Rivaroxaban, a Direct Factor Xa Inhibitor, Ameliorates Hypertensive Renal Damage Through Inhibition of the Inflammatory Response Mediated by Protease‐Activated Receptor Pathway

Figures

• 

  Figure 1. Effects of rivaroxaban on coagulation activity. The 12‐ to 16‐week‐old Ren‐TG and WT mice were administered with or without 6 or 12 mg/kg of rivaroxaban for 1 month. A, Plasma factor Xa in the WT and Ren‐TG mice administered with or without 12 mg/kg of rivaroxaban. B and C, Anti–factor Xa activity, and prothrombin time in WT and Ren‐TG mice administered with or without rivaroxaban at 6 and 12 mg/kg. Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. D, Relationship between anti–factor Xa activity and rivaroxaban concentration in the blood (white circle; WT with rivaroxaban at 6 mg/kg, n=5, white square; WT with rivaroxaban at 12 mg/kg, n=5, solid circle; Ren‐TG with rivaroxaban at 6 mg/kg, n=3, solid square; Ren−TG with rivaroxaban at 12 mg/kg, n=5). *P<0.05, **P<0.01. Sample sizes are given on each bar. Ren‐TG indicates renin transgenic; WT, wild‐type.

• 

  Figure 2. Effects of rivaroxaban on UAE. A and B, UAE in the WT and Ren−TG mice treated with or without 12 mg/kg of rivaroxaban for 1 month (A) and for 4 months (B). Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. **P<0.01. Sample sizes are given on each bar. C, Effects of genotype, age, and drug on UAE were evaluated by 3‐way ANOVA. Their interactions were evaluated by both 2‐way and 3‐way ANOVA. Age was used as a categorical (5 months old for 1‐month rivaroxaban treatment and 8 months old for 4‐month rivaroxaban treatment). ns indicates not significant; Ren‐TG, renin transgenic; UAE, urinary albumin excretion; WT, wild‐type.

• 

  Figure 3. Effects of rivaroxaban on glomerular hypertrophy. A through F, Representative images (×200) of the kidneys stained with PAS in WT and Ren‐TG mice with or without rivaroxaban at 6 and 12 mg/kg for 4 months. G, Comparison of glomerular tuft surface area among the groups. Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. *P<0.05, **P<0.01. Sample sizes are given on each bar. PAS indicates periodic acid‐Schiff; Ren‐TG, renin transgenic; WT, wild‐type.

• 

  Figure 4. Histologic examination with Masson trichrome staining. A through F, Representative images (×400) of the kidneys stained with Masson trichrome in WT and Ren‐TG mice with or without rivaroxaban at 6 and 12 mg/kg for 4 months. Ren‐TG indicates renin transgenic; WT, wild‐type.

• 

  Figure 5. Effects of rivaroxaban on GBM and podocyte foot process effacement. A through F, Representative images of GBM thickness and the podocyte foot process effacement by electron microscopy (×19 000) in the WT (A and D), Ren‐TG (B and E), and Ren‐TG treated with 12 mg/kg of rivaroxaban for 4 months (C and F). G and H, Quantification of GBM thickness (the ratio to the WT mice) (G) and open slit pore number per 100 μm (H). Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. **P<0.01. Sample sizes are given on each bar. GBM indicates glomerular basement membrane; Ren‐TG, renin transgenic; WT, wild‐type.

• 

  Figure 6. Effects of rivaroxaban on renal expressions of PAR‐2 and proinflammatory genes. A, Comparison of the renal gene expression of PAR‐2 among the WT and Ren‐TG mice with or without 12 mg/kg of rivaroxaban for 1 month. B and C, Representative bands of PAR‐2 by western blotting (molecular weight markers are shown) and relative levels of renal protein expression of PAR‐2. D through F, Comparisons of the renal gene expression of TNF‐α (D), PAI‐1 (E), and MCP‐1 (F). Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. *P<0.05, **P<0.01. All data are normalized to GAPDH and are described as a ratio to the WT mice. Sample sizes are given on each bar. GAPDH, glyceraldehyde‐3‐phosphatase dehydrogenase; MCP‐1, monocyte chemoattractant protein‐1; PAI‐1, plasminogen activator inhibitor‐1; PAR‐2, protease‐activated receptor‐2; Ren‐TG, renin transgenic; TNF‐α, tumor necrosis factor‐α; WT, wild‐type.

• 

  Figure 7. Effects of rivaroxaban on podocyte damage induced by angiotensin II stained by phalloidin. A, Arrow indicates normal actin filament structure in the control. B, Treatment with angiotensin II (1.0 μmol/L) for 24 hours resulted in reorganization of the actin cytoskeleton with disruption of central stress fibers (stars) and its accumulation on the cell periphery (arrowhead). C, Rivaroxaban (500 μg/L) partially ameliorated actin reorganization induced by angiotensin II. D, Comparison of the gene expression of synaptopodin in human podocytes. Data are normalized to GAPDH and are described as a ratio to the control. Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. *P<0.05, **P<0.01. Sample sizes are given on each bar. GAPDH indicates glyceraldehyde‐3‐phosphatase dehydrogenase.

• 

  Figure 8. Effects of rivaroxaban on proinflammatory response induced by angiotensin II in human podocytes. Cells were stimulated with angiotensin II (1.0 μmol/L) for 6 hours with the presence or absence of rivaroxaban (500 μg/L). A through C, Comparisons of the gene expressions of MCP‐1 (A), TNF‐α (B), and interleukin‐6 (C). Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. *P<0.05, **P<0.01. D through F, Representative images of the human podocytes stained with p65 nuclear factor–κB in the control (D), stimulation with angiotensin II (1.0 μmol/L) for 1 hour (E), and rivaroxaban+angiotensin II (F). Sample sizes are given on each bar. MCP‐1, monocyte chemoattractant protein‐I; TNF‐α, tumor necrosis factor‐α.

• 

  Figure 9. Role of PAR‐2 signaling on proinflammatory genes in human podocytes. A, Effect of rivaroxaban (500 μg/L) on the gene expression of PAR‐2 in human podocytes in the presence or absence of angiotensin II (1.0 μmol/L). B, Validation of knockout of PAR‐2 by small interfering RNA (si PAR‐2) in human podocytes. C through E, Human podocytes were treated with control small interfering RNA (si cont) or small interfering PAR‐2 in the presence or absence of angiotensin II (1.0 μmol/L), and the gene expressions of MCP‐1 (C), TNF‐ α (D), and synaptopodin (E) were quantified. Data are presented as means±SD. Statistical analysis was performed by 1‐way ANOVA, followed by the Tukey's honest significant difference test. *P<0.05, **P<0.01. Sample sizes are given on each bar. MCP‐1 indicates monocyte chemoattractant protein‐I; PAR‐2, protease‐activated receptor‐2; TNF‐α, tumor necrosis factor‐α.