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. 2012 Jun 22;31(15):3363–3374. doi: 10.1038/emboj.2012.169

Figure 6.

Figure 6

Sykki mice produce anti-insulin autoantibodies and develop age-related glomerulonephritis. (A) Permeabilized Hep-2 cells were incubated with Sykctrl or Sykki mouse sera, stained with anti-IgM-FITC or anti-IgG-FITC and analysed for Hep-2 reactivity via epifluorescence microscopy (Zeiss Axioscope, × 10, scale bar=30 μm). (A, left image and middle panel) Representative images indicating prominent cytoplasmic Hep-2 reactivity (IgM depicted) of ki sera. (A, right) Numbers below pie charts indicate Hep-2 reactive sera (IgM+ and IgG+)/total number of animals analysed. (B) Kidney cryosections of ctrl and ki mice (8 weeks) were stained with anti-IgM-FITC (upper panel), anti-IgG-FITC (lower panel) and anti-C3 to assess glomerular immune complex and complement deposition (× 20/ × 63, scale bar=100 μm). (C) Periodic acid Schiff staining of kidney paraffin sections from Sykctrl (i, ii) and Sykki (iii, iv) mice at the age of 60 weeks (× 63, scale bar=50 μm) showed significant Sykki glomerular mesangial hypercellularity (*), mesangial matrix accumulation (arrows) and basement membrane increase (**). (D) Quantification of glomerular diameter and (E) proteinuria in Sykctrl and Sykki mice. (F) dsDNA-specific IgM titres were comparable between Sykctrl and Sykki mice, while (G) insulin-specific IgM titres were elevated in ki mice (8–12 weeks). (H) Basal blood glucose levels of 14-week-old Sykctrl and Sykki mice were measured after 16 h of chow starvation and (I) 0, 15, 30, 60 and 120 min after intraperitoneal injection of D-glucose (2 g/kg). (J) To test whether the anti-insulin-specific IgM titres in Sykki mice were aggravated upon TLR-4 stimulation of MZ B cells, sorted B220+CD19+ splenic B cells were incubated with LPS for 36 h and analysed for spot enlargement upon stimulation. *P<0.01, **P<0.01, ***P<0.001; n=18/19 (A), 3 per age (B, C), 4 (D, ∼100 glomeruli total), 12 (E), 8 (F), 18 (G), 8 (H, I) and 3 (J) per group; error bars indicate means±s.e.m. BSA, bovine serum albumin; ctrl, control; Ig, immunoglobulin; LPS, lipopolysaccharide; ns, not significant.