Modeling Crescentic Glomerulonephritis in Mice via Bovine NC1 Domain of Type IV Collagen: A Low-Mortality Approach

Crescents are characteristic histopathological findings in ANCA-associated vasculitis (AAV) and anti-GBM antibody-mediated rapidly progressive glomerulonephritis. They consist of cellular and interstitial aggregates filling Bowman’s space, and nephrons with crescent formation lose their ability to filter plasma and produce primary urine. Although crescents are associated with poor prognosis, their formation mechanism remains unclear, and no definitive treatment has been established. Animal models are essential for elucidating disease mechanisms and developing therapies. Existing mouse models of crescentic glomerulonephritis include administration of heterologous anti-GBM antibodies (e.g., from rabbits) and BSA nephritis, in which mice are immunized with bovine serum albumin (BSA) to induce anti-BSA IgG, followed by BSA injection to deposit immune complexes in glomeruli. The former raises concerns about pathophysiological relevance due to the use of xenogeneic antibodies, while the latter suffers from strain-dependent variability and high mortality. Our group previously employed the BSA model but encountered significant mouse loss, limiting research efficiency. Therefore, we aimed to establish a high-grade crescentic glomerulonephritis model with low mortality.

Eight-week-old C57BL/6 mice, including uninephrectomized individuals, were subcutaneously immunized with 200 μg of bovine type IV collagen NC1 domain (Collagen Engineering Workshop, Hino, Tokyo) emulsified in complete Freund’s adjuvant, administered every two weeks for a total of eight injections. Negative controls received PBS with adjuvant under the same schedule. Urine and blood samples were collected at 1, 3, and 5 weeks after the final antigen injection. Organs were harvested for histopathological analysis of the kidneys using light microscopy and immunofluorescence with anti-mouse IgG antibodies.

No mice died during the protocol. Mild proteinuria (qualitative 1+) was observed in control and non-nephrectomized mice, whereas uninephrectomized mice immunized with collagen showed marked exacerbation of proteinuria. Light microscopy revealed no changes in controls, and mesangial proliferation without crescent formation in non-nephrectomized mice. In contrast, uninephrectomized, collagen-immunized mice exhibited crescent formation and interstitial inflammatory cell infiltration. Immunofluorescence showed linear IgG deposition along glomerular capillary walls.Type IV collagen NC1 domain successfully induced anti-GBM-like autoantibodies in mice. The target epitope of anti-GBM antibodies is the NC1 domain, a hidden antigen of type IV collagen. It is known that antibody formation alone does not cause nephritis; a second hit, such as infection or inhalation of foreign substances, is required for disease onset. While it remains unclear whether uninephrectomy constitutes a second hit, increased antibody delivery to the kidney and abrupt elevation of GFR may have exposed self-antigens.

Immunization with type IV collagen NC1 domain (Collagen Engineering Workshop) enabled the establishment of a low-mortality mouse model of anti-GBM antibody-mediated crescentic glomerulonephritis.