Renal and Vascular Effects of the Allosteric Transglutaminase 2 Modulator LDN-27219 in One-Kidney DOCA–Salt Mice

Figures

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  Figure 1. Illustration of the closed and open conformation of transglutaminase 2 (TG2) including stimuli able to induce a shift between the two states. ROS, reactive oxygen species. See text for further details.

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  Figure 2. Flowchart of the experiment. Randomization of C57/BL6 mice into DOCA and sham groups and into treatment groups with LDN-27219 or vehicle. Three mice in the DOCA group were euthanized after surgery but before the start of treatment. Three mice died during the treatment period in the DOCA vehicle group, while one mouse died during the treatment period in the sham LDN-27219 group.

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  Figure 3. Urine albumin/creatine ratio (logarithmically transformed) for DOCA mice treated with LDN-27219 (n = 12) or vehicle (n = 10) and sham mice treated with LDN-27219 (n = 9) or vehicle (n = 10). Data were analyzed with 2-way ANOVA and are expressed as mean ± SEM. ns = non significant.

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  Figure 4. (A) Representative images of the Picrosirius red collagen staining (arrows) visualized under circularly polarized light from the different groups with (B) corresponding individual and mean quantification of positive picrosirius red staining percentage areas. Data were analyzed with 2-way ANOVA and are expressed as mean ± SEM. ns = non significant.

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  Figure 5. (A) Kidney TG2 mRNA expression based on qPCR and (B) averaged kidney TG2 protein expression (TG2/total protein) based on Western blotting in DOCA, and sham mice treated with LDN-27219 or vehicle. (C) Representative blots. The 2-way ANOVA was non-significant for both TG2 mRNA and TG2 protein. Data show individual results and mean ± SEM.

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  Figure 6. (A) Native PAGE with representative blots showing two bands corresponding to the open and closed conformation of TG2 and (B) average percentage of kidney TG2 protein present in the closed formation in DOCA mice treated with LDN-27219 (n = 12) or vehicle (n = 10) and in sham mice treated with LDN-27219 (n = 9) or vehicle (n = 10). Data were analyzed with 2-way ANOVA and are expressed as mean ± SEM. ns = non significant.

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  Figure 7. Renal mRNA expression based on qPCR for (A) fibronectin, (B) collagen 1α1, (C) collagen 3α1, (D) α–smooth muscle actin (αSMA) and (E) transforming growth factor beta (TGFβ) in DOCA mice treated with LDN-27219 (n = 12) or vehicle (n = 10) and in sham mice treated with LDN-27219 (n = 9) or vehicle (n = 10). The 2-way ANOVA was only significant for αSMA. Data show individual values and mean ± SEM. ns = non significant.

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  Figure 8. (A) Average constriction of mesenteric small arteries to noradrenaline (NA) in DOCA mice treated with LDN-27219 (n = 12) or vehicle (n = 10) and (B) average constriction to NA in sham mice treated with LDN-27219 (n = 9) or vehicle (n = 10). The responses are normalized to the tension obtained with KPSS. Data are mean ± SEM.

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  Figure 9. Mesenteric resistance artery relaxation to acetylcholine (ACh) and sodium nitropruside (SNP) following pre-constriction of the vessels with phenylephrine to achieve a tension corresponding to 60–80% of that induced by KPSS. Responses were normalized to the tension obtained with KPSS. The upper panel (A,B) shows data for DOCA mice treated with LDN-27219 (n = 12) or vehicle (n = 10), and the lower panel (C,D) shows data for sham mice treated with LDN-27219 (n = 9) or vehicle (n = 10). Data are mean ± SEM.