bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 416: Chloride Ion Contributes to the Hypertension Induced by Central NaCl
Authors: Van Huysse James , Hou Xiaohong , Wu Hengwei , Lingrel Jerry B
Published: July 2021

Previous studies in salt-sensitive animal strains suggest that an increase in the level of sodium in the cerebrospinal fluid (CSF [Na]) is crucial for the development of hypertension induced by a high salt diet. Although chronic intracerebroventricular (icv) infusions of NaCl-rich artificial CSF (NaCl-rich aCSF) are frequently used to study the mechanisms of the hypertension caused by high salt diet and/or elevated CSF [Na], the contribution of chronic increased CSF [Cl] to the hypertensive response has not been examined. In the present study, we compared the effects on blood pressure of chronic (10 day) icv infusions of NaCl-rich aCSF vs. similar infusions in which excess Na+ or Cl- was substituted with another ion in wild-type (WT) mice and in knockout/knockin mice with a ouabain-resistant
endogenous
Na, K-ATPase α2 subunit (α2
R/R
). The latter mice are known to lack a pressor response to icv NaCl-rich aCSF. Mean arterial pressure (MAP) and heart rate (HR) on day 10 of infusion were (mean ± S.E.M., n = 4-5 per group:

These studies suggest that both elevated CSF [Na] and CSF [Cl] contribute to the pressor response to increased CSF [NaCl] via a common mechanism: binding of endogenous ouabain to the Na, K-ATPase α2 isoform.
Abstract 421: The Subfornical Organ (SFO) Mediates CSF Na+ -induced Pressor Response via Activation of AT1 Receptors
Authors: Tiruneh Missale A , Huang Bing S , Leenen Frans H
Published: July 2021

In salt-sensitive rats on high salt or rats with icv infusion of Na
+
, the increase in CSF [Na
+
] leads to activation of the brain renin-angiotensin-aldosterone system and thereby to sympatho-excitation and hypertension. We tested whether the SFO and AT
1
receptors in the SFO play a crucial role in mediating the Na
+
-induced responses. In conscious Wistar rats, intra-SFO infusion of Na
+
-rich aCSF increased BP in a dose-related manner, whereas mannitol with the same osmolarity had no effects. Intra-SFO infusion of the AT
1
receptor blocker candesartan (cand.,10 μg) abolished pressor responses to intra-SFO infusion of Ang II (80 ng) or Na
+
-rich aCSF (0.45-0.6 M NaCl), and prevented 50% of the BP increase induced by icv infusion of Na
+
-rich aCSF (0.3 M NaCl, 4 μl/min for 6 min). In another set of Wistar rats, electrolytic lesion of the SFO prevented 50-65% of BP increases induced by icv infusion of Na
+
-rich aCSF or Ang II (5 ng/min). These data suggest that the SFO neurons are Na
+
-sensitive and via AT
1
receptors mediate a major part of the pressor response to CSF Na
+
.

Data=means±SE (n=5-7). *p<.05 vs vehicle or sham lesion.
Abstract 423: Associations Between Improved Fitness and a Marker of Hypertension Associated Neuro Degenerative Disease S 00B
Authors: Kretzschmar Jan , Feairheller Deborah L , Sturgeon Kathleen , Diaz Keith M , Williamson Sheara T , Veerabhadrappa Praveen , Ling Chenyi , Perkins Amanda , Grimm Heather , Lee Hojun , Babbitt Dianne , Crabbe Deborah L , Kashem Abul M , Brown Michael D
Published: July 2021

Neurodegenerative diseases such as Alzheimer’s have recently been linked to hypertension (HTN). S100B is a marker of cerebral injury and the progression of neuro-degeneration. Serum levels of S100B are known to be increased after acute brain trauma, with the progression of chronic neuro-degenerative diseases, and even in the presence of HTN. Optimization of aerobic fitness has long been implicated in the treatment and prevention of HTN. However, no study has investigated whether aerobic exercise training (AEXT) has an effect on the prevention of hypertension associated neurodegenerative disease markers such as S100B.

METHODS:
18 participants ages 40-62 were assessed. To be considered for inclusion participants had to be free of any cardiovascular disease, non-smoking and sedentary. After inclusion participants underwent blood draws, blood pressure (BP) measurements, flow mediated dilation (FMD) analysis using Doppler ultrasound and aerobic exercise testing by measuring VO2max. Participants then followed AEXT (3days/week, 65% of VO2max, 40 minutes/day) for 6 months. S100B serum levels were assessed using an ELISA Assay (Milipore, USA).

Results:
AEXT did not significantly change serum S100B levels or BP. However, VO2max fitness levels (24.55 ± 1.01 vs. 27.52 ± 1.59 mL/kg/min) and FMD percentage (6.47 ± 0.9 vs. 9.75 ± 1.09 %) increased significantly (p≤0.008 and p≤0.048 respectively). Although there were no significant correlations between S100B and the above variables before and after AEXT, S100B levels were inversely correlated with VO2max after the six months of AEXT (r = -.594, p≤0.009). Also, participants who improved their VO2max level by at least 3ml/kg/min (N=10) had significantly (p≤0.008) lower S100B levels (45.43 ± 11.9 vs. 64.19 ± 14.16 pg/mL) after the completion of AEXT than participants who did not (N=8).

Conclusion:
Although AEXT did not lower BP, it did improve endothelial function. Also, people who responded to AEXT, had significantly better S100B levels. Additionally, the fact that S100B was inversely correlated to VO2max, as was the case after AEXT, implicates a potential role for the improvement of aerobic fitness in the prevention of HTN-associated neurodegenerative diseases.
Abstract 425: High Sodium Excretion is not Associated with Reduced Vasodilation in Healthy African American Adults
Authors: Ling Chenyl , Diaz Keith M , Kretzschmar Jan , Feairheller Deborah L , Sturgeon Kathleen M , Williamson Sheara T , Veerabhadrappa Praveen , Perkins Amanda , Lee Hojun , Crabbe Deborah L , Kashem Abul , Grimm Heather , Brown Michael D
Published: July 2021

Background:
High dietary sodium (Na) intake has been shown to be related to the development of endothelial dysfunction and hypertension. Urinary sodium excretion in people with normal renal function can indirectly reflect the level of dietary sodium intake. The purpose of the study was to test whether high urine Na excretion was independent from other risk factors and related to the compromised endothelial-dependent (fiow-mediated dilation [FMD]) and/or endothelium-independent (nitroglycerin-induced dilation [NMD]) in normotensive African Americans. Normotensives were studied in order to eliminate the influence of hypertension.

METHODS:
Urine was collected for 24 hr in 30 subjects aged 41-63 yrs with normal blood pressure (125.7 ± 9.7/81 ± 7.1 mmHg). 16 subjects had Na excretion <150 mmol (99.6 ± 24.8 mmol) and were classified as the Normal Na excretion group and 14 subjects had Na excretion ≥150 mmol/g (186.5 ± 27.1 mmol) and were classified as the High Na excretion group. FMD was measured after 5-min cuff occlusion and NMD was measured after application of 0.8 mg nitroglycerin using Doppler ultrasound. The maximum FMD- and NMD-diameters and FMD/NMD ratio were calculated.

Results:
There were no differences in other dietary factors, age, body mass index, blood pressure, or physical activity levels between the groups. Plasma concentrations of cholesterol, glucose, antioxidant status and inflammatory markers did not differ between groups. FMD (Normal: 7.4 ± 2.9% vs. High: 9.5 ± 3.6%; p=0.09) and NMD (Normal: 17.9 ± 6.3% vs. High: 27.8 ± 18.1%; p=0.07) and true peak FMD/NMD ratio (Normal: 0.45 ± 0.22 vs. High: 0.38 ± 0.11; p=0.26) were not different between groups. No significant correlations were found between 24hr Na excretion and FMD (r=0.332; p=0.073), NMD (r=0.356; p=0.053) or FMD/NMD ratio(r=0.112; p=0.554) in the entire study group.

Conclusion:
High Na excretion was not associated with reduced endothelial responsiveness in healthy African Americans with normal blood pressure. These results suggest that high Na intake may not be predictive of endothelial dysfunction in putatively healthy populations.
Abstract 454: Endoplasmic Reticulum Stress Response in Pressure-Overloaded Ischemic-Reperfused Hearts
Authors: Mozaffari Mahmood , Liu Jun Yao , Baban Babak
Published: July 2021

An integral component of endoplasmic reticulum (ER) stress-induced apoptosis is expression of growth arrest- and DNA damage inducible protein 153 (GADD153); this protein is normally expressed in low levels but its expression markedly increases following sustained stress to the ER. GADD153 regulates both apoptosis and inflammatory response. We previously showed that pressure overload exacerbates myocardial ischemia reperfusion (IR) injury. In this study, we tested the hypothesis that pressure overload regulates ER stress response manifested as increased GADD153 expression thereby upregulating inflammatory cytokine production and contributing to worsening of myocardial IR injury. Accordingly, Langendorff-perfused rat hearts were subjected to global IR protocol with perfusion pressure set at 80 or 160 cmH
2
O; normoxic hearts served as controls. Compared to normoxia, an IR insult increased expressions of pro-inflammatory cytokine (interleukin (IL)-17) and GADD153 in association with increased cell death. In the ischemic-reperfused hearts, pressure overload a) increased expression of GADD153, b) reduced interleukin (IL)-10 but increased IL-17 and c) increased annexin V immunostaining as well as apoptotic and necrotic cell death. Collectively, the results suggest that pressure overload exacerbates cell death in the isolated ischemic-reperfused heart involving regulation of ER stress response and inflammation.
Abstract 476: Renal Sensory Reinnervation Following Acute Renal Denervation.
Authors: Kopp Ulla , Mulder Jan , Hölt Tomas , Knuepfer Mark M
Published: July 2021

It is known that efferent sympathetic nerves reinnervate the kidney following renal denervation (DNX). As there is evidence for lack of cardiac sensory reinnervation following heart transplantation, it has been thought that renal sensory nerves do not reinnervate following renal DNX. This absence of renal sensory reinnervation has been suggested to contribute to the chronic blood pressure reduction (3 years) in humans following renal DNX. However, whether renal sensory reinnervation occurs following acute renal DNX has not been thoroughly examined. Therefore, we assessed renal sensory innervation at multiple time points from 4 days to 12 wks following unilateral surgical DNX + application of phenol to the renal artery in normal Sprague Dawley rats. The contralateral innervated (INN) kidney served as control. Sensory innervation was determined as optical density (ImageJ) of the sensory nerves identified by immunohistochemistry using antibodies against the neuropeptides substance P and calcitonin-gene-related peptide (CGRP) in the renal pelvic wall, as it is a major site of renal sensory innervation. In INN kidneys, optical density of the sensory nerve fibers in the renal pelvic wall was unchanged over the 4 days - 12 wk period (n=17). In DNX kidneys, optical density of the substance P/CGRP containing sensory nerves was 3%, 30%, 50 % and 100% of the control contralateral INN kidney at 4 days, 2 wks, 4 wks and 12 wks, respectively, after DNX. Linear regression of optical density (DNX/INN) versus time yielded similar intercept and slope values for the substance P and CGRP containing sensory nerves (R
2
= 0.82 and 0.77, respectively, P<0.001 for both).

Conclusion:
in normal rats, sensory reinnervation of the renal pelvic wall is complete at 12 wks following acute surgical renal DNX.
Abstract 483: The Signaling Pathways of Nicotine-induced ERK1/2 Phosphorylation in Rat Mesangial Cells
Authors: Hua Ping , Feng Wenguang , Rezonzew Gabriel , Chumley Phillip H , Jaimes Edgar A
Published: July 2021

Tobacco smoking is associated with accelerated progression of chronic kidney disease of different etiologies including diabetes and hypertension. However, the mechanisms involved are not well understood. We have previously reported that nicotine, a biologically active compound present in high concentrations in tobacco, induces cell proliferation and fibronectin production in mesangial cells which are prevented by ERK1/2 inhibition (AJP’05). In these studies we determined whether rat mesangial cells (MC) express nicotine receptors and characterized the signaling pathways that lead to ERK1/2 phosphorylation in response to nicotine. MC were grown in DMEM with 15% FBS in the presence of 0.4 mg/ml G418 and starved for 24 hours in DMEM without FBS before treatment. We first demonstrated that MC are endowed with several nicotinic Ach receptor (nAChR) subunits including α2-7 and β1-4 as assessed by western blot. Treatment of rat MC with nicotine at 10
-7
M caused a time-dependent ERK1/2 phosphorylation which peaked after 10 min of stimulation( N=3). Several protein kinase inhibitors were then used to identify the upstream kinases that mediate nicotine-induced ERK1/2 phosphorylation. The calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (10
-7
M) decreased the ERK1/2 phosphorylation level by ∼57% (0.41 of 0.95) as compared to nicotine. The PKC inhibitor Go6983 at 10
-9
M, the PKA inhibitor H89 (10
-8
M) and the EGFR inhibitor AG 1478 (10
-7
M) also inhibited ERK1/2 phosphorylation by 60% (0.38 of 0.95), 48% (0.63 of 1.20) and 68% (0.40 of 1.23) respectively as compared to nicotine. Given the role of the nicotine receptors as agonist-regulated Ca
2+
channels, we determined the effects of Ca
2+
channel blockade on nicotine induced ERK1/2 phosphorylation. Treatment of MC with the calcium channel Verapamil (10
-9
M) resulted in 33% (0.49 of 0.73) inhibition of ERK1/2 phosphorylation as compared to nicotine. In summary, we have determined in these studies that rat MC are endowed with several nAChR subunits and that ERK1/2 phosphorylation in response to nicotine requires CaMK II, PKA, PKC and EGFR. In addition, we have demonstrated that these effects require Ca
2+
consistent with the role of the nAChR as agonist-regulated Ca
2+
channels in MC.
Abstract 489: Mechanisms of Carbon Monoxide Attenuation of Tubuloglomerular Feedback
Authors: Ren YiLin , D'Ambrosio Martin A , Wang Hong , Garvin Jeffrey L , Carretero Oscar A
Published: July 2021

Tubuloglomerular feedback (TGF) is an autoregulatory mechanism of the renal microcirculation in which the macula densa (MD) senses NaCl concentration in the lumen of the nephron and sends a signal that controls glomerular filtration rate by constricting the afferent arteriole (Af-Art). We have shown that MD depolarization is sufficient for inducing TGF. Carbon monoxide (CO), either endogenous or exogenous, is known to inhibit TGF, at least in part
via
cGMP. However, whether cGMP-independent mechanisms are involved, and where in the TGF cascade CO exerts its inhibitory effect, remain unknown. Thus we hypothesize that CO, acting
via
both cGMP-dependent and -independent mechanisms, attenuates TGF by acting downstream from MD cell depolarization.
In vitro
, microdissected rabbit Af-Arts and their attached MD were simultaneously perfused and TGF was measured as the decrease in Af-Art diameter. Depolarization of the MD was induced by switching luminal KCl from 4 to 50 mM in the presence of the potassium ionophore valinomycin, while adding the CO-releasing molecule CORM-3 to the MD perfusate at non-toxic concentrations. CORM-3 blunted depolarization-induced TGF at a concentration of 50 μM, from 3.6±0.4 to 2.5±0.4 μm (P<0.01), and completely abolished it at a concentration of 100 μM, to 0.1±0.1 μm (P<0.001, n=6). Similar results were found with 100 μM CORM-3 when depolarization was induced by nystatin (3.0±0.2 vs. 0.4±0.2 μm,
P
<0.001, n=6). This indicates that CO inhibits TGF acting downstream from depolarization. When cGMP generation was blocked with the guanylate cyclase inhibitor LY-83583 (1 μM) added to the MD, CORM-3 no longer had an effect on depolarization-induced TGF at 50 μM (2.9±0.4 vs. 3.0±0.4 μm), but retained partial inhibitory effect on TGF at 100 μM (1.3±0.2 μm,
P
=0.02, n=9). This suggests that CO acts
via
cGMP at low concentrations, but additional mechanisms of action may be involved at higher concentrations. Finally, we confirmed that cGMP inhibits TGF downstream from MD depolarization by adding the degradation-resistant cGMP analog dibutyryl-cGMP (500 μM), which attenuated depolarization-induced TGF (from 3.9±0.5 to 0.6±0.2 μm,
P
<0.01, n=6). Our results could help explain the physiological role of CO in controlling the renal microcirculation.
Abstract 496: Deep Sequencing of microRNAs in Medullary Thick Ascending Limbs Isolated From Fibrotic and Non-Fibrotic Regions of a Kidney
Authors: Kriegel Alison J , Liu Yong , Liu Pengyuan , Liang Mingyu
Published: July 2021

Renal medullary fibrosis is a pathological hallmark of Dahl salt-sensitive (SS) rats fed a high-salt diet. The development of fibrosis is localized and progressive. We examined alterations of microRNA expression in medullary thick ascending limbs (mTALs) during the progression of fibrosis within a kidney. mTALs were isolated using laser capture microdissection from fibrotic and non-fibrotic regions of the same kidney in SS rats fed a 4% NaCl diet for 14 days (n=5) or from SS rats maintained on a 0.4% NaCl diet (n=2). microRNA expression profiles were examined using next-generation sequencing. microRNA abundance profiles were highly reproducible across rats (r
2
=0.93 to 0.99). Detectable microRNAs included 130 known rat microRNAs, 64 microRNA homologs, 89 newly predicted rat microRNAs, and 13 newly predicted microRNA homologs. Four known rat microRNAs, 4 newly predicted rat microRNAs, and 1 microRNA homolog exhibited p values of less than 0.1 (paired t-test) between mTALs isolated from fibrotic and non-fibrotic regions in rats fed the high-salt diet (n=5). Seventeen microRNAs exhibited p values of less than 0.05 (unpaired t-test) in mTALs isolated from non-fibrotic kidney regions of SS rats fed the high-salt diet (n=5) compared to mTALs from SS rats fed the 0.4% NaCl diet (n=2). Thirteen microRNAs were identified when mTALs isolated from fibrotic regions were compared to SS rats fed the 0.4% NaCl diet, 6 of which overlapped with microRNAs identified from non-fibrotic regions. The 4 known rat microRNAs identified from the comparison of fibrotic and non-fibrotic regions within a kidney included 2 members of the miR-30 family (miR-30a and miR-30b-5p). Both miR-30’s were down-regulated in mTALs isolated from fibrotic regions compared to non-fibrotic regions. We previously found that miR-30b was down-regulated in human kidney epithelial cells treated with the pro-fibrotic factor TGFβ1. miR-30a or miR-30b was not identified in comparisons of SS rats on the 4% and 0.4% NaCl diets. The study was the first to examine genome-wide molecular profiles in tissue regions in different stages of injury progression within a kidney. The result suggests that down-regulation of the miR-30 family in mTALs might contribute to the progression of renal medullary fibrosis in SS rats.
Abstract 501: PVN Gαi2 Proteins are Required to Maintain Fluid and Electrolyte Homeostasis and Long-term Blood Pressure Regulation
Authors: Wainford Richard D , Kuwabara Jill T
Published: July 2021

Hypothesis: Hypothalamic PVN GPCR-activated Gαi
2
protein-gated pathways mediate the natriuretic and diuretic responses evoked by acute volume expansion (VE) and are required for fluid & electrolyte homeostasis and MAP regulation during elevated dietary salt-intake.

METHODS:
Male Sprague-Dawley rats received a 7-day bilateral PVN infusion of a scrambled (S) or Gαi
2
oligodeoxynucleotide (ODN-300ng/side/day) or a control i.c.v. infusion of a Gαi
2
ODN (600ng/day) prior to an acute i.v. isotonic saline VE (5% body weight). Separate rats infused i.c.v. or PVN with a Gαi
2
ODN were maintained on a normal 0.4% (NS) or high 8% NaCl (HS) diet for 7-days - MAP, 24h metabolic balance, plasma norepinephrine (NE) and PVN Gαi
2
protein levels were then determined (N=6/group).

Results:
Bilateral PVN Gαi
2
ODN infusion evoked PVN specific down-regulation of Gαi
2
proteins; in contrast i.c.v. Gαi
2
ODN infusion did not alter brain Gαi
2
protein levels. PVN specific Gαi
2
protein down-regulation attenuated the natriuretic (peak UNaV [μeq/min] PVN S 30±4, i.c.v Gαi
2
34±4 vs. PVN Gαi
2
16±3, P<0.05) and diuretic (peak UV [μl/min] PVN S 320±22, i.c.v Gαi
2
336±27 vs. PVN Gαi
2
175±26, P<0.05) responses to an acute i.v. VE. In chronic studies HS-intake evoked a significant endogenous 3-fold site-specific increase in PVN Gαi
2
proteins in i.c.v. Gαi
2
ODN infused rats. Following bilateral PVN Gαi
2
ODN infusion, rats maintained on HS retained sodium (24h Na
+
balance [meq] PVN Gαi
2
+ NS 0.3±0.2, i.c.v. Gαi
2
+ HS 0.5±0.2 vs. PVN Gαi
2
+ HS 2.3±0.4, P<0.05), exhibited global sympathoexcitation (plasma NE [nmol/L] PVN Gαi
2
+ NS 48±6, i.c.v. Gαi
2
+ HS 17±4 vs. PVN Gαi
2
+ HS 75±9, P<0.05) and developed salt-sensitive hypertension (MAP [mmHg] PVN Gαi
2
+ NS 124±3, i.c.v. Gαi
2
+ HS 126±3, PVN Gαi
2
+ HS 141±2, P<0.05).

Conclusion:
PVN Gαi
2
protein-gated pathways contribute significantly to the diuretic and natriuretic responses to acute VE. Further, dysregulation of sympathetically driven sodium retaining mechanisms occurred following PVN Gαi
2
protein down-regulation evoking salt-sensitive hypertension. We conclude PVN Gαi
2
-subunit protein-gated pathways are required to appropriately regulate renal sodium excretion to facilitate sodium homeostasis and maintain a salt-resistant phenotype.
Abstract 504: 23Na Magnetic Resonance Imaging-Determined Tissue Sodium Increases With Age.
Authors: Kopp Christoph , Linz Peter , Dahlmann Anke , Hammon Matthias , Jantsch Jonathan , Müller Dominik N , Schmieder Roland E , Cavallaro Alexander , Eckardt Kai-Uwe , Uder Michael , Luft Friedrich C , Titze Jens
Published: July 2021

Hypertension is the most common cardiovascular risk factor and systolic blood pressure increases continuously with age. Increased vascular stiffness, nitric oxide, renin-angiotensin aldosterone system and salt intake have been implicated. We have developed
23
Na magnetic resonance imaging (
23
Na MRI) and showed that aldosterone influences tissue Na
+
storage. We reasoned that Na
+
might accumulate in tissues with age. We investigated tissue Na
+
of the lower leg in 113 subjects with 3T
23
Na MRI. Tissue Na
+
concentration was calibrated by solutions containing 10 to 40 mmol/l of Na
+
. Prior to
23
Na MRI measurements, a medical history was taken, blood and urine samples were obtained and blood pressure was analyzed in seated position. The age of the 113 subjects analyzed by
23
Na MRI ranged from 22 to 90 years. We studied 44 women and 69 men. Hypertension was diagnosed in 46 subjects (29 men and 17 women). Men showed an exponential increase in tissue Na
+
with age (muscle R
2
= 0.54, skin R
2
= 0.33). In contrast, tissue Na
+
in women was only slightly affected by age and increased linearly (skin R
2
= 0.40, muscle R
2
= 0.13). To compare subpopulations, we allocated the participants into three different age groups <50, 50-65 and >65 years. A gender difference in skin tissue Na
+
could be found throughout all groups with higher levels in men (14.3 vs. 18.1 (<50 yrs), 19.0 vs. 24.5 (50-65 yrs), 20.1 vs. 27.4 mmol/l (>65 yrs); p < 0.05). Skin Na
+
content was elevated in hypertensive women compared to age-matched controls (17.2, n = 12 vs. 21.0 mmol/l, n = 17; p<0.05). We could not identify such a difference in hypertensive versus normotensive men. In line with our previous results, older hypertensive patients receiving an aldosterone antagonist revealed muscle Na
+
values below age-matched subjects (17.6, n = 6 vs. 21.3 mmol/l, n = 24; p < 0.05). We conclude that aging is associated with Na
+
tissue accumulation with a pronounced gender difference. Skin Na
+
overload occurs in hypertensive women but not in men. Aldosterone antagonists may reset the age-dependent muscle Na
+
overload to levels of young healthy controls.
Abstract 510: Altered c-Src Activity by Aldosterone in Vascular Smooth Muscle Cells From SHR Involves PDGFR, C-terminal Src Kinase (Csk) and Csk-binding Protein (CBP).
Authors: Callera Glaucia E , Montezano Augusto , Yogi Alvaro , Touyz Rhian M
Published: July 2021

We previously demonstrated that non-genomic signaling by aldosterone through c-Src-dependent pathways is up-regulated in SHR vascular smooth muscle cells (VSMCs). In the present study we examined molecular mechanisms underlying aldosterone-mediated aberrant regulation of c-Src, by focusing on processes that control c-Src catalytic activation as well as recruitment of enzyme regulators in SHR VSMCs. Through intramolecular interactions, c-Src undergoes conformational changes that determine its activation state. The inactive conformation occurs when the phosphorylated Tyr527 binds to the SH2 domain of the kinase. In cultured VSMCs from WKY aldosterone (100 nM) induced c-Src Tyr527 phosphorylation (153.5 ± 13.6 %), whereas this response was blunted in SHR cells indicating the lack of the regulatory loop of the kinase. C-terminal Src kinase (Csk) is a cytosolic kinase that catalyzes c-Src Tyr527 phosphorylation. This kinase is recruited to the membrane by the phosphorylated form of its transmembrane adaptor protein Csk-binding protein (CBP), in order to regulate c-Src activity. Aldosterone-stimulated VSMCs from SHR displayed reduced Csk content in the membrane fractions and lower levels of CBP phosphorylation compared with cells from WKY. Phospho-caveolin-1 (Cav 1 Tyr14) serves as a docking protein for recruiting Csk to membrane microdomains for the negative regulation of c-Src. Aldosterone induced Cav-1 phosphorylation increase in cells from WKY with a reduced response in those from SHR (176.4 ± 18.0 vs 116.3 ± 8.2 %). PDGFR is a potent activator of c-Src catalytic activity through the phosphorylation of the Tyr216 in the SH2 domain of the kinase, which overrides the inhibitory action of the Tyr 527. Aldosterone induced an increase in phosphorylation of both PDGFR (186.6 ± 26.2 vs 281.9 ± 26.7 %) and c-Src Tyr216 (145 ± 10.1 vs 200 ± 22.2 %) in VSMCs from SHR versus WKY. These events were inhibited by the MR antagonist, eplerenone and the PDGFR inhibitor, AG1296. Our findings demonstrate that key regulators of c-Src activity, specifically PDGFR, Csk and CBP, are altered in SHR VSMCs. These data identify new targets of interest in cellular processes associated with aldosterone-mediated vascular hypertrophy in hypertension.
Abstract 515: Aldosterone Modulates Endothelial Paracellular Permeability by Opening Intercellular Junctions
Authors: Kirsch Torsten , Klinge Uwe , Park Joon-Keun , Haubitz Marion , Haller Hermann , Fiebeler Anette
Published: July 2021

Aldosterone (Ald) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Ald alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells (HUVEC) were exposed to Ald (10
-9
mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions, and activation of intracellular signaling pathways were determined. Ald increased endothelial paracellular permeability for molecules up to 70 kDa within 60 min. A transient loss of cortical actin with formation of actin stress fibers, and disruption of continuous adherens and tight junction strands, accompanied the changes. Mineralocorticoid receptor (MR) blockade, inhibition of RhoA or disruption of extracellular regulated protein kinase (ERK) 1/2 signaling pathways attenuated the Ald-related effects. Moreover, the observed changes of the actin cytoskeleton and intercellular junction architecture were accompanied by a rapid dephosphorylation of protein kinase B (AKT) as well as deactivation of endothelial NO synthase (eNOS). Ex vivo tracer flux experiments with Evans Blue (EB)-conjugated albumin demonstrated concordant response to Ald in freshly isolated umbilical arteries. In summary, Ald leads to increased permeability of endothelial monolayer or isolated arteries. These alterations are associated with a temporal opening of Intercellular junctions and diminished eNOS activity, and depend on activation of the RhoA/ERK pathway. We conclude that these mechanisms could contribute to Ald-induced vasculopathy.
Abstract 519: Endothelin-1-Induced Oxidative Stress and Inflammatory Cell Infiltration Play a Role in High-Fat Diet Induced-Atherosclerosis in Apolipoprotein E Knockout Mice
Authors: Li Melissa W , Paradis Pierre , Schiffrin Ernesto L
Published: July 2021

Background:
Endothelin (ET)-1 plays an important role in generation of reactive oxygen species (ROS) and inflammation in the vasculature. ET-1has been implicated in the pathogenesis of atherosclerosis since plasma and tissue ET-1 are increased in human and animal models of atherosclerosis. We observed that ET-1 overexpression exacerbates high-fat diet (HFD)-induced atherosclerosis in
apolipoprotein E
knockout (

apoE
-/-

) mice. We hypothesized that ET-1-induced ROS and inflammation contribute to the development of atherosclerosis.

Design and methods:
Eight-week-old male transgenic mice overexpressing preproET-1 in the endothelium (eET-1),

apoE
-/-

, eET-1/

apoE
-/-

and wild type mice were fed a HFD for 8 weeks. Aortic atherosclerotic lesions were quantified using Oil Red O staining. ROS production using dihydroethidium staining and monocyte/macrophage and T cell infiltration using immunofluorescence with MOMA-2 and anti-CD4 antibodies, respectively, were determined in perivascular fat, media and plaque in ascending aortic sections.

Results:
eET-1/

apoE
-/-

presented 3.8-fold more atherosclerotic lesions in whole aorta compared to

apoE
-/-

(
P
<0.01). ET-1 overexpression caused 2.6-, 1.9- and 1.9-fold increase in ROS production in perivascular fat, media and plaque of

apoE
-/-

, respectively (
P
<0.05). ET-1 overexpression increased monocyte/macrophage infiltration by 5- and 8-fold in perivascular fat and media, respectively (
P
<0.05). CD4
+
T cell infiltration was observed in perivascular fat and plaque of 3 and 5 of 6 eET-1/

apoE
-/-

compared to 0 and 1 of 6

apoE
-/-

, respectively (
P
<0.05).

Conclusions:
These results suggest that ET-1 play an important role in progression of atherosclerotic lesions by increasing the oxidative stress and monocyte/macrophage and T cell infiltration in the atherosclerotic aorta, including the perivascular fat.
Abstract 52: Enhanced Phosphodiesterase 5 (PDE5) in Thick Ascending Limbs of Dahl Salt Sensitive Rats Blunts NO-induced Inhibition of Transport
Authors: Haque Mohammed Z , Ares Gustavo R , Ortiz Pablo A
Published: July 2021

In normal rats, NO inhibits thick ascending limb (TAL) NaCl reabsorption via cGMP. In Dahl Salt Sensitive (SS) rats NO fails to decrease TAL NaCl absorption. However, the mechanism for this defect is unknown. NO stimulates soluble guanylyl cyclase to produce cGMP. The intracellular concentrations of cGMP and its effect are limited by PDE5, a cGMP-specific phosphodiesterase. We hypothesize that enhanced PDE5 expression in SS TALs blunts the inhibitory effect of NO on transport by decreasing cGMP concentration. We isolated TALs from SS and Dahl Salt-Resistant (SR) rats fed normal salt and measured PDE5 expression by Western blot. PDE5 expression was 210±20% higher in SS TALs compared to SR (
p
<0.05). We then studied whether PDE5 inhibition with vardenafil (25 nM) enhances the inhibitory effect of NO on transport by measuring Na transport-related oxygen (O
2
) consumption in TALs. In SR TALs, the NO donor spermine-NONOate (NO) decreased O
2
consumption by 7.9±2.3% (p<0.05). Adding vardenafil to the bath further decreased O
2
consumption by 7.7±2.3% (p<0.05). In contrast, in SS TALs NO failed to decrease oxygen consumption (5±2.5% from baseline). However in the presence of a PDE5 inhibitor NO, inhibited oxygen consumption by 15.5±2.5 % (p<0.05, n=7). We then tested whether NO-stimulated cGMP production is decreased in SS TALs. To measure production, we inhibited all phosphodiesterases with IBMX. NO enhanced cGMP production in both SS and SR TALs, but production was higher in SS than SRs (delta SS: 4.0±1.0 vs SR: 1.9±0.6 fmoles/μg, p<0.05). Finally we tested whether the decreased effect of NO in SS TALs is related to enhanced cGMP degradation by PDE5. During PDE5 inhibition NO-stimulated cGMP levels were 4-fold greater in SS than SR TALs (Δ SS: 1.9±0.7
vs
SR: 0.4±0.2 fmoles/μg, p<0.05). We concluded that: a) NO-induced inhibition of transport is blunted in SS TALs due to abnormally enhanced PDE5 expression and cGMP catabolism; and b) the blunted transport response to NO in SS TALs is not caused by decreased cGMP production. PDE5 inhibition in TALs could provide protection against salt-sensitive hypertension.
Abstract 525: Nanomolar Ouabain Increases Na/Ca Exchanger-1 Expression and Enhances Ca2+ Signaling in Human Arterial Myocytes: A Mechanism That Links Salt to Increased Vascular Resistance?
Authors: Linde Cristina I , Antos Laura K , Golovina Vera A , Blaustein Mordecai P
Published: July 2021

The mechanisms by which excess dietary NaCl raises blood pressure (BP) in hypertension are unresolved. Much evidence indicates that endogenous ouabain (EO, an adrenocortical hormone and Na
+
pump blocker) plays a role. In rodents, arterial smooth muscle cell (ASMC) Na
+
pumps with an α2 catalytic subunit (ouabain EC
50
≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na
+
pumps have an α1 subunit (ouabain EC
50
≈5 μM). Human α1 Na
+
pumps, however, have high ouabain affinity (EC
50
≈ 20 nM). Therefore, we studied the expression, distribution and function of Na
+
pump α subunit isoforms in ASMCs from surgically-recovered human artery segments. Immunoblots and immunocytochemistry reveal that human ASMCs (hASMCs) express α1 and α2 Na
+
pumps. As in rodents, α2, but not α1, pumps are confined to plasma membrane (PM) microdomains adjacent to sarcoplasmic reticulum, SR (identified with ER Tracker, an SR stain, and anti-SR Ca
2+
pump-2, SERCA2, antibodies). Na/Ca exchanger-1 (NCX1) and TRPC6 (component of receptor-operated channels, ROCs) co-localize with α2 in the PM microdomains. Incubation (72 hr) with 100 nM ouabain (blocks nearly all α1 and α2 pumps) was toxic to most cultured hASMCs, but 10 nM ouabain (blocks 90-95% of α2 and <<50% of α1 pumps) increased NCX1 and SERCA2 expression by 45±10% and 31±6%, respectively (
P
<0.05;
n
=5 patients). Ca
2+
transients were measured with fura 2: The 72 hr, 10 nM ouabain pre-treatment increased 10 μM ATP-induced SR Ca
2+
release in 0Ca
2+
media by 27±8% (
P
<0.05;
n
=3), and increased Ca
2+
influx when external Ca
2+
was restored (ATP still present) by 96±15% (
P
<0.01;
n
=3 patients). This Ca
2+
influx was likely mediated primarily by ROCs and store-operated Ca
2+
channels. Thus, α2 Na
+
pumps, NCX1, ROCs, and the SR are structurally and functionally linked. Freshly-isolated myocytes from the arteries of several rat hypertension models display comparable protein expression and Ca
2+
signaling changes. We conclude that the same molecular mechanisms regulate long-term Ca
2+
homeostasis and signaling in human and rodent ASMCs. These ouabain/EO-modulated mechanisms underlie the ‘whole body autoregulation’ associated with increased vascular resistance and elevation of BP in human hypertension.
Abstract 528: Angiotensin-ii Induces Strain-dependent Renovascular Remodeling in Mice
Authors: Pushpakumar Sathnur , Kundu Sourav , Coley Denise , Givvimani Srikanth , Tyagi Suresh C , Sen Utpal
Published: July 2021

Activation of renin-angiotensin system and production of Angiotensin-II plays an important role in several pathological processes leading to end stage renal disease. Sustained hypertension induces renovascular remodeling in both intra and extra-renal vasculature by altering extracellular matrix (ECM) components. Recent studies in vascular remodeling have shown strain-dependent phenotypic variation in carotid artery and in pulmonary hypertension indicating a genetic basis for disease susceptibility. We hypothesized that sensitivity to develop hypertension to angiotensin-II infusion and subsequent renovascular remodeling will vary depending on the genetic background. C57BL/6J (WT) and TIMP2
-/-
mice were used in this study. Osmotic pumps loaded with Angiotensin-II (Ang-II) were introduced into a dorsal subcutaneous pocket for delivery at 250 ng. kg
-1
. min
-1
. Pumps loaded with saline served as controls for both groups. Blood pressure, renal vascular blood flow, renal vascular density, collagen and elastin deposition, oxidative stress and tissue levels of MMP-2 and MMP-9 were determined.

Results:
TIMP2
-/-
mice had higher baseline mean blood pressure (130.5±6.4 mm Hg) compared to WT mice (111.07±7.3 mm Hg). After 4 weeks of Ang-II treatment, mean arterial pressure increased to 150.33±11.9 in TIMP2
-/-
mice compared to 129.9±5.7 mm Hg in WT mice. The renal cortical blood flow was reduced in TIMP2
-/-
mice (1125 flux units) compared to WT (1350 flux units) and untreated controls. Barium angiography demonstrated decreased renal vascular density in TIMP2
-/-
mice compared to WT group. Peri-glomerular and vascular collagen deposition was increased in TIMP2
-/-
and WT, whereas elastin was reduced and disrupted in TIMP2
-/-
renal vasculature. Intracellular reactive oxygen species production was predominant in TIMP2
-/-
group than in WT or control animals.

Conclusion:
Our results suggest that in Ang-II induced hypertension, renovascular response is strain dependent and TIMP2
-/-
mice appear to be more susceptible to end organ damage than WT mice.
Abstract 529: Atorvastatin and Sildenafil Reduce Vascular Remodeling and Oxidative Stress in 2K1C-hypertension
Authors: Guimaraes Danielle A , Rizzi Elen , Ceron Carla , Martins-Oliveira Alisson , Gerlach Raquel , Tanus-Santos Jose Eduardo
Published: July 2021

Atorvastatin (ATORVA) and sildenafil (SILD) exert beneficial effects on cardiovascular diseases through their pleiotropic effects. Hypertension-induced vascular hypertrophy is associated with oxidative stress and matrix metalloproteinase (MMP) up-regulation. We evaluated the effects of ATORVA and SILD on vascular changes induced by MMPs and on oxidative stress and MMP activity in 2 kidney-1 clip (2K1C) hypertension. Sham and 2K1C rats were treated with vehicle, ATORVA (50 mg/kg), SILD (45 mg/kg) or both for 8 weeks. ATORVA, SILD, or both drugs exerted antihypertensive effects (systolic blood pressure: 148±6, 156±2, and 138±4 mmHg, respectively,
vs.
200±4 mmHg in 2K1C untreated rats; P<0.05). All treatments prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats (P<0.05). Aortas from 2K1C rats showed higher MMP-2 levels when compared with sham and all treatments attenuated 2K1C hypertension-induced increases in MMP-2 levels (both P<0.05). Increased gelatinolytic activity (20.6±0.9
vs.
13.9±1.2 A.U.; arbitrary units) co-localized with upregulated aortic MMP-2 expression (8.1±0.3
vs.
5.4±0.4 A.U.) in 2K1C
vs.
control rats. ATORVA, SILD, or both drugs lowered gelatinolytic activity to 14.6±0.5, 14.6±1.0 and 14.5±0.8 A.U. and lowered the aortic MMP-2 expression to 5.8±0.3, 5.9±0.5 and 5.5±0.5 A.U., respectively. However, these drugs had no
in vitro
effects on human recombinant MMP-2 activity (P>0.05). Hypertension induced oxidative stress assessed with dihydroethidium probe (7.9±1.2
vs.
16.5±1.5 A.U. in sham
vs
2K1C), which was attenuated by ATORVA, SILD or both (9.9±0.3, 10.4±1.5 and 9.6±0.6 A.U., respectively). Plasma malondialdehyde levels paralleled dihydroethidium results. Treatment with ATORVA or SILD, or both, exert antihypertensive effects and prevents 2K1C hypertension-induced vascular remodeling and oxidative stress. These effects were associated with lower MMP-2 levels possibly resulting of antioxidants effects. Our results suggest that ATORVA and SILD may prevent the vascular alterations of hypertension.Supported by FAPESP, Cnpq.
Abstract 612: Apelinergic System within the Kidney is Altered in Diabetic Mice
Authors: Kalea Anastasia , Ye Minghao , Wysocki Jan , Hsieh Ivy , Batlle Daniel
Published: July 2021

Background:
The Apelin receptor (APJ)-Apelin system has been implicated in cardiovascular function but little is known regarding diabetic kidney disease. Apelin’s effects appear related, in part, to an interaction with angiotensin II. Apelin, like angiotensin II, is a substrate for ACE2, which we previously showed to localize in tubular and glomerular epithelial cells (podocytes). Here we wanted to investigate the localization of this peptide within the glomerulus and the overall expression of apelin and its receptor APJ within normal and diabetic mouse kidney.

METHODS:
We employed RT real time PCR, western blot, immunostaining, tissue and cell confocal microscopy to study apelin and APJ in kidneys from female
C57BLKS
mice (8 wks of age) and in cultured immortalized podocytes. Mouse podocytes were stimulated with Pyr
1
Apelin-13 (100nM) for 0, 1, 5, 15 and 60min and tested for phospho-status of AKT, p70S6K and ERK. Kidneys from 8 weeks old
db/db
mice were also studied.

Results:
Both Apelin and APJ were abundantly present in the kidney being expressed in most glomerular tufts, proximal tubules, and arterioles. In glomeruli, apelin colocalized with nephrin, PECAM-1, and a-SMA indicating its presence in podocytes, endothelial cells, and mesangial cells. APJ, by contrast, did not colocalize with PECAM-1 but also colocalized with several podocyte markers. In the proximal tubule, the APJ receptor colocalized with ACE2. Apelin and APJ mRNA were also expressed in cultured mouse podocytes where apelin-13 induced AKT, p70S6K and ERK rapid signaling (at 15min). By RT real time PCR, kidney cortex from
db/db
mice (n=6) had reduced APJ (0.19±0.02 vs 0.62±0.08, p< 0.005) and pre-proapelin mRNA levels (0.12±0.08 vs 1.13±0.04, p< 0.001) as compared to
db/m
controls (n=6).

Conclusions:
Apelin and its receptor are widely expressed in the kidney, including podocytes where apelin-13 is involved in AKT, p70S6K and ERK signaling. Reduced apelinergic activity in the diabetic kidney could be possibly compensatory to the known overactivity of the RAS.
Abstract 634: Selective Genetic Deletion of the Brain-Specific Intracellular Renin Isoform (Renin-1b) Results in Altered Metabolic and Fluid Balance Regulation
Authors: Folchert Matthew D , Liu Xuebo , Grobe Justin L , Sigmund Curt D
Published: July 2021

An evolutionarily-conserved mRNA initiating with an alternative first exon (exon 1b) of the renin gene codes for an active renin enzyme that should be targeted to the cytoplasm instead of the cellular export apparatus. This intracellular renin (icREN) is expressed primarily in brain and is regulated independently from the traditional secreted isoform of renin (sREN), which is encoded by a mRNA initiated at the traditional first exon (exon 1a). We have developed two mouse models containing conditional alleles of either exon 1a (knocking out sREN and preserving icREN) and exon 1b (knocking out icREN and preserving sREN), thus allowing for global and site-selective knockout (KO) of the individual renin isoforms. icREN-KO mice (n=5, vs n=8 littermate controls) exhibit a trend toward reduced body mass (25.1±2.3 vs 29.0±1.0 g) despite normal food intake (3.2±0.3 vs 2.9±0.9 g/day). During a two-bottle choice between water and 0.15 M NaCl, icREN-KO mice exhibit, at baseline, normal total fluid intake (3.4±0.8 vs 2.8±0.4 mL/day), total sodium intake (0.7±0.2 vs 0.6±0.1 mEq/day), and preference for 0.15 M NaCl (47.6±11.0 vs 42.8±9.5 %). Following an 18-hour dehydration, icREN-KO mice exhibit a trend toward increased water intake (1.5±0.3 vs 1.1±0.2 mL/2hr) that is exaggerated when normalized to their smaller body size (63±15 vs 37±7 mL/kg/2hr). Despite normal 5-hour fasted blood glucose (202±6 vs 214±11 mg/dL), icREN-KO mice exhibit markedly improved glucose tolerance (2 mg/kg ipGTT; 18,540±4,937 vs 33,003±2,578 mg/dL*min, P=0.02). Ongoing studies include measurement of resting metabolic rate under baseline conditions, and assessment of all of the above endpoints following chronic treatment of icREN-KO mice with deoxycorticosterone acetate and high dietary sodium (DOCA-salt treatment). Interestingly, DOCA-salt treated C57BL/6 mice also exhibit improved glucose tolerance (39,901±2,198 vs 31,712±1,722 mg/dL*min, P<0.01) and exhibit a 60% decrease in icREN mRNA expression in the brain. These data introduce for the first time a mouse with selective ablation of intracellular renin and provide the first experimental support for physiologically-relevant functions of intracellular renin.
Abstract 655: Ischemia-induced Epoxyeicosatrienoic Acid Release Protects Female Rats From Acute Kidney Injury
Authors: Dragun Duska , Hoff Uwe , Blum Maximilian , Bubalo Gordana , Fechner Mandy , Falck John R , Schneider Wolfgang , Luft Friedrich C , Schunck Wolf-Hagen
Published: July 2021

Females are naturally protected against ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) in various clinical and experimental settings. However, the underlying mechanisms are unknown. We hypothesized that female protection may be conferred by enhanced production of cytochrome P450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) that promote vasodilation as well as antiinflammatory and antiapoptotic pathways in the kidney. To test this hypothesis, we first analyzed the renal CYP-eicosanoid profile by liquid chromatography tandem mass spectrometry in male and female Lewis rats. Ischemia was induced through 45 min of left renal vessel clamping after right nephrectomy (n=6-8 per group). In non-ischemic controls, male and female kidneys stored almost identical amounts of EETs as well as 20-hydroxyeicosatetraenoic acid (20-HETE), both predominantly esterified into phospholipids, under basal non-ischemic conditions. 45 min of ischemia induced a massive release of EETs from membrane stores in females but not males. The free renal EET-levels reached 70.2±20.1 in females compared to only 4.6±1.3 ng/g in males. After ischemia, the ratio of free EETs to free 20-HETE was about 1:1 in females and 1:3 in males. Next, we proved the functional importance of EETs in renal protection by pretreating males with a synthetic EET-agonist (12-HUDE) and females with a selective EET-antagonist (14,15-EEZE-mSI). As analyzed two days after reperfusion, the EET-agonist protected males against loss of creatinine clearance (1.03±0.18 vs. 0.26±0.02 ml/min, p<0.01 vs. vehicle, compared to 1.28±0.06 ml/min in sham control). Females were rendered susceptible to I/R-injury by the EET-antagonist (creatinine clearance: 0.25±0.05 vs. 0.67±0.04; p<0.01 vs. vehicle, compared to 0.81±0.04 ml/min in sham control). Changes in inflammatory cell infiltration and tubular apoptosis paralleled these effects on renal function. Our results indicate that female rats are protected against renal I/R-injury by enhanced ischemia-induced EET-release and demonstrate that renal protection can be transferred to males using synthetic EET-agonists.