A dexamethasone prodrug reduces the renal macrophage response and provides enhanced resolution of established murine lupus nephritis

We evaluated the ability of a macromolecular prodrug of dexamethasone (P-Dex) to treat lupus nephritis in (NZB × NZW)F1 mice. We also explored the mechanism underlying the anti-inflammatory effects of this prodrug. P-Dex eliminated albuminuria in most (NZB × NZW)F1 mice. Furthermore, P-Dex reduced the incidence of severe nephritis and extended lifespan in these mice. P-Dex treatment also prevented the development of lupus-associated hypertension and vasculitis. Although P-Dex did not reduce serum levels of anti-dsDNA antibodies or glomerular immune complexes, P-Dex reduced macrophage recruitment to the kidney and attenuated tubulointerstitial injury. In contrast to what was observed with free dexamethasone, P-Dex did not induce any deterioration of bone quality. However, P-Dex did lead to reduced peripheral white blood cell counts and adrenal gland atrophy. These results suggest that P-Dex is more effective and less toxic than free dexamethasone for the treatment of lupus nephritis in (NZB × NZW)F1 mice. Furthermore, the data suggest that P-Dex may treat nephritis by attenuating the renal inflammatory response to immune complexes, leading to decreased immune cell infiltration and diminished renal inflammation and injury

Anticitrullinated protein antibody (ACPA) in rheumatoid arthritis: influence of an interaction between HLA-DRB1 shared epitope and a deletion polymorphism in glutathione s-transferase in a cross-sectional study

Abstract Introduction A deletion polymorphism in glutathione S-transferase Mu-1 (GSTM1-null) has previously been implicated to play a role in rheumatoid arthritis (RA) risk and progression, although no prior investigations have examined its associations with anticitrullinated protein antibody (ACPA) positivity. The purpose of this study was to examine the associations of GSTM1-null with ACPA positivity in RA and to assess for evidence of interaction between GSTM1 and HLA-DRB1 shared epitope (SE). Methods Associations of GSTM1-null with ACPA positivity were examined separately in two RA cohorts, the Veterans Affairs Rheumatoid Arthritis (VARA) registry (n = 703) and the Study of New-Onset RA (SONORA; n = 610). Interactions were examined by calculating an attributable proportion (AP) due to interaction. Results A majority of patients in the VARA registry (76%) and SONORA (69%) were positive for ACPA with a similar frequency of GSTM1-null (53% and 52%, respectively) and HLA-DRB1 SE positivity (76% and 71%, respectively). The parameter of patients who had ever smoked was more common in the VARA registry (80%) than in SONORA (65%). GSTM1-null was significantly associated with ACPA positivity in the VARA registry (odds ratio (OR), 1.45; 95% confidence interval (CI), 1.02 to 2.05), but not in SONORA (OR, 1.00; 95% CI, 0.71 to 1.42). There were significant additive interactions between GSTM1 and HLA-DRB1 SE in the VARA registry (AP, 0.49; 95% CI, 0.21 to 0.77; P < 0.001) in ACPA positivity, an interaction replicated in SONORA (AP, 0.38; 95% CI, 0.00 to 0.76; P = 0.050). Conclusions This study is the first to show that the GSTM1-null genotype, a common genetic variant, exerts significant additive interaction with HLA-DRB1 SE on the risk of ACPA positivity in RA. Since GSTM1 has known antioxidant functions, these data suggest that oxidative stress may be important in the development of RA-specific autoimmunity in genetically susceptible individuals

A dexamethasone prodrug reduces the renal macrophage response and provides enhanced resolution of established murine lupus nephritis.

We evaluated the ability of a macromolecular prodrug of dexamethasone (P-Dex) to treat lupus nephritis in (NZB × NZW)F1 mice. We also explored the mechanism underlying the anti-inflammatory effects of this prodrug. P-Dex eliminated albuminuria in most (NZB × NZW)F1 mice. Furthermore, P-Dex reduced the incidence of severe nephritis and extended lifespan in these mice. P-Dex treatment also prevented the development of lupus-associated hypertension and vasculitis. Although P-Dex did not reduce serum levels of anti-dsDNA antibodies or glomerular immune complexes, P-Dex reduced macrophage recruitment to the kidney and attenuated tubulointerstitial injury. In contrast to what was observed with free dexamethasone, P-Dex did not induce any deterioration of bone quality. However, P-Dex did lead to reduced peripheral white blood cell counts and adrenal gland atrophy. These results suggest that P-Dex is more effective and less toxic than free dexamethasone for the treatment of lupus nephritis in (NZB × NZW)F1 mice. Furthermore, the data suggest that P-Dex may treat nephritis by attenuating the renal inflammatory response to immune complexes, leading to decreased immune cell infiltration and diminished renal inflammation and injury

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

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

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

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