新月体IgA肾病致病性IgA1铰链区糖型鉴定及其应用研究

Crescentic IgA nephropathy(IgAN) is a severe form of IgAN disease, and also one of the most common reason of rapidly progressive glomerulonephritis. Its pathogenesis is still unclear. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region O-glycans (Gd-IgA1), and its anti-glycan autoantibodies play a pivotal role in IgAN. Our prior studies showed that the IgA1 from crescentic IgAN showed much different antigenicity to the anti-glycans, as compared to non-crescentic IgAN. This difference was not related to the Gal defficient levels of IgA1. Thus we hypothesize that the specific site of glycosylation defect (glycoforms) has influence its antigenicity with increased pathogenicity. In this study we aim to identify the specific glycoforms of IgA1 through activated ion-electron capture.dissociation mass spectrometric analysis, and then synthesize this glycopeptide in vitro. Rats are immunized with this peptide to obtain specific monoclonal antibody. Gd-IgA1 ELISA for specifically detecting this glycoform is consequently constructed to evaluate its relevance with the crescents forming in IgA nephropathy and also its association with patient prognosis. We also evaluate the complement activation of this specific glycoforms in vitro.

新月体IgA肾病是IgA肾病的一个重症类型，也是引起急进性肾炎的常见原因。IgA1铰链区存在5个O-糖结合位点，尽管该区域半乳糖缺失是引起IgA肾病发病的关键机制，但是IgA1沉积后如何引起广泛的新月体形成，目前仍然缺乏相关的研究。我们前期工作发现来自新月体IgA肾病病人血浆IgA1分子针对抗糖抗体的抗原性与非新月体IgA肾病存在显著的差异，并且其肾脏局部补体激活能力明显增强，这种抗原性差异与半乳糖缺失的IgA1水平无关，因此我们推测IgA1分子铰链区不同位点半乳糖缺失，即糖型的差异是改变IgA1分子抗原性、并增强其补体激活和致病性的原因。本研究中我们通过质谱分析鉴定新月体IgA肾病IgA1分子的特异型糖型，并体外合成该糖肽后免疫大鼠获取其单克隆抗体，用于该糖型的检测；进而通过体外补体激活试验以及体内观察该糖型与新月体及治疗反应的相关性，最终明确该糖型及其单克隆抗体致病性及临床应用价值。

新月体型IgA肾病（IgAN）常在数周或数月内进展至ESRD，是IgAN中的重型患者，然而目前IgAN中新月体形成机制不清。目前已经公认肾脏沉积IgA1分子铰链区存在O糖基化缺陷是引起IgAN发病的关键机制，目前已知该铰链区存在9 个潜在O-糖结合位点，然而IgAN中具体糖型结构差别仍然不清并且是否参与广泛新月体形成仍然缺乏相关研究。本研究中通过质谱分析解析了新月体IgAN中IgA1分子O糖的糖型结构，通过开发人群和验证人群两步法分析我们发现IgA1铰链区O糖的核心糖GalNAc数目减少是引起IgA肾病发病并且新月体形成的重要因素，GalNAc数目与新月体形成比例呈负相关，而既往研究所发现的半乳糖缺失是继发于GalNAc核心糖数目下降导致；在新月体IgAN患者尿液补体包括旁路和凝集素途径补体激活片段显著高于非新月体IgAN，其中C4d水平与新月体比例呈现正相关，这些结果提示IgA1分子GalNAc数目下降所致糖结构改变可能通过局部补体过度激活导致肾脏损伤、新月体形成；继而我们建立了化学酶法检测IgA1铰链区半乳糖缺失和核心糖新方法，可以替代复杂的质谱分析技术，该方法有望将糖结构检测应用于临床，成为新的生物标志物用于疾病诊断和判断预后，具有很好的临床转化应用前景。