Journal: Gut Microbes

Article Title: Gut microbiota signatures in primary aldosteronism and functional identification of an aldosterone-degrading gut bacterium

doi: 10.1080/19490976.2026.2657047

Figure Lengend Snippet: Identification of aldosterone-associated microbial species across all subjects. (a) Bar plot showing significant associations between microbial species and plasma aldosterone concentration (PAC) in all subjects (partial Spearman’s rank correlation, adjusting for age, sex, and BMI; * p < 0.05, ** p < 0.01, *** p < 0.001). (b) Co-abundance network showing the correlations among PAC-associated species. Red edges represent significant positive associations and blue edges represent negative associations (Spearman correlation; p < 0.05, |rho| > 0.3). (c) Bar plot showing the degree centrality of species within the co-abundance network.

Article Snippet: Subsequently, on Day 0, the VEH + PBS group was infused with vehicle (5% DMSO in saline), whereas the ALD + PBS and ALD + RG groups were infused with aldosterone (600 μg/kg/day; MedChemExpress, Monmouth Junction, NJ, USA) for 14 days using ALZET osmotic minipumps (model 2004; DURECT Corporation, Cupertino, CA, USA), with all three groups receiving 1% NaCl (MeilunBio, Dalian, China) in the drinking water., During this period, the VEH + PBS and ALD + PBS groups received sterile PBS, whereas the ALD + RG group received R. gnavus (2 × 108 CFUs in 200 μL PBS) by oral gavage once daily.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Gut Microbes

Article Title: Gut microbiota signatures in primary aldosteronism and functional identification of an aldosterone-degrading gut bacterium

doi: 10.1080/19490976.2026.2657047

Figure Lengend Snippet: Degradation of aldosterone and diverse steroid hormones by Ruminococcus gnavus in vitro . (a) Aldosterone concentrations in culture supernatants of control medium, Ruminococcus gnavus , and Bacteroides fragilis after 24 hours of incubation in medium containing 25 μM aldosterone ( n = 3 per group). *** p < 0.001. (b) Growth curves of R. gnavus in culture medium containing dimethyl sulfoxide (DMSO), 10 μM, 25 μM, or 50 μM aldosterone ( n = 4 per group). (c-d) Concentrations of aldosterone (c) and 3α,5β-tetrahydroaldosterone (d) in R. gnavus culture supernatants after 24 hours of cultivation in medium containing DMSO, 10 μM, 25 μM, or 50 μM aldosterone, respectively ( n = 4 per group). *indicates a significant difference compared to the DMSO group. # indicates a significant difference between groups with and without R. gnavus at the same aldosterone concentration. ***and ### , p < 0.001. (e-f) Concentrations of aldosterone (e) and 3α,5β-tetrahydroaldosterone (f) in R. gnavus culture supernatants supplemented with 50 μM aldosterone at 0, 2, 6, and 12 hours, compared to control medium containing aldosterone alone ( n = 3 per group). *indicates a significant difference compared to the medium-only group at the corresponding time point. ** p < 0.01, *** p < 0.001. For samples in which 3α,5β-tetrahydroaldosterone was not detectable, values were recorded as zero for visualization purposes. (g) Proposed conversion of aldosterone to 3α,5β-tetrahydroaldosterone by R. gnavus . (h) Concentrations of the corresponding steroid hormones in R. gnavus culture supernatants supplemented individually with different steroid hormone substrates, compared to control medium containing the corresponding hormone alone ( n = 3 per group). *** p < 0.001. THAldo, 3α,5β-tetrahydroaldosterone; 21C, C21 steroid hormones; 19C, C19 steroid hormones; 18C, C18 steroid hormones; 11-DOC, 11-deoxycorticosterone; 17α-OHP, 17α-hydroxyprogesterone; 21-DOC, 21-deoxycorticosterone; 21-DOX, 21-deoxycortisol; DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; 11β-OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione. Data are presented as mean ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (a), two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons test (b) and (e-f), two-way ANOVA followed by Tukey’s multiple comparisons test (c-d), and unpaired two-tailed Student’s t-test (h).

Article Snippet: Subsequently, on Day 0, the VEH + PBS group was infused with vehicle (5% DMSO in saline), whereas the ALD + PBS and ALD + RG groups were infused with aldosterone (600 μg/kg/day; MedChemExpress, Monmouth Junction, NJ, USA) for 14 days using ALZET osmotic minipumps (model 2004; DURECT Corporation, Cupertino, CA, USA), with all three groups receiving 1% NaCl (MeilunBio, Dalian, China) in the drinking water., During this period, the VEH + PBS and ALD + PBS groups received sterile PBS, whereas the ALD + RG group received R. gnavus (2 × 108 CFUs in 200 μL PBS) by oral gavage once daily.

Techniques: In Vitro, Control, Incubation, Concentration Assay, Two Tailed Test

Journal: Gut Microbes

Article Title: Gut microbiota signatures in primary aldosteronism and functional identification of an aldosterone-degrading gut bacterium

doi: 10.1080/19490976.2026.2657047

Figure Lengend Snippet: Validation of R. gnavus -mediated degradation of aldosterone and multiple steroid hormones in germ-free mice. (a) Experimental design. (b) Quantification of R. gnavus colonization in fecal samples by quantitative real-time PCR (qPCR) targeting R. gnavus -specific 16S rRNA gene sequences. (c-d) Fecal (c) and serum (d) aldosterone concentrations in germ-free (GF) mice and GF mice colonized with R. gnavus . (e) mRNA expression levels of aldosterone-responsive genes related to intestinal epithelial ion transport in the distal colon. (f) Fecal concentrations of steroid hormones. 21C, C21 steroid hormones; 19C, C19 steroid hormones; 18C, C18 steroid hormones; 11-DOC, 11-deoxycorticosterone. For (b-c) and (e-f), n = 9 per group. For (d), n = 6 for the PBS group and n = 7 for the R. gnavus group. Data are presented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, on Day 0, the VEH + PBS group was infused with vehicle (5% DMSO in saline), whereas the ALD + PBS and ALD + RG groups were infused with aldosterone (600 μg/kg/day; MedChemExpress, Monmouth Junction, NJ, USA) for 14 days using ALZET osmotic minipumps (model 2004; DURECT Corporation, Cupertino, CA, USA), with all three groups receiving 1% NaCl (MeilunBio, Dalian, China) in the drinking water., During this period, the VEH + PBS and ALD + PBS groups received sterile PBS, whereas the ALD + RG group received R. gnavus (2 × 108 CFUs in 200 μL PBS) by oral gavage once daily.

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: Gut Microbes

Article Title: Gut microbiota signatures in primary aldosteronism and functional identification of an aldosterone-degrading gut bacterium

doi: 10.1080/19490976.2026.2657047

Figure Lengend Snippet: Attenuation of exogenous aldosterone-induced physiological alterations by R. gnavus in vivo . (a) Experimental design. (b) Quantification of R. gnavus colonization in fecal samples by qPCR targeting R. gnavus -specific 16S rRNA gene sequences. (c-g) Serum aldosterone concentrations (c), fecal aldosterone concentrations (d), systolic blood pressure (e), serum sodium (f), and serum potassium (g) in antibiotic (ABX)-treated mice receiving VEH + PBS ( n = 5), ALD + PBS ( n = 6), or ALD + RG ( n = 6). VEH, vehicle; ALD, aldosterone; RG, Ruminococcus gnavus ; Serum Na + , serum sodium; Serum K + , serum potassium. For (b-g), data are presented as mean ± SEM. Statistical analysis was performed using One-way ANOVA with Tukey’s multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, on Day 0, the VEH + PBS group was infused with vehicle (5% DMSO in saline), whereas the ALD + PBS and ALD + RG groups were infused with aldosterone (600 μg/kg/day; MedChemExpress, Monmouth Junction, NJ, USA) for 14 days using ALZET osmotic minipumps (model 2004; DURECT Corporation, Cupertino, CA, USA), with all three groups receiving 1% NaCl (MeilunBio, Dalian, China) in the drinking water., During this period, the VEH + PBS and ALD + PBS groups received sterile PBS, whereas the ALD + RG group received R. gnavus (2 × 108 CFUs in 200 μL PBS) by oral gavage once daily.

Techniques: In Vivo

Journal:

Article Title: Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells * S⃞

doi: 10.1074/jbc.M809460200

Figure Lengend Snippet: Aldosterone increases oxidant stress in VSMC. A, vascular smooth muscle cells were exposed to aldosterone (ALDO) (10-9-10-7 mol/liter) or vehicle control (V) for 24 h, and ROS accumulation was measured by 6-carboxy-2′,7′ dichlorodihydrofluorescein diacetate ester (DCF) fluorescence (n = 3). B, hydrogen peroxide (H2O2) generation was evaluated by Amplex Red assay fluorescence (n = 3) *, p < 0.01 versus vehicle control. Data are presented as mean ± S.E.

Article Snippet: Aldosterone (Steraloids) and apocynin (Cayman) were dissolved in dimethyl sulfoxide (10 nmol/liter), which also served as the vehicle control.

Techniques: Control, Fluorescence, Amplex Red Assay

Journal:

Article Title: Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells * S⃞

doi: 10.1074/jbc.M809460200

Figure Lengend Snippet: Aldosterone and NO· byproduct formation. A, vascular smooth muscle cells were exposed to ALDO (10-7 mol/liter) or vehicle control (V) in the presence or absence of cytokines (lipopolysaccharide (LPS) = 30 μg/ml, interleukin 1-β (IL-1-β) = 50 ng/ml, interferon-γ (IFN-γ) = 50 ng/ml) for 24 h to induce iNOS expression. The effect of ALDO on iNOS protein expression was determined by Western blotting (n = 3), and as shown in B, the influence of ALDO on formation (n = 4) was measured by 2,3 diaminonaphthalene fluorescence. To confirm iNOS as the source of formation, cells were treated with AMT (100 μmol/liter), a specific iNOS inhibitor. ND, non-detectable. C, the influence of aldosterone and NADPH oxidase on the ratio in iNOS-expressing cells was assessed (n = 6). Apo, apocynin. D, peroxynitrite formation was assessed by examining anti-3-nitrotyrosine immunohistochemistry (n = 3). *, p < 0.01 versus vehicle control, **, p < 0.001 versus ALDO; Representative images are shown. Data are presented as mean ± S.E.

Article Snippet: Aldosterone (Steraloids) and apocynin (Cayman) were dissolved in dimethyl sulfoxide (10 nmol/liter), which also served as the vehicle control.

Techniques: Control, Expressing, Western Blot, Fluorescence, Immunohistochemistry

Journal:

Article Title: Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells * S⃞

doi: 10.1074/jbc.M809460200

Figure Lengend Snippet: Aldosterone and GC subunit expression and activity. A, GC α1- and β1-subunit protein expression in VSMC exposed to either ALDO(10-7 mol/liter) or vehicle control (V) for 24 h was assessed by Western blotting (n = 3). B and C, the effect of aldosterone (10-7 mol/liter) on GC activity under basal conditions (B) and iNOS-expressing cells in the presence or absence of NADPH oxidase inhibition with apocynin (Apo)(3 × 10-5 mol/liter) (n = 4) (C). D, cGMP levels were determined in iNOS-expressing cells (n = 4). *, p < 0.02 versus vehicle control, **, p < 0.04 versus iNOS, #, p < 0.03 versus ALDO. Representative blots are shown. Data are presented as mean ± S.E.

Article Snippet: Aldosterone (Steraloids) and apocynin (Cayman) were dissolved in dimethyl sulfoxide (10 nmol/liter), which also served as the vehicle control.

Techniques: Expressing, Activity Assay, Control, Western Blot, Inhibition

Journal:

Article Title: Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells * S⃞

doi: 10.1074/jbc.M809460200

Figure Lengend Snippet: Disulfide bond formation and MS-MS spectra of peptide CTDADKGK oxidation products. A, vascular smooth muscle cells were exposed to 10-7 mol/liter ALDO or vehicle control (V) for 24 h, free thiols were blocked with iodoacetamide and N-ethylmaleimide, disulfide bonds were reduced, and previously oxidized (now reduced) cysteinyl thiols were labeled with polyethylene glycol-conjugated maleimide (molecular mass 10 kDa). Disulfide bond formation specific to the β1-subunit of GC is detected by Western immunoblot. For each disulfide bond, a 20-kDa increase in the molecular mass occurs, resulting in a shift in band location on Western immunoblot. Aldosterone treatment resulted in the formation of two disulfide bonds not present in the vehicle control (n = 3). A representative blot is shown. Cys122 oxidation products were examined by exposing the peptide CTDADKGK to H2O2 (200 μmol/liter) and FeSO4 (0.2 mmol/liter) at pH 5.9 for 30 min and mass spectrometry analysis. Three major oxidation products were identified by enhanced MS1 scan, and subsequently, MS-MS spectra were acquired for definitive identification of cysteinyl sulfinic acid ([M+2H]2+ = m/z 435) (B), cysteinyl sulfonic acid ([M+2H]2+ = m/z 443) (C), and peptide disulfide ([M+2H]2+ = m/z 836) formation (D).

Article Snippet: Aldosterone (Steraloids) and apocynin (Cayman) were dissolved in dimethyl sulfoxide (10 nmol/liter), which also served as the vehicle control.

Techniques: Tandem Mass Spectroscopy, Control, Labeling, Western Blot, Mass Spectrometry

Journal: bioRxiv

Article Title: Renal mechanotransduction is an essential regulator of renin

doi: 10.1101/2023.11.04.565646

Figure Lengend Snippet: a , Plasma renin levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals (Mann–Whitney: ** p = 0.0019, U = 48; n = 20 Pdgfrb WT and 13 Pdgfrb CreERT2 mice). b , Plasma aldosterone levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals (Mann–Whitney: p = 0.8653, U = 84; n = 11 Pdgfrb WT and 16 Pdgfrb CreERT2 mice). c , Plasma angiotensin II (ATII) levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals (Mann–Whitney: p = 0.1932, U = 21; n = 7 Pdgfrb WT and 10 Pdgfrb CreERT2 mice). d , Systemic blood pressure (systolic/SAP; diastolic/DAP; and mean arterial pressure/MAP) measured using the VPR system in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals (two-tailed nested t -tests (left to right): ** p SAP = 0.0013, t = 3.899, d.f. = 16; ** p DAP = 0.0056, t = 3.197, d.f. = 16; ** p MAP = 0.0027, t = 3.546, d.f. = 16; n = 10 Pdgfrb WT and 8 Pdgfrb CreERT2 mice). e , Plasma renin levels in Piezo2 fl/fl ; Ren WT versus Piezo2 fl/fl ; Ren Cre animals (Mann–Whitney: ** p = 0.0047, U = 3; n = 6 Ren WT and 8 Ren Cre mice). f , Systemic blood pressure measured in Piezo2 fl/fl ; Ren WT versus Piezo2 fl/fl ; Ren Cre animals (two-tailed nested t -tests (left to right): * p SAP = 0.0147, t = 2.716, d.f. = 17; ** p DAP = 0.0097, t = 2.913, d.f. = 17; ** p MAP = 0.0060, t = 3.137, d.f. = 17; n = 9 Ren WT and 10 Ren Cre mice). Each experiment was performed on at least two independent cohorts of mice, and error bars represent mean ± s.e.m.

Article Snippet: ELISA was performed using the indicated assays for the following analytes according to the manufacturer’s instructions: renin (LSBio, LS-F508-1), aldosterone (Tecan, RE52301), angiotensin II (Ray Biotech, EIA-ANGII-1), albumin (Abcam, ab108792).

Techniques: Clinical Proteomics, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Renal mechanotransduction is an essential regulator of renin

doi: 10.1101/2023.11.04.565646

Figure Lengend Snippet: a , Plasma aldosterone levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals after seven days of captopril (Mann–Whitney: p = 0.7664, U = 45; n = 11 Pdgfrb WT and 9 Pdgfrb CreERT2 mice). Experiment was performed on at least two independent cohorts of mice, and error bars represent mean ± s.e.m.

Article Snippet: ELISA was performed using the indicated assays for the following analytes according to the manufacturer’s instructions: renin (LSBio, LS-F508-1), aldosterone (Tecan, RE52301), angiotensin II (Ray Biotech, EIA-ANGII-1), albumin (Abcam, ab108792).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Renal mechanotransduction is an essential regulator of renin

doi: 10.1101/2023.11.04.565646

Figure Lengend Snippet: a , The subcutaneous PEG model of hypovolemia. b , Plasma renin levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals six hours following PEG (Mann– Whitney: *** p = 0.0003, U = 53; n = 17 Pdgfrb WT and 19 Pdgfrb CreERT2 mice). c , Plasma aldosterone levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals six hours following PEG (Mann–Whitney: * p = 0.0257, U = 48; n = 12 Pdgfrb WT and 16 Pdgfrb CreERT2 mice). d , Plasma ATII levels in Piezo2 fl/fl ; Pdgfrb WT versus Piezo2 fl/fl ; Pdgfrb CreERT2 animals six hours following PEG (Mann–Whitney: ** p = 0.0043, U = 0; n = 5 Pdgfrb WT and 6 Pdgfrb CreERT2 mice). e , Plasma renin levels in Piezo2 fl/fl ; Ren WT versus Piezo2 fl/fl ; Ren Cre animals six hours following PEG (Mann–Whitney: ** p = 0.0025, U =18; n = 13 Ren WT and 10 Ren Cre mice). f , Plasma aldosterone levels in Piezo2 fl/fl ; Ren WT versus Piezo2 fl/fl ; Ren Cre animals six hours following PEG (Mann– Whitney: **** p < 0.0001, U =2; n = 15 Ren WT and 10 Ren Cre mice). g , Plasma renin levels in Piezo2 fl/fl ; FoxD1 WT versus Piezo2 fl/fl ; FoxD1 Cre animals six hours following PEG (Mann–Whitney: * p = 0.0360, U =14; n = 8 FoxD1 WT and 9 FoxD1 Cre mice). h , Plasma aldosterone levels in Piezo2 fl/fl ; FoxD1 WT versus Piezo2 fl/fl ; FoxD1 Cre animals six hours following PEG (Mann–Whitney: * p = 0.0418, U =12; n = 7 FoxD1 WT and 9 FoxD1 Cre mice). Each experiment was performed on at least two independent cohorts of mice, and error bars represent mean ± s.e.m.

Article Snippet: ELISA was performed using the indicated assays for the following analytes according to the manufacturer’s instructions: renin (LSBio, LS-F508-1), aldosterone (Tecan, RE52301), angiotensin II (Ray Biotech, EIA-ANGII-1), albumin (Abcam, ab108792).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Renal mechanotransduction is an essential regulator of renin

doi: 10.1101/2023.11.04.565646

Figure Lengend Snippet: a , Experimental strategy to chemically ablate sympathetic efferent neurons and inhibit prostaglandin synthesis prior to induction of hypovolemia. b , Plasma renin levels in Piezo2 fl/fl ; Ren WT versus Piezo2 fl/fl ; Ren Cre animals after chemical sympathectomy and indomethacin treatment and six hours following saline or PEG (two-way ANOVA: *** p interaction = 0.0004, F(1,29) = 15.81; Tukey’s multiple comparisons (left to right): * p = 0.0307, ** p = 0.002, **** p < 0.0001, **** p < 0.0001; n = 8 Ren WT saline, 8 Ren Cre saline, 9 Ren WT PEG, and 8 Ren Cre saline). c , Plasma aldosterone levels in PEG-treated animals from b (Mann–Whitney: *** p = 0.001, U = 4; n = 9 Ren WT and 8 Ren Cre mice). Each experiment was performed on at least two independent cohorts of mice, and error bars represent mean ± s.e.m. d , Schematic of model for role of PIEZO2 in regulation of renin in JG cells.

Article Snippet: ELISA was performed using the indicated assays for the following analytes according to the manufacturer’s instructions: renin (LSBio, LS-F508-1), aldosterone (Tecan, RE52301), angiotensin II (Ray Biotech, EIA-ANGII-1), albumin (Abcam, ab108792).

Techniques: Clinical Proteomics, Saline, MANN-WHITNEY

Journal: Journal of Cell Communication and Signaling

Article Title: Spironolactone rescues renal dysfunction in obstructive jaundice rats by upregulating ACE2 expression

doi: 10.1007/s12079-018-0466-2

Figure Lengend Snippet: BDL induces renal dysfunction. a, TBil increased in BDL rats compared to the SO rats. b, Scr increased in BDL rats compared to the SO rats. c, Scr was decreased after spironolactone administration. d, ICAM-1 expression decreased after spironolactone administration. Total Bilirubin levels and Scr levels in different groups was examined by using an automated biochemistry analyzer. The ICAM-1 expression was examined by ELISA. SO group: Sham-operated group; BDL group: Bile Duct Ligation group; *P < 0.05 vs. SO group, #P<0.05 vs. BDL group (n = 7 for each group at each time point)

Article Snippet: Aldosterone was measured using the Rat ALD ELISA Kit (Elabscience, Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Journal of Cell Communication and Signaling

Article Title: Spironolactone rescues renal dysfunction in obstructive jaundice rats by upregulating ACE2 expression

doi: 10.1007/s12079-018-0466-2

Figure Lengend Snippet: ELISA analysis of ACE2, renin, AngII, Ang-(1–7) and ALD in kidney tissue after spironolactone administration

Article Snippet: Aldosterone was measured using the Rat ALD ELISA Kit (Elabscience, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: RNLS adapts upward in the APA and promotes aldosterone production in vitro. (a) Distribution of RNLS immunoreactivity in different types of adrenocortical pathology sections. NFA: n = 7; APA: n = 14; AAG: n = 14; scale bar = 50µm. Localised magnification is shown in the box below. (b) Quantitative statistical analysis of RNLS shown in the positive area of a. (c) NCI-H295R cells were stimulated with RNLS (0.5, 1, 2, or 4μg/ml) for 12 h, and the levels of aldosterone production in the cell supernatant were measured. (d) Cells were treated with 4 μg/ml RNLS, and the level of aldosterone production in the supernatant was measured at different times. (e) EdU detection of cell proliferation after treatment with 4μg/ml RNLS. Nuclei are shown in blue, and red-stained EdU-positive cells with blue-stained nuclei in the merged layers are shown in purple; scale bar = 50 μm. (f) Statistical analysis of the percentages of EdU-positive cells in e. (g) CCK-8 assay for determining cell activity. (h) Photograph of plate colony formation after three weeks of 4 μg/ml RNLS treatment. (i) Statistical analysis of the plate colony formation rate in h. NFA: non-functioning adenoma; APA: aldosterone-producing adenoma; AAG: APA-adjacent adrenal gland.

Article Snippet: The cell supernatants were collected after drug intervention, centrifuged to remove the cellular precipitate, and assayed for aldosterone levels (ALPCO Diagnostics, 11-AD2HU-E01).

Techniques: In Vitro, Staining, CCK-8 Assay, Activity Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Effect of RNLS on aldosterone synthase expression in human adrenocortical cells. (a) Schematic diagram illustrating the process of aldosterone synthesis. The orange ovals represent mitochondria, and the blue mesh represents the endoplasmic reticulum. (b) WB analysis of the protein levels of aldosterone synthesis-related enzymes after NCI-H295R cells were treated with RNLS for 16h. The protein blots in b were statistically analysed for (c) CYP11B2, (d) CYP21A2, (e) HSD3B2, (f) StAR, and (g) CYP11A1. The qRT-PCR was performed to measure the mRNA levels of aldosterone synthesis-related enzymes, including (h) CYP11B2, (i) CYP21A2, (j) HSD3B2, (k) StAR, and (l) CYP11A1.

Article Snippet: The cell supernatants were collected after drug intervention, centrifuged to remove the cellular precipitate, and assayed for aldosterone levels (ALPCO Diagnostics, 11-AD2HU-E01).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Impact of RNLS on the level and activity of aldosterone synthase-related transcription factors. (a) Schematic representation of regulatory element sites within 1000 bp upstream of the HSD3B2, CYP21A2, and CYP11B2 promoters. Transcription factors known to bind to each regulatory element are shown in the table below. (b) The transcription factor NR4A2 was detected via qRT-PCR following 16 h of RNLS intervention. (c–e) NR4A2 was silenced for 48 h with small interfering RNA in NCI-H295R cells, followed by treatment with 4μg/ml RNLS for 16 h. The mRNA levels of related aldosterone synthases, including (c) CYP11B2, (d) CYP21A2, and (e) HSD3B2, were subsequently detected via qRT-PCR. (f) The protein expression of ATF/CREB family members and their phosphorylation levels were detected via WB. Quantitative statistical analysis of the phosphorylated protein to total protein ratios in f for (g) ATF1, (h) ATF2, (i) CREB, and (j) CREM. The members of the ATF/CREB family were silenced with the corresponding small interfering RNA, followed by the addition of RNLS, and the mRNA levels of the relevant aldosterone synthases, including (k) CYP11B2, (l) CYP21A2, and (m) HSD3B2, were detected via qRT-PCR.

Article Snippet: The cell supernatants were collected after drug intervention, centrifuged to remove the cellular precipitate, and assayed for aldosterone levels (ALPCO Diagnostics, 11-AD2HU-E01).

Techniques: Activity Assay, Quantitative RT-PCR, Small Interfering RNA, Expressing, Phospho-proteomics

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Effect of cAMP signalling inhibition on the RNLS regulation of aldosterone synthase expression and aldosterone production. NCI-H295R cells were pre-treated with the PKA inhibitor H-89 and then incubated with 4 μg/ml RNLS for 16 h. (a) Cellular supernatant aldosterone levels were assayed by ELISA. The mRNA levels of related aldosterone synthases, including (b) CYP11B2, (c) CYP21A2, and (d) HSD3B2, were detected by qRT-PCR. (e) WB was used to examine the protein expression of the relevant aldosterone synthases. The relative expression of proteins in e was quantified, and the results are shown for (f) CYP11B2, (g) CYP21A2, and (h) HSD3B2. (i) WB assay for PKA phospho-substrate proteins. (j) Quantification of the p-PKA substrate in i. (k) qRT-PCR analysis of NR4A2. (l) WB detection of ATF/CREB family member proteins and their phosphorylated proteins. The phosphorylated protein to total protein ratio of ATF/CREB family members in l was quantified and statistically analysed, as shown for (m) ATF1, (n) ATF2, (o) CREB, and (p) CREM.

Article Snippet: The cell supernatants were collected after drug intervention, centrifuged to remove the cellular precipitate, and assayed for aldosterone levels (ALPCO Diagnostics, 11-AD2HU-E01).

Techniques: Inhibition, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Role of PMCA4b in aldosterone production stimulated by RNLS. NCI-H295R cells were pre-treated with siPMCA4b and then incubated with 4μg/ml RNLS for 16 hours. (a) Cellular supernatant aldosterone levels were assayed by ELISA. The mRNA levels of related aldosterone synthases, including (b) CYP11B2, (c) CYP21A2, and (d) HSD3B2, were detected via qRT-PCR. (e) WB was used to examine the protein expression of aldosterone synthase. The relative expression of proteins in e was quantified, and the results are shown for (f) CYP11B2, (g) CYP21A2, and (h) HSD3B2. (i) WB assay for PKA phospho-substrate proteins and PKACA. (j,k) Quantification of the p-PKA substrate and PKACA in i. (l) qRT-PCR analysis of NR4A2. (m) WB detection of ATF/CREB family member proteins and their phosphorylated proteins. The phosphorylated protein to total protein ratios of the ATF/CREB family members were quantified and statistically analysed as shown for (n) ATF1, (o) ATF2, (p) CREB, and (q) CREM.

Article Snippet: The cell supernatants were collected after drug intervention, centrifuged to remove the cellular precipitate, and assayed for aldosterone levels (ALPCO Diagnostics, 11-AD2HU-E01).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing