Hou Ling, Du Yue, Zhao Chengguang, Wu Yubin
Department of Pediatric Nephrology, Shengjing Hospital of China Medical University, Shenyang, 110004, Liaoning, China.
Int Urol Nephrol. 2018 Sep;50(9):1729-1741. doi: 10.1007/s11255-018-1956-0. Epub 2018 Aug 16.
We sought to investigate the role of PAX2 in renal epithelial-to-mesenchymal transition (EMT), examining the influence of PAX2 on ADAM10 expression during renal EMT and ADAM10 expression in fibrotic kidneys.
A rat renal tubular epithelial cell line, NRK52E, was transfected with lentivirus carrying PAX2, and E-cadherin and α-SMA expressions were measured. The influence of PAX2 on ADAM10 promoter activity was evaluated using chromatin immunoprecipitation (CHIP) and dual-luciferase reporter assay. We also treated NRK52E with ADAM10-specific over-expression vector and inhibitors and measured E-cadherin and α-SMA expression. In vivo, Wistar rats (n = 36) were subjected to unilateral ureteral obstruction (UUO) (n = 18) or sham surgery (n = 18), with tissues from post-operative day 3, 7, and 14 days examined, and PAX2/ADAM10 activity measured. ADAM10 expression was also assessed in kidneys from patients with chronic kidney disease (CKD).
In NRK52E overexpressing PAX2, ADAM10 and α-SMA levels were increased, while E-cadherin levels were decreased. CHIP and dual-luciferase reporter assay showed that PAX2 directly bound to a specific site within the ADAM10 promoter, and over-expression of PAX2 significantly activated ADAM10 transcription. NRK52E with ADAM10 over-expression also significantly decreased E-cadherin and increased α-SMA levels. In the fibrotic kidneys of rats with UUO, E-cadherin levels were increased and α-SMA levels were decreased, and expression of PAX2 and ADAM10 increased. ADAM10 expression also elevated in the renal tissues of CKD patients.
PAX2 directly increased expression of ADAM10, the presence of which contributed to EMT in renal tubular epithelia and hence plays an important role in renal fibrosis.
我们试图研究PAX2在肾上皮-间充质转化（EMT）中的作用，探讨PAX2对肾EMT过程中ADAM10表达的影响以及其在纤维化肾脏中的表达情况。
用携带PAX2的慢病毒转染大鼠肾小管上皮细胞系NRK52E，检测E-钙黏蛋白和α-平滑肌肌动蛋白（α-SMA）的表达。采用染色质免疫沉淀（CHIP）和双荧光素酶报告基因检测法评估PAX2对ADAM10启动子活性的影响。我们还用ADAM10特异性过表达载体和抑制剂处理NRK52E，并检测E-钙黏蛋白和α-SMA的表达。在体内，将Wistar大鼠（n = 36）分为单侧输尿管梗阻（UUO）组（n = 18）和假手术组（n = 18），检测术后第3、7和14天的组织，测定PAX2/ADAM10活性。还评估了慢性肾脏病（CKD）患者肾脏中ADAM10的表达。
在过表达PAX2的NRK52E中，ADAM10和α-SMA水平升高，而E-钙黏蛋白水平降低。CHIP和双荧光素酶报告基因检测显示，PAX2直接与ADAM10启动子内的特定位点结合，PAX2的过表达显著激活ADAM10转录。过表达ADAM10的NRK52E也显著降低了E-钙黏蛋白水平并增加了α-SMA水平。在UUO大鼠的纤维化肾脏中，E-钙黏蛋白水平升高，α-SMA水平降低，PAX2和ADAM10的表达增加。CKD患者肾组织中ADAM10的表达也升高。
PAX2直接增加ADAM10的表达，其存在促进肾小管上皮细胞的EMT，因此在肾纤维化中起重要作用。
