(Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

Figures

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  Figure 1. PRR is up-regulated in human and mouse fibrotic livers and in activated HSCs. (A) Left: Representative Western blot of hepatic PRR from patients without liver fibrosis (normal), and with mild and severe fibrosis (Ishak scores 1~3 and 4~6, respectively). The Western blots of hepatic PRR from other patients are shown in Figure 2A. Right: Quantification of hepatic PRR expression of the total patients (normal, n = 12; mild fibrosis, n = 15; severe fibrosis, n = 14). ∗P < .05 vs normal group; #P < .05 vs mild fibrosis group. (B) Plasma renin levels in patients without liver fibrosis, and with mild and severe fibrosis. ∗P < .05 vs normal group. (C) Immunohistochemical staining for PRR in normal and fibrotic human livers with various etiologies, and dual immunofluorescence staining in normal and fibrotic human livers (PRR, red; α-SMA, green; diamidino-2-phenylindole [DAPI] nuclear stain, blue). (D) Western blot analysis of PRR level in the livers of mice treated with TAA (n = 6) or vehicle (n = 5). (E) Hepatic dual immunofluorescence in TAA mouse fibrosis model (PRR, red; α-SMA, green; DAPI, blue). (F) mRNA expression of PRR in quiescent (n = 5) and culture-activated (n = 8) HSCs. ∗P <…

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  Figure 2. PRR expression in human livers and mouse primary cells. (A) Western blot of hepatic PRR from patients without liver fibrosis (normal), and with mild and severe fibrosis (Ishak scores 1~3 and 4~6, respectively). (B) PRR expression in mouse primary hepatocytes, quiescent hepatic stellate cells (qHSCs), KCs, and sinusoidal endothelial cells (SECs) isolated from normal mice (n = 5−6 in each group). (C) PRR immunofluorescence in mouse kidney (PRR, red); diamidino-2-phenylindole [DAPI] nuclear stain, blue). Scale bar: 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  Figure 3. PRR mRNA expression of primary liver cells in TAA-injured liver. PRR mRNA expression of primary liver cells isolated from TAA mice treated with lentivirus-delivered scramble or PRR shRNA (n = 9−10 in each group). The expression level in the primary cells isolated from TAA mice treated with scramble virus was assigned arbitrarily as 1. ∗P < .05 between groups.

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  Figure 4. Hepatic PRR knockdown did not alter hepatic inflammation in TAA-injured liver. Vehicle or TAA-administered mice were treated with lentivirus-delivered scramble or PRR shRNA (n = 6−8 in each group). (A) Liver necroinflammation was analyzed by H&E staining using a modified Ishak scoring system. (B) Serum ALT levels. (C) mRNA levels of Tnf, Il6, and Ccl2. (D) F4/80 immunostaining of liver sections. (E) Myeloperoxidase (MPO) staining of liver sections. (F) NKp46 immunostaining. (G) Hepatic levels of Va14-Ja18 mRNA. ∗P < .05 vs mice treated with vehicle and scramble virus. Scale bar: 100 μm. Arrows indicate positive cells. FOV, field of view.

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  Figure 5. The effects of PRR knockdown in RAW 264.7 cells. (A) PRR knockdown in RAW 264.7 cells using siRNA. (B) mRNA expression of Il1β, Il6, Tnf, and Ccl2 in RAW 264.7 cells treated with LPS (100 ng/mL, 24 h) (n = 6 in each group). ∗P < .05.

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  Figure 6. Hepatic PRR knockdown ameliorated TAA-induced liver fibrosis with suppression of ERK/TGF-β1/Smad3 pathway. Vehicle or TAA-administered mice were treated with lentivirus-delivered scramble shRNA or PRR shRNA (n = 6–8 in each group). (A) Western blot of hepatic PRR levels (left) and expression of hepatic renin mRNA (right) in the 4 groups. (B) Representative images of liver sections after Sirius red staining for the quantification of positively stained area. Scale bar: 100 μm. (C) Hepatic hydroxyproline levels. (D) Western blot of α-SMA with quantitative analysis. (E) Hepatic transcript expression of Col1a1, PAI-1 (Serpine1), Timp1, and Fn1. The expression levels in mice treated with vehicle and scramble virus were assigned arbitrarily as 1. (F) Western blot analysis of phosphorylated and total ERK1/2 (p/t-ERK1/2), TGF-β1, phosphorylated Smad3 (p-Smad3), and Smad3 in mouse livers. ∗P < .05 vs mice treated with vehicle and scramble virus. #P < .05 vs mice treated with TAA and scramble virus.

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  Figure 7. α-smooth muscle actin (α-SMA) expression in thioacetamide (TAA)-injured liver. Vehicle or thioacetamide (TAA)-administered mice were treated with lentivirus-delivered scramble or PRR shRNA (n = 6−8 in each group). (A) Hepatic immunofluorescence of PRR (red), α-SMA (green), and DAPI (diamidino-2-phenylindole, nuclear stain, blue) and (B) α-SMA immunohistochemical staining of liver sections in the 4 groups. ∗P < .05 vs mice treated with vehicle and scramble virus. #P < 0.05 vs mice treated with TAA and scramble virus. Scale bars: 100 μm.

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  Figure 8. Hepatic PRR knockdown attenuated MCD diet–induced liver fibrosis. (A) PRR mRNA expression in quiescent HSCs from normal chow (NC)-fed mice and in vivo–activated HSCs isolated from mice fed MCD diet for 12 weeks (n = 6 in each group). ∗P < .05 vs the quiescent HSC group. (B) Hepatic dual immunofluorescence of PRR and α-SMA in mice fed with NC or MCD diet. Scale bar: 100 μm. (C–H) MCD diet–fed mice were treated with lentiviral scramble shRNA or PRR shRNA (n = 6–8 in each group). Mice fed the NC diet (vehicle) and that received lentiviral-scramble shRNA served as controls (n = 6). (C) Western blot analysis of hepatic PRR levels and transcript expression of hepatic renin. (D) Sirius red staining of liver sections and quantification of the positively stained areas. Scale bar: 200 μm. (E) Hepatic hydroxyproline levels. (F) Western blot for α-SMA with quantitative analysis. (G) Hepatic transcript expression of profibrotic genes. The expression levels in the mice treated with vehicle and lentiviral scramble shRNA were assigned arbitrarily as 1. (H) Western blot analysis of phosphorylated and total ERK1/2, TGF-β1, p-Smad3, and Smad3 in mouse livers. ∗P < .05 vs mice treated with vehicle…

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  Figure 9. Hepatic PRR knockdown did not alter hepatic inflammation in the MCD diet model. The MCD diet–fed mice were treated with lentivirus-delivered scramble or PRR shRNA (n = 6−8 in each group). Mice fed with a normal chow diet (vehicle) and that received scramble virus were served as normal control (n = 6). (A) Serum ALT levels. (B) The mRNA levels of Tnf, Il6, and Ccl2. (C) NAFLD scores were analyzed by H&E staining. (D) Hepatic triglyceride levels, and (E) F4/80 immunostaining of liver sections. (F) Myeloperoxidase (MPO) staining of liver sections. (G) NKp46 immunostaining. (H) Hepatic levels of Va14-Ja18 mRNA. ∗P < .05 vs mice treated with vehicle and scramble virus. Arrows: positive cells. Scale bar: 100 μm. DAPI, diamidino-2-phenylindole; FOV, field of view.

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  Figure 10. PRR suppression inhibited expression of profibrotic genes and the ERK/TGF-β1/Smad3 pathway in LX-2 cells. Knockdown of PRR expression using shRNA reduced the PRR protein level in LX-2 cells (LX-2-shRNA, n = 6) compared with scrambled sequences (LX-2–scramble, n = 6). (A) mRNA expression of profibrotic genes including ACTA2, COL1A1, SERPINE1, PTGS2, TIMP1, and FN1 was analyzed in LX-2 cells with or without PRR knockdown. Significant difference compared with LX-2-scramble cells. ∗P < .05. (B) Expression of the ERK/TGF-β1/Smad3 pathway in LX-2 cells with or without PRR knockdown analyzed by Western blot. (C) TGF-β1 production in LX-2–scramble or LX-2–shRNA cells treated with control medium (vehicle), renin or prorenin (10-8 mol/L) for 24 hours (n = 6/group for the renin experiment and n = 10/group for the prorenin experiment). The levels of TGF-β1 were expressed as fold changes relative to the levels of TGF-β1 from LX-2–scramble cells treated with vehicle. ∗P < .05 vs LX-2–scramble cells treated with vehicle. #P < .05 vs LX-2–scramble cells treated with renin or prorenin. (D) LX-2 cells were co-incubated with renin (10-8 mol/L), the ERK inhibitor (U0126; 50 μmol/L), and the TGF-β1…

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  Figure 11. Myofibroblast-specific knockdown of PRR mitigated liver fibrosis in TAA-injured mice. Mice administered TAA randomly received injections of lentivirus expressed PRR shRNA (n = 11) or scrambled sequences (n = 9) under α-SMA promoter (labeled as SMA-miR-shPRR or SMA-miR-scramble, respectively). (A) Structure of lentiviral SMA-EGFP-miR-PRR shRNA vector. (B) Myofibroblast-specific delivery was confirmed in vivo. Hepatic immunofluorescence of GFP, α-SMA, and diamidino-2-phenylindole (DAPI). Scale bar: 100 μm. (C) HSCs isolated after lentiviral injection, arrowheads indicate GFP (+) cells and arrows indicate GFP (-) cells. Scale bars: 50 μm. Western blot analysis of isolated HSCs confirmed successful knockdown of PRR. (D) Sirius red staining of liver sections along with the quantification of positively stained area. Scale bar: 100 μm. (E) Hepatic hydroxyproline levels. (F) Western blot analysis of hepatic α-SMA expression. (G) Hepatic transcript levels of Col1a1, PAI-1 (Serpine1), Timp1, and Fn1. The expression levels in mice treated with vehicle and SMA-miR-scramble virus were assigned arbitrarily as 1. (H) Western blot analysis of PRR, phosphorylated and total ERK1/2, TGF-β1,…

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  Figure 12. PRR mRNA expression in primary cells and renin mRNA expression in livers of myofibroblast-specific PRR knockdown mouse models. (A) PRR mRNA expression in primary hepatocytes, KCs, and SECs isolated from TAA mice treated with SMA-miR-scramble or SMA-miR-shPRR lentivirus (n = 10 in each group). The expression level in the primary cells isolated from TAA mice treated with SMA-miR-scramble virus was assigned arbitrarily as 1. Hepatic renin mRNA expression in (B) TAA-injured mice or (C) MCD diet–fed mice treated with SMA-miR-scramble or SMA-miR-shPRR lentivirus. (D) PRR mRNA expression in primary hepatocytes, KCs, and SECs isolated from TAA mice treated with SMA-scramble or SMA-PRR-His lentivirus (n = 10 in each group). The expression level in the primary cells isolated from TAA mice treated with SMA-scramble virus was assigned arbitrarily as 1.