Min Seo-Yun, Ha Dong-Soo, Ha Tae-Sun
Department of Pediatrics, Chungbuk National University College of Medicine, Cheongju, Korea.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.
Kidney Res Clin Pract. 2018 Sep;37(3):210-221. doi: 10.23876/j.krcp.2018.37.3.210. Epub 2018 Sep 30.
Puromycin aminonucleoside (PAN) is a known podocytotoxin. PAN-induced nephrosis is a widely used animal model for studying human idiopathic nephrotic syndrome. Abnormal protein accumulation associated with podocyte-specific endoplasmic reticulum (ER) stress damages cells structurally and functionally, which in turn induces apoptosis and severe proteinuria. In the present study, we investigated the effect of PAN on ER stress and apoptosis in podocytes .
Mouse podocytes were cultured and treated with various concentrations of PAN. ER stress markers were then evaluated by western blotting, and apoptosis was evaluated by fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.
PAN treatment increased ER stress markers such as activating transcription factor (ATF) 6α and caspase-12 in a dose-dependent manner at 12 and 24 hours, respectively. These markers were reduced by chemical chaperones, such as sodium 4-phenylbutyric acid and tauroursodeoxycholic acid. PAN treatment also increased 78 kD glucose-regulated protein (GRP78)/binding immunoglobulin protein (BiP) at the earlier stage of 12 hours. PAN significantly induced podocyte apoptosis in concentration- and time-dependent manners, as seen using FACS and TUNEL assays. This result was improved by Nox4 siRNA, ATF6 siRNA, and chemical chaperones. LY294002, a PI3-kinase inhibitor, significantly boosted ER stress and apoptosis. PAN-induced ER stress increased oxidative stress and subsequently induced apoptosis, and could be mitigated by inhibition of PI3-kinase signaling.
Our findings suggest that PAN induces ER stress in podocytes mainly through the GRP78/BiP, ATF6α, and caspase-12 pathways, which trigger apoptosis via induction of oxidative stress. This stress is mitigated by inhibiting PI3-kinase signaling.
嘌呤霉素氨基核苷（PAN）是一种已知的足细胞毒素。PAN诱导的肾病是研究人类特发性肾病综合征广泛使用的动物模型。与足细胞特异性内质网（ER）应激相关的异常蛋白质积累会在结构和功能上损害细胞，进而诱导细胞凋亡和严重蛋白尿。在本研究中，我们调查了PAN对足细胞内质网应激和细胞凋亡的影响。
培养小鼠足细胞并用不同浓度的PAN处理。然后通过蛋白质印迹法评估内质网应激标志物，通过荧光激活细胞分选（FACS）和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）试验评估细胞凋亡。
PAN处理分别在12小时和24小时以剂量依赖的方式增加了内质网应激标志物，如活化转录因子（ATF）6α和半胱天冬酶-12。这些标志物被化学伴侣，如4-苯基丁酸和牛磺熊去氧胆酸降低。PAN处理在12小时的早期阶段也增加了78 kD葡萄糖调节蛋白（GRP78）/结合免疫球蛋白蛋白（BiP）。如使用FACS和TUNEL试验所见，PAN以浓度和时间依赖的方式显著诱导足细胞凋亡。Nox4 siRNA、ATF6 siRNA和化学伴侣改善了这一结果。PI3激酶抑制剂LY294002显著增强了内质网应激和细胞凋亡。PAN诱导的内质网应激增加了氧化应激并随后诱导细胞凋亡，并且可以通过抑制PI3激酶信号传导来减轻。
我们的研究结果表明，PAN主要通过GRP78/BiP、ATF6α和半胱天冬酶-12途径诱导足细胞内质网应激，这些途径通过诱导氧化应激触发细胞凋亡。通过抑制PI3激酶信号传导可减轻这种应激。
