P0473LONGITUDINAL CHANGES OF IGA1 O-GLYCOFORM IN IGA NEPHROPATHY

Abstract

Abstract Background and Aims Elevation of circulatory IgA1 with galactose-deficient (Gd) hinge-region (HR) O-glycans (Gd-IgA1) has been detected in most IgA nephropathy (IgAN) patients based on lectin ELISA. However, new approaches are needed for molecular-level characterization of IgA1 HR glycoform(s) in IgAN. We established a high-throughput method for analysis of IgA1 HR O-glycoforms using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). To identify IgAN-associated IgA1 HR O-glycoforms and to assess their changes after therapy (with or without corticosteroids (CS)), we profiled IgA1 HR glycopeptides from sera collected at two time points (before and after therapy) from Japanese IgAN patients. Method Of the 10 Japanese IgAN patients recruited, 4 received CS treatment (CS group) and 6 have not (non-CS group). Japanese healthy volunteers (HC, n=10) were recruited as controls. Serum IgA1 was purified by affinity chromatography from HC and IgAN patients before and after therapy. After neuraminidase treatment and trypsin digestion, IgA1 HR glycosylation heterogeneity was analyzed by LC-HRMS. The relative abundance (RA, %) for each glycopeptide was calculated as percentage to the total IgA1 HR glycopeptide. The amount of each glycopeptide was then calculated by multiplying serum IgA concentration (mg/dL) by RA. Results Approximately 60% of IgA1 HR O-glycoforms in IgAN patients and HC were Gd O-glycoforms; these glycoforms contained one to three Gd-glycan(s), designated as 1 Gd-glycoform, 2 Gd-glycoform and 3 Gd-glycoform, respectively. In IgAN patients, the RA of non Gd-IgA1 glycoforms was elevated (P=0.002) and correlated with proteinuria (g/gCr) at renal biopsy (P=0.039, R=0.657). The amounts of non Gd- and 1Gd-glycoforms were higher in IgAN patients compared to HC (each P<0.001). After several years of follow up (2.77 years (1.44-3.85)), the RA of non Gd-glycoforms decreased in CS group of IgAN patients (P=0.039) whereas it remained unchanged in the non-CS group (P=0.488). The amount of non Gd-glycoforms exhibited similar trends, i.e., decreased in CS group (P=0.068) whereas it remained unchanged in the non-CS group (P=0.943). Conclusion This study profiled serum IgA1 for IgAN-associated IgA1 HR O-glycoforms at the molecular level and assessed their changes in response to CS vs. non-CS therapy. IgA1 HR O-glycoforms altered by treatment may serve as a biomarker(s) for monitoring patients’ responses to therapy.