Background: Altered connexin expression and/or function is linked to the development and progression of secondary microvascular complications associated with diabetes. Despite this, we know little for the role of these small membrane proteins in the diabetic kidney. This study examines if glucose-evoked changes in TGF-β1 modulate connexin expression and gap junction-mediated intercellular communication in diabetic nephropathy.
Methods: Biopsy material was isolated from patients with diabetic nephropathy and stained for connexin-26 and connexin-43. Changes in expression, were corroborated by immunoblot analysis in model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5 mmol/l) +/− TGF-β1 (210 ng/ml) or high glucose (25 mmol/l) for 7 days. ELISA was used measured TGF-β1 secretion and paired-patch electrophysiology recorded junctional conductance in control versus TGF-β1 treated (10 ng/ml) HK2 cells.
Results: Connexin-26 expression was significantly up regulated in biopsy material from patients with diabetic nephropathy, compared to normal control (102 700±6226 vs 21 030±4727; n=5, P<0.01). Similarly, connexin-43 expression increased to 116 300±5908 as compared to control 21 460±10 920 (n=5, P<0.01). In response to high glucose (25 mmol/l) treatment for 7 days, HK2 cells increased TGFβ1 secretion to 994.4±43.6 pg/ml compared to 5 mmol/l glucose (334±14.9 pg/ml; n=3; P<0.01). Immunoblot analysis confirmed that TGFβ1 (10 ng/ml) up-regulates expression of connexin-26 and connexin-43 to 203.9±7.5% and 151.1±7.1% respectively compared to control (n=4; P<0.001). Whole cell paired-patch electrophysiology was used to determine the junctional conductance between coupled HK2 cells ± TGF-β1 (10 ng/ml). TGF-β1 produced decreased junctional conductance to 0.42±0.2 nS compared to control 4.5±1.3 nS (n=5; P≤0.05).
Conclusion: Expression of connexin-26 and connexin-43 increased in biopsy material isolated form patients with diabetic nephropathy, changes corroborated in HK2 cells treated chronically with TGF-β1. Despite this gain in expression, gap junction mediated intercellular conductance was reduced, a feature linked to increased hemi-channel activity.
07 Nov 2016 - 09 Nov 2016