Arctigenin suppresses renal interstitial fibrosis in a rat model of obstructive nephropathy
Graphic abstract
Introduction
Chronic kidney disease (CKD) has emerged as a worldwide public health problem with a rapid growth in prevalence (Couser et al., 2011). Progressive tubulointerstitial fibrosis (TIF) is the common pathological presentation of nearly all kinds of CKD leading to end-stage renal failure (Liu, 2006). Although tremendous efforts have been made to prevent or retard the progression of TIF, specific drug therapies to delay the progression of TIF toward end-stage renal failure are limited.
CKD can initially manifest as inflammatory responses characterized by infiltration of immune cells, mainly monocytes/macrophages and T lymphocytes, into the glomeruli and tubulointerstitium (Lopez-Novoa and Nieto, 2009). On one hand, these inflammatory cells secrete various pro-inflammatory cytokines and chemokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1), which further contribute to recruitment of circulating inflammatory cells. This creates a malignant positive feedback loop of inflammation (Diamond et al., 1994). On the other hand, activated macrophages produce profibrotic cytokines such as transforming growth factor-β1 (TGF-β1), which have been shown to induce activation of matrix-producing myofibroblasts (Leask and Abraham, 2004). The stimulated myofibroblasts cause accumulation of extracellular matrix (ECM) proteins and lead to complete destruction of renal parenchyma and irreversible renal failure. Renal inflammation—and in particular the accumulation of macrophages in renal interstitium—are a critical element in the mechanism responsible for the initiation and development of renal fibrogenesis. Therefore, inhibiting inflammatory responses may significantly attenuate renal TIF.
Recent studies show that a significant portion of synthetically active myofibroblasts arise from renal tubular epithelial cells (TECs) via the epithelial-mesenchymal transition (EMT) in fibrotic kidney diseases (Liu, 2010). The pro-fibrogenic effect of inflammation depends, at least partially, on triggering EMT (Wynn, 2008). Several studies have shown that sustained stimulation of pro-inflammatory cytokines TNF-α or IL-1 can induce EMT in the epithelial cell lines (Takahashi et al., 2010). During EMT, differentiated TECs lose their epithelial characteristics and undergo multiple biochemical changes, which enable them to assume a mesenchymal phenotype. This phenotypic conversion involves the de novo synthesis of mesenchymal cytoskeletal biomarkers such as α-smooth muscle actin (α-SMA), fibronectin and vimentin, a down-regulation of epithelial biomarkers such as E-cadherin and zonula occludens-1, and the acquisition of a fibroblastic morphology with a concomitant invasive phenotype. TGF-β1 has been identified as the main inducer of EMT and consequent interstitial ECM production in kidney and other organ systems (Acloque et al., 2009, Pan et al., 2015). The intracellular Smad pathway is important for TGF-β1 to initiate tubular EMT and fibrotic responses (Lan and Chung, 2011). To prevent progression of TIF, the inhibition of tubular EMT mediated by TGF-β1/Smad signaling pathway may be helpful.
Unilateral ureteral obstruction (UUO) is a well-established experimental model used to elucidate pathological mechanisms of chronic obstructive nephropathy (Chevalier et al., 2009). Oxidative stress plays a central role in the progression of renal damage in obstructed nephropathy (Kawada et al., 1999). Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the capacity of intrinsic antioxidant defense. In UUO kidneys, the superoxide anion (O2•−) and its derivative hydrogen peroxide (H2O2) are increased, while the antioxidant enzyme catalase (CAT) and copper-zinc superoxide dismutase (SOD1) mRNA correspondingly decreased (Ricardo et al., 1997). Increased ROS concentrations can cause tubulointerstitial injury by increasing lipid peroxidation, hydrogen peroxides, leukocyte activation, DNA breakdown, protein oxidation, and apoptosis. Furthermore, ROS generated in TECs induce nuclear factor kappa B (NF-κB) activation (Gloire et al., 2006) leading to transcription of some pro-inflammatory mediators such as MCP-1, TNF-α, INF-γ, and IL-1β, infiltration of monocytes/macrophages, proliferation of fibroblasts, and ECM accumulation in the renal interstitium. Initial oxidative stress and inflammation contribute to the progression of renal fibrosis. Thus, antioxidant and/or anti-inflammatory agent have been used to protect the kidney from injury induced by UUO.
Burdock (Arctium lappa L.) has been used in traditional and folk medicine in oriental countries for centuries. Various experimental models have shown that arctigenin (ATG) is a main bioactive ingredient derived from dried fruit of A. lappa. ATG exhibits anti-inflammatory (Hyam et al., 2013), anti-oxidant (Zhang et al., 2015), and antineoplastic (Awale et al., 2006) properties in various disorders. Our preliminary study demonstrated that oral administration of 95% (v/v) hydroalcoholic extract from A. lappa containing a higher amount of ATG and its glycoside Arctiin versus aqueous and petroleum ether extracts, exhibited the strongest inhibitory effect on ECM component synthesis in the obstructive kidneys of rats with UUO (Supplementary Fig. S1). Our previous data also showed that ATG could reverse TGF-β1-triggered renal tubular EMT-like phenotypic changes in vitro using human proximal tubular epithelial cells (HK-2 cells)—mainly due to the inhibition of TGF-β1-induced up-regulation of MCP-1 (Li et al., 2015). Because the regulatory role of ATG in renal fibrosis in vivo has not yet been studied, we studied here a 14-day UUO rat model to assess the anti-fibrotic efficacy of ATG and further delineated the potential molecular mechanisms by which ATG elicits its effects on experimental renal fibrosis.
Section snippets
Chemicals
Arctigenin (MW: 372.41) was provided by Nanjing Zelang Medical Technology Co. Ltd. (Nanjing, China). The purity of ATG was determined to be > 8% using high performance liquid chromatography (HPLC; Supplementary Fig. S2). Losartan was purchased from Merck Sharp & Dohme Ltd. (Hangzhou, China). Antibodies against the following proteins were used: NF-κB p65 (Signalway Antibody, Pearland, TX, USA); fibronectin (BD Biosciences, San Jose, CA, USA); Smad2/3, phosphorylated Smad2/3 (p-Smad2/3) and
ATG attenuated progressive fibrosis in the obstructed kidneys induced by UUO
To investigate whether ATG can suppress renal TIF in vivo, we examined interstitial fibrotic lesions and collagen accumulation in UUO kidneys. As shown in Fig. 1A, compared with sham-operated controls, the UUO caused obvious tubulointerstitial injury consisting of tubular dilatation and epithelial atrophy, interstitial inflammatory cell infiltration, and a marked dilation of interstitial spaces. This interstitial expansion was accompanied by a noticeable increase in collagen deposition as shown
Discussion
Renal TIF has emerged as a common feature of progressive CKD because it eventually progresses to end-stage renal failure requiring dialysis or kidney transplantation (Liu, 2006). The activation of inflammatory cascade is an early and characteristic feature of CKD. It is widely accepted that sustained inflammation initiates the progression of renal injury in CKD, which eventually leads to the destruction and collapse of the renal parenchyma replaced by fibrotic scar tissues. An intrinsic
Conclusion
For the first time, we evaluate the anti-fibrotic effect of ATG using the UUO model. We found that ATG could attenuate tubular injuries and accumulation of ECM proteins in the kidneys of UUO rats. We also noted that ATG could suppress the infiltration of macrophages via inactivation of NF-κB pathway in the progression of renal TIF while inhibiting pro-inflammatory cytokine (TNF-α, IL-1β, and INF-γ) and chemokine (MCP-1) release. Meanwhile, ATG could block the TGF-β1-induced tubular EMT process,
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81001675), Macao Science and Technology Development Fund (No. 007/2014/AMJ), Research Committee of the University of Macau (No. MYRG2014-00089-ICMS-QRCM), Chongqing Natural Science Foundation (No. cstc2014jcyjA10030).
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These authors contributed equally to this work.