1,25-dihydroxyvitamin D3 ameliorates lupus nephritis through inhibiting the NF-κB and MAPK signalling pathways in MRL/lpr mice

Abstract

Background: Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). However, the aetiology and pathogenesis of LN remain unknown. 1,25-dihydroxyvitamin D3 [1,25-(OH)2-VitD3] is the active form of vitamin D, and it has been shown to perform important functions in inflammatory and immune-related diseases. In this study, we investigated the time-dependent effects of 1,25-dihydroxyvitamin D3 and explored the underlying mechanism in MRL/lpr mice, a well-studied animal model of LN.

Methods: Beginning at 8 weeks of age, 24-h urine samples were collected weekly to measure the levels of protein in the urine. We treated female MRL/lpr mice with 1,25-dihydroxyvitamin D3 (4 μg/kg) or 1% DMSO by intraperitoneal injection twice weekly for 3 weeks beginning at the age of 11 weeks. The mice were separately sacrificed, and serum and kidney samples were collected at the ages of 14, 16, 18, and 20 weeks to measure creatinine (Cr) levels, blood urea nitrogen (BUN) levels, histological damage, immunological marker (A-ds DNA, C1q, C3, IgG, IgM) levels, and inflammatory factor (TNF-α, IL-17, MCP-1) levels. Furthermore, the nuclear factor kappa B (NF-κB) and the mitogen-activated protein kinase (MAPK) signalling pathways were also assessed to elucidate the underlying mechanism.

Results: We found that MRL/lpr mice treated with 1,25-dihydroxyvitamin D3 displayed significantly attenuated LN. VitD3-treated mice exhibited significantly improved renal pathological damage and reduced proteinuria, BUN, SCr, A-ds DNA antibody and immune complex deposition levels (P < 0.05) compared with untreated MRL/lpr mice. Moreover, 1,25-dihydroxyvitamin D3 inhibited the complement cascade, inhibited the release of proinflammatory cytokines, such as TNF-α, IL-17, and MCP-1, and inhibited NF-κB and MAPK activation (P < 0.05).

Conclusion: 1,25-dihydroxyvitamin D3 exerts a protective effect against LN by inhibiting the NF-κB and MAPK signalling pathways, providing a potential treatment strategy for LN. Interestingly, the NF-κB and MAPK signalling pathways are time-dependent mediators of LN and may be associated with lupus activity.

Keywords: 1,25-dihydroxyvitamin D3; Lupus nephritis; MRL/lpr mice; NF-κB and MAPK signalling pathways.

Fig. 1

The experiment schedule

Fig. 2

1,25-dihydroxyvitamin D3 reduced proteinuria and protected renal function. A MRL/lpr mice in the control groups. B MRL/lpr mice in the VitD3-treated groups. C The body weight of the experimental MRL/lpr mice. D Urine protein time-course studies. E Serum creatinine levels. F Blood urea nitrogen levels. Values are expressed as means ± SD (n = 6/group). & VS. mice at the previous week. * VS. the control group at the same time. # VS. the control group at 14 weeks. △VS. the treated group at 14 weeks. ^*#△& p < 0.05, ^**##△△&&p < 0.01, ^{***###△△△&&&}p < 0.001, ^&&&&P<0.0001

Fig. 3

1,25-dihydroxyvitamin D3 ameliorated glomerular, tubular, and interstitial injuries. A Histopathological features of the renal tissues determined by HE, Masson (400×, Scale bar = 50 μm). B Histopathological features of the renal tissues determined by immunohistochemistry (400×, Scale bar = 50 μm). C Scoring results. Values are expressed as means ± SD (n = 3/group). * VS. the control group at the same time. # VS. the control group at 14 weeks. △VS. the treated group at 14 weeks. ^*#△ p < 0.05, ^**##△△p < 0.01, ^{***###△△△}p < 0.001

Fig. 4

1,25-dihydroxyvitamin D3 decreased deposition of IgG/M in MRL/lpr mice. A The intensity of immunofluorescence. B-D Immunofluorescence findings for renal tissues. 3×, Scale bar = 500 μm (upper panel) and 400×, 50 μm (lower panel). Values are expressed as means ± SD (n = 3/group). * VS. the control group at the same time. # VS. the control group at T1. △VS. the treated group at T1. ^*#△ p < 0.05, ^**##△△p < 0.01, ^{***###△△△}p < 0.001

Fig. 5

1,25-dihydroxyvitamin D3 reduced lupus activity and inhibited the pro-inflammatory cytokines. A The concentrations of A-ds DNA and C3 in the serum. B-D The concentrations of TNF-α, IL-17, and MCP-1 in the serum and kidneys. Values are expressed as means ± SD (n = 6/group). * VS. the control group at the same time. # VS. the control group at 14 weeks. △VS. the treated group at 14 weeks. ^*#△ p < 0.05, ^**##△△p < 0.01, ^{***###△△△}p < 0.001

Fig. 6

1,25-dihydroxyvitamin D3 inhibited the NF-κB/MAPKs signaling pathways. A The mRNA levels by RT-PCR. B, C The protein levels by Western blot, bands 1–8 represent the control groups at 14, 16, 18, 20 weeks and the VitD3-treated groups at 14, 16, 18, 20 weeks respectively. The samples derive from the same experiment and the gels/blots were processed in parallel. Data are presented as means ± SD (n = 3/group). * VS. the control group at the same time. # VS. the control group at 14 weeks. △VS. the treated group at 14 weeks. ^*#△ p < 0.05, ^**##△△p < 0.01, ^{***###△△△}p < 0.001, ^{****####△△△△}p < 0.0001