Journal: Journal of Cellular Physiology

Article Title: Vacuolar H + ‐ATPase and Megalin‐Mediated Prorenin Uptake: Focus on Elements Beyond the (Pro)Renin Receptor

doi: 10.1002/jcp.31518

Figure Lengend Snippet: Efficacy of V‐ATPase subunit knockdown, and its effect on prorenin uptake. (A) V‐ATPase subunit and accessory protein expression after their individual knockdown by siRNA in BN16 cells. A nontargeting siRNA was used a negative control. Data (individual data points and mean) have been expressed versus control and represent three independent experiments. (B) cellular prorenin levels after incubating siRNA‐transfected BN16 cells with recombinant human prorenin for 4 h. Data (individual data points of three triplicate independent experiments and mean) have been expressed versus control. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Article Snippet: Next, they were incubated with 100 U/L recombinant human prorenin (a gift of Actelion Pharmaceuticals, Allschwil, Switzerland) in PBS at 37°C for 4 h. After incubation, the medium was removed, and the cells were washed twice with ice‐cold 0.5% BSA in PBS and once with ice‐cold PBS.

Techniques: Knockdown, Expressing, Negative Control, Control, Transfection, Recombinant

Journal: Frontiers in bioscience (Landmark edition)

Article Title: C3aR Antagonist Alleviates C3a Induced Tubular Profibrotic Phenotype Transition via Restoring PPARα/CPT-1α Mediated Mitochondrial Fatty Acid Oxidation in Renin-Dependent Hypertension.

doi: 10.31083/j.fbl2810238

Figure Lengend Snippet: Fig. 3. Coincubation with recombinant rat renin increases the cleavage fragments of C3. (A) Representative western blotting

Article Snippet: HK2 cells were treated with 10% serum from healthy volunteer 7 (H7) supplemented with recombinant renin (1.2 μg/mL) or PBS at 37 °C for 24 h. Aliskiren (10 μM, MedChemExpress, Monmouth Junction, NJ, USA), a specific renin inhibitor, was preincubated with recombinant renin for 1 h before addition.

Techniques: Recombinant, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: 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.

Article Snippet: PRR-shRNA–transfected LX-2 and scrambled shRNA-transfected LX-2 cells were treated with recombinant human renin or prorenin (Cayman Chemical, Ann Arbor, MI) at the indicated dose (10 -8 mol/L) and times., Culture supernatants were collected, and the level of TGF-β1 was determined by enzyme-linked immunosorbent assay (R&D Systems, Inc, Minneapolis, MN).

Techniques: Knockdown, shRNA, Western Blot, Expressing, Staining, Virus

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: 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 and scramble virus. # P < .05 vs mice fed with MCD diet and scramble virus. DAPI, diamidino-2-phenylindole; NV, normal mice treated with vehicle; p-ERK, phosphorylated extracellular signal regulated kinase; p-SMAD3, phosphorylated SMAD3; t-ERK, total extracellular signal regulated kinase.

Article Snippet: PRR-shRNA–transfected LX-2 and scrambled shRNA-transfected LX-2 cells were treated with recombinant human renin or prorenin (Cayman Chemical, Ann Arbor, MI) at the indicated dose (10 -8 mol/L) and times., Culture supernatants were collected, and the level of TGF-β1 was determined by enzyme-linked immunosorbent assay (R&D Systems, Inc, Minneapolis, MN).

Techniques: Knockdown, Expressing, In Vivo, Isolation, Immunofluorescence, shRNA, Western Blot, Staining, Virus

Journal: Cellular and Molecular Gastroenterology and Hepatology

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

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: 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 neutralizing antibody (19D8; 10 μg/mL) for 2 hours. The phosphorylation of ERK was evaluated by Western blot. Ctrl, control cells incubated with vehicle. ( E ) LX-2 cells were co-incubated with renin (10 -8 mol/L) and U0126 (50 μmol/L) for 6 hours. The levels of TGF-β1, p-Smad3, and Smad3 were determined by Western blot. ( F ) LX-2 cells were co-incubated with renin (10 -8 mol/L) and 19D8 (10 μg/mL) for 6 hours. The phosphorylation of Smad3 was determined by Western blot. ( G ) Western blot analysis of hepatic ERK/TGF-β1/Smad3 pathway from patients without liver fibrosis (normal), and with mild and severe fibrosis (Ishak scores 1~3 and 4~6, respectively). ( H ) PRR mRNA expression in LX-2 cells incubated with vehicle, TNF-α (100 ng/mL), epidermal growth factor (EGF, 5 ng/mL), platelet-derived growth factor-BB (PDGF-BB, 5 ng/mL), TGF-β1 (10 ng/mL), renin (10 -8 mol/L), or prorenin (10 -8 mol/L) for 24 hours (n = 6/group). ∗ P < .05 vs LX-2 cells treated with vehicle. ( I ) PRR mRNA expression in primary mouse HSCs incubated with renin (10 -8 mol/L) or prorenin (10 -8 mol/L) for 24 hours (n = 5/group). ∗ P < .05 vs primary HSCs treated with vehicle. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-ERK, phosphorylated extracellular signal regulated kinase; p-SMAD, phosphorylated SMAD; t-ERK, total extracellular signal regulated kinase.

Article Snippet: PRR-shRNA–transfected LX-2 and scrambled shRNA-transfected LX-2 cells were treated with recombinant human renin or prorenin (Cayman Chemical, Ann Arbor, MI) at the indicated dose (10 -8 mol/L) and times., Culture supernatants were collected, and the level of TGF-β1 was determined by enzyme-linked immunosorbent assay (R&D Systems, Inc, Minneapolis, MN).

Techniques: Expressing, Knockdown, shRNA, Western Blot, Control, Incubation, Phospho-proteomics, Derivative Assay