ghibelli [5]). Oxidant stress-related pathways could also play a role in the accelerated apoptosis of leukocytes from HD patients. In T lymphocytes from CRF patients not yet on HD, Matsumoto et al. [2] first proposed the association between an accelerated apoptosis and increased Fas expression. The Fas system, homologous to the tumour necrosis factor- (TNF-
) system, can stimulate the generation of reactive oxygen species and drives death messages by means of redox-sensitive transcription elements. In another study, Fernandez-Fresnedo et al. [6] demonstrated decreased expression of Bcl-2 in B lymphocytes of HD patients. This oncogene product consists of a thiol-containing protein that is able to block apoptosis triggered by different stimuli through a radical-scavenging mechanism. Other recent findings by Carracedo et al. [7] demonstrated that non-biocompatible materials for HD lead to leukocyte aggregation and apoptosis via G-protein-mediated signalling and protein kinase C (PKC) activation. This pathway is known to lead to the formation of bioactive lipids and reactive species such as lipid-centred radicals and hydroperoxides. Moreover, we preliminarily observed a significant decrease of the apoptotic rate in PBMLs when patients on regular HD were supplemented with the natural antioxidant vitamin E [8].
Therefore, although an increased apoptotic susceptibility of immune cells can co-exist with overall conditions of oxidant stress in uraemia and dialysis, it is not clear whether specific oxidant stress-related changes of these cells are present and could be a cause of apoptosis. Recent evidence suggests that the depletion of intracellular SHi via active extrusion of glutathione (GSH) can represent a specific trigger and signalling event in the apoptosis of mononuclear cells [5].
This study investigates the presence of a defect in SHi as a possible mechanism for the increased rate of apoptosis in PBMLs of end-stage renal disease (ESRD) patients. These parameters were also investigated in healthy control PBMLs and human monocyte leukaemia cells exposed to uraemic plasma and ultrafiltrate obtained from ESRD patients. The role of antioxidants in preventing abnormal apoptosis and SHi disturbances in PBMLs of HD patients was assessed both in vitro by using vitamin E and the thiol supplier N-acetyl-cysteine (NAC) and in vivo in patients treated with a vitamin E-modified dialysis membrane.
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Subjects and methods |
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In the patient subgroups, the presence of severe leukopenia, significant changes in the relative counts of leukocyte subpopulations and specific complement-related disorders were excluded by appropriate laboratory investigation. The HD patients showed at inclusion in the study a mean Kt/V value (weekly) of 3.9 ± 0.7 and the residual creatinine clearance (CrCl, ml/min) was <1 in all the patients. Other routine biochemical parameters in HD and CAPD patients were: blood urea nitrogen (mg/dl) 149.6 ± 43.4, creatinine (mg/dl) 9.8 ± 1.9, haematocrit (Hct) (%) 33.0 ± 3.6, cholesterol (mg/dl) 195.4 ± 35.2, triglycerides (mg/dl) 175.5 ± 123.2, total proteins (g/dl) 6.7 ± 0.4 and albumin (g/dl) 4.1 ± 0.3. Recombinant human erythropoietin supplementation was provided following standard protocols for the patients with the most severe cases of anaemia (Hct target, 30%).
The group of HD patients included 21 subjects treated with cuprammonium rayon (CR) filters for at least 3 months. After informed consent, 15 of these patients were involved in a two-step prospective study aimed at evaluating the effect on PBML apoptosis of a multilayer vitamin E-coated filter (Excebrane series, Terumo Co., Japan) that was observed to increase both plasma and blood cell vitamin E levels in a significant number of the HD patients [9]. The vitamin E covalently bound to the Excebrane surface was in the form of dl--tocopheryl-acetate, and the release of vitamin E during each dialysis session (4 h) was assumed to be <2 mg (information provided by the manufacturer). After baseline evaluation carried out when the patients were on CR filters (step A), they were changed to a 1-month treatment with Excebrane (step B). The same evaluations as in step A were repeated at the end of step B. Routine biochemical parameters did not change significantly during the protocol.
The remaining patients (n = 9) had their plasma studied for pro-apoptogenic activity. They were treated with CR, polysulfone (PS) and polymethylmethacrylate (PMMA) filters (n = 5, n = 2 and n = 2, respectively).
Blood cells
Samples were taken under fasting conditions before and after dialysis in the second dialysis session of the week in HD patients, and before the first exchange of the morning on the third day of the week in the case of the CAPD patients. Two blood samples (5 ml each) were drawn from the antecubital vein into green-top heparinized (for biochemistry tests) or EDTA-containing (for apoptosis-related assays) tubes using the vacutainer technique. The blood was centrifuged immediately (2800 r.p.m. for 10 min at 4°C), the plasma was carefully removed and the buffy coat was used to prepare PBMLs by density gradient separation with Lymphoprep, according to the procedure recommended by the manufacturer (Nycomed Pharma As, Oslo, Norway). The ring of mononuclear cells obtained was collected carefully and washed three times in RPMI 1640. The cells were then used immediately for the biochemistry tests or resuspended (1 x 106 cells/ml) in the same medium supplemented with 10% fetal calf serum (FCS) and antibiotics as described in [10] before being placed in culture under standard conditions (5% CO2, 90% humidity and temperature of 37°C). In the in vitro cultures of PBMLs, the numbers of monocytes and lymphocytes were scored over time.
Aliquots of plasma were maintained at 20°C or used immediately both for the biochemistry assays or added to the cell culture media of the U937 human monocytemacrophage leukaemia cell line, as detailed below.
The blood cells were collected from heparinized blood and prepared for the determination of vitamin E and lipid composition as previously described [9].
Laboratory determinations
Apoptosis assay: general methods. The quantitative analysis of apoptosis in both PBMLs and U937 cells in culture was carried out routinely by scoring the number of cells displaying typical nuclear morphology as measured by fluorescence microscopy after staining with specific probes for the nucleic acids. Both Hoechst 33342 and propidium iodide were used according to the procedures previously published [9,10]. These two probes, together with the annexin V test, were also used to confirm the data obtained from fluorescence microscope analyses by cytofluorimetry (FACScan, Becton-Dickinson). The cytofluorimetric analyses of the samples were performed after 24 h of culture, and a good correlation between the microscopy and cytofluorimetry data was observed (r = 0.898). The choice of routinely evaluating chromatin condensation and cell morphology as apoptotic hallmarks was due to the fact that they are early forced steps easy to perform in routine laboratories. In some experiments, cell sample morphology was also assessed by light microscopy, as previously published [10]. Microscopy measurements were performed by two different operators in more than five fields including 100 cells or more. In PBMLs and U937 cells, the number of apoptotic cells was evaluated at intervals of 12 or 24 h up to 120 h. Data were expressed as a percentage of apoptotic cells.
Cytosolic free thiol (SHi) assay. An aliquot of PBMLs (from 5 x 105 to 2 x 106 cells) was lysed and homogenized using sonication, as previously described [9]. After centrifugation at 14 000 r.p.m. for 5 min in a Beckman microspin centrifuge at 4°C, the supernatant (cytosolic fraction) was processed immediately for analysis of free SHi [11].
Assay of vitamin E, fatty acids and lipoperoxidative subproducts in plasma and blood cells. Plasma vitamin E levels of the patients in subgroup 2 (treated with the vitamin E-modified filter) were determined by HPLC analysis [8,9]. Some preliminary experiments were also performed to determine the levels of vitamin E and arachidonic acid (C20:4) in cells from patients dialysed with CL-E and in cells cultured in the presence of vitamin E-acetate. The lipoperoxidation end-product malondialdehyde (MDA) was measured using the thiobarbituric acid (TBA) test coupled with direct determination by HPLC as described in Buoncristiani et al. [11].
Assay of the pro-apoptotic and pro-oxidant activity of uraemic plasma and antioxidant supplementation of U937 cells. To investigate the effect of uraemic plasma on mononuclear leukocyte apoptosis, we used the U937 human monocytemacrophage leukaemia cell line. In some experiments, mononuclear leukocytes (1 x 106 cells/ml) from healthy donors (blood was kindly provided by Dr Picardi, Transfusion Center, ASL Urbino Hospital, Italy) or HD patients as prepared above were also used. Preliminary experiments and data in the literature [10,12] demonstrate that the apoptosis of these cells shows similar biochemical and morphological features. Thus, U937 can be used with a good approximation as a reference cell system (reporter cells) to investigate the underlying mechanisms of apoptosis in PBMLs. The cells (1 x 106 cells/ml), cultured as described above, were exposed for up to 120 h to uraemic and control plasma at a final concentration ranging from 5 to 50% (v/v). This test was designed to quantify the pro-apoptotic activity of the plasma and other biological fluids including dialysis fluids.
In some experiments, the cells were cultured in the presence of 70 µM R,R,R--tocopheryl-acetate (vitamin E-acetate) in agreement with Devaraj et al. [13], or in the presence of the thiol supplier NAC [12,14] at the final concentration of 0.5 mM. Then, apoptosis and SHi were measured over a 24 h time period. TNF-
in the serum and culture medium was measured by immunoassay (R&D Systems Europe, Abingdon, Oxon, UK).
Assay of the pro-apoptotic activity of the ultrafiltrate on U937 cells. To investigate further uraemic solutes responsible for the apoptotic triggering of mononuclear leukocytes and their removal with HD, U937 cells were exposed to ultrafiltrate produced using the F10HPS low-flux PS dialyser (Fresenius Medical Care, Cremona, Italy). Briefly, in seven patients after the initial 10 min of HD carried out with a standard procedure, the circuit was stopped and emptied of dialysate. The circuit was recommenced in pure ultrafiltration mode and ultrafiltrate was collected after 10 min. Standard HD was recommenced. Twenty minutes before the end of the treatment (4 h), the same procedure was performed to collect further ultrafiltate. Fifty ml of blood from healthy subjects (n = 3) was harvested and put through a circuit producing ultrafiltrate with the same procedure, and was used as control. Since the dialysis membrane used shows a nominal cut-off of 5200 Da, only small and middle molecular weight solutes were obtained with the ultrafiltrate.
Each solution after 1/1 (v/v) dilution with fresh RPMI was added to U937 cells (1 x 106/ml) and incubated with gentle shaking for 48 h.
Statistics
Statistical analysis was carried out using the paired t-test and non-parametric one-way ANOVA. Values were expressed as means ± SD. Correlation between variables was determined by linear regression analysis; P > 0.05 was considered not significant.
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Results |
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Figure 1 A and Table 1 show the incidence of apoptosis in PBMLs of uraemic patients (HD and CAPD) and healthy controls. In the PBMLs of both healthy controls and chronic uraemic patients (HD and CAPD), the rate of apoptosis increased linearly over time; however, in the HD group, the apoptosis rate was more than twice that of controls and CAPD patients. The PBML apoptosis levels at 24 h assessed by fluorescence microscopy and cytofluorimetry were comparable, with a value in HD patients of 16 ± 6 vs 4 ± 3% in healthy controls (P < 0.01).
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Neither diabetes (n = 8 in HD patients) nor smoking (n = 9 in HD patients and n = 4 in healthy controls) affected the apoptotic rate and SHi of PBMLs (data not shown).
In vivo protection from apoptosis by vitamin E
Table 2 shows the levels of apoptosis, as well as plasma and blood cell vitamin E and SHi, both before and after 1 month of treatment with CL-E in a subgroup of chronic HD patients. The noticeable increase in plasma vitamin E observed in these subjects (from 14.6 ± 4.9 to 26.8 ± 6.5 mg/l) was related to a decrease in PBML apoptosis from 18.7 ± 3.0 to 14.0 ± 4.1% (P < 0.05, r = 0.960). During the time period in which CR filters were used, the levels of plasma vitamin E (15.6 ± 3.5 mg/l) did not differ from those of healthy controls. Similarly, SHi before treatment with CL-E were normal or slightly decreased. After CL-E treatment, total blood thiols increased significantly while SHi increased without reaching the statistical cut-off required for significance (Table 2). The plasma ratio of arachidonic acid/total triglycerides in these patients increased by 220% after treatment with vitamin E-modified filters.
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When cells were incubated for 24 h with plasma collected in pre-HD, after 30 min of HD and in post-HD, apoptosis was, respectively, 19 ± 8, 28 ± 10 and 15 ± 7% (Figure 3A). In U937 cells exposed for 24 h to uraemic plasma collected at different time points during HD, the levels of apoptosis and the decrease in SHi were linearly correlated (r = 0.689, P < 0.01) (Figure 3B).
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The in vitro effect of vitamin E and NAC on apoptosis and SHi of PBML.
The incubation of U937 cells with uraemic plasma, but not with healthy control plasma, resulted in an increased rate of apoptosis and cell membrane lipid oxidation, and a decrease of SHi (Table 3, Figures 2 and 3). Similar data but with a higher variability due to a high percentage of necrosis at 24 h were also observed in PBMLs from healthy controls (not shown). The in vitro supplementation of these cells with a concentration of vitamin E similar to that reached in the serum of patients on CL-E (see above), i.e. 70 µM, decreased the apoptotic rate observed at 24 h of culture in the presence of uraemic plasma from 14 ± 6 to 9 ± 5% (P < 0.01) and significantly increased SHi (P < 0.05) (Table 3). The levels of arachidonic acid in the cell membrane increased by 170%, and TBA reactants were lowered by 49 ± 16% after the in vitro supplementation of PBMLs with vitamin E. Similar results were obtained for the spontaneous apoptosis of PBMLs, that was inhibited by >60% in the presence of 70 µM vitamin E.
Also, the supplementation of the culture medium with 0.5 mM of the thiol supplier NAC dramatically reduced the apoptotic rate of U937 cells induced by the plasma of HD patients under increased SHi concentrations (Figure 4).
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Discussion |
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Conditions of oxidative stress could play a key role in this increased apoptotic rate as they participate in both the initiation and signalling of the apoptotic process of mononuclear cells elicited by apparently diverging stimuli such as oxidants, proinflammatory cytokines and drugs [5,12,15]. Recently, a model in which oxidant stress can represent a definite strategy for mononuclear cells to commit suicide has been proposed. This model is based on the evidence that both oxidant stress-related and -unrelated apoptotic stimuli elicit cell thiol perturbations through the active extrusion of the main cytosolic thiol, i.e. reduced glutathione [5,10]. This early event sustains the apoptotic signalling together with other events such as changes in the control of cytosolic Ca2+ homeostasis and mitochondrial potential that result in cytocrome c release and further oxidant stress. This controlled disruption of SHi homeostasis can be reversed with a full rescue of cell function and viability once apoptotic signalling would not reach late steps. Accordingly, thiol suppliers such as NAC [12,14] or other antioxidants such as vitamin E [8,16] could be used to down-regulate apoptosis in different cell models.
In this study, we provide evidence of the association between an increased in vitro apoptotic rate and decreased SHi in PBMLs of chronic HD patients. To our knowledge, this is the first evidence of this oxidant stress-based mechanism in the accelerated apoptosis of PBMLs in ESRD patients. The following results support this evidence: (i) in HD patients, the levels of PBML apoptosis after 24 h of culture were more than twice as high as in PD patients and controls, and SHi levels were lowered by >40%; (ii) U937 cells exposed to uraemic plasma showed a time-dependent increase of apoptosis and SHi depletion; (iii) the same relationship between loss of SHi and extent of apoptosis was observed when PBMLs from both controls and HD patients were studied in vitro for spontaneous apoptosis or apoptosis induced by uraemic plasma; (iv) the susceptibility of PBMLs to undergo apoptosis and SHi loss was inversely correlated with the concentration of vitamin E in the culture medium; and (v) in HD patients, a 1-month treatment with CL-E dialysers increased blood vitamin E, decreased the levels of apoptosis in circulating PBMLs and partial restoration of total blood and cell SH.
Unfortunately, the modality of cell preparation and the investigation procedures used did not permit to study the apoptosis and SHi of leukocyte subsets. However, lymphocytes are known to predominate largely in PBML preparations (90%) and, after 24 h in culture, monocytes progressively reduced to <5% of total cells as a function of cell manipulation and adhesion to culture dishes. Therefore, our data on PBMLs from patients and controls mainly reflect the apoptotic rate and SHi levels of lymphocytes. However, as suggested by the results obtained using the monocytemacrophage cell line U937, apoptotic pathways of monocytes and lymphocytes largely overlap particularly as regards the role played by the SHi-related apoptotic signalling [5].
An increased apoptotic rate in lymphocytes from HD patients was demonstrated previously in both the T and B subsets [2,6], and the anti-apoptotic oncogene product Bcl-2 was suggested to play a role in the increased susceptibility to apoptotic death of B lymphocytes [6]. The fact that this factor contains a critical cysteine residue and is known to provide its anti-apoptotic function through an antioxidant-like mechanism gives further support to our evidence that SHi homeostasis perturbations may represent an underlying mechanism for the increased apoptosis of PBML in HD patients.
This increased apoptotic rate could originate from different untoward events associated with uraemia and dialysis. Other than a poor membrane biocompatibility [7], pro-apoptotic solute accumulation [3,17] and defective growth factor production [such as interferon-, interleukin (IL)-4 and IL-10] (see Fernandez-Fresnedo et al. [6] and references therein) could be considered as apoptotic stimuli in HD patients. In this study, we demonstrated that in HD patients, both plasma and ultrafiltrate contain low and middle molecular weight solutes capable of triggering apoptosis in mononuclear leukocytes. Interestingly, the pro-inflammatory monokine TNF-
was observed to accumulate strongly in the blood of HD patients at different time points during the dialysis, but its levels correlated with apoptosis only before HD. This preliminary evidence suggests that during HD, other factors could take part in the oxidant stress-related apoptosis of PBMLs. In this context, complement-related factors, peptides already studied as regards their apoptotic role in uraemic polymorphonuclear leukocytes, glycation and oxidation end-products, and bioactive lipids could play a role [3,4,7,17], and will be the subject of further investigation.
Assuming that oxidant stress could be a relevant underlying mechanism for the increased PBML apoptotic rate in HD patients, we tested vitamin E-modified dialysis membranes (or CL-E) as a possible strategy to constrain, at least partially, this HD-associated drawback. We preliminarily observed that CL-E might affect PBML function and apoptosis, with several mechanisms involving both biocompatibility and bioreactivity properties of these dialysers [5,8]. Biocompatible membranes are known to decrease mononuclear cell apoptosis, pro-inflammatory cytokine generation [4] and accumulation of the DNA oxidation marker 8-OH-deoxy-guanosine in leukocytes [18]. In this context, CL-E membranes were observed to be comparable with synthetic membranes [18]. CL-E has been proposed to provide a protection against oxidant stress and leukocyte activation through the combined effect of increasing, at least in part, blood levels of vitamin E and providing a lower cell activation and antioxidant protection on the blood surface of the dialysis membrane. Moreover, recent studies (reviewed in Galli [19]) showed that CL-E could improve whole blood antioxidant systems adversely affected by non-biocompatible dialy-sers and of key importance in blood cell protection, such as plasma and blood cell thiols and vitamin C.
The relevance and reliability of these findings are supported by the in vitro anti-apoptotic activity of vitamin E on PBMLs of HD patients and controls, and in U937 cells exposed to uraemic plasma. In this context, it is important to consider that other than as a lipophilic antioxidant useful to protect cells against oxidant stress [13], vitamin E has non-antioxidant properties that could help in preventing uncontrolled mononuclear cell activation and apoptosis in HD due to non-biocompatible materials (reviewed in Galli [19]).
In agreement with Heidenreich et al. [1] and Martin-Malo et al. [4], our data demonstrate that an accelerated PBML apoptosis can be found in chronic HD patients but is almost absent in PD patients. This observation provides further support for a role of the oxidant stress as an underlying mechanism for PBML apoptosis in HD patients. In fact, the incidence of oxidant stress has been described to be higher in HD than in PD patients mainly as a consequence of dialysis-induced leukocyte activation [8]. Again, PD can provide a better control of the uraemic toxicity as regards both the continuity and efficacy to remove a broad range of toxins in the low and middle molecular weight range. These may include solutes described to trigger U937 cell activation via specific receptor-dependent pathways, and that are not removed efficiently with conventional high-flux dialysers, such as protein glycation and oxidation end-products [17,20].
In the present study, we found that a significant fraction (approximately two-thirds) of the pro-apoptotic substances contained in the plasma before HD could be lowmiddle molecular weight solutes (largely removed by membranes with a cut-off of 1030 kDa). TNF- and its homologues could fall into this class of solutes as they might justify an increased cell death rate and oxidative stress [14] in cells that express receptors for this class of pro-inflammatory cytokines, such as mononuclear leukocytes. However, the pro-apoptotic activity of the uraemic plasma was maximal at 30 min of dialysis, i.e. the time at which the peak of complement-mediated leukocyte activation is observed, while serum TNF-
showed a good correlation with the pro-apoptotic activity of the uraemic plasma only before HD. The absence of such a correlation at 30 min and after HD could suggest that different pro-apoptotic factors, such as complement factors and bioactive lipids, could be recruited during HD. Interestingly, the correlation between the apoptotic rate and SHi reduction in mononuclear cells exposed to uraemic plasma is maintained despite the fact that the amount and type of apoptotic stimuli may change as a function of the dialysis time. This evidence is in agreement with the in vitro finding that oxidant stressunder the outward appearance of a decrease in SHiaccompanies the apoptotic machinery independently from the type of apoptotic trigger [5,10].
In conclusion, PBMLs of HD patients but not PD patients show an increased apoptotic rate and a lower content of SHi. These defects correlate with and appear to be the consequence of a series of factors that in part are related to the uraemic toxicity and in part derive from the untoward effects of the HD procedures. As further evidence in support of a causeeffect relationship between this increased apoptotic rate and a fall in SHi in mononuclear leukocytes, it has been observed here that both the lipophilic antioxidant vitamin E and the thiol supplier NAC in vitro can reduce to different extents these changes in PBMLs of HD patients as well as in U937 cells exposed to uraemic plasma and ultrafiltrate. Similar results were also obtained in vivo when plasma vitamin E and SHi were increased by the treatment with vitamin E-modified dialysers. These results highlight the potential role of antioxidant-based strategies to constrain the abnormal apoptosis in different mononuclear leukocyte subsets of chronic HD patients.
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Acknowledgments |
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References |
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