Reversal of Lupus Nephritis by SAP Treatment Figure 5. pSAP administration in the late stage of SLE disease ameliorates ALD-DNA-induced lupus nephritis. BALB/c mice were immunized subcutaneously with ALD-DNA (50 mg/mouse) or PBS for total 3 times in 4 weeks. Mice were administrated intramuscularly with pSAP (100 mg/mice) from week 8 after initial immunization (when sever lupus nephritis has been established) and injected every 2 weeks for total 4 times. (A) The dynamics of SAP level in serum of lupus mice injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) were determined by ELISA assay every 2 weeks. (B) The dynamics of circulating DNA level in serum of lupus mice injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) were determined by ELISA assay every 2 weeks. (C) The ratios of SAP to DNA in SLE murine model injected with pSAP (ALD- DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) at week 12 after initial immunization. Data are means 6 SD from 10 mice in each group. ***, P,0.001. (D) Serum anti-dsDNA IgG levels were measured by ELISA assay every 2 weeks. (E) Anti-dsDNA IgG antibody titers in serum of pSAP-treated lupus mice (ALD-DNA plus pSAP) or pcDNA3-treated lupus mice (ALD-DNA plus pcDNA3) were detected by ELISA assay at week 14 after the initial ALD-DNA immunization. n = 10. (F) Urine protein levels of the mice were assessed by BCA Protein Assay Kit (Thermo Fisher Scientific) every 2 weeks. n = 10. (G) The deposition of IgG-containing IC in glomeruli at week 12 after initial immunization. Imagines (6200) are representative of at least 10 mice in each group. (H) Mean glomerular fluorescence intensity (arbitrary units) was determined for IgG in ALD-DNA-immunized lupus mice and control mice at week 12 after initial immunization. n = 10. NS, not significant. (I) 12 weeks after initial immunization, nephritic pathological changes were shown by H&E staining of renal tissues surgical resected from the mice. Imagines (6200) are representative of at least 10 mice in each group. (J) The kidney score was assessed using paraffin sections stained with H&E. n = 10. ***, P,0.001. doi:10.1371/journal.pone.0022659.g005 Figure 6. pSAP administration in the late stage of SLE disease reduces inflammation in kidneys of mice. BALB/c mice were immunized subcutaneously with ALD-DNA (50 mg/mouse) for total 3 times in 4 weeks. Mice were administrated intramuscularly with pSAP (100 mg/mice) from week 8 after initial immunization and injected every 2 weeks for total 4 times. (A) 12 weeks after initial immunization, the infiltration of leukocyte populations in kidneys of treated mice were assessed by flow cytometry. Decreased CD45+ cells, CD19+ cells, CD4+ cells, and F4/80+ cells were found in kidneys of pSAP-treated lupus mice. Data are representative of results obtained in three independent experiments. n = 10. (B) At week 12, the kidney tissues were collected and homogenized, the expression of TNF-a, IL-1b, IL-6, IL-10, IL-12, and MCP-1 were determined by ELISA assay. Data are means 6 SD of three independent experiments. n = 8. ** P,0.01, *** P,0.001. (C) At week 12 after initial immunization, expression of TNF-a, IL-1b, IL-6, IL-10, IL-12, and MCP-1 in serum of the mice were determined by ELISA assay. Data are means 6 SD of three independent experiments. n = 8. * P,0.05, ** P,0.01, *** P,0.001. doi:10.1371/journal.pone.0022659.g006 PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22659