Blocking connexin-43 (CX43) hemichannels negates glucose-associated inflammation in clonal proximal tubular epithelial cells

Cliff, CL, Squires, PE, Williams, BM , Potter, JA and Hills, CE (2021) Blocking connexin-43 (CX43) hemichannels negates glucose-associated inflammation in clonal proximal tubular epithelial cells. In: Diabetes UK AGM, 19-30 April 2021, Online.

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Introduction: Inflammation has been linked to hemichannel-mediated release of adenosine triphosphate (ATP) and downstream fibrosis in various tissue type-specific complications of diabetes. In the current study we examined how inflammatory cytokines alter markers of renal tubular injury ahead of evaluating the efficacy of the connexin-43 (Cx43) hemichannel blocker Tonabersat in negating these effects.
Methods: Human Kidney (HK-2) proximal tubule epithelial cells were cultured in low (5mM) or high (25mM) glucose ± interleukin (IL)-1β (10ng) and tumour necrosis factor alpha (TNFα; 10ng) for 48 hours. Immunoblotting determined expression of key proteins, whilst carboxyfluorescein (200µM) dye uptake examined hemichannel activity in treated cells +/- Tonabersat (10µM).
Results: Under high glucose conditions, IL-1β and TNFα significantly increased hemichannel activity to 161±16%, (P<0.05, n=5) an effect which was negated when co-incubated with hemichannel blocker Tonabersat (123±8.5%, n=5). Moreover, stimulation with IL-1β and TNFα increased expression of the inflammatory protein IL-6 (206±42%, P<0.05, n=5) and the extracellular matrix proteins, fibronectin (365±81%, P<0.05, n=4), collagen-I (516±36%, P<0.05, n=4) and collagen-IV (830±217%, P<0.01, n=5) as compared to low glucose control. The effect was augmented at high (25mM) glucose for IL-6 (491±164%, P<0.05, n=5), fibronectin (455±112%, n=6), collagen-I (725±85%, P<0.05, n=4) and colIagen-IV (868±77%, P<0.001, n=6).
Conclusion: We demonstrate that IL1β and TNFα increase expression of inflammatory and profibrotic proteins in glucose treated HK-2 cells. Paralleled by a Tonabersat-sensitive increase in Cx43 hemichannel activity, we believe that blocking hemichannels has therapeutic potential and protects against changes associated with tubular injury in the diabetic kidney.

Keywords:connexin, hemichannel, diabetic nephropathy, Tonabersat
Subjects:C Biological Sciences > C130 Cell Biology
B Subjects allied to Medicine > B120 Physiology
Divisions:College of Science > School of Life Sciences
ID Code:46011
Deposited On:20 Sep 2021 11:52

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