bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 482: Renoprotective Effects of Exercise Training in Dahl Salt-sensitive Rats
Authors: Sakata Yoshiko , Ito Osamu , Sakuyama Akihiro , Rong Rong , Suda Chihiro , Muroya Yoshikazu , Kohzuki Masahiro
Published: July 2021

Exercise training (Ex) slows the progression of hypertension and renal failure in some animal models. We and other groups have reported that Ex ameliorated hypertension and renal dysfunction in spontaneously hypertensive rats. Since little has been reported on the effects of Ex on renal disorder in salt-sensitive hypertension, we investigated the effects of Ex on blood pressure and renal function in Dahl salt-sensitive (DS) rats. Six-week-old male DS rats were divided into three groups: 1) Normal salt (0.5% NaCl) diet (NS) (NS group, n=10), 2) High salt (8% NaCl) diet (HS) (HS group, n=11), 3) HS plus moderate Ex with treadmill running (HE group, n=11). After 8 weeks, HS induced severe hypertension, and Ex did not affect systolic blood pressure (114±3, 209±6 and 205±8 mmHg in the NS, HS and HE groups, respectively). Plasma creatinine was significantly higher in the HS and HE groups than in the NS group, but not different between the HS and HE groups (0.22±0.01, 0.44±0.03 and 0.40±0.03 mg/dl). Urinary protein and albumin significantly increased in the HS and HE groups, but these were significantly lower in the HE group compared with the HS group (Protein: 15.9±1.6, 432.8±36.8 and 327.2±22.3 mg/day. Albumin: 3.9±0.8, 195.2±15.2 and 154.2±11.1mg/day). Urinary TBARS, an index of oxidative stress, significantly increased in the HS and HE groups, but this was significantly lower in the HE group compared with the HS group (0.30±0.02, 0.75±0.06 and 0.53±0.03 mg/day); there was no significant difference in plasma TBARS among all groups. CYP4A1 and CYP4A2 protein expressions in the outer medulla were significantly lowered by 37% and 48% in the HS group compared with the NS group, and were significantly elevated by 40% and 150% in the HE group compared with the HS group. There was no significant difference in cortical CYP4A1 protein expression among all groups. Although there was no difference in cortical CYP4A2 between the NS and HS groups, it was significantly increased by 35% in the HE group compared with the HS group. These results suggest that Ex attenuated HS-induced proteinuria independently of blood pressure or plasma creatinine level in DS rats. The improvement of oxidative stress and CYP4A expression in the kidneys may contribute to the renoprotective effects of Ex in DS rats.
Abstract 488: Atorvastatin Inhibits Src/ROS-mediated Redox Sensitive And Pro-Inflammatory Actions of Aldosterone in Vascular Smooth Muscle Cells: Focus on Statins Pleiotropic Effects
Authors: Bruder-Nascimento Thiago , Callera Glaucia , Montezano Augusto , He Yeng , Antunes Tayze , Tostes Rita de Cassia , Touyz Rhian
Published: July 2021

Statins, described as cholesterol-lowering drugs, are now recognized to induce effects throught pleiotropic actions, including oxidative stress reduction and protein geranylgeranylation inhibition. Mechanisms whereby aldosterone associates with c-Src and related redox signaling molecules involve lipid rafts. This study aims to identify whether c-Src/NADPH oxidases (Nox) pathway is a statin-sensitive target independent of membrane cholesterol depletion mechanisms. In order to discriminate statin pleiotropic effects from its classic cholesterol synthesis inhibition, VSMCs from WKY were treated with 1 uM atorvastatin for 60 min or 72h prior 100 nM aldosterone stimulations respectively. Aldosterone-induced c-Src phosphorylation (% vehicle, 267 ± 35) was inhibited by long (150 ± 23 %) and short (114 ± 33 %) term atorvastatin treatment. To restore cholesterol synthesis, cells were incubated with its intermediate, mevalonate (100 μM). Mevalonate reload restored c-Src phosphorylation (287 ± 27 %) induced by aldosterone in atorvastatin long term-treated VSMCs. Geranylgeranyl-pyrophosphate (GGPP) was used to prevent protein geranylgeranylation inhibition. GGPP reposition recovered the effects of aldosterone on c-Src phosphorylation in both short (268 ± 22 %) and long (203 ± 35 %) term VSMCs treated with atorvastatin. Aldosterone-induced increase of Nox1, 2 and 4 expression and the associated ROS-generation (215 ± 38 %) were inhibited by long and short term atorvastatin incubation. Aldosterone-induced RAC1/2 and p47phox translocation (cytosol to membrane) was prevented by atorvastatin treatments. Aldosterone stimulation increased Nox1 and p47phox content in cholesterol-enriched fractions, an effect inhibited by atorvastatin short term treatment. Atorvastatin also prevented the increase of redox-signaling phosphorylation (ERK1/2, p38 and JNK) and pro-inflammatory markers (VCAM-1 expression, NFkB p65 phosphorylation) by aldosterone. We demonstrate that atorvastatin influences c-Src/Nox-mediated effects of aldosterone involving lipid rafts through classic and pleiotropic actions independent of cholesterol depletion. This study identifies a novel mechanism for statins in aldosterone-associated vascular injury.
Abstract 49: Targeting Endothelial Cells with HO-1 Attenuated Vascular and Adipocyte Dysfunction in Mice Fed High Fat Diet
Authors: Kim Dong Hyun , Getty Morghan S , Stec David E , Abraham Nader G
Published: July 2021

Introduction:
Obesity or high fat diet (HF) are risk factors for the development of hypertension.

We examined the hypothesis that targeting the vascular system with human heme oxygenase -1 (HO-1) may attenuate both vascular and adipocyte dysfunction in vivo and in vitro.

Methods:
Lentivirus (Lenti) construct expressing human HO-1 under the control of endothelium specific promoters VE-cadherin (VECAD-HO-1) and VECAD −GFP (control) were used to treat mice, using a bolus injection into the renal artery, and kept on a high fat diet for 26 weeks. Human HO-1 gene expression was sustained for 9 months. For in vitro studies, human EC were cultured with Lenti- VECAD-HO-1 and added to Lenti-VECAD-GFP or control. The conditioned media (CM) from ECs was harvested and 10% CM was added to adipogenic media to measure paracrine effect on adipogenesis in human Mesenchymal Stem Cells (MSCs) derived adipocytes. Signaling molecules were measured by western blot.

Results:
Lentiviral transduction with VECAD-HO-1 construct attenuated the increase in blood pressure (from 149.9 ± 2.4 to 118 ± 2.0 mmHg, p<0.01) and prevented body weight gain by 39% (p<0.05) in obese mice and increased plasma adiponectin (from 2.9 ± 0.2 to 6.5 ± 0.1 μg/ml, p<0.05). CM derived from EC lenti HO-1 decreased adipogenesis compared to control (from 17.0 ± 0.2 to 10.2 ± 0.1, p<0.05) and resulted in an increase of β-catenin and Wnt but a decrease in PPARγ (p<0.05). These beneficial effects were reversed by treating the cell with SnMP, an inhibitor of HO activity (p<0.01). EC-HO-1 increased angiopoietin-1 (ANG-1), vascular endothelial growth factor A (VEGF), and platelet derived growth factor (PDGF)-AA,−BB (p<0.05). The addition of ANG-1 to adipogenic media resulted in the inhibition of adipogenesis (8.1 ± 0.1, p<0.01).

Conclusion:
Targeting HO-1 to ECs resulted in the attenuation of blood pressure and the prevention of body weight gain resulting in increased ANG-1, PDGF, and VGEF levels and the reprogramming of MSCs derived adipocytes to produce healthy and smaller adipocytes with the release of adiponectin.
Abstract 495: GSTM1 Deficiency Exacerbates Hypertension in the Remnant Model of Chronic Kidney Disease
Authors: Cechova Sylvia , Chan Rosa , Koller Beverly , Le Thu H
Published: July 2021

There is a general consensus that oxidative stress is a factor in the progression of chronic kidney disease (CKD). Hence, genetic variants that affect the capacity to handle oxidative stress may influence the outcomes of CKD. One important class of enzymes that has evolved to combat the damaging effects of reactive oxygen species is the glutathione-S-transferases. In particular, the μ class isoform 1 (GSTM1) has emerged as a potential modifier of multiple chronic diseases in humans. Approximately 30%-50% of humans are completely deficient of the GSTM1 enzyme because of homozygous inheritance of the
GSTM1
null allele,
GSTM1(0).
We have identified the
GSTM1
gene as a modifier of disease progression in hypertensive nephrosclerosis (HN). In an ancillary study of the African American (AA) Study of Hypertension and Kidney Disease (AASK) Trial, we reported that participants carrying one (
1/0
) or two (
0/0
) null alleles had 1.7- and 2-fold higher risk of the composite outcome of a 50% decline in the glomerular filtration rate (GFR), dialysis, or death relative to those with two active alleles.

Here,
the objective of our study
was to determine the consequence of deletion of
Gstm1
on the course of chronic kidney disease induced by reduction of renal mass (RRM) model in mice. We generated
Gstm1-/-
(KO) mice on the 129S6 background through conventional gene targeting strategy. By radiotelemetry,
Gstm1
KO mice displayed a modest but significantly higher baseline systolic blood pressure (SBP) compared to their wild type (WT,
Gtsm1 +/+
) littermates: KO (n = 5): 138.8 ± 1.3; WT (n = 5): 132.1 ± 1.1, p < 0.01. Baseline urinary isoprostane (ng/100 mg of body weight) was significantly higher in KO mice than WT mice (15.1 ± 2.9; WT: 8.0 ± 0.8, p < 0.04). Four weeks after sub-total nephrectomy,
Gstm1
KO mice developed significantly more severe hypertension than WT mice. The average SBP over a 2 week recording by radiotelemetry was 154.0 ± 3.2 mm Hg in KO mice (n = 5), and 142.3 ± 4.2 in WT mice (n = 3), p < 0.01. The effects of deletion of
Gstm1
on kidney function and histopathology are under investigation.

In conclusion, loss of GSMT1 increases oxidative stress and exaggerates hypertension in the murine model of chronic kidney disease.
Abstract 508: Resistance Exercise Training Performed Prior Diabetes Mellitus Suppresses Renal and Skeletal Muscle Abnormalities
Authors: Silva Kleiton A , Santos Ralmony A , Arlotti Marcelo R , Jorge Luciana , Cunha Tatiana S , Arita Danielle Y , Schor Nestor
Published: July 2021

Prior study of our group shown that previous aerobic exercise training improved the damage caused by diabetes mellitus on renal and cardiovascular system. Resistance exercise training, also known as strength training, is traditionally performed to gain muscle mass; however, it is not clear whether this type of exercise modulates renal system. Additionally, it is also unknown whether previous resistance exercise training can potentially influence the kidney and skeletal muscle. Wistar rats were submitted to resistance exercise training in an apparatus developed especially to this type of exercise (8 - 12 climbs/day, 5 days/week, 12 weeks). Previous resistance exercise trained group (PTD) performed for 4 weeks before the establishment of the disease and after this period they were followed by 8 weeks of resistance exercise training. Additional trained groups such as trained diabetic (TD) and trained control (TC) groups were followed by 8 weeks of resistance exercise training. Control groups were also followed (control - C, diabetes - D). We have found that PTD suppressed abnormalities linked to renal system such as, water consumption and amount of urine PTD=71mL vs. DT=127mL vs. D=138mL (measured during metabolic cage period), as well as attenuated proteinuria and kidney weight. Regarding to skeletal muscle, PTD group had increased muscle weight (extensor digitorium longus - EDL; C=192mg, D=116mg, TD=106mg and PTD=126mg; Tibialis anterior, C=780mg, D=496mg, DT=450mg and PTD=535mg); we also found a great muscle force level in the PTD group (C=573g, CT=1037, D=414g, TD=737g and PTD=825g), suggesting a protective effect of previous exercise in this group. PTEN was suppressed in PTD group and Akt and 4EBP1 (upstream and downstream of mTOR) were activated in PTD group, measured by western blot. These data suggest that, resistance exercise performed prior the establishment of the diabetes mellitus can protect kidney from diabetic nephropathy and skeletal muscle from atrophy. The mechanisms by which kidney and skeletal muscle have been improved are linked to mTOR signaling pathway. Further studies will be performed to confirm the potential involvement of this signaling pathway.

Support:
FAPESP, CAPES, CNPq.
Abstract 522: HEALS Program Implementation in African American Churches for Hypertension Control and Prevention
Authors: Dodani Sunita , Arora Sahel , Mahin Collisa , Butterfield Ryan
Published: July 2021

African Americans (AAs) living in the “Stroke Belt” (Southeastern US including northern Florida) are at elevated risk for stroke. Our long-term goal is to eliminate this ethnic/regional health disparities by targeting a major modifiable risk factor for stroke, hypertension (HTN). Here we present the results of HEALS (Healthy Eating And Living Spiritually)- a faith-based, socio-culturally tailored HTN control intervention modified from PREMIER and DASH programs for AA churches.

Methods:
4 church members were trained as program leaders who delivered the 3-months intervention followed by 9 months booster sessions. AA Church members of 25-75 yrs of age with known or newly diagnosed HTN (including pre-HTN as per JNC-VII) were recruited, who received HEALS program.

Results:
AAs with known (84%) or newly diagnosed HTN (16%) joined the program. Majority (80%) were on anti-HTN drugs. At the baseline, only three (8%) had HTN controlled (>120/80 mm Hg), 13 (35%) had pre-HTN levels, 15 (40.5%) were stage 1 and 6 (16%) in Stage 2. After receiving 12 wks HEALS sessions, mean reduction in systolic and diastolic blood pressure were -13.64 mm Hg (p =0.005) and -6.12 mm Hg (p=0.01) respectively. The study is currently in maintenance phase.

Conclusion:
If successful, the findings from this study will provide much-needed information on the translation and sustainability of evidence-based lifestyle modification in HTN members in community-based settings, particularly within churches, which represent the most influential institution in the community lives of AA
Abstract 532: BIA 5-1058, Beyond Blood Pressure, Improves Cardiometabolism and Decrease End-organ Damage
Authors: Igreja Bruno , Pires Nuno M , Wright Lyndon C , Soares-da-Silva Patrício
Published: July 2021

The sympathetic nervous system (SNS) can alter blood pressure (BP) by modulation of cardiac output, peripheral vascular resistance and renal function. One strategy for controlling sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH). BIA 5-1058 is a new peripheral DβH inhibitor that decreases NE levels in sympathetically innervated tissues and slows down the drive of SNS.

In order to evaluate the cardiometabolic effects of BIA 5-1058 in aged spontaneously hypertensive rats (SHR), 12 male SHR 50-week-old were randomized into two groups and one group was treated with BIA 5-1058 (30 mg/Kg/day) mixed in the diet for 9 weeks. During week 8 of treatment, blood pressure (BP) and heart rate (HR) were measured by tail cuff. At the end of the study, 24-hour urine and plasma was collected and organs weight was recorded.

BIA 5-1058 treatment reduced systolic BP (224±5 vs 183±8 mmHg, p<0.05) with no significant effect on HR. The heart/body weight ratio was decreased in animals treated with BIA 5-1058 (3.66±0.08 vs 3.45±0.04 mg/g, p<0.05), while the ratio kidney/body weight was unchanged. BIA 5-1058 significantly decreased plasma levels of the inflammatory markers CRP (447.3±7.1 vs 401.2±7.1 μg/ml, p<0.05), MCP-1 (90.3±10.5 vs 58.2±9.0 pg/ml, p<0.05) and ASAT (91.7±8.0 vs 67.3±4.0 U/l, p<0.05) but no significant effect on ALAT was observed. Concerning lipid metabolism, there was a decrease in plasma triglycerides (0.94±0.05 vs. 0.70±0.05 mmol/l, p<0.05) and free fatty acids levels (0.23±0.03 vs 0.11±0.01 mmol/l, p<0.05) induced by BIA 5-1058, while total cholesterol plasma levels was similar in both groups. BIA 5-1058 significantly reduced the 24-hour urine excretion (13.5±1.6 vs 8.9±1.2 ml, p<0.05), but had no significant effect in the amount of protein excreted in urine nor in the creatinine clearance rate.

In conclusion, the new DBH inhibitor, BIA 5-1058, presents cardiometabolic benefits in aged SHR.
Abstract 546: L-NAME Attenuates the Cardiotonic Steroids-induced Monolayer Permeability in Lymphatic Endothelial Cells
Authors: Castor Juan , Horvat Darijana , Cromer Walter E , Kuehl Thomas J , Zawieja David C , Uddin Mohammad N
Published: July 2021

Objective:
Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na
+
/K
+
ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome
in vivo
and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture.

Methods:
LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1μM before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of β-catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure β-catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests.

Results:
MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1μM L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The β-catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VE-cadherin and LYVE-1 protein expression, but MBG did not.

Conclusions:
We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the β-catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.
Abstract 547: Excess Glucose Dowregulates Factors for Invasion and Induces an Anti-angiogenic Milieu in First Trimester Cytotrophoblast Cell
Authors: Cawyer Chase , Horvat Darijana , Jones Richard O , Allen Steven R , Kuehl Thomas J , Uddin Mohammad N
Published: July 2021

Objective:
Diabetes mellitus is a known risk factor for the development of preeclampsia (preE). First trimester cytotrophoblastic (CTB) cells invade the decidua using the serine protease plasmin. The enzymatic conversion from the inactive plasminogen to the active form plasmin, is performed by urokinase plasminogen activator (uPA), which is regulated by plasminogen activator inhibitor 1 (PAI-1) in CTBs. Disruption of placental development, setting the stage for preE development is believed, in part, to be caused by a dysfunction of the invasive nature of CTBs. The aim of this study is to determine whether excess glucose induces anti-invasive and anti-angiogenic effects in CTBs
in vitro
.

Methods:
Human CTB cells (Sw. 71) were derived from first trimester placenta. The CTB cells were treated with 45, 135, 225, 495 or 945 mg/dL glucose for 72h. Thereafter, cell lysates were utilized to measure uPA and PAI-1 protein expression and phosphorylation of p38 MAPK by western blot. The mRNA expression of uPA and PAI-1 in glucose-induced CTB cell lysates was measure by qPCR. Levels of angiogenic and anti-angiogenic factors (sFLT-1, sENG, VEGF165, PlGF) and the level of inflammatory cytokine IL-6 were measured in the culture media by the commercially available kits. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests.

Results:
Both uPA and PAI-1 protein and mRNA expression were downregulated (p<0.05) in CTB cells treated with >135 mg/dL glucose as compared to those in basal (45 mg/dL). The anti-angiogenic factors (sENG and sFLT-1) were upregulated, while the angiogenic factors (VEGF165 and PlGF) were downregulated in the presence of >135 mg/dL glucose. The level of IL-6 was higher (p<0.05) in the presence of glucose >135 mg/dL compared to basal (45 mg/dL) conditions. The p38 MAPK phosphorylation was upregulated (p<0.05) in excess glucose-treated CTBs.

Conclusions:
Exposure of CTB cells to glucose at levels associated with diabetes causes a disruption in the invasive profile of CTB cells by decreasing the synthesis of uPA and PAI-1 (both protein and mRNA expression); by downregulating expression of VEGF165 and PlGF; and by upregulating expression of sENG, sFLT-1, and IL-6. Moreover, glucose induced a stress signaling in CTBs.
Abstract 553: Prostaglandin E2 EP3 Receptor Downregulates COX2 Expression in the Medullary Thick Ascending Limb (mTAL) Induced by Hypertonic NaCl Intake
Authors: Hao Shoujin , Vio Carlos P , Cespedes Carlos , Quiroz-Munoz Mariana , Ferreri Nicholas R
Published: July 2021

We recently showed that a novel negative feedback mechanism involving PGE
2
acting on EP3 receptors regulates cyclooxygenase-2 (COX-2) expression in the thick ascending limb (TAL) induced by the selective COX-2 inhibitors, celecoxib and rofecoxib. In the present study we tested the hypothesis that inhibition of EP3 facilitates COX-2 expression in the TAL induced by ingestion of hypertonic NaCl or exposure of mTAL cells to hypertonic media. COX-2 protein expression in the outer medulla (OM) increased 2.2±0.3 fold in mice given free access to 1% NaCl in the drinking water for 3 days compared with tap water; p<0.05. The increase was associated with a 4-fold elevation in COX-2 mRNA accumulation (tap water: 0.5±0.1 vs 1% NaCl: 1.9±0.4; p<0.05), and higher PGE
2
production by freshly isolated mTAL tubules (tap water: 87.6±9.4 vs 1% NaCl: 203.3±19.3 pg/mg protein; p<0.05). EP3 mRNA levels also increased approximately 2-fold in OM of mice ingesting 1% NaCl (tap water: 0.7±0.2 vs 1% NaCl: 1.3±0.2). Administration of a selective EP3 receptor antagonist (L-798106: 20μg/kg/day) for 2 days increased COX-2 mRNA accumulation in mTAL tubules (L-798106: 2.1±0.1 fold change vs control; p<0.05), and the elevation in COX-2 protein expression induced by 1% NaCl was increased an additional 50% in mice given L-798106. COX-2 mRNA accumulation in primary cultures of mTAL cells increased 2-fold in response to media made hypertonic by addition of NaCl (400 mosmol/kg H
2
O), compared with cells incubated in isotonic media. L-798106 increased COX-2 mRNA 2-fold in isotonic media and 4-fold in cells exposed to 400 mosmol/kg H
2
O. PGE
2
production by mTAL cells increased approximately 4-fold in response to challenge with 400 mosmol/kg H
2
O for 9 hr, and was inhibited in cells transiently transfected with a lentivirus shRNA construct (EGFP-C2-ex5) to silence COX-2 (286.1±14.8 to 64.3±5.2 pg/mg protein; p<0.05). Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3 receptor expression, which attenuates COX-2 activity in this segment of the nephron. Moreover, PGE
2
signaling via EP3 receptors in the TAL may be part of a mechanism that regulates mTAL COX-2 expression and function in response to diverse stimuli.
Abstract 567: Combined Treatment With Irbesartan and Vitamin D Retards Renal Injury in Salt-loaded Uni-nephrectomized Stroke-prone Spontaneously Hypertensive Rats
Authors: Takenaka Tsuneo , Inoue Tsutomu , Ohno Yoichi , Miyazaki Takashi , Nishiyama Akira , Ishii Naohito , Suzuki Hiromichi
Published: July 2021

Vitamin D diminishes renin expression. In addition, we previously reported that vitamin D increased renal expression of klotho in rats with normal kidney function. In the present study, effects of vitamin D on renal injury was assessed in four groups of rats (n=6-8 for each group); uni-nephrectomized stroke-prone spontaneously hypertensive rats fed high salt (6%) diet as a control (C), those treated with irbesartan (100 mg/kg/day) (I), rats treated with calcitriol (30 ng/kg/day) (V), and rats treated with both irbesartan and calcitriol (I+V). Six weeks later, rats were killed with over-anesthesia, and harvested right kidney for analysis. As shown in table, systolic blood pressure (SBP) in C (p<0.05) was higher than I and I+V groups. While renal angiotensin II (AngII) concentration was lower in I and I+V groups than C, plasma AngII levels of I and V groups are higher and lower than C (p<0.05), respectively. In addition, urine albumin/creatinine ratio (Alb/Cr) was lower in I and I+V group than C, and 8-epi-prostaglandin F2α (PGF2α) excretion was reduced in V and I+V groups (p<0.05). Immunoblot analysis revealed that compared to C, renal klotho expression was preserved in V and I+V group (p<0.05). Creatinine clearance (Ccr) was elevated in I+V group than C. The present data indicate that irbesartan effectively decreases blood pressure with reductions in renal AngII and albuminuria. Our findings demonstrate that vitamin D enhances klotho expression, suppressing oxidative stress and albuminuria without substantial changes in renal AngII. The current results may provide evidence that renal renin-angiotensin system is regulated independently of circulating one.
Abstract 569: Both Young and Old ACE2-transgenic Mice are More Resistant to Cardiac Dysfunction Induced by Myocardial Infarction
Authors: Zhang Juan , Qi Yanfei , Jeffery Colleen , Espejo Andrew , Raizada Mohan , Katovich Michael
Published: July 2021

Background:
Increased activity of the classic renin-angiotensin system (RAS) is associated with cardiovascular diseases (CVD) such as heart failure and hypertension. Angiotensin-converting enzyme 2 (ACE2) is reported to provide a protective role in CVD. In addition, there is a shift in the balance of the ACE2/Angiotensin-(1-7) axis towards the ACE/Angiotensin II axis during aging, making the cardiovascular system more vulnerable to damages. We hypothesized that global ACE2-overexpression could correct the imbalance between the two axes by providing a cardiovascular protective role against heart failure both in young and aged mice.

Methods:
Both young (10 weeks old) and old (12 months old) ACE2-transgenic and wild type (WT) mice underwent myocardial infarction surgery. Cardiac function was measured using echocardiography four weeks after MI. The number of circulating inflammatory cells (CD11b) in the blood for these animals was also measured using flow cytometry.

Results:
In the MI animals, the WT-old mice had a significant reduction in ejection fraction (47.67±4.23%: from 72.63±8.52% to 24.96±4.29%). However, the reduction in EF for ACE2-old mice was 37% lower (26.26±1.90%: from 60.06±7.56% to 29.18±5.66%) than that of WT-old mice. Meanwhile, the MI-induced decrease in EF of 14.60±1.56% for ACE2-young mice (from 59.09±8.38% to 44.49±6.82%) also was significantly less than the 26.97±1.21% reduction observed in WT-young mice (from 53.23±7.67% to 26.26±6.46%). Thus, ACE2 overexpression provides some protection of cardiac function from ischemia-induced injury in both young and old animals. Moreover, the circulating monocytes and macrophages in the blood of ACE2-young MI mice (22.65±6.86%) was also less than which observed in the WT control (31.37±4.90%), suggesting that there is less cardiac damage in these animals.

Conclusions:
Collectively, our observation suggests that global ACE2 overexpression has a consistent cardiac protection both in the young and old and as a result, the heart may be more resistant to heart failure damage during aging. This protective effect may be partly due to its systematic anti-inflammatory effect.
Abstract 600: Intrarenal Ghrelin Receptor Antagonism Inhibits cAMP Mediated Angiotensin II Sodium Reabsorption in Normal Rats
Authors: Kemp Brandon A , Howell Nancy L , Padia Shetal H
Published: July 2021

An interaction between angiotensin II (Ang II) and ghrelin has been established in many tissues relevant to cardiovascular control, but nothing is known about their relationship within the kidney. Intrarenal ghrelin receptors (GRs) localize to the collecting duct (CD) where they couple to an adenylyl cyclase second messenger system to increase cAMP and ENaC-dependent Na+ reabsorption. Ang II also stimulates the activity of ENaC in the CD (independent of aldosterone), via actions at AT1Rs. The following studies seek to determine whether CD GRs are an important mechanism of Ang II-induced antinatriuresis. Uninephrectomized Sprague-Dawley rats received 3 cumulative 1h renal interstitial (RI) infusions of vehicle 5% dextrose in water (D5W, N=8), Ang II (2 ng/kg/min, N=8), Ang II + D-LYS-GHRP-6, a highly selective GR antagonist (D-LYS, 2, 4, 6 μg/min, N=8) or D-LYS alone (N=8). Urine Na+ excretion rate (UNaV) was measured each hour and compared to baseline, during which only vehicle was infused. RI fluid was collected each hour for cAMP determinations. RI Ang II induced a significant antinatriuresis (UNaV was reduced by 34% at 1h, P<0.01; by 46% at 2h, P<0.001; and by 56% at 3h, P<0.001 from baseline). Ang II-induced antinatriuresis was accompanied by a significant increase in RI cAMP levels from a baseline value of 2.97±0.56 pmol/mL to 10.9±2.2, 13.4±2.2, and 15.3±2.7 pmol/mL after 1h, 2h, and 3h respectively (all P<0.01). However, each of these effects of RI Ang II infusion was abolished by concurrent GR blockade with D-LYS. These data suggest that intact intrarenal GR activity is necessary for Ang II-induced Na+ reabsorption in vivo. Furthermore, since cAMP fails to increase in response to Ang II when GRs are blocked, (and GRs are known to signal via cAMP in the kidney), these data strongly suggest that one of the mechanisms of Ang II-induced Na+ reabsorption in the kidney is via GR-induced increases in cAMP.
Abstract 602: Mass Spectrometry Based Characterization of Angiotensin Metabolism in ACE2 Deficient Mice Treated with RAS Blockers
Authors: Oliver Domenig , Manzel Arndt , Stegbauer Johannes , Gurley Susan B , Antlanger Marlies , Kovarik Johannes , Linker Ralf , Saemann Marcus , Poglitsch Marko
Published: July 2021

The Renin-Angiotensin-System is a peptide hormone cascade being responsible for the regulation of blood pressure and fluid balance through a sophisticated signaling network. During recent years the local Renin-Angiotensin-System (RAS) moved increasingly into the focus of research as the expression of multiple angiotensin processing enzymes had been reported for tissues. Nevertheless, scientific progress in this field is hampered due to the obvious presence of technical caveats in the analysis of the local RAS, which are indicated by a tremendous variation of tissue angiotensin levels reported in the literature. Hence, in order to assess the functions of the local RAS in different tissues, a solid and reproducible assay for the measurement of endogenous angiotensin peptide levels in tissues would be essential. We developed a novel mass-spectrometry-based method allowing the quantification of up to 10 angiotensin metabolites in tissue samples simultaneously. This novel tool was tested for applicability in murine studies by measuring tissue and plasma levels of angiotensin metabolites in wildtype and ACE2 knockout mice treated with various RAS-interfering drugs. The study provided deep insights into the systemic and tissue specific RAS revealing drug specific responses. As expected, the knockout of ACE2 resulted in increased Angiotensin II levels in hearts of knockout animals while the kidneys were only moderately affected. Enalapril treatment significantly lowered Angiotensin II levels in all investigated tissues. Surprising tissue specific effects were observed regarding other angiotensin metabolites, which underlines the importance of the comprehensive analysis of the RAS for conclusive interpretation of pharmacologic studies and allows to draw conclusions about expression levels of RAS-enzymes in the tissue of interest. Mass spectrometry based multiplex quantification of tissue angiotensin peptides (Tissue RAS-Fingerprinting) is a potent analytic tool for applications in basic science and drug development. Multiplex analysis of the tissue RAS provides new insights that might lead to the development of novel drugs and therapeutic concepts for the treatment of cardiovascular diseases in the future.
Abstract 61: Hypertension in a Rat Model of Postmenopausal Hyperandrogenemia (Polycystic Ovary Syndrome): Roles of the Renin-Angiotensin and Sympathetic Nervous Systems
Authors: Maranon Rodrigo O , Romero Damian G , Bui Bich , Zhang Huimin , Zhang Howei , Hosick Karen , Smith Andrew , Reckelhoff Jane F
Published: July 2021

Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in premenopausal women. PCOS is characterized by hyperandrogenemia that does not abate after menopause. Hyperandrogenemic postmenopausal women (HA-PMW) are hypertensive, but the mechanisms responsible have not been elucidated. Hypertension in PMW is less well controlled than in age-matched men. We have developed a model, the hyperandrogenemic postcycling female (PMHAF) rat, aged 13-14 mos, that exhibits many of the characteristics found in HA-PMW. In the present studies we tested the hypothesis that the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) contribute to hypertension in PMHAF rats. Female Sprague-Dawley rats were implanted with dihydrotestosterone (7.5mg/90d) or placebo pellets beginning at 4 wks of age, and pellets were changed every 85 d. At 12-13 mos of age, PMHAF rats were obese compared to placebo controls, and CT scans showed that both total adipose (TAF) and visceral adipose (VAF) were increased in PMHAF vs placebo controls (n=4/grp) (TAF: 7.9±0.5 vs 5.46±1.00 AU/g BW, p<0.05; VAF, 4.60±0.30 vs 3.45±0.07 AU/g BW, p<0.05). PMHAF rats (n=3/grp) were implanted with radiotelemetry transmitters, and 2 wks later, baseline MAP was measured for 5 d. Then rats were given losartan (40mg/kg/d) or tap water placebo for 5 days, and then tap water was returned. After additional 5 d and return of MAP to baseline, rats were implanted with minipumps containing terazocin and propranolol (10 mg/kg/d for each drug, sc, for 7 d) or saline vehicle. Baseline MAP in PMHAF rats was (128±9 vs 105±9 mmHg, p<0.05). Losartan reduced MAP more in PMHAF than placebo controls (111±9 vs 95±7 mmHg, p<0.05). Intrarenal angiotensinogen and ACE1 mRNA levels were increased by 8 fold and 2 fold, respectively, in PMHAF rats vs placebo controls (p<0.05). α
1
-β
1,2
-Adrenergic blockade reduced MAP by 13% in PMHAF rats, the same level as in placebo controls, and had no effect in placebo controls (110±12 vs 102±5 mmHg, p<0.05 vs baseline MAP). These data suggest that both the RAS and SNS are upregulated in PMHAF rats and contribute to their hypertension. These data also suggest that multiple drug therapy is likely to be necessary to control MAP in PMW with hyperandrogenemia. Supported by PO1HL51971.
Abstract 614: Premenopausal Women May Not Be Protected Against Early Vascular Disease In The Presence Of Diabetes.
Authors: Garcia Nestor H , Perez Hernan A , Spence J. David , Armando Luis J
Published: July 2021

Coronary heart disease (CHD) is the leading cause of death among women. Currently, global risk assessment derived by a Framingham risk equation is used to identify women at increased risk, but still we do not detect them early enough to decrease the rate of cardiovascular events. Perhaps we overestimate their protection during the premenopausal and early postmenopausal years, and overestimate the sensitivity of risk scores.

Methods:
A descriptive cross-sectional study in primary prevention in 1256 women (age 19-84) from Argentina. FRS with body mass index (FRSbmi) and PTP-TPA were used for the evaluation. TPA by ultrasound was determined as previously described by Spence et al (Stroke 2002;33;2916-2922). Patients were divided into DMII (n=293) and control groups (n=963) and then each group was divided according to age, <40, 40-50, 50-60 and older than 60.

Results:
No difference was observed between the incidence of smoking, Hypertension or presence of early family cardiovascular event in DM II and control group. DM II patients had higher TPA vs. control group at all ages. FRSbmi was higher in the DM II group at all age groups. PTP-TPA score for DM II <40, 40-50, 50-60, >60 groups were 10±3, 27±4, 48±3, 73±1 respectively while in control group were 3±1, 9±1, 23±1, and 50±1 respectively. These data indicate that DM II women in premenopausal or first years of menopausal age (40-50 years) are at intermediate or high risk to develop a cardiovascular event while non-diabetic women reach this PTP-TPA risk after age 50.

CONCLUSION:
In our population, diabetic women after age 40 should be considered at possibly high risk of cardiovascular events compared with a non-diabetic group. Direct assessment of atherosclerotic burden, such as TPA, should be used early in this population, even in the presence of menstrual cycles, instead of relying on traditional risk scores.
Abstract 623: Angiotensin Receptor Activation Contributes to Glucose Intolerance and Increased Insulin Resistance Independent of Elevated Systolic Blood Pressure, Adiposity and Dyslipidemia in a Model of Metabolic Syndrome
Authors: Lee Andrew Y , Rodriguez Ruben R , Martinez Bridget , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy
Published: July 2021

Activation of the renin-angiotensin system (RAS) leads to an increase in blood pressure and onset of insulin resistance (IR); however, the contributions of increased blood pressure and AT1 activation independently on the manifestation of IR are not well defined. The goal of this study was to determine the contribution of elevated blood pressure, independent of RAS, to the onset of IR in a model of metabolic syndrome. To address the hypothesis that AT1 activation, and not elevated blood pressure independently, is the principal contributor of obesity-associated IR, we measured changes in systolic blood pressure (SBP), adiposity, plasma triglycerides (TG), glucose tolerance, and insulin resistance index (IRI) in four groups of rats: 1) lean strain-control Long Evans Tokushima Otsuka (LETO; n=5), 2) obese Otsuka Long-Evans Tokushima Fatty (OLETF; n=7), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg; n=8), and 4) OLETF + calcium channel blocker (CCB; 5 mg amlodipine/kg; n=7). ARB treatment alleviated the obesity-related increase in mean SBP, while the decrease with CCB remained 10.2 mmHg greater than LETO. ARB (0.51 g/100g body mass) and CCB (0.64 g/100g body mass) reduced mean relative retroperitoneal fat mass and mean plasma triglycerides (28.4 and 27.4 mg/dL respectively) compared to OLETF, but both remained greater (1.7, 1.57 g/100g body mass and 28.94, 29.94 mg/dL respectively) than LETO. ARB improved glucose tolerance by 4605.94 of 34965.94 and mean calculated IRI by 5581429.5 of 17398363, but CCB had no detectable effect on either. Despite relatively similar reductions in SBP, adiposity and plasma TG (principal components of metabolic syndrome), CCB did not improve glucose tolerance and IRI (additional metrics of metabolic syndrome), while ARB did, demonstrating that AT1 activation is the primary factor contributing to the development of impaired glucose metabolism and regulation during metabolic syndrome, independent of the hypertension, adiposity and dyslipidemia. Thus, targeting RAS to improve the consequences of metabolic syndrome appears to be prudent and effective.
Abstract 629: Differential Expression Of Angiotensin-(1-12)/chymase In Human Atrial Tissue
Authors: Nagata Sayaka , Varagic Jasmina , Simington Stephen W , Ahmad Sarfraza , VonCannon Jessica L , Wang Hao , Groban Leanne , Kon Neal D , Dell'Italia Louis J , Ferrario Carlos M
Published: July 2021

Proangiotensin-12 [Ang-(1-12)] is a C-terminal extended form of Ang I [Ang I-Leu-Tyr] originally isolated from the rat small intestine. Additional studies showed that while both ACE and chymase cleaved Ang II from Ang-(1-12) in the rat heart, chymase but not ACE metabolized Ang-(1-12) in the human heart. This study characterized the expression of Ang-(1-12)/chymase in human atrial tissue to gain an insight into the potential role of this Ang II-forming substrate in cardiac disease. Left (LA; n=16) and right (RA; n=14) portions of atrial appendages were obtained from humans undergoing heart surgery. Quantitative Ang-(1-12) immunohistochemistry (Image J software) was performed using a high affinity purified antibody directed to the COOH-terminus of the full length of human Ang-(1-12) in all samples (LA: male=13 and female=3; RA: male=9 and female=5). Chymase activity was determined by HPLC in plasma membranes from left (males=7 and females=3) and right atria (males=7 and females=3). Quantitative analysis of Ang-(1-12) expression revealed significantly higher Ang-(1-12) expression in LA (Intensity: 35.37±6.24 units) versus RA (19.38±2.38 units, p=0.03) appendages. Likewise, chymase activity was higher in the LA (52.02±5.20 fmol/mg/min) compared to RA (18.09±3.05, p<0.0001) tissues. Expression of Ang-(1-12) in atria did not correlate with subject’s age, disease process or medications. The novel demonstration of higher Ang-(1-12) expression and chymase activity in human LA reveals a critical role of this tissue Ang II forming axis in modulating the diastolic and systolic properties of this cardiac cavity.
Abstract 633: Aerobic Training Induces Time-course Changes Of Ace2-Ang(1-7)-Mas Receptor Axis And Reactive Oxygen Species In The Heart Of Spontaneously Hypertensive Rats
Authors: Silva junior Sebastião D , Casarini Dulce E , Michelini Lisete C
Published: July 2021

Objective:
Since we showed recently that T causes time-dependent changes on brain RAS and oxidative stress of hypertensive rats, we sought now to investigate the sequential effects of T on ACE2-Ang(1-7)-MasR axis and reactive oxygen species (ROS) production in the left ventricle (LV) of SHRs.

Methods:
Male SHR and WKY aged 12 weeks underwent maximal exercise test (ET) and were allocated to T (50-60% of maximum exercise capacity, 1 h/day, 5 days/week) or sedentary (S) groups for 8 weeks. Subgroups of rats were cannulated at weeks 0 (S0), 1 (T1), 2 (T2), 4 (T4) and 8 (S8 and T8) for measurement of resting pressure (AP) and heart rate (HR), followed by saline perfusion and harvesting of the heart. ACE2 and MasR expression (Western Blotting) and ROS production (Dihydroethidium staining) were measured in the LV.

Results:
At the beginning of protocols SHR vs WKY exhibited higher MAP and HR (169±1 mmHg, 351±9 b/min, corresponding to 44% and 7% increases), elevated MasR (1,22±0,03 vs 1,00± 0,01 AU) but similar ACE2 expression (1,06±0,04 AU). ROS production was higher in SHR vs WKY (78±4 x 103 vs 66±1 x 103 AU). T increased treadmill performance (+0,87±0.10 km/h) and decreased resting HR (-15% at T4-T8) in both groups; MAP was only reduced in SHR (-10% at T8). T caused no change in the WKY group but increased ACE2 expression in the SHR (~1.14±0.03 au, T1-T4 P<0.05 vs WKY), with a biphasic response in Mas receptor expression: an early fall and a late increase (1,02±0,07 AU at T2-T4 and 1,20±0,04 AU at T8, respectively, P<0.05). In the SHR, T also caused an early normalization in ROS production (65±1 x 103 AU at T2) which was maintained up to T8. Again T did not change ROS production in the WKY group.

Conclusions:
Augmented ACE2 expression and reduced ROS production in the heart of the SHR are early responses to training, which are followed by a late increase in MasR expression. Data suggest that the prompt normalization of ROS production is mediated by ACE2-induced reduction of angiotensin II content in the LV of the trained SHR.
Abstract 643: Heterozygous Endothelial Endothelin-1 Overexpression Potentiates Endothelium-Dependent Contractions in the Carotid Arteries of Obese Mice
Authors: Baretella Oliver , Chung Sookja K , Xu Aimin , Vanhoutte Paul M
Published: July 2021

Elevated plasma levels of the vasoconstrictor peptide endothelin-1 (ET-1) are associated with cardiovascular risk factors such as obesity, diabetes and hypertension, in which endothelium-dependent contractions are prominent. Exogenous ET-1 promotes the release of endothelium-derived contracting factors (EDCF), but the role of endogenously produced ET-1 in these processes is unknown. Therefore, mice with
tie-1
promoter driven endothelium-restricted heterozygous overexpression of ppET-1 (TET+/-) and WT littermates were kept on standard chow as lean controls or administered a high fat diet for 30 weeks to induce obesity. At sacrifice, fasting glucose levels were significantly elevated in obese animals (8.3±0.3 vs. 5.3±0.2 mmol/l in lean controls,
n
=6,
P
<0.001). Isometric tension was measured in aortic and carotid arterial rings in wire myographs. In phenylephrine-contracted aortic rings, endothelium-dependent and −independent relaxations to acetylcholine and sodium nitroprusside, respectively, were unaltered between groups. In carotid arteries, the potency of phenylephrine to evoke contractions was greater in preparations from obese TET+/- mice (

pD
2

6.71±0.07 vs. lean TET+/- 6.34±0.13,
n
=5-6,
P
<0.05), whereas there was no change in the contractile response to the α
1
-adrenergic agonist by diet-induced obesity in WT littermates. The augmented EDCF responses to acetylcholine of quiescent carotid arterial rings of obese animals were further potentiated by TET+/- (

E
max

51.3±1.1% vs. 40.6±1.3% KCl in WT obese,
n
=6,
P
<0.001). The production of 6-keto PGF
1α
− the stable metabolite of prostacyclin − was increased significantly in preparations from obese TET+/- mice (238.4±30.0 vs. 127.0±12.7 pg/mL in obese WT littermates,
n
=4-6,
P
<0.05). In the presence of L-NAME, TP receptor activation by U46619 was more effective in obese TET+/- (

E
max

151.8±5.4% vs. 126.1±4.7% KCl in WT obese,
n
=4-6,
P
<0.05). Overall, TET+/- had no effect on relaxations in obese animals, but contractile responses − EDCF-mediated ones in particular − were facilitated. The present results suggest that this is due to an increased production of vasoconstrictor prostanoids possibly combined with an augmented responsiveness of the underlying vascular smooth muscle.