Background Adrenomedullin (ADM) is a peptide comprising 52 amino acids which is renowned for its potent vasodilating effect. It has also been shown to confer an anti-inflammatory milieu while regulating the immune system. While our previous clinical study demonstrated a negative association between proteinuria and serum ADM levels in patients with lupus nephritis, the potential therapeutic effect of ADM in lupus nephritis has not been explored in an in vivo system.
Objectives To evaluate the effects of ADM in nephritis, proliferation, activation and apoptosis of splenic lymphocytes and autoantibody profile in MRL/Faslpr mice before and after their onset of lupus-like disease.
Methods ADM (27mcg in 0.2ml normal saline [NS]) or 0.2ml NS (control treatment) was injected intraperitoneally into 6-week old (pre-lupus) and 12-week old (adult group) MRL/Faslpr mice thrice weekly for 18 weeks and 6 weeks respectively. After completing respective treatments, urine was collected and quantified for protein and red cell levels semiquantitively using Siemens Multistix based on the manufacturer's instruction. All cervical and axillary lymph nodes (LN) were dissected and weighed. The numbers of splenic B (B220+) and T lymphocytes (CD4+ and CD8+) and macrophages (CD11b+) were assessed by flow cytometry. Kidneys were studied by immunofluorescence for depositions of C3, C1q, anti-dsDNA and immunoglobulins (Ig)A, IgM and IgG. Serum levels of anti-dsDNA, anti-Sm, anti-ribosomal-P, anti-SSA and anti-SSB autoantibodies were measured by the AtheNA Multi-Lyte immunoassay. Finally, the effect of ADM on splenic B and T cells was assessed in vitro in the absence and presence of CpG or CD3/CD28. Activation, proliferation and apoptosis were studied following 24 and 72 hours. Statistical analyses between ADM and control treated data were completed using the Mann-Whitney U test.
Results Proteinuria and the LN weight were significantly lower in adult MRL/Faslpr mice after 6-week treatment of ADM compared to controls (p=0.007 and 0.033 respectively). Similarly, lower LN weight was noted in the pre-lupus mice after ADM treatment (p=0.036). However, there was no difference in proteinuria in the pre-lupus group. No difference in the serum autoantibody levels and the numbers of splenic CD4+, CD8+ and CD11b+ cells were found in both adult and pre-lupus groups when compared with their respective NS-treated groups. Less renal immunofluorescent depositions of IgG, IgM, IgA, C1q, C3 and anti-dsDNA were detected in the adult ADM-treated group but not in the pre-lupus group as compared with the NS-treated adult mice. In vitro study revealed no evidence of alteration in activation, apoptosis and proliferation of splenic B220+, CD4+ and CD8+ cells by ADM treatment.
Conclusions Exogenous ADM treatment reduced LN weight, proteinuria and renal Ig and complement depositions in adult MRL/Faslpr mice without affecting the levels of autoantibodies and the count, proliferation, activation and apoptosis of splenic lymphocytes. Such potentially beneficial therapeutic effects were not demonstrated in mice before the lupus development. Further exploration of how ADM alleviates lupus nephritis with special focuses on the phenotypes and functions of regulatory lymphocytes and dendritic cells are warranted.
Disclosure of Interest None declared