Robo2缺失小鼠肾小管前体细胞衰老导致肾脏囊肿的分子机制研究

Robo2 is an important cause of the pathogenic gene of urinary system defects. We found that Robo2 knockout mice (Robo2-/-) kidney cyst is not caused by vesicoureteral reflux, but from the renal tubule development defect. In the Robo2-/- in early stage of renal tubule development, showed the first discovery of senescence related markers beta -gal staining and cell gap junction defects. But the mechanism causing these cell senescence, gap junction which are associated with kidney development is not clear. This study intends to spectral analysis and verification and renal tubular cell senescence and gap junction gene defect related key through gene expression; co-precipitation, protein mass spectrometry analysis for interaction with Robo2 molecules using immune fluorescence. By detecting gap junction protein in the Robo2-/- and changes of gap junction gene on MDCK cells after siRNA intervention, observation of senescence changes to determine the candidate molecular phenotype, Robo2 regulation of gap junctions and gap junctions: molecular mechanisms of senescence effect relationship. The use of specific senescence gene p21 tracer kidney cyst development process, and by P21 gene modified mouse to resecue, developmental pathways analytic aging renal tubular precursor cells forming kidney cyst. This study will elucidate the Robo2 deletion resulted in gap junction defects caused by renal tubular precursor cell senescence and renal developmental defects of the pathogenic mechanism, put forward a new understanding of kidney development and senescence. The study will provide the basis for potential therapeutic target for early intervention.

Robo2是导致泌尿系统缺陷的重要致病基因。我们首次发现Robo2缺失小鼠（Robo2-/-）肾囊肿源于肾小管发育缺陷，并出现衰老相关标志β-gal阳性及细胞缝隙连接缺陷。但引起这些细胞衰老、缝隙连接等发育重要环节的机制不清楚。本研究拟通过基因表达谱分析并验证与肾小管衰老以及细胞缝隙连接缺陷相关的关键基因；利用免疫共沉淀、蛋白质谱分析寻找与Robo2相互作用分子。通过检测Robo2-/-缝隙连接蛋白变化及对MDCK细胞以缝隙连接基因siRNA干预，观察候选衰老分子变化，确定Robo2调控缝隙连接分子机制以及缝隙连接－衰老作用关系。利用特异性衰老基因p21示踪肾囊肿形成过程，并以P21基因修饰鼠进行挽救试验，解析衰老肾小管前体细胞形成肾脏囊肿的发育途径。本研究将阐明Robo2缺失引发肾小管前体细胞衰老导致肾脏发育缺陷的根本机制，提出对肾小管发育与衰老的新认识，为疾病早期干预靶点提供研究基础。

遗传性肾囊性疾病的特征是肾小管上皮细胞原发纤毛的缺陷和肾小管上皮的异常，最终导致肾囊肿的发展。但是，从肾小管上皮异常导致囊肿发生的机制尚不十分清楚。在本报告中，我们证明了Robo2在调节上皮发育（包括纤毛生成，极化和分化）中的关键作用。我们发现Robo2缺乏会导致肾囊性囊肿，并且囊细胞在管状上皮中显示出纤毛和极性缺陷。小于最终分化的囊细胞继续增殖。我们进一步确定了Robo2与p53以及极性和睫状蛋白（Par3，PKCς，ZO-2和Claudin-2）协同作用来调节这些过程。 Robo2通过肾上皮细胞中的IRSp53 / MIM同源域与Baiap2（也称为IRSp53）结合。这种结合使Robo2可以通过Baiap2磷酸化Ser166上的MDM2，并维持p53稳态。 Robo2-Baiap2复合物的破坏导致MDM2进行去磷酸化，导致高水平的活性p53，并通过p21引发p53介导的细胞衰老，并降低ZO-1，ZO-2，PKCς，Par3，和Claudin-2蛋白，导致上皮发育的缺陷，包括纤毛发生，极化和分化。重要的是，与敲除Robo2的肾脏相比，双敲除Robo2和p53可以挽救肾脏中的所有上皮缺陷。综上所述，目前的结果表明，Robo2缺乏症会导致肾脏囊性疾病，这主要取决于肾脏中有缺陷的Robo2-Baiap2整合信号传导。该研究阐明了Robo2缺失引发肾小管前体细胞衰老导致肾脏发育缺陷的根本机制，提出对肾小管发育与衰老的新认识，为疾病早期干预靶点提供研究基础.