The aims of this study were to investigate the time-dependent effects of 1,25-dihydroxyvitamin D3 on LN in MRL/lpr mice and to evaluate the effects on the regulation of NF-kB and MAPK signalling. Our study showed that 1,25-dihydroxyvitamin D3 treatment ameliorates lupus nephritis in MRL/lpr mice, as shown by improved renal function, decreased immune complex deposition, reduced inflammatory cytokine production, and diminished NF-κB and MAPK pathway activation.
Skin lesions are one of the typical symptoms of SLE, and 72–85% of patients develop skin lesions during the course of SLE . Skin lesions are characterized by epidermal atrophy, hyperkeratosis, liquefactive degeneration at the epidermal basal cell layer, infiltration of inflammatory cells and free melanin pigment . It was observed that the MRL/lpr mice began to show different degrees of skin lesions at the age of 8 weeks, especially in the skin of the head, back, mouth and nose. However, 1,25-dihydroxyvitamin D3 treatment significantly attenuated the degree of skin lesion formation. The pathogenesis of skin lesions in SLE, which needs to be further investigated, may involve deposition of IgG molecules related to lupus, immune cells, cytokines, and intracellular molecules in the skin, as well as exposure to ultraviolet light .
Multiple factors, including genetic susceptibility and environmental effects, contribute to the pathogenesis of LN, and the production of autoantibodies, the circulation of ICs, and severe proteinuria are the most common manifestations. The effect of 1,25-dihydroxyvitamin D3 on LN was confirmed by renal pathology and immunofluorescence in MRL/lpr mice. H&E and Masson’s staining showed that 1,25-dihydroxyvitamin D3 treatment significantly attenuated the histological damage to the kidney, and the kidney scores of the 1,25-dihydroxyvitamin D3-treated mice were lower than those of the DMSO-treated mice due to reduced mesangial proliferation, basement membrane thickening, interstitial fibrosis, and inflammatory infiltration. Moreover, clinical renal remission also relies mainly on improvements in proteinuria and autoantibodies . One study indicated that serum A-ds DNA concentration was closely correlated with the severity of kidney damage because glomerular collagen is bound by DNA and A-ds DNA antibodies can be eluted from nephritic kidneys . Lupus is closely related to defects in apoptosis clearance , and studies have shown that altering the levels of autoantigen clearance could promote the production of anti-nuclear autoantibodies in SLE . The dysfunction in the efficient removal of apoptotic material may be caused by failure of the autophagic process in immune cells . Previous studies have revealed that VitD3 may improve macrophage phagocytic defects and increase their number . This finding strongly supports our observation that 1,25-dihydroxyvitamin D3 reduces A-ds DNA antibody levels in LN. However, the effective therapies that are currently in use do not always effectively decrease the A-ds DNA antibody levels in serum. In our study, serum A-ds DNA levels were markedly increased over time after disease onset, which may be associated with lupus activity, while 1,25-dihydroxyvitamin D3 treatment notably reduced A-ds DNA antibody levels, suggesting that 1,25-dihydroxyvitamin D3 ameliorated LN and inhibited lupus activity.
A previous study suggested that not only the classical pathway but also the alternative pathway is involved in the pathogenesis of LN . Complement factors are bound in the kidney with immune complex deposition or formation . The deposition of C3, which is a component of the classical pathway, the alternate pathway, and the complement mannose pathway, is characteristic of LN. C1q is the first subcomponent of the C1 complex associated with the classical pathway of complement activation . Due to activation by C1q and other unknown mechanisms, complement factor C3 is deposited in the glomerulus [34, 35], inducing the proliferation of glomerular mesangial cells and ultimately leading to nephropathy . Circulating C3 levels are usually decreased due to its consumption, and these levels are associated with disease . A recent study showed that a C3 blocker effectively improved proteinuria and renal function in MRL/lpr mice , which strongly suggests the importance of exploring the effects of 1,25-dihydroxyvitamin D3 on C3 levels in LN. In our study, the serum level of C3 decreased, and the deposition of C1q and C3 increased over time, which may be associated with lupus activity. Through complement profiling, our results showed that 1,25-dihydroxyvitamin D3 prevents the consumption of C1q and C3, suggesting that 1,25-dihydroxyvitamin D3 acts via the inhibition of the complement cascade to some extent. Furthermore, deposition of immune reactants also plays a pivotal role in the occurrence, development, and prognosis of LN . IgG and IgM are immunoglobulins with different isotypes that recognize renal antigens. They play key regulatory roles in many biological processes, such as activation of complement cascades, regulation of gene expression and proliferation of cells . They induce phenotypic changes in glomerular resident cells, leading to kidney damage in LN . Additional evidence showed that 1,25-dihydroxyvitamin D3 treatment inhibited local IgG/M deposition in the kidney to protect glomerular mesangial cells. Together, these results indicated that 1,25-dihydroxyvitamin D3 was responsible for improving clinical presentations and reversing the pathological damage caused by LN.
TNF-α and its receptors are involved in the pathogenesis of autoimmune diseases, and TNF-α has both the effects of controlling autoimmunity and promoting inflammation . TNF-α contributes to the pathogenesis of LN as it promotes the activation and differentiation of macrophages, and its levels are increased in active LN and correlate with disease activity . IL-17 has the potential to induce the production of additional inflammatory cytokines and chemokines and to promote the recruitment of inflammatory cells, such as monocytes and neutrophils, to the inflamed organ; additionally, it is involved in the activation of many proinflammatory pathways . IL-17 promotes immune hyperactivation and participates in kidney damage in LN by inducing inflammation that triggers LN development . High serum levels of IL-17 have been observed in active LN and correlate with lupus activity . In MRL/lpr mice, mice lacking IL-17 showed greatly improved survival and were largely protected from the development of glomerulonephritis . C-C motif chemokine ligand 2 (CCL2), also known as MCP-1, and its chemokine receptor CCR2 promote LN by driving intrarenal inflammation via the recruitment and activation of proinflammatory leukocyte subsets. CCl2-deficient MRL/lpr mice are protected from LN . In addition, several studies have shown that TNF-α can induce the expression of MCP-1 [46, 47] and that IL-17 can upregulate MCP-1 . In our study, the levels of TNF-α, IL-17, and MCP-1were gradually upregulated with time, demonstrating that these proteins may be responsible for lupus activity. However, 1,25-dihydroxyvitamin D3 significantly reduced the levels of TNF-α, IL-17, and MCP-1 both in the serum and kidneys of MRL/lpr mice. These findings demonstrated that 1,25-dihydroxyvitamin D3 has a protective effect against LN by suppressing proinflammatory cytokine release to some extent.
To further investigate the mechanism by which 1,25-dihydroxyvitamin D3 protects against inflammation, the activation of the NF-κB pathway was examined. The NF-κB pathway is known to trigger inflammatory responses, and it controls the expression of several genes, such as proinflammatory cytokines, including TNF-α, and is essential for self-reactivity . Inflammation caused by activation of the NF-κB pathway plays an important role in the pathogenesis of LN . In resting cells, IκBα prevents the translocation of p65 into the nucleus. The IκB kinase (IKK) complex is activated by various stimuli and then phosphorylates and degrades the IκBα protein, releasing the p65 heterodimer to regulate the transcription of various genes . Many methods of inhibiting NF-κB have demonstrated that this inhibition results in LN remission. In our study, the expression of molecules related to the NF-κB signalling pathway was markedly upregulated in the kidneys of MRL/lpr mice over time, demonstrating that activation of the NF-κB signalling pathway is an important time-dependent mediator of kidney damage in MRL/lpr and may be responsible for lupus activity. However, 1,25-dihydroxyvitamin D3 evidently inhibited the NF-κB signalling pathway. Additionally, we found that the expression of the proinflammatory cytokine TNF-α was significantly reduced in the VitD3-treated groups compared to the control groups. To a certain extent, our data further proved that the NF-κB signalling pathway and downstream proinflammatory cytokine TNF-α, which may be associated with lupus activity, were inhibited by 1,25-dihydroxyvitamin D3.
MAPK are a family of protein-serine/threonine kinases that mediate the basic biological process of inflammatory and innate immune responses, including the ERK1/2, JNK [1,2,3], and p38 (α, β, γ and δ) . In response to inflammatory signals, MAPK are activated by the phosphorylation of ERK1/2, JNK, and p38 . Furthermore, NF-κB may be downstream of MAPK, and studies have shown that the P38MAPK signalling pathway is involved in the activation of NF-κB . There may be crosstalk between the signalling pathways. Under normal conditions, NF-kB is located in the cytoplasm. Once stimulated by exogenous inducers or inflammatory cytokines, NF-κB is released from the cytoplasmic complex and translocates to the nucleus to regulate the expression of inflammatory mediators . Moreover, in our study, we found that the MAPK signalling pathway was also markedly activated in the kidneys of MRL/lpr mice over time, suggesting that activation of the MAPK signalling pathway is another important time-dependent mediator of LN in MRL/lpr mice and may also be associated with lupus activity. Therefore, decreasing the activation of the MAPK and NF-kB signalling pathways may be beneficial to LN. In this study, 1,25-dihydroxyvitamin D3 significantly inhibited the phosphorylation of ERK1/2, JNK1/2, and p38. Thus, our data showed that 1,25-dihydroxyvitamin D3 significantly inhibited the NF-κB and MAPK signalling pathways to ameliorate lupus activity, providing potential targets for LN treatment.