The beneficial effects of treatment with all-trans-retinoic acid plus corticosteroid on autoimmune nephritis in NZB/WF mice

Abstract

Corticosteroids are highly effective anti-inflammatory or immunosuppressive drugs used commonly to treat human systemic lupus erythematosus (SLE). All-trans-retinoic acid (ATRA), which belongs to a class of retinoids that exert immunomodulatory and anti-inflammatory functions, can also suppress the development of lupus nephritis in an animal model. However, both agents can inflict serious adverse effects. Here, we have asked whether ATRA can serve as a steroid-sparing drug in the treatment of lupus nephritis. To examine the efficacy of combining predonisolone (PSL) with ATRA, we treated intraperitoneally New Zealand black/white F1 (NZB/W F1) mice with PSL, ATRA or both agents. Survival rate and proteinuria were determined once a month. Cytokine and anti-DNA antibody production were determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Renal histopathology was observed by haematoxylin and periodic acid Schiff (PAS), immunoperoxidase and immunohistochemical assay. Survival rate and proteinuria were improved in all experimental groups, and were much improved in the mice receiving the combination of ATRA and PSL (P <0.05). A single administration of ATRA reduced the Th1 [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], and a Th2 (IL-4) cytokine level, as effectively as administration of PSL. ATRA also suppressed the expression of inducible nitric oxide synthetase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) in the kidney. The combination of PSL and ATRA significantly reduced IgG2 (especially IgG2b)-specific anti-DNA antibody levels in comparison with administration of either agent alone. These data suggest that ATRA might have the potential to act as a new therapeutic and steroid-sparing drug against lupus nephritis.

Fig. 1

(a) Survival rate of the experimental and control mice. (b) Proteinuria was graded semiquantitatively monthly (0, none; 1, 30–99 mg/dl; 2, >100–299 mg/dl; 3, >300–999 mg/dl; 4, >1000 mg/dl or more of albumin). PSL (0·3 mg) + ATRA, n = 11 versus PSL (0·3 mg), n = 9; ATRA, n = 9; control, n = 16 (*P < 0·05) and PSL (0·6 mg) + ATRA, n = 12 versus PSL (0·6 mg), n = 8; PSL (0·3 mg) + ATRA; PSL (0·3 mg); ATRA; control (#P < 0·03).

Fig. 2

Expression of mRNAs for cytokines in the CD4⁺ T cells after 5 months (n = 8, *P < 0·05, **P < 0·005, ***P < 0·001 versus control mice, †P < 0·05; PSL (0·6 mg or 0·3 mg) alone versus combination of each dose of PSL + ATRA).

Fig. 3

Expression of mRNAs for MCP-1 and iNOS in the kidney after 5 months (n = 8; *P < 0·05 versus control mice; **P < 0·005).

Fig. 4

Effects of ATRA on the serum levels of IFN-γ and IL-12 at 8 months (n = 8, *P < 0·05, **P < 0·005, ***P < 0·001 versus control mice).

Fig. 5

Effects of ATRA on the serum levels of anti-DNA antibody of various isotypes at 8 months (n = 8, *P < 0·05, **P < 0·005 versus control mice, †P < 0·05; PSL (0·6 mg or 0·3 mg) alone versus combination of each dose of PSL + ATRA).

Fig. 6

Pathological and immunohistochemical findings of the kidney. (A) PAS and immunohistochemical staining. Representative photographs (a–e) of a mouse given PSL (0·6 mg) + ATRA (b,e) and control (a,c,d) are shown. (b) PAS (magnification × 400), (c) CD4⁺, (d) F4/80⁺, (e) CD4⁺ and (f) F4/80⁺ cells. T cells and macrophages (brown) are present in the periglomerular, interstitial and perivascular areas (magnification ×200). Control mice (a) had glomerulosclerosis (GS), leukocytic infiltrates (arrow), tubular atrophy (TA) and casts (magnification ×400). In contrast, the renal histology of all the experimental groups of mice remained nearly normal. (B) Renal pathology was evaluated by scoring in the PAS-stained sections. Renal pathology was evaluated by scoring in the PAS-stained sections (n = 8, *P < 0·05, **P < 0·005, ***P < 0·001 versus control mice).