紧密连接破坏及其相关ZO-1/ZONAB信号途径激活在I型肾单位肾痨肾间质纤维化的作用

Nephronophthesis (NPHP) is the main genetic cause of renal failure up to the third decade of life. Diffuse interstitial fibrosis is one of the main pathological characteristics, and its pathogenesis is not clearly understood. Up to now, there is no effective treatment for NPHP. NPHP type I is the most common hereditary form of NPHP. NPHP1 is the pathogenic gene of NPHP type I. NPHP1 is not only a component of cillia, but also important for the formation of cell junctions. Our previous study indicated that NPHP1 knockdown could disrupt on the formation of tight junction and adhesion junction of MDCK cells and could induce epithelial-mesenchymal transdifferentiation (EMT). The tight junction associated ZO-1/ZONAB signaling pathway was also activated. We also tried to use dexamethasone to stabilize the cell junctions secondary to the NPHP1 knockdown, the EMT and the activation of ZO-1/ZONAB signaling pathway were inverted. EMT is a key factor attributes to renal fibrosis. In the present study, we'll try to silence the expression of NPHP1 in MDCK cells by transfecting reconstructed lentiviral shRNA in vitro, and make mouse model of NPHP1 gene knockout. Then siRNA and reconstructed lentiviral shRNA are applied to knockdown the expression of ZONAB in vitro and in vivo, respectively; or dexamethasone is administrated to stabilize the tight junctions. For doing these, to observe the EMT and renal interstitial fibrosis both in vitro and in vivo. And to further clarify the pathogenesis of renal fibrosis in NPHP type I. It may provide a new idea and a new theory for the treatment of NPHP.

肾单位肾痨(NPHP)是30岁前肾衰竭的主要遗传因素，显著弥漫肾间质纤维化是其主要病理特征之一，发生机制尚未清楚，目前无有效治疗手段。I型NPHP是最主要的遗传类型，近年发现其致病基因NPHP1不仅影响纤毛结构和功能，还与细胞连接形成有关。我们前期研究发现，敲低NPHP1表达可影响肾小管上皮细胞紧密连接和粘附连接形成，诱导上皮细胞转分化(EMT)，激活ZO-1/ZONAB信号途径；用地塞米松稳定细胞连接可抑制该信号途径激活，抑制EMT。EMT是肾间质纤维化的重要因素。本项目进一步通过体外重组慢病毒转染MDCK细胞，沉默NPHP1表达，以及体内敲除NPHP1构建I型NPHP小鼠模型；用siRNA和重组慢病毒shRNA在体外、体内分别干扰ZONAB表达，或用地塞米松干预，观察体内外肾组织细胞发生EMT及肾间质纤维化情况。以进一步揭示NPHP肾间质纤维化的发生机制，为其治疗提供新思路和理论依据。

肾单位肾痨(NPHP)是30岁前肾衰竭的主要遗传因素，显著弥漫肾间质纤维化是其主要病理特征之一，发生机制尚未清楚，目前无有效治疗手段。I型NPHP是最主要的遗传类型，近年发现其致病基因NPHP1不仅影响纤毛结构和功能，还与细胞连接形成有关。在本研究中，我们成功筛选出了有效干扰MDCK细胞NPHP1表达的siRNA（d-1352）。使用siRNA（d-1352）干扰MDCK细胞降低NPHP1表达后，上皮细胞连接相关的E-cadherin、β-catenin表达被抑制，细胞失去上皮细胞特性，变成长梭形，迁移能力增强，MMP-2、MMP-9和α-SMA表达升高，获得成肌纤维细胞特性，促进MDCK细胞向间质细胞转分化（EMT）。同时细胞ZO-1表达下降，ZONAB表达升高，并由细胞膜转移聚集到细胞核，提示激活了ZO-1/ZONAB信号通路。糖皮质激素干预可以稳定NPHP1敲低MDCK细胞的细胞连接，抑制紧密连接相关ZO-1/ZONAB信号途径激活，抑制NPHP1敲低MDCK细胞发生EMT。同时敲低NPHP1和ZONAB的表达，可抑制单一敲低NPHP1表达时激活的ZO-1/ZONAB信号通路后，可部分抑制单一敲低NPHP1表达时诱导的上皮细胞连接破坏和上皮细胞间质转分化（EMT），但未完全阻止EMT。以上实验结果提示ZO-1/ZONAB信号通路可能在NPHP1肾间质纤维化中起重要作用。同时，我们通过CRISPR/Cas9技术成功构建NPHP1基因敲除的MDCK细胞株和NPHP1基因大片段缺失的小鼠模型。通过重组慢病毒转染在NPHP1基因大片段缺失小鼠模型中敲低ZONAB表达，阻断ZO-1/ZONAB信号通路，观察小鼠肾组织纤维化的改善情况，这部分研究仍在进行中。本研究课题的完成将进一步揭示NPHP肾间质纤维化的发病机制，为其阻止和延缓NPHP疾病进展的治疗提供新思路和理论依据。