利用斑马鱼肾脏损伤模型研究GADD45B和APLP1基因对足细胞损伤的影响及分子机制

It is well known that podocyte injury is an early event leading to glomerular disease. However, the molecular mechanism of early podocyte injury in glomerular disease remains unclear. In order to better understand the pathogenesis of early podocyte injury in glomerular disease, two mouse models of glomerular disease were generated and characterized : 1) TGFbr1(AAD):NPHS2-rtta (by crossing transgenic mouse line of constitutively active TGF-beta receptor type 1 (TGFbr1(AAD) into the NPHS2-rtta transgenic line and 2) Dicer f/f:NPHS2-Cre. To identify the common genes that may be responsible for the similar phenotypes of the two models, we performed microarray analyses of the glomeruli using both mouse models. 20 genes were identified to be regulated similarly in these two glomerular disease models. Next we determined if those genes identified in mouse glomerular disease models are clinically relevant. Through real-time RT-PCR analysis with laser micro dissected glomeruli and histological studies, we identified growth arrest and DNA damage gene 45B (GADD45B) and amyloid like protein gene (APLP1) were regulated in the same way as in the animal models. The conservative regulation of the genes is highly suggestive of that they play a critical role in the development of glomerular diseases. In this proposed project, we will be conducting functional studies on the genes by utilizing zebra fish as in vivo animal model. Pod::NTR-mCherry transgenic fish or puromycin nucleotide microinjection fish will be used as inducible podocyte injury model. MultiSite gateway strategy and Uas/Gal4 system will be used for target gene expression and podocyte specific transgenic animal model construction. In addition, CRISP/Cas9 will be used to knockdown gene expression. We will be assessing the morphological and functional changes in the kidney with GADD45B and APLP1 over-expression/knock-down through in situ hybridization, immunofluorescence, electron microscopy and proteinuria detection via GFP-tagged vitamin D-binding protein transgenic fish. The effect of GADD45B and APLP1 overexpression/know-down on the apoptosis and methylated profiles in podocytes will be obtained and analyzed to undermine the mechanism of GADD45B and APLP1 on podocyte injury.The identified molecular related with podocyte injury will be validated for diagnostice biomarker in glomerulare disease. The project will highlight the mechanism of podocyte injury and glomerular disease.

足细胞损伤是肾小球疾病发生发展的关键环节。课题组前期利用TGFbr1(AAD)和Dicer fl/fl:NPHS2-Cre两种足细胞损伤相关肾小球疾病模型，筛选发现一组一致性变化的差异表达基因，其中上调基因GADD45B和APLP1在病例对照研究中进一步得到证实,提示其可能与肾小球疾病的发生发展高度相关。本项目以GADD45B和APLP1为靶基因，利用斑马鱼模型技术平台，结合通路克隆、Uas/Gal4和CRISP/Cas9等系统，分别构建靶基因在足细胞上特异性过表达和基因敲除的斑马鱼模型；分析特异性提高或抑制靶基因表达对肾脏组织形态、结构和功能的影响；体内外研究相结合，系统比较靶基因过表达或抑制对凋亡信号通路和足细胞损伤基因甲基化的影响，解析靶基因与足细胞损伤发生、发展的关系及其分子基础；对筛选到的候选足细胞损伤标志物进行临床验证和转化评估。研究结果对深入阐明肾小球疾病分子机制有积极意义。

足细胞损伤是肾小球疾病发生发展的关键环节, 是导致蛋白尿、肾小球硬化及肾功能进行性恶化的重要病理基础。我们利用高通量测序技术和多源数据整合筛选策略发现GADD45B和TYRO3可能参与足细胞损伤的发生发展进程。本研究应用斑马鱼和小鼠模型技术平台，从动物、细胞和分子水平探讨GADD45与TYRO3作用及机制，对GADD45和TYRO3作为疾病治疗靶点进行评估，研究结果对深入阐明肾小球疾病和足细胞损伤分子机制、发现治疗肾小球疾病的潜在新药靶点有积极意义。. 本研究发现：.1..FSGS患者肾小球GADD45B基因和蛋白表达水平显著高于正常对照组，并与FSGS患者白尿、血白蛋白及血肌酐水平密切相关；体内外研究发现，高表达GADD45B显著加重足细胞受损（凋亡，足突融合），而抑制GADD45表达则能有效改善足细胞损伤；ROS-GADD45B-p38MAPK 活化参与GADD45B介导足细胞损伤。.2..雷公藤甲素有效缓解足细胞损伤斑马鱼模型的水肿、蛋白尿和足突融合，抑制足细胞凋亡；雷公藤甲素通过抑制NF-κB/ GADD45B信号传导减弱蛋白尿和足细胞凋亡；在斑马鱼足细胞中特异性过表达gadd45b消除了雷公藤甲素的保护作用。.3..TYOR作为足细胞保护性因子，在维持正常足细胞功能中发挥关键作用。TYRO3在肾小球中特异性表达于足细胞；大量白蛋白尿的糖尿病肾病患者和FSGS患者肾小球TYRO3mRNA水平下降。斑马鱼研究显示，利用Morpholino敲低tyro3可破坏肾小球滤过屏障发育，导致斑马鱼眼周和卵黄囊水肿；敲除Tyro3明显加重糖尿病肾病小鼠模型和阿霉素肾病小鼠模型的白蛋白尿和肾小球损伤，而足细胞特异性过表达TYRO3过表达则显著改善糖尿病肾病，阿霉素肾病和HIV相关肾病（HIVAN）小鼠的白蛋白尿和肾损伤。进一步机制研究发现，足细胞内TNF-α/ NF-κB通路活化可抑制TYRO3表达，从而影响TYRO3下游AKT抗凋亡信号通路。