Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of (pro)renin receptor in albumin overload-induced nephropathy in rats

doi: 10.1152/ajprenal.00071.2018

Figure Lengend Snippet: Analysis of (pro)renin receptor (PRR)/soluble (pro)renin receptor (sPRR) expression. A: quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of PRR in the renal cortex. The expression was normalized by β-actin. B: immunoblotting analysis of full-length PRR and sPRR in the renal cortex. Shown are representative blots with densitometry data. β-Actin was used as an internal control. C: ELISA analysis of urinary sPRR excretion. D: ELISA analysis of plasma sPRR. Data are means ± SE; n = 6/group. *P < 0.05 vs. control (CTR).

Article Snippet: Total rat prorenin/renin concentrations in urine were measured by using a commercial ELISA kit (RPRENKT-TOT; Molecular Innovations).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells

doi: 10.4196/kjpp.2022.26.6.479

Figure Lengend Snippet: Cells on day 2 postconfluence were incubated in Ca 2+ -free DMEM containing 1 mM EGTA (–Ca 2+ DMEM) (A, left panel) and then in 2 mM Ca 2+ -containing DMEM and 10% FBS (+Ca 2+ DMEM) successively for 1 h each or in the reverse order (A, right panel, p < 0.001, n = 6). In the next series of experiments, cells were incubated in +Ca 2+ DMEM for 1 h (control) and then another 1 h in +Ca 2+ DMEM + 10 % FBS containing either calmidazolium (3 × 10 −5 M) (B, n = 6), ML-7 (6 × 10 −5 M) (C, n = 6), isoproterenol (10 −8 –10 −5 M) (D, n = 6) or forskolin (3 × 10 −5 M) plus IBMX (10 −4 M) (E, n = 6). The rate of active renin secretion was determined by radioimmunoassay for ANG I in (A) and (B) or by ELISA in (C), (D), and (E). ANG I, angiotensin I; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum. Samples within groups were compared using paired Student’s t-tests and between groups using unpaired Student’s t-tests (A–C, E) or ANOVA (D), *p < 0.001 vs. control, except between samples at the two highest concentrations, which were compared by unpaired Student’s t-tests. † p < 0.001.

Article Snippet: Recently, renin ELISA kits have become available; secreted active renin and inactive renin (prorenin) were measured using commercial mouse renin ELISA kits from Ray Biotech and Molecular Innovations, respectively.

Techniques: Incubation, RIA Assay, Enzyme-linked Immunosorbent Assay, Modification

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells

doi: 10.4196/kjpp.2022.26.6.479

Figure Lengend Snippet: Cell lysates from cells grown to 70%–80% confluence in the presence of 10% FBS (A, lane 1), cells at day 2 postconfluence in the continuous presence of 10% FBS (A, lane 2) or in the absence of 10% FBS for two days after achieving 100% confluence (A, lane 3). After the aspiration of the incubation media, cells were lysed and centrifuged. Supernatant protein (60 μg) was resolved by SDS-PAGE on 7% or 15% acrylamide slab gels followed by Western blotting for N-cad, MRTF-A, sm MHC, nm MHC, and PP1β and then analyzed by densitometry. In the case of MRTF-A, a nuclear fraction was prepared, and 100 μg was resolved (B, upper panel). p-values were obtained by ANOVA. To assess nuclear localization of MRTF-A, cells were plated onto coverslips and fixed and permeabilized as described in the Methods section. Cells were then immunostained with primary MRTF-A antibody (1:100) and then with FITC-labelled goat anti-mouse secondary antibody to MRTF-A (1:1,000). Nuclei were stained with propidium iodide (DAPI) (B, lower panel). The density of preconfluent cells was arbitrarily set to 1. Fluorescence was analyzed by an image analyzer. Lane 1, preconfluent cells; lane 2, postconfluent cells in the presence of 10% FBS. p-values were obtained by unpaired Student’s t-tests (n = 4). Scale bar: 10 μM. (C) Postconfluent cells were incubated in DMEM + 10% FBS with or without CCG-1423 (5 × 10 −6 M), an inhibitor of nuclear translocation of MRTF-A, for 1 h and then with ML-7 (6 × 10 −5 M) for another 1 h. Secreted renin was measured by ELISA (C, n = 5). MRTF-A, myocardin related transcription factor-A; sm MHC, smooth muscle myosin heavy chain; nm MHC, nonmuscle myosin heavy chain; PP1β, protein phosphatase 1β; FBS, fetal bovine serum. NS, no significant difference. p-values within groups were obtained by paired Student’s t-tests, and those between groups were obtained by ANOVA.

Article Snippet: Recently, renin ELISA kits have become available; secreted active renin and inactive renin (prorenin) were measured using commercial mouse renin ELISA kits from Ray Biotech and Molecular Innovations, respectively.

Techniques: Incubation, SDS Page, Western Blot, Staining, Fluorescence, Translocation Assay, Enzyme-linked Immunosorbent Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells

doi: 10.4196/kjpp.2022.26.6.479

Figure Lengend Snippet: Cells at ~70% confluence were transfected with control plasmids (A, lane 1) or the MRTF-A gene was knocked out using MRTF-A HDR CRISPR-associated 9 (Cas9) (A, lane 2) for three days. On day 2 postconfluence, the expression of MRTF-A, N-cad, sm MHC, nm MHC, and PP1β in the supernatant fraction (80 μg) was determined as described in the legend of . p-values were obtained by unpaired Student’s t-tests. (B) Control and knockout cells were incubated before and after stimulation with ML-7 (6 × 10 −5 M) for 1 h each, and secreted active renin was measured by ELISA (B, n = 4). MRTF-A, myocardin related transcription factor-A; HDR, homology directed repair; CRISPR, clustered regularly interspaced short palindromic repeats; sm MHC, smooth muscle myosin heavy chain; nm MHC, nonmuscle myosin heavy chain; PP1β, protein phosphatase 1β. NS, no significant difference. p-values were obtained either by paired Student’s t-tests (within groups) or by ANOVA (between groups).

Article Snippet: Recently, renin ELISA kits have become available; secreted active renin and inactive renin (prorenin) were measured using commercial mouse renin ELISA kits from Ray Biotech and Molecular Innovations, respectively.

Techniques: Transfection, CRISPR, Expressing, Knock-Out, Incubation, Enzyme-linked Immunosorbent Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells

doi: 10.4196/kjpp.2022.26.6.479

Figure Lengend Snippet: Cells were transfected with control plasmids (A, lane 1) or SRF HDR CRISPR/Cas9 plasmids (A, lane 2) as described in the legend of . Then, the expression of each protein was determined in the supernatant fraction (80 μg). Both control and knockout cells were incubated with ML-7 (6 × 10 −5 M) before and after stimulation for 1 h each, and secreted active renin was measured by ELISA (B, n = 5). SRF, serum response factor; HDR, homology directed repair; CRISPR, clustered regularly interspaced short palindromic repeats. p-values were obtained either by paired Student’s t-tests (within groups) or by ANOVA (between groups).

Article Snippet: Recently, renin ELISA kits have become available; secreted active renin and inactive renin (prorenin) were measured using commercial mouse renin ELISA kits from Ray Biotech and Molecular Innovations, respectively.

Techniques: Transfection, CRISPR, Expressing, Knock-Out, Incubation, Enzyme-linked Immunosorbent Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Cell-cell contacts via N-cadherin induce a regulatory renin secretory phenotype in As4.1 cells

doi: 10.4196/kjpp.2022.26.6.479

Figure Lengend Snippet: Cells were transfected with control (lane 1) or siRNA of PP1β plasmids as described in the legend for . The expression of PP1β (A, upper panel) and cytosolic pMLC 20 level (A, lower panel) were determined using supernatant protein (60 μg). p-values were obtained by unpaired Student’s t-tests (n = 4). Active renin secretion was determined before and after stimulation by ML-7 (6 × 10 −5 M) for 1 h each, and secreted renin was measured by ELISA (B). PP1β, protein phosphatase 1β; pMLC 20 , phosphorylated 20 kDa myosin light chain. p-values were obtained either by paired Student’s t-tests (within groups) or by ANOVA (between groups) (n = 6).

Article Snippet: Recently, renin ELISA kits have become available; secreted active renin and inactive renin (prorenin) were measured using commercial mouse renin ELISA kits from Ray Biotech and Molecular Innovations, respectively.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay