bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P140: Alamandine Signaling in Cardiomyocytes in Health and Disease
Authors: Jesus Itamar C , Scalzo Sergio , Rocha-Resende Cibele , Santos Robson A , Guatimosim Silvia
Published: March 2022

Alamandine is a new component of the renin-angiotesin system generated from angiotensin A or angiotensin-(1[[Unable to Display Character: –]]7). Its biological actions include vasodilation, and antihypertensive effects. In the heart, the molecular pathways activated by alamandine have not been characterized. Our goal was to investigate the signaling pathways activated by alamandine in ventricular myocytes isolated from both healthy and hypertensive models. Cardiomyocytes isolated from C57BL/6 mice and from rats that overexpress renin (TGRmRen) were treated with 100nmol/L alamandine. Intracellular nitric oxide (NO) and Ca
2+
levels were recorded in cells loaded with DAF-FM and Fluo4-AM, respectively. Protein phosphorylation was assessed by western-blot. In cardiomyocytes, alamandine induced an increase in phosphorylation of PDK1 (arbitrary units (a.u.): control=0,20±0.06 versus alamandine=0,42±0.08 n=6, p<0.05) and Akt Ser473 (a.u.: control= 0,40 ±0,01 versus alamandine= 0,52±0,10 n=5 p<0.05). Moreover, alamandine induced a significant increase in NO (Fluorescence a.u.: control=4.8±0.8 n=44 versus alamandine=12.19±1.47 n=85 p<0.05), without any effect on Ca
2+
transient amplitude. Ventricular myocytes treated with alamandine showed decreased GSK3β phosphorylation (a.u.: control=0,62±0.19 versus alamandine=0,50±0.25 n=5 p<0.05) and increased phosphorylation of ERK1/2 (a.u.: control=0,48±0.24 versus alamandine= 0,80±0.29, n=7 p<0.05) when compared to control. Conversely, TGRmRen cardiomyocytes exposed to alamandine showed increased GSK3β phosphorylation and decreased phosphorylation of ERK1/2. The effect of alamandine on Akt phosphorylation was preserved in TGRmRen cardiomyocytes. Furthermore, alamandine treatment prevented both cardiomyocyte hypertrophy (area (μm
2
): control=447±23 n=58; angiotensin II= 609±26 n=70; angiotensin II+alamandine= 491±31 n=45 p<0.05), and nuclear translocation of GRK5 induced by 100nmol/L angiotensin II. Our data show that alamandine signaling in cardiomyocytes changes according to different pathophysiological condition, and includes the activation of cardioprotective pathways. These data highlight the therapeutic potential of alamandine in the heart.
Abstract P142: Regulation of Brain Derived Neurotrophic Fractor (BDNF) Expression by Angiotensin II in the Adrenals and the Brain
Authors: Wang Hong-Wei , Lu Jiao , Ahmad Monir , Leenen Frans H
Published: March 2022

Circulating Ang II activates central angiotensinergic pathways, and an amplifying aldosterone (aldo)-MR-ENaC-AT
1
R neuromodulatory pathway which are critical for several forms of hypertension. We hypothesized that in addition Ang II increases BDNF expression in both the CNS and adrenal which plays a balancing role against Ang II excitatory actions. In the 1
st
exp, Wistar rats were sc infused with Ang II at the high dose of 500 ng/kg/min for 7days. In the 2
nd
exp, rats were treated with regular salt diet (0.4% NaCl), high salt diet (2% NaCl), sc Ang II at the low dose of 150 ng/kg/min, or sc Ang II with high salt diet for 14 days. In the 3
rd
exp, MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT
1
R blocker (losartan) or vehicles (Veh) were icv infused combined with Ang II-salt. In the 4
th
exp, rats were sc infused with aldo (1μg/hr) with saline as drinking fluid. mRNA levels of BDNF were measured by real-time qPCR. In the adrenals, Ang II dose-related increased BDNF mRNA, and Ang II-salt further enhanced BDNF mRNA. High dose Ang II increased BDNF mRNA in the RVLM but not in the PVN. Central blockades markedly decreased Ang II-salt induced BDNF expression in the adrenal cortex. In contrast, saline alone or aldo-saline decreased BDNF mRNA in the adrenal cortex with no effect in the PVN. Central blockades lower Ang II-salt induced BDNF expression in the adrenal, indicating that a central regulatory mechanism plays a role in adrenal BDNF expression. Activation of BDNF by Ang II -modulated by salt-, may provide an important balancing mechanism both centrally and peripherally for Ang II associated hypertension.
Abstract P145: Immuno-neutralization of Endogenous Ouabain Lowers Blood Pressure in Angiotensin II-dependent Models
Authors: Chen Ling , Hamlyn John M , Blaustein Mordecai P
Published: March 2022

Circulating angiotensin II (Ang II) activates the brain RAAS. The activated brain RAAS increases sympathetic drive and elevates plasma endogenous ouabain (EO). Increased heart rate and vascular constriction as a result of increased sympathetic nerve activity are widely recognized, but the functional significance of the high EO, when Ang II is elevated, is unknown. We employed DigiFab, fab fragments that bind, and selectively immuno-neutralize ouabain, to determine its acute and chronic effects on BP (telemetry) in two rodent models with elevated Ang II: 1. Normal mice infused with Ang II (350 ng/kg/min; sc minipump) and fed high salt (HS; 4% NaCl) to raise BP; and 2. Normal rats fed low salt (LS; 0.04% NaCl) to activate the peripheral RAAS. CroFab, which immuno-neutralizes crotalus toxin, was used to control for protein and excipients. Neither acute DigiFab nor CroFab (10-40 mg/kg ip) lowered mean daytime BP (MBP) in control mice fed a normal salt (0.4% NaCl) diet. Both Ang II+HS and LS elevated plasma EO (from 0.07±0.09 to 1.30±0.71,
n
=8, and from 0.21±0.05 to 0.70±0.19 nM,
n
=8, respectively, both
P
<0.05; radioimmunoassay). The Ang II+HS increased mouse MBP from 104 ± 1 at baseline, to 123 ± 1 mm Hg (
P
<0.001;
n
=8) at 16 days. Acute DigiFab treatment (10 mg/kg, ip injection at 0 and 4 hrs) reversibly lowered MBP to 112 ± 2 mm Hg (
P
<0.001) within 2-3 hr, but CroFab (10 mg/kg, ip at 0 and 4 hrs) had no effect in the same mice (MBP = 120 ± 2 mm Hg). Chronic DigiFab treatment (sc minipump, 10.7 mg/kg/day x 7 days) in mice with Ang II+HS hypertension (MBP = 133 ± 1 mm Hg;
n
=8) reversibly lowered MBP (125 ± 2 mm Hg,
P
<0.01 vs CroFab, 136 ± 1 mm Hg), When Ang II was elevated by 16 days of low salt in rats, MBP was 107 ± 2 mm Hg. Acute DigiFab treatment (10 mg/kg, ip at 0 and 4 hrs) reversibly lowered MBP to 98 ± 2 mm Hg (
P
<0.001;
n
=6), whereas CroFab (10 mg/kg, ip at 0 and 4 hrs) did not in the same animals (MBP = 106 ± 1 mm Hg).

Conclusion:
Increased circulating EO augments the elevation of BP in Ang II+HS hypertension, and also helps to sustain BP during salt restriction. EO is a previously unrecognized functional component of the physiological and pathological actions of Ang II.
Abstract P146: Serum Soluble (Pro)Renin Receptor Levels in Patients Undergoing Hemodialysis
Authors: Morimoto Satoshi , Amari Yoshifumi , Kimura Shihori , Bokuda Kanako , Watanabe Daisuke , Yatabe Midori , Yatabe Junichi , Ando Takashi , Ichihara Atsuhiro
Published: March 2022

Background:
The (pro)renin receptor [(P)RR] plays an important role in regulating the tissue renin-angiotensin system (RAS) through the non-proteolytic activation of prorenin, the precursor of renin. (P)RR is cleaved by furin to generate soluble (P)RR [s(P)RR], which is secreted into the extracellular space, and serum s(P)RR has been reported to reflect the status of tissue RAS. Hemodialysis (HD) patients have poor prognosis due to increased prevalence of cardiovascular diseases resulting from severe atherosclerosis. It is speculated that activation of tissue RAS by (P)RR may be associated with this condition, it remains speculative. In the present study, we investigated the relationships between serum s(P)RR levels and background factors including indices of atherosclerosis in HD patients. Methods: Serum s(P)RR levels were measured in 258 maintenance HD patients and these values were compared with 25 subjects with normal renal function. Western blot analysis was done using HD waste water samples, and clearance of s(P)RR through the membrane of dialyzer was examined. Furthermore, relationships between serum s(P)RR levels and background factors were assessed in HD patients. Results: Serum s(P)RR levels were significantly higher in HD patients (30.4±6.1 ng/ml) than those in subjects with normal renal function (16.5±4.3, P<0.0001). s(P)RR was detected in HD waste water by Western blot analysis, and the clearance of s(P)RR and creatinine were 56.9±33.5 and 147.6±9.50 ml/min, respectively. Serum (P)RR levels were significantly higher in those with ankle-brachial index (ABI) of < 0.9, an indicator of severe stenosis or obstruction of lower limb arteries, than those of ≧0.9 (32.2±5.9 and 30.1±6.2 ng/ml, respectively; P<0.05). The association between low ABI and high serum s(P)RR levels were observed even after the correction with age, history of smoking, HbA1c, and LDL-C. Conclusions: Serum s(P)RR levels are significantly higher in HD patients when compared with subjects with normal renal function, although s(P)RR are dialyzed to some extent. High serum s(P)RR levels may be associated with atherosclerosis independent of other risk factors, suggesting that serum s(P)RR could be used as a marker for atherosclerotic condition in HD patients.
Abstract P149: Distinct Profiles of Brain Medullary Metabolites Detected by
1
H-Magnetic Resonance Spectroscopy Correlate with Indices of Autonomic Function and Visceral Fat in Healthy Adults
Authors: Diz Debra I , Olson John , Bourland J. Daniel , Kitzman Dalane , Carr John Jeffrey , Kritchevsky Stephen B , Groban Leanne , Shaltout Hossam A
Published: March 2022

Higher forebrain myoinositol (mIns), a marker of glial inflammation/proliferation as detected by proton magnetic resonance spectroscopy (MRS), correlates with systemic inflammation. Elevated circulating markers of inflammation are associated with lower baroreceptor reflex sensitivity (BRS) and heart rate variability (HRV) as well as visceral fat or central obesity. To determine whether transmitters/metabolites in a cardiovascularly (CV) relevant brain region correlate with age-related declines in BRS and HRV and markers of abdominal fat, autonomic profiles were determined by spectral and sequence analysis from continuous blood pressure and HR values in the supine position of healthy subjects 22 - 76 yrs old (12F, 4M); subjects later underwent a single voxel (10 x 7 x 20mm) proton MRS scan of dorsal medulla on a 3T magnet (n = 11; 9F, 2M) and measures of abdominal fat by computerized tomography (CT) (n = 9; 7F,2M). The mean arterial pressure was 88 ± 3 mm Hg, HR 64 ± 3 beats/min and BMI 27 ± 1 kg/m
2
. Glutamate (Glu) correlated directly with vagal (HF
RRI
r = 0.72, p < 0.02) and inversely with sympathetic (LF
RRI
r = -0.72, p < 0.02) control of HR. Markers of Glu metabolism and neuronal integrity/activity, N-acetyl-aspartate acid (NAA) + N-acetyl aspartyl glutamate (NAAG), did not correlate with age, but did correlate inversely with BRS (Seq ALL: r = -0.69, P < 0.02), HRV (rMSSD: r = -0.76, p < 0.008) and directly with HR (r = 0.68, p < 0.03). Total visceral fat had a negative correlation with BRS (Seq Up: r = -0.70, p < 0.02). mIns and markers of demyelination and reduced axonal integrity such as Glycerophosphocholine (GPC) and total choline containing compounds (GPC+PCh) exhibited striking positive correlations with percent visceral fat (r = 0.97, 0.81 and 0.82, P < 0.02). BMI and GPC correlated with HR (r = 0.55, 0.72, p < 0.04), but neither these nor mIns or choline compounds correlated with autonomic function. Thus, in healthy adults, Glu concentration in dorsal medulla directly correlates with cardiac vagal function, whereas markers of Glu metabolism inversely correlate with BRS and HRV. In contrast, markers of glial inflammation directly associate with increases in visceral adiposity, but not autonomic dysfunction. P30-AG21332, Farley Hudson, Hypertension & Vasc Res Ctr
Abstract P163: Endothelin-1 induces Epithelial-Mesenchymal Transition (EMT) in Human Renal Tubular Cells via Activation of RhoA /ROCK Kinase and Inhibition of YAP Pathways
Authors: Seccia Teresa Maria , Caroccia Brasilina , Gioco Francesca , Piazza Maria , Buccella Valentina , Montini Barbara , Guidolin Diego , Guerzoni Eugenia , Pagnin Elisa , Ravarotto Verdiana , Calò Lorenzo , Belloni Anna Sandra , Rossi GianPaolo
Published: March 2022

We tested the hypothesis that tubulo-interstitial fibrosis (TIF), the final outcome of most kidney diseases, involves activation of epithelial mesenchymal transition (EMT) through angiotensin II- and endothelin-1 (ET-1)-mechanisms.

In a transgenic model of fulminant angiotensin II-dependent hypertension with early prominent renal damage, the TG(mRen2)27 rat (TGRen2), we found that the development of renal damage implied a decrease of the epithelial marker E-cadherin and an increase of the mesenchymal markers alpha SMA and vimentin, which indicated the occurrence of EMT. As treatment with the angiotensin II type 1 receptor antagonist irbesartan, or with the mixed ET-1 receptor antagonist bosentan, prevented these changes an involvement of both angiotensin II and ET-1 in EMT is suggested.

To confirm this contention we exposed HK-2 human kidney tubular cells to ET-1. This showed that ET-1 blunted the expression of E-cadherin, increased that of alpha SMA and vimentin, enhanced the synthesis of collagen, and also the activity of metalloproteinases (MMP). These changes implicated activation of Rho-kinase signaling pathway and de-phosphorylation of Yes-associated protein (YAP).

Hence, ex vivo and in vitro experiments demonstrated that EMT is a fundamental process in the TIF that accompanies the development of angiotensin II-dependent hypertension. Moreover, they suggested that EMT involves ET-1 acting via ETA and ETB receptors through activation of Rho-kinase and de-activation of YAP pathways.
Abstract P167: Conditional Deletion of the Na+/H+ Exchanger 3 in the Proximal Tubule of the Kidney Promotes Pressure Natriuresis in Mice
Authors: Li Xiao C , Soleimani Manoocher , Nguyen Hoang , Li Hong , Roman Richard J , Zhuo Jia L
Published: March 2022

The development and progression of most, if not all, forms of hypertension appear to converge on a final common pathway or mechanism, i.e., increased renal salt retention due to significantly impaired pressure natriuresis responses. Yet the factors responsible for resetting the pressure natriuresis response in hypertension remain to be determined. The present study tested the hypothesis that the upregulation of the sodium and hydrogen exchanger 3 (NHE3) impairs while selective knockout of this transporter in the proximal tubule promotes pressure natriuresis and lowers blood pressure in mice. Proximal tubule-specific NHE3 knockout mice (PT-NHE3-KO) were generated by the Cre/Lox approach, and confirmed by complementary PCR, Western blot, and immunofluorescent imaging of NHE3 expression in the proximal tubule of the kidney, respectively. No abnormal histological phenotypes were observed in small intestines and the kidney of PT-NHE3-KO mice. Compared with wild-type mice (WT, n=12), PT-NHE3-KO mice (n=10) had significantly lower basal systolic blood pressure (WT: 116 ± 3 mmHg versus PT-NHE3-KO: 103 ± 3 mmHg, p<0.01). Mean intra-arterial pressure was also significantly lower in anesthetized PT-NHE3-KO mice (WT: 91 ± 3 mmHg versus PT-NHE3-KO: 77 ± 5 mmHg, p<0.01) (n=8 each). The lower basal blood pressure in PT-NHE3-KO mice was associated with higher basal urine flow (WT: 0.81 ± 0.09 ml/day versus PT-NHE3-KO: 1.12 ± 0.06 ml/day, p<0.05), urinary sodium (WT: 120.1 ± 17.5 μmol/day versus PT-NHE3-KO: 197.1 ± 24.5 μmol/day, p<0.01), potassium (WT: 171.7 ± 26.0 μmol/day versus PT-NHE3-KO: 310.3 ± 24.3 μmol/day, p<0.01), and chloride excretion (WT: 125.7 ± 22.5 μmol/day versus PT-NHE3-KO: 222 ± 29.2 μmol/day, p<0.05) (n=8-12 for each group). In response to ~25 mmHg increase in renal perfusion pressure in both strains, urinary sodium excretion was increased by 4-fold in WT mice (n=12, p<0.01), whereas it increased by 7-fold in PT-NHE3-KO mice (n=10, p<0.01). We concluded that NHE3 in the proximal tubule of the kidney plays an important role in the regulation of proximal tubular sodium reabsorption and blood pressure homeostasis, and that selective deletion of NHE3 in the proximal tubule promotes the pressure natriurestic response and lowers blood pressure in mice.
Abstract P170: Portfolio Analysis on Sex and Gender Differences Research in Hypertension at the National Heart, Lung and Blood Institute
Authors: Maric-Bilkan Christine , Galis Zorina
Published: March 2022

Objectives:
While epidemiological studies show that blood pressure levels and the prevalence of hypertension are subject to sex-differences, the mechanisms responsible for this sexual dimorphism are poorly understood. To gain a better understanding of funding trends and topics in the field of sex/gender differences in hypertension research, we performed a portfolio analysis of National Heart, Lung and Blood Institute (NHLBI) awards related to “sex differences” active between FY1991-2014.

Analysis:
A list of NHLBI awards (including subprojects) active in FY1991-2014 was obtained through an NIH RePORTER search using the following terms: “sex differences”, “gender differences”, “X chromosome”, “Y chromosome”, “sex hormones”, “preeclampsia”, “pregnancy hypertension” and “menopause”. The abstracts and specific aims of all the awards were read by staff in order to eliminate “false positives” (grants unrelated to sex/gender differences). Applications were further analyzed and categorized according to specific disease focus (e.g. hypertension, cardiovascular disease, preeclampsia etc.).

Results:
The number of NHLBI research awards related to sex and gender differences in hypertension being funded has progressively increased from FY 1991 (40 awards) to 2014 (140 awards), for a cumulative total of 2231 awards. Similarly, the overall dollar investment has also progressively increased from $6.1 million (FY 1991) to $76.3 million (FY2014), for a cumulative $837 million. According to the disease focus, in FY 2014, only 15% of active awards directly relate to sex/gender differences in hypertension, with 55% of awards relating to cardiovascular disease, 19% to pregnancy-related hypertension and 11% to other diseases (e.g. pulmonary hypertension).

Conclusions:
NHLBI is funding an increasing number of awards related to sex/gender differences in hypertension. However, the majority of these awards are only broadly related to hypertension per se, more being related to other cardiovascular diseases and pregnancy-related conditions. Our findings warrant more detailed analyses to identify potential knowledge gaps in need of support to further this important research field.
Abstract P173: Serum Soluble and Placental (Pro)Renin Receptor in Normal and Preeclamptic Pregnancy
Authors: Choudhury Saiara , Stucker Jamie E , Reyes Michelle , Afroze Syeda H , Drever Nathan , Allen Steven R , Zawieja David C , Kuehl Thomas J , Suzuki Fumiaki , Uddin Mohammad N
Published: March 2022

Objective:
Preeclampsia (preE), a syndrome of hypertension, proteinuria and edema, has many elusive triggers. The renin-angiotensin system (RAS) has been implicated in preE pathogenesis. Recently, we have demonstrated that (pro)renin levels are increased in preE patients and that levels of (pro)renin and (pro)renin receptor ((P)RR) are elevated in a rat model of preE. It has also been demonstrated that high circulating levels of soluble (P)RR at delivery is associated with preE. We evaluated the placental expression of (P)RR in preE patients as well as in a nonhuman primate. We also evaluated the serum levels of soluble (P)RR in preE patients and in a rat model of preE.
Study Design:
(1) Blood and placental samples were collected from 15 normal pregnant (NP) and 15 preE consenting patients in an IRB approved prospective study. (2) We used NP rats ( n=10) and pregnant rats receiving weekly injections of desoxycorticosterone acetate and whose drinking water was replaced with 0.9% saline (preE, n=10). (3) The placental samples from owl monkey (both early and term, NP, n= 2) were collected. The (P)RR expression was measured both by western blotting (WB) and Immunohistochemistry (IHC) with anti-ATP6IP2 (detect (P)RR). The extracellular-signal-regulated kinase ½ (ERK ½) expression was evaluated by WB. The levels of serum soluble (P)RR were measured by a commercially available ELISA kit.
Results:
The placental expression of (P)RR was higher (p<0.05) in preE compared to NP patients. The ERK1/2 expression was higher (p<0.05) in preE placenta compared to NP. The soluble (P)RR levels were higher in preE (preE patients: 29.2 ± 4.5; PDS rats: 16.9 ± 1.9 ng/mL) compared to NP (NP human: 19.3 ± 4.2; NP rats: 10.4 ± 3.7 ng/mL). The early placenta of owl monkey expressed higher (P)RR compared to term.
Conclusions:
These data suggest that increased expression of (P)RR and ERK1/2 in preE placenta is related to the occurrence of preE. These data also reconfirmed that high level of circulatory soluble (P)RR is associated with preE. The higher expression of (P)RR in early pregnancy compared to term placenta in owl monkey suggests that the (P)RR is important for normal placental development. The expression of (P)RR in nonhuman primates reveals the approach of future studies on owl monkey preE model.
Abstract P175: Impairment of Autonomic Function Precedes Blood Pressure Elevation in Rat Model of Pre-eclampsia
Authors: Shaltout Hossam A , Yamaleyeva Liliya M , Bader Michael , Dechend Ralph , Brosnihan Bridget
Published: March 2022

Hypertensive disorders of pregnancy including pre-eclampsia affect about 5-7% of all pregnant women and can cause severe acute morbidity, long-term disability and death among mothers and babies. Impairment of heart rate variability predicts risk of cardiovascular disease and all cause mortality. In this study we investigated the hypothesis that impairment of the autonomic function at early time point during pregnancy precedes and may lead to the elevation of blood pressure.

To test this hypothesis, blood pressure and heart rate were recorded under 2% isoflurane in 7 day pregnant rats generated via mating of the transgenic female rat containing the human angiotensinogen gene with the male transgenic containing human renin (hREN), the hAGTхhREN rat (TgA, n=8) which develop increased blood pressure, proteinuria and increased sensitivity to angiotensin II in the last half of gestation. The reverse mating of male containing the human angiotensinogen gene with the female transgenic containing human renin (TgR, n=8) which do not develop preeclampsia, and pregnant SD (n=8) rats at day 7 of gestation were used as controls.

By the 7th day of pregnancy, TgA rats had significantly impaired heart rate variability measured as root of mean successive differences (rMSSD) compared to SD and TgR (2.39 ±0.3 ms vs. 3.4 ± 0.2 in SD or 3.4 ±0.1 in TgR) and impaired baroreflex sensitivity measured as HF alpha (1.2±0.3 ms/mmHg vs. 2.5±0.4, SD or 1.7±0.26, TgR)

There was no difference in systolic arterial pressure or heart rate among the three groups at this time point but diastolic pressure was higher in TgA and TgR (91± 3.5 vs. 85±2.4 or vs. 75 ±2 mmHg in SD rats)

Although the predictive value of impaired baroreflex and heart rate variability in preeclampsia development needs further investigation, our findings suggest that these changes at early pregnancy precede and may contribute to the significant rise in pressure in the last half of gestation in this rat model of pre-eclampsia which may have significant clinical implications.
Abstract P176: Predicting Preeclampsia Using Copeptin in Women at Low Risk of Disease
Authors: Santillan Donna , Majors Benjamin , Scroggins Sabrina , Devor Eric , Zamba Gideon , Pierce Gary , Leslie Kimberly , Sigmund Curt , Grobe Justin , Santillan Mark
Published: March 2022

Preeclampsia annually kills 76,000 mothers and 500,000 babies worldwide often due to delay in diagnosis secondary to the lack ofsimple, early gestation tests. Elevated circulating copeptin (CPP), the pro-segment of vasopressin, is associated with preeclampsia (PreE). We have demonstrated that CPP is robustly predictive of PreE as early as the 6
th
week of gestation in all mothers. Development of PreE is increased 3 fold by a history of PreE. Currently, no test robustly predicts PreE in women without a history of PreE. To evaluate if CPP is predictive in this low risk setting when a predictor is most needed, a nested case-control study was performed to evaluate the predictive characteristics of CPP of women with and without a history of PreE. Maternal plasma CPP concentrations throughout gestation were measured by ELISA. Univariate comparisons were performed. Receiver operating characteristic (ROC curves were constructed to determine sensitivity, specificity, positive and negative predictive values for particular cutoffs. Multivariable logistic regression was performed to control for confounding to examine if CPP was significantly predictive of PreE. Apart from a difference in prior history of PreE, no significant demographic or clinical differences were observed between groups. In all trimesters, CPP predicted PreE similarly or better in women with no history of PreE as evidenced by an elevated ROC Area Under the Curve in comparison to values of women with a history of Pre (1
st
trimester: 0.96 vs. 0.85; 2
nd
trimester: 0.95 vs. 0.94 ; 3
rd
trimester: 0.82 vs. 0.67; p<0.05). Despite controlling for significant covariates such as maternal age, BMI, diabetes, chronic hypertension, and twin gestation, logistic modeling demonstrate that trimester specific CPP cutoffs throughout gestation are significantly associated with the development of PreE in women with no history of PreE (all models p< 0.001). Our data clearly support copeptin as an early predictor of preeclampsia in a low risk cohort. The ability to predict PreE in a low risk cohort with CPP is clinically significant as women in whom the diagnosis of preeclampsia is delayed or missed may now receive the appropriate interventions.
Abstract P179: miR-214 Reduces Termination Efficiencies of Alternative Transcriptional Termination Sites
Authors: Liu Yong , Yang Chun , Liu Pengyuan , Liang Mingyu
Published: March 2022

Alternative polyadenylation signals are genomic sites where transcription can be terminated, resulting in the production of transcripts often with shorter 3’ sequences. While it is clear that alternative transcriptional termination impacts the action of microRNAs, it is not known whether the reverse is true. We identified miR-214, a microRNA upregulated in the kidneys of Dahl salt-sensitive rats, as a potent regulator of the use of alternative transcriptional termination sites. miR-214 targeted human and rat cleavage and polyadenylation specific factor 4 (CPSF4), a component of the CPSF complex that plays a key role in transcriptional termination. miR-214 reduced the termination efficiency of the classic alternative termination site in human cyclin D1 gene from 66% to 47% (P<0.05). Of genes in HeLa cells that showed large abundance shifts between transcript isoforms with shorter and longer 3’ sequences in response to miR-214, a significantly greater portion (69%, 35 of 51 pairs ) shifted to longer isoforms (P<0.01). Interestingly, an alternative polyadenylation site was predicted and confirmed in the genomic region upstream of the sequence encoding miR-214 itself. The use of the alternative polyadenylation site correlated with decreased miR-214 abundance in rat tissues in vivo. The termination efficiency of the alternative termination site was reduced from 60% to 32% (P<0.001) by miR-214. The termination efficiency was not reduced by miR-214 if CPSF4 had been knocked down by siRNA. These data indicate that miR-214 can relieve alternative polyadenylation-dependent transcriptional termination signals including one that influences its own expression.
Abstract P183: Myeloid-Derived Suppressor Cells Prevent Cyclosporine A-Induced Hypertension, Endothelial Dysfunction, and Renal Injury in Mice
Authors: Chiasson Valorie L , Bounds Kelsey R , Pakanati Abhinandan R , Aziz Bilal , Roman Marcos H , Mitchell Brett M
Published: March 2022

Cyclosporine A (CsA) is an immunosuppressive drug used to treat focal segmental glomerulosclerosis, reduce autoimmune diseases, and prevent allograft rejection; however a limitation of CsA is that it can induce vascular and renal injury as well as hypertension. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature granulocytes, macrophages, and dendritic cells that suppress pro-inflammatory immune responses by secreting anti-inflammatory cytokines, inhibiting innate and adaptive immune cells, and inducing regulatory T cells (Tregs). We hypothesized that adoptive transfer of MDSCs would prevent CsA-induced vascular and renal injury and hypertension. Daily treatment of male C57BL6/J mice for 1 week with CsA (50 mg/kg/day, i.p. injection) significantly increased systolic blood pressure (Day 7 SBP in mmHg: Con=97±2 vs. CsA=145±3, p<0.05 vs. Con), decreased aortic endothelium-dependent relaxation responses, increased aortic fibronectin levels, increased renal glomerular mesangial expansion, increased renal fibronectin levels, and decreased splenic Treg levels. Adoptive transfer of 1 million MDSCs by i.p. injection on days 1, 4, and 7 partially prevented the CsA-induced rise in systolic blood pressure (CsA+1M MDSCs=120±3 mmHg) and the detrimental vascular and renal effects. However, adoptive transfer of 2 million MDSCs fully prevented the CsA-induced hypertension (CsA+2M MDSCs=105±1 mmHg) and vascular and renal effects. The anti-hypertensive and vascular and renal protective effects of 2 million MDSCs were independent of Treg induction as splenic Treg levels remained significantly decreased. These data suggest that MDSCs can prevent the hypertension and toxicity caused by CsA independent of Tregs and may be a therapeutic target for CsA-treated patients.
Abstract P185: Cardiometabolic and Inflammatory Biomarkers in the Dahl Salt-sensitive Rat
Authors: Igreja Bruno , Pires Nuno , Wright Lyndon C , Soares-da-Silva Patrício
Published: March 2022

The Dahl salt-sensitive (Dahl/SS) rat is a genetic model of salt-sensitive hypertension that develops left ventricle (LV) hypertrophy after five to six weeks and cardiac failure with LV dilation and contractile dysfunction after 10 to 12 weeks of high-salt intake. The aim of the present study was to evalute cardiometabolic and inflammatory biomarkers in the Dahl/SS rat fed a normal-salt diet (NS-0.3% NaCl) and a high-salt diet (HS-8% NaCl).

For that purpose, eight week-old Dahl/SS rats were randomized into two groups, one group received a HS and the other a NS diet for 10 weeks. In the last week of treatment, 3 out of 10 animals from the HS diet group died. All animals from the NS diet group survived (n=8). The high-salt diet induced heart and kidney hypertrophy, as shown by higher heart/body weight (3.86±0.05 vs 2.96±0.05 mg/g, p<0.05) and kidney/body weight ratios (5.23±0.29 vs 3.41±0.06 mg/g, p<0.05), as compared to the NS diet group counterparts. Rats in the HS diet group presented proteinuria (273.1±26.8 vs 34.5±2.6 mg/day, p<0.05), hypercholesterolemia (4.45±0.23 vs 3.07±0.22 mmol/l, p<0.05), reduced levels of free fatty acids (0.23±0.02 vs 0.35±0.02 mmol/l, p<0.05) and normal levels of triglycerides in plasma (1.87±0.29 vs 1.28±0.15 mmol/l, p>0.05), as compared to animals on a NS diet. Insulin plasma levels in the HS group were lower than in the NS group (1.82±0.13 vs 4.03±0.40 ng/ml, p<0.05), though the glucose plasma levels were unchanged (162±5 vs 150±7 mg/dl, p>0.05). The plasma levels of monocyte chemoattractant protein (MCP)-1 were higher in the HS than in LS diet group (105.0±10.4 vs 67.6±2.8 pg/ml, p<0.05). A multiplex inflammatory cytokine assay was used to profile expression of 23 inflammatory mediators. The plasma levels of nine inflammatory mediators, including IL-1β, IL-4 and VEGF, were significantly increased in rats in the HS group.

In conclusion, HS diet feeding in the Dahl/SS rat deteriorate cardiometabolic and inflammatory biomarkers.
Abstract P195: Transforming Growth Factor beta 1-Mediated Repression of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene Expression and Function Involving TGF-beta 1 and Delta EF1 in Vascular Smooth Muscle Cells
Authors: Sen Anagha , Kumar Prerna , Pandey Kailash N
Published: March 2022

The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPR-A) produces second messenger cGMP, which lowers blood pressure and prevents cardiovascular events. The objective of the present study was to examine the repressive effect of transforming growth factor (TGF-β1) in the regulation of Npr1 (coding for GC-A/NPRA) gene expression and function. The rat thoracic aortic vascular smooth muscle cells (RTASMC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and treated with TGF-β1. The luciferase assay results showed that TGF-β1 significantly repressed Npr1 promoter activity in a dose- and time-dependent manner by 82% and 85% (2.5 ng/ml, 25.51 ± 2.2 and 24 h, 7.0 ± 0.6 vs. untreated control 70.06 ± 4.6, p < 0.001, respectively). Treatment with TGF-β1 decreased NPRA mRNA and protein levels by 62% (treated, 0.42 ± 0.05 vs. control, 0.9 ± 0.02, p < 0.01) and 55% (treated, 9603 ± 860 vs. untreated, 22211 ± 1449, p < 0.01), respectively. TGF-β1 attenuated ANP-dependent intracellular accumulation of cGMP by 59% (TGF-β1 + ANP-treated, 8.66 ± 0.9 pmol/1x 106 cells vs. ANP-treated cells 23.51 ± 2.2; p < 0.001). Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TGF-β1 enhanced the recruitment of transcription factor delta EF1 (δEF1) to form a transcriptional repressor complex with their binding sites in Npr1 promoter. Western blot analysis showed significant increase in δEF1 protein expression by 2.4-fold (treated, 907.9 ± 36.5. vs. untreated, 378.5 ± 10.3; p < 0.001) and phosphorylation of mothers against decapentaplegic homolog 2/3 (SMAD2/3) proteins by 2.3-fold (treated, 620.9 ± 10.6 vs. untreated, 269 ± 9.7; p < 0.01) in TGF-β1-treated cells. Collectively, the present results demonstrate that TGF-β1 represses Npr1 gene transcription and expression by interactive actions of δEF1 and phosphorylated SMADs. Together, the present results suggest that TGF-β1 and δEF1 inhibit Npr1 gene expression and function, which may be critical in regulating the blood pressure and cardiovascular homeostasis.
Abstract P196: Chemerin Activates Procontractile Pathways in Isolated Arteries
Authors: Darios Emma , Thompson Janice , Wren Nguyen , Torres Rosa , Kelly Aisha , Watts Stephanie W
Published: March 2022

In the vasculature, the adipokine chemerin is present in perivascular adipose tissue and causes arterial contraction through activation of the heptahelical receptor ChemR23. We presently test the hypothesis that ChemR23 is linked to procontractile signaling pathways in isolated arteries. The isolated, endothelium-denuded thoracic aorta of both male and female rats was used for measurement of isometric contraction and biochemical analyses. The peptide analog chemerin-9 (1 nm -3 μM) caused concentration-dependent contraction in both isolated aorta from male and female, with a maximum of ~80% of a phenylephrine (10 μM) contraction. The L type voltage dependent calcium channel inhibitors verapamil (1 μM) and nifedipine (1 μM), as well as the protein kinase C (PKC) inhibitor chelerythrine chloride (10 μM), abolished chemerin-9 induced contraction relative to vehicle-incubated tissues (male and female). Next most effective in inhibiting chemerin-9-induced contraction were the Rho Kinase inhibitors Y27632 (1 μM) and fasudil (1 μM), both effective in male and female aorta. The p38 MAPK inhibitor SB203850 (10 μM) and phosphoinositide-3-kinase inhibitor LY294002 (10 μM) were significantly less effective in reducing chemerin-9-induced contraction in both sexes, reducing maximum contraction in male and female by less than 50%. Finally, the phospholipase C inhibitor U73122 (5 μM) and Erk MAPK inhibitor PD098059 (1 μM) did not shift or reduce chemerin-9-induced contraction compared to control. Western analyses of chemerin-9-contracted tissues validated activation of phosphoinositide-3-kinase and lack of activation of the Erk MAPKs. These data are the first to associate ChemR23 with L type channel and Rho Kinase activation, and support the hypothesis that ChemR23 taps into some but not all of the well-recognized contractile pathways. This is important knowledge for understanding the mechanisms by which chemerin could support obesity-associated hypertension
Abstract P197: Extracellular Ubiquitin Blocks CXCL12-Induced Proliferation of Cardiac Fibroblasts
Authors: Jackson Edwin K , Mi Eric , Ritov Vladimir B , Gillespie Delbert G
Published: March 2022

CXCR4 receptors mediate in part hypertension-induced cardiac fibrosis. This suggests that endogenous CXCR4-receptor agonists stimulate cardiac fibroblast (CF) proliferation; however, this possibility is unexplored. Although CXCL12 (aka SDF-1α) is the best described endogenous CXCR4 agonist, ubiquitin
1-76
(U-76) exists in the extracellular compartment (10 to 100 nM) and is reported to be a CXCR4 agonist. Therefore, we investigated the ability of both CXCL12 and U-76 to stimulate growth of rat CFs. Low concentrations of CXCL12 (10 nM x 4 days) increased CF proliferation (from 38,973 ± 384 to 44,429 ± 774 cells/well, n=6, p<0.0001), and this effect was augmented by sitagliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor (% increase by CXCL12: without sitagliptin, 14 ± 2 (n=6); with 1 μM of sitagliptin, 38 ± 9, n=6, p<0.0001). This finding is consistent with the known ability of DPP4 to metabolize CXCL12, i.e., sitagliptin inhibits the metabolism of CXCL12 and enhances CXCL12-induced effects. Not only did U-76 at low physiological concentrations (10 nM) not stimulate CF proliferation, U-76 surprisingly nearly abolished CF proliferation induced by CXCL12 + sitagliptin (% increase by CXCL12 + sitagliptin: without U-76, 58 ± 5, n=12; with U-76, 10 ± 7, n=12, p<0.0001). Ubiquitin
1-74
(U-74), a metabolite of U-76, inhibited the pro-growth effects of CXCL12 + sitagliptin 10-fold more potently than did U-76. CFs expressed insulin degrading enzyme (IDE) mRNA (qRT-PCR), protein (western blot), and activity (IDE activity assay) and inhibition of IDE with the newly discovered potent IDE inhibitor 6bK (1 μM) prevented U-76 from blocking the growth effects of CXCL12 + sitagliptin. Analysis by mass spectrometry (selected ion monitoring) demonstrated that CFs converted U-76 to U-74 and this conversion was attenuated by 6bK. Conclusions: CFs express IDE and therefore convert U-76 to U-74; U-74 blocks CXCR4 receptors thus protecting against CXCL12 + DPP4 inhibitor induced CF proliferation. Implications: In patients with low IDE activity (due to genes or drugs) who are treated with DPP4 inhibitors, U-74 (CXCR4 antagonist) levels would be low and CXCL12 (CXCR4 agonist) levels would be high, thus possibly creating a “perfect storm” for CF over-proliferation and cardiac fibrosis.
Abstract P202: Angiotensin Receptor-binding Protein ATRAP as a Possible Candidate for Therapeutic Strategy of Functionally Selective Modulation of Angiotensin Receptor Signaling
Authors: Tamura Kouichi , Wakui Hiromichi , Azushima Kengo , Uneda Kazushi , Kobayashi Ryu , Ohki Kohji , Haku Sona , Haruhara Kotaro , Kinguchi Sho , Ohsawa Masato , Maeda Akinobu , Kanaoka Tomohiko , Umemura Satoshi
Published: March 2022

Drug discovery targeting GPCRs is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. Therefore, if possible, functionally selective modulation of receptor signaling may be a safer, better tolerated, and more efficacious therapeutic strategy. Recent research progress is reported in the field of functional selectivity such as receptor biased ligands, and receptor binding molecules for the therapy of cardiovascular and renal pathophysiology.

The renin-angiotensin system including plays a critical role in the regulation of cardiovascular and renal function in physiological homeostasis via activity of its effector AT1R. While exaggerated activation of AT1R promotes organ damages by BP elevation and insulin resistance via enhancement of oxidative stress, inflammation and fibrotic response, genetic total inactivation of the renin-angiotensin system components, such as angiotensinogen, renin and AT1R, reportedly results in significant hypotension and in renal morphological alteration even under baseline condition from birth, indicating that baseline AT1R signaling activity is indispensable for the maintenance of cardiovascular and renal physiology.

In the course of an investigational search for a sophisticated means to regulate AT1R signaling at local tissue sites, we have focused our analysis on the AT1R-associated protein (ATRAP;
Agtrap
gene), which is a molecule that directly binds to the carboxyl[[Unable to Display Character: ‐]]terminal domain of AT1R. In contrast to the classical components of the renin-angiotensin system (i.e. angiotensinogen, renin and AT1R), alteration of ATRAP expression exerts no evident effects on baseline BP and renal morphology
in vivo
such as in ATRAP-transgenic mice and ATRAP-deficient mice. However, accumulating results in these mice indicate that ATRAP exerts inhibitory effects on the exaggerated activation of tissue AT1R signaling in response to pathological stimuli, in order to protect cardiovascular and renal tissues under pathological stimuli, in spite of no influence of ATRAP on physiological AT1R signaling.
Abstract P203: ICV Angiotensin-(1-7) Decreased TNF-α and Increased IL-10 in the Hypothalamus of (mRen2)27 Transgenic Hypertensive Rats
Authors: Kangussu Lucas M , Oliveira Marilene L , Santos Robson A , Campagnole-Santos Maria J
Published: March 2022

Hypertensive rats subjected to chronic intracerebroventricular (ICV) infusion of angiotensin-(1-7) [Ang-(1-7)] presented attenuation of arterial hypertension, improvement the baroreflex sensitivity and restoration of cardiac autonomic tonus. In the present study we evaluated whether chronic increase in Ang-(1-7) in the brain modulates inflammatory mediators in the hypothalamus of the transgenic hypertensive rats that present overexpression of renin [(mRen2)27; TGR]. Sprague Dawley (SD) and TGR were subjected to 14 days of ICV infusion of Ang-(1-7) (200 ng/h) or 0.9% sterile saline (0.5 μl/h) through osmotic mini-pumps. The animals were euthanized by decapitation and the hypothalamus was quickly removed and frozen on dry ice. Cytokine levels were evaluated through Elisa assay and enzymes of RAS were measured by fluorimetric assays. As expected, levels of pro-inflammatory cytokines (IL-1α, IL-6 and TNF-α) were increased in TGR (48 ± 3.5 pg/mg, 61 ± 2.7 pg/mg, 76 ± 4.3 pg/mg, respectively) as compared to SD rats (23 ± 1.8 pg/mg, 38 ± 3.4 pg/mg, 39 ± 3.5 pg/mg, respectively), while IL-10 was not altered. Interestingly, ICV infusion of Ang-(1-7) reduced levels of TNF-α (48 ± 3.4 pg/mg vs 76 ± 4.3 pg/mg in untreated TGR) and increased the levels of IL-10 (32 ± 2.6 pg/mg vs 19 ± 1.2 pg/mg in untreated TGR), without affecting IL-1α or IL-6 levels. No difference was found in ACE activity in plasma, on the other hand, the increased ACE activity in the hypothalamus of TGR (207 ± 25.5 nmoles His-Leu/ min/ mg of protein vs 173 ± 13.0 nmoles His-Leu/ min/ mg of protein, in SD rats) was significantly reduced (134 ± 7.9 nmoles His-Leu/ min/ mg of protein) by ICV infusion of Ang-(1-7). These data show that long term increase in Ang-(1-7) levels in the brain modulates inflammatory mediators in the hypothalamus, suggesting a possible additional mechanism for Ang-(1-7) antihypertensive action in the central nervous system.
Abstract P206: Structural Analysis of Glycosyl Chain at 14th Amino Acid Of Human Angiotensinogen in Plasma
Authors: Nagata Sayaka , Tokashiki Mariko , Kitamura Kazuo
Published: March 2022

The Renin angiotensin system (RAS) is a major regulator of body fluid balance and control of blood pressure. The protease enzyme renin secreted from kidneys cleaves speci[[Unable to Display Character: ﬁ]]cally angiotensinogen (Aogen) circulating in the blood to produce angiotensin I (Ang I). Human Aogen is a heterogeneous glycoprotein constitutively secreted by the liver. In addition, human Aogen contains four putative asparagine-linked glycosylation sites (Asn 14, 137, 271, 295) and contains four cysteines (Cys 18, 138, 232, 308), with Cys18 and Cys138 linked by a disulfide bridge. The glycosyl chains and cysteines position are very important for binding of the renin.

Big angiotensin-25 (Bang-25) is a consisting of 25 amino acids with glycosyl chain (14th amino asid) and added cysteine (18th amino acid), which we recently isolated from human urine. To compare glycosyl chain of Bang-25 and Aogen, we analyzed of structure glycosyl chain at position 14th amino acid of human Aogen in plasma.

To determine of glycosyl chain at position 14th amino acid, we performed lysyl endopeptidase digestion and reduction on human plasma Aogen. Then, Aogen after digest was purified by reverse-phase high-performance liquid chromatograpy (RP-HPLC), and glycosyl chain structure analyzed by the two-/three-dimensional HPLC mapping method.

We show that plasma Aogen has three types of glycosyl chain at position 14th amino acid. One glycosyl chain structure is identical to Bang-25 in urine. N-linked glycosylation on 14th amino acid of Aogen plays an important role about renin reaction. In addition, Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleave by renin. These results suggest that the structure of the glycosyl chain at position 14th amino acid of the human Aogen may be involved in the substrate specificity for renin or chymase.
Abstract P212: A Meta Analysis of Somatic KCNJ5 Mutations in 1636 Primary Aldosteronism Patients
Authors: Rossi Gian Paolo , Lenzini Livia , Rossitto Giacomo , Maiolino GIuseppe , Letizia Claudio , Funder John
Published: March 2022

Background:
We meta-analysed the available studies reporting on KCNJ5 mutations in Aldosterone Producing Adenoma (APA) to determine the clinical characteristics of APA patients with a mutation of the KCNJ5 gene.

Methods:
We applied the PICO strategy using predefined terms (Population: primary aldosteronism patients with aldosterone producing adenoma; Intervention: adrenalectomy, sequencing for KCNJ5 mutations; Control: APA without KCNJ5 mutations; Outcome: clinical and pathological correlates of KCNJ5 mutations) to extract relevant studies from the PubMed, Scopus, Web of Science e Cochrane databases until January 2015. To allow for independent replication of the results, we elected to use the commercially available software.

Results and Conclusions:
By this PICO strategy we could identify 13 studies involving a total of 1636 patients (age 49 years ± 4; 55% females). The overall prevalence of KCNJ5 mutations was 43%; it was lower frequency (p<0.003) in European, USA and Australian studies (35%) than in Japanese and Chinese studies (63%), and correlated (r=0.70, p=0.008) with the mean daily urinary sodium excretion. The meta-analysis comparison of wild-type and KCNJ5 mutated patients demonstrated that the latter were younger (45±3 vs 52±5 years), had higher plasma aldosterone levels (42±8 vs 33±8 ng/dl), bigger tumors (16.1±6.4 vs 14.9±7.4 mm) and were more females (67% vs 44%) (p<0.05 for all). At variance, no significant effect of KCNJ5 mutations on systolic and diastolic blood pressure, and on serum potassium could be found.

Therefore, the meta-analysis of a large dataset comprising all studies available thus far showed that features associated with the presence of KCNJ5 mutations in PA due to APA entail young age, female gender, bigger tumor size and more prominent hyperaldosteronism.