Abstract

Background and aims: In diabetic nephropathy, pivotal to early tubular injury is loss of expression of cell adhesion protein; epithelial (E)-cadherin. Co-localized with Ecadherin at sites of cell-cell contact, connexins (Cx) oligomerise into hexametric hemichannels that can form gap-junctions. Intercellular adhesion is a pre-requisite for gap junction formation and in the absence of neighbouring cells, uncoupled Cx-hemichannels permit local paracrine release of adenosine triphosphate (ATP). Recent studies link elevated ATP levels to increased tissue fibrosis in disease and altered Cx expression
&/or function is linked to the development and progression of secondary complications of diabetes. The current study examines if glucose-evoked changes in Cx-mediated ATP release drive extracellular matrix (ECM) remodelling and increased fibrosis.
Materials and methods: Biopsy material was isolated from patients with diabetic nephropathy and stained for Cx-26 and Cx-43. Changes in expression of key candidate proteins were examined by immunoblotting in model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) +/- TGF-β1 (10ng/mL) or high glucose (25mmol/L) for 7days. Lastly to assess the effects of ATP, cells were cultured in non-hydrolysable ATPγS (1-100μM) for 48hrs. ELISA and cytokine arrays measured protein secretion (TGF-β1, Interleukin-6 (IL-6) and Beta Nerve Growth Factor (β-NGF)). Lastly, Carboxyfluorescein uptake assays determined hemichannel activity in control vs TGF-β1 treated (10ng/mL) HK2 cells.
Results: Cx-26 expression was significantly up regulated in biopsy material from patients with diabetic nephropathy, compared to normal control (102700±6226 versus 21030±4727; n=10, P<0.01). Similarly, Cx-43 expression increased to 116300±5908 ascompared to control 21460±10920 (n=10, P<0.01). In response to high glucose (25mmol/L) treatment for 7days, HK2 cells increased TGFβ1 secretion to 994.4±43.6pg/ml compared to 5mmol/L glucose (334±14.9pg/ml; n=3; P < 0.01). Immunoblot analysis confirmed that TGFβ1 (10ng/mL) up-regulates expression of Cx-26 and Cx-43 to 203.9±7.5% and 151.1±7.1% respectively compared to control (n = 4; P<0.001). Dye uptake in TGFβ1-treated cells increased to 430±18% at day 7. Immunoblotting confirmed that ATPγs up-regulated Collagen I to 177±12%, 182±21%, and 187±21% (n=3 P<0.01) and Collagen IV to 233±26%, 344±18.5%, and 390±10% compared to control (n=3 P<0.001) at 1, 10 and 100 μM. In addition, ATPγs significantly increased expression of Fibronectin to 274±47%, 350±23%, and 433±81% of control (n=3 P<0.01) at 1, 10 and 100 μM. Array analysis of supernatant from ATPγs treated cells confirmed a significant increase in secretion of both IL-6 and β-NGF to 261±24 and 194±44 respectively compared to control (n=3 P<0.01).
Conclusion: Expression of Cx-26 and Cx-43 increased in biopsy material isolated from patients with diabetic nephropathy, changes corroborated in HK2 cells treated chronically with TGF-β1. This increased expression was linked to increased heme-channel mediated ATP release. ATP increased whole cell expression and secretion of key of ECM
components and pro-fibrotic markers, suggesting that increased hemichannel-mediated ATP release may exacerbate fibrosis in the diabetic kidney.