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. 2013;8(2):e54442.
doi: 10.1371/journal.pone.0054442. Epub 2013 Feb 4.

Effects of SGLT2 inhibition in human kidney proximal tubular cells--renoprotection in diabetic nephropathy?

Usha Panchapakesan  1 , Kate Pegg, Simon Gross, Muralikrishna Gangadharan Komala, Harshini Mudaliar, Josephine Forbes, Carol Pollock, Amanda Mather
Affiliations

Effects of SGLT2 inhibition in human kidney proximal tubular cells--renoprotection in diabetic nephropathy?

Usha Panchapakesan et al. PLoS One. 2013.

Abstract

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.

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Conflict of interest statement

Competing Interests: Boehringer Ingelheim provided the Empagliflozin and funding but had no role in the study design, data analysis and data interpretation of the manuscript. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. The regulation of SGLT2.
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.
Figure 2
Figure 2. The regulation of SGLT1.
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.
Figure 3
Figure 3. The effect of SGLT2inh on inflammatory markers.
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.
Figure 4
Figure 4. The effect of SGLT2inh on AP-1.
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.
Figure 5
Figure 5. The effect of SGLT2inh on CIV.
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.
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Grants and funding

The authors are grateful to the National Health and Medical Council of Australia for funding this study (1004926). The authors would like to thank Boehringer Ingelheim for providing the empagliflozin and funding for the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.