USAG-1对Alport综合征足细胞的损伤作用及分子机制研究

Alport syndrome is a hereditary renal disease, characterized by hematuria, proteinuria, and progressing renal failure. Males with X linked Alport syndrome progress inevitably to ESRD, with ESRD risks of 90% by age 40. It has been shown that podocyte injury was involved in Alport syndrome. Recently, USAG-1 (uterine sensitization–associated gene-1) has been proved to be an important factor in the progressing of Alport syndrome. Ablation of Usag1 in Col4a3–/– mice led to normalization of glomerular basement membrane (GBM) ultrastructure, less albuminuria and preserved renal function. We hypothesized that USAG-1 contributes to podocyte injury via activation of Wnt signaling pathway in Alport syndrome. Col4a3–/– mice was treated with a neutralizing antibody against USAG-1. Electron microscopy was used to identify podocyte injury and immunohistochemistry was used to identify the distribution of USAG-1. Real time PCR and western blot was used to detect the expression of nephrin, synaptopodin, USAG-1 andβ-catenin. Furthermore, the activation of Wnt signaling and podocyte injury was detected by overexpression of USAG-1 or adding recombinant USAG1 in cultured podocytes in vitro. Podocyte injury was detected by blocking Wnt signaling with Dickkopf-1 (Dkk1) or Wnt inhibitory factor-1 (WIF-1). This study may provide a new insight in mechanism of disease progression for Alport syndrome. USAG-1 may sever as a new potential theraputic target for the treatment of Alport syndrome in future.

Alport综合征是一种遗传性进行性肾炎，男性X连锁显性遗传的患者40岁前发生终末期肾脏病的比例高达90%。足细胞损伤、蛋白尿进行性加重与Alport综合征疾病进展密切相关。研究发现敲除USAG-1的Alport综合征小鼠蛋白尿减轻、肾小球硬化比例减少，但USAG-1是否通过足细胞发挥作用及可能的机制远未澄清。本研究应用USAG-1抗体干预Alport综合征小鼠，检测疾病不同阶段USAG-1的表达与足细胞损伤的关系，证明USAG-1在Alport综合征中对足细胞的损伤作用；体外培养足细胞，通过过表达USAG-1、用USAG-1重组蛋白处理足细胞，检测Wnt信号通路关键分子，应用Wnt信号通路特异性抑制剂，进一步检测足细胞损伤指标，揭示USAG-1导致足细胞损伤的分子机制。本课题将从新的角度加深对Alport综合征疾病进展机制的理解，为寻求新的治疗靶点提供重要理论依据。

Alport 综合征是一种预后严重不良的遗传性慢性肾脏疾病，临床进展过程表现为单纯血尿，微量白蛋白尿，大量蛋白尿，直至肾衰竭。然而目前防止肾衰竭的药物治疗远远不够。所以寻求新的靶点或通路来阻止甚至逆转Alport 综合征疾病进展是急需要解决的临床和科研问题。足细胞的损伤和功能紊乱是Alport综合征疾病进展的背后驱动力，然而在Alport综合征中导致足细胞损伤的因素和分子机制远未澄清。USAG-1在Alport综合征疾病进展过程中具有关键作用，很可能成为新的阻止疾病进展的靶向。足细胞是否是USAG-1在肾小球发挥作用的靶细胞呢？目前尚无研究报道USAG-1是否对足细胞有作用及可能的分子机制。.我们通过Alport综合征小鼠模型（Col4a3-/-小鼠），应用免疫蛋白印迹方法（Western blot）检测疾病不同阶段（初期、中期、晚期）小鼠肾组织USAG-1的表达，发现随着疾病进展小鼠肾组织USAG-1的表达增加，Wnt1和Activeβ-catenin信号分子表达增加。同时检测了嘌呤霉素大鼠肾病模型（足细胞损伤的动物模型），发现在蛋白尿最重的第十天（也是足细胞损伤最重的阶段）USAG-1的表达增加，Wnt1和Activeβ-catenin信号分子表达增加。为进一步探讨USAG-1对足细胞的损伤作用和机制，我们通过体外培养小鼠足细胞（MPC5），过表达USAG-1，应用MTT法检测足细胞损伤情况，发现过表达USAG-1导致足细胞活性显著降低，足细胞特异性标志物nephrin表达降低。通过足细胞过表达USAG-1，并应用Wnt/β-catenin通路特异性抑制剂Dkk1不同浓度干预，发现足细胞活性在应用Dkk1后呈剂量依赖性上调。以上实验结果提示USAG-1通过调节Wnt信号通路引起足细胞损伤，参与Alport综合征疾病的进展。目前国内外并没有USAG-1导致足细胞损伤及机制的研究报道，本研究创新性以USAG-1对Alport综合征疾病进展中足细胞的损伤作用为切入点，揭示和证实USAG-1通过Wnt信号通路导致足细胞损伤，为进一步理解Alport综合征疾病进展的分子机制和探索Alport综合征新的治疗靶点提供了理论数据。