Molecular Basis of Pathogenicity of IgA1-containing Immune Complexes

描述（由申请人提供）：伊加肾病（IgAN）是最常见的原发性肾小球肾炎，也是终末期肾衰竭的重要原因。它是一种系膜增生性肾小球肾炎，定义为IgA 1系膜沉积。虽然人们推测IgAN的发病机制是由循环免疫复合物（IC）的沉积驱动的，但由于缺乏IgAN的动物模型，这一直难以证明。我们开发的新方案，允许在体外形成工程IC和建立一个被动的小鼠模型的IgAN提供了一个前所未有的机会，阐明IgAN的病理生理学和确定潜在的治疗靶点。在 IgAN是循环IgA 1的一部分，具有半乳糖缺陷型O-聚糖（Gd-IgA 1），存在于与聚糖特异性自身抗体结合的循环IC中。我们现在已经表征了存在于这些IC中的Gd-IgA 1和抗Gd-IgA 1自身抗体，并使用靶向蛋白质组学方法来定义与这些IC相关的血清因子，并可能有助于其致病作用，并确定IC的系膜细胞受体。这些研究的合理扩展需要分析这些致病性IgAN IC对导致系膜细胞活化的信号传导事件的分子效应。使用Gd-IgA 1 IC刺激的人肾小球系膜细胞的创新肽底物微阵列平台，全球激酶活性profilin鉴定了强大的酪氨酸激酶活性，作为IC诱导的三种主要途径中的信号传导的主要参与者。用天然和工程化IC获得了类似的结果。用这些IC刺激后的系膜细胞反应是典型的IgAN，但不同于通过使用IC获得的那些缺乏致病IC中的所有组分。使用蛋白激酶抑制剂证明了与发病机制的关联，这证实了其中一种抑制剂在体外以及在IgAN的被动小鼠模型中完全阻断IC介导的系膜细胞增殖。这些数据表明，Gd-IgA 1的IC代表了一个关键的打击，通过激活系膜细胞通过特定的信号转导通路的发病机制的IgAN的假设，推论是，这种IC驱动信号在系膜细胞可以被阻断的小分子量抑制剂的蛋白激酶，因此代表了一个可行的治疗靶点（S）。基础和临床研究人员团队已经开发出强大的蛋白质组学、激酶组学和细胞学方法，用于生成初步数据，现在将通过以下方法来验证这一假设：1）确定来自IgAN患者血清的激活人系膜细胞的含Gd-IgA 1的IC的特征; 2）表征由含Gd-IgA 1的IC激活的系膜细胞的信号通路;和3）使用IgAN的动物模型确定关键蛋白激酶的小分子量抑制剂对体内肾小球系膜细胞活化的功效。相关性：结果将揭示IgAN的发病机制，并确定IgAN疾病特异性治疗的治疗靶点以及潜在的反应/预后生物标志物。

DESCRIPTION (provided by applicant): IgA nephropathy (IgAN) is the most common primary glomerulonephritis and an important cause of end-stage kidney failure. It is a mesangioproliferative glomerulonephritis defined by IgA1 mesangial deposits. Although it has been speculated for some time that the pathogenesis of IgAN is driven by deposition of circulating immune complexes (IC), this has been difficult to prove due to the lack of animal models of IgAN. Our development of new protocols that permit formation of engineered IC in vitro and establishment of a passive mouse model of IgAN provide an unprecedented opportunity to elucidate the pathophysiology of IgAN and identify potential therapeutic targets. In IgAN, a fraction of circulating IgA1 has galactose-deficient O-glycans (Gd-IgA1) and is present in circulating IC bound by glycan-specific autoantibodies. We now have characterized Gd-IgA1 and the anti-Gd-IgA1 autoantibodies that are present in these IC and used targeted proteomic approaches to both define the serum factors that associate with these IC and may contribute to their pathogenic effects and to identify the mesangial-cell receptors for the ICs. Rational extension of these studies required analysis of the molecular effects of these pathogenic IgAN ICs on the signaling events that lead to mesangial-cell activation. Global kinase-activity profilin using an innovative peptide substrate microarray platform of human mesangial cells stimulated with Gd-IgA1 IC identified robust tyrosine kinase activity as a major player in IC-induced signaling in three predominant pathways. Similar results were obtained with native and engineered ICs. The mesangial-cell responses after stimulation with these IC were typical of IgAN but differed from those obtained by using IC lacking all of the components in pathogenic ICs. An association with pathogenesis was demonstrated using protein-kinase inhibitors, which confirmed that one of the inhibitors completely blocked IC- mediated mesangial cell proliferation in vitro as well as in vivo in the passive mouse model of IgAN. These data suggest the hypothesis that Gd-IgA1-containing ICs represent a key hit in the pathogenesis of IgAN by activating mesangial cells through specific signaling pathways; the corollary is that this IC-drive signaling in mesangial cells can be blocked by small-molecular-mass inhibitors of protein kinases and thus represents a feasible therapeutic target(s). The team of basic and clinical investigators that has developed the powerful proteomic, kinomic, and cellular approaches used to generate the preliminary data will now test this hypothesis by: 1) Defining the characteristics of Gd-IgA1-containing ICs from sera of patients with IgAN that activate human mesangial cells; 2) Characterizing the signaling pathways activated by Gd-IgA1-containing ICs in mesangial cells; and 3) Determining the efficacy of small-molecular-mass inhibitors of key protein kinases on mesangial cell activation in vivo using the animal model of IgAN. Relevance: The results will shed light on the pathogenesis of IgAN and identify therapeutic targets for disease-specific treatment of IgAN as well as potential response/prognostic biomarkers.