Supplementary Material for: Uromodulin and α1-Antitrypsin Urinary Peptide Analysis to Differentiate Glomerular Kidney Diseases

<em>Background/Aims:</em> Glomerular kidney disease (GKD) is suspected in patients based on proteinuria, but its diagnosis relies primarily on renal biopsy. We used urine peptide profiling as a noninvasive means to link GKD-associated changes to each glomerular entity. <i>Methods:</i> Urinary peptide profiles of 60 biopsy-proven glomerular patients and 14 controls were analyzed by combining magnetic bead peptide enrichment, MALDI-TOF MS analysis, and ClinProTools v2.0 to select differential peptides. Tentative identification of the differential peptides was carried out by HPLC-MS/MS. <i>Results:</i> The HPLC-MS/MS results suggest that uromodulin (UMOD; m/z: 1682, 1898 and 1913) and α₁-antitrypsin (A1AT; m/z: 1945, 2392 and 2505) are differentially expressed urinary peptides that distinguish between GKD patients and healthy subjects. Low UMOD and high A1AT peptide abundance was observed in 80–92% of patients with GKD. Proliferative forms of GKD were distinguished from nonproliferative forms, based on a combination of UMOD and A1AT peptides. Nonproliferative forms correlated with higher A1AT peptide levels – focal segmental glomerulosclerosis was linked more closely to high levels of the m/z 1945 peptide than minimal change disease. <i>Conclusion:</i> We describe a workflow – urinary peptide profiling coupled with histological findings – that can be used to distinguish GKD accurately and noninvasively, particularly its nonproliferative forms.

**背景/研究目的：** 肾小球肾病（Glomerular kidney disease, GKD）可通过蛋白尿疑似诊断，但确诊主要依赖肾活检。本研究采用尿液肽谱分析作为无创手段，将GKD相关的分子变化与各肾小球病变类型关联起来。
**方法：** 本研究纳入60例经活检证实的肾小球肾病患者与14例健康对照者，联合磁珠肽富集、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析与ClinProTools v2.0软件筛选差异肽段；通过高效液相色谱-串联质谱（HPLC-MS/MS）对差异肽段进行初步鉴定。
**结果：** HPLC-MS/MS分析结果显示，尿调蛋白（uromodulin, UMOD；m/z：1682、1898和1913）与α₁-抗胰蛋白酶（α₁-antitrypsin, A1AT；m/z：1945、2392和2505）为区分GKD患者与健康受试者的差异表达尿液肽段。80%~92%的GKD患者呈现UMOD肽丰度降低、A1AT肽丰度升高的特征。结合UMOD与A1AT肽谱，可区分增生性与非增生性GKD亚型。非增生性GKD与更高水平的A1AT肽相关——局灶节段性肾小球硬化（focal segmental glomerulosclerosis）与m/z 1945肽段高水平的关联性强于微小病变型肾病（minimal change disease）。
**结论：** 本研究建立了一套尿液肽谱分析联合组织病理学检测的工作流程，可精准且无创地鉴别肾小球肾病，尤其是其非增生性亚型。