Background/Purpose:

Lupus nephritis (LN) is a serious manifestation of systemic lupus erythematosus (SLE) that adds substantial morbidity and mortality. Passive transfer of pre-formed nephritogenic antibodies to susceptible mice, or nephrotoxic serum nephritis (NTN), results in immune-mediated nephritis that closely models LN. We have previously shown that the BTK inhibitor BI-BTK-1 reverses the functional and structural kidney disease seen following induction of NTN. In the current study, we elucidated the effects of BI-BTK-1 on various immune cell populations to determine the mechanism of protection, as well as the pathways by which BI-BTK-1 reverses established nephritis. Furthermore, we assessed the effects of early and late withdrawal of treatment on the course of nephritis.  

Methods:

For induction of NTN, 11-week old 129 mice were immunized with rabbit IgG in CFA, and five days later passively transferred with nephrotoxic rabbit anti-mouse antibodies. To assess the effect of early (starting day 4) and late (starting day 8) treatment with BI-BTK-1 on immune cell infiltration and kidney pathology, we sacrificed BI-BTK-1 or control treated mice on days 7, 10, and 13 (n=5 per group) (Figure 1, “Treatment Delay” experiment). Kidneys were processed for flow cytometry and histology to assess the kinetics of different immune cells populations within the kidney over the course of nephritis. In a separate experiment, we began treating mice with BI-BTK-1 on day 4, and compared the effects of withdrawing treatment early (day 6) and late (day 9) on disease remission and flare (Figure 1, “Treatment Withdrawal” experiment).  

Results:

We found that early treatment with BI-BTK-1 appears to prevent the development of nephritis, and late treatment appears to reverse established disease, in murine NTN. Interestingly, early treatment withdrawal (day 6) resulted in increasing proteinuria comparable to sick control mice. Late withdrawal (day 9), however, provided significant protection against the development of proteinuria and kidney dysfunction (increased BUN) throughout the two week duration of the study (p<0.001 versus sick control, p<0.01 versus early withdrawal). In the treatment delay experiment, flow cytometric analysis revealed a significant reduction of kidney infiltrating M1 (inflammatory) macrophages by day 13 in both early and late treatment, whereas control treated mice exhibited decreased M2 macrophages. Both early and late treated mice had reduced kidney B220+ cells, whereas only mice treated early showed a reduction in neutrophils and CD4+ cells. Renal histology and RT-PCR results are pending. 

Conclusion:

Our study demonstrates the multiple cell types affected by treatment with BTK inhibition in LN. Furthermore, these results shed light upon the effects of withdrawing treatment, which may eventually have important implication for treatment decisions in human disease.  

Figure 1. Timeline