Background: The generation during hemodialysis of activated complement fragments and reactive oxygen species, including nitric oxide (NO), may affect peripheral blood mononuclear cell (PBMC) function. Currently, little is known about signal transduction pathways involved in PBMC activation. Jun N-terminal kinase (JNK) is a novel mitogen-activated protein (MAP) kinase phosphorylated and activated in response to oxidative stress and directly involved in cell activation.
Methods: The present study evaluated the activation of JNK in PBMCs isolated from eight uremic patients undergoing, in a randomized manner, three month-subsequent periods of hemodialysis with a low-flux cellulose acetate (CA) and a vitamin E-modified cellulose membrane (CL-E). After each period of treatment, PBMCs were harvested before (T0), during (T15) and after three hours (T180) of dialysis. At the indicated time points, plasma C5b-9 generation by ELISA and inducible NO synthase (iNOS) gene expression by in situ hybridization were evaluated also. The activation of JNK was studied by Western blotting using a specific monoclonal anti-phospho-JNK antibody, which recognizes the activated form of JNK.
Results: At T0, a significant increase in plasma C5b-9 levels was found in CA patients compared to CL-E-treated patients. During hemodialysis, C5b-9 levels rose more significantly in CA patients than in CL-E patients and returned to baseline values only in CL-E patients. At the same time, in CA patients an increased iNOS gene expression was observed at T180 together with a striking activation of JNK. By contrast, PBMC from CL-E-treated patients showed undetectable levels of phospho-JNK and a significant reduction in iNOS expression. Interestingly, incubation of PBMCs with normal human plasma (10%), activated by contact with a cellulosic membrane, induced a time-dependent increase in JNK phosphorylation that was completely inhibited by blocking complement cascade activation.
Conclusion: Our data suggest that JNK phosphorylation is strikingly increased in PBMCs obtained from CA-treated patients and may represent a key cellular event in PBMC activation during dialysis with bioincompatible membranes. The activation of this signaling enzyme, mediated by active complement fragments and PBMC-dialyzer interaction, can be significantly reduced by the use of vitamin E-coated membrane.