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 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

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

Metabolic and physiological parameters of UUO mice treated with Telmisartan or PXS64.

<p>Metabolic and physiological parameters of UUO mice treated with Telmisartan or PXS64.</p

Reduction in cellular infiltration and inflammatory markers in UUO kidneys exposed to telmisartan or PXS64.

<p>Untreated UUO kidneys showed increased F4/80, CD68 and CD45 positively stained cells as compared to the sham operated control animals. PXS64 significantly reduced F4/80 and CD45 positive stained cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g006" target="_blank">Fig. 6C and E</a>) with a trend to a reduction in CD68 cells, although this was not statistically significant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g006" target="_blank">Fig. 6D</a>). Telmisartan treated kidneys showed a reduction in F4/80 positive cells but no difference in CD45 or CD68 stained cells, suggesting a differential action of PXS64 and telmisartan in modifying cellular infiltration. Results are presented as mean showed (n = 8, *P < 0.05 vs. UUO, ** P < 0.01 vs. UUO). Magnification x 400.</p

PXS64 suppressed TGF-β1-Smad signaling pathways in HK2 cells.

<p>Phosphorylated Smad2 expression was markedly increased when HK2 cells were exposed to 100ng/ml latent TGF-β1 for 48 hours. This was suppressed when PXS64 was added to the media, which strongly suggests that PXS64 suppresses TGF-β1-Smad2 rather than AKT or ERK signaling pathways (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g003" target="_blank">Fig. 3A, B and C</a>). Results are expressed as mean ± SEM (n = 3, *P < 0.05, ** p<0.01 vs. appropriate control).</p

PXS64 reduced pSmad2 staining in UUO kidney cortex.

<p>Unilateral ureteric ligation increased tubular pSmad2 expression in the kidney cortex, which was markedly reduced when treated with PXS64 and telmisartan (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g007" target="_blank">Fig. 7</a>). Results are presented as Mean +/-SEM. (n = 8).</p