Supplementary Material for: Response Predictors to Calcineurin Inhibitors in Patients with Primary Membranous Nephropathy

Background: Currently, there is an urgent need to find ways of identifying primary membranous nephropathy (PMN) patients who are likely to benefit from calcineurin inhibitors (CNI) or who are resistant to them. In this study, we employed nano-HPLC-MS/MS analysis to identify serum biomarkers that predict the clinical response to CNI therapy in PMN patients. Methods: The endpoint was complete remission (CR) after CNI treatment. PMN patients were grouped into no-remission (NR) or CR groups to screen predictive candidates using the nano-HPLC-MS/MS analysis. Results: Compared with NR patients, 3 upregulated proteins and 5 downregulated proteins were found to present a twofold change in CR patients’ serum. Serum amyloid A1 protein (SAA1) was further validated by ELISA; it was decreased in patients in the NR group compared with patients in the CR group, but SAA1 in patients in these groups was lower than in healthy controls and minimal change disease patients. The area under the receiver operating characteristic (ROC) curve of SAA1 was used to distinguish PMN NR patients from those in remission and was 0.901, with a sensitivity of 78.3% and specificity of 86.8%, similar to that of the phospholipase A2 receptor (PLA2R) antibody. Combining SAA1 with the PLA2R antibody, the area under the ROC curve was 0.956, which was higher than that of SAA1 or the PLA2R antibody alone. Conclusions: Serum SAA1 may be a candidate PMN biomarker that can be used to discriminate CNI NR cases from remission patients. The combination of SAA1 and the PLA2R antibody increases the accuracy of diagnosis.

研究背景：目前亟需建立筛选方法，以识别出可从钙调神经磷酸酶抑制剂（calcineurin inhibitors, CNI）治疗中获益，或对该类药物产生耐药性的原发性膜性肾病（primary membranous nephropathy, PMN）患者。本研究采用纳米高效液相色谱-串联质谱（nano-HPLC-MS/MS）分析技术，筛选可预测PMN患者对CNI治疗临床应答的血清生物标志物。研究方法：本研究以CNI治疗后完全缓解（complete remission, CR）为研究终点，将PMN患者分为无缓解（no-remission, NR）组与CR组，通过纳米高效液相色谱-串联质谱分析筛选潜在预测标志物。研究结果：与NR组患者相比，CR组患者血清中共筛选出3种上调蛋白与5种下调蛋白，其表达量变化幅度达2倍。进一步通过酶联免疫吸附试验（enzyme linked immunosorbent assay, ELISA）验证血清淀粉样蛋白A1（serum amyloid A1, SAA1）的表达水平：NR组患者血清SAA1水平低于CR组，且上述两组患者的SAA1水平均低于健康对照者及微小病变肾病患者。以SAA1的受试者工作特征（receiver operating characteristic, ROC）曲线下面积区分PMN NR患者与缓解患者，其曲线下面积为0.901，灵敏度为78.3%，特异度为86.8%，与磷脂酶A2受体（phospholipase A2 receptor, PLA2R）抗体的区分效能相近。将SAA1与PLA2R抗体联合检测后，ROC曲线下面积升至0.956，显著高于单一检测SAA1或PLA2R抗体的效能。研究结论：血清SAA1可作为潜在PMN生物标志物，用于区分CNI治疗无应答患者与缓解患者；联合检测SAA1与PLA2R抗体可提升诊断准确率。