RIF is the common pathway in chronic renal diseases regardless of the initial cause. Therefore preventing RIF could slow down the pace of renal function deterioration . Recent documents show that hepatocyte growth factor (HGF), angiotensin-converting enzyme inhibitors (ACEI), and erythropoietin all could antagonize RIF and improve renal function [6, 29, 30]. Traditional Chinese medicine has a rich history of treating chronic renal diseases over thousands of years, and some herbal medicine and extracts have proven to contain anti-renal fibrosis properties both in vivo and in vitro. It is well recognized that tubular epithelial cells could differentiate into myofibroblasts via a process referred to as EMT, which plays a pivotal role in renal interstitial fibrogenesis . Since specific therapies of preventing the progression of chronic renal diseases are still lacking, seeking drug candidates or herbal compounds that can effectively prevent or inhibit EMT would represent one of the main strategies in the treatment of RIF.
Tubular EMT is a process during which renal tubular epithelial cells lose their epithelial phenotype and acquire new mesenchymal features and consists of four correlated steps with highly regulated mechanisms : (1) Loss of epithelial cell adhesion through down-regulation of E-cadherin. (2) De novo α-SMA expression and actin reorganization, which may provide a structural foundation not only for defining the morphology of the transformed cells, but also for them to migrate, invade, and even acquire the capacity for contractility. (3) Disruption of tubular basement membrane, and (4) Enhanced cell migration and invasion. The latter 2 steps are mainly regulated by increased activities of MMP-2 and MMP-9 which would specifically break down native type IV collagen and laminin, the principal proteins found in the TBM, thereby disrupting TBM structural and functional integrity. Although recent evidence with genetic manipulation indicates that kidney epithelial cells do not become myofibroblasts in vivo, most documents still support the phenomenon of tubular EMT and its important role in renal fibrogenesis .
In this study, we found that α-SMA expression was increased significantly around tubules in the fibrotic kidney induced by HgCl2, using immmunohistochemistry staining, when compared with the normal kidney. In contrast, E-cadherin expression decreased around tubular areas. Although we have not confirmed if these α-SMA positive tubular cells come from the epithelium or whether they can synthesize collagen, the increased α-SMA expression around tubules and MMP-2 activity in kidney support EMT in vivo. Also, the in vitro results clearly show that human proximal tubular epithelial cells, represented by the HK-2 cell line, had undergone EMT after TGF-β1 incubation, as evidenced by the loss of E-cadherin, activation of α-SMA, acquisition of a spindle-like morphology, F-actin re-organization, and an increase in the activities of MMP2/9. More interestingly, Sal B could inhibit tubular EMT in fibrotic kidneys, as evidenced by the reversal of increased α-SMA and decreased E-cadherin expression, as well as preventing renal collagen deposition and fibrosis.
Vit E is a potential antioxidant agent and can prevent organ fibrosis [33–35]. In our previous study, we found that chronic mercury intoxication induced RIF with obvious characteristics of oxidative stress through the depletion of intracellular thiols . Vit E exhibited anti-renal fibrosis effects through its mechanism of action, which protects the kidney from oxidative injury. Here we used Vit E as a control as well, and the results showed that Vit E also blocked EMT in vivo, but the effects were not as strong as Sal B. With the EMT cell model induced by TGF-β1 in HK-2, our data in vitro demonstrated that Sal B blocked EMT in HK-2 cells, and these results are consistent with our in vivo results as well as in vitro results from other reports . However, in our experiment, reasonable dosages f Sal B (1 μM and 10 μM) were utilized, which produced no obvious toxicity, and were used effectively in other experiments . In addition, SB-431542 , a potential and specific inhibitor of TβR-I kinase was used as an active control. The results revealed that Sal B, as well as SB-431542, significantly reversed EMT induced by TGF-β1 in HK-2 cells, as evidenced by the restoration of diminished E-Cadherin and CK-18 expression, blockage of α-SMA expression, cell morphology transformation, F-actin reorganization, and down-regulation of MMP-2 and MMP-9 activation. These data not only confirms Sal B's effects in preventing or reversing tubular EMT in vivo and in vitro, but also implies a mechanism of action involved with the regulation of MMP-2/9. MMP-2/9, in particular MMP-2, have pivotal roles in initiating renal fibrosis by degrading TBM components and disrupting its integrity . It was reported that genetic expression of active MMP-2 in the renal proximal tubule is sufficient to generate the entire spectrum of EMT in the absence of superimposed injury. The rat RIF model induced by HgCl2 displayed a remarkable feature of oxidative stress, which could promote MMP-2 expression and activity . Our previous studies showed that Sal B had anti-oxidant effects, and in this study, we found that Sal B can also inhibit MMP-2 activity in the diseased kidney and subsequently protect tubular basement membrane and prevent EMT and renal fibrosis.
TGF-β1 is the most potent inducer in initiating and completing the entire EMT course . Its biological responses are primarily dependent on regulation via the TβR and Smads signaling pathway. TGF-β1 signals are transduced by transmembrane serine/threonine kinase type I and type II receptors, as well as intracellular mediators known as Smads [40–42]. Upon TGF-β1 stimulation, the activated TβR1 associates with R-Smads (Smad2 and Smad 3) and also phosphorylats them. Such phosphorylation of Smad-2/3 induces their association with their common partner Smad4 and subsequent translocation into the nucleus, where they control the transcription of TGF-β1 responsive genes [43, 44]. As an inhibitory Smad, Smad 7 prevents or attenuates TGF-β signaling mainly through interaction with activated TβRI and prevention of R-Smad phosphorylation.
In this study, we found that the protein expression of TGF-β1 and TβRI and the phosphorylation levels of Smad 2 and Smad 3 were significantly increased in the fibrotic kidney, compared with ones in the normal kidney. In cultured HK-2 cells, TGF-β1 dramatically up-regulated TβRI expression and phosphorylated levels of Smad 2 and Smad 3, but down-regulated Smad7 expression, consistent with the data in vivo, and confirmed the characteristics of TGF-β/Smads signaling in EMT . The down-regulation of Smad7 protein by TGF-β1 is most likely due to the promotion of Smad7 degradation . In the rat model, Sal B and Vit E suppressed TGF-β1 and TβR-I expression and restored phosphorylation of Smad2 and Samd3 in fibrotic kidneys. Also, in cultured HK-2 cells, Sal B and SB-431542 significantly down-regulated the expression of TβR-I and p-Smad2/3, while restoring the diminished expression of Smad7 secondary to TGF-β1 stimulation. Although we did not find the action of Sal B to be dose dependent, the effects of 10 μM of Sal B are much more prominent than 1 μM of Sal B, and almost had a similar effect as the SB-431542 inhibitor, indicating that Sal B can antagonize TβR-I function as well as its protein expression. The above results indicate that Sal B can down-regulate TGF-β/Smads signaling pathway, via suppressing TGF-β1 expression, inhibiting TβR-I expression and function, down-regulating Smad 2/3 phosphorylation, and preventing the down- regulation of Smad7 by TGF-β1. As stated above, MMP-2 has a role in renal fibrosis, especially during early pre-fibrotic stage. However, MMP-2 and MMP-9 are not only stimulated by oxidative stress, but more importantly up-regulated by TGF-β1 through signal mediators such as Smads and ILK . Our data also showed that MMP-2/9 activities increased in HK-2 cells after TGF-β1 incubation, but were reversed by Sal B and SB-431542. Therefore, the TGF-β1/Smads transduction pathway plays a key role in tubular EMT and Sal B's pharmacological mechanisms of action against EMT and renal fibrosis.