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

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WT1 mRNA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(10 μg/L)、AngII（10-9 mol/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、AngII 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。

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

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 WT1 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 mol/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.