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

Cliff, Chelsey L, Williams, Bethany, Chadjichristos, Christos E , Squires, Paul and Hills, Claire (2022) 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. In: Diabetes UK AGM, March 28-April 1, Online/London.

Full content URL: https://onlinelibrary.wiley.com/toc/14645491/2022/...

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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
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Abstract

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-1β (IL1β; 10ng) and tumour necrosis factor alpha (TNFα; 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 IL1β (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 IL1β 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.

Keywords:inflammasome, NLRP3, diabetes, diabetic nephropathy, Connexin-43
Subjects:A Medicine and Dentistry > A100 Pre-clinical Medicine
B Subjects allied to Medicine > B120 Physiology
Divisions:College of Science > School of Life Sciences
ID Code:49109
Deposited On:05 May 2022 15:13

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