Peritonitis induces native and EDA+ fibronectin synthesis in human peritoneal mesothelial cells through PI3K and MAPK activation

Abstract

BACKGROUND: Peritonitis is a severe complication of peritoneal dialysis (PD), which could lead to progressive structural and functional deterioration of the peritoneum and PD failure. EDA+ fibronectin (FN) is induced during disease states, but its expression in PD associated peritonitis has not been investigated. We examined peritoneal expression of native and EDA+ FN in an experimental model of peritonitis and their association with mesothelial epithelial-to mesenchymal transition. METHODS: Male C57BL/6 mice were challenged with PBS or lipopolysaccharide (LPS, 500mg) by intraperitoneal injection for 2, 3 and 6 times (n=6), after which time the parietal peritoneum was excised for further studies. Confluent, growth arrested human peritoneal mesothelial cells (HPMC) were stimulated with spent peritonitis PD fluid, LPS, or exogenous TGF-β1 or CTGF (growth factors that are increased during peritonitis) either alone or in combination, for periods up to 72h to investigate their effect on cell morphology, and FN, collagen and SNAIL synthesis. RESULTS: Mice exposed to six, but not fewer, challenges of LPS exhibited mesothelial denudation, influx of infiltrating cells and substantial submesothelial thickening attributed to increased collagen and native and EDA+ FN deposition. Peritonitis PD fluid induced phenotypic changes, and SNAIL, collagen I, and native and EDA+ FN synthesis in HPMC. Exogenous TGF-β1, but not CTGF, significantly increased native and EDA+ FN by 4.3- fold and 6.2-fold respectively (P < 0.05 for both). Cells co-stimulated with TGF-β1 and CTGF showed synergistic increase of native and EDA+ FN. This was mediated in part through PI3K, ERK and p38 MAPK activation. TGF-β1 and CTGF neutralizing antibody significantly decreased native and EDA+ FN synthesis, but had no effect on collagen I or SNAIL expression in HPMC. CONCLUSIONS: Our data demonstrated that peritonitis, especially when recurrent, induced progressive peritoneal deposition of collagen, and native and EDA+ FN. TGF-β1 and CTGF synergistically induced native and EDA+ FN synthesis in HPMC, and play important roles in peritoneal fibrosis induced by bacterial peritonitis. Funding: Government Support - Non-U.S.