The kinetics of glomerular deposition of nephritogenic IgA

Abstract

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.

Figure 1. A single injection of serum from gddY mice induced glomerular IgA deposition with activation of glomerular podocytes and endothelial cells.

(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.

Figure 2. Kinetics of injected fluorescently labeled IgA in a fluorescence molecular tomography system.

A fluorescence molecular tomography system (FMT) is capable of resolving size and concentration of fluorochromes in deep tissue in vivo. 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.

Figure 3. IgA from gddY mice is deposited along the glomerular capillary wall in a focal and segmental manner.

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.

Figure 4. Glomerular IgA deposits in old gddY mice did not disappear after bone marrow transplantation (BMT) despite improvement in proteinuria.

BM cells from healthy Balb/c mice were transplanted into young (8 weeks) and old (20 weeks) gddY mice at an early stage of disease. Although proteinuria was present in the young and old recipients 12 weeks after BMT, fluorescence analysis still detected glomerular IgA deposition in old gddY recipients but not in young gddY recipients (left panels). Electron microscopy still detected paramesangial dense deposits showing fibrous and lattice structures in the old recipients (right panel).