Tang Xun, Rong Guang, Bu Yang, Zhang Shaojie, Zhang Min, Zhang Jun, Liang Xiujie
Department of Nephrology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, China.
Cell Physiol Biochem. 2015;35(2):816-28. doi: 10.1159/000369740. Epub 2015 Jan 30.
In chronic kidney disease (CKD), the accumulation of advanced oxidation protein products (AOPPs) is prevalent. Hypertrophy and epithelial-to-mesenchymal transition (EMT) of tubular cells are associated with the pathogenesis of CKD. However, whether AOPPs induce tubular-cell hypertrophy and EMT is unclear. In this study, we investigated the effect of AOPPs on human proximal tubular cells (HK-2 cells) and the mechanisms underlying tubular-cell hypertrophy and EMT in vitro.
The mRNA and protein expression of CCAAT/enhancer-binding protein-homologous protein (CHOP), glucose-regulated protein (GRP) 78, p27, α-smooth muscle actin (α-SMA) and E-cadherin were evaluated by quantitative real-time PCR and western blot, respectively. Cell cycle was detected by flow cytometry. Bicinchoninic acid method was performed to measure total protein content.
AOPP treatment upregulated total protein expression, caused an increase in the percentage of G1-phase cells, and induced the overexpression of p27 and α-SMA, lowered the expression of E-cadherin. Furthermore, AOPP treatment induced the overexpression of GRP78 and CHOP. Moreover, the aforementioned effects were reversed following the treatment of cells with an NADPH oxidase inhibitor, a reactive oxygen species (ROS) scavenger, or salubrinal, which is an inhibitor of ER stress, whereas these effects were produced after exposure to thapsigargin, an inducer of ER stress.
Our results suggest that AOPPs induced HK-2-cell hypertrophy and EMT by inducing ER stress, which was likely mediated by ROS. These findings could facilitate the development of novel therapeutic strategies for suppressing the progression of CKD.
在慢性肾脏病（CKD）中，晚期氧化蛋白产物（AOPPs）的蓄积很普遍。肾小管细胞肥大和上皮-间质转化（EMT）与CKD的发病机制相关。然而，AOPPs是否诱导肾小管细胞肥大和EMT尚不清楚。在本研究中，我们在体外研究了AOPPs对人近端肾小管细胞（HK-2细胞）的影响以及肾小管细胞肥大和EMT的潜在机制。
分别通过定量实时PCR和蛋白质印迹法评估CCAAT/增强子结合蛋白同源蛋白（CHOP）、葡萄糖调节蛋白（GRP）78、p27、α-平滑肌肌动蛋白（α-SMA）和E-钙黏蛋白的mRNA和蛋白表达。通过流式细胞术检测细胞周期。采用二辛可宁酸法测定总蛋白含量。
AOPPs处理上调了总蛋白表达，导致G1期细胞百分比增加，诱导p27和α-SMA的过表达，降低E-钙黏蛋白的表达。此外，AOPPs处理诱导GRP78和CHOP的过表达。而且，在用NADPH氧化酶抑制剂、活性氧（ROS）清除剂或内质网应激抑制剂沙芦比诺处理细胞后，上述作用被逆转，而在用内质网应激诱导剂毒胡萝卜素处理后产生了这些作用。
我们的结果表明，AOPPs通过诱导内质网应激诱导HK-2细胞肥大和EMT，这可能由ROS介导。这些发现可能有助于开发抑制CKD进展的新型治疗策略。
