PP2A调控YB-1表达致足细胞损伤及蛋白尿发生的分子机制

Proteinuria is not only the most common feature of kidney disease, but also the important risk factor for development of end stage renal disease and fatal cardiovascular events in human beings. .Recently, we identified that protein phosphatases 2A (PP2A) is expressed in podocyte of glomeruli from human and mouse kidney, furthermore, conditional knock-out of PP2A in glomerular podocyte of mice presented with the reduced size of both kidney, glomerular ultra-structure abnormality, and gross proteinuria, which indicated that PP2A may play a key role in maintaining structural and functional integrity of podocyte in glomerular filtration barrier. Further, by protein chips, we identified that, Y-box protein-1 (YB-1) is highly expressed in isolated podocyte from mice with conditional knock-out of PP2A, implying that YB-1 might be the candidate target molecule of PP2A in podocytes. .On the basis of these exciting findings, our project try to identify the expression pattern of YB-1 in podocyte in mice and patients with proteinuria, the morphological and functional alteration in cultured podocyte with YB-1 knock-down or over-expression, and the phenotypes of YB-1 transgenic mice, or YB-1 knockout mice with conditional knock-out of PP2A in glomerular podocyte. .Our purpose of present project is to identify the important role and molecular mechanisms of PP2A & YB-1 in maintaining structural and functional integrity of podocyte and glomerular filtration barrier, which might be invaluable in the treatment strategy for patients with proteinuria and glomerular nephritis.

蛋白尿不仅是各类肾小球疾病常见临床表现，也是疾病进展及出现严重并发症的重要危险因素。本小组长期从事肾小球性蛋白尿发生的足细胞机制研究，近来发现，蛋白磷酸酶2A(PP2A)足细胞特异性敲除小鼠出现双肾体积缩小、肾小球结构改变及大量蛋白尿等表型，提示PP2A对维系足细胞功能具有重要意义。在此基础上，采用磷酸化蛋白芯片比较足细胞相关蛋白质差异表达，发现Y-box protein-1(YB-1)分子表达有显著差异。因此，我们计划，分析PP2A足细胞特异性敲除小鼠和蛋白尿病人肾组织足细胞YB-1的表达(体内)，分析培养足细胞上敲低/过表达YB-1后足细胞形态及吞噬、侵袭、凋亡等变化(体外)，在PP2A足细胞特异性敲除小鼠建立YB-1转基因及基因敲除模型后分析蛋白尿等表型变化(Rescue实验)，以此分析PP2A/YB-1与足细胞及肾小球滤过膜功能完整性的关系，为蛋白尿的临床治疗提供新思路和新靶点。

蛋白尿不仅是各类肾小球疾病常见临床表现，也是疾病进展及出现严重并发症的重要危险因素。本团队长期从事肾小球性蛋白尿发生的足细胞机制研究，前期研究发现蛋白磷酸酶2A(PP2A)足细胞特异性敲除小鼠出现双肾体积缩小、肾小球结构改变及大量蛋白尿等表型，提示PP2A对维系足细胞功能具有重要意义。在此基础上，采用磷酸化蛋白芯片比较足细胞相关蛋白质差异表达，发现YB1（Y-box protein-1）分子及BASP1（Brain Acid Soluble Protein 1）表达有显著差异。通过临床实验，细胞及动物实验，我们发现足细胞水平敲低YB1表达后影响足细胞增殖，而细胞骨架未受明显影响，YB1基因足细胞特异性敲除小鼠的足细胞数量也出现显著变化。同时，免疫荧光结果显示在紫癜性肾炎、狼疮性肾炎及原发性肾病综合征（Primary Nephrotic syndrome，PNS）患者肾组织BASP1表达明显上调，在糖尿病肾病小鼠模型肾小球及肾小管的表达上调。BASP1在足细胞表达敲低后，足细胞的粘附、迁移、吞噬等功能增强。细胞骨架免疫荧光染色结果显示BASP1敲低可缓解细胞松弛素D以及HG对足细胞骨架F-肌动蛋白（F-actin）以及podocin表达的影响。动物体内实验中，我们发现从4周龄起，BASP1足细胞特异敲除小鼠肾脏体积以及重量均较野生对照小鼠增大。BASP1敲除可以减缓足细胞的凋亡，在BASP1敲除对足细胞的保护机制中，我们发现敲低BASP1可能是通过影响P53/FAS/IGFBP3通路以及促进凋亡调节蛋白（Bcl-2 apoptotic regulator，BCL-2）抗凋亡因子来减少足细胞的凋亡。该研究阐述了肾小球局部足细胞损伤的新分子机制，为临床大量蛋白尿及肾小球疾病患者的治疗提供了新的干预靶点。

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