Characterizing the proteome of pathogenic IgA1-containing immune complexes in IgA Nephropathy

项目摘要 伊加肾病（IgAN）是一种自身免疫性肾小球肾炎，经常导致肾衰竭。Igan是 其特征是与半乳糖缺陷型免疫球蛋白A1（Gd-IgA 1）结合的产量增加 特异于Gd-IgA 1的免疫球蛋白G（IgG）自身抗体，以形成IgA 1相关的免疫复合物（IgA 1- IC）与血液中的其他蛋白质。这些复合物存款在肾小球中并通过以下途径引起肾损伤： 激活肾小球系膜细胞增殖和肾小球毛细血管破裂， 尿没有全球范围的筛查，只能通过肾活检进行诊断。我们知道 免疫球蛋白和不确定的血液蛋白是必需的肾活动的高 分子量IgA 1-ICs，但我们不知道什么浓度，以及每种蛋白质的变异性，也不知道比例 形成复合物所必需的免疫球蛋白。我研究的主要目标是量化和建立 免疫球蛋白和血液蛋白在IgA 1-IC的组成中的比例在患者的血清中， 与健康对照相比，IgAN具有相反的临床谱。我们的合作团队观察到， 将血清添加到免疫球蛋白成核因子中对于致病性IC的形成是必要的 激活人类培养的系膜细胞。本提案的总体目标是， 血清IgA 1-IC的浓度将决定免疫球蛋白和其他血液蛋白的化学计量， IgA 1-IC在进展和稳定的IgAN患者中的形成。在我最近提交的 在一篇手稿中，我确定了21个高质量的血液蛋白靶点，这些靶点特异性地富集在从IgAN分离的IgA 1-IC中 病人血清当比较蛋白质的鉴定及其在IgA 1-IC中相应的富集时 对于其它未复合形式的IgA 1对照级分（单体和多聚伊加），我们观察到， 按分子形式聚类的组分，显示了我们的样品制备和血清创新分馏的价值。 基于初步数据，我假设IgA 1与IgG自身抗体的比例是离散的，可以确定 通过靶向LC-MS定量分析，多聚IgA 1是参与免疫应答的IgA 1的主要分子形式。 复合物形成和补体级联激活因子以较高的浓度存在于复合物组合物中。 比免疫球蛋白沉积到肾脏之前。在全球大流行期间，世界 了解到疾病筛查的重要性，疾病表现的可变性，以及每天的恐惧， 免疫力低下的人群生活在其中。作为伊加肾病的研究人员，我已经意识到这些 IgAN患者群体在COVID-19之前的实际情况。这激发了我的奉献精神， 在IgAN领域的问题，并了解这一关键部分的发病机制的IgA 1-IC形成的， 目前，使用血液生物标志物的微创筛查方法或疗法的未来发展， 不可预知的疾病

Project Abstract IgA Nephropathy (IgAN) is an autoimmune glomerulonephritis that frequently results in kidney failure. IgAN is characterized by elevated production of galactose-deficient immunoglobulin A1 (Gd-IgA1) being bound by an immunoglobulin G (IgG) autoantibody specific for Gd-IgA1 to form IgA1-associated immune complexes (IgA1- ICs) with additional proteins in blood. These complexes deposit in the glomerulus and cause kidney injury by activating mesangial cell proliferation and breakdown of glomerular capillaries allowing protein or blood in the urine. There is no worldwide screening and can only be diagnosed by renal biopsy. We know that the immunoglobulins and the undefined blood proteins are necessary to the nephritogenic activity of the high molecular weight IgA1-ICs, but we do not know what concentration, and variability of each protein nor the ratio of immunoglobulins necessary for complex formation. The primary goal of my study is to quantify and establish the ratios of immunoglobulins and blood proteins in the composition of IgA1-ICs in the serum of patients with IgAN of opposing clinical spectrum in comparison to healthy controls. Our collaborative team has observed that the addition of serum to the immunoglobulin nucleating factors is necessary for the formation of pathogenic ICs that activate human cultured mesangial cells. The overarching goal of this proposal is that quantitative analysis of serum IgA1-ICs will determine stoichiometry of immunoglobulins and other blood proteins involved in the formation of IgA1-ICs in both progressing and stable disease patients with IgAN. In my recently submitted manuscript, I identified 21 high-quality blood protein targets specifically enriched in IgA1-ICs isolated from IgAN patient serum. When comparing the identification of proteins and their corresponding enrichment in the IgA1-ICs to the other uncomplexed forms of IgA1 control fractions (monomeric and polymeric IgA), we observed that each fraction clustered by molecular form, showing the value of our sample prep and innovative fractionation of serum. Based on preliminary data, I hypothesize the ratio of IgA1 to IgG autoantibody is discreet and can be determined by targeted LC-MS quantitative analysis, polymeric IgA1 is the predominant molecular form of IgA1 involved in complex formation and complement cascade activation factors are present in complex composition at a higher ratio than immunoglobulins before deposition into the kidney. Throughout the global pandemic, the world has learned of the importance of disease screening, the variability of disease presentation, and the daily fear that immunocompromised populations live in. Being a researcher in IgA Nephropathy, I have been aware of these realities in IgAN patient populations prior to COVID-19. This has fueled my dedication to answering important questions in the IgAN field and understanding this crucial part of pathogenesis of IgA1-IC formation for the sake of future development of less-invasive screening methods or therapies using blood biomarkers for this currently unpredictable disease.