In vitro effect of uremic serum on barrier function and inflammation in human colonocytes

ABSTRACT Introduction: In chronic kidney disease (CKD), it has been suggested that alterations within the gut are associated with an inflammatory state and uremic toxicity. Studies suggest that uremia may impair the function of the intestinal barrier via the promotion of increased intestinal permeability. To understand the mechanisms that are involved in intestinal barrier damage in the setting of uremia, we evaluated the in vitro effect of uremic serum on transepithelial electrical resistance (TER), inflammation, and apoptosis in intestinal epithelial cells (T84). Methods: Pools of serum from healthy individuals, patients not on dialysis, and patients on hemodialysis (Pre-HD and Post-HD) were prepared. T84 cells were incubated for 24 h in medium, of which 10% consisted of the pooled serum from each group. After incubation, the TER was measured and the following parameters were determined by flow cytometry: expression of toll-like receptors (TLRs), production of reactive oxygen species (ROS), and apoptosis. The level of IL-6 in the culture supernatant was determined by ELISA. Results: No difference was observed among the groups with respect to TER, apoptosis, and ROS or the expression of TLR-2, TLR-4, and TLR-9. IL-6 secretion was higher (p < 0.001) in cells that were incubated with pre- and post-HD serum. Conclusion: The results that were obtained from this model suggest that uremic serum per se does not seem to impair the integrity of intestinal epithelial cells. The increased IL-6 secretion by cells that were incubated with HD serum suggests a potential effect of uremia in the intestinal inflammatory response.

摘要
引言：在慢性肾脏病（chronic kidney disease, CKD）中，已有研究表明肠道内部的改变与炎症状态及尿毒症毒性相关。现有研究提示，尿毒症可通过促进肠通透性升高而损伤肠屏障功能。为阐明尿毒症背景下肠屏障损伤的相关机制，本研究评估了尿毒症血清对肠上皮细胞（T84）的跨上皮电阻（transepithelial electrical resistance, TER）、炎症反应及凋亡的体外影响。
方法：分别收集健康个体、未透析患者及血液透析患者（透析前Pre-HD与透析后Post-HD）的血清并制备混合血清池。将T84细胞在含10%各组混合血清的培养基中孵育24小时。孵育结束后，检测跨上皮电阻，并通过流式细胞术测定以下参数：Toll样受体（toll-like receptors, TLRs）的表达水平、活性氧（reactive oxygen species, ROS）的生成量及细胞凋亡情况。采用酶联免疫吸附实验（ELISA）检测培养上清液中IL-6的水平。
结果：各组在跨上皮电阻、细胞凋亡、活性氧生成以及TLR-2、TLR-4、TLR-9的表达水平上均无显著差异。经透析前及透析后血清孵育的细胞，其IL-6分泌水平更高（p < 0.001）。
结论：本模型所得结果提示，尿毒症血清本身似乎并不会损伤肠上皮细胞的完整性。经透析血清孵育的细胞IL-6分泌升高，提示尿毒症可能对肠道炎症反应存在潜在影响。