Table 1.
Screening of 17 autoantigens associated with LN using a biotinylated human autoantigen library.
Fig 1.
Immunoprecipitation of RRP8 and TNP1 with sera from patients with rheumatic diseases.
Reprisentative photographs are shown. (A) RRP8 (50.7 kDa) (B) TNP1 (GST-tagged, 32.4 kDa). LN; active lupus nephritis, SLE; systemic lupus erythematosus without active lupus nephritis, DM; dermatomyositis, SSc; systemic sclerosis, cont; healthy control.
Fig 2.
Double immunofluorescence of RRP8 or TNP1 and IgG or C3 in renal sections from patients with LN.
Formalin-fixed, paraffin-embedded sections of biopsy or autopsy specimens from LN patients were processed for double immunofluorescence staining. Representative photographs are shown. The specimens were stained with Alexa 546-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with FITC-conjugated anti-IgG or anti-C3 antibodies (green). (A) The same glomeruli were stained with periodic acid-Schiff. (B) Co-presence of RRP8 and C3 was detected along the sub-epithelial area of the glomeruli showing a dotted pattern (arrowhead) in LN1. Also in Autopsy1, both RRP8 and IgG signals were detected in the mesangial area as well as in the sub-epithelial area (arrowhead). (C) Co-presence of TNP1 and IgG was revealed along the basement membrane showing a linear pattern in LN2, as was the case for TNP1 and C3 in Autopsy2. The bold and strong autofluorescence in each of the panels is mainly due to red blood cells.
Table 2.
Deposition of RRP8 and/or TNP1 in renal sections obtained by biopsy and autopsy from LN patients.
Fig 3.
Serum levels of anti-RRP8 (A) and anti-TNP1 (B) antibodies in patients with rheumatic diseases.
Cut-off values for positivity (9.1 units for anti-RRP8 antibody and 10.6 units for anti-TNP1 antibody) are indicated by the dotted lines. LN, lupus nephrits; SLE, systemic lupus erythematosus without LN; DM, dermatomyositis; PM, polymyositis; SSc, systemic sclerosis; MCTD, mixed connective tissue disease; RA, rheumatoid arthritis; SS, Sjögren syndrome; BD, Behçet’s disease; AAV, anti-neutrophil cytoplasmic antibody-associated vasculitis.
Fig 4.
Relationship between anti-RRP8 or anti-TNP1 antibody and clinical findings in SLE patients.
(A) anti-RRP8 antibody and anti-dsDNA antibodies, (B) Anti-RRP8 antibody and SLEDAI, (C) anti-TNP1 antibody and anti-dsDNA antibodies, (D) anti-TNP1 antibody and SLEDAI. SLEDAI, systemic lupus erythematosus disease activity index. Correlations were expressed as Spearman rank correlation coefficients. P values of <0.05 were considered significant.
Table 3.
Correlation between anti-RRP8 or anti-TNP1 antibody level and clinical findings in SLE patients.
Fig 5.
Changes in antibody levels and clinical findings in patients with lupus nephritis.
▲ LN1, ♦ LN2, ● LN3, ◊ LN4, ○ LN5, □ LN6, ■ LN7. (A) Anti-RRP8 antibody, (B) anti-TNP1 antibody, (C) SLEDAI, (D) anti-dsDNA antibody, (E) C3, and (F) C4. The active and inactive phases of LN were defined as 4≤ and 0 of the renal SLEDAI score, respectively.
Fig 6.
Measurement of proteinuria (A) and anti-RRP8 (B), anti-TNP1 (C) or anti-dsDNA (D) antibodies in murine serum samples.
Proteinuria was assessed before sacrifice. The urine diluted 1:10 was examined and graded with a score of 0 (<30mg/dL); 1 (30-99mg/dL); 2 (100-299mg/dL); or 3 (300-999mg/dL). Serum samples were assayed by ELISA using purified human RRP8 or TNP1 protein. RRP8- or TNP1-mice; C57BL/6 mice injected repeatedly with RRP8 or TNP1, respectively. normal; normal C57BL/6 mice.
Fig 7.
Double immunofluorescence of RRP8 or TNP1 and IgG in renal sections from RRP8-injected or TNP1-injected mice.
Cryostat kidney sections from mice were analyzed by double immunofluorescence staining with a combination of anti-human RRP8 or anti-human TNP1 antibody and anti-mouse IgG antibody. The specimens were stained with Alexa 568-conjugated anti-RRP8 or anti-TNP1 antibodies (red) and with Alexa 488-conjugated anti-murine IgG antibody (green). (A) Photomicrograph of representative glomeruli of RRP8- and TNP1-injected mice (periodic acid-Schiff staining). Both manifested the slight enlargement of glomeruli with thickening of capillary wall (arrows) as well as the endocapillary proliferation with leukocytes (arrow head). (B) RRP8 signals coexisted with IgG signals in the sub-endothelial (arrows) and sub-epithelial (arrowhead) regions, exhibiting a granular pattern. On the other hand, TNP1 signals coexisted with IgG signals in the sub-epithelial region (arrows).
Fig 8.
Immunoelectron microscopy of kidney sections from RRP8-injected (A and B) or TNP1-injected (C and D) mice.
Photographs show the characteristic dense deposit in the subendothelial part especially at around the border of pericapillary and perimesangial areas. Panel B (anti-RRP8) and Panel D (anti-TNP1) show the high-power view of the deposition which manifests the adjacent (within 30nm) of RRP8 or TNP1 represented by 10-nm-gold particles and IgG represented by 5-nm-gold particles (circle area). *, electron-dense deposit; PD, podocyte; GBM, glomerular basement membrane.
Fig 9.
Analysis of infiltrating cells (A) and cytokine expression (B) in the kidneys of RRP8-injected and TNP1-injected mice.
(A) Quantitative analysis of mononuclear cells in glomerular and tubulointerstitial areas was performed in control, RRP8-injected and TNP1-injected mice. Values are the mean and SD of infiltrating and parenchymal cells (glomerular, cell counts in 20 random glomeruli; and tubulointerstitial, 10 random fields at x400 magnification). (B) qRT-PCR analysis was performed on total RNA prepared from kidneys of all mice of each group. Results are calculated as a ratio of cytokine expression to the expression of HPRT1. * no positive signal with 45 cycles.