Yap蛋白在常染色体显性多囊肾病囊肿进展中的双重作用机制研究

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. However, due to the fact that the pathogenesis of the disease is still unclear and until now there is no effective treatment, ADPKD is still a serious threat to human health and life. Recent studies reveal that YAP protein is significantly upregulated and translocated to nucleus，this indicates that YAP might have a role in promoting the growth of cysts with an increasing expression of downstream target genes in polycystic kidney disease models. In our previous research, we found that there is gene interaction effects between yap and pathogenetic gene pkd2 in zebrafish model of polycystic kidney disease. And the cysts in zebrafish embryos caused by pkd2 knockdown can be reduced by increasement of phosphorylated cytoplasmic YAP protein. This indicates that YAP may have a dual function in regulating cyst progression. Based on our previous research, we will further explore the structural and functional relationship between YAP and PC2, clarify the exact role and mechanism of YAP in the regulation of cyst growth. By utilizing various biological techniques such as pulse-chase experiments, co-immunoprecipitation, GST pull-down, calcium flow imaging, gene cloning, et al, we will prove the hypothesis that YAP play as a dual functional regulator on the cyst progression. In order to verify the safety and efficacy of YAP-target therapy, we will use YAP-target drugs to treat zebrafish, gene knockout mouse and spontaneous mutation rat. Our study will further elucidate the pathogenesis of polycystic kidney disease, and will provide a cutting-edge theory and experimental basis for finding novel intervention targets in the polycystic kidney disease.

常染色体显性多囊肾病是最常见的遗传性肾病，至今发病机制不明，没有治疗药物，严重威胁人类的健康和生命。新近研究发现多囊肾病中YAP蛋白表达上调，核转位，靶基因转录上调，提示YAP可能具有促囊肿生长的作用。我们在前期研究中发现多囊肾病斑马鱼模型中yap与多囊肾病致病基因pkd2具有基因互作效应，而增加胞浆磷酸化YAP蛋白水平可降低pkd2缺失斑马鱼胚胎的囊肿比例，提示YAP可能对囊肿进展具有双重调控作用。本项目拟在前期研究基础上采用脉冲示踪实验、免疫共沉淀、GST pull-down、钙流成像等技术明确YAP与PC2互相作用的结构学基础；运用基因克隆、显微注射等技术开展其功能学研究；采用YAP干预药物在模式生物及基因敲除动物等多种疾病模型上验证其作为新型治疗靶点的安全性和有效性，最终证实YAP对多囊肾病的双重调控假设，进一步阐明疾病的发病机制，为寻找治疗多囊肾病的全新靶标提供理论和实验依据。

.常染色体显性多囊肾病是最常见的遗传性肾病，严重威胁人类的健康和生命，但至今无有效阻断疾病进展的药物。开展多囊肾病分子发病机制研究将有助于寻找多囊肾病的干预靶点，具有潜在的治疗应用前景。我们在前期工作中发现多囊肾病斑马鱼模型中存在YAP蛋白的异常，yap不仅与多囊肾病致病基因pkd2具有基因互作效应，且YAP蛋白表达、磷酸化水平及定位的不同状态可分别促进或抑制囊肿生长，对囊肿进展具有双重调控作用。因此本研究首先观察了YAP的表达情况，在斑马鱼胚胎发育中yap mRNA在前肾肾管中持续表达，而Yap蛋白在肾管上皮细胞顶端膜及胞浆聚积。在斑马鱼胚胎中下调yap表达会出现心包水肿、前肾囊肿、体轴轻微弯曲等类似“纤毛病”的表型。yap morphants前肾肾管各个节段的细胞群体迁移存在缺陷，侧基底膜细胞极性存在异常，向顶端膜分布且眼及肾管的纤毛形态存在缺陷，肾管多纤毛基体的接列和单纤毛基体的顶端膜迁移异常。过表达可磷酸化Yap蛋白能够部分抑制pkd2缺失导致的肾囊肿形成。进一步研究发现，PC2通过Scribble蛋白调控YAP，最终Polycystin 2-Scribble-YAP通过Hippo通路调控ADPKD肾囊肿形成。本研究明确了Scribble是YAP与PC2相互作用的结构和功能基础，PC2通过Scribble调控YAP蛋白亚细胞定位，从而影响ADPKD肾囊肿的发生发展，阐明了YAP对多囊肾病囊肿形成的双重调控机制是通过Polycystin 2-Scribble-YAP通路实现的。本课题证实了YAP与多囊肾病异常纤毛以及细胞极性紊乱之间的关系，进一步证实并完善了多囊肾病的纤毛致病学说和极性紊乱学说；本课题在不同模型上证实了干预胞浆磷酸化YAP水平可以明显抑制多囊肾病囊肿形成，初步证实了YAP作为全新多囊肾病治疗靶点的潜在应用价值。