Fig. 7

Connexin 43-hemichannels amplify and perpetuate inflammation by an ATP-P2 × 7R autocrine feedback loop. With priming of the NLRP3 inflammasome linked to increased Cx43 expression and inflammation a known gating stimulus of connexin hemichannels, we determined the presence of a feedback loop in which Cx43-NLRP3 activation perpetuates inflammation by an ATP-P2 × 7R autocrine feedback loop. We assessed Cx43 hemichannel mediated carboxyfluorescein dye uptake (a&b) and real time ATP release (a&c) in TGF-β1 treated cells which had been pre-incubated with inhibitors to either NLRP3 (CY-09) or caspase 1 (AC-YVAD-CMK). Inhibition of the NLRP3 inflammasome decreased hemichannel mediated dye uptake (a&b) and ATP release (c), suggesting that targeting Cx43-hemichannels offers a potential therapeutic strategy to break the cycle of inflammatory events in tubules of the diseased kidney. The implications of blocking these pathways was further assessed by evaluating expression of extracellular matrix proteins and markers of injury in TGF-β1 treated primary tubule cells pre-incubated with either a Cx43 hemichannel (Peptide5), NLRP3 (CY-09) or caspase 1 (AC-YVAD-CMK) inhibitor (d&e). An ANOVA and Tukeys post-test was used for all analysis, excluding analysis of transcriptomic data where an unpaired t-test with Welch’s correction was used *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001