Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Sci Rep. 2013;3:1041. doi: 10.1038/srep01041. Epub 2013 Jan 9.
We examined whether p38 MAPK plays role in calcium oxalate monohydrate (COM) crystal-induced tight junction disruption. Polarized MDCK cells were pretreated with or without 20 μM SB239063 (p38 MAPK inhibitor) for 2-h, and then incubated with 100 μg/ml COM crystals for up to 48-h. Western blotting showed increased level of phospho-p38, not total p38, in COM-treated cells, whereas SB239063 pretreatment successfully maintained phospho-p38 at its basal level. COM crystals also caused decreased levels of two tight junction proteins, zonula occludens-1 (ZO-1) and occludin. Immunofluorescence study revealed disruption of tight junction, redistribution, and dissociation of ZO-1 and occludin. Moreover, transepithelial resistance (TER) showed defective barrier function, whereas Western blotting for Na(+)/K(+)-ATPase-α1 revealed defective fence function of tight junction in COM-treated cells. All these expression and functional defects were successfully prevented by SB239063 pretreatment. These findings indicate that COM crystals cause tight junction disruption in distal renal tubular epithelial cells through p38 MAPK activation.
我们研究了 p38MAPK 是否在一水合草酸钙（COM）晶体诱导的紧密连接破坏中发挥作用。极化的 MDCK 细胞用或不用 20μM SB239063（p38MAPK 抑制剂）预处理 2 小时，然后用 100μg/ml COM 晶体孵育长达 48 小时。Western blot 显示 COM 处理的细胞中磷酸化 p38 的水平增加，而不是总 p38，而 SB239063 预处理成功地将磷酸化 p38 维持在基础水平。COM 晶体还导致两种紧密连接蛋白，封闭蛋白-1（ZO-1）和闭合蛋白的水平降低。免疫荧光研究显示紧密连接的破坏、重分布和 ZO-1 和闭合蛋白的解离。此外，上皮细胞跨膜电阻（TER）显示出有缺陷的屏障功能，而 Western blot 检测到 Na(+)/K(+)-ATPase-α1 显示 COM 处理的细胞中紧密连接的篱笆功能有缺陷。所有这些表达和功能缺陷都可以通过 SB239063 预处理成功预防。这些发现表明，COM 晶体通过 p38MAPK 激活导致远端肾小管上皮细胞紧密连接破坏。
