Abstract
Renal interstitial fibrosis is characterized by increased extracellular matrix (ECM) synthesis. Epithelial-mesenchymal transition (EMT) in kidneys is driven by regulated expression of fibrogenic cytokines such as transforming growth factor-beta (TGF-β). Yam, or Dioscorea alata (DA) is an important herb in Chinese medicine widely used for the treatment of clinical diabetes mellitus. However, the fibrosis regulatory effect of DA is unclear. Thus, we examined TGF-β signaling mechanisms against EMT in rat fibroblast cells (NRK-49F). The characterization of DA water-extracts used various methods; after inducing cellular fibrosis in NRK-49F cells by treatment with β-hydroxybutyrate (β-HB) (10 mM), we used Western blotting to examine the protein expression in the TGF-β-related signal protein type I and type II TGF-β receptors, Smads2 and Smad3 (Smad2/3), pSmad2 and Smad3 (pSmad2/3), Smads4, Smads7, and EMT markers. These markers included E-cadherin, alpha-smooth muscle actin (α-SMA), and matrix metalloproteinase-2 (MMP-2). Bioactive TGF-β and fibronectin levels in the culture media were determined using ELISA. Expressions of fibronectin and Snail transcription factor, an EMT-regulatory transcription factor, were assessed by immunofluorescence staining. DA extract dose-dependently (50–200 µg/mL) suppressed β-HB-induced expression of fibronectin in NRK-49F cells concomitantly with the inhibition of Smad2/3, pSmad2/3, and Smad4. By contrast, Smad7 expression was significantly increased. DA extract caused a decrease in α-SMA (α-smooth muscle actin) and MMP-2 levels, and an increase in E-cadherin expression. We propose that DA extract might act as a novel fibrosis antagonist, which acts partly by down regulating the TGF-β/smad signaling pathway and modulating EMT expression.
Highlights
The incidence of chronic kidney disease (CKD) is rapidly increasing in industrialized countries, partly due to increases in disorders such as obesity, diabetes, and peripheral artery disease [10,21]
We further investigated the fibrosis-regulatory effects of Dioscorea alata (DA) extract on renal fibroblast cells. b-hydroxybutyrate (HB)-induced production of fibronectin and transforming growth factor-beta (TGF-b) provided an in vitro model for renal fibrosis
Because TGF-b is a potent fibrogenic cytokine inducer for renal fibroblasts, it is reasonable to assume that DA extract may contain certain active compounds with potential for fibrosis-inhibition
Summary
The incidence of chronic kidney disease (CKD) is rapidly increasing in industrialized countries, partly due to increases in disorders such as obesity, diabetes, and peripheral artery disease [10,21]. Tubulointerstitial fibrosis is the common pathway in progressive renal disease; it leads to functional deterioration and eventual loss of renal function irrespective of the diverse initial causes [12,24,25]. Tubulointerstitial fibrosis is involved in the accumulation of extracellular matrix components and loss of tubular architecture. Proximal tubular epithelial cells play a central role in renal tubulointerstitial fibrosis [1,3]. A critical step in the pathogenesis of tubulointerstitial fibrosis is epithelial mesenchymal transition (EMT), whereby renal tubular epithelial cells change phenotypically and functionally into myofibroblasts [26]. The occurrence of EMT in the kidneys provides a significant therapeutic target; it is important to prevent tubular epithelial cells from undergoing EMT to prevent tubulointerstitial fibrosis
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