Mononuclear leukocyte apoptosis in haemodialysis patients: the role of cell thiols and vitamin E

Francesco Galli1,2, Lina Ghibelli3, Umberto Buoncristiani4, Valeria Bordoni2,5, Vincenzo D’Intini5, Serena Benedetti2, Franco Canestrari2, Claudio Ronco5 and Ardesio Floridi1

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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. An increased apoptotic rate of peripheral blood mononuclear leukocytes (PBMLs) in haemodialysis (HD) patients has been reported in several studies, but its underlying mechanisms remain poorly understood. Oxidant stress is a well known cause of cell damage, and several lines of evidence suggest that it might influence the induction and signalling steps of mononuclear cell apoptosis through different mechanisms so as to provoke disturbances of the intracellular pool of thiols (SHi). In this study, we investigated the in vitro apoptotic rate and SHi of PBMLs in end-stage renal disease (ESRD) patients on HD or peritoneal dialysis (PD).

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 low–middle 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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
A multifactorial impairment of the immune function is diagnosed commonly in chronic renal failure (CRF), and is particularly exacerbated in chronic haemodialysis (HD) patients.

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-{alpha} (TNF-{alpha}) 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.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and clinical study
A group of 40 ESRD patients (18 males and 22 females) and an age-matched group of healthy controls (n = 10; five males and five females) were studied after informed consent. Thirty patients were on chronic HD with a standard three times a week dialysis protocol for an average time of 62 ± 38 months (range: 28–122), and showed a mean age of 69 ± 7 years (range: 58–78). Ten patients were on PD by a standard procedure (8 l/day in four exchanges) for an average time of 44 ± 8 months (range: 37–58) and had a mean age of 68 ± 5 years (range: 62–78). Primary renal diseases were chronic glomerulonephritis (n = 15), chronic pyelonephritis (n = 9), polycystic kidney (n = 5), nephroangiosclerosis (n = 9) and unknown (n = 2). Patients showing conditions of immunodeficiency, acute and chronic infections, malnutrition, hyperparathyroidism and iron overload at the enrolment were excluded. All pharmacological treatments influencing the immune function and antioxidant status (including antioxidant supplements) were also considered as exclusion criteria.

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-{alpha}-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 monocyte–macrophage 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 monocyte–macrophage 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-{alpha}-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-{alpha} 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.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Apoptosis and thiol loss in the PBMLs of uraemic patients undergoing dialysis
Total leukocyte counts in both HD and CAPD patients were slightly greater than in healthy controls (P = NS). The percentage of total (T+B) lymphocytes was lower in HD patients (24.2 ± 3.6; P < 0.01) and CAPD patients (27.8 ± 4.4; P < 0.05) compared with controls (33.9 ± 6.4), while the relative granulocyte count was higher in HD patients than in CAPD and controls (67.5 ± 6.8 vs 56.2 ± 7.1 and 53.9 ± 4.5; P < 0.05 in both). Also, the number of monocytes was slightly but not significantly greater in HD patients than in the other two groups (data not shown). In scoring the number of monocytes and lymphocytes in PBML cultures, no significant difference was observed between patients and controls. Cell counts within 24 h of culture were not significantly different from baseline values (loss of <10% in both the cell subpopulations). When incubated in the presence of FCS alone or FCS+plasma for >24 h, the number of monocytes decreased by 35–58% as activation and adhesion to culture dishes occurred, while lymphocyte number did not change significantly. All the data were corrected for this change in monocyte number.

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).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. (A) Time course and mean levels of apoptosis in PBMLs in culture obtained from HD patients (n = 30), CAPD patients (n = 10) and healthy controls (n = 10). The cells were cultured in RPMI 1640 medium and were examined for the levels of apoptosis at different time points by fluorescence microscopy after propidium iodide staining. The data at 24 h of culture were also determined by cytofluorimetry (see Results and Subjects and methods for further details). (B) Free thiol (SHi) levels in the cytosol of PBMLs assessed by spectrophotometric analysis at 24 h of culture of a subgroup of the same subjects as in (A) (HD, n = 12; CAPD, n = 9; controls, n = 10). *P < 0.01: HD vs CAPD and controls.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Levels of apoptosis and cytosolic thiols in PBMLs of healthy control subjects and chronic HD or CAPD patients after 24 h of culture

 
PBML levels of SHi are shown in Table 1. In the subgroup on HD, the SHi concentrations were < 0.12 µmol/mg of cytosolic protein (–40% vs the mean value of healthy controls), and PBML apoptosis was >10%. Both these values were significantly different from CAPD and healthy control values. These latter two groups showed comparable levels of both SHi and apoptosis. A negative correlation between the levels of apoptosis and SHi was observed at 24 h of culture in both patients and controls (r = –0.648, P < 0.001) (Figure 1B).

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.


View this table:
[in this window]
[in a new window]
 
Table 2. Levels of plasma vitamin E, PBML and total blood thiols, and PBML apoptosis in chronic HD patients (n = 15) before and after a 1-month period of treatment with vitamin E-modified filters (CL-E)

 
Effect of the uraemic plasma and ultrafiltrate on the apoptosis of mononuclear cells
The time course of apoptosis in U937 cells cultured in the presence of pre-HD plasma is shown in Figure 2. The data show that in the plasma of HD patients, but not healthy controls, are present factors able to trigger apoptosis in this cell model (see also Table 3). A sudden increase between 24 and 48 h of culture was observed featuring an ‘apoptotic burst’; this test could be used to characterize the presence in uraemic blood samples of high loads of apoptosis-triggering substances.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Time course of apoptosis in U937 cells exposed to plasma of HD patients and controls. Aliquots of plasma used in these experiments were obtained from patients immediately before HD and from healthy controls (n = 6 in both cases), showing levels of apoptosis at 24 h of culture, respectively, of >14 and <5%. The monocyte–macrophage human leukaemia cell line U937 was cultured as described in Materials and methods in the presence of plasma (20% v/v). SHi levels in these cells were assessed after 24 h (see Table 3). *P < 0.05 and **P < 0.01 vs baseline evaluation.

 

View this table:
[in this window]
[in a new window]
 
Table 3. Apoptosis, cytosolic thiols (SHi) and lipid peroxidation (as thiobarbituric acid reactants or TBARs) in U937 cells incubated with plasma (20% v/v) from healthy subjects and HD patients before and after vitamin E supplementation

 
Before dialysis, the serum of HD patients compared with controls contained more than five times higher levels of TNF-{alpha} (in pg/ml: 7.8 ± 2.9 vs 1.4 ± 0.5 pg/ml; P < 0.001). These levels did not change significantly after 30 min and post-HD (7.3 ± 2.8 and 8.5 ± 4.2 pg/ml). In pre-HD, a positive correlation between U937 apoptosis induced by uraemic plasma and levels of TNF-{alpha} was observed (r = 0.742, P = 0.002). This correlation was lost when plasma samples collected after 30 min and post-HD were studied (data not shown).

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).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3. Levels of apoptosis (A) and SHi (B) in U937 cells exposed to plasma from HD subjects. Plasma was collected before, after 30 min and at the end of dialysis. Experiments were carried out as described in Figure 2. *P < 0.01 vs tests carried out on plasma collected before and after HD.

 
The ultrafiltration of plasma collected before and after 30 min of HD (mini-concentrator membrane cut-off of 10 kDa) lowered the apoptosis at 24 h by 39 ± 18 and 23 ± 12%, respectively (n = 5). The ultrafiltrate procured from dialysed subjects (see Subjects and methods) produced an apoptosis of 22.9 ± 13.8 and 13.8 ± 6.6%, respectively, at the beginning and after the dialysis (control value = 2.7 ± 1.7%).

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).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. The effect of the thiol supplier N-acetyl-cysteine (NAC) on the apoptotic rate and SHi of U937 cells exposed to plasma from HD patients. Aliquots of plasma used in these experiments were obtained immediately before dialysis from four HD patients showing levels of apoptosis at 24 h of culture of 16 ± 5%. NAC was supplemented in the culture medium at a concentration of 0.5 mM. Experimental conditions were the same as in Figures 2 and 3.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
A growing body of evidence demonstrates that both mononuclear and polymorphonuclear leukocytes of HD patients show a characteristic accelerated rate of apoptosis, and this can play a key role in the defective immune response of these patients [13]. This could be the consequence of a chronic and extensive challenge to immune cells by different and possibly converging stimuli that arise from the accumulation of uraemic toxins, the use of bio-incompatible dialysis membranes, and the progressive worsening of the overall clinical condition.

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 monocyte–macrophage 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-{gamma}, 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-{alpha} 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 low–middle molecular weight solutes (largely removed by membranes with a cut-off of 10–30 kDa). TNF-{alpha} 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-{alpha} 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 stress—under the outward appearance of a decrease in SHi—accompanies 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 cause–effect 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.



   Acknowledgments
 
We thank Dr Giorgio Brandi for helpful discussion and assistance in fluorescence microscopy studies, and Miss Jessica Bianchi for technical work.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Heidenreich S, Schmidt M, Bachmann J, Harrach B. Apoptosis of monocytes cultured from long-term hemodialysis patients. Kidney Int 1996; 49: 792–799[ISI][Medline]
  2. Matsumoto Y, Shinzato T, Amano I et al. Relationship between susceptibility to apoptosis and Fas expression in peripheral blood T cells from uremic patients. A possible mechanism for lymphopenia in chronic renal failure. Biochem Biophys Res Commun 1995; 215: 98–105[CrossRef][ISI][Medline]
  3. Jaber BL, Cendoroglo M, Balakrishnan VS, Perianayagam MC, King AJ, Pereira BJ. Apoptosis of leukocytes: basic concepts and implications in uremia. Kidney Int 2001; 78: S197–S205
  4. Carracedo J, Ramirez R, Madueno JA et al. Cell apoptosis and hemodialysis-induced inflammation. Kidney Int 2002; 80: 89–93[CrossRef]
  5. Coppola S, Ghibelli L. GSH extrusion and the mitochondrial pathway of apoptotic signalling. Biochem Soc Trans 2000; 28: 56–61[ISI][Medline]
  6. Fernandez-Fresnedo G, Ramos MA, Gonzalez-Pardo MC, de Francisco AL, Lopez-Hoyos M, Arias M. B lymphopenia in uraemia is related to an accelerated in vitro apoptosis and dysregulation of Bcl-2. Nephrol Dial Transplant 2000; 15: 502–510[Abstract/Free Full Text]
  7. Carracedo J, Ramirez R, Martin-Malo A, Rodriguez M, Aljama P. Nonbiocompatible hemodialysis membranes induce apoptosis in mononuclear cells: the role of G-proteins. J Am Soc Nephrol 1998; 9: 46–53[Abstract]
  8. Galli F, Rovidati S, Benedetti S et al. Lipid peroxidation, leukocyte function and apoptosis in hemodialysis patients treated with vitamin E-modified filters. Contrib Nephrol 1999; 127: 156–171[Medline]
  9. Galli F, Rovidati S, Chiarantini L, Campus G, Canestrari F, Buoncristiani U. Bioreactivity and biocompatibility of a vitamin E-modified multi-layer hemodialysis filter. Kidney Int 1998; 54: 580–589[CrossRef][ISI][Medline]
  10. Ghibelli L, Coppola S, Rotilio G, Lafavia E, Maresca V, Ciriolo MR: Non-oxidative loss of glutathione in apoptosis via GSH extrusion. Biochem Biophys Res Commun 1995; 216: 313–320[CrossRef][ISI][Medline]
  11. Buoncristiani U, Galli F, Rovidati S, Albertini MC, Campus G, Canestrari F. Oxidative damage during hemodialysis using a vitamin E-modified dialysis membrane: a preliminary characterization. Nephron 1997; 77: 57–61[ISI][Medline]
  12. Malorni W, D’Ambrosio A, Rainaldi G, Rivabene R, Viola M. Thiol supplier N-acetylcysteine enhances conjugate formation between natural killer cells and K562 or U937 targets but increases the lytic function only against the latter. Immunol Lett 1994; 43: 209–214[CrossRef][ISI][Medline]
  13. Devaraj S, Li D, Jialal I. The effects of alpha tocopherol supplementation on monocyte function. Decreased lipid oxidation, interleukin 1 beta secretion, and monocyte adhesion to endothelium. J Clin Invest 1996; 98: 756–763[Abstract/Free Full Text]
  14. Talley AK, Dewhurst S, Perry SW et al. Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA. Mol Cell Biol 1995; 15: 2359–2366[Abstract]
  15. Fernandes RS, Cotter TG. Apoptosis or necrosis: intracellular levels of glutathione influence mode of cell death. Biochem Pharmacol 1994; 48: 675–681[CrossRef][ISI][Medline]
  16. Ricciarelli R, Tasinato A, Clement S, Ozer NK, Boscoboinik D, Azzi A. {alpha}-Tocopherol specifically inactivates cellular protein kinase C{alpha} by changing its phosphorylation state. Biochem J 1998; 15: 243–249
  17. Cohen G, Rudnicki M, Walter F, Niwa T, Horl WH. Glucose-modified proteins modulate essential functions and apoptosis of polymorphonuclear leukocytes. J Am Soc Nephrol 2001; 12: 1264–1271[Abstract/Free Full Text]
  18. Tarng DC, Huang TP, Liu TY, Chen HW, Sung YJ, Wei YH. Effect of vitamin E-bonded membrane on the 8-hydroxy 2'-deoxyguanosine level in leukocyte DNA of hemodialysis patients. Kidney Int 2000; 58: 790–799[CrossRef][ISI][Medline]
  19. Galli F. Vitamin E-modified dialysers. Contrib Nephrol 2002; 137: 95–105[ISI][Medline]
  20. Ando M, Gafvels M, Bergstrom J, Lindholm B, Lundkvist I. Uremic serum enhances scavenger receptor expression and activity in the human monocytic cell line U937. Kidney Int 1997; 51: 785–792[ISI][Medline]
Received for publication: 24. 9.02
Accepted in revised form: 28. 2.03





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Disclaimer
Request Permissions
Google Scholar
Articles by Galli, F.
Articles by Floridi, A.
PubMed
PubMed Citation
Articles by Galli, F.
Articles by Floridi, A.