MP011
THE INVOLVEMENT OF P38 MAPK SIGNALING IN THE IMPAIRED NEUTROPHIL BACTERICIDAL ACTIVITY OF HEMODIALYSIS PATIENTS

Introduction and Aims: Mortality from infections has been reported to be higher in hemodialysis (HD) patients than that in healthy subjects. Previous studies reported that vascular access was the main route of bacterial infection in HD patients and also pointed that the most common micro-organism were Staphylococcus aureus (S.aureus). To protect the host from bacterial infection, innate immunity of neutrophils have been thought to play central roles in the pathogenesis of the infection. This far, the dysfunction of neutrophils against bacterial infection in HD patients was reported. However the precise mechanism of neutrophil dysfunction in HD patients against bacteria remains unclear. In this study, we investigated the impacts of neutrophil inflammatory signaling against bacterial infection in HD patients.

Methods: Four HD patients and eight hypertensive (HT) patients without CKD were recruited for this study. None of HD patients had diabetes, cardiovascular disease and cancer. RNA was extracted from whole blood using the PAXgene Blood RNA System Kit. Microarray analysis by using the GeneChip scanner 3000 (Affymetrix, Santa Clara, CA) was performed to assess global gene expression in leukocytes. Neutrophils from another eight HD patients and healthy subjects were isolated by density gradient centrifugation from 10cc heparinized peripheral blood of. Neutrophil bactericidal function was evaluated by S.aureus growth rate after co-culture of it with neutrophils. Reactive oxygen species (ROS) production and myeloperoxidase (MPO) activities were analyzed by 2’, 7’-Dichlorodihydrofluorescein and Aminophenyl fluorescein intensity. Phosphorylation of signaling molecules against bacteria were also evaluated by western blot analysis.

Results: There were no difference at age and nutrition states such as serum albumin levels, cholesterol profile between HD and HT patients. The number of lymphocytes was lower in HD patients than HT patients (1699 ± 494/μL, 816 ± 45.4/μL, respectively; p<0.05). Microarray analysis showed the impairment of p38 mitogen activated protein kinase (MAPK) signaling in leukocytes of HD patients. In addition to that, phosphorylation rate of p38 MAPK of neutrophils stimulated by S.aureus or fMLP was also lower in HD patients than control. S.aureus growth rate was higher in neutrophils from HD patients than that from healthy subjects (490.1 ± 147.1%, 144.5 ± 49.4%, respectively; p<0.05). The levels of ROS from neutrophils after co-culture with S.aureus were lower in HD patients than those in healthy subjects (24.9 ± 4.6 RFU, 34.8 ± 10.5 RFU, respectively; p < 0.05). On the other hand, there was no difference of MPO activity between both groups. To confirm the importance of p38 MAPK, we examined the effects of the selective p38 MAPK inhibitor SB202190 on neutrophil bactericidal function. The treatment with SB202190 suppressed bacterial killing function as well as ROS production in neutrophils of healthy subjects.

Conclusions: Impairment of p38 MAPK signaling pathway contribute to the suppression of neutrophil bacterial killing function in HD patients through the dysregulation of ROS production.