Inhibition of intern A2B1 negates collagen-1 induced changes in connexion-mediated hemichannel activity in an in vitro model of diabetic nephropathy

Aims: Tubulointerstitial fibrosis, the underlying pathology of diabetic nephropathy, is characterised by extracellular matrix accumulation. Aberrant connexin (Cx) expression and increased adenosine triphosphate (ATP) have been linked to inflammation and fibrosis, and we have previously reported that glucose-evoked transforming growth factor-beta1 (TGF-b1) increases connexin mediated hemichannel ATP release. While extracellular matrix modification has been linked to altered cell phenotype, this study explores a role for collagen-I in regulating connexin-mediated hemichannel activity. Methods: Human kidney (HK-2) proximal tubule cells were cultured on collagen-I (50μg/ml) +/- TGF-b1 (10ng/ml) +/- anti-a2b1 integrin neutralising antibody (2.5μg/ml) for 48h. Uncoated plastic served as the control. The expression of candidate proteins was determined by immunoblotting. Carboxyfluorescein (200μM) dye uptake confirmed hemichannel activity. Results: TGF-b1 decreased Cx26 to 57.0 +/- 3.4% and increased collagen-I expression to 187.0 +/- 3.1% as compared to control (n-4, p < 0.01). Co-incubation of HK-2 cells with TGF-b1 + a2b1 (neutralising antibody against the principle collagen integrin), restored Cx26 expression to 76.2 +/- 6.1% (n-4, p < 0.01). In support of this data, when cultured on collagen-I, Cx26 expression decreased to 58.7 +/- 10.1%; and a further 31.0 +/- 8.9% (n-4, p < 0.01) in the presence of TGF-b1. When cultured on collagen-I +/- TGF-b1, HK-2 cells demonstrated increased carboxyfluorescein uptake to 260.0 +/- 10.9% and 182.5 +/- 8.1%, respectively, while incubation with neutralising a2b1 antibody significantly reduced dye uptake by 27.0 +/- 3.7% and by 24.0 +/- 2.6%, respectively, as compared to uncoated control (n-4, p < 0.01). Conclusion: Collagen-I +/- TGF-b1 decreases Cx26 expression, an effect paralleled by increased hemichannel activity. Inhibition of integrin a2b1 negates collagen/TGF-b1 induced dye uptake, suggesting a role for collagen-I in regulation of connexin-mediated communication.