Division of Nephrology, No 3 People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Nephrol Dial Transplant. 2011 May;26(5):1516-22. doi: 10.1093/ndt/gfq620. Epub 2010 Oct 5.
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.
在中国血液透析（HD）中心，常规透析液（CD）（粉末状碳酸氢钠用反渗透水手动溶解后用于透析）的使用较为常见。然而，这种准备方法存在降解和污染的风险，可能会对宿主防御产生负面影响。商业上可获得的高纯度透析液（HPD）可能会降低 HD 患者的炎症并改善营养状况。然而，HPD 是否会影响免疫细胞尚不清楚。本研究旨在探讨这些透析液在体外对 U937 单核细胞凋亡的影响及其可能的机制。
用两种不同类型的透析液孵育 U937 细胞后，通过流式细胞术测量细胞凋亡。用 Hoechst 33258 荧光染色观察细胞形态变化。通过 RT-聚合酶链反应和 Western blot 测定蛋白激酶 C-δ（PKC-δ）的表达。用商业酶联免疫吸附试验测定 U937 细胞在暴露于 CD 或 HPD 后不同时间细胞因子的水平。
CD 中的细菌（66 ± 6 CFU/mL）比 HPD（7 ± 3 CFU/mL）多，而内毒素水平没有差异。与暴露于 HPD 和磷酸盐缓冲盐水（PBS）的细胞相比，CD 孵育 24 和 48 小时后 U937 单核细胞凋亡更多，而 HPD 和 PBS 之间无显著差异。CD 孵育 24 和 48 小时后，U937 细胞中 PKC-δ mRNA 和蛋白的表达增强，PKC-δ 的蛋白水解切割增加，可被 PKC-δ 的特异性抑制剂 rottlerin 抑制。此外，CD 暴露细胞的培养上清液中白细胞介素 6（4.09 ± 0.36 vs 2.73 ± 0.38 pg/mL，P < 0.01，24 小时；4.28 ± 0.32 vs 2.83 ± 0.32 pg/mL，P < 0.01，48 小时）和肿瘤坏死因子-α（3.45 ± 0.79 vs 2.44 ± 0.39 pg/mL，P < 0.05，24 小时；4.60 ± 0.57 vs 2.50 ± 0.37 pg/mL，P < 0.01，48 小时）水平明显高于 HPD。
CD 中含有更多的细菌污染，以 PKC-δ 依赖的方式增加单核细胞凋亡，并诱导更多的细胞炎症。这些发现表明，透析液的杂质可能是 CD 治疗患者炎症升高的一个重要决定因素。
