Aberrantly Glycosylated IgA1 in Glomerular Immune Deposits of IgA Nephropathy

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In IgA nephropathy, abnormal O-glycosylation of IgA1 molecules contributes to mesangial IgA1 deposition and the development of glomerular injury; however, direct in situ demonstration of aberrantly O-glycosylated IgA1 within glomerular immune deposits has not been reported. This study investigated the presence of abnormally glycosylated IgA1 in situ and its spatial relationship with complement within the immune deposits and correlated these features with glomerular lesion severity. Immunofluorescence and confocal microscopy were used to evaluate 19 consecutive renal biopsies, and the severity of glomerular lesions were also scored. Aberrantly glycosylated IgA was observed within the immune deposits, and its amount was found to correlate with both the severity of glomerular lesions and the amount of C3c on the surface of the deposits. These results demonstrate that qualitative and quantitative evaluation of aberrantly glycosylated IgA can be performed on routine renal biopsy samples. Its presence in immune deposits likely influences the spatial organization of IgA and C3c, thereby contributing to the glomerular inflammatory response in IgA nephropathy. IgA nephropathy (IgAN) is the most common form of human primary glomerulonephritis, characterized by mesangial deposition of IgA molecules, chiefly of the IgA1 subclass.1,2 The disease displays a wide range of clinical presentations, leading to progressive renal failure in a substantial number of patients.3,4 The mechanism of mesangial deposition of IgA1 and the initiation of the ensuing inflammatory glomerular damage are still unclear, but a role has been suggested for altered O-glycosylation, a common posttranslational modification of cell surface proteins. Several studies have demonstrated a defect in glycosylation of serum IgA1 in patients with IgAN, with reduced galactose and/or sialic acid content, leading to increased exposure of preterminal GalNac.5–7 Such alterations may favor complex formation,8 and, indeed, it has been shown that undergalactosylated IgA1-containing immune complexes bind more efficiently to mesangial cells than circulating immune complexes from healthy control subjects.9,10 Alternatively, it has been proposed that undergalactosylated IgA1 could directly deposit in the mesangium, independent of any requirement for immune complex formation.7,11 Furthermore, binding of IgA1 eluted from isolated glomeruli of patients with IgAN to lectins specific for terminal GalNac was markedly higher as compared with serum IgA1, suggesting that abnormal IgA1 molecules are more likely to deposit in the kidney.12–14 Finally, enhanced mesangial deposition may result from the defective hepatic clearance of IgA1 with reduced terminal galactosylation.15 Regardless of the mechanisms involved, deposited IgA1 molecules likely contribute to the development of renal damage by direct interaction with resident glomerular cells16,17 and by complement activation18–21; however, the range of pathologic features of human IgAN is variable, ranging from minimal mesangial alterations to severe proliferative glomerulonephritis with necrotizing lesions and crescent formation, and it is widely accepted that there is little, if any, correlation between the amount of IgA or of complement fraction(s) deposited in mesangial areas and the severity of glomerular lesions.22 We showed previously that the severity of glomerular inflammatory lesions is influenced by the variations of the spatial relationship of IgA and complement within the immune deposits23; in this line, it is likely that the extent of some intrinsic defects of IgA molecules such as lack or aberrant glycosylation may be relevant.24,25 Evidence for mesangial deposition of aberrantly glycosylated IgA molecules is still very limited, however; it relies on elution techniques using isolated glomeruli, therefore requiring collection of nephrectomy specimens13,14 or pooling of several different biopsy specimens.12 As a matter of fact, direct, in situ evidence of glomerular deposition of aberrantly glycosylated IgA molecules in IgAN is still lacking. We undertook a study of the three-dimensional (3-D) organization of immune deposits and of the in situ demonstration of glycosylation pattern of IgA deposited in the mesangium in IgAN to show whether these features are of significance in determining the severity of histologic lesions, possibly interacting with complement and modifying the inflammatory response. Our investigation was carried out by using fluorescence (immuno)histochemistry and confocal microscopy on renal tissue from an unselected cohort of patients with IgAN. Histologic severity of glomerular lesions was compared by qualitative and quantitative analysis with the ratio of IgA and C3c present in the immune deposits and with the presence of terminal D-galactose by the study of in situ binding of a specific lectin. RESULTS Glomerular Inflammatory Index Details of light microscopic findings are summarized in Table 1. In particular, glomerular lesions ranged from minimal increase of mesangial cellularity (Figure 1A) to severe proliferative lesions, with crescent formation and focal necrosis of glomerular tuft (Figure 1B). Glomerular inflammatory index (GII) values ranged from 1 to 8, with a mean value of 2.75 ± 0.45. Six cases had a GII = 1, which corresponded to extremely mild lesions; in such cases, crescents or areas of necrosis of the glomerular tuft were never observed. The GII was ≥6 in two cases, indicating severe inflammatory lesions; the remaining 10 cases had a GII ranging from 2 to 5, representative of moderate histologic damage. Confocal Microscopy Analysis 3-D Surface Analysis. Results of 3-D Surface analysis are detailed in Table 2. Confocal microscopy of the double-stained specimens resulted in an optimal visualization of the glomerular immune deposits, with superior resolution of their external surface and identification of their features. The total surface areas of the 3-D reconstructed deposits were calculated in each case; values ranged from 5.96 to 59.35 square pixels (mean value 26.07 ± 3.18). Notably, area values in all glomeruli of each single case were uniformly distributed. The external surface of each immune deposit was composed by a mixture of IgA and C3c, with their proportions varying in the different cases (Figure 2). The ratio of the surface area occupied by IgA over total surface area (rIgA) ranged from 0.13 to 0.98 (mean value 0.75 ± 0.05), whereas the ratio of the surface area occupied by C3c over total surface area (rC3c) ranged from 0.01 to 0.86 (mean value 0.24 ± 0.05). The vast majority of the cases had higher rIgA values when compared with rC3c; only three cases (9, 14, and 17) displayed significantly higher values for rC3c. It is interesting that the same cases displayed also moderate or severe GII (6, 3, and 7, respectively) and lower values of Peanut lectin staining intensity (see next paragraph). Statistical analysis showed no correlation between total surface area of immune deposits and GII (r2 = 0.01; NS; Figure 3A); instead, a strong direct correlation existed between rC3c and GII (r2 = 0.25; P = 0.028; Figure 3B), whereas rIgA inversely correlated with GII (r2 = 0.25; P = 0.028; Figure 3C). 3-D Maximum Intensity Analysis. Results of 3-D maximum intensity analysis are also detailed in Table 2. Confocal microscopy of the specimens double-stained with IgA and Peanut lectin revealed that a strong and diffuse reactivity for the latter was present in all glomeruli, after Neuraminidase digestion (Figure 4); in particular, capillary walls displayed the highest intensity, which was present, however, also in the mesangial areas. Staining for IgA molecule was evident in the mesangium and, more rarely, along the capillary walls; the two signals were at times co-localized, with variable staining intensity of the two fluorochromes. IgA positivity was therefore used to identify specifically the immune deposits, and confocal las

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