Protein Transduction Domain Recombinant Bone Morphogenetic Protein-7 Attenuates Peritoneal Dialysis-Related Epithelial-Mesenchymal Transition of Peritoneal Mesothelial Cells

Abstract

Background: Epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) has a role in the process of peritoneal fibrosis, a serious complication in peritoneal dialysis (PD) patients. Bone morphogenetic protein-7 (BMP-7) is known as an EMT inhibitor. Accumulating evidences showed BMP-7 prevented and reversed high-glucose induced EMT on HPMCs, in association with reversal of peritoneal fibrosis. However, because of the short half-life of recombinant human BMP-7 (rhBMP-7), mainly due to rapid clearance and enzymatic degradation, large amount of this protein is needed to use as therapeutic purpose. On the other hand, adenovirus-mediated transfection of BMP-7 has virus-related toxicity and difficulty to titrate. Therefore, the new solution is needed to overcome these limitations in clinical setting. Many studies have been suggested that protein transduction domains (PTDs) were attractive delivery tool for protein delivery into cells. Purpose: The aim of this study was to produce a BMP-7 polypeptide-based on a PTD to transduce the protein into cells followed by furin-mediated protein cleavage and secretion of active form out of cells. In addition, this study was undertaken to evaluate the impacts of PTD-BMP-7 on TGF-β1-induced EMT in cultured HPMCs. Furthermore, the effects of PTD-BMP-7 on EMT and peritoneal fibrosis were investigated in animal models of PD. Materials and Methods: Tissue regenerative polypeptide 2 (TRP2) consisted of PTD, tissue regenerative domain, furin activation domain, and BMP-7 were generated for an indirect delivery system of BMP-7. For in vitro studies, HPMCs were treated with TGF-β1 (2 ng/ml) to induce EMT. The effects of TRP2 on EMT and fibrosis were evaluated by treating TGF-β1-stimulted HPMCs with TRP2 (100 ng/ml). In vivo, PD catheters were inserted into 18 Sprague-Dawley (SD) rats, and these rats were infused intraperitoneally for 4 weeks with either saline (control group, n = 6), 4.25% PD fluid (PD group, n = 6), or 4.25% PD fluid with TRP2 (30 μg/kg per week) (PD+TRP2 group, n = 6). After 4 weeks, these rats were sacrificed and the peritoneal tissues were removed. EMT and fibrosis-related molecules such as type I collagen, fibronectin, α-smooth muscle actin (α-SMA), Snail, and E-cadherin in HPMCs and the rat 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 fibronectin, α-SMA, and Snail expression, while it reduced E-cadherin expression in HPMCs (P < 0.01). TRP2 treatment ameliorated TGF-β1-induced fibronectin, α-SMA, and Snail expression and restored E-cadherin expression in HPMCs (P < 0.05). In vivo, in rats treated with PD solution for 4 weeks, the expression of fibronectin, α-SMA, and Snail was significantly increased in the peritoneum, while E-cadherin expression was significantly decreased (P < 0.05). The thickness of submesothelial layer and the intensity of Masson’s trichrome staining of the PD group were significantly increased compared to the control group. These changes were significantly abrogated by the intraperitoneal administration of TRP2. Conclusion: These findings suggest that TRP2 be an effective therapeutic method for peritoneal fibrosis in PD patients.