Abstract

Background and aims: Efficient tubular function within the proximal nephron relies on inter-cellular communication between epithelial cells via connexin-mediated gap-junctions. In early diabetic nephropathy, a loss of epithelial (E)-cadherin mediated cell-adhesion instigates a series of events that culminate in disrupted cell-cell coupling, cell-to-cell communication and dissociation of the epithelia. In the current study, we utilized model epithelial cells from human renal proximal tubules, to demonstrate a role for the glucose associated beta1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) on connexin expression, gap-junction intercellular communication and hemi-channel activity.
Materials and methods: Connexin-26 (Cx26) and connexin-43 (Cx43) expression were assessed by immunoblot analysis in human kidney (HK2) tubular epithelial cells treated with low glucose (5mmol/L) +/- TGFβ1 (10ng/mL) for 48hr or 7days. Whole cell paired-patch electrophysiology assessed junctional conductance between TGFβ1 treated HK2 cells, whilst carboxyfluorescein (200M) uptake determined hemi-channel opening at 48hr and 7day, with ATP bio-sensing measuring real-time nucleotide release.
Results: Immunoblot suggests a biphasic change in connexin expression following acute (48hr) and chronic (7day) exposure with TGFβ1 (10ng/mL). At 48hr, Cx26 was down regulated to 58.3±5.7% and Cx43 to 48.1±3.8% of control. Conversely, 7day exposure to the pro-fibrotic cytokine, increased Cx26 and Cx43 expression to 203.9±7.5% and 151.1±7.1% respectively (n = 4; P<0.001). Despite time-dependent differences in connexin expression, gap junctional intercellular communication (GJIC) decreased from 4.5±1.3nS (n=5) between control cells to 1.15±0.9nS (P<0.05, n=5) and 0.42±0.2nS (P<0.05, n=5) in cells treated with TGFβ1 at either 48hr or 7day. Removing extracellular calcium opens connexin-mediated hemi-channels and allows uptake of the membrane-impermeant dye carboxyfluorescein. Dye uptake in to TGFβ1-treated cells increased to 346±33% compared of control at 48hr and 430±18% at day 7. These effects were negated by pre-incubation with the hemi-channel blocker carbenoxolone (200µM, 30mins; 110±5.4% of control at 48hr and 64±2.6% at 7day; P<0.001, n=3). ATP bio-sensing confirmed that the TGF1 increased ATP hemi-channel release (1.99±0.47µM compared to 0.29± 0.06µM, P<0.01, n=3).
Conclusion: The current study suggests that despite biphasic changes in connexin expression following acute (48hr) and chronic (7day) exposure to the glucose-dependant pro-fibrotic cytokine TGF1, hemi-channel mediated ATP release increases at the expense of GJIC as cells attempt to maintain cell-to-cell communication. Linked to fibrosis in the diabetic kidney, a switch favouring local increases in purinergic signalling, may actually exacerbate disease progression in diabetic nephropathy.