Tonabersat breaks the cycle of Cx43- hemichannel mediated activity and NLRP3 inflammasome activation in a model of diabetic nephropathy

Introduction: Our previous findings link connexin-43 (Cx43)-hemichannel mediated ATP release to an increase in the nod-like receptor protein-3 (NLRP3) inflammasome, via nuclear factor kappa-B (NF?B)-mediated priming and purinergic P2X7 receptor (P2X7R) activation. Evidence from studies examining diabetic retinopathy suggests that NLRP3 priming indirectly regulates Cx43-hemichannel activity through increased NF?B binding to the Cx43 promoter. In the diabetic kidney, this feedback loop remains to be established.Method: Human primary proximal tubule epithelial cells (hPTECs) were cultured in 5mM or 25mM D-glucose and treated with interleukin (IL)1? (10ng) and tumour necrosis factor-alpha (TNF?; 10ng) ± Cx43 hemichannel blocker Tonabersat (50?M), ± Caspase-1 inhibitor YVAD-CMK (10?M/mL), ± NF?B inhibitor BAY117082 (5?M) for 48hrs (n=3-6). Real-time qPCR and western blotting examined mRNA and protein expression respectively, whilst carboxyfluorescein dye-uptake and ATP release assessed Cx43-hemichannel activity.Results: Inhibition of NLRP3 priming and activation decreases interleukin (IL)1? (35±13%; P<0.01 and 55±10%; P<0.001 respectively) and generates a priming-dependent reduction in granulocyte-colony stimulating factor (G-CSF) expression (40±10%; P<0.001). Similarly, blocking Cx43-hemichannels with Tonabersat reduced IL1? (61±5.5%; P<0.001) and G-CSF (52±7.9%; P<0.001) expression, with inhibition of NLRP3 reducing Cx43 mRNA and protein expression (29±5.0%; P<0.01 and 34±9.1%; P<0.01 respectively), hemichannel-mediated dye-uptake (37±2.7%; P<0.001) and ATP release (32±8.7%; P<0.05).Discussion: In combination with previous data demonstrating a role for Cx43-hemichannel activity in mediating NLRP3 priming and activation, we establish the presence of a cycle of events in which NLRP3-induced inflammation and Cx43-hemichannel mediated activity reciprocally exacerbate the activation of each other. This vicious cycle is blocked by Tonabersat.