Anti-glomerular basement membrane (GBM) disease is a rare but life-threatening autoimmune disorder that is characterized by rapidly progressive glomerulonephritis with or without pulmonary hemorrhage. Kidney biopsy of anti-GBM disease has shown that immunoglobulin G (IgG) linearly deposits along the GBM, which is usually accompanied by linear or granular deposition of complement 3 (C3) (1). This indicates that complement activation may participate in the pathogenesis of anti-GBM disease. The pathways of complement activation in anti-GBM disease are mainly studied by passive injection of heterologous antibodies against GBM. It has been found that the complement system is activated in human anti-GBM disease through classical and alternative pathways (2), which plays an aggressive role in the pathogenesis of kidney injury by proinflammatory effect or cell lysis effect (3, 4). Multiple studies also showed that low serum C3 levels at diagnosis are associated with poor kidney outcomes in ANCA-associated vasculitis (5) and IgA nephropathy (6). However, to the best of our knowledge, few studies have analyzed the effect of serum C3 levels as a prognostic parameter in anti-GBM disease.

Therefore, this study aimed to investigate whether patients with low serum C3 levels at diagnosis have different clinical and histopathological features or outcomes compared to patients with normal C3 levels in anti-GBM disease.

This is the first retrospective study to explore the relationship between serum C3 levels measured at diagnosis and the risk of kidney failure in patients with anti-GBM disease. We observed that patients with low C3 levels had more severe glomerular sclerosis and poorer kidney survival than normal C3 levels. Serum C3 may be a protective factor for kidney outcomes. Furthermore, we confirmed that C3 was associated with kidney outcomes by univariable and multivariable Cox regression. Meanwhile, there was a difference in the Kaplan–Meier curve between the low C3 and the normal C3 groups. Therefore, we thought that serum C3 at diagnosis has a protective effect on kidney prognosis in patients with anti-GBM disease. In addition, our work suggested that serum albumin and oliguria/anuria were considered the prominent interactive factors that affect the relationship between C3 and the risk of kidney failure by the interaction analysis.

The complement system plays an essential role in immune-mediated glomerulonephritis. C3 deposition of the glomerular capillary wall can be shown by immunofluorescence, which is consistent with the assumption that complement activation participated in the generation of kidney damage of human anti-GBM disease. Previous studies of humans and animals both have found that the complement system is activated through the classical and alternative pathways in anti-GBM disease (2, 8). Some researchers supported the idea that antibody-directed complement activation initiates a cascade involving cytokine production, upregulation of adhesion molecules and chemokine production, and recruitment and activation of platelets and neutrophils, which ultimately lead to this early glomerular injury (9–12). Ma et al. have found that the complement cascade played a pathogenic role in kidney injury, as shown by the possible proinflammatory effect of C5a and/or cell lysis effect of C5b-9 in 20 patients with kidney biopsy-confirmed anti-GBM disease (8).

Several animal models have been employed to better understand the complement system’s role in the development of anti-GBM disease. Hammer et al. discovered that the primary stage of nephrotoxic serum nephritis produced by rabbit nephrotoxic serum appears to a great extent, but not wholly, upon the participation of serum complement (13). Sheerin et al., using C3-deficient mice, found that complement acts synergistically with heterologous antibodies, resulting in neutrophil infiltration and glomerular injury in experimental anti-GBM disease. Their studies also supported that classical and alternative pathways of the complement system are involved in the development of anti-GBM disease (14). Moreover, in the different phases of anti-GBM disease, the role of complement is contradictory. They thought that the effect of complement in the pathogenesis of glomerular disease might be dependent on the stage of the disease (15). More importantly, they also compared the contribution of systemic and local production of C3, concluding that circulating C3 is a critical factor in reducing the glomerular accumulation of immune complexes, while local synthesis of C3 did not have a major influence on this aspect of glomerular disease (16).

Our study found that lower C3 levels are associated with poorer kidney survival. There were no statistical differences between the two groups of patients who received the standard induction therapy including glucocorticoids and cyclophosphamide combined with a plasma purification technique. Even after adjusting for age, gender, hypertension, GBM, eGFR, KRT at onset, and crescents, C3 levels remained significantly associated with kidney survival. We further found that the C3 level was linearly related to the changing trend of kidney failure, and the risk of kidney failure gradually decreased as the C3 levels increased, which would suggest that serum C3 levels at diagnosis of anti-GBM disease could be an independent protective factor, but this needs further confirmation.

In addition, we found that serum albumin level was considered the prominent interactive factor that affects the association between C3 and the risk of kidney failure by the interaction analysis, which may be related to complement involvement in the pathogenesis of proteinuria (17). Considering that both albumin and C3 may be related to the nutritional status of patients and are potential systemic inflammatory response proteins, there may be other factors at play resulting in a concurrent decrease in both albumin and C3 (and maybe C4), which, in turn, results in worse outcomes that need to be further explored. As for C3, there was no protective role in patients with oliguria or anuria, which may be explained by oliguria or anuria being a stronger predictor of kidney survival in patients with anti-GBM disease compared with other variables, and almost all patients with initial oliguria or anuria end up entering kidney failure (18). Serum C4 levels and local synthesis of C3 have not been found to be associated with kidney outcomes of the disease in our study. We discovered that oliguria/anuria and peak SCr were risk factors for kidney failure in multivariable regression analysis, similar to previous reports.

Although C3 has been rarely studied in human anti-GBM disease, the crucial role of complement in pathogenesis has been explored in IgA nephropathy (6), C3 glomerulonephritis (19), membranous nephropathy (20), and other nephritis (21). Many recent studies demonstrated that C3 levels are correlated to patient and kidney survival in ANCA-associated vasculitis as well (22–24). The critical role of C3 at the intersection of all complement activation pathways and its synergistic effect in multiple immune and inflammatory networks have facilitated the development of C3-based therapies (25). Now, C3 intervention is emerging as a viable therapeutic strategy for rare inflammatory kidney diseases, such as C3 glomerulopathy (26).

Nevertheless, this study still has some limitations, including the small sample size and the single-center design. Moreover, some patients did not have a kidney biopsy at diagnosis, thus limiting the histological analysis. Despite these limitations, we demonstrated clearly that serum C3 plays a protective role in the progress of anti-GBM disease and that low serum C3 level at diagnosis is associated with poor kidney outcomes.

In conclusion, this study retrospectively compared the clinicopathological features and outcomes between the low C3 group and the normal C3 group, and found that patients with low serum C3 levels had a higher proportion of glomerular sclerosis progressing into kidney failure. Serum C3 is closely related to the kidney outcomes of patients with anti-GBM disease, and the pathogenesis of C3 in the disease needs to be further studied.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Independent ethics committee of Jinling Hospital. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

JW and HZ contributed to the conception of the study. WL screened the data, and MZ analyzed and interpreted the data. FX completed the pathological analysis. MZ, JW, YJ, and CJ contributed to the follow-up. MZ finished the manuscript. JW and HZ supervised and edited the manuscript. All authors contributed to the work and approved the submitted version for publication.

This work was supported by the National Key Research and Development Project of China (2021YFC2501302) and the Jiangsu Clinical Research Center Project (YXZXA2016003).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.