Periostin-binding DNA aptamer treatment ameliorates peritoneal dialysis-induced peritoneal fibrosis

Abstract

Background: Peritoneal fibrosis is a major complication, leading to peritoneal structural changes and ultrafiltration failure in peritoneal dialysis (PD) patients. The epithelial-mesenchymal transition (EMT) of the peritoneal mesothelial cells (PMCs) and peritoneal accumulation of extracellular matrix (ECM) proteins are key features of peritoneal fibrosis in which transforming growth factor (TGF)-β1 is known to play a crucial role. Periostin, an ECM protein increased by the activation of the TGF-β1 pathway, induces the expression of ECM genes, such as collagen and fibronectin, by activating integrin-related intracellular signaling pathways. Accumulating evidence has suggested that periostin expression is related to the development of various fibrotic disorders. Recently, a number of studies have demonstrated that aberrant periostin expression is accompanied in PD-related peritoneal fibrosis. Therefore, regulating the expression and function of periostin in human peritoneal mesothelial cells (HPMCs) may have an effect on the development and progression of peritoneal fibrosis in PD patients. Purpose: This study was undertaken to evaluate the impact of periostin inhibition by a novel aptamer-based inhibitor on TGF-β1-induced EMT in cultured HPMCs. In addition, the effects of the periostin-binding aptamer on EMT and peritoneal fibrosis were also investigated in an animal model of PD. Methods and Materials: To regulate the functional role of periostin in HPMCs, periostin-binding DNA aptamer that was generated to target human periostin was used. For in vitro studies, primary HPMCs were treated with TGF-β1 (2 ng/ml) to induce EMT. The role of periostin in TGF-β1-induced EMT in cultured HPMCs was evaluated by periostin siRNA transfection (100 nM). To validate whether periostin-binding DNA aptamer specifically targets periostin, the fluorescence intensity of Cy3-tagged periostin-binding DNA aptamer was assessed by fluorescence-activated cell sorting analysis. The effect of regulating periostin function through periostin-binding DNA aptamer on EMT and fibrosis was also evaluated by treating TGF-β1-stimulated HPMCs with periostin-binding DNA aptamer. In vivo, PD catheters were inserted into 48 C57BL/6 mice, and these mice were infused intraperitoneally for 4 weeks with either saline (C group, n=12), saline with periostin-binding DNA aptamer (C+periostin aptamer group, n=12), 4.25% PD solution (PD group, n=12), or 4.25% PD solution with periostin-binding DNA aptamer (PD+periostin aptamer group, n=12). After 4 weeks, the mice were sacrificed and the peritoneal tissues were removed. The mRNA levels and protein expression of periostin, fibronectin, α-smooth muscle actin (α-SMA), Snail, and E-cadherin in HPMCs and the mouse peritoneum were evaluated by quantitative real-time polymerase chain reaction and western blotting, respectively. Peritoneal fibrosis was assessed by Masson’s trichrome staining. Results: In vitro, TGF-β1 treatment significantly up-regulated periostin, fibronectin, α-SMA and Snail expression, while it reduced E-cadherin expression in HPMCs (P < 0.01). When Cy3-tagged periostin-binding DNA aptamer was introduced to HPMCs, the Cy3 fluorescence activity was significantly increased in TGF-β1-stimulated HPMCs. In contrast, the activity was significantly decreased in periostin-knocked down HPMCs. Periostin siRNA treatment ameliorated TGF-β1-induced periostin, fibronectin, α-SMA, and Snail expression and restored E-cadherin expression in HPMCs (P < 0.05). Similarly, periostin-binding DNA aptamer also attenuated fibronectin, α-SMA, and Snail up-regulation and E-cadherin down-regulation in TGF-β1-stimulated HPMCs (P < 0.05). In vivo, in mice treated with PD solution for 4 weeks, the expression of periostin, fibronectin, α-SMA, and Snail was significantly increased in the peritoneum, while the E-cadherin expression was significantly decreased (P < 0.05). The thickness of submesothelial layer and the intensity of Masson’s trich...