Background

5-Lipoxygenase activity is enhanced in peripheral blood mononuclear cells (PBMC) from end-stage renal disease (ESRD) patients on maintenance hemodialysis (HD), leading to lipoperoxidation and reactive oxygen species formation. These effects are prevented by vitamin E, which inhibits 5-lipoxygenase activity. The present study was designed to test the possibility that 5-lipoxygenase activation might cause mitochondrial damage and cytochrome c release, ultimately leading PBMC to apoptosis.

Methods

Apoptosis, mitochondrial uncoupling, and cytochrome c release were investigated in PBMC from 16 healthy volunteers and 16 ESRD patients on maintenance HD with cuprammonium rayon (CL-S) membranes in a two-step crossover study: after a four-week treatment with vitamin E-coated cuprammonium rayon (CL-E) membranes, and again after a four-week treatment with oral vitamin E.

Results

Compared to healthy controls, PBMC from ESRD patients showed a threefold increase in mitochondrial uncoupling and cytochrome c release (within 4 and 8 hours, respectively), followed by a threefold increase in apoptotic body formation (within 48 hours). Regardless of the administration route, vitamin E reduced mitochondrial uncoupling, cytochrome c release and apoptosis of mononuclear cells, as did the 5-lipoxygenase inhibitor eicosatetraynoic acid. Conversely, the cyclooxygenase inhibitor indomethacin was ineffective.

Conclusions

Reported data suggest that the 5-lipoxygenase branch of the arachidonate cascade is only responsible for mitochondrial disruption and apoptosis of PBMC of ESRD patients, and that vitamin E may be helpful in the control of oxidative stress-related disease in these subjects, independent of the administration route.

METHODS

Patients

After informed consent was obtained, sixteen ESRD patients (aged 54 5 years) under maintenance HD from 60 5 months, and sixteen age-matched healthy controls (52 5 years) were enrolled into the study. ESRD patients and healthy controls were not matched for concurrent cardiovascular disease. However, neither ESRD patients nor healthy controls experienced acute myocardial infarction or cerebrovascular accident before the beginning of the study. Nine subjects in the patient group and three in the control group had taken antihypertensive medications, three subjects in the patient group, and one in the control group, had taken -hydroxymethylglutaryl-GA (HMGCoA) reductase inhibitors. All ESRD patients received a standard bicarbonate dialysis schedule of four hours, three times weekly, using 1.3 square-meter cuprammonium rayon membranes (Clirans®S15, CL-S), for at least six months. The cause of ESRD was chronic glomerulonephritis in nine patients, nephroangiosclerosis in five, and polycystic kidney disease in two. Healthy controls showed normal parameters of renal function. All patients and healthy controls were nonsmokers for at least one year. Both ESRD patients and controls were on a free diet with a normal and constant intake of essential fatty acids, and none of them took any drug with an established or potentially oxidizing effect. In particular, any treatment with HMGCoA reductase or ACE inhibitors, or with angiotensin (AT) receptor blockers, was suspended in both ESRD patients and healthy controls for at least one month before the beginning of the study. When needed, blood pressure was kept under control with alternative drugs. We performed a two-step crossover study. In the first step, PBMC were isolated from ESRD patients treated with CL-S filters before and after a four-week treatment with a 1.3 square-meter Excebrane® multi-layer vitamin E-coated cuprammonium rayon (Clirans®E15, CL-E) dialyzer, and then after another four-week washout with CL-S filters. In the second step, PBMC were isolated from the same patients treated with CL-S filters before and after a four-week treatment with oral vitamin E 600 mg/daily (Ephynal tabs, 300 mg bid; Roche, Milan, Italy), and then after another four-week washout with CL-S filters only. Both Clirans®S15 and Clirans®E15 membranes are brands from Terumo Corporation (Tokyo, Japan).

Evaluation of cell death, mitochondrial uncoupling, and cytochrome c release

PBMC (1 x 10⁶/test) were isolated as described⁴, washed in phosphate-buffered saline, and cultured for up to 48 hours in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum⁷. Apoptotic body formation in dead cells was then quantified by flow cytometric analysis in a FACScalibur Flow Cytometer (Becton Dickinson), stained with propidium iodide (50 g/mL, pretreated also with 15,000 U/mL RNase to reduce noise; from Sigma Chemical Co., St. Louis, MO), as reported⁹. Control PBMC were treated with vehicle alone and contained less than 4.0 1.0 apoptotic bodies for every 100 cells analyzed⁹. Mitochondrial uncoupling of PBMC was evaluated by flow cytometric analysis in a FACScalibur Flow Cytometer, using the fluorescent probe JC-1 (20 M, dissolved in dimethylsulfoxide; from Molecular Probes, Eugene, OH)⁹.

The amount of cytochrome c released into the cytosol of PBMC was quantified by enzyme-linked immunosorbent assay, previously validated by Western blot analysis⁹. Anti-cytochrome c monoclonal antibodies (PharMingen, San Diego, CA), diluted 1:250, and goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (Bio-Rad Laboratories, Hercules, CA), diluted 1:2000, were used as primary and secondary antibodies, respectively.

RESULTS

Apoptosis and mitochondrial damage in PBMC from ESRD patients

PBMC from ESRD patients showed a maximum 3.3-fold increase in apoptotic body formation, compared to PBMC from healthy controls, after 48 hours of culture Table 1. However, in cells isolated from patients treated with vitamin E (either filter-bound or supplied orally) apoptotic bodies were only twofold higher than in control PBMC Table 1. Mononuclear cells from ESRD patients also showed mitochondrial uncoupling, which reached a 3.5-fold maximum in healthy controls after 4 hours, and increased cytochrome c release with a threefold maximum after 8 hours of culture Table 1. Again, vitamin E prevented mitochondrial uncoupling and cytochrome c release, regardless of its administration route Table 1. Moreover, 50 M vitamin E or 10 M eicosatetraynoic acid (ETYA), a selective 5-lipoxygenase inhibitor⁶, significantly reduced apoptotic body formation, mitochondrial uncoupling, and cytochrome c release in mononuclear cells from post CL-S patients. Conversely, 100 M indomethacin, a selective inhibitor of cyclooxygenase, which converts arachidonic acid into prostanoids⁵, was ineffective Table 1. Finally, in PBMC of the post CL-S patients, the caspase-3 inhibitor Z-DEVD-FMK⁹ significantly reduced apoptosis, but not mitochondrial uncoupling or cytochrome c release, when used at 50 M Table 1.

Table 1 - Mitochondrial uncoupling, cytochrome c release, and apoptosis of PBMC from ESRD patients on maintenance HD.

Full table

DISCUSSION

Recently, we have shown that membrane lipid peroxidation and cytosolic ROS formation occur in PBMC of ESRD patients on maintenance HD, mainly due to the activation of 5-lipoxygenase⁴. We have also shown that in the same patients vitamin E prevents 5-lipoxygenase-mediated oxidative stress by directly inhibiting enzyme activity⁴. Activation of the lipoxygenase pathway has been associated to the early phase of PCD in several mammalian cells⁶. Here, we extend these findings to mononuclear cells of uremic patients, where apoptosis has been observed⁷, but not elucidated. Remarkably, PCD of mononuclear cells was paralleled by mitochondrial uncoupling and cytochrome c release (i.e., by disruption of mitochondrial integrity) Table 1, which is consistent with the reported ability of lipoxygenase to make "pore-like" structures in organelle membranes⁹. Moreover, cytochrome c release usually triggers caspase activation, a common step in several unrelated apoptotic pathways, and indeed, inhibition of caspase-3, which is upstream of the caspase cascade^6,9, blocked cell death Table 1. Taken together, it can be proposed that activation of 5-lipoxygenase is pivotal in the execution of apoptosis in PBMC from ESRD patients, according to the scheme depicted in Figure 1. The scheme also shows that orally or intramuscularly supplied vitamin E, as well as the filter-bound form, increases the plasma level of vitamin E⁸, which in turn inhibits 5-lipoxygenase activity and prevents mitochondrial damage and PCD. It seems noteworthy that 5-lipoxygenase-dependent oxidative stress may not only be related to, but somewhat specific for, ESRD patients under maintenance HD. This hypothesis could explain the beneficial effects of the administration of high doses of vitamin E in ESRD patients with preexisting cardiovascular disease, reported in the Secondary Prevention with Antioxidants of Cardiovascular Disease in Endstage Renal Disease (SPACE) study¹⁰, which, however, were neither confirmed in non-ESRD patients, nor in the general population. Yet, the relatively simple experimental model of the study reported here (i.e., ESRD patients versus non-ESRD healthy subjects) did not take into account any concurrent cardiovascular disease; further investigations are deemed necessary to ascertain whether 5-lipoxygenase activation and subsequent apoptosis, if ever present, are related to this type of pathologic condition. In conclusion, these data might be relevant for the treatment of various oxidative stress-related diseases that still account for a significant morbidity and mortality among ESRD patients¹¹.

Figure 1.

Interplay between vitamin E, 5-lipoxygenase, mitochondrial damage, and apoptosis of mononuclear cells. ESRD patients on maintenance HD show enhanced 5-lipoxygenase (5-LOX) activity in peripheral blood mononuclear cells, which leads to increased membrane lipoperoxides and cytosolic reactive oxygen species (ROS), and to reduced mitochondrial membrane potential (). The increased cytochrome c (cyt c) release from mitochondria activates the caspase cascade, which, together with lipoperoxide and ROS formation, triggers nuclear alterations and ultimately leads to apoptosis. Oral or intramuscular (IM) administration of vitamin E, as well as the filter-bound form, increases the plasma level of vitamin E, which prevents ESRD/HD-induced apoptosis by directly inhibiting 5-LOX activity.

Full figure and legend (93K)

References

References

Acknowledgments

The authors express their gratitude to Dr. Monica Bari and Dr. Natalia Battista for their skillful assistance with PBMC culture and treatment, and to Dr. Simone Manca di Villahermosa for his experienced clinical cooperation. This investigation was partly supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica – Consiglio Nazionale delle Ricerche (MURST-CNR Biotechnology Program L. 95/95), Rome.