PMC

SGLT1 expression is not significantly altered with 30 mM high glucose and SGLT2 inhibition after 72 hours (A). GLUT2 expression is not altered with 30 mM high glucose and SGLT2 inhibition (B). HK2 cells were incubated with 5 mM (ctrl), 30 mM high glucose, and the SGLT2 inhibitor empagliflozin at 100 nM and 500 nM final concentration. SGLT1 protein expression was assessed using western blot. Normalized results are expressed as mean±SEM, n = 4–6.

SGLT2inh significantly reverses high glucose induced AP-1 binding. To assess the level of AP-1 binding, HK2 cells were incubated for up to 72 h with 5 mM (ctrl) media, 30 mM high glucose and SGLT2inh at 100 nM and 500 nM. AP-1 binding was assessed using EMSA. High glucose induced AP-1 binding and the SGLT2inh at both concentrations significantly inhibited this increase. Normalized results are expressed as mean±SEM, n = 3.* p<0.05 versus control; † p<0.05 versus 30 mM Glu.

SGLT2inh reverses high glucose induced CIV expression. To assess the level of CIV expression, HK2 cells were incubated for up to 48 h with 5 mM (ctrl) media, 30 mM high glucose and SGLT2inh at 100 nM and 500 nM. High glucose induced CIV expression and the SGLT2inh at 100 nM significantly inhibited this increase. Although SGLT2inh at 500 nM reduced CIV expression, it did not reach statistical significance. Normalized results are expressed as mean±SEM, n = 4. * p<0.05 versus control; † p<0.05 versus 30 mM Glu.

SGLT2 expression is not regulated by high glucose (A) but is increased with TGFβ₁(B) in HK2 cells after 48 hours (B). Chromatin immunoprecipitation assay showed significantly increased binding of phosphosmad3 to the relevant binding site in the promoter region of the SGLT2 gene with TGFB treatment compared to control. Results were normalized to input DNA and expressed as % input of 3 separate experiments where input represents the amount of chromatin used. Amplified PCR products were also analysed on a agarose gel (C). HK2 cells were incubated with 5 mM (ctrl), 30 mM high glucose, 0.5 ng/ml TGFβ1 and the SGLT2 inhibitor empagliflozin at 100 nM and 500 nM final concentration. SGLT2 protein expression was assessed using western blot. Normalized results are expressed as mean±SEM, n = 4–6. * p<0.05 vs control.

SGLT2inh significantly reverses high glucose induced TLR4 expression at 24 hours (A) and NF-κB binding at 72 hours (B). This was specific to blockade of glucose entry into the cell as another stimulus of NF-κB binding like HMGB1 was not affected by SGLT2 inhibition (C). SGLT2inh also reduced high glucose induced IL-6 secretion (D). HK2 cells were exposed to 5 mM (ctrl), 30 mM high glucose, 50 ng/ml recombinant HMGB1 and the SGLT2 inhibitor empagliflozin at 100 nM and 500 nM final concentration. TLR4 expression was assessed with western blot and NF-κB binding was measured using EMSA. For the HMGB1 experiments, cells were pretreated with the SGLT2 inhibitor for 24 hours then exposed to recombinant HMGB1 for 2 hours. IL-6 was measured in the supernatant using a commercial ELISA kit Normalized results are expressed as mean±SEM, n = 5.* p<0.05 vs control; † p<0.05 versus 30 mM Glu.