Nephrin在肾脏足细胞发育中的作用及机制研究

Nephrin is essential to glomerular filtration and to the health of podocyte foot processes. Although many years of research efforts have established about nephrin, but its role and mechanism in podocyte is still unclear. We generated a transgenic mice line with doxycycline inducible RNA interference–mediated nephrin knockdown. Our data establish that after completion of kidney development, under the basal condition and in acquired glomerular diseases, nephrin is required to maintain slit diaphragm integrity and slit diaphragm–mediated signaling to preserve glomerular function and podocyte viability in adult mice.The nephrin gene, Nphs1, is mutated in congenital nephrotic syndrome of the Finnish type (CNF), that leads to heavy proteinuria in utero with the absence of podocyte slit diaphragm(SD). Due to the perinatal lethality of Nphs1 deletion mice, the complete knowledge on nephrin in podocyte development is far from concluded and additional information of the mechanism of nephrin is certainly required to gain. So here we try to study the expression and function of nephrin in podocyte development by using our inducible RNA interference–mediated nephrin knockdown transgenic mice. Furthermore, we will investigate the interaction between nephrin and other slit diaphragm proteins, and the possible signaling pathways of nephrin in podocyte. Our study may provide a new theoretical and experimental basis for the mechanism of CNF.

Nephrin对保护肾小球滤过屏障具有不可或缺的作用，但其具体功能及机制尚未完全清楚。本研究前期建立了doxycycline可逆性调控，RNAi介导的nephrin 基因knockdown小鼠模型。首次证明在无其他因素干预下和成年获得性肾病中，nephrin对维持足细胞裂孔膜(SD)完整性和信号转导有重要作用。已知nephrin基因突变可造成大量蛋白尿，即芬兰型先天性肾病综合征(CNF)，而nephrin基因敲除小鼠在围生期均死亡。导致我们对nephrin在足细胞发育中的作用及机制至今仍知之甚少。本研究拟在胚胎不同时期进行nephrin knockdown，分析nephrin表达量及时间点与足细胞损伤的关系，探讨nephrin与其他SD成分蛋白的相互作用及信号转导通路，阐明nephrin对足细胞发育影响机制。以期深入了解足细胞病理生理，为CNF发病机制提供可靠实验依据，为防治提供新靶点。

（一）Nephrin特异性表达于肾小球足细胞，是一种重要的足细胞裂孔膜结构蛋白，其基因突变可导致先天性肾病综合征。目前没有合适的Nephrin基因敲除小鼠模型，Nephrin对肾脏足细胞发育的影响及机制目前尚未完全清楚。本项目建立了可逆性调控siRNA干扰介导的nephrin基因敲减小鼠模型，研究结果提示nephrin在肾脏胚胎发育期间对足细胞发育和维持肾小球滤过膜完整性和正常肾脏结构具有重要作用。本部分研究结果为足细胞靶向治疗寻找新靶点提供可靠的实验依据，具有科学性、创新性，重要的社会意义和潜在的经济价值，未来具有良好的应用前景。.（二）我国人口老龄化日益严重，CKD在老年人中的发病率逐年升高，研究并阐明其发病机制非常重要。本项目建立了可逆性调控siRNA干扰介导的足细胞特异性 Sirt1基因敲减小鼠模型，诱导anti-Sirt1siRNA表达后饲养26-28个月形成衰老相关肾损伤模型。结果发现足细胞特异性Sirt1基因敲低增加老年小鼠尿白蛋白/尿肌酐比值，加重衰老相关肾小球硬化，并使足细胞数量明显减少。 Sirt1基因敲低后老年小鼠足细胞特异性标志物（nephrin、podocin、synaptopodin和WT1）表达显著下降；肾小球衰老相关蛋白p14ARF和p16INK4a 表达增加，并通过调节PGC1α、PPARγ、FOXO3、FOXO4及NF-κB通路在衰老相关肾损伤发病机制中的发挥作用。本项目研究结果提示Sirt1在衰老相关肾损伤发病机制中对足细胞受损有特殊作用，是治疗衰老相关肾损伤的潜在治疗药物靶点。足细胞特异性Sirt1基因敲低加重衰老相关肾损害。Sirt1通过调节PGC1α、、PPARγ、FOXO3、FOXO4及NF-κB通路发挥作用。本课题对阐明衰老相关肾损伤发病机制及治疗具有重大的推动作用，为将来的治疗衰老相关肾损伤药物的开发提供理论和实验依据，具有一定的应用前景和潜在经济价值，未来具有良好的临床转化应用前景。