Supplementary Material for: Relapse of Nephrotic Syndrome after Adrenocorticotrophic Hormone-Induced Remission: Implications of Adrenocorticotrophic Hormone Antibodies

Background: Prolonged use of corticosteroids continues to be the mainstay in the management of most proteinuric glomerulopathies, but is limited by extensive side effects. Alternative medications such as adrenocorticotrophic hormone (ACTH) have been recently used to treat refractory glomerulopathies and have shown superior outcomes when compared with steroids. However, the clinical responsiveness to ACTH therapy varies considerably with a number of patients exhibiting de novo or acquired resistance. The underlying mechanism remains unknown. Methods: A patient with steroid-dependent focal segmental glomerulosclerosis (FSGS) developed severe steroid side effects impacting quality of life and was converted to repository porcine ACTH therapy. Immediate response in the form of remission of nephrotic syndrome was noted followed by relapse in 10 weeks. Suspecting the role of some ACTH-antagonizing factors, the patient’s serum was examined. Results: Immunoblot-based antibody assay revealed high titers of de novo IgG antibodies in the patient’s serum that were reactive to the porcine corticotropin with negligible cross-reactivity to human corticotropin. In vitro, in cultured B16 melanoma cells that express abundant melanocortin receptors, addition of the patient’s serum substantially abrogated the porcine corticotropin triggered signaling activity of the melanocortinergic pathway, marked by phosphorylation of glycogen synthase kinase 3β, thus suggesting a mitigating effect on the biological functionality of porcine corticotropin. Conclusion: ACTH is a useful alternative therapeutic modality for refractory proteinuric glomerulopathies like FSGS. However, as quintessential therapeutic biologics, natural ACTH, regardless of purity and origin, is inevitably antigenic and may cause the formation of neutralizing antibodies in some sensitive patients, followed by resistance to ACTH therapy. It is imperative to develop ACTH analogues with less immunogenicity for improving its responsiveness in patients with glomerular diseases.

背景：长期使用糖皮质激素仍是多数蛋白尿性肾小球疾病治疗的核心手段，但因其广泛的不良反应而应用受限。近年来，促肾上腺皮质激素（adrenocorticotrophic hormone, ACTH）等替代药物被用于治疗难治性肾小球疾病，且相较于糖皮质激素展现出更优的治疗效果。然而，患者对ACTH治疗的临床应答差异显著，部分患者会出现新发或获得性耐药，其潜在机制尚未明确。
方法：1例糖皮质激素依赖型局灶节段性肾小球硬化（focal segmental glomerulosclerosis, FSGS）患者出现严重的激素不良反应，显著影响其生活质量，遂转为长效猪源ACTH治疗。患者即刻出现肾病综合征缓解的应答，但在10周后病情复发。研究人员怀疑存在某些ACTH拮抗因子，遂对患者血清进行检测。
结果：基于免疫印迹的抗体检测显示，患者血清中存在高滴度的新发IgG抗体，该抗体可与猪促肾上腺皮质激素结合，但与人促肾上腺皮质激素的交叉反应可忽略不计。体外实验中，在表达丰富黑皮质素受体的培养B16黑色素瘤细胞中加入患者血清后，可显著阻断猪促肾上腺皮质激素触发的黑皮质素能通路信号活性，该效应以糖原合成激酶3β的磷酸化为标志性特征，提示患者血清对猪促肾上腺皮质激素的生物学功能具有抑制作用。
结论：ACTH是治疗FSGS等难治性蛋白尿性肾小球疾病的有效替代治疗手段。然而，作为经典的治疗性生物制剂，天然ACTH无论纯度与来源如何，均不可避免地具有免疫原性，可能在部分敏感患者体内诱导中和抗体的产生，进而导致ACTH治疗耐药。因此，开发免疫原性更低的ACTH类似物以改善肾小球疾病患者对ACTH治疗的应答率，具有重要的临床必要性。