Molecular Basis of Pathogenicity of IgA1-containing Immune Complexes

描述（由申请人提供）：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的沉积驱动的，但由于缺乏IgAN的动物模型，这一点很难证明。我们现在已经开发并验证了新的协议，允许在体外形成工程免疫复合物与IgAN的被动小鼠模型。这些创新与我们开发的灵敏的分析方法和广泛的合作，允许使用良好表征的临床cohot测试临床相关性，为阐明IgAN的病理生理学提供了前所未有的机会。在IgAN中，IgA1的一部分具有半乳糖缺陷型O-聚糖（Gd-IgA1），其存在于具有聚糖特异性自身抗体的循环免疫复合物中。我们已经定义了这些免疫复合物中存在的Gd-IgA1和抗Gd-IgA1自身抗体的子集，并使用蛋白质组学分析来定义与这些复合物相关的血清因子，并可能导致其致病作用。使用创新的肽底物微阵列平台对用Gd-IgA1免疫复合物刺激的人肾小球系膜细胞进行全球激酶活性分析，鉴定出强大的酪氨酸激酶活性，表明这些免疫复合物在肾小球系膜细胞中诱导的过程涉及蛋白酪氨酸激酶信号传导。蛋白激酶抑制剂的测试表明，抑制剂之一完全阻断免疫复合物介导的系膜细胞增殖在体外，以及在体内，在我们的新的被动小鼠IgAN模型。基于这些新的结果，我们提出了这样的假设：含有Gd-IgA 1的免疫复合物通过特定的信号通路激活系膜细胞，在IgAN的发病机制中发挥了关键作用。这一假设的推论是，系膜细胞中的这种免疫复合物驱动的信号传导可以被蛋白激酶的小分子量抑制剂阻断，因此代表了一个新的治疗靶点。我们将：1）确定来自IgAN患者血清的激活人肾小球系膜细胞的含Gd-IgA 1的免疫复合物的特征; 2）通过整体酪氨酸和丝氨酸/苏氨酸激酶组谱确定这些复合物在肾小球系膜细胞中激活的信号通路，并通过siRNA敲低实验证实;和3）鉴定在体外和体内阻断系膜细胞中免疫复合物所使用的特异性信号传导途径的蛋白激酶抑制剂。我们将选择小分子量激酶抑制剂来靶向关键 我们将研究由致病性免疫复合物刺激的信号通路，并将使用我们培养的人肾小球系膜细胞模型和IgAN的被动小鼠模型对有前景的抑制剂进行功效测试。相关性：对IgAN发病机制的进一步了解将为IgAN的疾病特异性治疗以及潜在的预后生物标志物确定治疗靶点。

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 the deposition of circulating immune complexes, this has been difficult to prove due to the lack of animal models of IgAN. We have now developed and validated new protocols that permit the formation of engineered immune complexes in vitro together with a passive murine model of IgAN. These innovations together with our development of sensitive methods of analysis and extensive collaborations that permit testing of clinical correlations using well- characterized clinical cohots provide an unprecedented opportunity to elucidate the pathophysiology of IgAN. In IgAN, a fraction of IgA1 has galactose-deficient O-glycans (Gd-IgA1) that is present in circulating immune complexes with glycan-specific autoantibodies. We have defined the subsets of Gd-IgA1 and the anti-Gd-IgA1 autoantibodies that are present in these immune complexes and have used proteomic analysis to define the serum factors that associate with these complexes and may contribute to their pathogenic effects. Global kinase-activity profiling using an innovative peptide substrate microarray platform of human mesangial cells stimulated with Gd-IgA1 immune complexes identified robust tyrosine kinase activity, suggesting that the processes induced by these immune complexes in mesangial cells involve protein-tyrosine kinase signaling. Testing of protein-kinase inhibitors indicated that one of the inhibitors completely blocked immune complex- mediated mesangial cell proliferation in vitro, as well as in vivo in our new passive murine model of IgAN. Based on these novel results, we propose the hypothesis that Gd-IgA1-containing immune complexes represent a key hit in the pathogenesis of IgAN by activating mesangial cells through specific signaling pathways. A corollary to this hypothesis is that this immune-complex-driven signaling in mesangial cells can be blocked by small-molecular-mass inhibitors of protein kinases and thus represents a new therapeutic target(s). We will: 1) Define the characteristics of the Gd-IgA1-containing immune complexes from sera of patients with IgAN that activate human mesangial cells; 2) Determine the signaling pathways activated by these complexes in mesangial cells by global tyrosine and serine/threonine kinome profiling and confirm by siRNA knock-down experiments; and 3) Identify inhibitors of protein kinases that block the specific signaling pathways used by the immune complexes in mesangial cells in vitro and in vivo. We will select small-molecular-mass kinase inhibitors to target the key signaling pathways stimulated by the pathogenic immune complexes and will perform efficacy testing of promising inhibitors using our cultured human mesangial cells model and passive mouse model of IgAN. Relevance: The improved understanding of the pathogenesis of IgAN will identify therapeutic targets for disease-specific therapy of IgAN as well as potential prognostic biomarkers.