Determination of severity of murine IgA nephropathy by glomerular complement activation by aberrantly glycosylated IgA and immune complexes

Abstract

The pathogenic roles of glomerular deposition of components of the complement cascade in IgA nephropathy (IgAN) are not completely clarified. To investigate the pathologic role of complement pathways in IgAN, two IgAN-prone mouse models were examined. Grouped ddY (gddY) mice showed significant high proteinuria, severe glomerular lesions, and extracellular matrix expansion compared with high serum IgA (HIGA) mice but with similar intensity of glomerular IgA deposition. Glomerular activation of the classical, lectin, and alternative pathways was demonstrated by significantly stronger staining for complement (C)3, C5b-9, C1q, C4, mannose-binding lectin (MBL)-A/C, MBL-associated serine protease-2, and factor B and properdin in gddY mice than in HIGA mice. Similarly, the serum levels of IgA-IgG2a/IgM and IgA-MBL-A/C immune complexes and polymeric IgA were significantly higher in gddY mice than in HIGA mice. Moreover, the serum levels of aberrantly glycosylated IgA characterized by the binding of Sambucus nigra bark lectin and Ricinus communis agglutinin I were significantly higher in gddY mice than in HIGA mice. This aberrancy in glycosylation was confirmed by monosaccharide compositional analysis of purified IgA using gas-liquid chromatography. This study is the first to demonstrate that aberrantly glycosylated IgA may influence the formation of macromolecular IgA including IgA-IgG immune complexes and subsequent complement activation, leading to full progression of IgAN.

Figure 1

Glomerular injury in gddY mice is more severe than that in HIGA mice. A: Proteinuria at 11 weeks [albumin creatinine ratio (ACR)]. B and C: Glomerular lesions and PAS-positive areas in glomeruli of gddY mice were significantly more severe than those in glomeruli of HIGA and control BALB/c mice. D–F: Glomerular type IV collagen (Col IV) expression in each IgAN-prone mouse. Expression of Col IV mRNA and protein was significantly higher in gddY mice than in HIGA mice. G and H: Glomerular IgA deposition in each IgAN-prone mouse. No significant difference was observed in glomerular IgA deposition in gddY and HIGA mice. Eleven mice were evaluated in each group. The bars represent mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2

Glomerular complement cascades were strongly activated in gddY mice. A: Common pathway: the positive area of glomerular C3 and C5b-9 staining in gddY mice was stronger than that in HIGA mice. B: Lectin pathway: the positive area of glomerular MBL-A, MBL-C, and MASP-2 staining was stronger in gddY mice than that in HIGA mice. C: Classical pathway: the positive area of glomerular C1q and C4 staining in gddY mice was stronger than that in HIGA mice. D: Alternative pathway: the positive area of glomerular factor B and properdin staining in gddY mice was stronger than that in HIGA mice. Eleven mice were evaluated in each group. The bars represent mean ± SD. *P < 0.01, **P < 0.001, and ***P < 0.0001.

Figure 3

Levels of IgA-IgG2a IC in sera and glomerular deposition were markedly increased in gddY mice compared with HIGA mice. A: The serum IgA level was significantly higher in HIGA mice than in gddY and control mice. B: However, the serum level of IgA-IgG2a IC was significantly higher in gddY mice than in HIGA and control mice. IgG2a (C) and IgM (D) were co-localized with C1q in the glomeruli of both IgAN-prone strains of mice. The intensity of IgG2a, IgM, and C1q staining was significantly higher in gddY mice than in HIGA mice. Eleven mice were evaluated in each group. The bars represent mean ± SD. *P < 0.01, **P < 0.001, and ***P < 0.0001

Figure 4

gddY mice showed greater activation of the lectin pathway. Serum levels of IgA–MBL-A IC (A) and IgA–MBL-C IC (B) were significantly higher in gddY mice than in HIGA mice. MBL-A (C) and MBL-C (D) were co-localized with IgA in both IgAN-prone strains of mice. Eleven mice were evaluated in each group. The bars represent mean ± SD. *P < 0.01, **P < 0.001.

Figure 5

gddY mice had higher amounts of circulatory aberrantly glycosylated IgA than HIGA mice. A: Levels of serum IgA were increased in HIGA mice (left panel). However, after adjustment for serum IgA levels, high-molecular-weight IgA was more abundant in gddY mice (right panel). The loaded samples under reducing conditions were developed with anti-IgA antibody (lower panels). B: Serum IgA in gddY mice showed significantly lower binding to lectin SNA than that in HIGA and BALB/c mice and significantly lower binding to RCA-I than that in BALB/c mice. Eleven mice were evaluated in each group. The bars represent mean ± SD. *P < 0.01.

Figure 6

The hypothesis of the role of complement pathways in murine IgAN. The findings of this study in gddY and HIGA mice suggest that the degree of aberrant glycosylation of IgA may determine the degree of IC formation and IgA polymerization and, therefore, influence subsequent glomerular injury through activation of the different complement pathways.