Iguratimod, also named T-614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1- benzopyran-4-one), has been considered to be a novel immunomodulator and widely used to treat RA by rheumatologists in China and Japan [39–41]. In present study, we observed the effect of iguratimod on RIF in LN. In vivo, iguratimod effectively inhibited RIF and its upstream pathological processes, including immune deposition along the tubular basement membrane, infiltration of inflammatory cells into renal interstitium, tubular injury and EMT; in vitro, iguratimod obviously reversed TGF-β1-induced EMT process through suppressing the activation of Smad and p38 MAPK signaling pathways.
The tubulo-interstitium accounts for 90% of the total renal volume and proximal renal tubular epithelial cells are the main constituent cell type [23]. RIF is a common pathological manifestation and the ultimate result of various chronic kidney diseases. Numerous studies have shown that it is the severity of interstitial damage, rather than glomerular injury, that is significantly associated with poor renal prognosis [33]. Therefore, the research and development of drugs for RIF is of great significance for the treatment of CKD. Yan Q et al also verified that MRL/lpr mice treated with iguratimod showed less proteinuria, less glomerular injury and vasculitis scores than vehicle-treated controls [15]. However, the effect of iguratimod on RIF in LN has not been studied. The present study found that iguratimod significantly alleviated the deposition of collagen fibers in the renal interstitium determined by Masson’s trichrome and sirius red staining in MRL/lpr mice. Quantitative real-time PCR and immunofluorescence verified that the expression level of COL-I decreased evidently after iguratimod treatment, further supporting the protective role of iguratimod on inhibiting the RIF. Moreover, iguratimod treatment decreased the levels of blood urea nitrogen and serum creatinine, although the latter is not statistically significant, which may be due to the small sample size. Therefore, iguratimod could be an effective drug on inhibiting the progress of RIF in LN.
Although RIF portends poor long-term renal prognosis, the mechanisms resulting in RIF in LN has received relatively less attention to date. The deposition of immune complex in the kidney mediated by autoantibodies is the initiating factor of LN, in which anti-dsDNA plays a key role [23]. Glomerular and tubulo-interstitial expression of fibronectin is increased in mice and patients with active LN, which colocalized with IgG deposition, suggesting a pathogenic link between autoantibodies deposition and increased ECM accumulation in LN [42–44]. Immune deposition within the tubulo-interstitium is associated with increased cytokine production, immune cell infiltration, and tubular atrophy [24]. Anti-dsDNA antibodies bind to resident renal cells, including PTECs, and could induce synthesis of ECM, which promotes the progress of renal fibrosis [45]. The present study found that iguratimod showed an inhibitory effect on the deposition of IgG along the tubular basement membrane in lupus mice, which contributes to prevent the occurrence of RIF in LN. The serum titer of anti-dsDNA in MRL/lpr mice was also reduced by iguratimod, another study also found that iguratimod delayed the appearance of autoantibody and decreased autoantibody titers [15]. Therefore, we speculate that the decrease of IgG deposition along the tubular basement membrane is partly due to the reduction of serum autoantibodies in serum. whether iguratimod can inhibit the binding of autoantibodies to target antigens is not clear, which deserves further study.
Anti-dsDNA antibodies can increase secretion of TNF-α, IL-6, IL-8 and monocyte chemoattractant protein-1 in renal tubular epithelial cells, contributing to the establishment of chemotactic gradients and tubulo-interstitial infiltration of inflammatory cells [45]. Activated inflammatory cells will release a large number of pro-fibrosis factors, which could transform the resting ECM producing cells into myofibroblasts, resulting in a large number of ECM accumulation, and eventually lead to RIF [26, 46–48]. Moreover, anti-dsDNA and inflammatory factors can lead to abnormal proliferation and apoptosis of renal tubular epithelial cells [23], resulting in tubular atrophy, casts, and dilation. After tubular injury, the function of movement, migration, secretion and transformation of renal tubular epithelial cells increased significantly, and the normal tubulo-interstitial structure was destroyed [30, 49, 50]. At the same time, a large number of chemokines, inflammatory factors, pro-fibrosis factors and matrix proteins were produced [33], which further aggravated the injury of renal tubular epithelial cells and RIF. In present study, iguratimod not only inhibited the renal interstitial infiltration of inflammatory cells, but also alleviated the tubular injury, which may be related to decreased RIF in iguratimod-treated MRL/lpr mice. The number of Ki-67 (+) cells and caspase-3 (+) cells all decreased in iguratimod-treated MRL/lpr mice, which further proved the protective role of iguratimod on renal tubules in LN, and also explained the reason for the reduction of renal tubular injury in mice treated with iguratimod.
In the process of RIF, tubular EMT is a key step [25, 51, 52]. Although a small number of renal interstitial myofibroblasts are from bone marrow, more myofibroblasts are derived by local tubular EMT during renal fibrogenesis [53]. Decreased expression of epithelial markers together with a concomitant increase in mesenchymal markers could be detected in renal biopsies from patients with LN and is associated with renal impairment, interstitial leukocyte infiltration, and interstitial fibrosis [30, 50]. In present study, iguratimod treatment decreased the FSP-1 expression and increased the E-cadherin expression in renal tubular epithelial cells of MRL/lpr mice. Moreover, in HK2 cells induced by TGF-β1, iguratimod not only reversed morphologic changes, but also prevented down-regulation of E-cadherin and up-regulation of fibronectin. So iguratimod could effectively inhibited tubular EMT in vivo and in vitro, thus blocking the key step of RIF.
TGF-β1 which has been identified as the key profibrogenic cytokine could regulate EMT process through multiple signaling pathways [54–56], such as Smad and p38 MAPK. Canonical Smad signaling pathway is considered to be the most important pathway in EMT and most of the TGF-β1-induced EMT appears to be dependent on this signaling pathway [36, 57]. Thus, blocking the TGF-β1/Smad signaling pathway may be promising for preventing RIF. In this study, iguratimod suppressed effectively TGF-β1-induced phosphorylation of Smad2/3 and subsequent translocation into the nucleus, suggesting that iguratimod could revers TGF-β1-induced EMT partly by inhibiting Smad signaling pathway.
Noncanonical TGF-β1 signaling pathways also has been verified to promoting EMT process, specifically through the activation of p38 MAPK [38]. Co-treatment with iguratimod resulted in a decrease in IL-17-induced phosphorylation of p38 MAPK in the cultured fibroblast-like synoviocytes [58]. In RANKL induced RAW264.7 cells, iguratimod failed to inhibit but to markedly delay the phosphorylation of p38 MAPK [59]. However, in mesenchymal stem cells cultured with recombinant human bone morphogenetic protein-2, iguratimod exposure accelerated the activation of p-p38 MAPK [60]. These results suggest that the effect of iguratimod on p38 MAPK signaling pathway may be influenced by cell type and other co-existing inducers. In our data, iguratimod demonstrated a negative regulation on the activation of p38 MAPK signaling pathway in TGF-β1 -induced HK2 cells, contributing to preventing the tubular EMT.