bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 18: Excess Dietary Salt-induced Blood Pressure Elevation Is Associated With Impaired Bile Acid Signaling And Human Monocyte Activation
Authors: Ishimwe Jeanne A , Ferguson Jane F , Kirabo Annet
Published: August 2021

Excess dietary sodium (Na
+
) is a major risk for hypertension and cardiovascular disease. We previously found that excess dietary salt induces gut microbial dysbiosis and leads to activation of antigen presenting cells and hypertension, but the mechanisms are not known. The gut microbiome metabolizes bile acids, and these have been implicated in activation of antigen presenting cells. We hypothesized that high dietary Na
+
-induced inflammation leading to hypertension is associated with impaired bile acids signaling. We measured plasma bile acids via a metabolomics analysis in 75 volunteers. Blood pressure was also monitored. Based on the recommendations by the American Heart Association, we classified daily Na
+
intake <2.3g as normal salt (NS, n=23), and high salt (HS, n=52) for subjects consuming ≥ 2.3g Na
+
. Spearman correlation was used to assess the relationship between Na
+
intake and blood pressure. We found that elevated diastolic (r=0.331, p=0.003), systolic (r=0.383, p<0.001) and mean arterial pressure (r=0.278, p=0.014) were associated with increased Na
+
intake. Five taurine and glycine-conjugated secondary bile acids including taurodeoxycholate (1.320 ± 0.238 vs. 2.820 ± 0.669; p=0.010); glycodeoxycholate (1.197± 0.154 vs. 1.998± 0.432; p= 0.033); glycodeoxycholate sulfate (1.037± 0.132 vs. 1.521± 0.192; p= 0.044); glycolithocholate (0.829± 0.094 vs. 1.622± 0.363; p= 0.006) and glycolithocholate sulfate (1.083± 0.127 vs. 1.598± 0.205; p= 0.030) were lower in the HS when compared to the NS group. Interestingly, using RNA sequencing in human monocytes, we found that the NR1H4 gene which encodes the farnesoid X receptor, a nuclear receptor activated by bile acids, was significantly downregulated by high salt treatment, and this was associated with a pro-inflammatory phenotype (9.80± 1.65 vs. 11.82± 1.64; adjusted p-value=0.043). Our findings suggest that high salt impairs bile acids signaling leading to inflammation and salt-induced hypertension.
Abstract 20: Association Of Systolic Blood Pressure Time-in-target Range With Adverse Kidney And Cardiovascular Outcomes In Adults With And Without Diabetes
Authors: Buckley Leo F , Baker William L , Van Tassell Benjamin W , Cohen Jordana , Alkhezi Omar , Bress Adam P , Dixon Dave L
Published: August 2021

Introduction:
Hypertension associates with both kidney and cardiovascular (CV) disease risk. Time-in-target range (TTR) associates with CV risk independent of mean SBP and SBP variability. We hypothesized that SBP TTR predicts both adverse kidney and CV outcomes.

Methods:
ACCORD BP and SPRINT trial participants with >=2 SBP measurements were eligible, except ACCORD standard BP lowering participants due fewer SBP measurements. SBP TTR for months 0-3 was calculated using Rosendaal linear interpolation with target ranges of 110-130 mm Hg and 120-140 mm Hg for participants in the intensive or standard arms, respectively. Adverse kidney outcomes included dialysis, kidney transplant, serum creatinine > 3.3 mg/dL, sustained eGFR of < 15 mL/min per 1.73 m
2
or sustained eGFR decline >40% after month 3. Adverse CV outcomes included myocardial infarction, stroke, heart failure and CV death. Cox proportional hazards regression models were used to estimate the association between TTR and adverse outcomes after demographics, clinical risk factors and baseline SBP adjustment

Results:
Participants with higher TTR were younger, less likely to have preexisting CV disease and had less albuminuria, higher eGFR and lower baseline SBP. In fully adjusted models accounting for baseline SBP, higher TTR independently associated with a lower risk of adverse kidney and CV outcomes (P-trend < .001 for each). Whereas the relationship between TTR and CV risk increased monotonically with higher TTR, the TTR association with kidney risk was greatest at the extremes of TTR (
Figure
).

Conclusions:
Further reductions in adverse kidney and CV outcomes may be achievable through sustained SBP control.
Abstract 38: Radiation Exposure And Coronary Atherosclerosis: Differential Effect Of The Radiation Site
Authors: Nepali Prerna R , Mathieu Mickael , Kitz Sarah , Nakauchi Chiharuko , Russell James , Monette Sebastien , Rimner Andreas , Stewart Fiona , Haimovitz-friedman Adriana , Jaimes Edgar A
Published: August 2021

Background:
Thoracic radiation is commonly used in breast cancer, Hodgkin’s lymphoma, head and neck and lung cancer patients. Accelerated coronary artery atherosclerosis is a common complication of thoracic radiation as a result of unintended cardiac radiation. It is unclear however whether specific areas of the heart are more susceptible to the effects of radiation (RT). In this study we hypothesize that
accelerated development of lesions post RT is dependent upon differential sensitivity of specific areas of the heart to the effects of RT.

Methods:
Male Apolipoprotein E knockout mice on a high fat diet received 16Gy cardiac RT targeted to the whole or partial (apical or basal) region of the heart at 9 or 16 weeks of age (n=5 per group). Atherosclerotic lesions in H&E stained slides and inflammatory infiltrates in the hearts by IHC were assessed 8 weeks following RT and compared to control unirradiated mice.

Results:
Our studies show that: (1) Subendocardial atherosclerotic lesions at the base of heart in mice irradiated at 9 weeks of age after basal irradiation (7.8±2.49) were comparable to whole heart irradiation (12.2±3.29). (2) A greater number of atherosclerotic lesions were present in the basal coronary arteries (29.33±5.48 vs 9±2.70) and basal subendocardial vasculature (6.66±2.07 vs 0.2±0.2) after irradiation of the cardiac base as compared to unirradiated controls in mice irradiated at 16 weeks of age. (3) Apical or whole heart irradiation had no impact on the development of lesions in the basal region of the hearts of older mice. (4) IL-6 was significantly increased in the serum of mice 6 hours post basal cardiac irradiation (105.10±17.56 pg/ml) when compared to unirradiated controls (29.85±11.63 pg/ml), demonstrating an acute inflammatory response. (5) Infiltration of inflammatory cells (CD45 and CD3) and enhanced expression of endothelial adhesion molecules (CD31) were differentially and locally regulated based upon the site of irradiation.

Conclusion:
Our results indicate that the base of the heart is more prone to development of RT induced atherosclerotic lesions likely due to acute and delayed inflammatory responses. Avoiding this area from direct radiation exposure may improve the quality of life for cancer patients receiving thoracic RT.
Abstract 50: Acceptability And Adherence To Home, Kiosk, Office Blood Pressure Measurement Compared To 24- Hour Ambulatory Monitoring In Primary Care
Authors: Thompson Matthew , Green Beverly , Ehrlich Kelly , Hall Yoshio , Anderson Melissa , McClure Jennifer , Margolis Karen L , Joseph Dwayne
Published: August 2021

Introduction:
Despite recommendations to use out of office blood pressure (BP) to confirm diagnosis of hypertension, there is little robust evidence on the acceptability of different methods for confirming hypertension. Most evaluations have involved small observational studies and have not compared measurement methods. The Blood Pressure Checks for Diagnosing Hypertension trial (BP-CHECK) trial compared the acceptability and adherence of clinic, home, kiosk and 24-hour ambulatory monitoring (ABPM) among adults with elevated BP who had not been diagnosed with hypertension.

Methods:
Adults presenting to Kaiser Permanente Washington primary care clinics with elevated BP but who did not have a hypertension diagnosis, were randomized to one of three arms: office, home and kiosk. ABPM was also conducted on all participants. Acceptability of each method to patients was measured using a validated questionnaire to calculate an overall score. Differences in mean acceptability by randomization arms and ABPM were calculated using linear regression models and Generalized Estimating Equations. Adherence was calculated as the proportion of participants completing their BP testing protocols and compared using chi-square tests.

Results:
A total of 510 individuals were randomized, with mean age 59 years, 80% white, 51% male, with mean BP of 150/88. The overall acceptability score was highest (i.e. most acceptable) for home (mean 6.2, SD 0.7) and lowest (i.e. least acceptable) for ABPM (mean 5.0, SD 1.0). Scores were intermediate for clinic (5.5, SD 1.1) and kiosk (5.4, SD 1.0) groups. Acceptability was significantly higher for all three intervention arms compared to ABPM. Adherence to the minimum number of BP readings pre-specified in the study protocol was higher for home (90.6%) and clinic (87.2%) groups than for the kiosk group (67.9%).

Conclusions:
Home BP measurement was the most acceptable method for diagnosing hypertension followed by measurement in the clinic or at kiosks, with ABPM the least acceptable, due to its discomfort, disturbance to sleep and to everyday activities. Although ABPM is considered the gold standard for confirming elevated BP, patients find home BP more acceptable.
Abstract 58: Angiotensin-(1-7) Mas Receptors In The Arcuate Nucleus Of The Hypothalamus Protect Against High Fat Diet-induced Insulin Resistance In Female Mice
Authors: Mehay Darren , Bingaman Sarah , Silberman Yuval , Arnold Amy
Published: August 2021

Angiotensin (Ang)-(1-7) is a protective hormone of the renin-angiotensin system that improves insulin sensitivity, glucose tolerance, and energy balance in obese rodents. Our recent findings suggest that Ang-(1-7) activates
mas
receptors (MasR) in the arcuate nucleus of the hypothalamus (ARC), a brain region critical to control of energy balance and glucose homeostasis, to induce these positive metabolic effects. The distribution of MasR in the ARC and their role in metabolic regulation, however, is unknown. We hypothesized: (1) MasR are expressed in the ARC; and (2) deletion of ARC MasR leads to worsened metabolic outcomes following high fat diet (HFD). To test this, male and female C57Bl/6J mice were fed a 60% HFD or matched control diet
ad libitum
for 12 weeks. RNAscope
in situ
hybridization was performed on coronal ARC sections in rostral-middle-caudal regions to determine percentage of MasR positive neurons (n=5/group). In a second experiment, we assessed body composition and insulin and glucose tolerance in transgenic mice with deletion of MasR in ARC neurons (MasR-flox with AAV5-hsyn-GFP-Cre). RNAscope revealed a wide distribution on MasR-positive cells throughout the rostral to caudal extent of the ARC. The average percentage of MasR positive neurons was increased in females versus males, with HFD tending to increase MasR expression in both sexes (control diet male: 11±2; control diet female: 17±3; HFD male: 15±5; HFD female: 24±2; p
sex
: 0.030; p
diet
: 0.066; p
int
: 0.615; two-way ANOVA). Deletion of MasR in ARC neurons worsened insulin sensitivity in HFD but not control diet females (area under the curve for change in glucose from baseline: -1989±1359 HFD control virus vs. 2530±1762 HFD Cre virus; p=0.016), while fasting glucose, glucose tolerance, and body composition did not change. There was no effect of ARC MasR deletion on metabolic outcomes in control diet or HFD male mice. These findings suggest females have more MasR positive neurons in the ARC compared to males, which may be a sex-specific protective mechanism for glucose homeostasis. While further studies are needed to explore the role of ARC MasR in metabolic regulation, these findings support targeting Ang-(1-7) as an innovative strategy in obesity.
Abstract 61: Cd4+t Cells Cause Hypertension, Increased Glucose, And Mitochondrial Dysfunction In A Novel Rodent Model Of Gestational Diabetes Mellitus
Authors: Deer Evangeline , Williams Jan Michael , Amaral Lorena M , Fitzgerald Sarah , Herrock Owen , Turner Ty , Campbell Nathan , Lamarca Babbette
Published: August 2021

Hypertensive (HTN) disorders of pregnancy increase risks for gestational diabetes mellitus (GDM) in pregnant women. GDM is characterized by hyperglycemia and β-cell dysfunction associated with increased inflammatory cytokines, oxidative stress, and activated CD4+ T cells. Streptozotocin (STZ) is used in nonpregnant rats to induce β-cell destruction causing features of diabetes. However, STZ is not ideal for pregnancy and leads to unsuccessful pregnancy outcomes, therefore other ways to establish animal models of GDM must be pursued. Previously, we showed CD4+T cells from a rat model of preeclampsia causes HTN and mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) rats. Therefore, we hypothesize CD4+ T cells from a diabetic rat model could cause mt dysfunction/ROS and pancreatic β-islet cell destruction and lead to increased glucose and HTN during pregnancy. To examine our hypothesis, we adoptively transferred CD4+ T cells from STZ Dahl diabetic rats into pregnant Sprague Dawley (SD) rats and measured GDM features. Circulating CD4+ T cells were isolated from STZ induced diabetic Dahl virgin female rats and injected into pregnant SD rats on gestational day (GD) 12. On GD19, blood pressure (MAP) and tissues were collected and glucose levels were measured after 2h fasting in STZ CD4+ T cell recipients (GDM) and NP controls. Mt respiration and mtROS was measured in isolated mitochondria. On GD19, MAP increased to 105±0.5 mmHg (n=4, p<0.05) in GMD pregnant rats compared to control NP rats 91±2.1 mmHg (n=3). Blood glucose levels were elevated in GDM rats (139 ± 7 mg/dl, n=4, p<0.05) compared to NP controls (94 ± 1 mg/dl, n=3). Placental state 3 (26.4±5.9 vs 53.9±1.7 pmol/sec/mg, p<0.05) respiration rates, indicative of ATP production, was reduced in GDM rats (n=4) compared to NP controls (n=3). Placental mtROS was significantly increased in GDM rats (190 ± 27.1 % gated, n=3, p<0.05) compared to NP rats (100 ± 2.7 % gated, n=3). Collectively, the data indicate adoptive transfer of STZ CD4+ T cells causes increased circulating glucose, placental mt dysfunction and mtROS and HTN during pregnancy. These data demonstrate the importance of CD4+T cells in mechanisms causing the pathophysiology of GDM, and also introduces a potential novel animal model of GDM.
Abstract MP01: Deacetylation Of Mitochondrial Cyclophilin D K166 Inhibits Cytokine-induced Oxidative Stress And Attenuates Hypertension
Authors: Dikalov Sergey I , Mayorov Vladimir , Fehrenbach Daniel , Ao Mingfang , Panov Alexander , Harrison David G , Dikalova Anna
Published: August 2021

We have previously reported that depletion Cyclophilin D (CypD), a regulatory subunit of mitochondrial permeability transition pore, improves vascular function and attenuates hypertension, however, specific regulation of CypD in hypertension is not clear. Analysis of human arterioles from hypertensive patients did not reveal alterations in CypD levels but showed 3-fold increase in CypD acetylation. We hypothesized that CypD-K166 acetylation promotes vascular oxidative stress and hypertension, and measures to reduce CypD acetylation can improve vascular function and reduce hypertension. Essential hypertension and animal models of hypertension are linked to inactivation of mitochondrial deacetylase Sirt3 by highly reactive lipid oxidation products, isolevuglandins (isoLGs), and supplementation of mice with mitochondria targeted scavenger of isoLGs, mito2HOBA, improves CypD deacetylation. To test the specific role of CypD-K166 acetylation, we developed CypD-K166R deacetylation mimic mutant mice. Mitochondrial respiration, vascular function and systolic blood pressure in CypD-K166R mice was similar to wild-type C57Bl/6J mice. Meanwhile, angiotensin II-induced hypertension was substantially attenuated in CypD-K166R mice (144 mmHg) compared with wild-type mice (161 mmHg). Angiotensin II infusion in wild-type mice significantly increased mitochondrial superoxide, impaired endothelial dependent relaxation, and reduced the level of endothelial nitric oxide which was prevented in angiotensin II-infused CypD-K166R mice. Hypertension is linked to increased levels of inflammatory cytokines TNFα and IL-17A promoting vascular oxidative stress and end-organ damage. We have tested if CypD-K166R mice are protected from cytokine-induced oxidative stress. Indeed,
ex vivo
incubation of aorta with the mixture of angiotensin II, TNFα and IL-17A (24 hours) increased mitochondrial superoxide by 2-fold in wild-type aortas which was abrogated in CypD-K166R mice. These data support the pathophysiological role of CypD acetylation in inflammation, oxidative stress and hypertensive end-organ damage. We propose that targeting CypD acetylation may have therapeutic potential in treatment of vascular dysfunction and hypertension.
Abstract MP03: ROCK2 Specific Inhibition Attenuates Angiotensin II-induced Hypertension In Mice
Authors: Fehrenbach Daniel J , Madhur Meena S
Published: August 2021

Hypertension, or an elevated blood pressure, is the primary modifiable risk factor for cardiovascular disease, the number one cause of mortality worldwide. We previously demonstrated that Th17 activation and interleukin 17A (IL-17A)/IL-21 production is integral for the full development of a hypertensive phenotype as well as the renal and vascular damage associated with hypertension. Rho-associated coiled-coil containing protein Kinase 2 (ROCK2) serves as a molecular switch upregulating Th17 and inhibiting regulatory T cell (Treg) differentiation. We hypothesize that hypertension is characterized by excessive T cell ROCK2 activation leading to increased Th17/Treg ratios and ultimately end-organ damage. We first showed
in vitro
that KD025, an experimental orally bioavailable ROCK2 inhibitor inhibits Th17 cell proliferation and IL-17A/IL-21 production. To determine if hypertensive stimuli such as endothelial stretch increases T cell ROCK2 expression, we cultured human aortic endothelial cells exposed to 5% (normotensive) or 10% (hypertensive) stretch with circulating human T cells and HLA-DR+ antigen presenting cells. Hypertensive stretch increased T cell ROCK2 expression 2-fold. We then tested the effect of ROCK2 inhibition with KD025 (50mg/kg i.p. daily)
in vivo
on angiotensin II (Ang II)-induced hypertension. Treatment with KD025 significantly attenuated the hypertensive response within 1 week of Ang II treatment (systolic blood pressure: 139± 8 vs 108±7mmHg) and this persisted for the duration of the 4 week study reaching blood pressures 20 mmHg lower (135±13mmHg) than vehicle treated mice (158±4mmHg p<0.05 effect of treatment 2-way Repeated Measures ANOVA). Flow cytometric analysis of tissue infiltrating leukocytes revealed that KD025 treatment increased Treg/Th17 ratios in the kidney (0.61±0.03 vs 0.79±0.08, p<0.05 student’s t-test). Thus, T cell ROCK2 may be a novel therapeutic target for the treatment of hypertension.
Abstract MP05: The Mechanism Of The Pvat Pro-inflammatory Micro-environment Formation During The Development Of High Fat Diet-induced Hypertension
Authors: Jin Yining , Kana Omar , Kumar Ramya , Nault Rance , Garver Hannah , Bhattacharya Sudin , Watts Stephanie W , Fink Gregory D , Bernard Jamie , Rockwell Cheryl
Published: August 2021

There is considerable evidence for a causative role for T cells in hypertension, including studies with immunosuppressive drugs and T cell-deficient models. Our previous studies showed that soluble mediators from mesenteric perivascular adipose tissue (mPVAT) modulate T cell function. Specifically, conditioned media from mPVAT (mPVAT-CM) from Dahl S rats on a high fat diet (HFD) promoted expression of the pro-inflammatory cytokines, IFNg, IL-17a and GM-CSF, by activated T cells. Furthermore, the Dahl S rats on HFD will later develop hypertension.

Hypothesis:
mPVAT is stimulated to produce immunomodulatory mediators that promotes Th1/17 differentiation preceding the development of HFD-induced hypertension. We conducted bulk RNA-seq on activated splenocytes cultured in mPVAT-CM from Dahl S rats on either control or HFD for 10 weeks. In accordance with our previous studies, PVAT-CM from HFD-fed rats significantly upregulated many genes associated with IFNg/IL-17 induction, including Mpeg1, Lyz2 and Tnfsf4 (5.0±1.78, 3.70±0.53 and 1.78±0.42 fold over Control diet, respectively). In contrast, Th2/Treg-associated genes, such as Ctla2a (-0.27±0.02) and Ccr4 (-0.41±0.03) were downregulated. We also performed single cell (sc) RNA-seq on the PVAT stromal vascular fraction (SVF) and found that acute inflammatory genes were enriched in the HFD group. Together with the bulk RNA-seq on mPVAT, these data strongly suggest that the pro-inflammatory mPVAT micro-environment may promote Th1/Th17 differentiation. To identify mediators in PVAT-CM that may induce Th1/Th17 differentiation, we compared the bulk RNA-seq on splenocytes cultured in PVAT-CM with bulk RNA-seq conducted on the whole mPVAT itself. We found that a T cell co-stimulatory receptor DPP4 (CD26), which is closely associated with T cell activation was significantly increased in mPVAT from HFD-fed rats (33.4±2.3 HFD vs. 15.3±1.8 Control diet). We also observed an increase in DPP4 global expression from mPVAT SVF in HFD-fed rats, as determined by scRNA-seq.

Conclusion:
The data suggest that HFD promotes the IFNg and IL-17a pathways in PVAT, which precedes hypertension in Dahl S rats and correlates with an increase in expression of DPP-4, a gene that promotes T cell activation. (NIH P01 HL070687).
Abstract MP10: Furin-Mediated Modification Is Required For Epithelial Sodium Channel-Activating Activity Of Soluble (Pro)Renin Receptor In Cultured Collecting Duct Cells
Authors: Zheng Huaqing , zou Changjiang , Yang Tianxin
Published: August 2021

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M1 cells. M1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin Mut) or without (WT) mutagenesis of furin cleavage site; empty vector (EM) served a control. As compared with EM, overexpression of WT and Furin Mut vectors induced a more than 16-fold comparable increase in the release of sPRR. Amiloride-sensitive short circuit current as assessed by Ussing chamber technique was elevated by overexpression of WT PRR which was reduced by 37% by Furin Mut (ENaC activity: 1.00
+
0.06 μA/cm
2
in EM, 1.67
+
0.05 μA/cm
2
in WT, and 1.04
+
0.07 μA/cm
2
in Furin Mut,
p
< 0.05). In parallel, the expression of α-ENaC but not β or γ subunit as assessed by immunoblotting and qRT-PCR analysis was elevated by WT PRR and this increase was blunted by Furin Mut. In a separate experiment, M1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin); empty vector was used as a control. Overexpression of cDNA for the two types of sPRR induced a significant and comparable increase in the release of sPRR. By Ussing chamber technique, ENaC activity was 1.00
+
0.09 μA/cm
2
in EM, 1.03
+
0.10 μA/cm
2
in sPRR-S1P, and 1.39
+
0.14 μA/cm
2
in sPRR-furin,
p
< 0.05. Lastly, 293 cells were pretreated for 1 h with furin inhibitor α1-antitrypsin Portland followed by transfection with empty vector, WT PRR, or Furin Mut vectors. sPRR in the condition medium was enriched by using protein centrifugal filter devices and applied to M1 cells for 10 min followed by measurement of ENaC activity. Pretreatment with furin inhibition attenuated ENaC-acting activity induced by overexpression of WT PRR. Overall, the three independent approaches consistently demonstrated that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na
+
transport in the CD cells.
Abstract MP14: Endothelial Cullin3 Mutation Causes Decreased Nitric Oxide (NO) Bioavailability And Vascular Dysfunction Through Protein Phosphatase 2A
Authors: Wu Jing , Fang Shi , Hu Chunyan , Otanwa Adokole , Brozoski Daniel , Lu Ko-Ting , Kumar Gaurav , Donato Silva Sebastiao , Wackman Kelsey , Agbor Larry , Nair Anand R , Sigmund Curt D
Published: August 2021

Mutations in
CULLIN3
gene (in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT) driven by a combination of renal tubular and vascular mechanisms. To test the importance of endothelial Cul3
in vivo
, we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE
ERT2
mice. The resultant mice (E-Cul3Δ9) developed arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, n=5-7, p<0.05) and a trend towards elevated nighttime blood pressure (peak systolic BP, E-Cul3Δ9 136±3 vs control 128±3 mmHg, n=9-11) that were not associated with any alterations in locomotion, food/water intake or sleep/wake behaviors. No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg
2
, n=5-9, p<0.05) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (max ACh relaxation: 69% vs 84%, n=5-7, p<0.05) and cerebral resistance basilar artery (41% vs 77%, n=4-6, p<0.05). However, no dilatory impairment in mesenteric resistance artery and no difference in smooth muscle function were observed, suggesting that the effects of Cul3Δ9 are arterial bed specific. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS and NO production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Therefore, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid (4nM), but not by a PP1 inhibitor tautomycetin (4nM). Thus,
CUL3
mutations in the endothelium may contribute to human HT in part through decreased endothelial NO bioavailability, arterial stiffening and secondary sympathoexcitation.
Abstract MP17: Neuropilin-1 Is A Novel Regulator Of Vascular Tone And Blood Pressure
Authors: Balasubbramanian Dakshnapriya , Lambrinos George , Cristofaro Vivian , Bigger-Allen Alexander , Wang Beibei , Gao Yao , Chen Hong , Adam Rosalyn M , Sullivan Maryrose P , Bielenberg Diane R
Published: August 2021

Introduction:
Neuropilin-1 (NRP1) is a transmembrane receptor present in vascular smooth muscle cells (VSMC) that mediates the inhibition of Rho signaling by binding the Class 3 Semaphorin (SEMA) ligand SEMA3A.

Hypothesis:
We hypothesize that loss of NRP1 in VSMC mitigates SEMA3A-induced Rho inhibition, thereby increasing vascular tone and blood pressure
in vivo
.

Methods:
Male and female adult mice (8-12 weeks) with inducible, smooth muscle cell-specific deletion of NRP1 (SM22a-Cre
ERT2
X Nrp1
flox/flox
) were examined. Following recombination using 4-hydroxy tamoxifen (SM-
NRP1
KO), systolic blood pressure (SBP) was measured using a tail cuff and compared to age- and sex-matched mice that did not receive tamoxifen (control). Aortic vascular reactivity and expression of key proteins in the Rho signaling cascade were measured using
ex vivo
tension myography and western blotting, respectively.

Results:
SBP was significantly increased in SM-
NRP1
KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Contractile responses in aortas of SM-
NRP1
KO mice to phenylephrine (p=0.025), KCl (p=0.012), and the thromboxane agonist U44619 (p=0.019) were significantly enhanced compared to controls. Expression of total myosin light chain and LIMK-2 proteins were increased in SM-
NRP1
KO compared to control aortas.
In vitro
, treatment of murine primary VSMC expressing NRP1 with SEMA3A decreased angiotensin II-induced Rho-GTP activation. Additionally, control and SM-
NRP1
KO mice (starting at 2 weeks post-recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM-
NRP1
KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, mRNA expression of atrial natriuretic peptide was increased in hearts of angiotensin II-infused SM-
NRP1
KO mice.

Conclusion:
Our data suggest that VSMC NRP1 regulates basal tone and blood pressure, and that loss of NRP1 causes hypertension and exacerbates cardiac dysfunction.
Abstract MP19: Endogenous 5-hydroxytryptamine May Regulate Skeletal Muscle Vascular Resistance In Normal Healthy Rats
Authors: Fink Gregory D
Published: August 2021

Infusing low doses of 5-hydroxytryptamine (5-HT) into normal rats causes chronic (weeks to months) hypotension and a fall in total peripheral resistance. These effects are mediated by activation of 5-HT
7
receptors. Therefore, we generated 5-HT
7
receptor knockout rats (5-HT
7
KO) to explore possible cardiovascular effects of 5-HT
7
receptors under normal and pathophysiological conditions. We previously reported that healthy 5-HT
7
KO rats have normal blood pressure and total peripheral resistance at rest. This suggested that 5-HT
7
receptors plays no role in cardiovascular regulation under normal conditions. But total peripheral resistance is determined by multiple vascular beds that differ in their sensitivity to 5-HT. Others have indicated that 5-HT
7
receptors in the skeletal muscle vasculature are particularly sensitive to the effects of 5-HT. Therefore, we hypothesized that 5-HT
7
KO rats would show both reduced responsiveness to exogenous 5-HT and increased resting skeletal muscle vascular resistance. Experiments were performed in isoflurane-anesthetized, male Sprague-Dawley (SD) (n=6), 5-HT
7
wild-type (5-HT
7
WT) (n=5) and 5-HT
7
KO (n=6) rats at 7-8 months of age. Arterial pressure was measured with an aortic catheter. Blood flow to the hindquarters (mostly skeletal muscle) was measured with transit-time, ultrasound flowmetry. After 10 minutes of baseline hemodynamic measurements were obtained, 5-HT was infused iv at a rate of 25 μg/kg/min for 15 minutes, followed by a 15-minute recovery period. As expected, 5-HT
7
KO rats did not show a significant fall in hindquarter vascular resistance (HQVR) during 5-HT infusion, while SD and 5-HT
7
WT did. More importantly, HQVR at baseline was significantly (p < 0.05) higher in 5-HT
7
KO rats (16.0 ± 2.0 mmHg/ml/min) than in 5-HT
7
WT rats (10.9 ± 0.06 mmHg/ml/min) or SD rats (7.0 ± 0.03 mmHg/ml/min). These results support our hypothesis that in healthy (albeit anesthetized) rats, 5-HT
7
receptors reduce skeletal muscle vascular resistance.
Abstract MP20: Optogenetic Control Of Pip2 To Assess Mechanisms Regulating The Epithelial Sodium Channel
Authors: Archer Crystal R , Kaur Amanpreet , Mohamed Tarek , Stockand James D
Published: August 2021

The epithelial Na
+
channel (ENaC) plays a key role in Na
+
transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na
+
reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP
2
) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP
2
binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP
2
binding sites, by controlled depletion of PIP
2
. We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP
2
levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na
+
indicator, was used to monitor ENaC activity by tracking the relative intracellular Na
+
levels. Upon optogenetic-controlled depletion of PIP
2
, Na
+
levels decreased in cells expressing wt ENaC. Mutations to the PIP
2
sites of ENaC were expected to have no change in Na
+
levels upon PIP
2
depletion due to the disruption of PIP
2
binding. As a control, mutations to non-PIP
2
binding sites were included, and were expected to have decreased Na
+
levels similar to wt ENaC. Interestingly, mutation of each independent PIP
2
site resulted in only a small decrease of intracellular Na
+
, compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP
2
binding site, resulted in a significant increase of Na
+
upon PIP
2
depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na
+
fluctuations. These data suggest that the residues surrounding the PIP
2
binding sites play a significant role in the affinity of PIP
2
for ENaC. The role of these other domains in PIP
2
binding is still under investigation.
Abstract MP25: Defining The Role Of Renin Lineage Cells During Regeneration After Release Of Partial Ureteral Obstruction In Neonatal Mice.
Authors: Kusma Harinathan Vidya Nagalakshmi , Li Minghong , Gomez Ariel R , Sequeira-Lopez Maria Luisa S
Published: August 2021

Our previous study on a partial unilateral ureteral obstruction (pUUO) model in neonatal mice showed that the release of obstruction halts the progression of kidney damage and leads to a remarkable repair of the kidney with improvement in renal blood flow. In the current study, we aim to understand the role of mural cells of the renin lineage during kidney damage and repair in the neonatal pUUO model. Our results show a marked increase in renin-positive areas in kidneys obstructed for 3W (Sham-3W: 0.70±0.10%, n=3; Obstructed-3W: 1.82±0.43%, n=3). However, relief of obstruction at 1W restored the renin-positive areas to sham levels (Post-release-2W: 0.70±0.09%; n=3). Lineage tracing using

Ren1
d
Cre;mTmG

mice revealed a significant increase in GFP+ cells in the obstructed kidneys, with a decrease post-release. To understand further the dynamic changes in cells of renin lineage due to obstruction, we ablated the renin cells using DTA (Diphtheria toxin subunit A). We crossed the

DTA
fl/fl

mice with

Ren1
d
-DTA
het
;Ren1
d
Cre;mTmG

mice and performed pUUO in the resultant pups with DTA in the renin cells (DTA+). DTA+ animals showed thinning of the renal vasculature and a 90% reduction in renin-positive area compared to controls [Control: 0.70±0.10% (n=3); DTA+: 0.06±0.03% (n=3)]. In addition, there was no significant increase in the renin-positive area post-obstruction [Sham-3W: 0.06±0.04% (n=3); Obstructed-3W: 0.12±0.05% (n=4); Post-release-2W: 0.08±0.03% (n=4)]. These results indicate that ablation of renin cells abolished the obstruction-mediated surge in the renin expression. However, measurement of interstitial collagen positive area indicated that despite the absence of renin cells, the fibrotic damage due to obstruction recovered remarkably post-release [Collagen positive area: Sham-3W: 3.38±0.67% (n=3) Obstructed-3W: 62.98±31.50% (n=3); Post-release-2W: 10.93±5.46% (n=4)]. Similarly, vascular damage induced by persistent obstruction and recovery following the relief of obstruction was similar between the DTA+ and non-DTA animals. Our results imply that though the renin and renin lineage cells increase in obstructed kidneys, ablation of renin cells does not affect the regeneration capacity of kidneys following the relief of obstruction.
Abstract MP30: Circulating Sars-cov-2 Spike Protein 1 Causes Microarteriolar Oxidative Stress, Endothelial Dysfunction And Enhanced Thromboxane And Endothelin Contractility That Are Prevented By Spironolactone.
Authors: Wang Dan , Wilcox Christopher S
Published: August 2021

Introduction and hypothesis:
Following bodily entry, the SARS-CoV-2 virus undergoes pulmonary replication with release of circulating viral spike protein 1 (SP1) into the bloodstream. Uptake of SP1 by endothelial cells might provoke vascular dysfunction and thrombosis. We hypothesized that spironolactone could prevent microvascular complications from circulating SP1 in COVID-19.

Methods:
male C57Bl/6 mice received spironolactone (100 mg · kg
-1
· d
-1
PO x 3d) or vehicle and intravenous injections of recombinant full-length human SP1 (10 μg per mouse) or vehicle. They were euthanized after 3 days. Mesenteric resistant arterioles (n=4 per group) were dissected and mounted on isometric myographs. Acetylcholine-induced EDRF responses and L-NAME-inhibitable NO generation (DAF-FM fluorescence) were studied in pre-constricted vessels and contraction to endothelin 1 (ET1) or thromboxane (U-46, 619) and ET1-induced ROS (PEG-SOD inhibitable ethidium: dihydroethidium fluorescence) were studied by fluorescence microscopy in other vessels.

Results:
SP1 reduced acetylcholine-induced EDRF (17 ± 3 vs 27 ± 5 % mean ± sem; P < 0.05) and NO generation (0.21 ± 0.03 vs 0.36 ± 0.04, F
1
/F
0
; P < 0.05) while increasing contraction to ET1 (10
-7
mol·l
-1
: 124 ± 13 vs 89 ± 4 %; P < 0.05) and U-46, 619 (10
-6
mol·l
-1
:114± 5 vs 87± 6 %; P < 0.05) and ET1-induced ROS generation(0.30± 0.08 vs 0.09± 0.03; P < 0.05). Spironolactone did not modify any of these responses in vessels from normal mice but prevented all the effects of SP1.

Conclusion:
these preliminary studies provide a novel model to study COVID-19 vasculopathy. They indicate that spironolactone can provide protection from microvascular oxidative stress, endothelial dysfunction and enhanced contractility and might thereby moderate COVID-19 complications.
Abstract MP32: Renal Tubular Epithelial Cell-derived IL-1β Triggers The Inflammatory Response That Induces Salt Sensitivity In Diabetes
Authors: Veiras Luciana C , Bernstein Ellen , Khan Zakir , Okwan Derick , Cao Duoyao , Ahmed Faizan , Gibb David , Bernstein Kenneth E , Giani Jorge F
Published: August 2021

Renal inflammation, and specifically interleukin (IL)-6, induces salt-sensitivity in diabetic mice by upregulating the epithelial sodium channel (ENaC). However, the origin of this inflammatory response is still unknown. We hypothesize that, during diabetes, tubular epithelial cells initiate renal inflammation by releasing IL-1β that further activates renal macrophages to produce IL-6 and impair kidney function. A primary culture of renal tubular epithelial cells from wild-type (WT) mice was exposed to either low (LG, 5mM) or high (HG, 15mM) glucose media. HG-treated cells released higher levels of IL-1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and have higher expression of NLRP3 (4 ± 1 fold increase, P<0.001) and Caspase-1 (2.1 ± 0.2 fold increase, P<0.001) compared to LG-treated cells. No effect was observed using mannitol as an osmotic control. Tubular cells were also co-cultured with bone marrow-derived macrophages. Flow cytometry analysis revealed that HG-treated tubular cells induced an M1-type polarization of macrophages. This effect was blunted when macrophages were obtained from IL-1 receptor KO mice (IL1RKO) confirming a role of IL-1β. Here, the percentage of M1 macrophages (CD45
+
F4/80
+
CD80
+
) of total macrophages (CD45
+
F4/80
+
) decreased from 31 ± 4% to 10 ± 3% (P<0.01). To evaluate whether IL-1β-mediated macrophage activation plays a role in vivo, 2-mo-old diabetic db/db mice (n=6) were transplanted with bone marrow from IL1RKO mice. In these mice, named db/db-IL1RKO, immune cells cannot respond to IL-1β. Db/db receiving wild-type (WT) bone marrow (db/db-WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to a high salt diet (NaCl 4% w/w) for 4 weeks. Db/db-IL1RKO did not develop salt-sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal IL-6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05) and lower ENaC activity (0.4-fold decrease by amiloride test, P<0.05) compared to db/db-WT. In conclusion, our data show that tubular epithelial cells are a major source of IL-1β under high glucose conditions. This cytokine promotes M1 macrophage polarization resulting in higher renal IL-6 levels which increases ENaC activity leading to salt sensitivity.
Abstract MP33: Heterozygous Knockout Of Nfatc1 In Mice Is Protective Against Salt-sensitive Hypertension
Authors: Wang Yves T , Nguyen Nhu , Le Luan H , Cunningham Jason J , Franceschini Nora , Le Thu H
Published: August 2021

Nuclear Factor of Activated T Cells 1 (NFATC1) plays a role in the immune response and has been shown to be required for cardiac development. A recent trans-ethnic meta-analysis of genome-wide association studies (GWAS) found the
NFATC1
gene in humans was associated with estimated glomerular filtration rate (eGFR). Further studies in salt-sensitive mice showed a decrease in renal
Nfact1
mRNA expression during high-salt exposure, suggesting a link between NFATC1 and salt-sensitive hypertension. Here, we backcrossed a knockout mouse model of
Nfatc1
to a salt-sensitive 129S6 background for 6 generations. Heterozygous (Het) and wild-type (WT) male mice at 8-12 weeks of age were trained for blood pressure measurement by tail cuff manometry over 2 weeks, and measurements were taken over the following week while on standard chow (NSD), and again for 2+1 weeks while on a matched high-salt diet (HSD, 6% total NaCl). On NSD, there was no difference in SBP between WT and Het mice. However, while WT mice displayed an expected elevation in SBP on HSD, Het mice showed no change in SBP, resulting in a statistically significant difference between genotypes (140.7±11.1 mmHg vs 125.7±9.7 mmHg, p = 0.02, n = 8/6). This preliminary data suggests that down-regulation of
Nfatc1
under HSD may be a compensatory response to limit the rise in blood pressure during high salt consumption, and that NFATC1 may be a therapeutic target for salt-sensitive hypertension. Further studies are underway to delineate the molecular mechanism by which
Nfatc1
mediates salt-sensitivity.
Abstract MP35: Pharmacological Inhibition And Knockout Of K
ir
7.1 Does Not Affect The Development Of Salt-Sensitive Hypertension In The Dahl SS Rat
Authors: Zietara Adrian P , Levchenko Vladislav , Isaeva Olena , Palygin Oleg , Staruschenko Alexander
Published: August 2021

Ion channels play a vital role in the kidney’s ability to maintain electrolyte homeostasis, and mutations of certain inwardly rectifying K
+
channels (K
ir
) have been associated with various tubulopathies that can cause salt-wasting and as a consequence, low blood pressure. K
ir
7.1 is a member of the K
ir
family, that helps maintain resting membrane potential; however, its role in the kidney is not well understood. Immunohistochemistry in the kidney confirmed that K
ir
7.1 is expressed in both aquaporin 2 and sodium chloride co-transporter positive cells, suggesting it may affect distal tubule K
+
handling. We hypothesized that K
ir
7.1 is involved in renal K
+
homeostasis and may influence the development of salt-sensitive (SS) hypertension. To examine the role of K
ir
7.1 during the development of SS hypertension, we used both a genetic knockout of the gene encoding K
ir
7.1,
Kcnj13
, on the Dahl SS background and pharmacological inhibition; SS
Kcnj13-/-
was lethal and we used SS
Kcnj13+/-
. Mean arterial pressure (MAP) in response to a high salt diet was determined by telemetry or arterial catheter. In the genetic model, there was no difference in MAP development between wild type and SS
Kcnj13+/-
rats (153±6 vs. 159±7, mmHg; n=11 for each group) after 3 weeks on a 4% NaCl diet. Additionally, there were no differences in K
+
or Na
+
urinary excretion in WT vs. SS
Kcnj13+/-
. For the pharmacological studies, ML418, a novel and highly specific K
ir
7.1 inhibitor, was administered daily by i.v. infusion during 2 weeks of 8% NaCl diet. While no significant differences in MAP or K
+
excretion were observed between vehicle or drug treated rats, administration of ML418 had significantly increased urinary Na
+
excretion (197±54 vs. 333±21, Na/Cre, vehicle vs. ML418; n≥3 for each group, p=0.02). Moreover, equivalent short circuit current measurements of polarized mCCD cells demonstrated that K
ir
7.1 antagonism by ML418 reduced vectoral Na
+
transport. Overall, these results indicate that K
ir
7.1 can impact renal Na
+
homeostasis, but loss of basolateral K
ir
7.1 conductance is insufficient to alter development of hypertension, likely due to compensation by the primary basolateral K
ir
’s in the distal nephron such as K
ir
4.1 and K
ir
5.1.
Abstract MP45: Deletion Of The Transient Receptor Vanilloid-1 (TRPV1) Channel In Rats Attenuates 2 Kidney 1 Clip Hypertension
Authors: Stocker Sean D , DeLalio Leon J
Published: August 2021

Renal denervation lowers arterial blood pressure (ABP) in both clinical populations and multiple experimental models of hypertension. This therapeutic effect is partly attributed to the removal of overactive renal sensory nerves that increase sympathetic efferent activity and ABP. Renal sensory nerves highly express TRPV1 channels, and administration of the TRPV1 agonist capsaicin increases renal sensory nerve activity. However, the extent by which TRPV1 channels directly contribute to renal nerve dependent models of hypertension has not been tested. To test this hypothesis, we generated a novel TRPV1
-/-
rat using CRISPR/Cas9 and deletion of exon 3. Male and female TRPV1
-/-
and wild-type littermates (8-12 weeks) were instrumented with telemetry. At 2 weeks later, renovascular hypertension via renal stenosis was produced by placement of a PTFE cuff (0.16 x 0.22 inches, 1mm long) around the right renal artery. Male TRPV1
-/-
and wild-type rats had no differences in baseline mean ABP (99±2 vs 98±3 mmHg, respectively; n=7-9) or heart rate (390±7 vs 400±8 bpm, respectively). Renal stenosis significantly increased mean ABP in both groups; however, mean ABP was significantly lower at Day 28 in male TRPV1
-/-
versus wild-type rats (125±8 vs 155±2 mmHg, respectively: P<0.01). Ganglionic blockade with chlorisondamine (2.5mg/kg, sc) at Day 28 produced a smaller fall in mean ABP of male TRPV1
-/-
versus wild-type rats (-53±4 vs -86±3 mmHg, respectively; P<0.001). On the other hand, female TRPV1
-/-
and wild-type rats had no differences in baseline mean ABP (102±2 vs 104±1 mmHg, respectively; n=6-9) or heart rate (419±8 vs 410±7 bpm, respectively). Renal stenosis significantly increased mean ABP in both groups; however, there were no differences at Day 28 between female TRPV1
-/-
versus wild-type rats (117±8 vs 122±6 mmHg, respectively). Moreover, the increase in mean ABP was smaller in females versus males. The ganglionic blocker chlorisondamine produced similar depressor responses in female TRPV1
-/-
versus wild-type rats (-64±7 vs -65±7 mmHg, respectively). These findings illustrate a sex difference in renovascular hypertension in rats, but importantly indicate that TRPV1 channels contribute to the established phase of renovascular hypertension in male rats.