Abstract

Aim: Connexins (Cx) form gap-junctions or hemi-channels through which messengers, including adenosine triphosphate (ATP) can propogate. This study investigated a role for Transforming Growth Factor-beta1 (TGF-b1), a major pro-fibrotic cytokine associated with glucose-evoked renal fibrosis, in regulating Cx-mediated cell-to-cell communication.

Methods: Expression of P2Y1, P2Y6, Cx26 and Cx43 in TGF-b1 (10ng/mL, 48hrs) treated human proximal (HK2) tubule cells were assessed by immunoblot analysis. Calcium microfluorimetry assessed changes in cytosolic calcium ([Ca2+]i) in response to increasing concentrations of ADP and ATP (0.01-10µM). Cells, preloaded with ATP (10mM for 10mins), were used to measure real-time ATP release via bio-sensing.

Results:
TGF-b1 (10ng/mL, 48hrs) significantly decreased expression of Cx26 (63.5±8.8%, p<0.01,n=5), Cx43 (51.1±2.9%, p<0.0001,n=6), P2Y1 (23.6±1.4%, p<0.0001,n=4) and P2Y6 (41.9±2.0%, p<0.01,n=3) as compared to control. Basal-to-peak ADP (10µM) and ATP (10µM) evoked changes in [Ca2+]i were significantly reduced following TGF-b1 treatment (p<0.0001,n=3). Removing extracellular calcium opens hemi-channels and allows ATP release from HK2 cells. In TGF-b1 (10ng/mL) treated cells the amount of ATP release increased from 0.24µM± (control) to 2±0.47µM (p=0.0012, n=3).

Conclusions: TGF-b1 significantly decreased expression of Cx26, Cx43, P2Y1 and P2Y6 in HK2 cells and functionally reduced purinergic-evoked Ca2+-signalling. However, ATP bio-sensing suggests that the cytokine increases the amount of hemi-channel ATP release. Loss of connexin expression appears to impair GJ-mediated cell-communication but increases the amount of hemi-channel activity. This may represent a compensatory mechanism to maintain cell-to-cell communication in the diabetic kidney.

This work is supported by a grant from Diabetes UK (BDA: 11/0004215).