1. Volume 86, Issue 4, Pages 780-789 (October 2014)
The fungal lactone oxacyclododecindione is a potential new therapeutic substance in the treatment of lupus-associated kidney disease 
Jenny Henke, Gerhard Erkel, Christoph Brochhausen, Hartmut Kleinert, Andreas Schwarting, Julia Menke, Andrea Pautz 
Kidney International 
Volume 86, Issue 4, Pages (October 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Analysis of the expression of proinflammatory mediators in the kidney of MRL-Faslpr mice. MRL-Faslpr mice received intraperitoneal injections of 1mg/kg oxacyclododecindione (Oxa), 5mg/kg dexamethasone (Dex), or phosphate-buffered saline (PBS)/10% EtOH as solvent control every other day for 5 weeks. Each treatment group contained seven mice. (a) The data show the relative interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ mRNA expression of PBS-, Oxa-, or Dex-treated mice evaluated by quantitative real-time RT-PCR (qRT-PCR). (b) Western blot analyses were performed with specific anti-TNF-α and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and protein extracts of PBS-, Oxa-, and Dex-treated mice. The blot is representative of two other blots showing similar results. The quantitative analysis of TNF-α protein expression was performed with QuantityOne software (Bio-Rad). (c) The data show the relative S100A8 and SPP1 mRNA expression of PBS-, Oxa-, or Dex-treated mice determined by qRT-PCR. (d) The data show the relative S100A8 protein expression of PBS- and Oxa-treated mice measured by enzyme-linked immunosorbent assay experiments. All data represent the relative mRNA or protein expression (±s.e.m.) compared with PBS/10% EtOH-treated mice (***P<0.001; **P<0.01; *P<0.05 vs. PBS/10% EtOH-treated mice).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Analysis of chemokine and cytokine expression in the kidney of MRL-Faslpr mice. For the ‘mouse cytokine array’ proteome profile analyses, proteins were isolated from kidneys of phosphate-buffered saline (PBS)/10% EtOH- and oxacyclododecindione (Oxa)-treated animals (seven animals per group) 5 weeks after the first Oxa application. (a) The data show quantitative protein analyses of significantly regulated chemokines. (b) The data show protein analyses of significantly regulated cytokines. All data (means±s.e.m.) show the relative protein expression compared with PBS/10% EtOH-treated mice (***P<0.001 vs. PBS/10% EtOH-treated mice).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
Severity of disease and intrarenal infiltrates are ameliorated in MRL-Faslpr mice following oxacyclododecindione (Oxa) treatment. (a) Kidney histopathology of MRL-Faslpr following treatment with Oxa compared with phosphate-buffered saline (PBS)/10% EtOH-treated control MRL-Faslpr mice. We assessed the following: glomerular pathology by scoring each glomerulus and periglomerular area on a semiquantitative scale evaluating the periglomerular infiltrates, glomerular proliferative changes, and glomerular hypercellularity; interstitial/tubular pathology by evaluating the number of infiltrates and damaged tubules; and perivascular cell accumulation by scoring the number of cell layers surrounding most vessel walls. Representative photomicrographs of the kidney (periodic acid–Schiff reagent). Magnification × 20, inset magnification × 40. (b) Number of intrarenal CD4+ (T cells), CD68+ (expressed on monocytes and macrophages), and CD20+ (B cells) cells in Oxa-treated compared with PBS/10% EtOH-treated MRL-Faslpr mice. Data are given as mean+s.e.m. (*P<0.05; **P<0.01).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Oxacyclododecindione (Oxa) treatment results in reduced expression of proinflammatory cytokines in the kidney of MRL-Faslpr mice accompanied by an increase in intrarenal immunoregulatory CD4+ fork head box P3 (FoxP3+) cells. (a) Comparison of interferon (IFN)-γ, interleukin (IL)-17, and FoxP3 expression in CD4+ intrarenal cells in MRL-Faslpr mice following Oxa treatment compared with phosphate-buffered saline (PBS)/10% EtOH controls, as evaluated by flow cytometry. Data are given as mean+s.e.m. (**P<0.01). Representative fluorescence-activated cell sorting plots are shown. (b) Flow cytometric analysis of the frequency of intrarenal CD4+ leukocytes expressing IL-6 or FoxP3 demonstrated in combination with immunostaining results for IL-6+- or FoxP3+-infiltrating cells into the kidney of MRL-Faslpr mice following Oxa treatment compared with PBS/10% EtOH-treated controls. Representative photomicrographs of the kidney are shown; magnification × 40. Arrows indicate intrarenal FoxP3+ cells. Data are given as mean+s.e.m. (*P<0.05; **P<0.01).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Effect of oxacyclododecindione (Oxa) treatment on proteinuria. (a) Semiquantitative determination of proteinuria with test strips during the treatment period. Results showed a delayed increase in proteinuria under Oxa treatment (n=8) compared with the phosphate-buffered saline (PBS)/10% EtOH control (n=8). (b) Enzyme-linked immunosorbent assay experiments demonstrated decreased albumin/creatinine ratios in urinary samples of Oxa-treated mice compared with PBS/10% EtOH treatment. All data are given as mean±s.e.m. (***P<0.001; **P<0.01 vs. PBS/10% EtOH-treated mice).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of oxacyclododecindione (Oxa) treatment on antibody production and complement activity. (a) In kidney sections we detected fewer Immunoglobulin G (IgG) deposits in the glomeruli under Oxa treatment. Data were obtained by scoring 20 glomeruli for each probe as either positive or negative in the fluorescence staining (n=16). (b) Using enzyme-linked immunosorbent assay (ELISA) we detected the presence of bound IgG subclasses. In Oxa-treated mice, we detected significantly less total IgG, IgG2a, IgG2b, and IgG3. (c) In sera (n=16) we detected via double-stranded DNA (dsDNA) ELISA significantly less circulating antibodies under Oxa treatment. (d) In fluorescence staining, Oxa treatment displays a trend toward an improvement in C3 deposition in the glomeruli compared with phosphate-buffered saline (PBS)/10% EtOH control. Data were obtained as described in (a) (n=16). (e) In addition, in C3 ELISA assays we detected no significant change in C3 complement levels in the sera of Oxa-treated mice compared with untreated Balb/c control mice. All data are given as mean±s.e.m. (**P<0.01; *P<0.05 vs. PBS/10% EtOH-treated mice). NS, not significant.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Effect of oxacyclododecindione (Oxa) on kidney fibrosis. (a) The Elastica van Gieson staining in the kidney showed significantly less collagen deposition compared with the phosphate-buffered saline (PBS)/10% EtOH-treated group. Data are given as mean+s.e.m. (*P<0.05 vs. PBS/10% EtOH-treated mice).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions