miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor β1 (TGF-β1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-β1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-β1–induced α-SMA expression. These data demonstrate that TGF-β1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-β1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.     

Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells

Unregulated activity of myofibroblasts, highly contractile cells that deposit abundant extracellular matrix (ECM), leads to fibrosis. To study the modulation of myofibroblast activity, we used human adipose-derived mesenchymal stem cells (ADSCs), which have much potential in regenerative medicine. We found that ADSCs treated with TGF-β developed a myofibroblastic phenotype with increases in α-smooth muscle actin (α-SMA), a myofibroblast marker, and ECM proteins type I collagen and fibronectin. In contrast, treatment with bFGF had the opposite effect. bFGF-differentiated ADSCs showed marked down-regulation of α-SMA expression, collagen I, and fibronectin, and loss of focal adhesions and stress fibers. Functionally, bFGF-differentiated ADSCs were significantly more migratory, which correlated with up-regulation of tenascin-C, an anti-adhesive ECM protein, and vimentin, a pro-migratory cytoskeletal protein. On the other hand, TGF-β-differentiated ADSCs were significantly more contractile than bFGF-differentiated cells. Interestingly, cells completely reversed their morphologies, marker expression, signaling pathways, and contractility versus migratory profiles when switched from culture with one growth factor to the other, demonstrating that the myofibroblast differentiation process is not terminal. Cell differentiation was associated with activation of Smad2 downstream of TGF-β and of ERK/MAP kinase downstream of bFGF. Reversibility of the TGF-β-induced myofibroblastic phenotype depends, in part, on bFGF-induced ERK/MAP kinase signaling. These findings show that ADSC differentiation into myofibroblasts and re-differentiation into fibroblast-like cells can be manipulated with growth factors, which may have implications in the development of novel therapeutic strategies to reduce the risk of fibrosis.     

Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-β1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition.

Methods

To identify the mechanism of Arsenic trioxide’s (ATO)’s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-β1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen.

Results

Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-β1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-β1-mediated contractile response in NHLFs. ATO’s down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-β1-induced H₂O₂ and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-β1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression.

Conclusions

In summary, these data indicate that low concentrations of ATO inhibit TGF-β1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.     

The AMPK Agonist AICAR Inhibits TGF-β1 Induced Activation of Kidney Myofibroblasts

Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis in vivo and TGF-β1-induced renal fibroblasts activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-β1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-β1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-β1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-β1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-β1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.     

Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist.     

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.

Measurements and Main Results

In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.

Conclusions

We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.     

Macitentan inhibits the transforming growth factor-β profibrotic action,blocking the signaling mediated by the ETR/TβRI complex in systemic sclerosis dermal fibroblasts

IntroductionSystemic sclerosis (SSc) is a complex and not fully understood autoimmune disease associated with fibrosis of multiple organs. The main effector cells, the myofibroblasts, are collagen-producing cells derived from the activation of resting fibroblasts. This process is regulated by a complex repertoire of profibrotic cytokines, and among them transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) play a major role. In this paper we show that TGF-β and ET-1 receptors co-operate in myofibroblast activation, and macitentan, an ET-1 receptor antagonist binding ET-1 receptors, might interfere with both TGF-β and ET-1 pathways, preventing myofibroblast differentiation.MethodsFibroblasts isolated from healthy controls and SSc patients were treated with TGF-β and ET-1 and successively analyzed for alpha smooth muscle actin (α-SMA) and collagen (Col1A1) expression and for the Sma and Mad Related (SMAD) phosphorylation. We further tested the ability of macitentan to interfere with these process. Furthermore, we silenced ET-1 and endothelin-1 receptor A expression and evaluated the formation of an ET-1/TGF-β receptor complex by immunoprecitation assay.ResultsWe showed myofibroblast activation in SSc fibroblasts assessing the expression of α-SMA and Col1A1, after stimulation with TGF-β and ET-1. Macitentan interfered with both ET-1- and TGF-β-induced fibroblast activation. To explain this unexpected inhibitory effect of macitentan on TGF-β activity, we silenced ET-1 expression on SSc fibroblasts and co-immunoprecipitated these two receptors, showing the formation of an ET-1/TGF-β receptor complex.ConclusionsDuring SSc, ET-1 produced by activated endothelia contributes to myofibroblast activation using TGF-β machinery via an ET-1/TGF-β receptor complex. Macitentan interferes with the profibrotic action of TGF-β, blocking the ET-1 receptor portion of the ET-1/TGF-β receptor complex.     

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.     

Myofibroblast Differentiation and Enhanced Tgf-B Signaling in Cystic Fibrosis Lung Disease

Rationale

TGF-β, a mediator of pulmonary fibrosis, is a genetic modifier of CF respiratory deterioration. The mechanistic relationship between TGF-β signaling and CF lung disease has not been determined.

Objective

To investigate myofibroblast differentiation in CF lung tissue as a novel pathway by which TGF-β signaling may contribute to pulmonary decline, airway remodeling and tissue fibrosis.

Methods

Lung samples from CF and non-CF subjects were analyzed morphometrically for total TGF-β₁, TGF-β signaling (Smad2 phosphorylation), myofibroblast differentiation (α-smooth muscle actin), and collagen deposition (Masson trichrome stain).

Results

TGF-β signaling and fibrosis are markedly increased in CF (p<0.01), and the presence of myofibroblasts is four-fold higher in CF vs. normal lung tissue (p<0.005). In lung tissue with prominent TGF-β signaling, both myofibroblast differentiation and tissue fibrosis are significantly augmented (p<0.005).

Conclusions

These studies establish for the first time that a pathogenic mechanism described previously in pulmonary fibrosis is also prominent in cystic fibrosis lung disease. The presence of TGF-β dependent signaling in areas of prominent myofibroblast proliferation and fibrosis in CF suggests that strategies under development for other pro-fibrotic lung conditions may also be evaluated for use in CF.     

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin α_vβ₅-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin α_vβ₅, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin α_vβ₅ interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin α_vβ₅-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-α_vβ₅ interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin α_vβ₅ interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin α_vβ₅. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.     

Adiponectin Attenuates Lung Fibroblasts Activation and Pulmonary Fibrosis Induced by Paraquat

Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β₁, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β₁, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.     

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

Background

Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.

Methods

TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.

Results

The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.

Conclusions

TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.     

Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.     

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.