Dimethylfumarate Attenuates Renal Fibrosis via NF-E2-Related Factor 2-Mediated Inhibition of Transforming Growth Factor-β/Smad Signaling

TGF-β plays a key role in the development of renal fibrosis. Suppressing the TGF-β signaling pathway is a possible therapeutic approach for preventing this disease, and reports have suggested that Nrf2 protects against renal fibrosis by inhibiting TGF-β signaling. This study examines whether dimethylfumarate (DMF), which stimulates Nrf2, prevents renal fibrosis via the Nrf2-mediated suppression of TGF-β signaling. Results showed that DMF increased nuclear levels of Nrf2, and both DMF and adenovirus-mediated overexpression of Nrf2 (Ad-Nrf2) decreased PAI-1, alpha-smooth muscle actin (α-SMA), fibronectin and type 1 collagen expression in TGF-β-treated rat mesangial cells (RMCs) and renal fibroblast cells (NRK-49F). Additionally, DMF and Ad-Nrf2 repressed TGF-β-stimulated Smad3 activity by inhibiting Smad3 phosphorylation, which was restored by siRNA-mediated knockdown of Nrf2 expression. However, downregulation of the antioxidant response element (ARE)-driven Nrf2 target genes such as NQO1, HO-1 and glutathione S-transferase (GST) did not reverse the inhibitory effect of DMF on TGF-β-induced upregulation of profibrotic genes or extracellular matrix proteins, suggesting an ARE-independent anti-fibrotic activity of DMF. Finally, DMF suppressed unilateral ureteral obstruction (UUO)-induced renal fibrosis and α-SMA, fibronectin and type 1 collagen expression in the obstructed kidneys from UUO mice, along with increased and decreased expression of Nrf2 and phospho-Smad3, respectively. In summary, DMF attenuated renal fibrosis via the Nrf2-mediated inhibition of TGF-β/Smad3 signaling in an ARE-independent manner, suggesting that DMF could be used to treat renal fibrosis.


Introduction
The progressive accumulation of extracellular matrix (ECM) components in renal parenchyma leading to end stage renal disease is a characteristic feature of chronic kidney diseases [1,2]. A number of profibrotic growth factors, including transforming growth factor-beta (TGF-b), connective tissue growth factor, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), have been implicated in the pathogenesis of ECM accumulation [3][4][5]. Several lines of evidence from both animal and human studies have suggested a critical role for TGF-b in the development of renal fibrosis, and this evidence is supported by studies showing that TGF-b not only stimulates matrix protein generation but also inhibits matrix protein removal [6][7][8]. The upregulation of TGF-b expression has been demonstrated in a variety of renal diseases, including obstructive nephropathy. Increased TGF-b expression in the obstructed kidney stimulated genes involved in ECM protein accumulation including type 1 collagen and fibronectin [9]. Additionally, TGF-b stabilizes ECM proteins by stimulating the expression of plasminogen activator inhibitor 1 (PAI-1). Thus, the inhibition of TGF-b signaling has been included in several therapeutic approaches for preventing renal fibrosis [2,10].
Dimethylfumarate (DMF) is an orally bioavailable fumaric acid ester currently used for the treatment of psoriasis [11][12][13]. In addition, DMF attenuates multiple sclerosis, an immune-mediated inflammatory disease that attacks myelinated axons in the central nervous system [14][15][16][17][18] and inhibits tumor cell invasion [19][20][21]. DMF has also been shown to reduce airway smooth muscle cell proliferation through the induction of heme oxygenase (HO)-1 [22]. Recent studies have shown that DMF increases the expression of NF-E2-related factor 2 (Nrf2), which is repressed by binding to the inhibitor Keap1 in the cytoplasm [17,23,24]. Keap1, an Nrf2 inhibitor, consists of three protein domains, one of which, the intervening region (IVR) contains several thiol groups and DMF interacts with these thiol groups to induce a conformational change in Keap1. As a result of this conformational change, Nrf2 dissociates from Keap1 and enters the nucleus, where it induces the expression of various antioxidant/detoxification enzymes [23,[25][26][27].
Nrf2 is a basic leucine-zipper (bZip) transcription factor that protects a variety of tissues and cells against oxidative and electrophilic stress through antioxidant response element (ARE)mediated induction of diverse phase II detoxification and antioxidant enzymes, including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [26,28]. Recently several studies demonstrated that Nrf2 protects against streptozotocin-induced diabetic nephropathy [29] and mitigates cisplatininduced nephrotoxicity through the induction of antioxidant enzymes [30]. In addition, Nrf2 attenuates cyclosporin A-induced epithelial-mesenchymal transition in renal fibrosis via the induction of HO-1 [31] and inhibits inflammation and sclerosis in focal segmental glomerulosclerosis in mice [32]. Therefore, chemicals that modulate the activity of Nrf2 and regulate the TGF-b signaling pathway represent potential therapeutic agents for the treatment of renal fibrosis. Until now, protective effect of DMF, one of the Nrf2 activators, against renal fibrosis has not been investigated.
In this study, we examined the potential effects of DMF on TGF-b-stimulated ECM production in vitro and unilateral urethral obstruction (UUO)-induced renal fibrosis in vivo, and elucidated its underlying molecular mechanisms which are associated with Nrf2mediated inhibition of the TGF-b/Smad3 signaling pathway.

DMF inhibits TGF-b-stimulated PAI-1, a-SMA, fibronectin and type I collagen expression
Firstly, the effect of DMF on TGF-b-stimulated profibrotic genes and on ECM protein expression was examined in a normal rat renal fibroblast cell line (NRK-49F). As shown in Figure 1A-D, DMF inhibited TGF-b-stimulated PAI-1, a-SMA and fibronectin mRNA and protein expression in a dose-dependent manner. The expression of type I collagen mRNA and protein had not increased at 6 h after treatment with TGF-b (data not shown); however, its expression was induced at 24 h after TGF-b treatment, after which time DMF was seen to inhibit TGF-b-stimulated type I collagen expression in a dose-dependent manner ( Figure 1B). The inhibitory effect of DMF on TGF-b-stimulated PAI-1 protein expression was confirmed further in RMCs, a rat mesangial cell line ( Figure 1E). These data demonstrated that DMF inhibits TGF-b-stimulated profibrotic genes and ECM protein expression in rat kidney cell lines.

DMF inhibits the TGF-b/Smad signaling pathway
To determine whether DMF represses profibrotic genes and ECM protein expression through the inhibition of the TGF-bactivated Smad signaling pathway, we examined the effect of DMF on a luciferase reporter construct carrying the PAI-1 promoter (PAI-1-Luc), which contains three binding sites for Smad3. Its effect on (CAGA) 9 MLP-Luc promoter activity, a reporter construct that contains nine copies of the Smad3 binding site, was also investigated. As shown in Figure 2A and B, DMF inhibited TGF-b-stimulated PAI-1 promoter activity in AD-293 cells, a derivative of the commonly used HEK-293 human embryonic kidney cell line. DMF also inhibited the activity of (CAGA) 9 MLP-Luc promoter stimulated by either TGF-b or a constitutively active TGF-b type I receptor (ALK5).
Because phosphorylation of Smad3 is a critical step in ensuring the proper TGF-b signaling [33], we evaluated the effect of DMF on the phosphorylation of Smad3 (p-Smad3). As shown in Figure 2C-D, DMF treatment inhibited TGF-b-stimulated Smad3 phosphorylation in NRK-49F ( Figure 2C) and RMC cells ( Figure 2D). These data indicate that DMF negatively affects TGF-b-mediated transcription via the inhibition of Smad3 phosphorylation.

DMF-activated Nrf2 protein suppresses the TGF-bstimulated expression of profibrotic genes
Since DMF is a well-known activator of the transcription factor Nrf2, which has been implicated in the pathogenesis of renal fibrosis, we investigated whether Nrf2 mediates the suppression of DMF on TGF-b-stimulated profibrotic genes and ECM protein expression. As expected, treatment with DMF resulted in a rapid increase in the protein expression of Nrf2 ( Figure 3A). This increased expression of Nrf2 was maintained up to 24 h after cotreatment of DMF with TGF-b in NRK-49F cells ( Figure S1), although its expression levels gradually decreased from 1 h after treatment of DMF. DMF also induced the nuclear accumulation of Nrf2 in a dose dependent manner ( Figure S2). Because the p62mediated stabilization of Nrf2 has recently been suggested as an antioxidant-independent mechanism for Nrf2 activation [34], we investigated the effect of DMF on p62 expression and the involvement of p62 in DMF-induced Nrf2 expression. Unlike rapid induction of Nrf2 expression by DMF, p62 expression was augmented at 6 h and further enhanced at 12 h after DMF treatment ( Figure S3A), suggesting that DMF increases Nrf2 expression by a p62-independent mechanism. Consistently, a small interfering RNA (siRNA) against p62 (p62-siRNA) had little effect on DMF-increased Nrf2 expression, while it significantly diminished both basal and DMF-induced p62 expression in NRK-49F cells ( Figure S3B). Moreover, down-regulation of p62 expression in AD-293 cells did not reverse the inhibitory effects of DMF on (CAGA) 9 MLP-Luc activity and profibrotic gene expression stimulated by TGF-b ( Figure S3C-E).
Next, we examined whether Nrf2 suppresses TGF-b-stimulated ECM expression. The results showed that adenovirus-mediated overexpression of Nrf2 (Ad-Nrf2) decreased PAI-1, a-SMA and fibronectin mRNA and protein expression in NRK-49F cells ( Figure 3B and C). Ad-Nrf2 also decreased type I collagen mRNA expression at 24 h after TGF-b treatment ( Figure 3D). Additionally, we confirmed that DMF causes a rapid increase in expression of the Nrf2 protein in RMC cells ( Figure 3E) and that Ad-Nrf2 inhibits TGF-b-stimulated ECM expression in RMCs ( Figure 3F). Moreover, transient transfection with Nrf2 decreased PAI-1 promoter activities stimulated by either TGF-b treatment or ALK5 cotransfection in a dose-dependent manner ( Figure 3G). In contrast, promoter activity of NQO1, a well-known target gene of Nrf2 was markedly activated by transfection with Nrf2. These data demonstrate that Nrf2 activated by DMF suppresses the TGF-binduced expression of profibrotic genes.

Nrf2 inhibits the TGF-b/Smad signaling pathway
To determine whether Nrf2 inhibits TGF-b/Smad signaling, the effect of Ad-Nrf2 on TGF-b-stimulated Smad3 phosphorylation was investigated. The results revealed that Ad-Nrf2 inhibited TGF-b-stimulated Smad3 phosphorylation but had no effect on total Smad3 and Smad4 protein expression in NRK-49F cells ( Figure 4A) and RMC cells ( Figure 4B). To confirm further that the suppression of TGF-b/Smad3 activity and ECM protein expression by DMF is mediated by Nrf2, endogenous Nrf2 expression was down-regulated by transfecting AD-293 cells with a small interfering RNA (siRNA) against Nrf2 (Nrf2-siRNA). The Nrf2-siRNA successfully inhibited the expression of Nrf2 ( Figure 4C) and significantly blocked the DMF-induced suppression of TGF-b-stimulated (CAGA) 9 MLP-Luc promoter activity ( Figure 4D). Moreover, the Nrf2-siRNA reversed the inhibitory effects of DMF on TGF-b-stimulated type 1 collagen expression ( Figure 4E). These data suggest that Nrf2 mediates the inhibitory effect of DMF on TGF-b/Smad signaling pathway and TGF-bstimulated ECM protein expression.
Anti-fibrotic activity and Inhibitory effect of DMF on the TGF-b/Smad signaling are independent of induction of ARE-driven Nrf2 target genes A growing body of evidence indicates that reactive oxygen species (ROS) mediate TGF-b-induced renal fibrosis, while Nrf2 target genes, such as NQO1 and HO-1, prevent ROS-induced renal fibrosis [29][30][31]. As expected, DMF increased NQO1 and HO-1 mRNA expression in NRK-49F cells ( Figure 5A). Thus, we aimed to investigate the involvement of these antioxidant enzymes in the inhibition of TGF-b/Smad signaling and TGF-b-stimulated ECM protein expression by DMF. To determine whether the induction of NQO1 and HO-1 is required for the suppressive effect of DMF on the TGF-b/Smad signaling pathway, DMFinduced NQO-1 and HO-1 expression was knocked down by siRNAs against NQO-1 (NQO1-siRNA) or HO-1 (HO-1-siRNA) ( Figure 5B). However, neither NQO1 nor HO-1 siRNAs abolished the inhibitory effects of DMF on the TGF-b-stimulated (CAGA) 9 MLP-Luc promoter activity and ECM mRNA expression ( Figure 5C and 5D). In a good agreement with knock-down experiments, ES936 or SnPP, chemical inhibitors of NQO1 or HO-1, respectively did not reverse the suppressive effects of DMF on TGF-b/Smad signaling and ECM expression ( Figure S4). Furthermore, down-regulation of glutathione S-transferase (GST), one of other antioxidant response element (ARE)-dependent Nrf2 target genes using siRNA did not block the inhibition of TGF-bstimulated expression of profibrotic genes by DMF ( Figure S5). Collectively, these data indicated that the effects of DMF on TGFb-stimulated Smad signaling and expression of profibrotic genes

DMF ameliorates UUO-induced renal fibrosis
Finally, an experimental UUO-induced renal fibrosis model was employed to determine whether DMF attenuates renal fibrosis in vivo. As expected, the architecture of the tubules was dramatically altered in UUO kidneys, resulting in the development of interstitial fibrosis.
Expression of Nrf2 and NQO1 was markedly increased in the renal tubules and interstitial area in the DMF-treated UUO kidneys, compared with those of sham-operated or UUO control mice ( Figure 6A). Sirius Red and trichrome staining showed, in comparison with UUO control kidneys, that UUO kidneys of DMF-treated mice exhibited a marked decrease in renal fibrosis seven days after UUO ( Figure 6B). Immunohistochemical staining for type 1 collagen and a-SMA revealed that renal fibrosis in UUO kidneys of DMF-treated mice was less extensive than that in UUO control kidneys ( Figure 6C). Quantitative real-time PCR results showed that UUO markedly increased mRNA levels of a-SMA, fibronectin and type 1 collagen. In contrast, the UUO kidneys of DMF-treated mice showed a significant decrease in expression of all three ECM proteins analyzed ( Figure 6D). Moreover, DMF markedly reduced the UUO-induced nuclear staining of phosphorylated Smad3 in the renal tubular epithelial cells ( Figure 6E). Collectively, these results indicated that DMF prevents the progression of UUO-induced renal fibrosis in mice.

Discussion
This study demonstrated that DMF effectively inhibited TGF-bstimulated profibrotic genes and ECM protein expression in two cultured rat renal cell lines. In addition, DMF inhibited TGF-bstimulated Smad3 phosphorylation and attenuated profibrotic gene and ECM protein expression in UUO-induced kidneys. Furthermore, our results demonstrated that Nrf2 mediated the suppressive effects of DMF on TGF-b-stimulated profibrotic genes and ECM protein expression via an ARE-independent mechanism. Taken together, our data suggest the possibility that DMF could be used for the treatment of renal fibrosis.
As mentioned previously, TGF-b expression is increased in a variety of renal diseases including obstructive nephropathy, and it has been implicated as a major mediator of ECM protein accumulation in diabetic nephropathy and tubulointerstitial fibrosis [9,35,36]. TGF-b phosphorylates Smad2/3, which then translocates into the nucleus where it induces the expression of PAI-1 and ECM proteins such as fibronectin and type 1 collagen. Therefore, the suppression of TGF-b signaling has been included in several therapeutic approaches for preventing renal fibrosis [37,38]. In this study, we found that DMF attenuated TGF-bstimulated profibrotic gene expression via the inhibition of TGF-binduced Smad3 phosphorylation. Moreover, DMF inhibited UUO-induced ECM accumulation in vivo.
It is well known that DMF increases the levels of active Nrf2 [17,23,24]. Our previous study showed that sulforaphane-induced Nrf2 activation effectively inhibited hepatic fibrosis via the inhibition of TGF-b/Smad signaling. These data prompted us to examine whether Nrf2 mediates DMF-induced suppression of profibrotic genes and ECM protein expression in cultured renal cells induced by TGF-b and, as expected, DMF increased Nrf2 protein expression levels in cultured renal cells. Adenovirusmediated overexpression of Nrf2 successfully inhibited TGF-bstimulated profibrotic gene expression by inhibiting the TGF-b/ Smad signaling pathway. Moreover, knockdown of Nrf2 using an siRNA reversed the inhibitory effect of DMF on TGF-b/Smad3 signaling as well as on the profibrotic genes and ECM protein expression. Our previous study demonstrated that Nrf2 interacted physically with the Smad3 protein, and repressed p-Smad3 and the trapping of p-Smad3, in cultured hepatocytes [33]. Consistent with these results, interaction between Nrf2 and Smad3 was confirmed by co-immunoprecipitaiton assay in cultured AD-293 cells ( Figure S6). Additionally, DMF and Ad-Nrf2 inhibited ALK5-stimulated Smad3/4 reporter activity and Smad3 phosphorylation in cultured renal cell lines, implying that Nrf2 negatively regulates signaling molecules downstream of the TGF-b receptor. Taken together, these data suggested that DMF-induced Nrf2 might repress TGF-b-stimulated profibrotic gene and ECM protein expression via direct physical interaction with Smad3.
In recent reports, p62 interacts with the Nrf2-binding site on Keap1 and increased p62 competes with Nrf2 for the interaction with Keap1, resulting in stabilization of Nrf2 followed by transcriptional induction of ARE target genes [34]. In the present study, we found that DMF augmented p62 expression, but this increase in p62 expression by DMF occurred much later than that of Nrf2. Moreover, down-regulation of p62 expression did not affect DMF-induced Nrf2 expression, as well as repression of the TGF-b-stimulated (CAGA) 9 MLP-Luc activity and profibrotic gene expression by DMF. Taken together, Nrf2 activation by DMF is independent of p62 expression.
Increasing evidence indicates that, in addition to the TGF-bstimulated Smad pathway, other signaling pathways, such as ROS-induced redox sensitive transcription factor pathways, are also important in the initiation and progression of renal disease [39]. It is known that TGF-b induces ROS production, which mediates profibrotic responses through Smad-independent pathways [40,41], and thus the antioxidant activities of DMF are likely to act as potential antifibrotic agents. The antioxidant property of DMF functions via the Nrf2-dependent stimulation of antioxidant enzymes such as NQO1 and HO-1 [42] whose induction is reported to prevent the progression of fibrosis and to reverse established renal scarring in UUO rats [43]. On the basis of these data, we examined whether the DMF-induced upregulation of NQO1 and HO-1 was related to the DMF-induced suppression of profibrotic gene expression. However, intriguingly, the inhibition of ARE-driven Nrf2 target genes such as NQO1, HO-1 and GST did not reverse the DMF-induced suppression of the TGF-b/ Smad signaling pathway and profibrotic gene expression, indicating that induction of ARE-driven Nrf2 target genes are not involved in mediating the effects of DMF.
In conclusion, our data demonstrated that the upregulation of Nrf2 by DMF attenuated renal fibrosis via ARE-independent inhibition of the TGF-b/Smad signaling pathway. This study suggests that pharmacologic agents, such as DMF, may provide promising prospects for overcoming the doubtful efficacy and safety of antioxidant or immunomodulatory agents currently used to treat renal fibrosis.

Preparation of recombinant adenovirus
A full length mouse Nrf2 cDNA was inserted into the KpnI and XhoI sites of the pAdTrack-CMV shuttle vector. The recombinant adenoviral plasmid was generated as described previously [44], and recombinant adenoviruses were amplified in HEK-293 cells and subsequently purified.

Transfection of siRNAs, RNA isolation and RT-PCR
For siRNA transfection, 10 nmol/l rat Nrf2-siRNA, 10 nmol/l rat NQO1-siRNA, 10 nmol/l rat HO-1-siRNA and control siRNA duplexes were purchased from the Bioneer Corporation (Table 1). Cells were seeded onto 60 mm plates and simultaneously transfected with Lipofectamine TM RNAiMax reagent (Invitrogen). After incubation for 24 h, cells were starved for 12 h, and then pretreated with DMF for 1 h, cells were stimulated with TGF-b. Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions, and semiquantitative RT-PCR analysis was performed as described previously [44]. An aliquot (1 mg) of total RNA was reverse transcribed using the First Strand cDNA synthesis kit (Fermentas, EU) according to the manufacturer's protocol. The first strand cDNAs were amplified by PCR using gene-specific primers [33] to determine mRNA expression levels. Quantitative real-time PCR (qRT-PCR) was carried out using Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) with the StepOnePlus Real-Time PCR System (Applied Biosystems). The expression levels of b-actin (semi-quantitative RT-PCR) and GAPDH (qRT-PCR) were used as internal controls.

Western blot analysis
Western blot analysis was performed as described previously [44] using specific primary antibodies [anti-p-Smad3 (Ser423/ 425); anti-Smad3 and anti-Smad4 (Cell Signaling, Beverly, MA); anti-type I collagen; anti-a-SMA and anti-b-actin (Abcam, Cambridge, MA); anti-Nrf2 (Santa Cruz Biotechnologies, Santa Cruz, CA); and anti-PAI-1 and anti-fibronectin (BD Biosciences, San Jose, CA). For Western blot analysis of type I collagen, cell lysates were subjected to native polyacrylamide gel electrophoresis (native PAGE: non-heating and non-denaturing conditions). Quantitative analysis of p-Smad3 to total Smad3 ratio was conducted with NIH Image J software.

Ethics statement
All experimental procedures were carried out in accordance with the appropriate institutional guidelines for animal research.

Mouse UUO-induced renal fibrosis model
Eight-week-old male C57BL/6 mice (Hyochang Science, Daegu, Korea) were divided into three groups (sham-operated control, UUO mice and UUO mice treated with 25 mg/kg of DMF, n = 5/group) for each experimental regime. For UUO surgery, the animals were anesthetized with pentobarbital (50 mg/ kg) and the left ureter was doubly ligated. Sham animals underwent the same surgical procedures except for ligation. Following UUO, mice in the DMF-treated group were dosed with DMF (25 mg/kg/day) by gavage daily for seven days. All mice were sacrificed seven days after UUO surgery.

Immunohistochemical analysis
Kidney tissues were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) and paraffin embedded. Kidney sections (4 mm) were deparaffinized in xylene, rehydrated through descending grades of ethanol and subjected to immunohistochemical analysis using antibodies against Nrf2, NQO1 (Santa Cruz), type I collagen, a-SMA and p-Smad3 [33,45]. To evaluate renal fibrosis, sections were stained with Sirius Red (Sigma-Aldrich) and trichrome (Sigma-Aldrich) according to the manufacturer's instructions.

Statistical analysis
Data are expressed as means 6SEM. Statistical analyses were performed using an unpaired Student's t-test and a value of P,0.05 was considered statistically significant. Author Contributions