Emodin ameliorates glucose‐induced fibronectin synthesis in human peritoneal mesothelial cells by inhibiting PKC‐α activation

Abstract

Introduction Peritoneal dialysis (PD) is recognized as an effective treatment for patients with end‐stage renal failure. Conventional PD solutions are unphysiological, as they contain bio‐incompatible concentrations of glucose to provide the osmotic drive (Popovich et al. 1978) Thus, with increasing duration on PD, the peritoneum undergoes progressive structural and functional deterioration. Diabetiform alterations of the peritoneum, for example, the reduplication of the basement membrane, and increased matrix accumulation and deposition within the submesothelium are commonly observed in PD patients. These changes are mediated in part through activation of PKC and induction of TGF‐β1 (Ha et al. 2001). Emodin (3‐methyl‐1,6,8 trihydroxyanthraquinone) has previously been demonstrated to reduce cell proliferation and fibronectin synthesis in cultured mesangial cells (Kuo et al. 2001; Yao et al. 1994). How emodin modulates glucose‐induced fibronectin synthesis in human peritoneal mesothelial cells (HPMCs) has not been elucidated and thus constitutes the aim of this study.Materials and methods Confluent growth‐arrested HPMCs were cultured under physiological (5 mm) or elevated (30 mm) d‐glucose concentrations in the presence or absence of emodin (20 µg/ml) for periods up to 72 h. PKC‐α activation in HPMCs cultured under control and experimental conditions was determined by Western blot analysis of cytosolic and cell membrane extracts. Synthesis of fibronectin and TGF‐β1 was investigated by a combination of ELISAs, immunohistochemistry and Western blot analysis. Mannitol was used as the hexose control.Results Exposure of HPMCs to 30 mm d‐glucose resulted in the activation of PKC‐α commencing 12 h after stimulation, as demonstrated by the translocation of PKC‐α from the cytosol to the cell membrane. Maximum induction of PKC‐α was observed 48 h after stimulation and increased 1.4‐fold and 3.7‐fold in the cytosolic and membrane fractions, respectively, compared to 5 mm d‐glucose. Activation of PKC‐α was accompanied by the induction of TGF‐β1 secretion in a time‐dependent manner (3.72 ± 0.29 and 4.30 ± 0.50 pg/µg cellular protein at 24 h and 48 h, respectively, for 30 mm d‐glucose compared to 2.13 ± 0.23 and 2.65 ± 0.32 pg/µg cellular protein at 24 h and 48 h, respectively, for 5 mm d‐glucose, P < 0.001 at both time points) and increased fibronectin synthesis. Whilst emodin had no effect on constitutive PKC‐α, TGF‐β1 or fibronectin synthesis, emodin abrogated elevated glucose stimulation of PKC‐α, and decreased TGF‐β1 secretion and fibronectin synthesis to basal levels.Discussion Our findings demonstrate that emodin can ameliorate the undesirable effects of concentrated glucose on HPMCs through the suppression of PKC‐α activation. This study suggests that emodin may have therapeutic potential in the prevention or treatment of glucose‐induced structural and functional abnormalities in the peritoneal membrane.