Impact of treatment on tubulointerstitial inflammation and injury in (NZB × NZW)F1 mice.

<p>(A), Representative images of immunohistochemical staining of kidney sections from mice in the saline (n=13), Dex (n=13), and P-Dex (n=9) treatment groups are shown. Sections were stained with an anti-TLR9 antibody (brown) and counterstained with hematoxylin (blue). (B), Quantification of TLR9 staining is illustrated. (C), TLR9 transcript levels in the kidney were measured by quantitative RT-PCR. For the saline group, frozen kidneys were available for RNA extraction only from the 6 mice that survived until the 14-week time point. (D), Levels of LCN2 were measured in kidney lysates by ELISA. For the saline group, frozen kidneys were available for preparation of protein lysates only from the 6 mice that survived until the 14-week time point. Scale bars: 25 μm; the asterisk (*) indicates a statistically significant difference (<i>P</i> < 0.05) from the saline control group. </p

Impact of treatment on hypertension, splenomegaly and vasculitis in (NZB × NZW)F1 mice.

<p>(A), Mean arterial pressure was measured at pretreatment, 4-week, 8-week, 12 week time points via tail-cuff method. For the saline group, measurements were obtained only for the subset of mice surviving at each time point: 12 mice at 4-week time point; 11 mice at 8-week time point; 9 mice at 12-week time point (B), Spleen mass was determined at the time of sacrifice in each mouse. (C), A representative hematoxylin and eosin stained histological section illustrating a splenic vessel from each treatment group is provided. The arrow indicates perivascular fibrin deposits indicative of vasculitis. Scale bars: 50 μm; the asterisk (*) indicates a statistically significant difference (<i>P</i> < 0.05) from the saline control group. The double asterisk (**) indicates a statistically significant difference (<i>P</i> < 0.05) from the Dex group. The dagger (†) indicates a statistically significant difference (<i>P</i> < 0.05) from the pretreatment time point of the same treatment group. For saline and Dex treatments, n=13; for P-Dex treatment, n=9.</p

The effect of treatment on serum anti-dsDNA IgG and renal immune complexes.

<p>(A), Anti-dsDNA IgG levels for mice in saline (n=13), Dex (n=13), and P-Dex (n=9) treatment groups were determined via ELISA at pretreatment, 4-week, 8-week, and 12 week time points. For the saline group, serum was available for analysis only from the subset of mice surviving at each time point: 12 mice at 4-week time point; 11 mice at 8-week time point; 9 mice at 12-week time point (B), Levels of anti-dsDNA IgG of each subclass were determined via ELISA at the 12-week time point. For the saline group, serum was available for this analysis only from the 9 mice that survived to the 12-week time point (C), Representative sections of kidney from each treatment group are shown. Sections were stained for renal deposition of anti-dsDNA IgG via immunohistochemistry. (D), Quantification of immune complex staining is illustrated. Scale bars: 25 μm; The asterisk (*) indicates a statistically significant difference (<i>P</i> < 0.05) from the saline control group. The dagger (†) indicates a statistically significant difference (<i>P</i> < 0.05) from the pretreatment time point of the same treatment group. </p

Evaluation of treatment-induced side effects.

<p><b>At sacrifice, femurs were collected for endpoint analysis of bone quality</b>. (A), bone mineral density (B), bone volume/tissue volume and (C), trabecular number measurements in each treatment group are shown. (D), white blood cell counts and (E), total serum IgG levels were determined at pretreatment 4-week, 8-week and 12-week time points. For the saline group, measurements were obtained only for the subset of mice surviving at each time point: 12 mice at 4-week time point; 11 mice at 8-week time point; 9 mice at 12-week time point (F), Adrenal mass was determined at the time of sacrifice in each mouse. The asterisk (*) indicates a statistically significant difference (<i>P</i> < 0.05) from the saline control group. The double asterisk (**) indicates a statistically significant difference (<i>P</i> < 0.05) from the Dex group. The dagger (†) indicates a statistically significant difference (<i>P</i> < 0.05) from the pretreatment time point of the same treatment group. For saline and Dex treatments, n=13; for P-Dex treatment, n=9.</p

P-Dex ameliorates albuminuria, extends lifespan and attenuates development of severe nephritis and tubulointerstitial disease in (NZB × NZW)F1 females.

<p>(A), Albuminuria data for mice in saline (n=13), Dex (n=13), and P-Dex (n=9) treatment groups is illustrated at the pretreatment (PT) and 14-week time points. The incidence of albuminuria at the 14-week time point for each group is shown (in %) in upper right corner of each sub-section. For mice in the saline group that did not survive to the 14-week time point (n=7), the albuminuria reading shown is the last recorded value. (B), A Kaplan-Meier survival curve for each treatment group is shown. (C), The fraction of mice in each treatment group with mild, moderate and severe renal disease is shown. (D), A PAS stained histological section illustrating representative glomeruli from each treatment group are provided. Scale bars: 20 μm. (E), A representative PAS stained histological section illustrating the tubulointerstitium from each treatment group is provided. Scale bars: 40 μm. The asterisk (*) indicates a statistically significant difference (<i>P</i> < 0.05) from the saline control group. </p

Impact of treatment on renal macrophage infiltration in (NZB × NZW)F1 mice.

<p>(A), Representative confocal images of immunohistochemical staining of kidney sections from mice in the saline (n=13), Dex (n=13), and P-Dex (n=9) treatment groups are shown. Sections were stained with an anti-Iba1 antibody (red) and DAPI (blue). Negative control (no Iba1 antibody) and merged images are shown. (B), Quantification of Iba1 staining is illustrated. Scale bars: 50 μm; the asterisk (*) indicates a statistically significant difference (<i>P</i> < 0.05) from the saline control group. </p