bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P130: Association Between PFOA, PFOS and Obesity Among Children in a Nationally Representative Sample
Authors: Geiger Sarah D , Yao Ping , Rogers Elizabeth , Vaughn Michael , Qian Zhengmin
Published: June 2021

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are two types of perfluoroalkyl substances (PFASs) commonly used in the manufacturing process of many consumer products. Both have been detected in the blood of the majority of Americans. PFASs have been shown to be associated with intermediate cardiovascular disease (CVD) outcomes such as hypertension, hyperuricemia and dyslipidemia, but their relationship with obesity, a risk factor for intermediate and advanced CVD, remains largely unexplored. In this context, we examined the associations between PFOA and PFOS levels, and Body Mass Index (BMI) and waist circumference (WC) in a representative sample (N = 5,180) of US children. Our cross-sectional sample included participants aged 12-19 years from CDC’s National Health and Nutrition Examination Survey 1999-2000, 2003-2012. PFOA and PFOS were measured in ng/mL and modeled as quartiles of exposure, where quartile 1 is the referent group across models. Overweight/obesity was defined as age-, sex-specific BMI ≥85
th
percentile; abdominal obesity was defined as age-, sex-specific waist circumference ≥90
th
percentile. A multivariable model adjusting for age (years), sex (male, female), race/ethnicity (non-Hispanic white, non-Hispanic black, Mexican American, other), and annual household income category (<$25,000, $25,500-$54,999, $55,000 and over), revealed an inverse association between PFOS and overweight/obesity. Odds ratios (ORs) for overweight/obesity were 0.81 (95% Confidence Interval [CI] 0.14-0.58) for exposure quartile 2, 0.28 (0.11-0.58) for quartile 3, and 0.26 (0.15-1.05) for quartile 4 (p-trend=0.001). Results were similar for abdominal obesity where, for example, children in quartile 2 of PFOS exposure experienced a multivariable-adjusted OR of 0.42 (0.25-0.72; p-trend=0.023). PFOA was not found to be significantly associated with either outcome. Results are paradoxical in that PFASs may be protective against a risk factor for conditions with which PFASs are positively associated. Because we could not identify any temporal relationships between exposure and outcomes in this cross-sectional study, more studies with improved study design (such as a cohort study) are warranted to confirm the association.
Abstract P133: Melanocortin 4 Receptor is Required for the Effect of Testosterone Supplement on Metabolic Parameters and Blood Pressure
Authors: Maranon Rodrigo O , Dalmasso Carolina , Spradley Frank T , Granger Joey P , Juncos Luis A , Reckelhoff Jane F
Published: June 2021

Melanocortin 4 receptor (MC4R) activation causes appetite suppression and sympathetic nervous system activation. Blockade of MC4R increases food intake and in some cases, decreases BP. MC4R has been implicated as playing a role in obesity and hypertension. Previously we observed that testosterone supplementation in male Zucker rats improved insulin resistance and the characteristics of metabolic syndrome, but increased BP. Similarly, testosterone increases BP in male SHR, and blockade of the MC4R reduces BP in both young and old male SHR. In the present study we tested the hypothesis that testosterone supplementation (T) requires activated MC4R to attenuate metabolic parameters, and to increase BP. Male obese MC4R
-/-
and Wistar Hannover rats (WT) were treated with T (MC4R-KO+T vs WT+T: 22 mg/10 mm silastic pellet) or placebo (empty pellets; MC4R-KO+P, and WT+P), beginning at 10 wks of age for 6 weeks. In WT animals, T reduced body weight by 13% compared to placebo (367 gr vs 432gr; p<0.05), but had no effect on food intake (WT+P: 19±7 gr/d vs WT+T: 16±5 gr/d, n=3, p:NS). In MC4R-KO rats, body weight and food intake were similar in both placebo and T rats (BW: 555±20 gr vs 526±18 gr, and FI: 26±1 gr vs 28±2; n=3, p:NS, respectively). T reduced fat mass (measured by ECHO-MRI
®
) in WT+T by 38% compared to placebo (50±2.9 gr vs 31±3.2 gr; p<0.05); however, there was no effect on fat mass in MC4R-KO + T (149±8 gr vs 147±11 gr; n=3, p:NS). Lean mass (by ECHO-MRI
®
) was not affected by T in either MC4R-KO+T or WT+T (p:NS). Finally, unlike our previous studies in obese Zucker and SHR males, T failed to affect mean arterial pressure or heart rate (by telemetry) in MC4R-KO+T rats compared to control MC4R-KO+P (MAP: 122±2 mmHg vs 122±1 mmHg; n=3, p:NS; HR:309±5 bpm vs 320±17 bpm; n=3, p:NS). These data suggest that the effect of T on metabolic parameters and BP may be mediated at least in part by MC4R since in the absence of active MC4R, T is not able to affect either body composition or BP. Also, these data gave a potential new insight into the mechanism by which T contributes to BP control and adipose tissue regulation. More studies are necessary to clarify the role of MC4R in mediating the effects of testosterone. Supported by NIH-R01HL66072, R01HL69194, PO1HL51971 (JFR), 14POST18640015 (ROM).
Abstract P134: Hypertension in Postmenopausal Women: Role of Renin-angiotensin System and Eicosanoids
Authors: Patil Chetan N , Dalmasso Carolina , Maranon Rodrigo O , Yanes-Cardozo Licy L , Reckelhoff Jane F
Published: June 2021

Hyperandrogenemic postmenopausal women have elevated blood pressure (BP) and the mechanisms responsible for it have not been elucidated. We tested the hypothesis that activated renin-angiotensin system (RAS) in combination with cytochrome P450 (CYP 450) arachidonic acid metabolites in renal microvasculature contribute to the elevated BP in post-estrous cycling hyperandrogenemic female (PMHAF) rats. Female SD rats were implanted with DHT (7.5mg/90d) or placebo pellets (n=12/grp) beginning at 6 wks of age; pellets were changed every 85 d. At 14 months of age, baseline mean arterial pressure (MAP, telemetry) was measured in control and PMHAF rats. Each group was then divided into two groups, one group received non-specific CYP 450 inhibitor, 1-aminobenzotriazole (1-ABT; 100mg/kg/day, i.p.), and the other received Losartan (40mg/kg/day, P.O.) for 10 days. For the following 10 days all rats received both drugs. Baseline BP was significantly higher in PMHAF than controls (112±3 vs 126±3 mmHg, p=0.008). 1-ABT alone had no effect on BP in either control or PMHAF rats. Losartan alone reduced MAP in both groups (112±3 vs 97±3 mmHg, p<0.05 in control and 126±3 vs 100±3 mmHg, p<0.05 in PMHAF). In control rats first given 1-ABT, losartan had no effect on BP (112±3 vs 113±2 mmHg, p=NS). In control rats first given losartan, BP continued to decrease over the next 10 days (97±3 vs 87±2 mmHg, p=0.046). Just as in control rats, in PMHAF rats given 1-ABT first, losartan had no further effect on BP (130±2 vs 126±4 mmHg, P=NS). Unlike in control rats, PMHAF rats given 1-ABT after losartan, the BP increased to back to baseline levels (126±3 vs 116±5 mmHg, p=NS). This data suggests that BP in aging female control rats is mediated by the RAS and likely greater vasoconstrictor to vasodilator ratios of eicosanoids. BP control in PMHAF rats is more complex with the RAS playing a major role. Data suggests that vasodilators (EETs) in the renal microvasculature and 20-HETE in the tubules contribute to BP control which would explain the increase in BP back to baseline with 1-ABT + losartan. Future studies will be necessary to determine exact mechanisms responsible for the hypertension in PMHAF rats, and may explain why in hyperandrogenemic women BP is difficult to control to normotensive levels. PO1HL51971.
Abstract P138: Targeted Disruption of Regulated Endocrine Specific Protein (
Resp18
) in Dahl SS Rats Increases Blood Pressure and Renal Injury
Authors: Kumarasamy Sivarajan , Waghulde Harshal , Haller Steven , Mell Blair , Cheng Xi , Joe Bina
Published: June 2021

Essential hypertension is a complex polygenic trait. To understand the genetics of Blood pressure (BP) control, loci are mapped using populations derived by crossing several rat strains with the genetically hypertensive rat model, the Dahl SS rat (SS) and validated as BP quantitative trait loci (QTL). Genes located within the mapped BP QTLs are candidates as inherited loci controlling BP, but require further proof. One such mapped locus is on rat chromosome 9, wherein the proof for one of the candidate genes Regulated Endocrine Specific Protein-18 (
Resp18
), as a BP QTL, is currently inadequate. To ascertain the status of
Resp18
as a BP QTL, a custom targeted gene disruption model of
Resp18
was developed on the SS background. As a result of this ZFN mediated disruption, a 7 bp deletion occurred within exon 3 of the
Resp18
locus, resulting in a truncated protein with 111aa compared to the full length protein consisting of 175aa. Under a high salt dietary regimen, both systolic and diastolic BP of

Resp18
mutant

rats were significantly increased compared to SS rats (151±3 vs.170±6mmHg; 116±2vs.129±4mmHg n=10-12,
p
<0.05).

Resp18
mutant

rats demonstrated higher proteinuria compared to SS rats (221±14vs.268±14mg of protein/kg body weight/24hr; n=14-25,
p
<0.05). In vascular reactivity experiment,

Resp18
mutant

rat mesenteric arteries demonstrated significantly reduced relaxation as compared to SS rats (n=4,
p
<0.05). An associated decrease in sodium excretion and an increase in glucose excretion were also observed in urine samples of

Resp18
mutant

rats compared to SS rats (51±7.3vs.27±2.7meq/L/24hr; 10±0.3 vs. 14±1.4mg/dl/24hr,n=5-8,
p
<0.05).Renal histology examination revealed that

Resp18
mutant

rat kidneys showed increased fibrosis compared to SS rats. The median survival of

Resp18
mutant

rats was 259 days, which was significantly lower than the median survival of 309 days for the SS (n=8-16,
p
<0.05). In conclusion, the data suggest that
Resp18
is a gene associated with the development of hypertension, renal disease and increased mortality in the SS rats.
Resp18
is a molecule involved in the secretory pathway and thereby, future studies will be conducted to examine the mechanistic links between
Resp18
, its function in the secretory pathway and BP regulation.
Abstract P139: Stability of DNA Methylation Profiles in Human DNA Samples in Long Term Storage
Authors: Li Yingchuan , Kidambi Srividya , Liu Pengyuan , Leittl Michelle L , Liu Yong , Mattson David L , Cowley Allen W , Kotchen Theodore A , Liang Mingyu
Published: June 2021

Introductions:
Historic DNA samples represent a potentially highly valuable resource for epigenetic analysis. Reduced representation bisulfite sequencing (RRBS) is a cost-effective, near genome-wide method for quantifying DNA methylation levels at single base resolution. It is unknown whether historic DNA samples stored for a long time (15-20 years) under various conditions could maintain stable methylation profiles as determined by RRBS.

Methods:
We used 5 groups of DNA samples (n=4 in each group) to compare and evaluate the stability of DNA methylation profiles under standard storage conditions of 4°C since 1996. Group 1 had been extracted from EDTA-anticoagulated blood and stored at 4°C since 1996; Group 2 had been diluted from group 1 to 10ng/μl and stored at -20°C since 2009; Group 3 were extracted from citrate-anticoagulated blood stored at -80°C since 1996; Group 4 were extracted from fresh EDTA-anticoagulated blood; Group 5 were extracted from fresh citrate-anticoagulated blood. The samples in groups 1, 2 and 3 were from the same subjects. All DNA samples were processed for RRBS libraries and then sequenced.

Results:
The global methylation level of group 1 was lower than group 2 (36.88±4.12% vs 43.66±1.29%, p=0.04), but was not statistically different from group 3 (44.31±1.46%, p=0.07). The methylation level of groups 4 and 5 (40.34±3.73% and 38.64±1.94%) was not significantly different. In addition, group 4 and 5 were not significantly different from group 1. The correlation of methylation levels for all CpG sites between individual samples in group 1 (0.987±0.001) was higher than that in group 2 and group 3 (0.984±0.002, p=0.01, and 0.969±0.015, p=0.01) and not significantly different from group 4 or 5. For CpG sites within CpG islands (CGI), the correlation between individual samples was not significantly different between any of the 5 groups. Moreover, the samples from the same subject show high correlation levels between groups 1, 2, and 3 for all CpG sites (0.987±0.006) and CpG sites within CGI (0.983±0.009).

Conclusion:
DNA samples stored at 4°C for prolonged periods of time are amenable to RRBS analysis. In addition, there were no significant differences in the DNA methylation profiles between citrate- and EDTA-anticoagulated blood.
Abstract P142: Inflammation Upregulates Adipocyte Expression of (Pro)renin Receptor
Authors: Culver Silas A , Quadri Syed S , Siragy Helmy M
Published: June 2021

Obesity represents a state of chronic inflammation in adipose tissue and this inflammation contributes to obesity related comorbidities. Our laboratory has previously demonstrated that the (Pro)renin receptor (PRR) promotes the production of proinflammatory factors via the ERK-MAP kinase pathway. We have also shown that in the setting of renal inflammation, NF-κB increases renal expression of PRR by binding to the PRR promoter. In this study we hypothesized that inflammation increases expression of PRR in adipose tissue. We monitored changes in expression of PRR mRNA by RT-PCR and protein by western blot in 3T3-L1 adipocytes treated with 100ng/ml LPS or normal culture medium (control) for 24 hours. To confirm that LPS treatment effectively induced an inflammatory response in 3T3-L1 adipocytes, we monitored production of the inflammatory cytokine, IL-6, in culture media by luminex assay. Compared to control, LPS significantly increased IL-6 production (48.8 pg/mL in controls vs. 1877 pg/mL in LPS treated cells, p<0.05). Similarly, compared to control, LPS treatment increased adipocyte expression of PRR mRNA and protein by 120% and 62%, (p<0.05) respectively (figure 1). We conclude that inflammation increases adipocyte expression of PRR, suggesting that PRR may play a role in obesity related adipose tissue inflammation and obesity associated pathology.
Abstract P147: Production of Anti-inflammatory Peptide
N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP)
in Kidney
Authors: Romero Cesar A , Kumar Nitin , Rhaleb Nour-Eddine , Carretero Oscar A
Published: June 2021

Ac-SDKP is a natural peptide with anti-fibrotic and anti-inflammatory properties in vascular, myocardial and kidney tissues. It is released from thymosin B4 (TB4) by two step enzymatic reactions by meprin and the prolyl oligopeptidase enzymes (POP) and degraded by angiotensin converting enzyme (ACE). Treatment with ACE inhibitors (ACEi) increases Ac-SDKP plasma concentration. Kidney has been proposed as clearance organ for Ac-SDKP. Thus after Ac-SDKP filtration, 90% is degraded and only 10% is excreting in the urine. However, in rodents the decrease of glomerular filtration rate did not affect the amount of Ac-SDKP in urine and prevention of Ac-SDKP filtration at the glomerular level by using neutralizing antibodies also did no change the urinary Ac-SDKP content; This suggest that mechanisms other than only filtration can be present.
We hypothesized that Ac-SDKP is produced in the kidney.
We evaluated the presence of TB4 and POP mRNA by analyzing the trascriptome in each segment of the nephron using the public access NHI database ESBL. We confirmed kidney expression of POP enzyme by immunohistochemistry (IHC). Finally the stop flow pressure technique was used to evaluate the Ac-SDKP formation in different segments of the nephron, in normal condition, under POP inhibition (POPi) and ACEi. All experiments were performed in Sprague-Dawley rats.TB4 mRNA was present in all the nephron segments, mainly in the distal parts (1941±949.8 RPKM) in comparison with proximal tubule (101.4±73.7 RPKM) (p<0.05). POP enzyme mRNA was present in proximal tubule, loop of Henle (inner medulla) and distal nephron (distal, connecting and collecting tubules). POP enzyme expression by IHC was observed in the distal convoluted tubule in cortex and mainly in the medullary region. The Stop flow technique showed the high Ac-SDKP/Inulin ratio in the distal nephron: 10.5±0.8 vs. 4.2±0.1 in the proximal segments (p<0.01). POPi infusion into the kidney decreased Ac-SDKP/Inulin in comparison to the vehicle group in distal (10.5±0.8 vs 5.6±0.8, p<0.01) and proximal nephron segments (4.22±0.1 vs. 2.1±0.2, p<0.01). ACEi increased the Ac-SDKP content in all nephron segments, mainly in the distal part. We conclude that Ac-SDKP is synthetized in kidney, predominantly in the distal nephron.
Abstract P148: Connecting Tubule-glomerular Feedback (ctgf) in Renal Hemodynamics and Blood Pressure (bp) After Unilateral Nephrectomy (unx)
Authors: Romero Cesar A , Monu Sumit , Knight Robert , Carretero Oscar A
Published: June 2021

Afferent arteriole resistance is regulated in part by myogenic response, tubuloglomerular feedback (TGF) and connecting tubule-glomerular feedback (CTGF). CTGF dilates afferent arteriole in response to high sodium in connecting tubule (CNT) counteracting and shifting to the right the TGF response (resetting); CTGF increases renal blood flow and glomerular pressure, both favoring glomerular filtration and sodium excretion. CTGF is initiated by epithelial sodium channel (ENaC) in CNT and inhibited by Benzamil. Unilateral nephrectomy (UNx) is accompanied by TGF resetting, increase in renal blood flow (RBF) and single nephron GFR in the remnant kidney, without any changes in systemic BP. We evaluated the effects of CTGF in TGF resetting, RBF and BP after UNx. UNx was performed on Sprague-Dawley rats and 24h later TGF was evaluated
in vivo
by renal micropuncture techniques by measure stop flow pressure (Psf). CTGF was evaluated by the differences between TGF maximal responses (TGFmax) with or without tubular benzamil perfusion. Another set of animals received chronic infusion of benzamil directly into the kidney, that started 1 week before UNx. Renal blood flow (RBF) was measured by arterial spin labeling-MRI 24h before and 24h after the UNx. Direct BP measurement was performed before and 3 weeks after the UNx. After UNx, TGF resetting was observed in UNx rats (TGFmax 8±1 vs. 1±1 mmHg, Sham vs. UNx; p<0.05). This TGF resetting was inhibited by benzamil. RBF increased after the UNx in comparison to sham and this increase was prevented by chronic infusion of Benzamil into the kidney (Sham: 3±0.6; UNx: 4.6±0.3; UNx+Benzamil 3.5±0.6 ml/min/g tissue p<0.002). Basal mean BP values were not different between the vehicle or treated rats before the UNx; however 3 weeks after the UNx, rats receiving benzamil into the kidney showed higher mean BP values than vehicle (88±0.3 vs. 97±4 mmHg, p<0.01). We conclude that CTGF participates in TGF resetting and RBF regulation after UNx, and that could participate in BP regulation after UNx.
Abstract P156: Fiber-rich Diet Suppresses Lactobacillus And Increases Blood Pressure In The Shr Independently Of T-lymphocyte Immune Responses
Authors: Yang Tao , Colon-Perez Luis M , Magee Kacy L , Malphurs Wendi L , Vickroy Thomas W , Febo Marcelo , Zubcevic Jasenka
Published: June 2021

Introduction:
Gut dysbiosis has been linked to hypertension in both rodents and humans. Microbial metabolites such as propionate have been shown to regulate blood pressure (BP), while butyrate, one of the major fermented end-products of fiber, reportedly produces beneficial anti-inflammatory effects in multiple dysbiosis-related diseases. Therefore, we tested the impact of a fiber-rich, butyrolytic diet on BP regulation and immune responses in the spontaneously hypertensive rats (SHR).

Methods:
SHR (5 wo) were placed on either the fructooligosaccharides/inulin-rich diet (Fiber, N=6), or its calorie-matched control diet (Control, N=6) (Research Diets, Inc.) for 10 weeks. Baseline BP was measured by tail cuff every week for the duration of the study. Fecal samples were collected for HPLC analysis of butyrate, and
Lactobacillus
population by QPCR. Manganese-enhanced magnetic resonance imaging was used to monitor neural activity in cardioregulatory brain regions. Blood was analyzed for circulating lymphocyte populations previously implicated in BP control in the SHR (CD3
+
CD45
+
, CD4
+
CD25
+
, CD8
+
).

Results:
Fiber-rich diet produced an increase in fecal butyrate levels as early as five weeks (Control vs. Fiber, 4.9umol/g vs. 9.7umol/g, p=0.068, N=6). This was associated with contraction of fecal
Lactobacillus
(47.5% vs. 5.9%, p=0.0008, N=6). However, we observed significantly higher systolic BP (181.4mmHg vs. 201.7 mmHg, p=0.0088, N=6) in the fiber group compared with control, beginning with week 9 post-diet switch. Changes in neural activation were observed in the paraventricular nucleus of hypothalamus (PVN) (3.3 voxel vs. 7.3 voxel, p=0.26) and amygdala (93.3 voxel vs. 31 voxel, p=0.0059, N=3). No changes in circulating T-lymphocytes were observed between the two groups: CD3
+
CD45
+
(32.4% vs. 32.1% lymphocytes); CD4
+
CD25
+
(1.05% vs. 0.78% lymphocytes); CD8
+
(18.7% vs. 16.2% lymphocytes, N=6) at week 10 post-diet switch.

Conclusion:
Fiber-rich diet suppression of Gut
Lactobacillus
is associated with increase in BP in the SHR, independently of T-lymphocyte responses. The observed higher neural activity in PVN and lower in amygdala in the fiber group suggest direct effects of gut bacterial metabolites on brain cardioregulatory regions.
Abstract P158: Cullin3 Regulated Endothelial Function by Modulating eNOS Activity
Authors: Wu Jing , Lu Ko-Ting , Agbor Larry N , Hu Chunyan , Liu Xuebo , Mukohda Masashi , Nair Anand R , Sigmund Curt D
Published: June 2021

Pseudohypoaldosteronism type II (PHAII) patients expressing dominant negative cullin3 mutations exhibit increased renal NaCl reabsorption and develop hyperkalaemia, metabolic acidosis and hypertension. It is unclear whether loss of cullin3 function in extra-renal tissues contributes to the hypertensive phenotype. In the vasculature, endothelial Nrf2 stability is tightly regulated by cullin3-based E3 ubiquitin ligase via the redox-sensitive adaptor kelch-like ECH-associated protein 1. In the present study, we found that 24-hour treatment with a pan cullin inhibitor MLN4924 (1 μM) caused a 3-fold increase in Nrf2 protein in mouse lung endothelial cells (MLECs), while tert-butyl hydroperoxide (tBHP, 240 μM) had no effect on Nrf2 level. However, both MLN4924 and tBHP triggered time-dependent accumulation of Nrf2 in the nuclei, which peaked at 40 minutes following treatment. As a result, both treatments induced marked upregulation of antioxidant genes including NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate cysteine ligase (rate-limiting enzyme in glutathione synthesis), and catalase both in MLECs and primary mouse aortic endothelial cells (MAECs). Of note, MLN4924 upregulated Nox4 expression (1.0 ± 0.15 vs 1.7 ± 0.2) and tBHP upregulated Nox1 (1.0 ± 0.2 vs 4.8 ± 1.1), while MLN4924 and tBHP both markedly increased intracellular superoxide as determined by dihydroethidium staining. In addition, intracellular nitric oxide was decreased by half in MLN4924-treated MLECs. This redox imbalance was likely due to impaired eNOS expression and activation as MLN4924 caused a 25% reduction in total eNOS and a 75% reduction in phosphorylated eNOS, while tBHP lead to a 50% reduction in phosphorylated eNOS with no effect on total eNOS. This suggests that decreased eNOS activation contributed to the oxidative stress induced by these agents. These data imply that suppression of cullin3 in arterial endothelial cells may dampen endothelium-dependent vascular relaxation and contribute to the blood pressure elevation observed in PHAII patients with global loss of cullin3 function. Although cullin3 also negatively regulates Nrf2-mediated antioxidant responses in vascular endothelial cells, this likely occurs as a compensatory mechanism.
Abstract P162: Butyrate, a Microbial Metabolite, Attenuates Angiotensin II-induced Hypertension and Gut Dysbiosis
Authors: Kim Seungbum , Lobaton Gilberto O , Raizada Mohan K
Published: June 2021

Objectives:
We have previously established that hypertension (HTN) is associated with gut dysbiosis in rat models and patients with high blood pressure. Gut dysbiosis was associated with an increase in Firmicutes/Bacteriodetes ratio and a decrease in butyrate-producing microbial populations. This led us to hypothesize that infusion of butyrate would overcome gut microbial dysbiosis induced butyrate deficiency and reverse angiotensin II (Ang II)-induced HTN.

Design and Method:
Four groups of C57BL6 mice were infused with either saline, Ang II (1000ng/kg/min), Ang II + sodium butyrate (1mg/kg), or sodium butyrate alone for 4 weeks. Fecal samples were analyzed by 16S bacterial rDNA sequencing for gut microbiome identification. Intestinal leukocytes were analyzed using FACS.

Results:
Ang II induced increase in MAP was significantly attenuated in mice co-administrated with butyrate (Control, 102.1±5.7 mmHg; Ang II, 148.3±8.1 mmHg; Ang II+Butyrate, 120.5±11.2 mmHg). Microbiota analysis demonstrated a significant increase of gut dysbiosis in Ang II-HTN that was normalized by butyrate treatment (F/B ratio: Control, 2.6; Ang II, 5.4; Ang II+Butyrate, 2.0). Principal Coordinates Analysis indicated each group in the input phylogenetic tree had significantly different microbial communities. LEfSe analysis showed that there were decreases of beneficial bacteria such as
Lactobacillus
and
Bifidobacterium
, and increases of
Corynebacterium
and
Staphylococcus
at the genus level of Ang II-HTN mice. Furthermore, inflammatory status of the gut as evidenced by the level of mucosal T cells in lamina propria from these groups showed that there was an increase of CCR2
+
Th17 cells in Ang II-HTN mice, but not in butyrate co-treated mice (Control, 15.7%; Ang II, 28.4%; Ang II+Butyrate, 15%).

Conclusions:
These observations show that gut dysbiosis and the decrease of butyrate producing bacteria are associated with Ang II-HTN. Thus, supplementing butyrate in Ang II treated mice attenuated HTN and reversed gut dysbiosis, as well as normalizing the intestinal Th17. These data suggest butyrate producing bacteria could be considered as a novel probiotic therapy for hypertension.
Abstract P164: The Role of the Adrenoreceptors Beta 3 on Metabolic Syndrome Induced by Fructose
Authors: Dias Eduardo , Tavolari Alexandre , Souza Marina , Oliveira Renata , Fiorino Patricia , Ribeiro Miriam O , Farah Vera M
Published: June 2021

The aim of this study was to evaluate the role of the adrenoreceptor beta 3 (ARβ3) in the cardiovascular function on Metabolic Syndrome (MS) induced by fructose overload. Male mice after weaning (8-10g) were divided into 4 groups (n=7/group): FVB Control (C) and ARβ3 Knockout Control (Kβ3C), with free access to food and water for 8 weeks; FVB fructose (F) and ARβ3 Knockout fructose (Kβ3F), with free access to food and fructose added to the drinking water (10%) for 8 weeks. At the end of the protocol, intraperitoneal glucose tolerance test was performed. Triglycerides and HDL fractions were evaluated by colorimetric methods. The animals were submitted to catheterization of carotid artery. This catheter was connected to a transducer and continuous signals of blood pressure (BP) was recorded. The variability of the resultant signal was evaluated in frequency domain. Visceral fat deposits were collected, weighted and normalized as fat (mg)/body weight (g). Results: fructose increased the fasting glycemia in the knockout group (Kβ3C=93±3 vs. Kβ3F=122±7 mg/dL) and decreased the glucose tolerance in both fructose groups. Fructose increased the triglycerides and decreased HDL cholesterol. The uric acid increased in the knockout animals. There was an increase on visceral fat deposits only in FVB animals (C=10±2 vs. F=16±1 mg/g), as well as on blood pressure (C=123±4 vs. 136±2 mmHg) There was an increase on blood pressure variability for all experimental groups compared to Control (C=10.7±1 vs. F=36±11; Kβ3C=50±7; Kβ3F=24±4 mmHg
2
) as well as on peripheral sympathetic modulation (C=3.3±0.4 vs. F=8.1±0.1; Kβ3C=22.9±3; Kβ3F=9.2±2 mmHg
2
). Furthermore, Kβ3F showed a decrease on blood pressure variability and peripheral sympathetic modulation. Our data suggest that the hypertension induced by fructose is related with adiposity. Moreover, the blood pressure variability and its sympathetic modulation are dependent of the ARB3. The association between fructose and ARβ3 knockout leads to the loss of the sympathetic modulation.
Abstract P167: Combined Exercise Training is Better than Isolated Aerobic and Resistance Exercise Training for an Experimental Model of Metabolic Syndrome and Menopausal Rats
Authors: Conti Filipe F , Brito Janaina O , Bernardes Nathalia , Dias Danielle S , Irigoyen Maria C , De Angelis Kátia
Published: June 2021

The aim of this study was to verify the effects of three different moderate exercise training protocols (aerobic, resistance and combined (aerobic + resistance)) in a model of metabolic syndrome and menopause on a cardiovascular parameter and oxidative stress. Female SHR rats were divided into (n=8): hypertensive (H), hypertensive ovariectomized submitted to fructose overload (100g/L in drinking water) (HFO), aerobic trained hypertensive ovariectomized submitted to fructose overload (AHOF), resistance trained hypertensive ovariectomized submitted to fructose overload (RHOF) and combined trained hypertensive ovariectomized submitted to fructose overload (CHOF). Arterial pressure (AP) signals were directly recorded. Vascular autonomic modulation was evaluated by spectral analysis. The cardiac oxidative stress was evaluated by lipoperoxidation (LPO) determination. The association of fructose overload and hormone deprivation promoted an increase in AP (HOF: 174±4 vs. H: 146±4 mmHg), heart rate (HOF: 393±10 vs. H: 352±13 bpm), VAR-SAP (HOF: 77.8±11.9 vs H: 31.1±2.6 mmHg2), LF-SAP (HOF: 10.6±2.3 vs H: 5.0±0.9 mmHg2) and LPO, and reduced baroreflex sensitivity (tachycardia response: HOF: 1.06±0.06 vs. H: 1.91±0.17 bpm/mmHg). All exercise training protocols were able to reduce LPO and LF-SAP. It was noted that only the combined exercise training was able in reducing AP (CHOF: 158±4 mm Hg) and heart rate (CHOF: 303±5 bpm). The AP reduction noted only in the CHOF group may be associated with an improve in baroreflex sensitivity, represented by an increase of tachycardic response observed only in the CHOF (1.62±0.1 bpm/mmHg) and in the AHOF (1.54 ±0.07 bpm/mmHg) groups and a reduction of VAR-PAS observed only in the CHOF (30.31±3.85 mmHg
2
) and in the RHOF (31±2.65 mmHg
2
) groups. In conclusion, fructose overload induced impairment in hemodynamic, vascular autonomic control and increased oxidative stress in hypertensive rats submitted to ovarian hormones deprivation. However, all exercise training protocols showed a beneficial role. Moreover, the combined exercise training showed additional improvement, suggesting that this could be a better approach than isolated aerobic and resistance training.
Abstract P168: 20-HETE-mediated Neutrophil Adhesion Impairs Coronary Collateral Growth in Metabolic Syndrome
Authors: Joseph Gregory , Soler Amanda , Hutcheson Rebecca , Hunter Ian , Hutcheson Brenda , Bradford Chastity , Gotlinger Katherine H , Falck John R , Proctor Spencer , Schwartzman Michal L , Rocic Petra
Published: June 2021

Transient, repetitive myocardial ischemia (RI)-induced coronary collateral growth (CCG) is impaired in metabolic syndrome patients and animal models. Endothelial cell (EC) dysfunction and chronic inflammation are hallmarks of metabolic syndrome. We showed that while in normal animals (SD), RI induces transient infiltration of monocytes, associated with successful CCG, in metabolic syndrome rats (JCR), RI induces sustained accumulation of neutrophils, which contributes to compromised CCG. 20-hydroxyeicosatetraeonic acid (20-HETE) is a pro-inflammatory metabolite of arachidonic acid. Its role in the regulation of CCG is unknown. We hypothesized that enhanced 20-HETE-mediated neutrophil adhesion to ECs and consequent EC dysfunction and apoptosis result in impaired CCG in metabolic syndrome. P-selectin and ICAM-1 expression was increased ~40% in JCR vs. SD rats. This increase was prevented by 20-HETE antagonists, 20-SOLA or 20-HEDGE. 20-HETE antagonists also prevented neutrophil accumulation observed in JCR rats. Coronary arteries from JCR rats exhibited reduced endothelium (Ach)-dependent vasodilation (20% JCR vs. 50% of max. SD). RI-induced eNOS activation and NO production were likewise decreased (~60% and~70%, respectively) in JCR vs. SD rats. EC apoptosis (TUNEL) was severely increased in response to RI in JCR rats (~75% vs. SD). Neutrophil adhesion-blocking antibodies partially attenuated EC apoptosis (~70%) and EC dysfunction (~75% eNOS activation and NO production, 75% Ach-dependent vasodilation). 20-HETE antagonists fully reversed impaired endothelium-dependent vasodilation, eNOS activation, NO production and prevented EC apoptosis. Finally, impaired CCG in JCR rats (collateral-dependent blood flow, microspheres) was completely restored by 20-HETE antagonists (CZ/NZ flow was 0.76±0.07 in JCR+20-SOLA, 0.84±0.05 in JCR+20-HEDGE vs. 0.11±0.02 in JCR vs. 0.84±0.03 ml/min/g in SD rats) and partially restored by neutrophil-blocking antibodies (0.49±0.05 ml/min/g). Taken together, these results indicate that 20-HETE-dependent neutrophil adhesion and accumulation compromises EC survival and function leading to impaired CCG. 20-HETE antagonists could provide therapy for restoration of CCG in metabolic syndrome.
Abstract P171: Superoxide via Transcription Factor Sp3 Up-regulates Renal Angiotensin II Receptor Function
Authors: Saleem Mohammad
Published: June 2021

Oxidative stress is linked to the up-regulation of angiotensin II type 1 receptor (AT1R) function and hypertension. Here we tested the mechanism of oxidative stress-associated up-regulation of AT1 receptor function in human kidney HK2 cells. Diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, and hydrogen peroxide (H
2
O
2
)

increased DHE and DCFHDA fluorescence respectively [DHE fluorescence (Control vs DETC vs DETC + tempol vs H
2
O
2
: 0.5397 ± 0.07057, 1.463 ± 0.1671, 0.2661 ± 0.01776, 0.6406 ± 0.04821); DCFH fluorescence (Control vs H
2
O
2
vs 3-AT vs DETC: 555.9 ± 21.22, 673.5 ± 37.05, 736.1 ± 33.79, 427.7 ± 22.17)]. DETC increased nuclear levels of Sp3 protein that were attenuated with tempol (Control vs DETC, vs DETC + tempol: 0.50 ± 0.08, 1.28 ± 0.21, 0.52 ± 0.12 densities). However, H
2
O
2
had no significant effect on nuclear Sp3 (Control vs H
2
O
2,
vs H
2
O
2
+ tempol: Sp3, 0.50 ± 0.08, 0.68 ± 0.14, 1.04 ± 0.30 densities). In transfection studies, Sp3 plasmid increased (Control vs Sp3: 0.1165 ± 0.01, 0.3810 ± 0.03, densities) while Sp3 siRNA decreased (Control siRNA vs Sp3 siRNA: 1.11 ± 0.25, 0.64 ± 0.06, densities) the levels of AT1R protein in cell lysate. DETC increased AT1R protein expression that was attenuated by tempol, measured by immunoblotting (Control vs DETC vs DETC + tempol: 0.3535 ± 0.99, 0.9975 ± 0.19, 0.3250 ± 0.02 densities). Furthermore, DETC increased cell membrane levels of AT1R protein as measured by biotinylation and immunoblotting. This effect was attenuated by tempol. (Control vs DETC vs DETC + tempol: 100 ± 0.0, 189.8 ± 0.34.60, 102.3 ± 10.77 densities, percent change). Moreover, DETC increased PKC activity in response to angiotensin II, an indicator of AT1R function. This effect was attenuated with candesartan and tempol. (Control vs Ang II vs DETC + AngII vs DETC + Cand + AngII vs DETC + tempol: 100.0 ± 0.0, 162.0 ± 12.97, 224.9 ± 52.97, 126.0 ± 6.133, 73.32 ± 35.01 percent change) These results suggest that superoxide but not H
2
O
2
increase nuclear Sp3 protein that up-regulates renal AT1R expression and function.
Abstract P176: Maternal and Neonatal Outcomes in Normal and Preeclamptic Pregnancies - A Comparative Prospective Study
Authors: Kalagiri Ram R , Afroze Syeda H , Vora Niraj , Drever Nathan , Beeram Madhava R , Kuehl Thomas J , Zawieja David C , Uddin Mohammad N
Published: June 2021

Background:
Preeclampsia (PreE) is de novo development of hypertension and proteinuria after 20 weeks of gestation with multiple pathophysiologic triggers affecting 3-8% of pregnancies. PreE has a significant link to alterations of feto-placental stress that pass to the offspring causing detrimental effects. We assessed and compared the pregnancy outcomes between patients with and without PreE.

Methods:
We recruited 35 normal pregnant (NP) and 25 PreE consenting patients in an IRB approved prospective study at Scott & White Memorial Hospital, Temple, Texas. We evaluated maternal age, body mass index (BMI), blood pressures, proteinuria, and gestational age at delivery. We divided the PreE subjects into early (before 34 weeks) and late PreE (after 34 weeks) groups and compared their outcomes. Placental thickness and volumes were measured. We also evaluated neonates for intrauterine growth restriction (IUGR), gestational age at birth, anthropometric measurements including Ponderal Index (PI), length of hospitalization and other neonatal complications. Comparisons were performed using Student’s t test.

Results: Maternal:
The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were higher in PreE (SBP 166 ± 11; DBP 93 ± 10) compared to normal pregnancies (SBP 122 ± 10; DBP 74 ± 9; p <0.05 for each case). PreE mothers had higher urinary protein excretion (457mg/24h ± 140) compared to NP (160 mg/24h ± 44; p <0.05 for each case). We did not find any difference in body mass index (BMI).
Placenta:
The placental thickness in early PreE subjects was 25mm compared to 32mm in late PreE (p <0.05) and placental volume in early PreE 296 cm
3
compared to 393cm
3
(p <0.05).
Neonatal:
The average GA at delivery was lower in PreE (34.8 wks. ± 4) compared to NP (39.2 weeks ± 0.3; p <0.05). Average hospital stay for PreE babies were longer (20 days ± 5) compared to NP (2 days ± 1; p <0.05). The PreE babies were SGA with lower PI (2.28 ± 0.3) compare to the NP babies (2.95 ± 0.2; p <0.05). Gestational age at delivery in early PreE is 32.4 weeks vs 36.8 weeks in late PreE (p <0.05). About 56% of the infants who are born to early PreE are small for gestational age (SGA) and 30% of the infants who are born to late PreE are SGA. PreE babies had multiple complications compared to NP babies.
Abstract P182: The Novel Perivascular Adipose Tissue Adipokine, Chemerin, Signals Through G
i
- and Calcium-Dependent Mechanisms
Authors: Ferland David J , Darios Emma S , Neubig Richard R , Sjögren Benita , Truong Nguyen , Torres Rosa , Dexheimer Thomas S , Thompson Janice , Watts Stephanie W
Published: June 2021

Chemerin is an adipokine associated with inflammation, increased blood pressure, and may be a link between the pathologies of obesity and hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via the chemerin receptor and calcium flux in smooth muscle cells. Known mediators of the amplified arterial responsiveness seen in hypertension (L-type Ca
2+
channels, Src, and Rho kinase) were interrogated by isometric contraction of rat aortic rings in parallel with calcium kinetics of rat aortic smooth muscle cells. Western blots were also used to observe phosphorylation of Erk/MAPK. Chemerin-9 (nonapeptide of the chemerin S
157
isoform) caused a concentration-dependent contraction of isolated aorta (EC
50
100 nM) and elicited a concentration-dependent intracellular calcium response (EC
50
10 nM). Both calcium influx and isometric contraction, respectively, were reduced (units of “% of vehicle response”) by Pertussis toxin (G
i
inhibitor; 0±3% and 23±9%), verapamil (L-type Ca
2+
channel inhibitor; 38±20% and 23±4%), PP1 (Src inhibitor; 43±23% and 15±4%), and Y27632 (Rho Kinase inhibitor; 58±23% and 22±4%) but U73122 (PLC inhibitor) had little to no effect (71±31% and 71±12%). PD098059 (Erk/MAPK inhibitor) did not inhibit chemerin-9 induced contraction (117±19%) and phosphorylation did not change after chemerin-9 stimulation [1.12±0.14 (44 kDa) and 1.11±0.29 (42 kDa) fold-increase with chemerin-9 contraction compared to vehicle, p>0.05]. The chemerin receptor-selective antagonist CCX832 inhibited chemerin-9-induced calcium flux and aortic contraction and calcium flux (0.1±10.3% and 10±7%). These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through the G
i
-linked chemerin receptor to activate L-type Ca
2+
channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us one step closer to targeting chemerin as a unique form of therapy.
Abstract P184: Co-localization and Natriuretic Interdependence of Angiotensin AT2R and MasR in Obese Rat Kidney
Authors: Patel Sanket N , Ali Quaisar , Steckelings Ulrike Muscha , Hussain Tahir
Published: June 2021

The actions of angiotensin II type 2 receptor (AT
2
R) and receptor mas (MasR) are complex but show similar pro-natriuretic function; particularly AT
2
R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of previous reports, we tested hypothesis that AT
2
R and MasR are interdependent to produce natriuresis in obese rats due to potential physical interaction. Infusion of AT
2
R agonist C21 (5 μg/kg/min) in obese Zucker rats (OZR) caused diuresis/natriuresis which were attenuated by simultaneous infusion of the AT
2
R antagonist PD123319 (50 μg/kg/min) or the MasR antagonist A-779 (50 μg/kg/min). Similarly, infusion of MasR agonist Ang-(1-7) (110 fmol/kg/min) in OZR caused diuresis/netriuresis, which were attenuated by simultaneous infusion of A-779 or PD123319. Dual labeling of AT
2
R and MasR in OZR kidney slices revealed four-fold co-localization of AT
2
R and MasR (9.83 vs. 2.50 dual labeled cells/1600 μm
2
) compared with lean rats in which AT
2
R is not natriuretic. Moreover, the AT
2
R co-immunoprecipitates with MasR in cortical homogenate of OZR. Immunoblotting of AT
2
R and MasR with zero length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline in OZR cortical homogenate revealed a shift of AT
2
R (~62 kDa) and MasR (~54 kDa) bands upward with overlapping migration for their complexes (~160 kDa and 245 kDa) which were sensitive to the reducing β-mercaptoethanol. Similar observations were made in HK-2 cells, where glucose (25 mM) treatment enhanced the crosslinking. Collectively, the study reveals AT2R and MasR are co-localized and functionally interdependent in producing natriuretic response. Hyperglycemic oxidative stress affecting sulfhydryl groups present a potential mechanism of such physical interaction between these receptors. (Support: R01DK061578)
Abstract P185: Role of Ucp2 in the Oxidative/nitrosative Stress-mediated Hypertension Associated with Dj-1 Depletion
Authors: De Miguel Carmen , Hamrick William C , Asico Laureano , Jose Pedro , Cuevas Santiago
Published: June 2021

DJ-1
-/-

mice, relative to wild-type (WT) littermates, have increased blood pressure (BP) (

DJ-1
-/-

:130±4 vs WT:100±3 mmHg, n=6/8). and renal expressions of nitro-tyrosine (+76±31% of WT mice, n=5) and malondialdenyde (+63±23% of WT mice, n=4). Tempol, a superoxide dismutase mimetic, decreased the BP of

DJ-1
-/-

mice (

DJ-1
-/-

: before tempol:119±3; after tempol:100±1 mmHg vs WT, n=4) and renal malondialdehyde production (

DJ-1
-/-

: before tempol:+40±5%; after tempol:-24±5% vs WT, n=4) but increased serum nitrate/nitrite levels (+72±30%, n=4), indicating the presence of both oxidative and nitrosative stress. Lack of DJ-1 makes some cells vulnerable to endoplasmic reticulum (ER) stress. However, renal mRNA expression of ER stress markers, GRP94, ATF-4, ATF-6, sXBP-1, CHOP, caspase-12, and caspase-3 was not different between

DJ-1
-/-

and WT (n=7) mice. Markers of inflammation, IL-6, TNF α, MCP-1, NFκB, and T-cell and macrophage infiltration, were also not increased in the kidney of

DJ-1
-/-

mice. By contrast, renal mitochondrial (mt) HSP60, but not mtHSP40, was increased in

DJ-1
-/-

mice (2.9±0.1 fold, n=4) but there were no changes in the renal mRNA expressions of Nix/BNIP3L, BNIP3, PINK, FIS1, MFN1, MFN2, PPRC1, NRF-1, and PGC1, indicating that mt oxidative stress did not affect mt function. The renal expression of UCP2, which is involved in the control of mt-reactive oxygen species production, was elevated in

DJ-1
-/-

mice (4.1±1.1 fold of WT, n=4). Silencing UCP2 in mouse renal proximal tubule cells (-0.46.5±0.01 fold) increased the expression of ER stress and apoptosis markers CHOP (2±0.4 fold), ATF4 (2.6±0.6 fold), caspase-3 (2.3±0.4 fold), and caspase-12 (1.7±0.2 fold)(n=3). There were no differences in renal renin expression, sodium excretion, and serum creatinine between

DJ-1
-/-

and WT mice (n=5). There were no abnormalities in renal morphology, including fibrosis, in the kidneys of

DJ-1
-/-

mice. However, urinary KIM-1 was increased in

DJ-1
-/-

mice (148±22% of WT mice, n=4) and decreased by tempol (-58±3%, n=4); renal UCP2 expression was also partially normalized by tempol (1.8±0.2 fold of WT, n=4). UCP2 may protect from the development of renal ER stress and damage in the mt oxidative/nitrosative stress associated with DJ-1 depletion.
Abstract P188: (Pro)renin Receptor (PRR) Mediates High Fat Diet-induced Hypertension via Upregulation of Epithelial Sodium Channel & Aquaporin-2
Authors: Quadri Syed S , Culver Silas A , Siragy Helmy M
Published: June 2021

We hypothesized that the renal prorenin receptor (PRR) mediates high fat diet (HFD)-induced hypertension via enhancing expression of epithelial sodium channel (α-ENaC) and aquaporin-2 (AQP-2). C57BL/6 mice underwent right nephrectomy (n=12) and allocated to receive regular diet (RD, 12 kcal% fat, n=6) or a high-fat diet (HFD, 45 kcal% fat, n=6) for 10 weeks. HFD group received left renal subcapsular interstitial administration of PRR shRNA (n=3) or vehicle (n=3). BW, food and water intake, BP, UV and UNaV, renal interstitial fluid (RIF) angiotensin II (Ang II), and renal expressions of PRR, α-ENaC, AQP-2 were monitored. At baseline, there were no significant differences in BW, food and water intake, BP, UV or UNaV between different animal groups. At the end of the study, HFD mice had significant increase in food intake, systolic blood pressure (HFD 154.6 ± 2.181 mmHg, vs. RD 112.21 ± 4.684 mmHg, P<0.0.05), BW (HFD 43.2 ± 1.125 gm, vs. RD 31.9 ± 0.89654 gm, P<0.0.05), and significant reduction in UV (HFD 0.16 ± 0.042 ml, vs. RD 0.41 ± 0.061 ml, P<0.0.05) and UNaV (HFD 26.02 ± 3.652 μmol/day vs. RD 46.32 ± 5.236 μmol/day, P<0.0.05). Compared to RD, there were significant increases in mRNA and protein expressions of PRR (64% and 40%, P<0.01), α-ENaC (85% and 75%, P<0.05), AQP-2 (105% and 80%, P<0.05) in HFD alone mice respectively. Compared to HFD alone, HFD+ PRR shRNA treatment caused significant reductions in BP (108 ± 13.88 mmHg vs HFD 154.6 ± 2.181 mmHg), mRNA and protein expressions of PRR (33% and 50%, P<0.01), α-ENaC (49% and 56%, P<0.05), AQP-2 (30% and 29%, P<0.05) respectively. There were no changes in RIF Ang II between different animal groups. We conclude that PRR mediates HFD-induced hypertension via enhancing renal α-ENaC and AQP-2 expression independent of Angiotensin II.