The effect of SGLT2inh on inflammatory markers.

<p>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.</p

The regulation of SGLT2.

<p>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.</p

The effect of SGLT2inh on CIV.

<p>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.</p

The regulation of SGLT1.

<p>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.</p

Inhibition of Kidney Proximal Tubular Glucose Reabsorption Does Not Prevent against Diabetic Nephropathy in Type 1 Diabetic eNOS Knockout Mice

<div><p>Background and Objective</p><p>Sodium glucose cotransporter 2 (SGLT2) is the main luminal glucose transporter in the kidney. SGLT2 inhibition results in glycosuria and improved glycaemic control. Drugs inhibiting this transporter have recently been approved for clinical use and have been suggested to have potential renoprotective benefits by limiting glycotoxicity in the proximal tubule. We aimed to determine the renoprotective benefits of empagliflozin, an SGLT2 inhibitor, independent of its glucose lowering effect.</p><p>Research Design and Methods</p><p>We induced diabetes using a low dose streptozotocin protocol in 7–8 week old endothelial nitric oxide (eNOS) synthase knockout mice. We measured fasting blood glucose on a monthly basis, terminal urinary albumin/creatinine ratio. Renal histology was assessed for inflammatory and fibrotic changes. Renal cortical mRNA transcription of inflammatory and profibrotic cytokines, glucose transporters and protein expression of SGLT2 and GLUT1 were determined. Outcomes were compared to diabetic animals receiving the angiotensin receptor blocker telmisartan (current best practice).</p><p>Results</p><p>Diabetic mice had high matched blood glucose levels. Empagliflozin did not attenuate diabetes-induced albuminuria, unlike telmisartan. Empagliflozin did not improve glomerulosclerosis, tubular atrophy, tubulointerstitial inflammation or fibrosis, while telmisartan attenuated these. Empagliflozin did not modify tubular toll-like receptor-2 expression in diabetic mice. Empagliflozin did not reduce the upregulation of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor β1 and fibronectin mRNA observed in the diabetic animals, while telmisartan decreased transcription of MCP-1 and fibronectin. Empagliflozin increased GLUT1 mRNA expression and telmisartan increased SGLT2 mRNA expression in comparison to untreated diabetic mice. However no significant difference was found in protein expression of GLUT1 or SGLT2 among the different groups.</p><p>Conclusion</p><p>Hence SGLT2 inhibition does not have renoprotective benefits independent of glucose lowering.</p></div

Physical and clinical parameters.

<p>Data are mean ± standard error of mean.</p><p>* = P<0.05 vs control group,</p><p>** = P<0.001 vs control group,</p>#<p> = P<0.05 vs diabetic group.</p><p>Physical and clinical parameters.</p

Diabetic mice showed increased renal cortical transcription of inflammatory and fibrotic cytokines, which was not improved by empagliflozin.

<p>Real time PCR results for Renal cortical transcription of A) MCP-1, B) Collagen 4, C) TGFβ and D) Fibronectin relative to actin (Data are expressed as mean ± SEM with * = P<0.05 vs ctrl and ** = P<0.001 vs ctrl).</p

Diabetic animals demonstrated increased tubulointerstitial inflammation, which was not ameliorated by empagliflozin.

<p>Representative photographs of immunohistochemistry for tubulointerstitial F4/80 stain for activated macrophages in A) ctrl, B) ctrl + empa, C) dm, D) dm + empa, E) dm + tel groups (Magnification  = original X 400) and quantification of F4/80 positive cells in tubulointerstitium (F) which was done by calculating the average number of F4/80 positive cells per high power field (HPF) in each group. Representative photographs of immunohistochemistry for tubular TLR2 in G) ctrl, H) ctrl + empa, I) dm, J) dm + empa, K) dm + tel groups (Magnification  = original X 200) and quantification of tubular TLR2 expression by Image J (L). (Data are expressed as mean ± SEM with * = P<0.05 vs ctrl, ** = P<0.001 vs ctrl and # = P<0.05 vs dm).</p

Blood glucose levels among diabetic mice were matched throughout the study and showed similar glucose levels among the diabetic mice at 1, 2, 3, 4 and 5 months post diabetes induction and were significantly higher than control mice (A).

<p>Empagliflozin reduced insulin requirement in diabetic mice (B). The comparison was made between average insulin requirement per dose administered thrice weekly, before and after initiation of empagliflozin. Data are expressed as mean ± SEM with * = P<0.05 and ** = P<0.001 vs ctrl.</p

Empagliflozin increased renal GLUT1 transcription in comparison with untreated diabetic mice although renal protein expression of GLUT1 and SGLT2 were unchanged.

<p>Real time PCR results for A) GLUT1, B) GLUT2, C) SGLT1 and D) SGLT2 expressed relative to actin. Expression levels of renal GLUT1 protein (E and F) and SGLT2 (G and H) relative to actin (Data are expressed as mean ± SEM with # = P<0.05 vs dm).</p