TGFβ-induced podocyte signaling involves small GTPases.

<p>RT-PCR for TGFβ (<b>A</b>) in podocytes with and without exposure to stretch (24hours) in the absence or presence EHT (1µM) or SAR (1µM). (<b>B</b>) TGFβ protein expression detected by ELISA in podocytes with and without exposure to stretch (24hours) and treated with or without EHT (1µM) or SAR (1µM). Western blot for fibronectin (FN) (<b>C</b>) and quantification (<b>D</b>) produced by primary podocytes stimulated with or without TGFβ (10 ng/ml), in the absence or presence of EHT (1µM) or SAR (1µM). Annexin V staining after exposure with or without TGFβ (24h) (<b>E</b>) and its quantification (<b>F</b>) in podocytes treated with or without EHT (1µM) or SAR (1µM). Data represent the mean ± SEM of three independent experiments. *<i>P</i><0.05 <i>vs</i>. corresponding non-stretched control (A, B) or non-stimulated control (D, F); # <i>P</i><0.05 <i>vs</i>. stretched control (A, B) or TGFβ stimulated control (D, F).</p

GTPase inhibition attenuates 5/6Nx-induced renal histological changes.

<p>Representative PAS (left panel; scale bar 100 µm) and podocin (right panel;, brown color, scale bar 20 µm) stained kidney sections (A), glomerulosclerosis index (<b>B</b>), podocin staining quantification (<b>C</b>), and percentage of WT-1 positive stained glomeruli (<b>D</b>) in kidneys from sham and 5/6Nx mice non-treated or treated with EHT, SAR, statin and ACE inhibitors. Data represent means ± SEM. *<i>P</i><0.05 <i>vs</i>. corresponding sham control (n=5-7 for sham and n=8-18 for 5/6Nx mice); # <i>P</i><0.05 <i>vs</i>. 5/6Nx non-treated mice (n=9-18).</p

TGFβ mediates stretch-induced signaling in podocytes.

<p>Western blot for fibronectin (FN) (<b>A</b>) and quantification (<b>B</b>) of primary podocytes with and without stretch exposure in the absence or presence of anti-TGFβ neutralizing antibody (40 µg/ml). Annexin V activation (<b>G</b>) and its quantification (<b>H</b>) in podocytes with and without stretch-exposure treated with or without EHT (1µM) or SAR (1µM). The data shown are representative of three independent experiments and are means ± SEM (B, D). *<i>P</i><0.05 <i>vs</i>. corresponding non-stretched control; # <i>P</i><0.05 <i>vs</i>. stretched control. </p

Stretch increases Rac-1 and RhoA activity in murine podocytes.

<p>Expression of the podocyte proteins WT-1, nephrin and synaptopodin as well as smooth muscle α-actin in podocytes and rat smooth-muscle cells (<b>A</b>). Activation of Rac-1 (<b>B</b>) and RhoA (<b>C</b>) in primary podocytes in response to stretch (1h) with or without preincubation with EHT (1µM) or SAR (1µM). Data are means ± SEM. *<i>P</i><0.05 <i>vs</i>. corresponding non-stretched control; # <i>P</i><0.05 <i>vs</i>. stretched control (n=3 for Rac-1 assay and n=5 for RhoA assay). Phalloidin staining of the actin cytoskeleton in podocytes after 24-hour stretch with or without EHT (1µM) or SAR (1µM) treatment (<b>D</b>).</p

Stretch-induced signaling is sensitive to GTPase inhibition.

<p>Effect of stretch (24hours) on podocytes in the presence or absence of EHT (1µM) or SAR (1µM) on fibronectin mRNA expression (<b>A</b>), α-smooth muscle actin mRNA expression (<b>B</b>) fibronectin protein expression (<b>C</b>&<b>D</b>) and nephrin and WT-1 mRNA expression (<b>E</b>&<b>F</b>). ERK was used as reference for protein expression, polymerase 2a (Polr2a) as housekeeping gene for mRNA expression. Data represent the mean ± SEM of three independent experiments. *<i>P</i><0.05 <i>vs</i>. corresponding non-stretched control; # <i>P</i><0.05 <i>vs</i>. stretched control.</p

GTPase inhibition prevents 5/6Nx-induced renal fibrosis.

<p>RT-PCR analysis of renal TGFβ, fibronectin and smooth muscle α-actin (α -SMA) expression (<b>A</b>), and expression of podocyte specific markers podocin, nephrin and WT-1 (<b>B</b>) detected in renal cortex homogenates. Polymerase 2a (Polr2a) served as housekeeping gene. Data are expressed as mean ± SEM from four mice of each group. *<i>P</i><0.05 <i>vs</i>. corresponding sham control; # <i>P</i><0.05 <i>vs</i>. 5/6Nx control.</p

Plasma levels of hydroxyeicosatetraenoic (HETE) acids.

<p>Plasma level of 5-HETE, 12-HETE, 15-HETE and 20-HETE as determined by LC-MS/MS 8 weeks after initiation of treatment. Effects of placebo, sEH-inhibition by cAUCB, ACE-inhibition by ramipril (Ramip.) and CYP-inhibition by fenbendazole (Fenbe.) are shown on sham operated (−) and 5-6-Nx (+) animals. * p<0,05 vs. sham, # p<0,05 vs. placebo, § p<0.05 vs. placebo all groups (n = 5–8/group).</p

mRNA expression of arachidonic acid metabolizing enzymes.

<p>Relative mRNA expression (2∧-ΔΔCT) determined by realtime RT-PCR for the factors indicate and representative sEH Western blot (WB sEH) from the kidney of sham operated and 5/6-Nx mice at the end of the study. A: Determination of EET-producing systems, B: qRT-PCR and Western blot for sEH. GAPDH and β-actin were used as loading control for the Western blots. C: Expression of Lox enzymes on the RNA level. n = 6, *p<0.05.</p

EET plasma level.

<p>11,12-EET and 11,12-DHET plasma levels 8 weeks after initiation of treatment. Effects of placebo, sEH-inhibition by cAUCB, and CYP-inhibition by fenbendazole (Fenbe.) are shown on sham operated (−) and 5-6-Nx (+) animals. * p<0,05 vs. sham, # p<0,05 vs. placebo, § p<0.05 vs. placebo and cAUCB (n = 5–8/group).</p

Blood pressure and albuminuria.

<p>Blood pressure measured by tail cuff technique (A) and albuminuria (B) were determined 4 and 8 weeks after initiation of treatment. Effects of placebo, sEH-inhibition by cAUCB, ACE-inhibition by ramipril (Ramip.) and CYP-inhibition by fenbendazole (Fenbe.) are shown on sham operated (−) and 5-6-Nx (+) animals. * p<0,05 vs. sham, # p<0,05 vs. placebo (n = 10–12/group).</p