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: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: Generation and characterization of transgenic mice expressing the human (pro)renin receptor (hPRR) transgene under control of the neuron-specific rat synapsin 1 promoter (Syn-hPRR). A: schematic of the Syn-hPRR construct used to develop transgenic mice. B: representative RT-PCR gel showing transgene expression of hPRR and β-actin in various tissues of Syn-hPRR mice (founder line 57994-1). C: representative RT-PCR gel showing transgene expression of hPRR and β-actin in various tissues in nontransgenic (NT) littermates. B, brain; H, heart; K, kidney; Lu, lung; Li, liver; S, spleen; M, skeletal muscle; V, vessel; F, white adipose tissue (fat); P, pancreas; +, positive control (Syn-hPRR construct used as a template); −, negative control (no-template PCR). D: hPRR protein in the cortex, hypothalamus, and brain stem of Syn-hPRR mice and NT littermates (n = 3–4/group). E: hPRR mRNA levels in brain homogenates of Syn-hPRR mice and NT littermates (n = 3–4/group) determined by quantitative real-time RT-PCR). F: mouse PRR mRNA levels in brain homogenates of Syn-hPRR mice and NT littermates (n = 3/group) determined by quantitative RT-PCR. G: PRR protein immunofluorescence in the subfornical organ (SFO), paraventricular nucleus (PVN), nucleus of tractus solitarius (NTS), area postrema (AP), and rostral ventrolateral medulla (RVLM) in Syn-hPRR mice and NT littermates (n = 3/group). Note that images of gels in B and C (top) were from two different agarose gels.

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques: Transgenic Assay, Expressing, Control, Construct, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Quantitative RT-PCR, Immunofluorescence

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: NADPH oxidase (NOX)4 knockdown prevents the human prorenin-induced blood pressure (BP) elevation in mice overexpressing human (pro)renin receptor specifically in neurons (Syn-hPRR). A: relative NOX2 mRNA levels normalized to adenoviruse (Ad)-scrambled-shRNA (Ad-sc-shRNA) in hypothalamic tissues of Syn-hPRR mice (n = 3–5/group). B: relative NOX4 mRNA levels normalized to Ad-sc-shRNA in hypothalamic tissues of Syn-hPRR mice (n = 3–5/group). C: schematic showing the time frame of intracerebroventricular administration for the adenovirus protocol. D: change in mean arterial pressure (ΔMAP) over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of human prorenin (300 ng) in Syn-hPRR mice previously administered Ad-sc-shRNA (n = 3), Ad-NOX2-shRNA (n = 4), Ad-NOX4-shRNA (n = 4), or Ad-NOX2-shRNA + Ad-NOX4-shRNA (n = 4). E: ΔMAP over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of polyethylene glycol (PEG)-catalase (2.01 units) with or without coinfusion of human prorenin in Syn-hPRR mice (n = 3/group). *P ≤ 0.05 vs. Ad-sc-shRNA (A and B) vs. Ad-sc-shRNA and Ad-NOX2-shRNA (D) or vs. PEG-catalase (E); #P ≤ 0.05 vs. human prorenin. All BP recordings were obtained by telemetry from nonanesthetized, conscious, freely moving mice.

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques: Knockdown, shRNA

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: Blood pressure, heart rate (HR), and locomotor activity in mice overexpressing the human (pro)renin receptor specifically in neurons (Syn-hPRR) and nontransgenic (NT) littermates. A–C: summary graphs of mean arterial pressure (MAP; A), HR (B), and locomotor activity (C) in Syn-hPRR mice and NT littermates over a 48-h period as measured by radiotelemetry (n = 4/group). Gray, nighttime period; white, daytime period.

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques: Activity Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: Representative traces of the blood pressure (BP) and heart rate (HR) response to artificial cerebrospinal fluid (aCSF) and human (h) prorenin in nontransgenice (NT) littermates and mice overexpressing human (pro)renin receptor (hPRR) specifically in neurons (Syn-hPRR). A and C: representative traces of BP and HR in a NT mouse or Syn-hPRR mouse during intracerebroventricular infusion of aCSF. B and D: representative traces of BP and HR in a NT mouse or Syn-hPRR mouse during intracerebroventricular infusion of human prorenin (hProrenin).

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: Human prorenin induces an elevation of blood pressure (BP) in mice overexpressing human (pro)renin receptor (hPRR) specifically in neurons (Syn-hPRR). A: changes in mean arterial pressure (ΔMAP) over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of artificial cerebrospinal fluid (aCSF; n = 4) or human prorenin (300 ng, n = 9) with or without intracerebroventricular coinfusion of losartan (30 pmol, n = 6) or captopril (30 pmol, n = 3) in nontransgenic (NT) mice. B: ΔMAP over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of aCSF (n = 4) or human prorenin (n = 14) with or without intracerebroventricular coinfusion of losartan (30 pmol, n = 8) or captopril (n = 8) in Syn-hPRR mice. C: ANG II levels in the hypothalamus after intracerebroventricular infusion of either aCSF or human prorenin in NT or Syn-hPRR mice (n = 4/group). D: ΔMAP over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of human prorenin (300 ng) with different doses of PRO20 (1, 3, 10, 30, 100, and 300 μM) in Syn-hPRR mice (n = 4/group). E: dose-inhibition curve presented as a percentage of the maximal response. B: *P ≤ 0.05 vs. Syn-hPRR + aCSF; all BP recordings were obtained by telemetry from nonanesthetized, conscious, freely moving mice.

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques: Inhibition

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: Mice overexpressing human (pro)renin receptor specifically in neurons (Syn-hPRR) have a normal blood pressure (BP) response to ANG II or carbachol. A: change in mean arterial pressure (ΔMAP) over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of ANG II (300 ng, n = 3–5) with or without intracerebroventricular coinfusion of losartan (30 pmol, n = 5) in nontransgenic (NT) and Syn-hPRR mice. B: ΔMAP over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of artificial cerebrospinal fluid (aCSF; n = 4) or carbochol (300 ng, n = 3–4) in NT and Syn-hPRR mice. *P ≤ 0.05 vs. NT + ANG II or Syn-hPRR + ANG II (A) or vs. NT + aCSF or Syn-hPRR + aCSF (B).

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: Increased NADPH oxidase (NOX) activity contributes to human prorenin-induced elevated blood pressure (BP) in mice overexpressing human (pro)renin receptor specifically in neurons (Syn-hPRR). A: hypothalamic NOX activity in Syn-hPRR mice and nontransgenic (NT) littermates infused intracerebroventricular (0.3 µl/min) with artificial cerebrospinal fluid (aCSF), human prorenin (300 ng, n = 4–8/group), or human protenin (300 ng) + losartan (30 pmol) (n = 3/group) measured using a lucigenin-based luminescence assay and expressed as fold increases in relative luminescence units (RLUs). B: change in mean arterial pressure (ΔMAP) over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of human prorenin with or without diphenyleneiodonium (DPI; 30 or 300 pmol) in Syn-hPRR mice and NT littermates (n = 3–4/group). *P ≤ 0.05 vs. NT with the same treatment (A) or Syn-hPRR + human prorenin vs. NT + human prorenin (B); #P ≤ 0.05, Syn-hPRR + human prorenin + DPI (300 pmol) vs. Syn-hPRR + human prorenin. All BP recordings were obtained by telemetry from nonanesthetized, conscious, freely moving mice.

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques: Activity Assay, Luminescence Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system

doi: 10.1152/ajpheart.00310.2017

Figure Lengend Snippet: ERK phosphorylation mediates the prorenin-induced elevation of NADPH oxidase (NOX) activity and blood pressure (BP) in mice overexpressing human (pro)renin receptor specifically in neurons (Syn-hPRR). A and B: representative Western blots and summary data showing the ratio of phosphorylated (p-)ERK to total (t) ERK protein in Syn-hPRR mice and nontransgenic (NT) littermates (n = 4/group) infused intracerebroventricularly (0.3 µl/min) with artificial cerebrospinal fluid (aCSF) or human prorenin (hPro; 300 ng) for 10 min. C: change in mean arterial pressure (ΔMAP) over the course of a 10-min intracerebroventricular infusion (0.3 µl/min) of human prorenin with or without coinfusion of U-0126 (3 pmol) or GDC-0994 (0.3 pmol) in Syn-hPRR mice and NT littermates (n = 3–4/group). D: hypothalamic NOX activity, measured using a lucigenin luminescence assay, indicating the fold increase in relative light units (RLUs) for NT mice + intracerebroventricular aCSF (control) and Syn-hPRR mice infused intracerebroventricularly with human prorenin (300 ng) or coinfused with human prorenin and the ERK inhibitor U-0126 (3 pmol, n = 4). B: *P ≤ 0.05 vs. Syn-hPRR + aCSF; #P ≤ 0.05 vs. NT + human prorenin. C: *P ≤ 0.05 vs. NT + human prorenin; #P ≤ 0.05 vs. Syn-hPRR + human prorenin. D: *P ≤ 0.05 vs. NT + aCSF; #P ≤ 0.05 vs. Syn-hPRR + human prorenin.

Article Snippet: With the use of a NE-1002X syringe pump (New Era Pump Systems), mice were intracerebroventricularly infused (0.3 µl/min) for 10 min with the following reagents/reagent combinations: aCSF, human prorenin (300 ng), human prorenin + losartan (30 pmol), human prorenin + captopril (30 pmol), human prorenin + PRO20 (1, 3, 10, 30, 100, 300 µM), ANG II (300 ng), ANG II + losartan (30 pmol), carbochol (300 ng), human prorenin + DPI (30, 300 pmol), human prorenin + U-0126 (3 pmol), human prorenin + GCD-0994 (0.3 pmol, MedChem Express, Monmouth Junction, NJ), or human prorenin + polyethylene glycol-linked catalase (PEG-catalase; 0.67 U/µl).

Techniques: Activity Assay, Western Blot, Luminescence Assay, Control

Journal: Journal of the Endocrine Society

Article Title: Podocan Is Expressed in Blood and Adipose Tissue and Correlates Negatively With the Induction of Diabetic Nephropathy

doi: 10.1210/js.2017-00123

Figure Lengend Snippet: Correlation of renal podocan mRNA levels with amelioration of diabetic nephropathy. UNx-db/db mice at 18 weeks of age were orally administered the vehicle (0.5% methyl cellulose solution) or irbesartan (5, 20, and 50 mg/kg) every day for 8 weeks (n = 5 per group). Age-matched db/+ mice (n = 10) were also orally administered the vehicle every day for 8 weeks. (a) Body weight, (b) PG levels, and (c) UACR were measured. Then their kidneys were dissected, and (d) kidney weight, (e) podocan mRNA levels, and (f) renin 1 mRNA levels in the renal cortex were measured. Data are expressed as the mean ± SD. * P < 0.05 compared with the db/+ mice by Student t test or Aspin-Welch t test; # P ≤ 0.025 compared with the vehicle by one-tailed Williams test.

Article Snippet: Predesigned primers and probes (Applied Biosystems) were used for the quantification of gene expression for the following genes: human podocan, Hs_00542259m1; human collagen 1A1, Hs_00164004_m1; human fibronectin, Hs_00365052_m1; human CTGF, Hs_01026927_g1; human PAI-1, Hs_00167155_m1; mouse podocan, Mm00556334_m1; mouse TNF- α , Mm00443258_m1; mouse renin-1, Mm02342887_mH; mouse glyceraldehyde-3-phosphate dehydrogenase, Mm99999915_g1; and human cyclophilin, Hs00332817_m1.

Techniques: One-tailed Test