The Kinetics of Glomerular Deposition of Nephritogenic IgA

<div><p>Whether IgA nephropathy is attributable to mesangial IgA is unclear as there is no correlation between intensity of deposits and extent of glomerular injury and no clear mechanism explaining how these mesangial deposits induce hematuria and subsequent proteinuria. This hinders the development of a specific therapy. Thus, precise events during deposition still remain clinical challenge to clarify. Since no study assessed induction of IgA nephropathy by nephritogenic IgA, we analyzed sequential events involving nephritogenic IgA from IgA nephropathy-prone mice by real-time imaging systems. Immunofluorescence and electron microscopy showed that serum IgA from susceptible mice had strong affinity to mesangial, subepithelial, and subendothelial lesions, with effacement/actin aggregation in podocytes and arcade formation in endothelial cells. The deposits disappeared 24-h after single IgA injection. The data were supported by a fluorescence molecular tomography system and real-time and 3D in vivo imaging. In vivo imaging showed that IgA from the susceptible mice began depositing along the glomerular capillary from 1 min and accumulated until 2-h on the first stick in a focal and segmental manner. The findings indicate that glomerular IgA depositions in IgAN may be expressed under the balance between deposition and clearance. Since nephritogenic IgA showed mesangial as well as focal and segmental deposition along the capillary with acute cellular activation, all glomerular cellular elements are a plausible target for injury such as hematuria.</p></div

IgA from gddY mice is deposited along the glomerular capillary wall in a focal and segmental manner.

<p>Detailed kinetics of IgA deposition analyzed from 1 min to 2 h postinjection using confocal laser microscopy. Alexa Fluor 633-labeled IgA from gddY and Balb/c mice (red) and 500-kDa fluorescein-labeled dextran (green) were injected for analyzing kinetics of IgA deposition and visualizing blood vessel wall integrity, respectively. (a) IgA signals were detectable even after 1 min and accumulated up to 2 h in a focal and segmental manner in mice with IgA from gddY mice. In contrast, mice who received Balb/c IgA did not show a signal even after 2 h. (b)(c) Serial images of a glomerulus in mice with IgA from gddY mice showed that these IgA molecules accumulated on top of the initial aggregates along the glomerular capillaries. These aggregates were found in a focal and segmental manner but not in a diffuse and global manner.</p

A single injection of serum from gddY mice induced glomerular IgA deposition with activation of glomerular podocytes and endothelial cells.

<p>(a) Glomerular IgA deposits were found at 2 h in mice injected with serum from gddY mice but not from Balb/c mice. These fluorescent signals disappeared after 24 h in this single-injection model. (b) These deposits and clearance were confirmed using electron microscopy. Electron-dense deposits were mainly detected in paramesangial lesions. (c) Some glomeruli showed subendothelial and subepithelial deposits (*) with arcade formation in glomerular endothelial cells (**) and effacement and actin aggregation in podocytes (***) 2 h after the injection.</p

Kinetics of injected fluorescently labeled IgA in a fluorescence molecular tomography system.

<p>A fluorescence molecular tomography system (FMT) is capable of resolving size and concentration of fluorochromes in deep tissue <i>in vivo</i>. Fluorescein-labeled IgA samples from gddY and Balb/c mice were injected into nude mice and monitored from 10 min to 24 h postinjection by FMT. After 2 h, IgA signals in the liver and bladder were found in a similar manner in both the groups of nude mice. However, IgA signals in the kidneys clearly differed between them. Mice injected with gddY IgA showed strong signals in the kidneys, with a peak at 4 h.</p

Naturally Occurring Structural Isomers in Serum IgA1 <i>O</i>-Glycosylation

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of <i>O</i>-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr²²⁵, Thr²²⁸, Ser²³⁰, Ser²³² and Thr²³⁶ have been identified as the predominant sites of <i>O</i>-glycan attachment. The range and distribution of <i>O</i>-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 <i>O</i>-glycan heterogeneity by use of high resolution LC–MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 <i>O</i>-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the <i>O</i>-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable <i>O</i>-glycan chains attached to Ser²³⁰, Thr²³³ or Thr²³⁶ produce the predominant positional isomers, including <i>O</i>-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 <i>O</i>-glycoforms, define the single-site heterogeneity for all <i>O</i>-glycan sites in a single sample, and have implications for defining epitopes based on clustered <i>O</i>-glycan variability

Comparison between groups A and B before and after tonsillectomy.

<p>(A) The rate of change in hematuria was <1 in group A and was significantly lower than that in group B. (B) Tonsillar TLR9 expression in group A was significantly higher than that in group B. (C) The rate of change in proteinuria before and after tonsillectomy was not significantly different between groups A and B. (D) The rate of change in serum Cr before and after tonsillectomy was not significantly different between groups A and B.</p

Serum galactose-deficient-IgA1 and IgG autoantibodies correlate in patients with IgA nephropathy

<div><p>IgA nephropathy is an autoimmune disease characterized by IgA1-containing glomerular immune deposits. We previously proposed a multi-hit pathogenesis model in which patients with IgA nephropathy have elevated levels of circulatory IgA1 with some <i>O</i>-glycans deficient in galactose (Gd-IgA1, autoantigen). Gd-IgA1 is recognized by anti-glycan IgG and/or IgA autoantibodies, resulting in formation of pathogenic immune complexes. Some of these immune complexes deposit in the kidney, activate mesangial cells, and incite glomerular injury leading to clinical presentation of IgA nephropathy. Several studies have demonstrated that elevated circulatory levels of either Gd-IgA1 or the corresponding autoantibodies predict progressive loss of renal clearance function. In this study we assessed a possible association between serum levels of Gd-IgA1 and IgG or IgA autoantibodies specific for Gd-IgA1 in serum samples from 135 patients with biopsy-proven IgA nephropathy, 76 patients with other renal diseases, and 106 healthy controls. Our analyses revealed a correlation between the concentrations of the autoantigen and the corresponding IgG autoantibodies in sera of patients with IgA nephropathy, but not of disease or healthy controls. Moreover, our data suggest that IgG is the predominant isotype of Gd-IgA1-specific autoantibodies in IgA nephropathy. This work highlights the importance of both initial hits in the pathogenesis of IgA nephropathy.</p></div

Profiles of patients with immunoglobulin A nephropathy (IgAN) patients just before tonsillectomy.

<p>Data presented as Mean ± SD or.</p><p>*Median (interquartile range).</p>†<p>p<0.05 (compared with group A). BUN, blood urea nitrogen; Cr, creatinine; GdIgA1, galactose-deficient IgA1; HPF, high power field; IgA, immunoglobulin A.</p

Comparison between groups B1 and B2 before and after the first steroid pulse therapy session.

<p>(A) An improvement in hematuria was observed in group B1, but not in group B2. The rate of change in hematuria in group B1 was significantly lower than that in group B2. (B) The rate of change in proteinuria before and after the first steroid pulse therapy session was not significantly different between groups B1 and B2. (C) The rate of change in serum creatinine before and after the first steroid pulse therapy session was not significantly different between groups B1 and B2.</p

Serum levels of Gd-IgA1-specific IgG in IgAN patients with high-Gd-IgA1 or normal-Gd-IgA1 in comparison to CKD and healthy controls.

<p>We divided IgAN patients into two subgroups: patients with serum levels of Gd-IgA1 ≥ the 90^th percentile for healthy controls (high-Gd-IgA1 group; n = 56) and patients with levels Gd-IgA1 < the 90^th percentile for healthy controls (normal-Gd-IgA1 group; n = 79). Although serum levels of Gd-IgA1-specific IgG were significantly higher in IgAN patients with high Gd-IgA1 levels (<i>vs.</i> CKD controls; *P<0.0001, <i>vs.</i> healthy controls; **P<0.0001), IgAN patients with normal Gd-IgA1 levels also had elevated Gd-IgA1-specific IgG (<i>vs.</i> CKD controls; *P<0.0001, <i>vs.</i> healthy controls; **P<0.0001).</p