Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children's Hospital, Radboud University Medical Center, Nijmegen, Netherlands.
Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland.
Front Immunol. 2018 Apr 4;9:612. doi: 10.3389/fimmu.2018.00612. eCollection 2018.
Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.
补体系统替代途径的过度激活与肾脏疾病非典型溶血尿毒症综合征（aHUS）和 C3 肾小球病（C3G）有关。C3 肾炎因子（C3NeF）通过稳定补体关键酶复合物 C3 转化酶，在 C3G 发病机制中发挥重要作用。然而，检测这些自身抗体的检测方法的可靠性是有限的。因此，在这项研究中，我们验证和优化了一种原型溶血方法，用于在大型患者队列中稳健地检测和表征导致转化酶过度活跃的因素。该检测方法直接在血清的生理环境中评估转化酶活性，因此不仅限于检测稳定的自身抗体（如 C3NeF），还可以揭示导致转化酶活性延长的遗传变异。我们首先根据健康对照者的转化酶活性定义明确的截断值。接下来，我们评估了 27 例 C3G 患者样本，发现 16 例存在延长的转化酶活性，表明存在影响转化酶稳定性的因素。在 3 名患者中，过活跃的转化酶谱在疾病过程中持续存在，而在另一名患者中，增加的稳定性在缓解期正常化。在所有这 4 名患者中，过活跃的转化酶稳定性存在于纯化的免疫球蛋白（Ig）部分，表明存在自身抗体。相比之下，携带补体因子 B 突变 p.Lys323Glu 的家族性 aHUS 患者的 Ig 未显示转化酶稳定。然而，在血清中观察到延长的转化酶活性，并在受影响和未受影响的家族成员中与突变分离。总之，我们提出了一种稳健可靠的方法，用于检测、表征和随时间评估延长转化酶活性的因素（C3NeF 或某些突变）在患者群体中的作用。该检测方法可为疾病发病机制提供新的见解，并有助于开发更具个性化的治疗策略。
