High glucose-induced PKC activation mediates TGF-β1 and fibronectin synthesis by peritoneal mesothelial cells

Background. Progressive peritoneal fibrosis, membrane hyperpermeability, and ultrafiltration failure have been observed in long-term peritoneal dialysis (PD) using glucose as an osmotic agent. High glucose activates protein kinase C (PKC), which is one important signal pathway in the activation of transforming growth factor-β1 (TGF-β1) and fibronectin (FN). To gain a better understanding of mechanisms involved in peritoneal fibrosis, we examined the effects of high glucose on human peritoneal mesothelial cell (HPMC) TGF-β1 and FN mRNA expression and protein synthesis and determined the involvement of PKC in the high glucose-induced HPMC activation. Methods. Synchronized confluent HPMC were incubated with different concentrations of glucose with and without inhibition of PKC. PKC activity and diacylglycerol (DAG) levels were measured. The expression of TGF-β1 and FN mRNAs by HPMC was measured by Northern blot analysis. TGF-β1 protein was measured by enzyme-linked immunosorbent assay (ELISA) and mink lung epithelial cell growth inhibition assay. FN protein was measured by Western blot analysis and ELISA. Results. PKC activity and DAG levels in HPMC cultured under 50 mmol/L (high) glucose increased 2.3- and 2.0-fold, respectively, that of 5.6 mmol/L (control) glucose at 24 hours and this was sustained up to 72 hours. The expression of TGF-β1 and FN mRNA by HPMC cultured under high glucose increased 1.6- and 1.7-fold, respectively, that of control values at 24 hours. TGF-β bioaetivity as well as protein content in heat-activated conditioned media from high glucose was significantly higher than that of control values at 24 and 48 hours. FN protein also increased in response to high glucose, as measured by Western blot analysis and ELISA. PKC activator phorbol 12-myristate 13-acetate (PMA) induced 2.2- and 1.4-fold increase in TGF-β1 and FN mRNA expression, respectively. Depletion of PKC and calphostin C, a PKC inhibitor, effectively prevented both PMA and high glucose-induced, but not constitutive, expression of TGF-β1 and FN. Conclusion. The present data demonstrate that high glucose up-regulates TGF-β1 and FN synthesis by HPMC, and that this high glucose-induced up-regulation is largely mediated by PKC. These results suggest that activation of PKC by high glucose in conventional PD solutions may constitute an important signal for activation of HPMC, leading to progressive accumulation of extracellular matrix and eventual peritoneal fibrosis.