Leukocyte activation and the resulting oxidative stress induced by bioincompatible materials during hemodialysis impact the prognosis of patients. Despite multiple advances in hemodialysis dialyzers, the prognosis of hemodialysis patients with complications deeply related to oxidative stress, such as diabetes mellitus, remains poor. Thus, we re-evaluated the effects of hemodialysis on multiple reactive oxygen species using electron spin resonance-based methods for further improvement of biocompatibility in hemodialysis. We enrolled 31 patients in a stable condition undergoing hemodialysis using high-flux polysulfone dialyzers. The effects of hemodialysis on reactive oxygen species were evaluated by two methods: MULTIS, which evaluates serum scavenging activities against multiple hydrophilic reactive oxygen species, and i-STrap, which detects lipophilic carbon-center radicals. Similar to previous studies, we found that serum hydroxyl radical scavenging activity significantly improved after hemodialysis. Unlike previous studies, we discovered that scavenging activity against alkoxyl radical was significantly reduced after hemodialysis. Moreover, patients with diabetes mellitus showed a decrease in serum scavenging activity against alkyl peroxyl radicals and an increase in lipophilic carbon-center radicals after hemodialysis. These results suggest that despite extensive improvements in dialyzer membranes, the forms of reactive oxygen species that can be eliminated during dialysis are limited, and multiple reactive oxygen species still remain at increased levels during hemodialysis.

Dysfunction of polymorphonuclear leucocytes (PMNLs) in end-stage renal disease (ESRD) patients contributes to a diminished immune defence. The serum levels of leptin are elevated in patients with ESRD. We analysed in vitro effects of leptin on PMNLs from healthy subjects (HS; n = 12) and haemodialysis (HD) patients (n = 15) before and after HD.

PMNL oxidative burst and phagocytosis were tested by flow cytometry in whole blood. Chemotaxis of isolated PMNLs was assessed by the under-agarose method. To assess the involvement of leptin in PMNL signalling pathways, signal transduction inhibitors were used and the activity of intracellular kinases was investigated by western blotting, in vitro kinase assays and the Luminex technology.

Increasing the leptin level in the blood of HS leads to a reduced activation of the oxidative burst by Escherichia coli and phorbol 12-myristate 13-acetate. Activation of the oxidative burst is reduced in the blood of HD patients and the addition of leptin does not lead to further PMNL inhibition. Leptin at a concentration measured in HD patients significantly reduces the chemotaxis of PMNLs from HS but had no effect on PMNLs from ESRD patients before and also after HD treatment with high-flux dialysers. The phosphoinositide 3-kinase/Akt pathway is involved in the inhibitory effects of leptin.

In the presence of leptin, PMNLs from HS and HD patients respond differently to stimuli. The lack of response to leptin in PMNLs from HD patients cannot be influenced by HD.

Oxygen radicals have recently been attracting close attention because of their involvement in tissue damage and their close relationship to various clinical conditions. It has been suggested that hemodialysis increases oxidative stress, triggering the development of complications such as atherosclerosis and dialysis-related amyloidosis. We recently developed a dialyzer containing a highly functional polysulfone membrane on which vitamin E had been bonded (PS-ViE). The present study was undertaken to evaluate the biocompatibility of this membrane and to conduct other experiments on the membrane in vitro. Human blood was dialyzed with minidialyzers (300-600 cm(2) membrane area) made of PS-ViE, cellulose, or untreated polusulfone (PS), and the effects of the dialyzers on complements (C3a, C4a, and C5a), cytokines (IL-1beta and IL-8), and granulocyte elastase as well as their anti-oxidative activity were investigated (n = 6). The effect of PS-ViE on complement activation and its effects on cytokines were comparable to those of PS membrane, whereas granulocyte elastase following dialysis with the PS-ViE membrane tended to be lower than that seen with PS membrane. The effects of PS-ViE-induced methemoglobin, lipid peroxide, and oxygen radicals were significantly less than those of PS membrane, indicating the antioxidative activity of PS-ViE. Vitamin E-modified polysulfone membrane dialyzers were found to have favorable effects on the immune system and to express antithrombotic and antioxidative effects.

Vitamin E (Vit.E, alpha-tocopherol) is a natural biological antioxidant and antinflammatory agent, which protects cells from the effects of free radicals and inhibits inflammation. For such properties Vit.E has been used to improve the biocompatibility of materials such as cellulose membrane for hemodialysis. In this study granulocytes adhesion and activation have been studied after contact with normal cell culture grade polystyrene (PS) and Vit.E-coated polystyrene (Vit.E 0.1 and 0.3% (v/v)) using optical microscopy, flow cytometry and substrate zymography. Vit.E increased the number of adherent granulocytes both at 0.1% (11470 +/- 1064 cells/cm(2), P < 0.01) and 0.3% ( 13706 +/-818) cells/cm(2), P < 0.001) concentration compared to normal PS (5529+/-692 cells/cm(2)). The morphology of granulocytes adherent to Vit.E-PS appeared lightly altered and no differences have been observed in their respiratory burst compared to control granulocyte, while matrix metalloproteinase 9 or gelatinase B (MMP-9) release and activation were increased compared to the normal PS samples. Our data indicate that Vit.E-coated surface induced an increase in granulocytes adhesion and MMP-9 release in the absence of the typical oxidative stress, hallmark of granulocytes activation. A possible explanation of the phenomenon is that Vit.E modifies the surface protein adsorption thus increasing cell adhesion and in turn MMP-9 releasing.

Lipid peroxidation and oxidative stress are enhanced in peripheral blood mononuclear cells (PBMCs) from hemodialysis (HD) patients because of upregulation of the 5-lipoxygenase pathway of the arachidonate cascade. 5-Lipoxygenase activity is specifically inhibited by vitamin E both in vitro and in vivo regardless of its administration route.

The effect of arachidonate cascade enzymes and vitamin E on oxidative stress and apoptosis was investigated in PBMCs from 16 maintenance HD patients treated for at least 6 months with cuprammonium rayon membranes in a two-step crossover study: after a 4-week treatment with vitamin E-coated cuprammonium rayon membranes and again after a 4-week treatment with oral vitamin E. Control PBMCs were obtained from 16 healthy volunteers.

Membrane lipoperoxidation, cellular luminescence, membrane fluidity, and leukotriene B(4) content were significantly greater in PBMCs from HD patients; lipoxygenase was upregulated, but prostaglandin H synthase (PHS) was not affected. Regardless of administration route, vitamin E partially controlled lipid peroxidation and oxidative stress through direct inhibition of 5-lipoxygenase. Cultured PBMCs from HD patients showed a significant increase in apoptotic cells compared with controls. Vitamin E markedly reduced cell luminescence, membrane fluidity, and apoptosis, whereas the PHS inhibitor indomethacin was ineffective. Similar results were obtained with control PBMCs induced to apoptosis by hydrogen peroxide.

Reported data suggest that the 5-lipoxygenase branch of the arachidonate cascade is only responsible for membrane peroxidation, oxidative stress, and apoptosis of PBMCs of HD patients, and administration of vitamin E may be helpful in the control of oxidative stress-related disease in these subjects.