Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by inflammation of small to medium-sized blood vessels and the presence of autoantibodies against cytoplasmic proteinases in neutrophils and monocytes, with the most common autoantibodies targeting myeloperoxidase (MPO) and proteinase 3 (PR3).1 Kidney involvement, also known as ANCA-associated glomerulonephritis (AAGN), is one of the most common manifestations of AAV, occurring in more than half of the patients,2 and may lead to end-stage renal disease (ESRD) or death.3 Therefore, gaining a deeper understanding of the molecular mechanisms underlying the occurrence and progression of AAGN is of paramount importance for the early diagnosis and treatment of the disease, as well as for identifying new therapeutic targets.

Activation of neutrophils and monocytes induced by ANCA triggers activation of the alternative complement pathway, initiating an amplification loop of inflammation on the vessel wall, attracting and activating neutrophils, leading to respiratory burst, degranulation, extracellular trap formation, cell apoptosis, and necrosis.4 Studies have indicated that monocytes/macrophages play a significant role in the onset and progression of AAGN.5 Monocytes in the blood are precursors of tissue macrophages. Upon entering tissues, monocytes differentiate into macrophages, collectively forming the mononuclear phagocyte system. Macrophages influence the progression and fibrosis of renal diseases,6, 7 and depletion of macrophages or inhibition of macrophage influx can alleviate renal injury and fibrosis,8, 9 The renal histology in AAGN patients demonstrates significant infiltration of macrophages,10, 11 which not only present in early lesion sites such as fibrinoid necrosis and cellular crescents but also within seemingly normal glomeruli, being more prominent than other cell types, such as neutrophils.12 The expression of cytoplasmic proteases on the surface of apoptotic neutrophils impairs the phagocytic function of macrophages, leading to the release of inflammatory cytokines and immunogenic components, potentially resulting in increased activation of neutrophils, T cells, and B cells, and the subsequent production of ANCA, thus creating a vicious cycle5. Additionally, proliferation markers of macrophages can be detected in inflamed renal tissues, indicating that besides inflammatory recruitment, cellular proliferation is also one of the reasons for local macrophage infiltration.13 The proliferation of macrophages at inflammatory sites may be an important mechanism exacerbating damage. Considering the crucial role of macrophages in the immune activation and related pathological changes of AAGN, exploring the biological and clinical significance of macrophage-associated genes (MAGs) may also contribute to the advancement of molecular diagnosis and immunomodulatory therapies for AAGN.

In this study, we conducted a systematic investigation of MAGs in the kidneys of AAGN patients. Here, MAGs refer to genes identified from renal transcriptomic analyses whose expression is significantly associated with macrophage infiltration/activation signatures, rather than genes exclusively expressed by macrophages. Firstly, we verified the abundant infiltration of macrophages in AAGN renal tissues using clinical specimens and explored its correlation with renal prognosis. Subsequently, we utilized the Gene Expression Omnibus (GEO) database to identify key MAGs and employed single-sample gene set enrichment analysis (ssGSEA) to analyze the correlations of key MAGs and immune infiltration. We then confirmed the significant differential expression of these key MAGs in clinical samples and further analyzed their relationships with immune cells, clinical indicators, and renal prognosis. Given that glomerular endothelial injury is a key pathological event in AAGN, we next used HRGECs stimulated with patient serum to investigate whether these candidate genes participate in endothelial inflammatory responses under disease-relevant conditions. Finally, to further explore their functional roles, we performed preliminary functional validation by silencing or overexpressing the key MAGs in vitro. Our findings provide new insights into the inflammatory microenvironment in AAGN and offer potential therapeutic targets for this disease.