Involvement of NADPH oxidases in the Na/K‑ATPase/Src/ROS oxidant amplification loop in renal fibrosis

Abstract

The Na/K‑ATPase/Src complex is reportedly able to affect reactive oxygen species (ROS) amplification. However, it has remained elusive whether NADPH oxidases (NOXs) are involved in this oxidant amplification loop in renal fibrosis. To test this hypothesis, interactions between oxidative features and Na/K‑ATPase/Src activation were examined in a mouse model of unilateral urethral obstruction (UUO)‑induced experimental renal fibrosis. Both 1‑tert‑butyl‑3‑(4‑chlorophenyl)‑1H‑pyrazolo[3,4‑d]pyrimidin‑4‑amine (PP2) and apocynin significantly attenuated the development of UUO‑induced renal fibrosis. Apocynin administration attenuated the expression of NOXs and oxidative markers (e.g., nuclear factor erythroid 2‑related factor 2, heme oxygenase‑1,4‑hydroxynonenal and 3‑nitrotyrosine); it also partially restored Na/K‑ATPase expression and inhibited the activation of the Src/ERK cascade. Furthermore, administration of PP2 after UUO induction partially reversed the upregulation of NOX2, NOX4 and oxidative markers, while inhibiting the activation of the Src/ERK cascade. Complementary experiments in LLC‑PK1 cells corroborated the in vivo observations. Inhibition of NOX2 by RNA interference attenuated ouabain‑induced oxidative stress, ERK activation and E‑cadherin downregulation. Thus, it is indicated that NOXs are major contributors to ROS production in the Na/K‑ATPase/Src/ROS oxidative amplification loop, which is involved in renal fibrosis. The disruption of this vicious feed‑forward loop between NOXs/ROS and redox‑regulated Na/K‑ATPase/Src may have therapeutic applicability for renal fibrosis disorders.

Keywords: NADPH oxidases; Na/K‑ATPase; Src; oxidative stress; renal fibrosis.

Figure 1.

UUO-induced renal fibrosis and myofibroblast accumulation are attenuated by PP2 and apocynin treatment. (A) Representative images and data analysis of Sirius Red renal histology. (B) Representative images and data analysis of immunohistochemical staining for COL-I. (C) Representative images and data analysis of immunohistochemical staining for α-SMA (magnification, ×400; scale bars, 100 µm). (D) Representative western blot and data analysis of COL-I expression in kidney homogenates. (E) Representative western blot and data analysis of α-SMA expression in kidney homogenates. Values are expressed as mean ± SEM (n=3-5). ***P<0.001 vs. sham alone; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. UUO alone. COL-I, collagen I; α-SMA, α-smooth muscle actin; PP2, 1-tert-butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; Apo, apocynin; UUO, unilateral ureteral obstruction.

Figure 2.

Apocynin treatment downregulates Na/K-ATPase expression and inhibits Src/ERK activation. (A) Representative images and data analysis of immunohistochemical staining for Na/K-ATPase (magnification, ×400; scale bars, 100 µm). (B) Representative western blot and data analysis of Na/K-ATPase expression in kidney homogenates. (C) Representative western blot and data analysis of Src activation in kidney homogenates. Src activation was expressed as the p-Src/t-Src ratio. (D) Representative western blot and data analysis of ERK activation in kidney homogenates. ERK activation was expressed as p-ERK/t-ERK. Values are expressed as the mean ± SEM (n=3-5). **P<0.01, ***P<0.001 vs. sham alone; ^#P<0.05, ^###P<0.001 vs. UUO alone. Apo, apocynin; UUO, unilateral ureteral obstruction; p-, phosphorylated; t-, total.

Figure 3.

Src inhibition decreases expression levels of Nrf2, HO-1, 3-NT and 4-HNE. (A) Representative western blot and data analysis of Nrf2 and HO-1 content in kidney homogenates. (B) Representative western blot and data analysis of 3-NT and 4-HNE content in kidney homogenates. (C) Representative images and data analysis of immunohistochemical staining of 8-OHdG (magnification, ×400; scale bars, 100 µm). Values are expressed as the mean ± SEM (n=3-5). **P<0.01, ***P<0.001 vs. sham alone; ^#P<0.05, ^##P<0.001, ^###P<0.001 vs. UUO alone. 3-NT, 3-nitrotyrosine; 4-HNE, 4-hydroxynonenal; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; Apo, apocynin; HO-1, heme-oxygenase 1; Nrf2, nuclear factor E2-related factor 2; UUO, unilateral ureteral obstruction.

Figure 4.

Expression levels of NOX2 and NOX4 are reduced by PP2 and apocynin treatment. (A) Representative images and data analysis of immunohistochemical staining of NOX2. (B) Representative images and data analysis of immunohistochemical staining of NOX4 (magnification, ×400; scale bars, 100 µm). (C) Representative western blot and data analysis of NOX2 expression in kidney homogenates. (D) Representative western blot and data analysis of NOX4 expression in kidney homogenates. Values are expressed as the mean ± SEM (n=3-5). ***P<0.001 vs. sham alone; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. UUO alone. Apo, apocynin; UUO, unilateral ureteral obstruction; NOX, NADPH oxidase.

Figure 5.

Ouabain-induced ROS production in LLC-PK1 cells. (A) Time course of Src activation. Src activation was expressed as the p-Src/t-Src ratio. (B) Time course of ERK activation. ERK activation was expressed as the p-ERK/t-ERK ratio. (C) Representative fluorescence images of ROS production in ouabain-treated LLC-PK1 cells (scale bars, 100 µm). (D) DCF fluorescence intensity in ouabain-treated LLC-PK1 cells, as determined with a microplate reader. Representative western blots and quantitative data are provided. Values are expressed as the mean ± SEM (n=3). *P<0.05; **P<0.01; ***P<0.001 vs. Ctrl. Ctrl, control; p-, phosphorylated; t-, total; ROS, reactive oxygen species; DCF, dichlorofluorescein.

Figure 6.

ROS are involved in ouabain-induced activation of Na/K-ATPase/Src/ERK signaling. (A) Effects of PP2 on ouabain-induced Src activation. (B) Effects of PP2 on ouabain-induced ERK activation. (C) Representative fluorescence images of ROS production in PP2-pretreated LLC-PK1 cells (scale bars, 100 µm). (D) DCF fluorescence intensity in PP2-pretreated LLC-PK1 cells. (E) Representative fluorescence images of ROS production in NAC-pretreated LLC-PK1 cells (scale bars, 100 µm). (F) DCF fluorescence intensity in NAC-pretreated LLC-PK1 cells. (G) Effects of NAC on ouabain-induced Src activation. (H) Effects of NAC on ERK activation. Representative western blots and quantitative data are shown. Values are expressed as the mean ± SEM (n=3). *P<0.05; **P<0.01; ***P<0.001 compared with Ctrl; ^#P<0.05; ^##P<0.01; ^###P<0.001 compared with ouabain. Ctrl, control; NAC, N-acetylcysteine; PP2, 1-tert-butyl-3- (4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; ROS, reactive oxygen species; DCF, dichlorofluorescein.

Figure 7.

NOX2 is the source of ouabain-induced ROS production. (A) Time course of NOX2 expression. (B) Time course of p67phox expression. (C) Time course of p47phox expression. (D) Time course of NOX1 expression. (E) Time course of NOX4 expression. (F) Time course of NOX5 expression. (G) NOX2 expression was knocked down by siRNA. (H) Representative fluorescence images of ROS production in transfected LLC-PK1 cells with ouabain treatment (scale bars, 100 µm). (I) DCF fluorescence intensity in transfected LLC-PK1 cells with ouabain treatment. Representative western blots and quantitative data are provided. Values are expressed as the mean ± SEM (n=3). *P<0.05; **P<0.01; ***P<0.001 compared with Ctrl or siCtrl; ^##P<0.01 compared with ouabain or siCtrl + ouabain. Ctrl, control; siCtrl, control siRNA; siNOX2, NOX2 siRNA; siRNA, small inhibitory RNA; ROS, reactive oxygen species; NOX, NADPH oxidase; DCF, dichlorofluorescein.

Figure 8.

Roles of NOX2/reactive oxygen species in ouabain-induced Na/K-ATPase/Src/ERK signaling and cellular trans-differentiation. (A) Effects of PP2 on NOX2 expression. (B) Effects of NOX2 siRNA on ouabain-induced Src activation. (C) Effects of NOX2 siRNA on ouabain-induced ERK activation. (D) Effects of NOX2 siRNA on E-cadherin expression. Representative western blots and quantitative data are shown. Values are expressed as the mean ± SEM (n=3). *P<0.05; **P<0.01 compared with Ctrl; ^#P<0.05; ^##P<0.01 compared with ouabain or siCtrl + ouabain. Ctrl, control; siCtrl, control siRNA; siNOX2, NOX2 siRNA; siRNA, small inhibitory RNA; PP2, 1-tert-butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; NOX, NADPH oxidase.

Figure 9.

Schematic illustration of the mechanism of the Na/K- ATPase/Src/ROS oxidant amplification loop in renal fibrosis (created with FigDraw). CTS, cardiotonic steroids; NOXs, NADPH oxidases; ROS, reactive oxygen species.