Introduction: IgA nephropathy (IgAN) is the most prevalent primary glomerular disease worldwide. The disease course ranges from mild to progressive, with a high lifetime risk for the development of kidney failure for the majority of patients. Although the disease mechanisms are not yet fully understood, a four-hit hypothesis for IgAN pathogenesis has been proposed, emphasizing the critical role of galactose-deficient IgA1 (gd-IgA1). According to this hypothesis, an overproduction of gd-IgA1 leads to elevated gd-IgA1 serum levels (hit 1) which are recognized by anti-gd-IgA1 autoantibodies (hit 2), resulting in the formation of immunocomplexes (hit 3). These complexes deposit in the mesangium (hit 4) causing glomerular inflammation and kidney injury. Current literature on IgAN pathology is based on gd-IgA detected by lectin-based immuno-assays. In this study, we aim to further elucidate the role of serum immunoglobulin glycosylation in IgAN pathology using a novel mass spectrometric technology that allows detailed site-specific glycoprofiling.

Methods: We employed a nanoliquid chromatography (LC)- mass spectrometry (MS)-based workflow for simultaneous analysis of IgG and IgA glycopeptides generated from serum of 41 patients with IgAN in different disease stages and 41 age- and kidney function-matched controls. Using ELISA-based methods, we measured gd-IgA, IgG autoantibodies against gd-IgA, and IgA/IgG immunocomplexes.

Results: We reliably identified 92 N- and O- glycopeptides from different peptide backbones (IgG1, IgG2/3, IgA1, IgA2). Calculation of structural features including galactosylation, sialylation, and N-glycan complexity, confirmed previous observations that high gd-IgA ELISA results were not caused by low expression of O-linked GalNAcs or due to shielding of galactose epitopes by sialic acids. Gd-IgA levels measured with ELISA correlated well with the amount of galactoses as measured with LC-MS (p=1.3e-09). O-glycosylation in the hinge region differed significantly between IgAN patients and controls and was similar across different disease stages. Sulfation of N-glycans was increased in non-IgAN patients and correlated with kidney function as assessed with the estimated glomerular filtration rate (eGFR). Furthermore, IgG N-glycosylation correlated with eGFR regardless of having IgAN. Finally, IgG autoantibodies against gd-IgA and IgA1/IgG immunocomplex levels were similar between controls and IgAN patients.

Conclusions: Our comprehensive workflow combining glycome profiling of both IgG and IgA provides a detailed signature of IgAN and points towards candidate glycan motifs recognized in current immunoassays detecting gd-IgA and gd-IgA mediated immune-complexes. This provides insight into the nature and role of skewed immunoglobulin glycosylation in IgAN pathogenesis. Our results support the role of O-glycosylation in the formation of immune complexes, crucial in driving the inflammatory response in IgAN.