胚胎分化/发育基因pax2与WT1的序列启动在肾小管上皮细胞逆向分化中的意义

Impact of serial activation of embryonic pax2 and WT1 genes on transdifferentiation of renal tubular epithelial cells

目的:通过观察肾小管上皮细胞(TEC)胚胎分化/发育关键基因Wilm s肿瘤基因(WT1)和pax2的序列启动,探讨TEC逆向分化的分子机制及病理生理学意义。方法:从胚胎肾、肾脏损伤模型及体外细胞培养3方面入手,通过RT-PCR、免疫组织化学等技术,研究TEC逆向分化时,胚胎期控制TEC分化/发育的关键基因pax2和WT1的表达、pax2和WT1之间的关系以及它们与TEC逆向分化间的关系。结果:(1)胚胎期:pax2和WT1mRNA先后在胚胎11.5 d及14 d时开始出现表达并逐渐增强,出生14 d后仅有痕量表达。免疫组化显示,成年大鼠中仅肾小球足突细胞表达WT1,TEC中pax2和WT1表达阴性。(2)5/6慢性肾损伤模型:损伤第2周,部分TEC重新出现pax2和WT1的表达,第4周达高峰,两者表达趋势相吻合。pax2在第10周尚有一个新的表达高峰,随后逐渐下降至痕量。(3)TEC体外培养:经炎性因子IL-1α(10μg/L)、AngII(10-9mol/L)掺入培养后,TEC分别在0.5、24 h内重新表达pax2和WT1,随后α-SMA出现高表达,细胞呈现间充质样细胞特征。以WT1中和抗体、An-gII受体阻断剂封闭WT1和pax2基因的作用后,α-SMA表达水平明显降低,细胞基本保持原有特征不变。结论:控制胚胎肾分化/发育的关键基因pax2和WT1在成年肾遭受损伤时,可获得重新表达,呈现序列启动现象,TEC同时出现胚胎间充质细胞特征;pax2和WT1的重新表达与细胞浸浴环境(高浓度细胞因子)密切相关,可能作为内在启动机制参与TEC的逆向分化。

AIM： To investigate the mechanisms of transdifferentiation of renal tubular epithelial cells by the observation of serial activation of embryonic pax2 and WT1 genes in chronic renal failure model of 5/6 nephrectomized injury. METHODS ： Embryo of mice, cultured renal tubule cells and chronic renal failure rat model of 5/6 nephrectomized injury were established. The expressions of paired Box gene （pax2） and Willm＇s tumor gene （ WT1 ）, as well as α - smooth muscle actin （ α - SMA）, a phenotype of mesenchymal cells, were detected by RT - PCR and immunohistochemistry techniques. RESULTS： （ 1 ） Expressions of pax2 and WT1 mRNA began at 11.5 d and 14 d of embryo and increased gradually, and expression in trace quantity at 2 weeks after birth. Immunohistochemistry analysis revealed that WT1 only expressed in the glomerular podocytes and expressions of pax2 and WT1 in adult renal tubular cells were negative. （2） Deno - expression of pax2 and WTI in some tubular cells appeared at week 2 and peaked at week 4 in 5/6 nephrectomized rats, both showing a same trend of expression. However, pax2 showed another peak at week 10 afterwards. （3） Re - expressions of pax2 and WT1 in TEC at 0.5 and 24 h after treated with IL - 1α （ 10 μg/L） or AngII （ 10^-9 mob/L） were observed respectively, followed by upregulation of α - SMA expression, and mesenchymal cells characters were shown. The effects were inhibited by Ang II receptor 2 antagonist and WT1 antibody, respectively. CONCLUSION： Adult renal tubule cells acquire re - activation of embryonic pax2 and WT1 genes and phenotypes of mesenchymes when challenge with certain injuries. Deno - expression of pax2 and WT1 genes closely relates with high concentration of bioactive facors, such as AngII and IL - 1, which are involved in the mechanisms of renal tubule cells transdifferentiation.