OASIS overexpression in murine podocyte cell line. OASIS overexpression in murine podocyte cell line

Podocyte injury is involved in the onset and progression of various kidney diseases. We previously demonstrated that the transcription factor, old astrocyte specifically induced substance (OASIS) in myofibroblasts, contributes to kidney fibrosis, as a novel role of OASIS in the kidneys. Importantly, we found that OASIS is also expressed in podocytes; however, the pathophysiological significance of OASIS in podocytes remains unknown. Upon lipopolysaccharide (LPS) treatment, there is an increase in OASIS in murine podocytes. Enhanced serum creatinine levels and tubular injury, but not albuminuria and podocyte injury, are attenuated upon podocyte-restricted OASIS knockout in LPS-treated mice, as well as diabetic mice. The protective effects of podocyte-specific OASIS deficiency on tubular injury are mediated by protein kinase C iota (PRKCI/PKCι), which is negatively regulated by OASIS in podocytes. Furthermore, podocyte-restricted OASIS transgenic mice show tubular injury and tubulointerstitial fibrosis, with severe albuminuria and podocyte degeneration. Finally, there is an increase in OASIS-positive podocytes in the glomeruli of patients with minimal change nephrotic syndrome and diabetic nephropathy. Taken together, OASIS in podocytes contributes to podocyte and/or tubular injury, in part through decreased PRKCI. The induction of OASIS in podocytes is a critical event for the disturbance of kidney homeostasis. Overall design: We searched for genes whose expression is induced by the transcription factor OASIS in podocytes. Murine cultured podocytes were transfected with a lentivirus expressing the active form of Oasis/Creb3l1 or venus for 24 h. Post the lentiviral infection, the medium was changed to RPMI-1640 with 10% FBS. Forty-eight hours after treatment with the lentiviral vectors, total RNA was extracted from the OASIS-overexpressing podocytes, using the RNeasy® mini kit (Qiagen, Venlo, Netherlands). Gene expression was analyzed using the SurePrint G3 Mouse GE v2 8×60K Microarray (Agilent Technologies, Santa Clara, CA, USA).

足细胞（podocyte）损伤参与多种肾脏疾病的发生与进展。我们此前已证实，肌成纤维细胞中的转录因子旧星形胶质细胞特异性诱导物质（old astrocyte specifically induced substance，OASIS）可促进肾纤维化，这是OASIS在肾脏中的全新功能。值得注意的是，我们发现OASIS在足细胞中同样存在表达，但目前尚不清楚OASIS在足细胞中的病理生理意义。经脂多糖（lipopolysaccharide，LPS）处理后，小鼠足细胞中的OASIS表达水平升高。在经LPS处理的小鼠以及糖尿病小鼠中，特异性敲除足细胞中的OASIS可减轻血清肌酐水平升高与肾小管损伤，但对蛋白尿和足细胞损伤无明显改善作用。足细胞特异性OASIS缺失对肾小管损伤的保护作用，由蛋白激酶Cι（protein kinase C iota，PRKCI/PKCι）介导，而OASIS在足细胞中对PRKCI存在负调控作用。此外，足细胞特异性OASIS转基因小鼠会出现肾小管损伤与肾小管间质纤维化，同时伴随严重蛋白尿和足细胞变性。最后，微小病变型肾病综合征和糖尿病肾病患者的肾小球中，OASIS阳性足细胞数量有所增加。综上，足细胞中的OASIS可通过部分降低PRKCI的表达，参与足细胞和/或肾小管损伤。足细胞中OASIS的诱导表达是扰乱肾脏内环境稳态的关键事件。

实验整体设计：我们筛选了足细胞中受转录因子OASIS诱导表达的基因。将小鼠培养足细胞用携带活性形式Oasis/Creb3l1或venus的慢病毒转染24小时。慢病毒感染后，将培养基更换为含10%胎牛血清（fetal bovine serum，FBS）的RPMI-1640培养基。用慢病毒载体处理48小时后，使用RNeasy®迷你试剂盒（Qiagen，文洛，荷兰）从OASIS过表达的足细胞中提取总RNA。使用SurePrint G3 Mouse GE v2 8×60K Microarray（安捷伦科技，圣克拉拉，加利福尼亚州，美国）分析基因表达情况。