TGF-β1 Mediates Glucose-evoked Up-regulation of Connexin-43 Cell-to-cell Communication in HCD-cells

Abstract

Background/Aims: In the current study we examined if the multifunctional cytokine TGF-β1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD). Methods: RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-β1 and Cx43 in HCD-cells. The effect of TGF-β1 and high glucose (25mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-β1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca²⁺-microfluorimetry. Results: At 24hrs, high glucose (25mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-β1 (2ng/ml) at low glucose (5mM). Both high glucose and TGF-β1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-β. Furthermore, high glucose-evoked changes were inhibited by a TGF-β1-specific monoclonal antibody. Mannitol (25mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-β1 evoked changes in Cx43 expression were biphasic. An early (4-8hr) transient decrease in expression was followed by an increase in protein expression (12-24hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24hrs coincided with a TGF-β1 specific increase in touch-evoked transmission of Ca²⁺-signals between coupled cells. Conclusions: High glucose evoked a TGF-β1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.