Abstract
Purpose Hirudin, a polypeptide structure containing 65 amino acids, is a potent natural thrombin inhibitor with anticoagulant property extracted from Hirudo medicinalis. It has been reported to have anti-inflammatory and antifibrotic property. Here we explored the renoprotective effect of hirudin on unilateral ureteral obstruction (UUO) induced renal interstitial fibrosis (RIF). Methods Rats were randomly divided into five groups: sham group, UUO alone group, and three UUO + hirudin-treatment groups (10, 20, or 40 IU/kg/d, for 14 continuous days). At the end of the experiment period, animals were sacrificed. Pathologic changes in renal specimens were observed using hematoxylin and eosin (HE) staining and Masson staining. The expressions of collagen III (Col III), fibronectin (FN), α-smooth muscle actin (α-SMA), protease-activated receptor 1 (PAR-1), and proteins in the TGF-β1/Smad and NF-κB pathways in renal tissues were examined by immunohistochemistry and/or Western blotting. Results HE and Masson staining showed that hirudin-treated UUO rats had lower extent of renal injury and deposition of extracellular matrix (ECM) in renal interstitium than those in the UUO group. The results of immunohistochemistry and WB indicated decreased protein expressions of Col III, FN, α-SMA, PAR-1, and inflammatory markers such as tumor necrosis factor-α and interleukin-6 after hirudin treatment. Furthermore, hirudin reduced the expressions of transforming growth factor β1 (TGF-β1), phosphorylated-Smad2, and phosphorylated-Smad3 in the UUO model. In parallel, we found inhibited nuclear factor-κB (NF-κB) signaling after hirudin treatment, with downregulated protein expressions of P65, phosphorylated-P65, and phosphorylated-iκBα and increased iκBα. Conclusion Hirudin improves kidney injury and suppresses inflammatory response and ECM accumulation in UUO rats; its underlying mechanism may be associated with the inhibition of TGF-β1/Smad and NF-κB signaling.
Highlights
Renal interstitial fibrosis (RIF) characterized by the excessive deposition of extracellular matrix (ECM), alteration of tubular structure, and activation of fibroblasts is a common pathological pathway resulting in end-stage renal disease
Its activation has been reported to be involved in the process of inflammation and fibrosis [12,13,14]. e role of protease-activated receptor 1 (PAR-1) in RIF has been suggested by Waasdorp et al who demonstrated that PAR-1 knockdown ureteral obstruction (UUO) model could attenuate the accumulation of fibroblasts, macrophages, and relevant fibrosis response [15]
UUO rats were characterized by severe tubular dilatation or atrophy, interstitial fibrosis and inflammatory cell infiltration (HE staining), and substantial deposition of collagens in the renal interstitium (Masson staining), and such histological changes were obviously alleviated in hirudin-treated groups; semiquantitative positive area analysis further confirmed these observations (Figures 1(c)–1(e))
Summary
Renal interstitial fibrosis (RIF) characterized by the excessive deposition of extracellular matrix (ECM), alteration of tubular structure, and activation of fibroblasts is a common pathological pathway resulting in end-stage renal disease. Transforming growth factor β1 (TGF-β1)/Smads signaling plays an important role in activating myofibroblasts with excessive extracellular matrix (ECM) production in tubular interstitial [1, 2]. We believe that inhibiting TGF-β1/Smads and NF-κB signaling is beneficial in preventing or delaying the progression of RIF, at least in part, by restraining the accumulation of ECM and occurrence of inflammation induced by unilateral ureteral obstruction (UUO). Bao et al showed that hirudin can reduce the release of TNF-α and matrix metalloproteinase 12 by inhibiting the activation of PAR-1 in lipopolysaccharide induced lung injury rats [16]. We explored the potentially antifibrotic effect of hirudin on RIF in a well-established UUO renal fibrosis rats model and examined components in the TGF-β and NFκB signaling pathway to elucidate the underlying molecular mechanisms
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