Epitope Mapping of Human α3(IV)NC1-Induced Membranous Nephropathy in Mice

Background and aim: Primary membranous nephropathy (pMN) is the most common cause of nephrotic syndrome in adults. Recent studies suggested that immunization of DBA/1 mice with the main antigen of antiglomerular basement membrane disease (GBM) disease, α3(IV)NC1, could lead to MN lesions. This study aimed to explore the pathogenic epitopes for mouse MN.

Methods: Twenty-four linear peptides were synthesized spanning human α3(IV)NC1. Male DBA/1 mice aged 6-8 weeks were immunized with the peptides 200 μg/mouse in Freund's complete adjuvant subcutaneously and boosted 3 times with the peptides in Freund's incomplete adjuvant in weeks 3, 5, and 7. The blood and 24-h urine samples were assessed every 2 weeks. The kidneys were examined when the mice were sacrificed at 18 weeks.

Results: All the mice immunized with human α3(IV)NC1 and the 24 peptides produced circulating antibodies against the immunogens at 2 weeks and achieved the maximum titers at 8 weeks. About 5/6 (83%) mice immunized with α3(IV)NC1 and (3/6) 50% of the mice immunized with peptide 23 (α3141-154) showed proteinuria at 8-10 weeks and increased continuously. The kidneys showed granular depositions of IgG, C3, and C5b-9 along the glomerular capillary loops. The major IgG subclass was IgG1 (equivalent to human IgG4). GBM thickening with the formation of spikes and subepithelial electron-dense deposits were observed under electron microscope.

Conclusion: The linear peptide of α3141-154 could induce clinical and histopathological features of MN in DBA/1 mice, which might give clues to the mechanism of MN in combination with anti-GBM disease.