Conventional, but not high-purity, dialysate-induced monocyte apoptosis is mediated by activation of PKC- and inflammatory factors release

Abstract

Use of conventional dialysate (CD) (powdered sodium bicarbonate dissolved manually with reverse osmosis water before dialysis) is common in Chinese haemodialysis (HD) centres. However, this preparation carries the risk of degradation and contamination, potentially negatively impacting host defense. Commercially available high-purity dialysate (HPD) may decrease inflammation and improve nutritional status in HD patients. However, whether HPD affects immune cells is unclear. The purpose of this study was to investigate the in vitro effect of these dialysates on apoptosis in U937 monocytes and its possible mechanism. Following incubation with two different types of dialysate, U937 cell apoptosis was measured by flow cytometry. Cell morphological changes were observed by Hoechst 33258 fluorescence staining. The expression of protein kinase C-δ (PKC-δ) was assayed by RT-polymerase chain reaction and western blot. Cytokine levels in U937 cells after exposure to CD or HPD for an indicated time were assayed by commercial enzyme-linked immunosorbent assay. CD contained more bacteria (66 ± 6 CFU/mL) than HPD (7 ± 3 CFU/mL) while there was no difference in endotoxin levels. Compared with cells exposed to HPD and phosphate-buffered saline (PBS), U937 monocytes experienced more apoptosis when exposed to CD for 24 and 48 h, while there was no significant difference between HPD and PBS. Expressions of PKC-δ mRNA and protein in U937 cells were enhanced following exposure to CD for 24 and 48 h, with increased proteolytic cleavage of PKC-δ which could be inhibited by rottlerin, a specific inhibitor of PKC-δ. Moreover, the cultured supernatant in CD-exposed cells contained significantly higher levels of interleukin-6 (4.09 ± 0.36 vs 2.73 ± 0.38 pg/mL, P < 0.01, 24 h; 4.28 ± 0.32 vs 2.83 ± 0.32 pg/mL, P < 0.01, 48 h) and tumour necrosis factor α (3.45 ± 0.79 vs 2.44 ± 0.39 pg/mL, P < 0.05, 24 h; 4.60 ± 0.57 vs 2.50 ± 0.37 pg/mL, P < 0.01, 48 h) than those of HPD. CD, but not HPD, contained more bacterial contamination, increased monocyte apoptosis in a PKC-δ-dependent manner and induced more cell inflammation. These findings suggest that impurity of dialysis fluid may be an important determinant of the elevated inflammation seen in CD-treated patients.