To Örebro University

Aims: Tubulointerstitial fibrosis is the underlying pathology of diabetic nephropathy and develops in response to aberrant activation of multiple cell types within and around the proximal tubule of the kidney, including extracellular matrix (ECM) producing fibroblasts. Whilst we previously reported a role for connexin- 43 (Cx43) hemichannel activity in tubule inflammation, the function and extent to which fibroblast hemichannels contribute to this damage, remains to be determined.

Methods: Human kidney fibroblasts (TK173) were cultured in the glucose-evoked cytokine transforming growth factor-beta1 (TGFb1) ± Cx43 hemichannel blocker Tonabersat, for 48hrs. Immunoblotting determined protein expression, whilst carboxyfluorescein dye uptake and an ATP lite assay assessed hemichannel- mediated ATP release.

Results: TGFb1 significantly increased hemichannel-mediated dye uptake by 73.6±3.9%, (P < 0.001, n = 4) in TK173 cells compared to control, an effect reduced when co- incubated with Tonabersat (P < 0.01, n = 4). The profibrotic cytokine TGFb1 increased ATP release by 92.8±13.9%, with Tonabersat decreasing ATP release by 90.8±25.8% (P < 0.05, n = 4). Immunoblotting determined that TGFb1 increased expression of the ECM proteins, fibronectin (330.8±16.4%, P < 0.001, n = 5) and collagen I (42.9±4.6%, P < 0.001, n = 5), and the principal Wnt signalling mediator b- catenin (91.8±6.6%, P < 0.001, n = 5) compared to control. Tonabersat restored expression of fibronectin, collagen I and b-catenin by 98±29.6%, (P < 0.01, n = 5), 20±6.8%, (P < 0.05, n = 5), and 56.9±26.7%, (P < 0.05, n = 5) respectively.

Conclusion: These data suggest that glucose- evoked changes in TGFb1, increase hemichannel-mediated ATP release and downstream expression of fibrotic candidates in human renal fibroblasts. The study indicates that Cx43 hemichannels may represent a future therapeutic target for alleviating tubulointerstitial fibrosis in people with diabetic kidney disease.