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Figures
- Fig. 1.
NTN phenotypes in parental and congenic rat strains. (A) Percentage of glomeruli with crescent formation. (B) Macrophage infiltration quantified by the percentage of ED1+ cells per glomerular cross-section. (C) Measurement of proteinuria. ***P<0.001; *P<0.05 between LEW.WCrgn1,2 and LEW rats using oneway ANOVA with Newman-Keuls comparison test. WKY, LEW, LEW.WCrgn1, LEW.WCrgn2 rats, n=6; LEW.WCrgn1,2 rats, n=10, except for proteinuria, where n=9. (D) H&E staining (upper panels) and ED-1 (CD68) immunohistochemistry (lower panels) of glomeruli 10 days after injection with NTS. The H&E stain showed crescentic glomeruli in WKY rats and, for the first time, formation of glomerular crescents in a LEW-derived strain (LEW.WCrgn1,2), whereas LEW rats were resistant to crescent formation. ED-1 immunohistochemistry also showed a significant influx of glomerular macrophages in the LEW.WCrgn1,2 strain.
- Fig. 2.
Tnfα,Nos2 and Mmp12 expression from nephritic glomeruli. (A) Pro-inflammatory cytokine Tumour necrosis factor-α; (B) nitric oxide synthase-2; and (C) matrix metalloproteinase-12 expression measured in nephritic glomeruli 10 days after induction of NTN. Analyses carried out by qRT-PCR. ***P<0.001 between LEW.WCrgn1,2 and LEW rats using one-way ANOVA with Newman-Keuls comparison test. n=4 in all strains.
- Fig. 3.
EAG phenotypes and autoantibody responses in parental (WKY and LEW) and double congenic (LEW.WCrgn1,2) rat strains at day 28. (A) Percentage of glomeruli with crescents. (B) Macrophage infiltration quantified by the percentage of ED1+ cells per glomerular cross-section. (C) Measurement of proteinuria. (D) Serum circulating anti-GBM antibody levels, showing similar serological autoimmunity in all three strains. (E) Quantification of deposited anti-GBM antibody (graded 0 to 3+ intensity) in each rat strain assessed by direct immunofluorescence (IF) for anti-rat immunoglobulins. (A–E) Statistically significant differences in mean values between the LEW.WCrgn1,2 strain and LEW strain compared using a one-way ANOVA with Newman-Keuls comparison test. ns: non-significant. WKY and LEW rats, n=6; LEW.WCrgn1,2 rats, n=10. (F) Representative direct immunofluorescence images showing pattern and intensity of anti-GBM antibody deposition in glomeruli in each rat strain.
- Fig. 4.
Macrophage activation assays in BMDMs in parental (WKY and LEW) and double congenic (LEW.WCrgn1,2) rat strains. (A) Production of superoxide assessed by chemiluminescence over 90 minutes, following addition of PMA (1 μM) (n=3 in all strains). (B,C) Fc-receptor-mediated phagocytic activity of BMDMs assessed by incubating WKY, LEW and LEW.WCrgn1,2 BMDMs with beads opsonised with rabbit anti-BSA IgG, or unopsonised for 30 minutes. Cells were then fixed and beads in 100 BMDMs were counted (n=4 in all strains). *P<0.05 between LEW.WCrgn1,2 and LEW rats using one-way ANOVA with Newman-Keuls comparison test.
- Fig. 5.
Gene expression profiles in BMDMs in parental (WKY and LEW) and double congenic (LEW.WCrgn1,2) rat strains. (A) Pro-inflammatory cytokine Nos2; (B) anti-inflammatory cytokine Il10; (C) Lilrb3l; (D) Nov and (E) Arg1 expression in LPS-stimulated BMDMs (except Nov, for which BMDMs in basal state). Expression analyses were carried out by qRT-PCR. ***P<0.001; **P<0.01; P was non-significant (ns) between LEW.WCrgn1,2 and LEW rats using one-way ANOVA with Newman-Keuls comparison test. n=4 biological rats per strain with three technical replicates per rat. BMDMs were untreated (basal) or stimulated with 100 ng LPS for 4 hours.
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