Vitamin E-modified filters modulate Jun N-terminal kinase activation in peripheral blood mononuclear cells.
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.


CONCLUSIONS
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.
Division of Nephrology, Department of Emergency and Organ Transplantation, University of Bari, Polyclinic, Bari, Italy. g.pertosa@nephro.uniba.it
Giovanni
G
Pertosa
Giuseppe
G
Grandaliano
Michela
M
Soccio
Carmela
C
Martino
Loreto
L
Gesualdo
Francesco Paolo
FP
Schena
D000328:Adult; D000368:Aged; D000975:Antioxidants / Q000627:therapeutic use*; D015938:Complement Membrane Attack Complex / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D048031:JNK Mitogen-Activated Protein Kinases; D007676:Kidney Failure, Chronic / Q000188:drug therapy / Q000378:metabolism*; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008297:Male; D008567:Membranes, Artificial; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation / Q000187:drug effects; D006435:Renal Dialysis / Q000379:methods*; D014810:Vitamin E / Q000627:therapeutic use*
D000975:Antioxidants; D015938:Complement Membrane Attack Complex; D008567:Membranes, Artificial; D014810:Vitamin E; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases