A role for connexin-43 hemichannel-mediated adenosine triphosphate triphosphate (ATP) release in inflammation of the diabetic proximal tubule

Aim: Glucose-evoked transforming growth factor beta1 (TGF-b1) dysregulates connexin (Cx)-mediated gap-junction intercellular communication and increases hemichannel adenosine triphosphate (ATP) release. Linked with inflammation in diabetes, we investigated a role for ATP in activation of the NLRP3 inflammasome and production of inflammatory mediators in models of diabetic kidney disease. Methods: Human proximal tubule epithelial cells (HK2) were treated for 48 h with ATPyS (100micoM) or TGF-b1 (10ng/ ml) +/- NLRP3 inhibitor CY09 (10microM), +/- caspase1 inhibitor ACYVAD-CHO (10microM), +/-P2X7 inhibitor A438079 (50microM). Primary proximal tubule cells (hPTECs) were treated with TGF-b1 (10ng/ml) +/- Cx43 inhibitor peptide5 (25microM). Protein expression was determined by immunoblotting, whilst a GLO assay assessed caspase1 activity. Results: TGF-b1 upregulated NLRP3, caspase1, IL1b and IL18 to 285.1 +/- 59.62%, 211.2 +/- 17.06%, 261.6 +/- 33.8% and 163.3 +/- 17.75% respectively (n=3, p < 0.001); an effect replicated by ATPyS, with expression at 340.7 +/- 23.75%, 193.7 +/- 13.47%, 220.6 +/- 17.46% and 192.5 +/- 7.5% (n=3, p < 0.001). Caspase1 activity increased to 601.8 +/- 33.6% (TGFb1) and 415.4 +/- 58.7% (ATPyS) (n=3, p < 0.001). CY09 and ACYVAD-CHO restored expression of NLRP3, caspase1, IL1b and IL18 to 139.6 +/- .6% and 108 +/- 2.2%, 139.6 +/- 6.8% and 202.5 +/- 36.2%, 138.4 +/- 13.0% and 152.6 +/- 12.9%, and 160.8 +/- 6.% and 146.6 +/- 4.3% respectively (n=3, p < 0.001). A438079 negated TGF-b1-evoked upregulation, returning the expression of NLRP3 to 120.9 +/- 4.9%, caspase1 to 132.3 +/- 13.7%, IL1b to 160.0 +/- 39.7% and IL18 to 154.5 +/- 14.3% (n=3, p < 0.001). Caspase1 activity decreased to 243.2 +/- 34.2% and 210.2 +/- 22.0% respectively (n=3, p < 0.001). Finally, peptide5 negated TGF-b1-evoked changes in primary hPTECs, returning the expression of NLRP3 to 117.3 +/- 8.6%, caspase1 to 120.1 +/- 39.2%, IL1b to 105.9 +/- 16.8%, and IL18 to 122.8 +/- 18.7% (n=3, p < 0.001). Conclusions: TGF-b1-induced hemichannel ATP release activates the NLRP3 inflammasome, leading to inflammation. Inhibition of Cx43 highlights potential as a future therapeutic target.