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

PXS64 reduced extracellular matrix mRNA and protein expressions in the UUO.

<p>Real time PCR showing increased fibronectin and collagen IV mRNA levels in UUO mice vs. sham and reduced levels by telmisartan and PXS64 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g005" target="_blank">Fig. 5A and B</a>). Western blot and Hydroxy-proline showing fibronectin protein expression and collagen release in the UUO kidney, telmisartan and PXS64 treated mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g005" target="_blank">Fig. 5C and Fig. 5D</a> respectively). Western blot shows three lanes of each group representing three independent experiments of sham, UUO control, UUO+telmisartan and UUO+PXS64. Immunostaining of kidney tissue showing fibronectin and collagen IV protein expression in UUO mice in the absence and presence of telimisartan or PXS64 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g005" target="_blank">Fig. 5E and 5F</a>). Isotype specific IgG1 was used to confirm specificity of the staining (data not shown). (n = 8, *P < 0.05, ** P < 0.01).</p

PXS64 inhibited TGF-β1-induced fibrotic and inflammatory markers in HK2 cells.

<p>HK2 cells exposed to 100ng/ml latent TGF-β1 showed a significant increase in fibronectin and collagen IV mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2A and B</a>) and protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2D and E</a>) expression, which were suppressed when co-exposed to PXS-64. These cells had increased mRNA expression of MCP-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2C</a>), which was translated to increased secretory MCP-1 in the supernatant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2F</a>). There were no difference mRNA and protein expression of the fibrotic and inflammatory markers s in HK2 cells under basal conditions or when cells were exposed to PXS64 alone (*<i>P</i> < 0.05, ** p<0.01 vs. appropriate control) Results are expressed as mean ± SEM, n = 4</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

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 conversion of latent to active TGFβ1 in HK2 cells.

<p>Active transforming growth factor TGF-β1 was determined in the concentrated supernatant after 48 hours exposure to 100ng/ml latent TGF-β1 with or without 10μM PXS64. There was increased active TGF-β1 concentration when cells were exposed to excess latent TGF-β1, which was markedly suppressed on co-exposure with PXS64. This data suggest that conversion of latent to active TGF-β1 can be effectively inhibited by a cationic independent mannose 6-phosphate receptor inhibitor (**<i>P</i> < 0.01 vs. control). Results are expressed as mean ± SEM, n = 3.</p

Details of primers used for pro-fibrotic and pro-inflammatory markers.

<p>Details of primers used for pro-fibrotic and pro-inflammatory markers.</p

Glomerular number in offspring of SE dams.

<p>Kidney H&E stained sections from the offspring of Control (1^st column) and smoke exposed dams (SE, 2^nd column right panel) at postnatal (P)1 (A, B), P20 (C, D), and week (W)13 (E, F). Reduced glomerular numbers was shown in offspring from SE dams at birth (P1), early postnatal life (P20) and adulthood (W13). Mag. 20X. Closed arrows show mature and fully vascularized glomeruli and open arrows show underdeveloped glomeruli. Numbers of developed glomeruli are shown in the 3^rd column. n = 6–8. *p<0.05 and #p<0.01, maternal smoke exposure effect.</p

The effects of maternal smoke exposure on growth and development in offspring.

<p>Results are expressed as mean ± SEM, n = 11–23.</p><p>*p<0.05.</p><p>SE: smoke exposed.</p