Regulation of CCN2/CTGF and related cytokines in cultured peritoneal cells under conditions simulating peritoneal dialysis

Abstract

Continuous ambulatory peritoneal dialysis (CAPD) is a major treatment modality for end-stage renal failure. The peritoneal membrane exhibits pathological changes that correlate with the duration of dialysis. These changes are due to the exposure of the peritoneum to non-physiologic peritoneal dialysis solution (PDS) with a high glucose content, and containing potentially toxic substances including glucose degradation products (GDP) and advanced glycation end products (AGE). Connective tissue growth factor (CTGF/CCN2) is one of the determinants of progressive fibrosis and peritoneal membrane dysfunction in CAPD. In this study, we examined the CCN2 expression and its regulation in peritoneal resident cells using a cell culture model. The expression of transforming growth factor-beta (TGF-beta), CCN2 and vascular endothelial growth factor (VEGF) in human peritoneal mesothelial cells (HPMC), human peritoneal fibroblasts (HPF) or endothelial cell line EA.hy926 (EC) cultured with various PDS and their components was examined by quantitative PCR (qPCR). The modulation of CCN2 synthesis under the crosstalk between HPMC and HPF or EC was examined using a conditioned medium transfer system in which HPMC was exposed to conditioned media obtained from HPF or EC incubated with PDS and their components. The differential effects of TGF-beta, CCN2 and VEGF in inducing the expression of transcriptional factors as well as interleukin-6 (IL-6), matrix metallopeptidase 9 (MMP-9) and collagen I were examined by electrophoretic mobility-shift assay (EMSA) and qPCR. PDS and their components differentially modulated the expression of TGF-beta, CCN2 and VEGF in HPMC, HPF and EC. The expression of CCN2 by HPMC was significantly increased after cultured with a HPF-conditioned medium and an EC-conditioned medium. Neutralizing anti-TGF-beta antibodies reduced but not completely abolished the CCN2 synthesis in HPMC cultured with the HPF- or EC-conditioned medium. CCN2, TGF-beta and VEGF activated distinct transcriptional factors in HPMC, which resulted in divergent biological responses in terms of IL-6, MMP-9 and collagen I mRNA expression. AGE and GDPs in PDS differentially regulate the synthesis of CCN2 by peritoneal resident cells. The CCN2 synthesis by HPMC can be further amplified by TGF-beta released from HPF or EC. The differential activation of different transcriptional factors and diverse response of HPMC towards CCN2, TGF-beta and VEGF suggest that these cytokines/growth factors have an overlapping and distinct role on HPMC.