Metabolic and physical parameters of mice.

<p>Values are shown as mean ± SEM.</p><p>* = P<0.05 vs control</p><p>** = P<0.01 vs control</p><p>^## = P<0.01 vs diabetic</p><p>Metabolic and physical parameters of mice.</p

Linagliptin reduced tubular pSmad 2/3 expression (a marker of transforming growth factor beta activation) in diabetic mice and showed a trend towards reduced cortical pSmad 2 expression on western blot analysis in diabetic mice.

<p>A) Diabetic mice demonstrated increased pSmad2/3 nuclear expression with immunohistochemistry, which was reduced by linagliptin and telmisartan. Representative photographs for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X 200). Quantification was done by counting the number of positive nuclei at X200 magnification. Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, ## = P<0.01 vs dm. B) Diabetic mice showed a trend towards increase in pSmad 2/total smad2 expression compared to control mice and a reduction with both linagliptin and telmisartan. This trend was consistent with the immunohistochemistry findings but did not reach statistical significance. Quantification was done using Image J. Data are expressed as mean ± SEM, n = 6.</p

Linagliptin reduced fibronectin transcription and expression in diabetic mice.

<p><b>(A)</b> Diabetic mice demonstrated increased cortical fibronectin mRNA transcription by real time PCR. This was significantly reduced by linagliptin and telmisartan. <b>(B)</b> There was a significant increase in tubulointerstitial FN expression measured by immunohistochemistry in the diabetic animals and this was reduced with linagliptin. Representative photographs for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X 200). Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, # = P<0.05 vs dm, ## = P<0.01 vs dm.</p

M6P and linagliptin reduced high glucose induced CIM6PR:DPP4 interaction in HK2 cells.

<p><b>(A)</b> Proximity ligation assay demonstrating endogenous protein-protein interaction between membranous DPP4 and CIM6PR in HK2 cells visualised as individual fluorescent dots. This is increased in 30mM high glucose (HG) environment compared to control 5mM glucose (ctrl). M6P at 1μM and linagliptin at 30nM reduced the high glucose induced interaction. A quantitation of this is shown in <b>(B)</b>. Data is represented as mean ± standard error, n = 3. * = P<0.05 compared to 5mM glucose, # = P<0.05 compared to 30mM glucose.</p

Linagliptin partially reduced tubular atrophy in diabetic mice.

<p>Diabetic mice demonstrated tubular atrophy, which was partially reduced by linagliptin and telmisartan. Representative photographs of PAS stained sections of tubulointerstitium for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X 200). Quantification of tubular atrophy in all groups was done by counting the number of atrophic tubules per 400 tubule count (Data are expressed as mean ± SEM with * = P<0.05 vs ctrl).</p

Linagliptin did not reduce glomerulosclerosis in diabetic mice.

<p>Diabetic mice demonstrated increased glomerulosclerosis, which was improved by telmisartan but not by linagliptin as demonstrated by quantification of glomerulosclerosis by glomerulosclerotic index. Representative photographs of PAS stained sections for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X400). Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, ## = P<0.01 vs dm.</p

Effect of separate and additive inhibition of TLR2 and TLR4 signalling pathways on NF-ĸB activation.

<p>(A) Reduction in NF-ĸB p65 subunit expression when TLR2 and 4 inhibitors were used separately and further abrogation of NF-ĸB p65 subunit expression when the inhibitors were used additively in control media (B) An attenuation in NF-ĸB-DNA binding was observed when anti-TLR2-IgA and TAK-242 were used separately and further downregulation in NF-ĸB-DNA binding when both inhibitors were used additively in control media. Normalized results are expressed as mean ± SEM, n = 4. *P<0.05 versus HMEC-1 cells treated with DMSO + control IgA (control). **P<0.01 versus HMEC-1 cells treated with DMSO + control IgA (control). ^††P<0.01 versus HMEC-1 cells treated with Anti-TLR2-IgA. ^‡‡P<0.01 versus HMEC-1 cells treated with TAK-242.</p

The effect of recombinant HMGB1 on NF-ĸB p65 subunit expression and NF-ĸB-DNA binding.

<p>(A) Exposure to recombinant HMGB1 (500 ng/ml) in control media for 2 hours induced nuclear NF-ĸB p65 subunit expression and, (B) NF-ĸB-DNA binding was also induced with exposure to recombinant HMGB1 (500 ng/ml) in HMEC-1 cells. Normalized results are expressed as mean ± SEM, n = 5. *P<0.05 versus HMEC-1 cells cultured in the absence of recombinant HMGB1. **P<0.01 versus HMEC-1 cells cultured in the absence of recombinant HMGB1.</p

TLR2 and 4 expression in cells exposed to the defined experimental conditions for 72 hours.

<p>(A) Reduction in TLR2 expression with 11.2 mM glucose and (B) maximal TLR4 expression with fluctuating glucose conditions. Normalized results are expressed as mean ± SEM, n = 4. *P<0.05 versus HMEC-1 cells cultured with 5 mM glucose. **P<0.01 versus HMEC-1 cells cultured with 5 mM glucose.</p

The effect of recombinant HMGB1 on stimulating inflammatory cytokines and cell adhesion molecules.

<p>(A) Stimulating HMEC-1 cells with recombinant HMGB1 in control media for 2 hours induced the secretion of MCP-1 and IL-8 into the media (B) Exposure to recombinant HMGB1 also induced a moderate increase in ICAM-1. Normalized results are expressed as mean ± SEM, n = 3. *P<0.05 versus HMEC-1 cells cultured in control media. **P<0.01 versus HMEC-1 cells cultured in control media.</p