Supplementary Material for: Upregulation of Nestin Protects Podocytes from Apoptosis Induced by Puromycin Aminonucleoside

<i>Background:</i> Nestin is an intermediate filament protein widely used as a marker of stem cells or progenitor cells. Nestin is also highly expressed in the glomerular podocyte, a type of terminally differentiated epithelial cell. Little is known about the significance of nestin in podocytes. <i>Methods:</i> Puromycin aminonucleoside (PAN) was injected into the rats to produce a PAN nephrosis model. Transmission electronic microscopy and terminal dUTP nick end-labeling assay were used to examine the podocyte foot process (FP) effacement and apoptosis, respectively. A mouse podocyte cell line was cultured and incubated with PAN. Immunoblot was used to examine the level of nestin expression both in vivo and in vitro. Enhanced green fluorescence protein-tagged plasmids containing nestin shRNA were transfected into the cultured podocytes to silence nestin expression. F-actin arrangement within cultured podocytes was investigated by immunofluorescence, while the apoptosis rate was examined by both Hoechst stain and flow cytometry. <i>Results:</i> In the PAN-induced rat nephrosis model, podocyte nestin expression was increased in the absence of apparent podocyte apoptosis, even though the FP was significantly effaced. In the cultured mouse podocytes, PAN upregulated nestin expression in a time-dependent manner within 24 h of treatment. Notably, no significant apoptosis occurred, however knocking down nestin expression resulted in a remarkable derangement of actin cytoskeleton and an increase in apoptosis in the cultured podocytes 24 h after being incubated with PAN. <i>Conclusions:</i> Upregulation of nestin expression during PAN nephrosis could protect podocytes from apoptosis and that this process is mediated by maintaining the regular arrangement of actin cytoskeleton.

<i>背景：</i>Nestin是一种中间丝蛋白（intermediate filament protein），广泛用作干细胞或祖细胞的标志物。Nestin在肾小球足细胞——一类终末分化的上皮细胞——中同样呈高表达，目前对于足细胞中Nestin的生物学意义尚不明确。<i>方法：</i>向大鼠体内注射嘌呤霉素氨基核苷（Puromycin aminonucleoside, PAN）以构建PAN肾病模型。分别采用透射电子显微镜（Transmission electronic microscopy）与末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（terminal dUTP nick end-labeling, TUNEL）实验，检测足细胞足突（foot process, FP）融合与细胞凋亡情况。培养小鼠足细胞系并使用PAN进行干预。通过免疫印迹实验检测体内及体外培养样本中Nestin的表达水平。将携带Nestin短发夹RNA（short hairpin RNA, shRNA）的增强型绿色荧光蛋白（enhanced green fluorescence protein, EGFP）标记质粒转染至培养的足细胞中，以沉默Nestin的表达。采用免疫荧光实验观察培养足细胞内的肌动蛋白丝（F-actin）排布情况，同时通过Hoechst染色与流式细胞术分别检测细胞凋亡率。<i>结果：</i>在PAN诱导的大鼠肾病模型中，尽管足细胞足突出现显著融合，但在未观察到明显足细胞凋亡的情况下，足细胞内Nestin的表达水平升高。在培养的小鼠足细胞中，PAN可在处理后的24小时内以时间依赖性方式上调Nestin的表达。值得注意的是，此时未发生显著的细胞凋亡；然而，在使用PAN处理足细胞24小时后，沉默Nestin的表达会导致肌动蛋白细胞骨架出现显著紊乱，并使足细胞的凋亡率升高。<i>结论：</i>PAN肾病过程中Nestin表达的上调可对足细胞起到抗凋亡保护作用，这一过程通过维持肌动蛋白细胞骨架的正常排布得以实现。