Molecular Basis of Pathogenicity of IgA1-containing Immune Complexes

描述(申请人提供)：IgA肾病(IgAN)是最常见的原发性肾小球肾炎，也是终末期肾功能衰竭的重要原因。这是一种系膜增生性肾小球肾炎，以IgA1系膜沉积为特征。虽然一段时间以来，人们一直推测IgAN的发病机制是由循环免疫复合体(IC)的沉积所致，但由于缺乏IgAN的动物模型，这一点一直难以得到证实。我们开发了允许在体外形成工程化IC的新方案，并建立了被动的IgAN小鼠模型，这为阐明IgAN的病理生理机制和确定潜在的治疗靶点提供了前所未有的机会。在……里面 IGAN是循环中的一小部分免疫球蛋白A1，具有半乳糖缺陷型O-糖链(Gd-IgA1)，存在于循环中被糖特异性自身抗体结合的IC中。我们现在已经鉴定了这些IC中存在的Gd-IgA1和抗Gd-IgA1自身抗体，并使用靶向蛋白质组学方法来确定与这些IC相关的血清因子和可能有助于其致病作用的因素，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的IgAN IC对导致系膜细胞激活的信号事件的分子效应。利用Gd-IgA1 IC刺激的人肾小球系膜细胞创新的多肽底物微阵列平台，全球酪氨酸激酶活性分布蛋白发现，在IC诱导的三条主要通路中，强大的酪氨酸激酶活性是主要参与者。对原生和工程IC也得到了类似的结果。这些IC刺激后的系膜细胞反应是典型的IgAN，但不同于用IC刺激后所获得的不同于病理性IC的全部成分。蛋白激酶抑制剂被证明与发病机制有关，这证实了其中一种抑制剂在体外和体内完全阻断了IC介导的系膜细胞在被动的IgAN小鼠模型中的增殖。这些数据表明，含有Gd-IgA1的ICs通过特定的信号通路激活系膜细胞，是IgAN发病机制中的关键环节；由此推论，这种IC驱动的系膜细胞信号可以被小分子质量的蛋白激酶抑制剂阻断，从而代表了一个可行的治疗靶点(S)。开发出用于生成初步数据的强大蛋白质组学、运动学和细胞学方法的基础和临床研究团队现在将通过以下方式验证这一假设：1)确定IgAN患者血清中含有Gd-IgA1的ICs激活人肾小球系膜细胞的特征；2)鉴定由含有Gd-IgA1的ICs激活的系膜细胞所激活的信号通路；以及3)使用IgAN动物模型确定关键蛋白激酶的小分子质量抑制剂对体内系膜细胞激活的有效性。相关性：这些结果将阐明IgAN的发病机制，并确定针对疾病特异性治疗的治疗目标以及潜在的反应/预后生物标记物。

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.