The AMPK Agonist AICAR Inhibits TGF-β1 Induced Activation of Kidney Myofibroblasts

<div><p>Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis <i>in</i><i>vivo</i> and TGF-β1-induced renal fibroblasts activation <i>in</i><i>vitro</i>. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-β1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-β1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-β1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-β1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-β1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.</p></div

AICAR inhibits transforming growth factor-β1 (TGF-β1)-induced fibroblast-myofibroblast transformation.

<p>Cultured rat fibroblasts, NRK-49F cells were pre-incubated in the presence or absence of AICAR (0.05 mM–0.5 mM) for 30 mins. Then, these cells were stimulated with TGF-β1 (1 ng/mL) for 24 h before harvesting. The culture supernatant was subject to immunoblot analysis with antibodies against collagen I, collagen IV (A). The cell lysate was also subject to immunoblot analysis with antibodies against collagen I (B), collagen IV (C), α-SMA (D) and tubulin. Representative immunoblots from three independent experiments are shown. Each bar represents the mean ± S.E. of three independent experiments. *<i>P</i><0.05 versus the TGF-β1 group. C: control, T: TGF-β1, A: AICAR.</p

The AMPKα1 subunit is the target of the inhibitory effects of AICAR in TGF-β1-stimulated NRK-49F cells.

<p>Cultured NRK-49F cells were transfected with specific siRNA for AMPKα1 (10 nM, 100 nM) or AMPKα2 (10 nM, 100 nM) or a control siRNA for 24 h. After 24 h of incubation, transfected cells were pre-incubated with or without AICAR (0.5 mM) for 30 mins. Then, these cells were stimulated with TGF-β1 (1 ng/mL) for 24 h before harvesting. (A) The inhibitory effects of siAMPKα1 were evaluated by measuring phospho-AMPKα and total-AMPKα levels. Representative immunoblots from three independent experiments are shown. (B) The efficiency of different concentrations of siAMPKα1 (1 nM, 10 nM, 50 nM, 100 nM) on AMPKα1 mRNA expression were evaluated. Data are expressed relative to the expression in control cells without transfection. Each bar represents the mean ± S.E. of three independent experiments. *<i>P</i><0.05 versus the TGF-β1 group; #<i>P</i><0.05 versus the TGF-β1+ AICAR group; ##<i>P</i><0.05 versus the control group. C: control, T: TGF-β1, A: AICAR.</p

AICAR reduces the expression of α-smooth muscle actin (α-SMA), collagen I, and fibronectin.

<p>A unilateral ureteral obstruction (UUO) model was induced in adult male BALB/c mice. Sham animals had their kidney exposed but the ureter was not tied. Mice with UUO were administered intra-peritoneal AICAR (500 mg·kg⁻¹·day) or saline 1 day before the UUO surgery and daily thereafter. Obstructed kidneys were harvested 7 days after surgery. qPCR analysis of the mRNA expression of collagen I (A), fibronectin (B), TGF-β1 (C), TNF-α (D) and MCP-1 (E), in sham, UUO, and UUO + AICAR kidneys. Data are expressed relative to the expression in sham-operated kidneys. Kidney tissue lysates were also subjected to immunoblot analysis with specific antibodies against α-SMA, collagen I, fibronectin, and β-actin (F). Protein expression levels of α-SMA (G), collagen I (H) and fibronectin (I) were analyzed by western blotting, quantified by densitometry, and normalized to β-actin levels. Each bar represents the mean ± S.E. (n = 6 in each group). *<i>P</i><0.05 between the sham and UUO group; #<i>P</i><0.05 between the UUO and UUO + AICAR group.</p

The effects of AICAR on the inhibition TGF-β1-mediated kidney myofibroblast activation were associated with STAT3 down-regulation.

<p>Cultured NRK-49F cells were incubated with TGF-β1 (1 ng/mL) for 15 mins–180 mins in the presence or absence of AICAR (0.5 mM). (A) Cell lysates were subject to immunoblot analysis with antibodies against phospho-STAT3 (P-STAT3) and tubulin. (B) TGF-β1-induced α-smooth muscle actin (α-SMA) expression in NRK-49F cells is inhibited by JAK inhibitor and AICAR. Representative immunoblots from three experiments are shown. Each bar represents the mean ± S.E. of three independent experiments. *<i>P</i><0.05 versus the corresponding group (control or TGF-β1) at the same time duration after TGF-β1 treatment; #<i>P</i><0.05 versus the control group; ##<i>P</i><0.05 versus the TGF-β1 group. C: control, T: TGF-β1, A: AICAR.</p

The effects of AICAR in the non-Smad TGF-β pathway were associated with down-regulation of ERK 1/2.

<p>Cultured NRK-49F cells were incubated with TGF-β1 (1 ng/mL) for 15 mins–180 mins in the presence or absence of AICAR (0.5 mM). (A) Cell lysates were subject to immunoblot analysis with antibodies against phospho-Smad2 (P-Smad2), phospho-Smad3 (P-Smad3), and tubulin. (B) Cell lysates were also subject to immunoblot analysis with antibodies against phospho-ERK1/2 (P-ERK1/2), phospho-p38 (P-p38), phospho-JNK (P-JNK), and tubulin. Representative immunoblots from three experiments are shown. Each bar represents the mean ± S.E. of three independent experiments. *<i>P</i><0.05 versus the corresponding group (control or TGF-β1) at the same time duration after TGF-β1 treatment. C: control, T: TGF-β1, A: AICAR.</p

AICAR reduces renal fibrosis and the deposition of extracellular matrix (ECM) in obstructed kidneys.

<p>A unilateral ureteral obstruction (UUO) model was induced in adult male BALB/c mice. Sham-operated animals had their kidney exposed but the ureter was not tied. Mice with UUO were administered intra-peritoneal AICAR (500 mg·kg⁻¹·day) or saline 1 day before the UUO surgery and daily thereafter. Obstructed kidneys were harvested 7 days after surgery. (A) Photomicrographs illustrating Masson trichrome staining of kidney tissue from mice in various treatment groups. (B) The Masson trichrome-positive tubulointerstitial area (blue) relative to the total area from 10 random cortical fields was analyzed. Data are shown as the mean ± S.E. (n = 6 in each group). *<i>P</i><0.05 versus the sham group; #<i>P</i><0.05 versus the UUO group.</p

AMPKα1 silencing and compound C blocks the inhibitory effects of AICAR on TGF-β1-induced myofibroblast activation.

<p>Cultured NRK-49F cells were transfected with a siRNA specific for AMPKα1 (10 nM) or a control siRNA for 24 h. After 24 h of incubation, the transfected cells were pre-incubated with or without AICAR (0.5 mM) for 30 mins. Then, these cells were stimulated with TGF-β1 (1 ng/mL) for 24 h before harvesting. (A) Culture supernatant and cell lysates were subject to immunoblot analysis with antibodies against collagen I, collagen IV, α-SMA, and tubulin. (B) Cultured NRK-49F cells were pre-incubated with compound C (1.0 mM, 2.5 mM, or 5.0 mM) for 30 mins. Then, these cells were stimulated with TGF-β1 (1 ng/mL) for 24 h before harvesting. Immunoblotting of the cell lysates, showed that the inhibitory effects of AICAR on α-SMA expression were decreased by the treatment with Compound C in a dose-dependent manner. Representative immunoblots from three experiments are shown. Each bar represents the mean ± S.E. of three independent experiments. *<i>P</i><0.05 versus the control group; #<i>P</i><0.05 versus the TGF-β1 (+/− mock) group; ##<i>P</i><0.05 versus the TGF-β1 (+/− mock) + AICAR group. C: control, T: TGF-β1, M: mock, A: AICAR.</p