Fig 1.
Renal morphology in different disease stages.
Typical micrographs from minimal, moderate, and severe nephritis. Periodic acid-Schiff (PAS) staining in the left panel show increasing changes in glomeruli and tubulointerstitium with disease progression: endocapillary proliferation, infiltration of immune cells, formation of wire loops and hyaline thrombi, crescents, atrophy and sclerosis. The middle panel demonstrate immune electron microscopy (IEM) with anti-IgG antibodies (6 nm gold) showing increasing amounts of IgG-deposits (arrows) in mesangium and later also in the capillary glomerular basement membrane. In addition, podocyte effacement was apparent in moderate and severe nephritis. Immunofluorescence (IF) in the right panel shows IgG staining in glomeruli for all three groups, and also staining in the tubulointerstitium in mice with moderate and severe nephritis.
Fig 2.
General disease features of lupus nephritis.
Mice with minimal nephritis (Min, n = 9), moderate nephritis (Mod, n = 11) and severe nephritis (Sev, n = 5) were compared according to different disease parameters. 20/25 mice had end-stage sera and urine available for ELISA and proteinuria evaluation: Min n = 5, Mod n = 10, Sev n = 5. (A) Distribution of activity and chronicity indices of renal tissue in the different groups, presented as mean with standard deviations. Mean activity index was highest in the severe nephritis group, which also had chronic changes. (B) Mean anti-dsDNA antibody titer with standard deviations. Mice with severe nephritis had elevated anti-dsDNA antibody titer compared to minimal nephritis (p = 0.023). (C) Mean anti-RNP activity concentrations (U/ml) with standard deviations. (D) Urinary protein concentrations (μg/μl) with standard deviations. (E) Renal cytokine mRNA levels, presented as fold change relative to the mice with minimal nephritis. IL-10 was significantly upregulated in mice with severe nephritis compared to minimal nephritis (p = 0.041).
Table 1.
Distribution of various lupus nephritis parameters within different disease stages.
Fig 3.
IgG and DNA colocalization immune electron microscopy.
Renal sections from mice with minimal nephritis (upper panel) and severe nephritis (lower panel) were incubated with anti-IgG (5 nm gold) and anti-DNA (10 nm gold). IgG were restricted to EDS, and colocalized with DNA. Both IgG and DNA appeared with highest density in severe nephritis.
Table 2.
Correlations between different variables in FcγRIIB-/-.yaa mice.
Fig 4.
Renal expression of TLRs and interferon stimulated genes.
Different disease stages were compared: Minimal nephritis (Min, n = 9), moderate nephritis (Mod, n = 11), and severe nephritis (Sev, n = 5). (A) IHC demonstrated renal TLR7 expression in tubular epithelial cells in mice with different stages of FcγRIIB-/-yaa nephritis. The perinuclear staining pattern of tubular epithelial cells resembled the staining pattern of infiltrating immune cells, as demonstrated for moderate nephritis (enlarged section). (B) Renal mRNA levels of TLR7, TLR8 and TLR9 from FcγRIIB-/-yaa mice with different stages of nephritis, presented as fold change relative to the minimal nephritis group. Expression levels of TLR7 and TLR9 were significantly upregulated in mice with moderate nephritis compared to mice with minimal nephritis (p = 0.006 and p = 0.027, respectively). In mice with severe nephritis, TLR7 was significantly upregulated compared to minimal nephritis (p = 0.017). (C) Renal mRNA expression levels of the interferon stimulated genes Eif2ak2, Ifit1 and Mx1 in FcγRIIB-/-yaa mice with different stages of nephritis, presented as fold change relative to the minimal nephritis group. Mx1 was significantly upregulated in moderate nephritis compared to mice with minimal nephritis (p = 0.008).
Fig 5.
Different disease stages were compared: minimal nephritis (Min, n = 9), moderate nephritis (Mod, n = 11), and severe nephritis (Sev, n = 5). DNase I expression was downregulated in mice with severe nephritis on transcriptional, protein end enzyme activity levels. (A) Fold change of renal DNase I mRNA levels in mice with moderate and severe lupus nephritis relative to the minimal nephritis group, showing a highly significant downregulation in mice with severe nephritis compared to minimal nephritis and moderate nephritis (p<0.001 and p = 0.002, respectively). (B) Immunohistochemistry demonstrated tubular staining of DNase I in mice with minimal nephritis and moderate nephritis, and weak staining in mice with severe nephritis. (C) Renal DNase I enzyme activity measured by single radial enzyme diffusion. The enzyme activity was significantly lower in mice with severe nephritis compared to the minimal nephritis and moderate nephritis (p = 0.001 and p<0.001, respectively). (D) DNase I gel zymography showing enzyme activity of two typical samples from each group of mice. The enzyme activity was lower in mice with severe nephritis compared to the minimal and moderate nephritis group. Recombinant DNase I was used as a control (left band).