High glucose-induced PKC activation mediates TGF-beta 1 and fibronectin synthesis by peritoneal mesothelial cells

Background. Progressive peritoneal fibrosis, membrane hyperpermeability, an d ultrafiltration failure have been observed in long-term peritoneal dialys is (PD) using glucose as an osmotic agent. High glucose activates protein k inase C (PKC), which is one important signal pathway in the activation of t ransforming growth factor-beta1 (TGF-beta1) and fibronectin (FN), To gain a better understanding of mechanisms involved in peritoneal fibrosis, we exa mined the effects of high glucose on human peritoneal mesothelial cell (HPM C) TGF-beta1 and FN mRNA expression and protein synthesis and determined th e involvement of PKC in the high glucose-induced HPMC activation. Methods. Synchronized confluent HPMC were incubated with different concentr ations of glucose with and without inhibition of PKC. PKC activity and diac ylglycerol (DAG) levels were measured. The expression of TGF-beta1 and FN m RNAs by HPMC was measured by Northern blot analysis. TGF-beta1 protein was measured by enzyme-linked immunosorbent assay (ELISA) and mink lung epithel ial cell growth inhibition assay. FN protein was measured by Western blot a nalysis 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 (co ntrol) glucose at 24 hours and this was sustained up to 72 hours. The expre ssion of TGF-beta1 and FN mRNA by HPMC cultured under high glucose increase d 1.6- and 1.7-fold, respectively, that of control values at 24 hours. TGF- beta bioactivity as well as protein content in heat-activated conditioned m edia 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-m yristate 13-acetate: (PMA) induced 2.2- and 1.4-fold increase in TGF-beta1 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-beta1 and FN. Conclusion. The present data demonstrate that high glucose up-regulates TGF -beta1 and FN synthesis by HPMC, and that this high glucose-induced up-regu lation is largely mediated by PKC. These results suggest that activation of PKC by high glucose in conventional PD solutions may constitute an importa nt signal fur activation of HPMC, leading to progressive accumulation of ex tracellular matrix and eventual peritoneal fibrosis.