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Abstract: FR-PO183

Identification of a Novel Smad Target in SGLT2-Mediated CTGF/CCN2 Expression

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic


  • Pan, Xinlu, South West Thames Institute for Renal Research, London, United Kingdom
  • Baines, Deborah L., St George's, University of London, London, United Kingdom
  • Phanish, Mysore K., South West Thames Institute for Renal Research, London, United Kingdom
  • Dockrell, Mark E., South West Thames Institute for Renal Research, London, United Kingdom

Drugs inhibiting Sodium Glucose Transporter 2 (SGLT2) activity are providing unexpected and significant benefits by the reduction of a number of conditions including CVD and mortality in diabetic patients. The precise mechanisms underlying the benefits are not fully understood. Proximal tubule cell SGLT2 is believed to be the major site of action for these drugs. We have investigated putative signaling pathways in these cells regulating SGLT2 induced effects, and have now identified a target already known to be involved in cardiovascular disease and fibrosis.


PTECs (primary proximal tubule epithelial cells) were cultured on collagen IV. They were treated with D glucose 7 mM (control), 25mM (high) or 7 mM + 18 mM L glucose (osmotic control), +/- TGFβ1 at 0.75ng/ml. The cells were also administered Dapagliflozin and MEK Inhibitor U0126 (0.1μ -10μM). Western blotting was used to detect the level of Connective Tissue Growth Factor (CTGF/CCN2), phosphorylated extracellular signal regulated kinase 2 (ERK 2), Smad3, and Smad3 linker region serine 204 (LR) protein.


Our high glucose (HG) +TGFβ1 treated PTECs significantly upregulated CTGF/CCN2 protein (P*<0.05). This rise was significantly attenuated by Dapagliflozin (P**< 0.01). Hence, we investigated a potential convergence of the glucose and TGFβ signaling. TGFβ1 phosphorylated ERK 2 (42kDa) from 5 - 60 min after treatment with HG (P**<0.01), while HG treatment exclusively phosphorylated ERK 2 from 15 - 45 min (P*<0.05). Smad3 (52kDa) was phosphorylated by TGFβ1 from 5 - 60 min, +/- high glucose (P**<0.01). HG+TGFB1 treatment at 30 min caused a significant rise of LR (25kDa) phosphorylation (P*<0.05), which was significantly reduced in the presence of U0126 (P*<0.05).


SGLT2 mediates high glucose induced CTGF/CCN2 in the presence of TGFβ1. We have identified a novel alternative mechanism by the convergence of TGFβ1 and HG treatment; phosphorylation of a serine on the Smad3 LR. Hayashida (2013) showed that glucose mediated ERK activation of LR potentiated Smad regulated transcription in mesangial cells. As MEK inhibition was able to reverse LR phosphorylation in our PTECs, our data indicates an important role for ERK in facilitating the observed glucose mediated pro-fibrotic effect.