bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP11: Aldosterone Synthase Structure With Cushing’s Disease Drug Osilodrostat Provides Clues For Treatment Of Primary Aldosteronism
Authors: Brixius-Anderko Simone , Scott Emily E
Published: September 2020

Primary aldosteronism, the major form of secondary hypertension, often leads to cardiac disease. It develops due to excess steroid hormone aldosterone produced by aldosterone synthase, also known as cytochrome P450 11B2. CYP11B2 is 93% identical to cortisol-producing CYP11B1, which makes it difficult to design drugs specifically targeting CYP11B2. Osilodrostat (LCI699, Isturisa®) was initially developed as CYP11B2 inhibitor, but due to higher potency for CYP11B1 is now the first FDA-approved drug for CYP11B1-mediated Cushing’s disease. Thus, there is still no effective therapeutic option targeting CYP11B2 for primary aldosteronism. To determine aspects of the CYP11B2/osilodrostat interaction that could be improved to design a more selective CYP11B2 inhibitor, this project took a structure/function approach. First, osilodrostat affinity to CYP11B2 was examined using UV/vis spectroscopy. Osilodrostat induced a spectral change typical of ligand nitrogen coordination to the catalytic heme iron, establishing the binding location and mode, and revealed high CYP11B2 affinity. X-ray crystallography was used to solve the structure of CYP11B2 bound to osilodrostat. Consistent with the spectral analysis, osilodrostat binds in the active site with its imidazole nitrogen coordinating the heme iron. Additional interactions occur with the osilodrostat fluorinated benzonitrile. Osilodrostat binding was compared with that of its analog fadrozole to both CYP11B enzymes. CYP11B2 binding of osilodrostat is similar to
(R)
-fadrozole, but the fluorination of osilodrostat mediates additional active site interactions. Comparison with the previously-available CYP11B1 structure reveals CYP11B1 favors
(S)
-fadrozole due to distinct CYP11B active site architectures. These results suggest an opportunity to optimize inhibitor sterics to better discriminate between these two steroidogenic cytochrome P450 enzymes. Exploiting structural differences between the CYP11B enzymes should promote the design of therapeutics for the treatment of primary aldosteronism targeting CYP11B2, while reducing undesirable side effects due to off-target CYP11B1 inhibition.
Abstract MP13: TRPM7 Downregulation Contributes To Cardiovascular Injury And Hypertension Induced By Aldosterone And Salt
Authors: Rios Francisco J , Zou ZhiGuo , Neves Karla B , Nichol Sarah S , Camargo Livia L , Alves-lopes Rheure , Chubanov Vladimir , Gudermann Thomas , Montezano Augusto C , Touyz Rhian M
Published: September 2020

TRPM7 has cation channel and kinase properties, is permeable to Mg
2+
, Ca
2+
, and Zn
2+
and is protective in the cardiovascular system. Hyperaldosteronism, which induces hypertension and cardiovascular fibrosis, is associated with Mg
2+
wasting. Here we questioned whether TRPM7 plays a role in aldosterone- induced hypertension and fibrosis and whether it influences cation regulation. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600μg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo-salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel structure was assessed by pressure myography. Molecular mechanisms were investigated in cardiac fibroblasts (CF) from WT and M7+/Δ mice. Protein expression was assessed by western-blot and histology. M7+/Δ mice exhibited reduced TRPM7 expression (30%) and phosphorylation (62%), levels that were recapitulated in WT aldo-salt mice. M7+/Δ exhibited increased BP by aldo, salt and aldo-salt (135-140mmHg) vs M7+/Δ-veh (117mmHg) (p<0.05), whereas in WT, BP was increased only by aldo-salt (134mmHg). Mesenteric resistance arteries from WT aldo-salt exhibited increased wall/lumen ratio (80%) and reduced internal diameter (15%) whereas vessels from M7+/Δ exhibited thinner walls by reducing cross-sectional area (35%) and increased internal diameter (23%) after aldo-salt. Aldo-salt induced greater collagen deposition in hearts (68%), kidneys (126%) and aortas (45%) from M7+/Δ vs WT. Hearts from M7+/Δ veh exhibited increased TGFβ, IL-11 and IL-6 (1.9-fold), p-Smad3 and p-Stat1 (1.5-fold) whereas in WT these effects were only found after aldo-salt. Cardiac expression of protein phosphatase magnesium-dependent 1A (PPM1A), a Mg
2+
-dependent phosphatase, was reduced (3-fold) only in M7+/Δ mice. M7+/Δ CF showed reduced proliferation (30%) and PPM1A (4-fold) and increased expression of TGFβ, IL-11 and IL-6 (2-3-fold), activation of Stat1 (2-fold), Smad3 (9-fold) and ERK1/2 (8-fold) compared with WT. Mg
2+
supplementation normalized cell proliferation and reduced protein phosphorylation in M7+/Δ CF (p<0.05). Our findings indicate a protective role of TRPM7 in aldosterone-salt induced cardiovascular injury through Mg
2+
-dependent mechanisms.
Abstract MP14: Endothelial Mineralocorticoid Receptor Deletion Abrogates Leptin-induced Endothelial Dysfunction And Fetal Growth Restriction In Pregnant Mice
Authors: Faulkner Jessica L , Wright Derrian , Kennard Simone , Antonova Galina , Jaffe Iris Z , Belin De Chantemele Eric J
Published: September 2020

Obesity increases preeclampsia (PE) risk. Plasma leptin levels increase with body weight in women with PE, and in addition, placental ischemia, an initiating event of PE, also increases plasma leptin. Our lab has shown that hyperleptinemia induces endothelial dysfunction and hypertension in female mice via endothelial mineralocorticoid receptor (ECMR) activation and that high progesterone levels of pregnancy increases ECMR expression. We hypothesized that leptin infusion induces PE-like endothelial dysfunction in pregnant mice, which is abrogated by ECMR deletion. Pregnant mice were infused with leptin by miniosmotic pump (0.9mg/kg/day, s.c.) on gestation day (GD) 11-18. Leptin decreased pup wt (0.86±0.04g ECMR
+/+
vs 0.52±0.11 ECMR
+/+
+leptin, *P<0.05). and increased fetal resorption (3±39% ECMR
+/+
vs 42±32 ECMR
+/+
+leptin, P=0.09). ECMR deletion rescued pup weight (0.91±0.06g ECMR
-/-
vs 1.0±0.07 ECMR
-/-
+leptin) and protected fetal resorptions (2±2% ECMR
-/-
vs 0±0 ECMR
-/-
+leptin) in leptin-infused pregnant mice. In association, placental efficiency (pup/placenta ratio) decreased with leptin in ECMR
+/+
(9.7±0.7 ECMR
+/+
vs 7.9±0.6 ECMR
+/+
+leptin, *P<0.05), but not ECMR
-/-
(9.6±0.5 ECMR
-/-
vs 9.4±1.2 ECMR
-/-
+leptin) mice. Leptin infusion reduced endothelial function (acetylcholine-mediated relaxation) in aortas of ECMR
+/+
, but not ECMR
-/-
, pregnant mice (2-way ANOVA, repeated measures, *P<0.05). Relaxation responses to acetylcholine with LNAME showed restoration of endothelial function in ECMR
-/-
+leptin mice was due to preserved NO bioavailability. No changes in endothelial-independent sodium nitroprusside or contractile responses to phenylephrine, serotonin or KCl were observed. ECMR deletion reduced aorta IL-1β mRNA (0.5±0.1-fold change ECMR
+/+
+leptin vs 0.2±0.1-fold change ECMR
-/-
+leptin, P=0.06) levels and also placental growth factor mRNA (1.0±0.3-fold change ECMR
+/+
+leptin vs 01.3±0.1 ECMR
-/-
+leptin, P=0.05) in placental tissues. Collectively, these data indicate that ECMR deletion protects pregnant mice from adverse fetal growth and demise in association with increasing NO vascular bioavailability, reducing vascular inflammation and improving placental function in a leptin-infused model of PE.
Abstract MP15: Treatment With IL-25 Slows The Early Progression Of Proteinuria In Obese Dahl Salt-sensitive Rats
Authors: Poudel Bibek , Shields Corbin A , Ekperikpe Ubong , Brown Andrea K , Cornelius Denise C , Williams Jan M
Published: September 2020

Recently, we reported that the early progression of proteinuria in the obese Dahl salt-sensitive (SS
LepR
mutant) strain was associated with increased renal macrophage infiltration in the absence of hyperglycemia and elevations in arterial pressure. Macrophages (MØ) can be divided into two distinct phenotypes: M1-macrophages (classical; pro-inflammatory), and M2-macrophages (alternative; anti-inflammatory). M1-macrophages induce renal inflammation and fibrosis whereas M2-macrophages reduce renal inflammation and fibrosis. Moreover, previous studies have demonstrated that interlukin-25 (IL-25) converts resting MØ and M1 macrophages into M2 macrophages. Therefore, the objective of the current study was to examine whether treatment with IL-25 would reduce the early progression of proteinuria in SS
LepR
mutant rats by increasing renal M2 macrophages. Four week-old SS
LepR
mutant rats were separated into two groups (n=5/group): (1) vehicle (PBS) and (2) IL-25 (1μg/day, i.p.) for the first 10 days of the study. At baseline, proteinuria was similar in vehicle and IL-25 treated rats (81±18 vs. 66±8 mg/day, respectively). After 2 weeks, proteinuria was markedly reduced in the IL-25-treated group compared to the values measured in the vehicle treated rats (191±37 vs. 365±55 mg/day respectively; p<0.05 vs. vehicle). Interestingly, treatment with IL-25 was no longer effective in inhibiting the progression of proteinuria after 4 weeks (508±83 and 521±73 mg/day). When examining the possible phenotypes of the infiltrated macrophages in the kidneys, we did not detect any differences in M1 versus M2 macrophages between vehicle and IL-25 treated groups after 4 weeks. However, after 2 weeks of treatment, we did observe a tendency for M2 macrophages to be elevated in the kidneys of the IL-25 group. Overall, these data support our previous finding that the early progression of proteinuria in the obese SS
LepR
mutant strain is associated with renal macrophage infiltration and also suggest that IL-25 may be considered a therapeutic target for renal disease associated with obesity. This study was supported by DK109133.
Key Words: Obesity, Renal Disease, Macrophages, IL-25
Abstract MP17: Time Restricted Feeding Improves Cardiovascular Rhythms And Vascular Metabolism In Mice On A Chronic High Fat Diet
Authors: Pati Paramita , Zhang Dingguo , Colson Jackson , Bailey Shannon M , Gamble Karen L , Pollock David M , Pollock Jennifer S
Published: September 2020

Irregular timing of food intake increases hypertension and cardiometabolic disease risk. A chronic high fat diet (HFD) also disrupts circadian rhythms. We hypothesized that active period time restricted feeding (TRF) during the last 2 weeks in mice on a chronic HFD will improve blood pressure rhythm, diurnal variation of circulating plasma factors, and vascular metabolism. Mice (male 8-week old, C57BL/6J) were fed a normal diet (ND; 10% fat) or HFD (45% fat) for 20 weeks ad libitum. For the final 2 weeks, half of the HFD mice were subjected to TRF. Mean arterial pressure (MAP), heart rate (HR), and locomotor activity were assessed by telemetry. TRF significantly increased the active-inactive period difference in MAP and HR in in mice fed a HFD (ΔMAP: ND: 16±0.7 mmHg, HFD: 15±0.8 mmHg, HFD+TRF: 18±0.9 mmHg, n=6-8, p=0.01; ΔHR: ND: 68±5.1 bpm, HFD: 69±6.5 bpm, HFD+TRF: 113±7.9 bpm, n=6-8, p<0.01). Diurnal changes in locomotor activity are not different between groups. At the end of the study, plasma was collected at 4 hour intervals over a 24 hour period (ZT0 at 7AM; ZT12 at 7PM). Circulating levels of liver-derived mediators β-hydroxybutyrate (βHB) and insulin-like growth factor-1 (IGF-1) showed significant differences due to diet but not TRF (βHB, ZT21: ND: 0.16±0.01 mM, HFD: 0.20±0.02 mM, HFD+TRF: 0.19±0.01 mM, n=5-6, p=0.02; IGF-1, ZT5: ND: 232±18 ng/mL, HFD: 292±34 ng/mL , HFD+TRF: 371±14 ng/mL, n=5-6, p<0.01). Plasma leptin was significantly higher in mice on HFD and reduced by TRF at ZT12 (ND: 5.3±1.3 ng/mL, HFD: 22.5±2.9 ng/mL, HFD+TRF: 10.3±3.5ng/mL, n=5-6, p<0.01) and ZT17 (ND: 6.7±1.1 ng/mL, HFD: 32.5±3.0 ng/mL, HFD+TRF: 25.0±1.3 ng/mL, n=5-6, p<0.01). Plasma adiponectin was unchanged between all groups. TRF in HFD mice increased NAD
+
, important for metabolism, in renal vessels at ZT17 (HFD: 0.10±0.02 pmol/μg; HFD+TRF: 0.19±0.03 pmol/μg; n=5, p=0.03). Aortic NAD
+
at ZT1 was not affected by TRF in HFD mice (HFD: 1.83±0.35 pmol/μg, HFD+TRF: 1.35±0.35 pmol/μg, n=4, p=0.37). Our results indicate that TRF in mice on HFD increases the active-inactive period difference in MAP and HR and alters plasma metabolites, suggesting the timing of food intake on a chronic HFD improves cardiovascular rhythms with increased renal vascular metabolism and reduced leptin levels.
Abstract MP18: Mutagenesis Of Protease-mediated Cleavage Site In (pro)renin Receptor Induces Obesity And Type 2 Diabetes In Mice
Authors: Wang Fei , Chen Yan-ting , Zou Chang-jiang , Luo Renfei , Hekking Brittin , Feng Ye , Fu Ziwei , Yang Tianxin
Published: September 2020

Soluble (pro)renin receptor (sPRR), the extracellular domain of PRR, is generated by multiple proteases, including furin or ADAM19, and recently site-1 protease (S1P). We have previously reported that the therapeutic potential of histidine-tagged recombinant soluble (pro)renin receptor (sPRR), termed as sPRR-His, in a mouse obesity model induced by high-fat diet (DIO) for management of obesity and hyperglycemia. Conversely, inhibition of endogenous sPRR production by PF429242, an inhibitor of S1P in DIO mice aggravated diabetes and insulin resistance (Wang F et al. JCI Insight In press). However, the existence of the multiple substrates for S1P may impose confounding influence on the function of sPRR. The goal of the present study was to employ CRISPR strategy to mutagenize the overlapping recognition site for S1P and furin in PRR (termed as mutant mice) to examine its impact on metabolism. Mutant mice were fertile and developed normally with a 50% reduction plasma and tissue sPRR. 12-wk-old male mutant mice fed chow diet exhibited increased body weight (45.6 ± 4.8 g vs. 34.8 ± 2.7 g, n = 9, p < 0.05) and fat mass percentage ( fat mass/ body weight: 26% ± 4% vs. 12% ± 3%, n =9, p < 0.01) accompanied by reduced energy expenditure (0.35 ± 0.02 Kcal/kg/h vs. 0.54 ± 0.04 Kcal/kg/h, n = 4, p < 0.05) as assessed by calorimetry. The mutant mice developed hyperglycemia (fasting blood glucose: 172 ± 6.8 in mutant vs. 118 ± 5.4 mg/dL in WT, n = 15, p < 0.01), hyperinsulinemia (serum insulin: 48.7 ± 12.9 ng/ml vs. 3.7 ± 0.4 ng/ml, n = 6, p < 0.01), and impaired GTT and ITT. Additionally, sPRR-His supplement in the mutant mice nearly normalized blood glucose (132.4 ± 12.2 mg/dL) and insulin level (14.1 ± 5.9 ng/ml). Overall, these results support sPRR as an essential regulator of energy metabolism.
Abstract MP19: Smooth Muscle Cell Cytochrome B5 Reductase 3 Causes Heart Failure With Reduced Ejection Fraction Under Hypoxic Stress
Authors: Durgin Brittany G , Straub Adam C , Wood Katherine C , Hahn Scott A
Published: September 2020

Pulmonary hypertension causes increased pulmonary vascular resistance and right heart failure. Nitric oxide (NO) binds to its receptor soluble guanylyl cyclase (sGC) within vascular smooth muscle cells in its reduced heme (Fe
2+
) form to increase intracellular cGMP production, activate protein kinase G signaling, and induce vessel relaxation. In pulmonary hypertension, endothelial damage leading to decreased NO bioavailability combined with oxidation of the sGC heme (Fe
3+
) in vascular smooth muscle cells rendering it NO-insensitive results in vasonstriction. Notably, we have previously shown that cytochrome b5 reductase 3 (CYB5R3) in vascular smooth muscle cells is an sGC reductase (Fe
3+
to Fe
2+
) that maintains NO-dependent vasodilation in vascular disease. We therefore hypothesized that CYB5R3 confers protection in pulmonary hypertension. To test this, we subjected smooth muscle cell-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 21 days of continuous hypoxia (10% O
2
) and assessed vascular and cardiac function. We found that SMC CYB5R3 KO led to enhanced cardiac hypertrophy when compared to wild-type (WT) controls (n=8/ group). Specifically, SMC CYB5R3 KO mice had a larger right ventricle per tibia size, left ventricle mass, and Fulton index compared to WT (n=8/ group). Moreover, SMC CYB5R3 KO mice had a significantly impaired ejection fraction and fractional shortening, and increased left ventricular posterior wall pressure (n=3-5/group). No differences in right heart function or overall cardiac fibrosis were observed between groups (n=3-5/group). With respect to vascular function, hypoxic pulmonary arteries from SMC CYB5R3 KO mice also had a blunted response to sodium nitroprusside induced NO-dependent vasodilation though no difference in sGC activator BAY 58-2667 induced NO-independent vasodilation was observed as compared to WT (n=8-11/ group). No differences in pulmonary arterial sGC levels or medial area were observed between groups (n=6-7). Combined, these data implicate that loss of SMC CYB5R3 exacerbates cardiomyocyte hypertrophy and reduces cardiac function independent of pulmonary pressure differences.
Abstract MP20: Prepartum 1,3-butanediol Treatment Improves Placental Perfusion In The Preeclamptic Dahl Salt-sensitive Rat
Authors: Ishimwe Jeanne , Garrett Michael R , Sasser Jennifer M
Published: September 2020

Chronic hypertension increases the risk of developing superimposed preeclampsia (PE). Previous reports showed that 1,3-Butanediol (BD) lowers blood pressure (BP) in male Dahl salt-sensitive (S) rats and female congenic S.SHR(11) rats which exhibit increased kidney injury over Dahl S rats. The goal of this study was to test if attenuating hypertension before pregnancy through the placentation period via BD prevents the onset of PE. Female Dahl S rats (a spontaneous model of superimposed PE, 11-16 weeks old) were divided into two groups: BD treated (20% via drinking water) and control (ad libitum water). Animals received BD for 7 weeks, baseline BP measurements (telemetry) were taken, and both groups were then mated. On gestation day (GD) 12, treatment was stopped because pilot studies showed that treatment reduced water intake during late pregnancy. Both groups were maintained on low-salt rodent chow (Teklad 7034, 0.3% NaCl; n=8/group). At GD18 (late pregnancy), uterine artery resistance index (UARI) was measured via Doppler ultrasound, 24h urine was collected on GD19, and tissues were harvested on GD20. Mean arterial pressure was lower in the treated group at baseline (141.9 ± 4.1 vs. 165.7 ± 4.5 mmHg, p= 0.008), early (135.9 ± 3.4 vs. 168.9 ± 4.6 mmHg, p= 0.0003), mid (142.0 ± 5.2 vs. 170.8 ± 4.61, p= 0.0048) but not late pregnancy (144.9 ± 5.9 vs. 161.9 ± 4.5 mmHg, p= 0.165). Late pregnancy maternal body weight was similar between groups (299.3 ± 5.6 vs. 311.3 ±7.0 g, p=0.201). Treated dams had a lower UARI (0.71 ± 0.02 vs. 0.81 ± 0.02, p=0.008) and less fetal resorptions (1.12 ± 0.29 vs. 2.25 ± 0.41, p= 0.043). No harm to the fetus was noted as no differences in pup weight (2.20 ±0.05 vs. 2.20 ± 0.09, p= 0.994) and pup length (30.19 ±0.24 vs. 29.45 ± 0.28 cm, p=0.072) were observed. Placentas from treated dams had decreased VEGF levels via ELISA (276.2 ± 33.4 vs. 498.9 ± 16.8 pg/mL, p= 0.001), suggesting reduced placental ischemia. In this study, we observed slightly improved placental perfusion and lower fetal demise following prepartum BD treatment; however, the antihypertensive effects of BD were not sustained through late pregnancy when supplementation was stopped at mid-pregnancy.
Abstract MP25: Expression Of Urinary Somatic And Soluble N-domain Isoforms Of Angiotensin Converting Enzyme And Association With Different Nutritional Status And Cardiovascular Risk Profile
Authors: Cruz Nayara A , Oliveira Lilian C , Fernandes Fernanda B , Casarini Dulce E
Published: September 2020

Angiotensin converting enzyme (ACE) plays a dominant role in renal and cardiovascular diseases, obesity and diabetes. The somatic ACE (130-190 kDa) is composed of two homologous N- and C- domains. Two soluble N-domain isoforms have been described in human urine with 65 and 90KDa. Studies have supported that N-domain ACE with 90KDa is a biomarker for hypertension, pre-eclampsia and inflammation. We analyzed the expression of somatic and soluble N-domain ACE isoforms in urine of children and adolescents with different nutritional status and cardiovascular risk profile. The volunteers aged from 6 to 19 years were classified into four groups according to their BMI percentile; underweight (n=51), normal weight (n = 53), overweight (n=53) and obese (n=49). Waist-height-ratio (WHtR) was used to assess cardiovascular risk profile dividing the participants into normal risk (n=105) and high risk (n=101). The urines were concentrated 10-fold and dialyzed with Tris-HCl pH 8 and pure water. Then, we performed western blot analysis using 50μg of lyophilized urinary protein, using the ACE polyclonal antibody Y1. Protein detection was performed by chemiluminescent and analysis in Image Lab software utilizing total protein stain for normalization. ACE expression is augmented in obese children when compared with normal weight children (
0.09 vs 0.53 arbitrary units, p=0,04
). The higher cardiovascular risk group also presented increased expression of ACE (
0.27 vs 0.09 arbitrary units, p=0.046
). The 90KDa N-domain isoform is frequently found in the high cardiovascular risk children (
p= 0.02
). According to Spearman correlation test, the expression of 90 kDa N-domain ACE correlates positively with waist circumference, WHtR, BMI percentile and Z-score of BMI. Increased ACE expression in obese children contributes to higher cardiovascular risk once this enzyme biosynthesizes Angiotensin II which promotes blood pressure increase, sympathetic nervous system activation and release of glucocorticoids from adrenal gland. ACE expression is also augmented in children with high cardiovascular risk. Presence of 90 KDa N-domain ACE in urine of children and adolescents is a biomarker of poor prognostic for cardiovascular disease in childhood obesity.
Abstract MP31: Peripheral Vascular Dysfunction In A Model Of Small Vessel Disease Of The Brain (CADASIL) Involves Impaired Redox-sensitive Cyclic GMP/PKG Signaling
Authors: Neves Karla B , Morris Hannah , Alves-lopes Rheure , Montezano Augusto C , Touyz Rhian M
Published: September 2020

CADASIL, a monogenic condition due to
Notch3
mutations, is a very aggressive small vessel disease of the brain resulting in premature vascular dementia and stroke. Changes in cerebral vessels include vascular dysfunction and narrowing, and accumulation of granular osmiophilic material (GOM). It is not clear whether small peripheral arteries undergo similar damage. Therefore, our aim is to assess vascular dysfunction and associated mechanisms in mesenteric resistance arteries from CADASIL mice. Mesenteric arteries (MA) from male CADASIL-causing
Notch3
mutation (TgNotch3
R169C
) and wildtype (TgNotch3
WT
) mice (6 months old) were investigated. GOM deposits in MA from CADASIL mice were identified by electron microscopy. mRNA expression of
Notch3
(WT: 2.0±0.5 vs. 6.0±1.3) and its downstream target HeyL (WT: 1.1±0.4 vs. 2.9±0.6) was augmented in CADASIL mice (p<0.01), suggesting increased Notch3 activation. CADASIL mice exhibited endothelial-dependent (Emax 109.9±7.4 vs. 81.3±5.4) and -independent dysfunction (pD
2
7.8±0.1 vs. 6.8±0.3); effects associated with increased eNOS inhibition (p-Thr
495
) (1.8-fold increase) and decreased cGMP levels (1.2±0.2 vs. 0.59±0.2) (p<0.05). Plasma lipid peroxidation (0.8±0.1 vs. 2.0±0.3; p<0.05) and vascular reactive oxygen species (ROS) production (7.2±1.9 vs. 75.4±35.0; p<0.05) were increased in TgNotch3
R169C
mice; processes associated with upregulation of soluble guanylate cyclase (sGC) oxidation and decreased sGC activity. H
2
O
2
levels were decreased in TgNotch3
R169C
mice (1.9±0.2 vs. 1.1±1.9; p<0.05), which was associated with reduced activation of protein kinase G (PKG). Observations in TgNotch3
R169C
mice were recapitulated in human CADASIL, where ROS levels (0.8±0.1 vs. 4.1±2.7; p<0.05) and sGC oxidation were also increased. Our findings demonstrate that the vasculopathy associated with a CADASIL Notch3 gain-of-function mutation in peripheral small vessels involves reduction in eNOS activation and redox-sensitive processes leading to impaired sGC/cGMP signalling pathway. We identify a potential new therapeutic target in CADASIL, for which there are no disease-specific treatments.
Abstract MP35: Automated Office Blood Pressures For Making A Diagnosis Of Hypertension And The Effect Of Attendance And Rest Time
Authors: Green Beverly B , Anderson Melissa L , Cook Andrea J , Ehrlich Kelly , Hall Yoshio N , Margolis Karen L , Thompson Matthew J
Published: September 2020

Context:
Automated office blood pressure (AOBP) where three to five blood pressures (BP) are taken using an oscillometric device without an attendant in the room may decrease white coat effect.

Objective:
We evaluated the presence/absence of the attendant and varying amounts of rest time on BP; and the diagnostic performance of AOBP in making a new diagnosis of hypertension compared to daytime ambulatory blood pressure monitoring (ABPM).

Methods:
Participants included 130 adults aged 18-84 with no hypertension diagnosis or treatment and high BP (
>
140/90) prior to the study. Participants completing ABPM testing in the prior 24-hours were randomized either to Attended AOBP first, unattended second or; Unattended AOBP first, attended second
.
AOBP was subsequently performed twice, once after 5 minutes of rest and again after 15 minutes. Outcomes included within person difference between attended versus unattended BPs , and between 5 and 15 minutes of rest. Sensitivity and specificity of AOBP for making a new diagnosis of HTN was evaluated.

Results:
AOBP was significantly lower than mean daytime ABPM regardless of rest or attendance (systolic BP between -3.9 mmHg and diastolic BP -2.9 mm Hg lower). There were no significant within-person differences between AOBPs that were attended versus unattended (mean difference systolic 0.1 mmHg, CI -0.8, 1.1; diastolic 0.2 mmHg, CI -0.5, 0.8) or after five versus 15-minutes of rest (mean difference systolic -0.5 mmHg, CI -1.4, 0.5, diastolic 0.2 mmHg, CI -1.2, 0.0). Using daytime mean ABPM threshold of
>
135/85, the sensitivity and specificity of AOBP for making a new diagnosis of hypertension was 71.0% and 54.1%.

Conclusion:
Neither the presence/absence of an attendant nor rest time significantly impacted AOBP measurement. A single set of AOBP measurements may be insufficient for making a new diagnosis of hypertension.
Abstract MP44: Proteolytic Activation Of ENaC Mediates Fructose-Induced Salt-Sensitive Hypertension
Authors: Shukla Rashi , Hong Nancy , Henderson Ryan , Gonzalez-Vicente Agustin , Hopfer Ulrich , Garvin Jeffrey L
Published: September 2020

In nephrotic syndrome, glomerular injury leads to filtration of plasma proteases. Once in the urine, these enzymes cleave and activate the epithelial Na channel (ENaC) causing inappropriate Na retention and hypertension. Dietary fructose causes inappropriate Na retention and salt-sensitive hypertension but whether proteolytic activation of ENaC is involved, and the source of the proteases, is unknown. We hypothesized that dietary fructose increases expression and release of proteases from the proximal nephron, and that these enzymes activate ENaC thereby causing Na retention and salt-sensitive hypertension. To test this hypothesis we first measured proximal nephron protease expression and urinary protease excretion in rats on either a high-salt diet without fructose (high salt) or one containing 4% NaCl plus 20% fructose in the drinking water (high salt/fructose) for 7 days. High salt/fructose treatment increased proximal nephron expression of trypsin I, an enzyme known to cleave and activate ENaC, by 68±7% (p < 0.01). Urinary excretion of trypsin I was 8.4±1.3 arbitrary units/μg protein in rats fed high salt while it was 20.3±4.6 arbitrary units/μg protein in those on the high salt/fructose diet, 142% greater (p < 0.02). There was no difference in total urinary protein excretion between groups. Finally, we examined the effect of high salt/fructose and high salt plus 20% glucose) high salt/glucose) on blood pressure before and after oral amiloride. After 7 days the systolic blood pressure of rats on high salt/fructose was 148±6 mm Hg while it was only 124±5 in those on high salt/glucose (p < 0.02). Amiloride reduced systolic blood pressure in rats on the high salt/fructose diet from 148±6 to 134±5 mm Hg but had no significant effect on the high salt/glucose group. We conclude that proteolytic cleavage of ENaC contributes to fructose-induced salt-sensitive hypertension and that the source of the protease(s) is likely the proximal tubule rather than glomerular filtration.
Abstract MP47: Cardiac Compensation And Altered CREB/ERK Signaling Within The Arcuate Nucleus In The C57BL/6J Mouse Model Of Selective Leptin Resistance
Authors: Balapattabi Kirthikaa , Reho John J , Oliveira Vanessa , O'Meara Caitlin C , Wan Tina , Wu Jing , Sigmund Curt D , Grobe Justin L
Published: September 2020

Prolonged obesity causes “selective leptin resistance” (SLR), in which cardiovascular responses to leptin are intact but metabolic responses are lost. High fat diet (HFD) for 10 weeks causes SLR in C57BL/6J mice, as it attenuates metabolic (adipose) sympathetic nervous activity (SNA) responses to leptin but has no effect on cardiovascular SNA responses. Using single-nucleus RNAsequencing, we recently discovered disruption of leptin and CREB signaling specifically in Agouti-related peptide (
Agrp
) neurons of the arcuate nucleus of the hypothalamus (ARC) after 10-week HFD. Because C57BL/6J mice are generally resistant to diet-induced changes in blood pressure (BP), one goal of this study was to examine BP in this model. Further, we sought to examine effects of prolonged HFD on CREB signaling and
Agrp
mRNA within the ARC. Male C57BL/6J mice were maintained on chow (2920x) or HFD (D12451) from 8-18 weeks of age. Intake behaviors were studied in metabolic cages and bomb calorimetry; body composition via nuclear magnetic resonance; BP and HR via radiotelemetry; and cardiac function via echocardiography. ARC biopsies were analyzed for
Agrp
mRNA via qPCR and for CREB and ERK proteins using capillary electrophoresis-based Western. HFD increased caloric uptake, leading to increases in body and fat mass (p<0.05). HFD had no effect on BP (24h MAP: chow 110±2 vs HFD 103±9 mmHg, n=8 each), but heart rate (HR; 555±12 vs 601±3 bpm, p<0.05) and HR variability (low/high freq. ratio: 0.6±0.08 vs 0.8±0.09, p<0.05) were increased by HFD, consistent with increased cardiac SNA. Stroke volume (41±2 vs 18±3 uL, n=10 each, p<0.05) and thus cardiac output (13±0.9 vs 6±1 mL/min, p<0.05) were significantly reduced with HFD, explaining normal BP despite increased cardiovascular SNA. HFD increased plasma leptin (p<0.05), decreased CREB (pCREB/CREB ratio: 0.7±0.1 n=4, vs 0.4±0.1, n=5, p<0.05) but increased ERK (pERK/ERK ratio: 8.4±1.2 vs 12.5±1.1, p<0.05) phosphorylation, and failed to suppress
Agrp
mRNA in the ARC. We conclude that prolonged HFD increases cardiovascular SNA in C57BL/6J mice without effect on BP because of cardiac compensation. Further, this model causes SLR at the level of the ARC
Agrp
neuron, in a mechanism likely involving alterations in CREB- and ERK-mediated signaling.
Abstract P004: Endothelial Function In Rat Juxtamedullary Afferent Arterioles After Renal Ischemia-reperfusion Injury
Authors: Guan Zhengrong , FENG Wenguang , Remedies Colton , McEachen Bailey , Cook Anthony K , Inscho Edward W
Published: September 2020

Renal ischemia-reperfusion (IR) induced kidney injury exhibits reduced renal blood flow (RBF) and glomerular filtration rate (GFR). Both RBF and GFR are regulated by renal microvascular function. Our previous study showed that IR caused impaired afferent arteriole autoregulatory capability via excess ROS accumulation and inflammation in renal microvessels. Other mechanisms contributing to renal microvascular dysfunction in IR have not been fully defined. In this study, we conducted acetylcholine (ACh) concentration-response experiments (n=5-7/each group) to assess endothelial function using the
in vitro
blood-perfused juxtamedullary nephron preparation. IR was induced by 60 minutes of bilateral renal artery occlusion followed by 24 hours or 7 days of reperfusion. Renal microvessels (RMV) were collected at either 24 hours or 7 days post-surgery for mRNA analysis. Systolic blood pressure was normal in IR rats vs. sham ranging from 126-136 mmHg (>0.05) over 7 days. IR markedly increased plasma creatinine concentration (Cr) to 3.9±0.2 mg/dL at 24 hours post-IR (
P
<0.05 vs. 0.9±0.1 mg/dL in sham). Plasma Cr remained elevated in IR rats by day 7 of reperfusion (1.5±0.2 vs. 1.0±0.0 mg/dL in sham,
P
<0.05). Baseline afferent arteriole diameter was smaller in IR rats 24 hours post-IR and averaged 11.1±0.7 μm (P<0.05 vs. 14.2±0.7 μm in sham). Superfusion of ACh (10
-8
to 10
-4
M) caused concentration-dependent vasodilation in sham rats, increasing diameter to 23.5±2.6 μm (169±14% of the control,
P
<0.05) at 10
-4
M ACh. The ACh-induced vasodilation was not altered by IR insult and was indistinguishable from the sham responses. IR diameter reached a maximum of 19.7±0.9 μm (179±8% of the control) at 10
-5
M ACh. The ACh responses were also similar between sham and IR rats on day 7 post-IR. mRNA expression of endothelial markers, iCAM and vCAM, were not statistically different in RMV collected at 24 hours post-surgery between two groups, but was markedly elevated in IR RMV on day 7 post-IR (4-fold in iCAM and 2-fold in vCAM, P<0.05 vs. sham). In conclusion, upregulation of iCAM and vCAM mRNA expression in RMV 7 days post-IR suggests renal microvascular inflammation, but rat juxtamedullary afferent arterioles still maintain normal endothelium-dependent reactivity to ACh.
Abstract P010: D
1
R And D
5
R And Their Role In The Etiology Of Inverse Salt Sensitivity Of Blood Pressure
Authors: Gildea John J , Daley Michael G , Xu Peng , Schiermeyer Katherine A , Yue Wei , Carey Robert M , Jose Pedro A , Felder Robin A
Published: September 2020

Cardiovascular studies show increased morbidity and mortality in individuals consuming low sodium diets as well as high salt diets. The incidence of salt sensitivity of blood pressure (SS) in normotensives is approximately 18%, causing similar mortality and morbidity as hypertensives. Paradoxically, approximately 15% of normotensives demonstrate an increase in blood pressure on low sodium diets, known as inverse salt sensitivity (ISS). However, little is known about the morbidity and mortality associated with ISS, let alone the mechanisms behind this condition. Since dopamine regulates up to 75% of renal sodium handling, we hypothesized that the dopamine 1 and 5 receptors (D1R, D5R) were involved with the etiology of ISS. Using renal proximal tubule cells (RPTC) isolated from salt diet participant’s urine exposed to 90 mM salt (NaCl) (2 hr), we demonstrated reduced binding of the non-cell permeable D1-like antagonist (D1R and D5R) bodipy-530 SKF83566 (Fl-SKF) in the ISS RPTC when compared to salt resistant (SR) RPTC (ISS -13.9 ± 3.8% vs SR -1.1 ± 3.2%, n=12, p<0.05, t-test). Incubation in 190 mM NaCl for 2 hours and overnight (ON) increased Fl-SKF binding only in the ISS RPTCs but not the SR RPTC when compared to 140 mM NaCl (NS) (ISS 2 Hours +16.6 ± 6.2% and ISS ON +12.0 ± 2.8%, n=12, p<0.05 vs NS, t-test). ON incubation in 90 mM NaCl reduced Fl-SKF binding in both ISS and SR RPTC (ISS -15.2 ± 2.9% and SR -16.3 ± 2.3%, n=12, p<0.01, vs NS, t-test), and this effect was completely blocked by co-incubation with the angiotensin type 1 receptor (AT1R) antagonist losartan (LOS, 1 uM). The decrease in Fl-SKF binding in 90 mM NaCl was attributed to the D5R, and an increase in FL-SKF binding in 190 mM NaCl was verified to be due to an increase in plasma membrane D1R expression using antibodies directed to extracellular epitopes. A D5R specific monoclonal antibody developed in-house binds to the third extracellular loop of this receptor in order to measure these receptors selectively. Continued studies will be conducted with these cell lines with D1R and D5R knocked down to determine specific roles these receptors have in the novel ISS phenotype.
Abstract P011: Novel Roles Of Global, Intestinal, And Kidney Proximal Tubule Sodium And Hydrogen Exchanger 3 In Angiotensin Ii-induced Hypertension
Authors: Zhuo Jia L , Zhang Liang , Leite Ana , Li Xiao C
Published: September 2020

The present study used global (

Nhe3
-/-

), kidney-selective (tg

Nhe3
-/-

), and proximal tubule-specific Na
+
/H
+
exchanger 3 (NHE3)-deficient mice (PT-

Nhe3
-/-

) to test the hypothesis that NHE3 is required for the full development of angiotensin II (Ang II)-induced hypertension in mice. Four groups of adult male, age-matched wild-type (WT), global

Nhe3
-/-

, kidney-selective tg
Nhe3

-/-

and proximal tubule-specific

Nhe3
-/-

mice were infused with: a) saline; b) Ang II (10 pmol/min, i.v.); Ang II via an osmotic minipump for 2 weeks (1.5 mg/kg/day, i.p.); or treated with Ang II and losartan concurrently for 2 weeks (20 mg/kg/day, p.o.). Under basal conditions, global

Nhe3
-/-

, kidney-selective tg
Nhe3

-/-

and proximal tubule-specific
Nhe3

-/-

mice all showed significantly lower systolic, diastolic, and mean arterial pressure than wild-type mice (~15 ± 3 mmHg,
P
<0.01). The hypotensive phenotype in both global

Nhe3
-/-

and kidney-selective tg

Nhe3
-/-

mice was associated with abnormal intestinal structures, diarrhea, increased 24 h fecal Na
+
excretion, and salt wasting (
P
<0.01). By contrast, there were no differences in intestinal structures and fecal Na
+
excretion between wild-type and PT-

Nhe3
-/-

mice. PT-

Nhe3
-/-

mice showed significant diuretic and natriuretic responses compared with wild-type mice (
P
<0.01). Acute infusion of Ang II markedly increased arterial blood pressure in a time-dependent manner in wild-type mice, as expected (
P
<0.01), but the pressure response was attenuated in global

Nhe3
-/-

, kidney-selective tg

Nhe3
-/-

, and PT-

Nhe3
-/-

mice (
P
<0.01). Furthermore, the chronic pressor response to 2-week Ang II infusion was also significantly attenuated in

Nhe3
-/-

,

tgNhe3
-/-

, and PT-

Nhe3
-/-

mice, compared with wild-type mice (
P
<0.01). Finally, concurrent treatment with losartan completely blocked the acute and chronic pressor responses to Ang II in wild-type,

Nhe3
-/-

, tg

Nhe3
-/-

, and PT-

Nhe3
-/-

mice (p<0.01). Taken together, these data support the proof of concept that NHE3 in the small intestines and the proximal tubules of the kidney is required for maintaining basal blood pressure homeostasis and for the development of Ang II-induced hypertension. Supported by NIH grants, 2R01DK102429-03A1, 1R56HL130988-01, and 2R01DK067299-10A1.
Abstract P012: Cytochrome P450 1B1 Generated 17β-Estradiol Metabolite 2-Methoxyestradiol Inhibits 12/15-Lipoxygenase To Protect Against Angiotensin II-induced Hypertension, Autonomic Dysfunction And Renal Injury In Female Mice
Authors: Singh Purnima , Dutta Shubha Ranjan , Malik Kafait U
Published: September 2020

17β-estradiol (E2) via its cytochrome P450 (CYP) 1B1 generated metabolite 2-methoxyestradiol (2ME) protects from angiotensin (Ang) II-induced hypertension in female mice. Gene disruption of 12/15 lipoxygenase (LOX) that metabolizes arachidonate (A) into 12(S)- and 15(S)-HETE minimizes Ang II-induced hypertension in male mice. We performed this study to determine if 2-ME generated by CYP1B1 from E2 mediates its protective effect against Ang II-induced hypertension via LOX inhibition in female mice. Systolic blood pressure (SBP, mmHg, tail-cuff) in response to Ang II (700 ng/Kg/min, sc, miniosmotic pump, 2 weeks) was greater (
P
<0.05, n=4-5) in ovariectomized (OVX) than intact
Cyp1b1
+/+
/
Alox15
+/+
(174±2 vs 135±5), and intact or OVX-
Cyp1b1
+/+
/
Alox15
–/–
(120±4 and 125±5) mice. Moreover, SBP in response to Ang II was greater in trans-2,3',4,5'-tetramethoxystilbene (CYP1B1 inhibitor, 300 μg/Kg, every 3rd day, ip)-treated intact
Cyp1b1
+/+
/
Alox15
+/+
than
Cyp1b1
+/+
/
Alox15
–/–
(164±3 vs 132±3, n=6-8,
P
<0.05) mice. Also, mean arterial pressure (MAP, mmHg, radiotelemetry) and low frequency to high-frequency oscillation ratio (LF/HF ratio, power spectral analysis), the index of baroreflex sensitivity impairment, in response to Ang II was greater (
P
<0.05, n=4) in OVX-
Cyp1b1
+/+
/
Alox
15
+/+
(160±3; 2.9±0.1) than in OVX-
Cyp1b1
+/+
/
Alox15
–/–
(119±7; 2±0.1) mice. However, MAP and LF/HF ratio in response to Ang II was lower in 2ME (1.5 mg/g, every 3rd day, ip)-treated OVX-
Cyp1b1
+/+
/
Alox15
+/+
(108±3; 1.6±0.1) and OVX-
Cyp1b1
+/+
/
Alox
15
–/–
(107±2; 1.6±0.1) mice (n=4). Plasma12(S)-HETE level (ELISA), and renal injury assessed by urine albumin/creatinine ratio in response to Ang II was lower in 2ME than its vehicle (dimethyl sulfoxide)-treated OVX-
Cyp1b1
+/+
/
Alox
15
+/+
mice (1616±30 vs 2162±55, pg/mL, and 2.7±0.3 vs 1.9±0.1; n=4,
P
<0.05). Ang II did not increase 12(S)-HETE or albumin/creatinine ratio in
Cyp1b1
+/+
/
Alo
x
15
–/–
mice. These data suggest that that E2-CYP1B1 generated metabolite 2ME by inhibiting A-LOX signaling protects against Ang II-induced hypertension, impaired baroreflex, and renal injury in female mice. Therefore, LOX inhibitors could be useful in the treatment of Ang II-induced hypertension and associated renal injury in females.
Abstract P022: Evidence For A Physiological Mitochondrial Angiotensin Ii System In The Proximal Tubules Of The Kidney
Authors: Li Xiao C , Zhou Xinchun , Zhuo Jia L
Published: September 2020

Angiotensin II (Ang II) has long been implicated in mediating mitochondrial dysfunction in cardiovascular, hypertension, and kidney diseases. However, whether it is circulating and paracrine Ang II or intracellular mitochondrial Ang II that induces mitochondrial dysfunction has not been studied previously. The present study tested the hypotheses that overexpression of an intracellular angiotensin II (Ang II) fusion protein, mito-ECFP/Ang II, selectively in the mitochondria of mouse proximal tubule (mPCT) cells induces mitochondrial oxidative and glycolytic responses and elevates blood pressure via the Ang II/AT
1a
receptor/superoxide/NHE3 (the Na
+
/H
+
exchanger 3)-dependent mechanisms. A proximal tubule-selective, mitochondria-targeting adenoviral construct encoding Ad-sglt2-mito-ECFP/Ang II was used to test the hypotheses. The expression of mito-ECFP/Ang II was colocalized primarily with Mito-Tracker® Red FM in mPCT cells or with TMRM in kidney proximal tubules. mito-ECFP/Ang II markedly increased oxygen consumption rate (OCR) as an index of mitochondrial oxidative response (69.5%;
P
<0.01) and extracellular acidification rate (ECAR) as an index of mitochondrial glycolytic response (34%;
P
<0.01). The mito-ECFP/Ang II-induced OCR and ECAR responses were blocked by AT
1
blocker losartan (
P
<0.01) and a mitochondria-targeting superoxide scavenger mito-TEMPO (
P
<0.01). By contrast, the non-selective NO inhibitor L-NAME alone increased, whereas the mitochondria-targeting expression of AT
2
receptors (mito-AT
2
/GFP) attenuated the effects of mito-ECFP/Ang II (
P
<0.01). In the kidney, overexpression of mito-ECFP/Ang II in the mitochondria of the proximal tubules increased systolic blood pressure 12 ± 3 mmHg (
P
<0.01), and the response was attenuated in proximal tubule (PT)-specific PT-

Agtr1a
-/-

and PT-

Nhe3
-/-

mice (
P
<0.01). Conversely, overexpression of AT
2
receptors selectively in the mitochondria of the proximal tubules induced natriuretic responses in PT-

Agtr1a
-/-

and PT-

Nhe3
-/-

mice (
P
<0.01). Taken together, these results provide new evidence for a physiological role of proximal tubule mitochondrial Ang II/AT
1a
/superoxide/NHE3 and Ang II/AT
2
/NO/NHE3 signaling pathways in maintaining blood pressure homeostasis.
Abstract P028: Activation Of The Renin-angiotensin System Drives Renal Metabolic Abnormalities In Diabetic Nephropathy
Authors: Azushima Kengo , Kovalik Jean Paul , Ching Jianhong , Gurley Susan B , Coffman Thomas M
Published: September 2020

Activation of the renin-angiotensin system (RAS) is a major contributor to the pathogenesis of diabetic nephroathy (DN). However, the precise mechanisms of renoprotection associated with RAS blockade in DN are not entirely clear. The aim of this study is to examine whether metabolic effects of RAS blockade might contribute to renoprotection. We utilized a mouse model of DN combining severe type I diabetes (the Akita mutation) with a single-copy renin transgene (ReninTG) driven by the albumin promoter. Akita-ReninTG mice on a 129/Sv background (DN-susceptible mice) develop clinical features of human DN including high-grade albuminuria, renal interstitial inflammation and glomerulosclerosis, while Akita-ReninTG mice on a C57BL/6 background (DN-resistant mice) do not develop significant kidney disease. These two experimental groups were treated with the angiotensin receptor blocker (ARB) losartan 10 mg/kg/day for 12 weeks, and metabolic profiles in kidney tissues were examined using a targeted metabolomics assay. The DN-susceptible mice exhibited high-grade albuminuria that was significantly attenuated by ARB (Vehicle vs ARB: 1480±562 vs 193±42 μg/day,
p
=0.045), while DN-resistant mice had minimal albuminuria that was not affected by ARB (Vehicle vs ARB: 80±14 vs 75±14 μg/day,
p
=0.801). The metabolomics profiles of the DN-resistant mice were similar to C57BL/6 wild-type controls. By contrast, DN-susceptible mice exhibited broad reductions in even-chain acyl-carnitines and an abnormal profile of TCA cycle intermediates compared to 129/Sv wild-type controls, suggesting substantial impairments of renal mitochondrial fuel oxidation including altered fatty acid metabolism. RAS blockade had broad effects to correct this profile by increasing acetyl-carnitines generated from acetyl-CoA and concomitantly normalizing expression of genes associated with mitochondrial fatty acid metabolism including PPAR-α, PGC-1α, CPT1 and CPT2. ARB treatment restored TCA cycle activity to normal. These findings suggest that effects of RAS blockade re-establish normal fuel metabolism and mitochondrial fatty acid oxidation in kidney and may contribute to renoprotection.