Evaluating a role for Connexin-43 hemichannel mediated ATP release in priming and activation of the NLRP3 inflammasome in a human model of diabetic kidney disease

Aims: Sterile inflammation in diabetic nephropathy is mediated by the Nod-like receptor pyrin domain contain- ing-3 (NLRP3) inflammasome, an integral driver of the innate immune response that mediates tissue damage in multiple age-associated diseases. We recently demonstrated that increased connexin-43 (Cx43) hemichannel activity drives inflammation in nephropathy. Here we evaluate a role for Cx43 hemichannel-mediated ATP re- lease in NLRP3 priming and activation.Methodology: Primary proximal tubule epithelial cells (hPTECs) isolated from renal biopsy, were cultured in 5mM or 25mM glucose ± interleukin-1? (IL1?; 10ng) and tumour necrosis factor alpha (TNF?; 10ng) ± Cx43 hemichannel blocker Tonabersat for 48hrs. Carboxyfluorescein dye uptake and ATPlite assays assessed hemichannel mediated ATP release. Quantitative real time PCR (qRT-PCR), caspase Glo-1 and inflammation arrays assessed NLRP3 priming and activation. Results: Carboxyfluorescein dye uptake and ATP re- lease were increased in 25mM glucose+cytokine treated hPTECs by 61±3.2% (P < 0.001, N = 4) and 54±6.4% (P < 0.001, N = 6) respectively compared to control, effects attenuated by Tonabersat (P < 0.001, N = 6). Quantitative RT-PCR evaluated priming of the NLRP3 inflammasome with IL1? (97.7±0.69%, P < 0.001, N = 5) and NLRP3 (81±9.4%, P < 0.001, N = 5) expression increased in high glucose+cytokine treated cells compared to control. Tonabersat reduced expression by 51±8.4%, (P < 0.001, N = 5) and 57±5.3% (P < 0.001, N = 5) respectively. In 25mM glucose+cytokine treated hPTECs, Tonabersat decreased caspase1 activity by 24±6%, (P < 0.05, N = 3) and IL1? secretion by 18±5.3% (P < 0.01, N = 4) respectively compared to control.Conclusion: Cx43 hemichannel-mediated ATP release primes and activates the NLRP3 inflammasome, effects negated by Tonabersat. Our data highlights Cx43 hemichannels as a target for dampening NLRP3-induced inflammation in the diabetic kidney.