Crucial role of Rho-nuclear factor-kappaB axis in angiotensin II-induced renal injury

Abstract

This study was performed to determine the effectiveness of the Rho kinase inhibitor and NF-kappaB inhibitor in renal injury of ANG II-infused hypertensive rats. Male Sprague-Dawley rats, maintained on a normal diet, received either a sham operation (n = 7) or continuous ANG II infusion (120 ng/min) subcutaneously via minipumps. The ANG II-infused rats were further subdivided into three subgroups (n = 7 each) to receive one of the following treatments during the entire period: vehicle, Rho kinase inhibitor (fasudil; 3 mg.kg(-1).day(-1) ip), or NF-kappaB inhibitor (parthenolide; 1 mg.kg(-1).day(-1) ip). After 12 days of ANG II infusion, systolic blood pressure (BP; 208 +/- 7 vs. 136 +/- 3 mmHg), Rho kinase activity, NF-kappaB activity, renal ANG II contents (160 +/- 25 vs. 84 +/- 14 pg/g), monocytic chemotactic protein (MCP) 1 mRNA, interstitial macrophage infiltration, transforming growth factor-beta1 (TGF-beta1) mRNA, interstitial collagen-positive area, urinary protein excretion (43 +/- 6 vs. 11 +/- 2 mg/day), and urinary albumin excretion were significantly enhanced compared with the Sham group. While fasudil or parthenolide did not alter systolic BP (222 +/- and 190 +/- 21, respectively), both treatments completely blocked ANG II-induced enhancement of NF-kappaB activity, renal ANG II contents (103 +/- 11 and 116 +/- 21 pg/g, respectively), MCP1 mRNA, interstitial macrophage infiltration, TGF-beta1 mRNA, interstitial collagen-positive area, urinary protein excretion (28 +/- 6 and 23 +/- 3 mg/day, respectively), and urinary albumin excretion. Importantly, parthenolide did not alter ANG II-induced Rho kinase activation although fasudil abolished ANG II-induced Rho kinase activation. These data indicate that the Rho-NF-kappaB axis plays crucial roles in the development of ANG II-induced renal injury independently from BP regulation.

Fig. 1

A: temporal profile of systolic blood pressure (BP). Systolic BP was similar among the 4 groups before the treatments. However, systolic BP progressively and significantly increased (208 ± 7 for ANG II vs. 136 ± 3 mmHg for Sham at day 12). Fasudil (Ri) or parthenolide (Ni) did not alter systolic BP (222 ± 8 and 190 ± 21 mmHg at day 12, respectively). *P < 0.05 compared with the corresponding Sham group at that time period and P < 0.05 compared with the corresponding group at day 0. B: chronic ANG II infusion significantly increased Rho kinase activity (2.23 ± 0.20 for ANG II vs. 1.00 ± 0.12 arbitrary units for Sham). Importantly, while fasudil abolished ANG II-induced Rho kinase activation, parthenolide did not alter ANG II-induced Rho kinase activation (0.98 ± 0.22 for ANG II+fasudil and 2.07 ± 0.25 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group. C: for evaluation of NF-κB expression, mRNA levels of RelA (p65), a part of the NF-κB complex, were measured by real-time-PCR. Chronic ANG II infusion significantly increased RelA mRNA levels (1.60 ± 0.18 for ANG II vs. 1.00 ± 0.11 arbitrary units for Sham). Both treatments completely blocked ANG II-induced enhancement of RelA mRNA levels (0.95 ± 0.11 for ANG II+fasudil and 0.68 ± 0.06 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group. D: chronic ANG II infusion significantly increased NF-κB activity (2.25 ± 0.18 for ANG II vs. 1.00 ± 0.19 arbitrary units for Sham). Both treatments completely blocked ANG II-induced enhancement of NF-κB activity (0.98 ± 0.13 for ANG II+fasudil and 0.95 ± 0.10 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group. E: chronic ANG II infusion significantly increased kidney ANG II levels (160 ± 25 for ANG II vs. 84 ± 14 pg/g for Sham). Both treatments completely blocked ANG II-induced enhancement of kidney ANG II levels (103 ± 11 for ANG II+fasudil and 116 ± 21 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group.

Fig. 2

A: chronic ANG II infusion significantly increased intrarenal MCP1 mRNA levels (1.55 ± 0.15 for ANG II vs. 1.00 ± 0.13 arbitrary units for Sham). Both treatments completely blocked ANG II-induced enhancement of intrarenal MCP1 mRNA levels (1.16 ± 0.13 for ANG II+fasudil and 0.90 ± 0.05 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group. B–E: interstitial macrophage/monocyte infiltration was evaluated by CD68-positive cell number, which is a surface marker for macrophages and monocytes, using zinc-saturated, formalin-fixed, paraffin-embedded kidney samples from Sham (B), ANG II (C), ANG II+fasudil (D), and ANG II+parthenolide (E) groups. CD68-positive cells are stained brown. F: CD68-positive cell numbers were significantly increased by chronic ANG II infusion (28 ± 2 cells/mm²) compared with Sham (16 ± 1). Both treatments completely blocked ANG II-induced enhancement of interstitial macrophage/monocyte infiltration (23 ± 2 for ANG II+fasudil and 18 ± 1 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group.

Fig. 3

A: chronic ANG II infusion significantly increased intrarenal transforming growth factor (TGF)-β1 mRNA levels (1.52 ± 0.16 for ANG II vs. 1.00 ± 0.08 arbitrary units for Sham). Both treatments completely blocked ANG II-induced enhancement of intrarenal TGF-β1 mRNA levels (0.95 ± 0.12 for ANG II+fasudil and 0.74 ± 0.07 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group. B–E: interstitial collagen-positive area was stained by Picro-sirius red using zinc-saturated, formalinfixed, paraffin-embedded kidney samples from Sham (B), ANG II (C), ANG II+fasudil (D), and ANG II+parthenolide (E) groups. Collagen-positive area is stained pink. F: an established computer-aided semiautomatic quantification system demonstrates that the interstitial collagen-positive area was significantly increased by chronic ANG II infusion (1.00 ± 0.04%) compared with Sham (0.82 ± 0.05). Both treatments completely blocked ANG II-induced enhancement of interstitial collagen-positive area (0.84 ± 0.02 for ANG II+fasudil and 0.81 ± 0.04 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group.

Fig. 4

A: chronic ANG II infusion significantly increased urinary protein excretion (43 ± 6 for ANG II vs. 11 ± 2 mg/day for Sham). Both treatments completely blocked ANG II-induced enhancement of urinary protein excretion (28 ± 6 for ANG II+fasudil and 23 ± 3 for ANG II+parthenolide, respectively). B: chronic ANG II infusion significantly increased urinary albumin excretion (6.9 ± 1.7 for ANG II vs. 2.7 ± 0.5 mg/day for Sham). Both treatments completely blocked ANG II-induced enhancement of urinary albumin excretion (2.3 ± 0.4 for ANG II+fasudil and 3.5 ± 1.7 for ANG II+parthenolide, respectively). *P < 0.05 compared with the Sham group.

Fig. 5

Representative Western blot analysis. To elucidate at which level ANG II-induced Rho activation stimulates the NF-κB pathway, related kinase activities were evaluated by Western blot analysis. Chronic ANG II infusion significantly increased phosphorylation of IκBα (A; 1.49 ± 0.09 for ANG II vs. 1.00 ± 0.07 arbitrary units for Sham). Both treatments completely blocked ANG II-induced phosphorylation of IκBα (1.03 ± 0.02 for ANG II+fasudil and 1.05 ± 0.07 for ANG II+parthenolide, respectively). Chronic ANG II infusion did not change phosphorylation of IκBKα (B; 85 kDa, 0.99 ± 0.07 for ANG II vs. 1.00 ± 0.09 arbitrary units for Sham). Either treatment did not alter phosphorylation of IκBKα (1.00 ± 0.05 for ANG II+fasudil and 1.08 ± 0.07 for ANG II+parthenolide, respectively). Chronic ANG II infusion did not change phosphorylation of IκBKβ (B; 87 kDa, 0.94 ± 0.05 for ANG II vs. 1.00 ± 0.07 arbitrary units for Sham). Either treatment did not alter phosphor-ylation of IκBKβ (0.93 ± 0.09 for ANG II+fasudil and 1.00 ± 0.07 for ANG II+parthenolide, respectively). Chronic ANG II infusion did not change phosphorylation of NF-κB-inducing kinase (C; 0.90 ± 0.05 for ANG II vs. 1.00 ± 0.07 arbitrary units for Sham). Either treatment did not alter phosphor-ylation of NF-κB-inducing kinase (0.89 ± 0.07 for ANG II+fasudil and 0.94 ± 0.07 for ANG II+parthenolide, respectively). To verify equal loading, membranes were reprobed with β-actin antibody. Similar densities were observed among the 4 groups (D). These data clearly indicate that ANG II-induced Rho activation stimulates NF-κB pathway at phosphorylation of IκBα levels and that this mechanism is independent of IκBKα, IκBKβ, and NF-κB-inducing kinase.