In the kidney glucose is filtered by the glomerulus and reabsorbed by sodium glucose cotransporter 2 (SGLT2) in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological changes seen in Diabetic Kidney Disease (DKD) in response to elevated glucose. We developed a model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis. Western blotting was performed to detect cellular and secreted proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on D-glucose, 25mM, and TGF-β1, 0.75ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. Only the combination of high D-glucose and TGF-β1 treatment significantly upregulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phosphoSSXS-Smad3 serines 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this was significantly reduced by SGLT2 and MEK inhibition. We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 –ERK mediated promotion of TGF-β1/Smad3 signalling inducing pro-fibrotic growth factor secretion. Our data evinces support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.
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