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• W2108206148 abstract "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 laser scanning microscope evaluation of the signal intensity for Peanut staining was performed only in such areas of the glomerulus, thus avoiding analysis of Peanut lectin positivity related to non-IgA material (Figure 4). By this method, Peanut lectin staining intensity ranged from 1.0 to 94.9, with a mean value of 35.7 ± 4.42. Statistical analysis demonstrated that Peanut lectin intensity strongly correlated inversely with GII: Progressive increase of positivity was associated with reduction of glomerular inflammatory lesions (P < 0.01; Figure 5A). A weaker but nevertheless significant reverse correlation was also found between Peanut lectin intensity and rC3c: Higher values of the former were associated with lower rC3c (P = 0.05; Figure 5B). DISCUSSION Our study demonstrates in situ, for the first time, that variable amounts of aberrantly glycosylated IgA molecules are present in the immune deposits of IgAN and that such amounts correlate significantly with the severity of glomerular histologic lesions and with the amount of C3c exposed along the external surface of the immune deposits. We also show that the total amount of the glomerular immune deposits does not correlate with the histologic picture. In addition, the study reported here confirms by quantitative analysis that 3-D organization of the immune deposits strongly correlates with the severity of the glomerular histologic lesions in IgAN: Higher amounts of C3c exposed along the surface of the mesangial deposits are associated with a more severe pathologic phenotype. Abnormal O-glycosylation of serum IgA1 in IgAN is now well established. The available evidence points to a lack of terminal galactosylation and/or sialylation leading to increased frequency of truncated O-glycan chains (reviewed by Barratt et al.26). In recent years, elution studies using isolated glomeruli from nephrectomy specimens or from pooled renal biopsies have indicated that abnormally O-glycosylated IgA1 are deposited in the mesangium and suggested that aberrant IgA1 O-glycosylation is not an epiphenomenon but is pathogenic to IgAN.12–14 The method we used allowed us to visualize directly the in situ deposition of aberrantly glycosylated IgA in the mesangial areas corresponding to immune complex deposits; in addition, it is easily reproducible on routine renal biopsy specimens and, importantly, is also suitable for quantitative evaluation of the relative amounts of the different IgA molecules deposited. O-glycosylation is a common feature of all cell surface proteins, and standard histochemical techniques using lectins with appropriate O-glycan specificity to define the characteristics of IgA molecules within the immune deposits have shown to be unhelpful, being the immune deposits in close vicinity to the glycocalix of podocytes and endothelial cells. Multiple immunofluorescence coupled with confocal microscopy gives the unique opportunity to visualize simultaneously and to co-localize two (or more) fluorochromes in thick renal tissue samples, therefore bypassing the high “background” of standard lectin staining, which usually masks the signal given by aberrantly glycosylated IgA1. The data we show here are obtained by the use of one single lectin after sialic acid removal, and other abnormalities are likely to be present in the remaining pool of IgA molecules; in this line, a thorough analysis of carbohydrate residues in the immune deposits of IgAN by the method we have reported will be necessary. The correlation among amount of deglycosylated IgA, extent of surface exposure of C3c, and severity of histologic lesions is in line with the known properties of the aberrantly glycosylated IgA molecules. In vitro experiments as well as data from patients with IgAN indicate that degalactosylated IgA1 molecules bind C3 fragments with much higher affinity than normal IgA molecules27; patients with IgAN and severe renal injury display glomerular deposition of mannose-binding lectin (a major recognition molecule of the lectin activation pathway of complement) probably as a consequence to higher affinity with aberrantly glycosylated circulating IgA1.28 Amore and colleagues29,30 showed that IgA glycoforms with aberrant glycosylation pattern, as well in vitro desialylated or degalactosylated normal IgA, are able to modulate several mesangial cell functions, including proliferation state, rate of apoptotic death, nitric oxide synthase activity, expression of integrins, and activation of NF-κB. Recent evidence also exists that mesangial cells are activated by aberrantly glycosylated IgA1 via binding transferrin receptor, with induction of proliferation and release of IL-6 and TGF-β.31 Taken together, these data suggest an important role for the spatial organization of IgA and C3c within the immune deposits in influencing the glomerular inflammatory response in IgAN. It is tempting to speculate that the presence of variable amounts of aberrantly glycosylated IgA molecules in the immune deposits may be instrumental in modulating the exposure of complement fractions along the external surface; complement fraction(s), when “buried” inside the immune deposit (i.e., not exposed along the external surface) would be prevented from exerting most of their proinflammatory and cytolytic activities, thereby reducing the severity of the histologic damage. CONCISE METHODS Patients and Clinical Features Nineteen consecutive patients (14 male, five female) with histologically proven IgAN and with serum creatinine ≤1.5 mg/dl were enrolled for our investigation (Table 3). Mean age at the time of renal biopsy was 32 ± 13 yr (range 16 to 68 yr); serum creatinine was 1.07 ± 0.2 mg/dl, and estimated GFR was 98.4 ± 27.0 ml/min (Cockcroft and Gault formula). Episodes of macrohematuria were referred by 13 patients, and the known duration of clinical signs of IgAN was 22.9 ± 19.6 mo. Mean proteinuria was 1.1 ± 1.1 g/24 h at the time of renal biopsy. No therapy with steroid and/or immunosuppressive drugs was administered before or at the time of biopsy. Arterial hypertension was present in six patients, and it was treated with success with angiotensin-converting enzyme inhibitors (two of them received also a calcium channel blocker). Standard Histology and Immunofluorescence Renal tissue was obtained by percutaneous needle biopsy. Tissue cylinders were received within 15 min from the time of biopsy; specimens from each biopsy were divided into three portions and processed for routine light and electron microscopy and immunofluorescence. The severity of glomerular lesions was evaluated using an arbitrary inflammatory index (GII) derived mainly from the histologic criteria identifying IgAN subclasses,32,33 integrated with the histologic criteria currently used to define active glomerular lesions in lupus nephritis.34,35 All biopsy samples displayed at least nine glomeruli. In particular, our GII included the following: Cellular proliferation: Hypercellularity of glomerular tuft, mostly as a result of mesangial cells, but also endothelial cells and infiltrating monocytes Leukocyte exudation: Presence of three or more neutrophils per glomerulus Karyorrhexis and fibrinoid necrosis: Presence of pyknotic and fragmented nuclei and intensely eosinophilic material Cellular crescents: Considered only when occupying at least one fourth or more of glomerular capsular circumference For each lesion, the following scale of severity was used: Absence of lesion: 0 Involvement of 1 to 19%: 1 Involvement of 20 to 39%: 2 Involvement of 40 to 59%: 3 Involvement of 60 to 79%: 4 Involvement of 80 to 100%: 5 GII of each case was calculated as the total of figures representing presence and degree of single inflammatory lesions. Scoring was performed by at least two investigators during a single session; assigned value was the mean value of observed records. Confocal Microscopy Twenty-micron-thick sections from OCT-embedded tissues were cut in a cryostat, collected on adhesive-coated slides, briefly fixed (approximately 5 to 10 min) in acetone, air-dried, and brought to PBS. Two slides were obtained from each case (three sections per slide), and immunohistochemical double staining was performed as follows: Slide 1: Incubation with tetramethylrhodamine isothiocyanate–conjugated rabbit anti-human IgA (Dako Corp., Glostrup, Denmark), 30 min at room temperature, followed by FITC-conjugated rabbit anti-human C3c (Dako Corp.), 30 min at room temperature. Antisera were diluted 1:20 in PBS. Slide 2: Digestion with neuraminidase type X (Sigma Corp., St. Louis, MO), 2 h at 37°C, followed by incubation with Alexa Fluor 647–conjugated lectin from Arachis hypogaea (Peanut; Molecular Probes, Eugene, OR), 1:50 in PBS, 2 h at room temperature. This lectin is known to have a high affinity for nonreducing terminal D-galactose. After careful washing, sections were then incubated with tetramethylrhodamine isothiocyanate–conjugated rabbit anti-human IgA, 1:20 in PBS, 30 min at room temperature. Vectashield (Vector Laboratories, Burlingame, CA) was used as mounting medium to minimize bleaching of the fluorescence signal. All sections were examined with a Zeiss LSM 510 Meta confocal laser scanning microscope. Optical sections (i.e., images obtained from laser scans of subsequent x-y planes at various z positions in the specimen) were collected from three nonsclerotic glomeruli in each case. Briefly, for double-labeled specimens, the dual-channel mode was used, and sections were scanned simultaneously at both wavelengths (488/514 and 514/647, respectively), with laser intensity, confocal aperture, gain, and black level settings kept constant. Stacks of scanned sections were then analyzed using the AxioVision 4.4 imaging analysis software (Zeiss) for reconstruction of 3-D images and area measurements; values were expressed as square pixels. The fluorescence intensity at selected areas, linearly correlated with the number of pixels, was also quantitatively analyzed with the same software using the same stacks of optical sections; values were expressed as number of pixels. All scans were performed using the same objective (×63 oil immersion objective). The following parameters were considered in each case: Slide 1, 3-D surface analysis: By this mode, we calculated the total surface area of the immune deposits and the surface area of the immune deposit occupied by each fluorochrome. To compare the values of surface areas of IgA and C3c in the different cases, we divided in each glomerulus the area values of each fluorochrome by the value of the total surface area. We therefore obtained the ratio of surface area occupied by IgA over total surface area of the immune deposits (rIgA) and the ratio of surface area occupied by C3c over total surface area of the immune deposits (rC3c). Slide 2, 3-D maximum intensity analysis: By this mode, we evaluated the presence and extent of Peanut lectin positivity in the context of the immune deposits, indicated by anti-IgA positivity. At least 15 deposits were examined in each glomerulus, with a total of at least 45 deposits for each case. Final value of Peanut lectin positivity was expressed as the average value of signal intensity within the 3-D reconstructed immune deposits. All data were expressed as median ± SE, except for clinical parameters (median ± SD); statistical analysis was performed with Microsoft Excel software. Correlations were investigated by simple regression analysis (Pearson correlation coefficient); P ≤ 0.05 was considered significant. DISCLOSURES None.Figure 1: IgAN; representative histologic images of GII. (A) Mild GII; glomeruli display focal, minimal increase in mesangial cellularity (arrow), and diagnosis is made solely on the basis of IgA deposition at immunofluorescence (data not shown). (B) Severe GII, with mesangial hypercellularity, crescent formation, and focal necrosis of the tuft. Magnification, ×300 (periodic acid-Schiff).Figure 2: IgAN; double immunofluorescence staining with antibodies against IgA (red) and C3c (green); confocal microscopy analysis of the immune deposits and 3-D rendering with surface mode. External surface of mesangial deposits displays a mosaic of both fluorochromes. Surface exposure of IgA and C3c varies significantly in the different cases: In A (case 11), there is prevalence of exposed IgA, whereas in B (case 9), there is marked increase of exposed C3c, with several deposits lacking surface IgA.Figure 3: Correlations between GII and total surface area (TSA; A), GII and rIgA (B), and GII and rC3c (C).Figure 4: IgAN; case 15. Double immunofluorescence staining using Alexa Fluor 647–conjugated Peanut lectin (blue) and tetramethylrhodamine isothiocyanate–conjugated monoclonal anti-IgA antibody (red). Confocal microscopy analysis with maximum intensity mode. The co-localization of the two fluorochromes is shown in variable intensities of purple (A). A mesangial immune deposit is selected for quantitative analysis of fluorescence intensity (B and C); values of blue signal are recorded only when colocalized with red (D).Figure 5: Correlations between GII and Peanut lectin intensity (PNA; A) and rC3c and Peanut lectin intensity (PNA; B).Table 1: GIITable 2: Immunohistochemistry and lectin histochemistry of immune depositsaTable 3: Clinical featuresThis work was supported in part by a grant from Ministero dell' Istruzione, Universita[Combining Grave Accent] e Ricerca - COFIN. Part of this study was presented in abstract form at the XLIII European Renal Association–European Dialysis and Transplant Association Congress; July 15 through 18, 2006; Glasgow, Scotland. The technical assistance of F. Fanella, G. Bolognesi, and L. Virgilii is greatly acknowledged." @default.
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• W2108206148 title "Aberrantly Glycosylated IgA1 in Glomerular Immune Deposits of IgA Nephropathy" @default.
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