Fig. 6

Co-incubation of TGF-β1 treated renal tubule cells with Cx43 mimetic, Peptide 5, impairs hemichannel activity and ATP release. HK2 cells and hPTECs were cultured on either fluorodishes or glass coverslips in low-glucose (5mM) ± TGF-β1 (10 ng/mL) ± Peptide 5 (25 μM) for 48 h. A carboxyfluorescein uptake assay (a & c) assessed hemi-channel activity in HK2 and hPTEC cells respectively. Minimal dye uptake was observed in control cells, whilst strong dye loading occurred in TGF-β1 treated cells. Co-incubation with Peptide 5 attenuated dye loading, whilst a scrambled control had no significant effect. Pixel intensity of dye loading was quantified and compared to the low-glucose control (b & d) for 10 cells in 3 separate experiments. Biosensors were used to measure hemi-channel dependent release of ATP. Representative traces (−null) are shown (e). The amplitude of any major ATP peak was measured and compared to control recordings, where little response occurred in response to hemi-channel opening following the removal of calcium. TGF-β1-treated cells exhibit significant ATP in response to hemi-channel opening, an effect negated when co-incubated with Peptide 5 (25 μM) (f). Exogenous ATP was administered at the end of each experiment ensuring effective calibration. Data is expressed as mean ± SEM of multiple cells from 4 separate experiments, with key significances shown: **P < 0.01, ***P < 0.001