Qu Ning, Chen Lei, Liang Shanshan, Wei Meng, Sun Lingshuang, He Quan, Xue Jinhong, Wang Meng, Shi Kehui, Jiang Hongli, Liu Hua
Dialysis Department of Nephrology Hospital, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Department of Blood Transfusion, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Front Med (Lausanne). 2022 Apr 13;9:850966. doi: 10.3389/fmed.2022.850966. eCollection 2022.
Increasing evidence supports the idea that the disruption of epithelial tight junction proteins (TJPs) caused by accumulation of uremia toxins, such as homocysteine (Hcy), is one of the most important mechanisms underlying the damage of intestinal barrier function (IBF) in chronic kidney disease (CKD). Since the decrease of hypoxia inducible factor-1α (HIF-1α) is reported to be involved in Hcy-induced cell injury, and the upregulation of microRNA-223 (miR-223) plays a vital protective role in the impairment of IBF in the experimental colitis, we investigated the effect of HIF-1α stabilizer roxadustat on the disruption of TJPs induced by Hcy and CKD and the underlying mechanism.
Chronic kidney disease was induced in rats 5/6 nephrectomy. In a series of experiments, the rats were treated orally with roxadustat of different doses. The expression of tight junction proteins, HIF-1α, and miR-223 was analyzed in different groups by western blotting analysis, RT-qPCR techniques and immunofluorescence. A series of experiments with cultured Caco2 cells was performed.
The results showed that the expression of TJPs (occludin, claudin-1, and ZO-1) decreased significantly, accompanied by the reduction of HIF-1α and miR-223 in Hcy-treated Caco2 cells and colonic mucosa of uremic rats. The reduction of HIF-1α and miR-223 was reversed by roxadustat and the decrease of TJPs expression was attenuated in both Caco2 cells induced by Hcy and colon tissue of CKD rats. Furthermore, transfection with miR-223 mimics increased the expression of TJPs, while transfection with miR-223 inhibitor decreased their expression in Caco2 cells. MiR-223 inhibitor applied before roxadustat treatment partly diminished the effect of roxadustat on TJPs expression in Caco2 cells.
These results indicated that roxadustat attenuated the disruption of epithelial TJPs induced by Hcy in Caco2 cells and the damage of colonic epithelium in CKD rats through the upregulation of miR-223 induced by HIF-1α. A novel insight into the IBF dysfunction in CKD was provided, and it suggests a potential therapeutic use of roxadustat for the IBF dysfunction besides anemia in CKD.
越来越多的证据支持这样一种观点，即尿毒症毒素（如同型半胱氨酸，Hcy）的蓄积导致上皮紧密连接蛋白（TJPs）破坏，是慢性肾脏病（CKD）患者肠道屏障功能（IBF）损伤的最重要机制之一。由于据报道缺氧诱导因子-1α（HIF-1α）的减少与Hcy诱导的细胞损伤有关，且微小RNA-223（miR-223）的上调在实验性结肠炎的IBF损伤中起重要保护作用，我们研究了HIF-1α稳定剂罗沙司他对Hcy和CKD诱导的TJPs破坏的影响及其潜在机制。
通过5/6肾切除诱导大鼠慢性肾脏病。在一系列实验中，大鼠口服不同剂量的罗沙司他。采用蛋白质免疫印迹分析、RT-qPCR技术和免疫荧光法分析不同组中紧密连接蛋白、HIF-1α和miR-223的表达。对培养的Caco2细胞进行了一系列实验。
结果显示，在Hcy处理的Caco2细胞和尿毒症大鼠结肠黏膜中，TJPs（闭合蛋白、claudin-1和ZO-1）的表达显著降低，同时伴有HIF-1α和miR-223的减少。罗沙司他可逆转HIF-1α和miR-223的减少，且在Hcy诱导的Caco2细胞和CKD大鼠结肠组织中，TJPs表达的降低均得到缓解。此外，转染miR-223模拟物可增加Caco2细胞中TJPs的表达，而转染miR-223抑制剂则降低其表达。在罗沙司他治疗前应用miR-223抑制剂可部分减弱罗沙司他对Caco2细胞中TJPs表达的影响。
这些结果表明，罗沙司他通过上调HIF-1α诱导的miR-223，减轻了Hcy诱导的Caco2细胞中上皮TJPs的破坏以及CKD大鼠结肠上皮的损伤。为CKD中IBF功能障碍提供了新的见解，并提示罗沙司他除了对CKD贫血有治疗作用外，对IBF功能障碍也有潜在的治疗用途。
