Renal fibrosis is a key problem in the field of CKD research, and there has been no breakthrough for a long time. Thus, there is still a shortage of safe and effective anti-fibrotic drugs. In this study, we identified a significant reduction in renal fibrosis in UUO mice treated with SFN and SFN could reverse TGF-β1-induced fibroblasts activation and renal tubular epithelial cell trans differentiation in vitro. Mechanistically, we found that SFN had dual regulation effect on autophagy activity in renal fibroblasts and renal tubular epithelial cells through the mTOR pathway. As a natural compound derived from plants with a wide range of biological effects, SFN was explored as a promising nutritional therapy in a range of diseases[18]. SFN is of interest to researchers for its safety. According to the reported literature, SFN had no significant effects on body weight, food intake and health status of mice at therapeutic doses, while at very high doses, mice showed lesions of sedation and muscle damage[19]. In addition, some researchers have used SFN in clinical trials and found that SFN has almost no adverse effects at low dose[20, 21].
The salutary effect and underlying mechanism of SFN on renal fibrosis have not been fully elucidated. Several studies have demonstrated the renoprotecitve effect of SFN in kidney injury animal models and cultured renal intrinsic cells in vitro including cisplatin-induced nephropathy, diabetic nephropathy, ischemia/reperfusion-induced kidney injury, contrast-induced nephropathy[22–25]. As previous reported, the above renoprotecitve effect was mainly attributed to the anti-inflammation and antioxidant functionality of SFN[26–28]. Regarding UUO models, fewer studies have indicated that SFN alleviated UUO-induced renal fibrosis in rats by remission of mitochondrial dysfunction[29, 30]. Here, we used UUO mice model and found that SFN reduced renal collagen fiber deposition and expression levels of renal fibrosis marker α-SMA and FN. In vitro, TGF-β1 intervention upregulated the expression level of α-SMA in renal fibroblasts and tubular epithelial cells, while this change was significantly prevented by administration of SFN. Taken together, our results further confirmed the therapeutic role of SFN against renal fibrosis.
Furthermore, we investigated the mechanism by which SFN attenuates renal fibrosis. Autophagy is an important mechanism in kidney injury and repairment but its exact role in renal fibrosis was discrepant according to previous studies[14–17]. The effect of SFN on autophagy has been investigated in different cell types and disease contexts mainly including malignant tumor, neuronal damage, muscular injury. As previous reported, SFN may promote the activation of autophagy through signaling pathways such as the NRF2[31–34], ROS[26, 35], mTOR[36, 37], SIRT1[27], ERK[38] pathway. However, in contrast, some researchers have also found that SFN had an inhibitory effect on autophagy[39, 40] through NRF2 pathway[41, 24, 42] and ROS-related pathway[28]. In our study, we intervened NRK-49F cells and HK2 cells with TGF-β1, followed by SFN treatment at consistent timing and concentration, based on the fact that these two cells participant central events in the renal fibrosis process. In addition, although we chose different species sources for our in vivo and in vitro experiments, which is also based on previous literature[43, 44]. We observed that SFN decreased the expression of fibrosis markers in both two cell types. And interestingly, the activity of autophagy in these two cells appeared to be differently altered. We found that the activity of autophagy was upregulated in fibroblasts while downregulated in renal tubular epithelial cells after SFN treatment. This result was in line with the bidirectional and cell-specific nature of SFN in regulating autophagy as previous published. According to previous literature, pathological activation and proliferation of fibroblasts leading to massive production of extracellular matrix is the central event in renal fibrosis[45]. Previous literature reported that activation of autophagy could inhibit the proliferation of renal fibroblasts and development of renal fibrosis[46]. Therefore, the enhancement of autophagy in fibroblasts might partly mediated the relieving effect of SFN on renal fibrosis. On the other hand, renal tubular epithelial cells damage and mesenchymal trans differentiation are also important mechanism in renal fibrosis[47]. Our results indicated that SFN reduced the autophagy activity of renal tubular epithelial cells, which we assume plays a protective role in renal fibrosis. This perspective is supported by previous studies from two groups, in which they used kidney proximal tubule-specific knockout of autophagy-related 7 gene mice and found that suppressed autophagy in kidney proximal tubules could prevent UUO associated fibrosis[15, 17]. By contrast, Li et al. reported that kidney proximal tubule-specific knockout of autophagy-related 5 gene induced autophagy deficiency and exacerbated UUO-induced renal fibrosis[14]. Although autophagy in renal proximal tubular cells can be renoprotecive or detrimental depending on pathological settings, its role in renal fibrosis remains largely unclear.
mTOR has been recognized as a key factor in the regulation of autophagy[48]. Kim et al.[13] found that mTOR inhibited AMPK/ULK1 pathway by phosphorylation thereby suppressed the initiation of autophagy. Previous studies have also demonstrated the interaction of SFN with the mTOR signaling pathway[49]. Researchers found that SFN exerted neuroprotective effect through mTOR-dependent restoration of autophagy in preventing Parkinson's disease[37]. Our data indicated that SFN does act on the mTOR pathway and that this action is equally cell-specific. In NRK-49F cells, SFN treatment inhibited p-mTOR expression, whereas in HK2 cells, SFN up-regulated p-mTOR expression. The obtained results corroborated with the altered autophagy levels echoing each other.
In summary, we found an apparent effect of SFN in alleviating renal fibrosis. In terms of mechanism, we revealed the cell-specific regulation of SFN on autophagy activity. Many renal parenchymal cells are involved in the lesion formation during renal fibrosis, so it is necessary to investigate the role of different cells types respectively. Our data suggested that SFN eventually reached an inhibitory effect on renal fibrosis through inconsistent regulation of autophagy in renal tubular epithelial cells and fibroblasts. Results from the present study may provide a new perspective on the renal salutary effect of SFN and provide a preclinical rationale for exploring therapeutic potential of SFN to slow down renal fibrosis.