Increased Goodpasture antigen-binding protein expression induces type IV collagen disorganization and deposit of immunoglobulin A in glomerular basement membrane

Abstract

Increased expression of Goodpasture antigen-binding protein (GPBP), a protein that binds and phosphorylates basement membrane collagen, has been associated with immune complex-mediated pathogenesis. However, recent reports have questioned this biological function and proposed that GPBP serves as a cytosolic ceramide transporter (CERT(L)). Thus, the role of GPBP in vivo remains unknown. New Zealand White (NZW) mice are considered healthy animals although they convey a genetic predisposition for immune complex-mediated glomerulonephritis. Here we show that NZW mice developed age-dependent lupus-prone autoimmune response and immune complex-mediated glomerulonephritis characterized by elevated GPBP, glomerular basement membrane (GBM) collagen disorganization and expansion, and deposits of IgA on disrupted GBM. Transgenic overexpression of human GPBP (hGPBP) in non-lupus-prone mice triggered similar glomerular abnormalities including deposits of IgA on a capillary GBM that underwent dissociation, in the absence of an evident autoimmune response. We provide in vivo evidence that GPBP regulates GBM collagen organization and its elevated expression causes dissociation and subsequent accumulation of IgA on the GBM. Finally, we describe a previously unrecognized pathogenic mechanism that may be relevant in human primary immune complex-mediated glomerulonephritis.

Figures

Figure 1

NZW mice undergo age-dependent glomerulonephritis. A: Mason trichromic staining of glomerular sections representing the indicated mice and glomerulonephritis (n = 60). In this and following figures, young is <6 months and aged is >7 months. B: Glomerular ultrastructural pathological findings in aged NZW mice (n = 5). a and b: Electron-dense homogenous or vacuolated materials aligned on lamina densa of (para)mesangial GBM are denoted by arrows in b, which represents the boxed region in a analyzed at higher magnification. c: Electron-dense homogeneous material filling mesangium and paramesangium. d and e: Electron-dense vacuolated material occupying paramesangium (d) and capillary space (e). f: Electron-dense material associated with lamina rara of (para)mesangial GBM. In d–f, arrows and asterisks denote electron-dense homogenous and vacuolated material, respectively. Illustrated are selected images from NZW mice of 5, 10, 13, 9, and 9 months (left to right) (A) and 8 to 9 months (B). Scale bars: 1 μm (a, d, and f); 0.5 μm (b); 2 μm (c); 7 μm (e). Original magnifications, ×400 (A).

Figure 2

Glomerular type IV collagen disorganization induces capillary GBM dissociation in NZW mouse glomerulonephritis. Glomerular sections representing the indicated mice and glomerulonephritis were analyzed by standard indirect immunofluorescence (n = 15) (A) or by confocal microscopy (n = 7) (B). In this and following immunofluorescence studies, labels at left of the composites indicate stained protein(s). Graphs represent fluorescence intensity distributions in the regions indicated by arrows in merged images. C: Electron microscopy analysis showing capillary GBM dissociation in aged NZW mice (n = 5). Inset in left appears magnified in right. Arrows denote endothelial (left) and epithelial (right) components of capillary GBM. Illustrated are selected images from NZW mice of: 5.5, 9, and 8.5 months (left to right) (A and B) and 8.5 months (C). Scale bars: 1 μm (left); 0.5 μm (right). Original magnifications, ×400 (A).

Figure 3

GPBP is overexpressed in NZW mouse glomerulonephritis. A: Glomerular GPBP mRNA levels in individual NZW mice at the indicated range of age (months) were determined by real-time RT-PCR on laser-dissected glomerular microsections. The relative expression of GPBP in individual animals are represented by dots or circles and was calculated using 5-month-old mice as reference (GPBP expression = 1). Bars indicate the mean value within a series (P = 0.0143). Shown is comparative immunofluorescence analysis of representative glomerular sections of NZW mice of the indicated age (months) (n = 7). B and C: Confocal microscopy analysis of glomerular sections (left) or magnified portions thereof (right) representing the indicated mice and glomerulonephritis (n = 9). Graphs represent fluorescence intensity distributions in the regions indicated by arrows in merged images. Illustrated are selected images from NZW mice of 5.5 months (B) and 9 and 8.5 months (left to right) (C). Scale bars = 25 μm. Original magnifications, ×400 (A).

Figure 4

NZW mice develop age-dependent autoimmune response and glomerular immune complex deposits. A: The titration units (TUs) of the indicated circulating autoantibodies in individual mice of indicated age (months) and strain are represented by dots or circles. The bar indicates the mean value in each series (*P = 0.0031, **P < 0.0001). B: Glomerular sections representing the indicated mice and glomerulonephritis were analyzed by standard direct immunofluorescence (n = 12). C: Confocal microscopy analysis on glomerular lesions representing aged NZW mice undergoing the indicated glomerulonephritis (n = 8). Graphs represent fluorescence intensity distributions in the regions indicated by arrows in merged images. Illustrated are selected images from NZW mice of 5.5, 9, 8, 5.5, 10, 8, 5.5, 13, and 13 months (left to right and top to bottom) (B); 13 and 9 months (left to right) (C). Original magnifications, ×400 (B).

Figure 5

Transgenic hGPBP expression induces glomerulonephritis in non-lupus-prone mice. A: Similar amounts of kidney homogenates from the indicated mice were subjected to immunoprecipitation and Western blot analysis of immunoprecipitates using the indicated antibodies. Bars at left of composite denote the positions of 97- and 66-kDa protein standards (n = 30). B: Mason trichromic staining of glomerular sections representing the indicated mice and glomerulonephritis (n = 38). C: Ultrastructural pathological findings in Tg-hGPBP mice (n = 16). a: Arrows indicate endothelial (thin) and epithelial (thick) components in dissociated capillary GBM and box denotes a buttonhole-like disruption within the epithelial component. b: A horsetail-like unwinding of the lamina densa in capillary GBM. c: Electron-dense material within a disruption of the lamina densa. Arrows denote dissociated GBM components. d: Electron-dense homogenous or vacuolated materials aligned on the lamina densa of (para)mesangial GBM. e: Electron-dense material filling mesangium and paramesangium. f: Vacuolated material protruding in capillary wall. Illustrated are selected images from mice 8-month F4 (A); 8-month F4 (control and mesangial) and 12-month F3 (nodular) (B); and 8- (a–c) and 12-month (d–f) F3 (C). Scale bars: 0.2 μm (a); 0.5 μm (b, c, and f); 1 μm (d); 2 μm (e). Original magnifications, ×400 (B).

Figure 6

Transgenic hGPBP expression induces immune complex-mediated glomerulonephritis in non-lupus-prone mice. A: Comparative confocal microscopy analysis of glomerular sections (left) or magnified portions thereof (right) representing the indicated mice and glomerulonephritis. B: Representative glomerular sections from the indicated mice were analyzed by standard direct immunofluorescence. C: Confocal microscopy on selected glomerular sections of the indicated mice and glomerulonephritis. D: Representative IgA deposits in the indicated lesion of Tg-hGPBP mice. In C and D, IgA appears in green, α1-α2(IV) in blue, and α3(IV) in red. A–D: n = 38. Graphs represent fluorescence intensity distributions in the regions indicated by arrows in merged images. Illustrated are images taken from mice 8-month F4 (control and nodular) and 12-month F3 (mesangial) (A); 8-month F5 (B); 10-month F4, 14-month F3, and 7-month F5 (control, mesangial, and nodular, respectively) (C); and 7-month F5 (D). Scale bars = 25 μm. Original magnifications, ×400 (B).

Figure 7

Model for type IV collagen distribution in normal glomeruli and in GPBP-mediated glomerulonephritis. The clover-like structure represents a section of a renal glomerulus in which type IV collagen is depicted in an unaffected capillary (normal) or in capillary undergoing the indicated GPBP-mediated glomerulonephritis. In black, the α3.α4.α5(IV) collagen (epithelial GBM component). In red, membrane-organized α1.α1.α2(IV) collagen (endothelial GBM component). In blue, mesh-organized α1.α1.α2(IV) collagen (mesangial component). In white, virtual spaces resulting from defective fusion of epithelial and endothelial GBM components that support GPBP accumulation and IgA deposits on orphan epithelial wall. In mesangial glomerulonephritis, the mesangial component progressively replaces the endothelial component whereas in nodular lesions the endothelial component progressively substitutes mesangial component. In advanced glomerulonephritis, a component can entirely substitute the other. In both NZW and Tg-hGPBP mice, there were animals, glomeruli, and even capillaries displaying both types of lesion.