探討內質網蛋白TXNDC5在甲基乙二醛誘發之腹膜纖維化扮演的角色

Abstract

腹膜透析利用腹膜充當半透膜以過濾血液中的廢物和多餘水分，為末期腎病患者的腎臟替代療法之一。然而，腹膜透析液中所含之高濃度葡萄糖會於透析過程產生葡萄糖降解產物，長期暴露於此物質會導致腹膜纖維化。腹膜纖維化會導致腹膜間皮細胞的脫落以及間皮下層的增厚，進而改變腹膜功能並最終造成腹膜透析的超過濾功能喪失。目前雖然已知造成腹膜纖維化的主要機制是由TGF-β及其相關路徑引起的，但對於腹膜透析所導致的腹膜纖維化尚無有效的治療方法。最近，內質網蛋白TXNDC5已被發現可調控心臟、肺、腎和肝之纖維化，因此也被認為可能參與腹膜纖維化的發生。本研究的目的是探討TXNDC5在腹膜透析所導致之腹膜纖維化中的角色。透過腹膜厚度的顯著增加、TGF-β1、膠原蛋白 I 和 α-SMA 蛋白質表現量上升，可得知此甲基乙二醛誘導的wild-type C57BL/6 小鼠腹膜纖維化模型被成功地建立。此外，TXNDC5蛋白質與基因表現在甲基乙二醛誘導的腹膜中也顯著的增加。然而，剔除Txndc5並無法緩解由甲基乙二醛誘導的腹膜纖維化，我們仍觀察到增厚的腹膜及高表現量之α-SMA。次世代定序數據亦顯示，Txndc5剔除不會抑制與甲基乙二醛誘導之腹膜纖維化相關的基因與訊息傳遞路徑，這表明在腹膜透析所導致之腹膜纖維化也許透過不同於其他器官的TGF-β路徑。此外，在wild-type 及Txndc5-/-小鼠中皆有觀察到同時呈現α-SMA及cytokeratin雙重染色的間皮細胞，並且次世代定序分析結果顯示在甲基乙二醛下誘導之小鼠具上升之Snail基因表現量，間接指出發生間皮間質轉化的可能性。在甲基乙二醛誘導的小鼠腹膜上，觀察到Col1a1-GFP陽性細胞的增加，然而人類間皮細胞線在甲基乙二醛的處置下沒有表現出膠原蛋白表現量的變化。綜上所述，雖然TXNDC5 在甲基乙二醛誘導的wild-type小鼠腹膜纖維化中具高表現量，但是Txndc5剔除未能減緩甲基乙二醛誘導之腹膜纖維化。因此未來仍需要更多的研究探討TXNDC5在腹膜纖維化與其他實質器官纖維化之間的差異。

Peritoneal dialysis (PD) is one of the kidney replacement therapies that maintain the lives of patients with end-stage renal disease (ESRD), where the peritoneum acts as a semipermeable membrane to filter waste products and excess water from the blood. However, the high glucose contained in the PD fluid (PDF) leads to the formation of glucose degradation products (GDPs), which can cause peritoneal fibrosis (PF). PF denudes mesothelial cells from the basement membrane and thickens the sub-mesothelial compact zone, which changes the peritoneal function and results in ultrafiltration failure. Mesothelial-to-mesenchymal transition (MMT), a process that considers mesothelial cells as the main progenitors of myofibroblasts in PF, has been doubted in these years. In addition, though PF is known to be mediated by TGF-β and its associated pathways, no effective treatment can be applied. Recently, an endoplasmic reticulum protein, thioredoxin domain-containing protein 5 (TXNDC5), has proven to be a novel mediator in cardiac, pulmonary, renal, and hepatic fibrosis and is also considered involved in PF. The purpose of this study is to investigate the involvement of TXNDC5 in PF. In this study, methylglyoxal (MGO) successfully induced PF as identified by significantly increased peritoneal thickness and the elevation of TGF-β1, collagen I and α-SMA protein expression in wild-type C57BL/6 mice. Furthermore, TXNDC5 expression was upregulated in the MGO-induced peritoneum. However, the deletion of Txndc5 did not ameliorate the MGO-induced PF, as the thickened sub-mesothelial compact zone with collagen accumulation and high α-SMA expression were still observed. Next-generation sequencing (NGS) data revealed that Txndc5 knockout did not suppress genes associated with MGO-induced peritoneal fibrosis, suggesting distinctive signaling pathways that differed from other organs might be involved in MGO-induced PF. In addition, mesothelial-to-mesenchymal transition (MMT), a process that considers mesothelial cells as the main progenitors of myofibroblasts, might occur upon MGO-induced peritoneal fibrosis, as evidenced by double positive cells of cytokeratin and α-SMA shown in immunofluorescence staining. This is further supported by RNAseq analysis showing an increased Snail gene expression. An increase in Col1a1-GFP positive cells was also observed on the surface of the MGO-induced peritoneum in mice. However, MeT5A cells, a human mesothelial cell line, did not show changes in collagen I protein expression under MGO treatment in vitro. In conclusion, apart from the high expression of TXNDC5 in MGO-induced peritoneal fibrosis in wild-type mice, the deletion of Txndc5 failed to prevent MGO-induced PF. Further studies are needed to explore the differences in TXNDC5 involvement between PF and fibrosis in other solid organs.