bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P278: On the Origin of Brain Renin
Authors: van Thiel Bibi S , te Riet Luuk , Severs David , Uijl Estrellita , Garrelds Ingrid M , Qadri Fatimunnisa , Alenina Natalia , Bader Michael , Danser A
Published: June 2021

A renin-angiotensin system (RAS) in the brain is believed to contribute to blood pressure regulation. Given the presence of the blood-brain barrier, brain RAS activity most likely depends on synthesis of (pro)renin in the brain. In support of this concept, an intracellular, non-secreted form of renin has been described in the brain, depending on an alternative transcript of the renin gene. To what degree this intracellular truncated prorenin truly displays angiotensin (Ang) I-generating activity (AGA) is unknown. In the present study, we set out to quantify brain (pro)renin, both before and after buffer perfusion of the brain, in normal mice, renin knockout (KO) mice, DOCA-salt-treated mice (which have been reported to display brain RAS activation), and angiotensin II-infused mice. Brain nuclei were homogenized and incubated with excess angiotensinogen to detect AGA, both before and after acid activation of prorenin, with or without the renin inhibitor aliskiren to correct for non-renin-mediated AGA. Renin-dependent (i.e., aliskiren-inhibitable) AGA was readily detectable in brain nuclei, the highest AGA being present in brainstem (>thalamus ≈cerebellum ≈striatum ≈midbrain >hippocampus ≈cortex). Brain AGA increased non-significantly after prorenin activation, suggesting that brain prorenin levels are low or absent. Buffer perfusion reduced AGA in all brain areas by >60%. Plasma renin (expressed per mL plasma) was 40-800 x higher than brain renin (expressed per g tissue). Plasma prorenin levels resembled plasma renin levels. AGA was undetectable in plasma and brain of renin KO mice, both before and after prorenin activation. DOCA-salt and Ang II suppressed plasma renin, and parallel decreases were observed for brain renin. In conclusion, brain renin levels (per g tissue) correspond with the amount of renin present in 1-20 μL blood plasma. Brain renin disappears after buffer perfusion, and varies in association with plasma renin. This indicates that renin detected in brain nuclei represents plasma renin and/or locally activated plasma prorenin. DOCA-salt exposure does not selectively increase brain renin expression.
Abstract P280: Novel Insights into Transcriptional Regulation of the Human Angiotensinogen Gene by High-salt: A Study in Transgenic Mice
Authors: Kaw Meenakshi , Jain Sudhir , Perla Shravan Perla , Sirianni Natalie , Alakrawi Mariam Alakrawi , Puri Nitin , Yanamandra Sai Sushani , Kumar Ashok
Published: June 2021

Angiotensinogen is the substrate for the entire RAS cascade and polymorphisms leading to its overexpression are linked to hypertension. SNPs in the promoter of the hAGT gene are associated with hypertension. Importantly, these SNPs can further modulate the gene of interest in various physiological/environmental settings like the high-sodium diet. In this regard, the human angiotensinogen (hAGT) gene has polymorphisms in its 2.5Kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A is associated with human hypertension whereas Hap -6G/-217G reduces cardiovascular risk. We have engineered transgenic (TG) mice with these haplotypes (Hap -6A: -6A/-217A and Hap -6G: -6G/-217G) so as to examine the transcriptional regulation of the hAGT in an in vivo setting. This study is designed to study the effects of a high-sodium diet on the transcriptional milieu of renal tissues with consequential effects on the hAGT expression in our two haplotypes. Male TG mice were placed on 4% Na
+
diet for a period of 8 weeks. High-salt diet induces mineralocorticoid receptor (MR) and SGK-kinase expression in both haplotypes, equally. MR has been shown to bind to GRE elements in the hAGT gene. Importantly, MR-binding (ChIP assay) and hAGT induction are significantly (p<0.05) greater in the -6A haplotype males as compared to -6G males. High-salt also increased the expression of transcriptional regulators including CEBPβ and HNF4 (p<0.05) that are independent of haplotype. Complementary ChIP assay confirmed enhanced transcription factor (TF) binding to the chromatin of male -6A TG mice as compared to their -6G counterparts after high-salt diet treatment. Thus, we show here an effect of high-salt on cellular transcriptional apparatus that is haplotype-independent. However, increased TF affinity of the chromatin in -6A TG mice leads to higher salt-induced AGT levels in this haplotype than -6G. These observations could partly account for increased salt-sensitivity of some adult males that, in turn, is governed by the “risk” haplotype. Identifying these individuals with the -6A haplotype will help guide therapeutic lifestyle changes in patients with essential hypertension.
Abstract P286: Modulation Way Classical and Alternatives of Renin Angiotensin System (RAS) in Mesangial Cells After Exposure to Fructose
Authors: Yokota Rodrigo , Palomino Zaira J , Matsumoto Larissa E , Aragão Danielle S , Casarini Dulce E
Published: June 2021

Background:
Our group demonstrated previously that rats subjected to a high fructose diet showed increased activity of kidney ACE.In order to understand the effect of fructose in the forming process of angiotensin II (AngII) and angiotensin 1-7 (Ang1-7) affecting the levels of these effectors, the aim of this study was to evaluate the activities of the RAS enzymes responsible for the formation of Ang1-7 and AngIIof immortalized human mesangial cells (IMCH)exposed to low and high concentration of fructose.

Methods:
IMCH were divided into control (low glucose medium), Fructose 5 mM(medium + 5 mM Fructose) and Fructose 30mM(medium + 30 mM Fructose) groups. The enzymatic activity of ACE, chymase, cathepsin D, Neutral Endopeptidase (NEP) and ACE2 were evaluated using Z-Phe-His-Leu,Abz-AIKFFSAQ-EDDnp, Abz-AIAFFSRQ-EDDnp,Abz(d)-Arg-Gly-Leu,Mca-APK (DNP) as substrates respectively. Data were analyzed using ANOVA one way.

Results:
Among the AngIIforming enzymes, fructose decreased ACE activity in cell lysate.Both fructose concentrations increased chymase in the culture medium. No effect was observed on the activity of cathepsin D. When evaluating the activities of peptidases forming Ang-1-7,NEP activity was increasedin culture medium.We did not detected differences in ACE2 activity.

Conclusion:
The results suggest fructose modulates renal and systemic RAS in IMCH acting in the classical andalternative pathways of AngII formation. Additionally this sugar was able to increase NEP activity, forming Ang-1-7. Quantitation of angiotensin remains necessary to better understand how the end effector is available in IMCH and evaluate possible effects on glomerular physiology.
Abstract P290: Human Stomach Gastrin is Regulated by Sodium Human Stomach Gastrin Secretion is Regulated by Ingested Sodium and the Dopamine 1 Receptor
Authors: Xu Peng , Gildea John J , Zhang Chi , Wang Dora , Tran Hahn T , Jose Pedro A , Felder Robin A
Published: June 2021

Dietary sodium stimulates the renal excretion of sodium long before there is expansion of the extracellular fluid volume. We have previously shown that an increase in sodium concentration in the incubation buffer in human SW626 colon adenocarcinoma cells that express gastrin-secreting G- cells increased gastrin mRNA and protein. Thus, we hypothesized that increasing extracellular sodium would also increase gastrin expression in normal human stomach G-cells. The highest sodium concentration (170 mM) increased gastrin protein expression significantly (all cell lines were normalized to their gastrin expression at 90 mM, sodium). In normal human G-cells cultured in non-polarized conditions, the normalized gastrin expression in cells incubated in 143mM sodium was 0.98 ± 0.06; 170mM sodium increased gastrin expression 1.515 ± 0.106-fold (n=4 different cell lines, P<0.01 one-way ANOVA). Cultured human renal tubule cells demonstrate more physiologically relevant phenotype when grown in a 3D cell culture (3DCC) system. Therefore, we also studied human G-cells grown in 3DCC. Normal human G-cells grown in 3D also had a greater gastrin expression when incubated in 170 than143 mM sodium: 1.08 ± 0.12 vs. 0.62 ± 0.04, n=4, P<0.05, t-test). We have also shown that the dopamine type 1 receptor (D
1
R) is expressed in G-cells and may play a role in regulation of gastrin expression. We now report the presence of the complete dopamine biosynthetic pathway in human G-cells: tyrosine hydroxylase is expressed ubiquitously in the stomach while DOPA decarboxylase is expressed selectively in G-cells where D
1
R is also expressed. We have reported that in the kidney, gastrin, via CCKBR and dopamine, via D
1
R synergistically increase renal sodium excretion. Thus, the pathway that is involved in the increase in sodium excretion after the ingestion sodium involves sodium- and D
1
R-mediated increase in gastrin expression in G-cells, followed by secretion of gastrin into the circulation to interact with kidney mediated gastrin pathways.
Abstract P293: Endothelial Sodium Channel Activation Promotes Vascular Stiffness in Obese Female Mice
Authors: Aroor Annayya , Ramirez-Perez Francisco I , Jia Guanghong , Habibi Javad , DeMarco Vincent G , Garro Mona , Barron Brady J , Sun Zhe , Meininger Gerald A , Martinez-Lemus Luis A , Sowers James R
Published: June 2021

Obesity-associated arterial stiffening is an independent predictor of cardiovascular disease (CVD) events. Although premenopausal non-obese women are protected against CVD, aortic stiffening in obese women is more common than in men. This disproportionate increase in vascular stiffness in obese females may partly explain their loss of sex-related CVD protection. Recent studies have suggested a role for endothelial sodium channel (ENaC) activation in promotion of endothelial stiffness and suppression of flow-(nitric oxide) mediated vasodilation. Increased mineralocorticoid receptor (MR) activation mediated endothelial stiffness is promoted, in part, by ENaC activation. In this regard, we have recently reported increased aortic stiffness, MR and ENaC expression and endothelial dysfunction in female mice fed a high fat and high fructose diet (western diet [WD]). This increase in aortic stiffness was prevented by very low dose MR antagonism. Accordingly, we hypothesized that inhibition of MR-mediated ENaC activation by using a very low dose of the ENaC inhibitor, amiloride, would prevent arterial stiffening and vascular dysfunction in WD-fed female mice. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1mg/kg/day) for 16 weeks. Amiloride significantly attenuated WD-induced increases in aortic stiffness in vivo as measured by pulse wave velocity as well as in vitro endothelial stiffness as measured by atomic force microscopy. Moreover, incubation of aortic explants with very low dose of amiloride (1 μM) inhibited WD-induced aortic stiffness in aorta explants from WD-fed female mice. Amiloride also prevented WD-induced impairment in acetylcholine-induced aortic vasodilatation and flow-mediated dilation in mesenteric arteries. Taken together, these observations support a role for ENaC activation in diet-induced vascular stiffening in obese females.
Abstract P296: Effective Arterial Elastance and Hypertensive Response to Stress
Authors: Kieu Andy , Widlansky Michael
Published: June 2021

Effective arterial elastance (Ea) has shown to be of importance in resting hypertension, though its role is unclear in hypertensive response to exercise or dobutamine. Prior studies have shown that individuals with hypertensive response to exercise (HRE) have diastolic dysfunction as shown on echocardiogram, but there are limited data to show if there are any correlations with Ea.

In this retrospective cohort study, we reviewed 415 stress echocardiograms (treadmill and dobutamine) that were performed between 2009 and 2011 that were completed on the same day as a full resting TTE at our institution. Following exclusion of subjects with coronary artery disease, resting LV ejection fraction less than 50%, resting regional wall motion abnormalities and inducible ischemia, 182 subjects who underwent treadmill stress echocardiogram (TSE) and 149 subjects who underwent dobutamine stress echocardiogram (DSE) were left for analysis. We determined univariate and multivariate predictors of a HTN response to each type of stress test by logistical regression. Covariates included age, sex, resting systolic pressure, resting diastolic pressure, resting hypertension, pulse pressure, Ea, septal e’, septal a’, mitral E/e’, mitral inflow E/A ratio, LV mass index, and left atrial size.

In the TSE group, 26 subjects had hypertensive responses compared to 11 in the DSE group. Resting hypertension was predictive of HRE in both groups. Ea was not predictive of HRE in the TSE group, but was predictive for those in the DSE group (OR 5.91, p=0.03).

Increasing resting Ea predicts hypertensive responses to dobutamine, but not exercise. Traditional diastolic Doppler parameters are not significantly associated with hypertensive responses to either stressor.
Abstract P297: Distinct Profiles of Extracellular Vesicles are Elevated During Evolution of Hypertension
Authors: Erdbruegger Uta , Howell Nancy L , Rudy Christine , Carey Robert M , Le Thu H
Published: June 2021

Despite the significant deleterious impact of hypertension (HTN), the blood pressure (BP) threshold at which to treat remains a matter of debate. Early and non-invasive biomarkers of end-organ damage in HTN are needed to optimize treatment for patients with HTN. Circulating microparticles (MPs) are potential candidate biomarkers to reflect early end-organ damage in HTN. MPs are submicron vesicles shed from various cell types in the blood and carry markers from their parent cells. We hypothesize that these MPs show a distinct pattern during evolution of HTN in spontaneous hypertensive rats (SHR), a model of essential HTN. MPs were generated from platelet poor plasma from tail vein of SHR before development of HTN (7 weeks (wk) of age), at time of development (9 wk) and severe HTN (12 wk). WKY rats served as control. Enumeration and phenotyping of MPs was performed with imaging flow cytometry using CD42 (platelet marker), CD31 (PECAM), CD105 (S-endoglin), CD45 (leukocyte) and Annexin V (AnV) as surface markers. Compared to WKY (n=3), aII SHR (n=3) had a significant increase in systolic BP by 40mmHG (by radiotelemetry) (p=0.02). All types of MPs increased at wk 9 compared to wk 7 and then decreased again at wk 12 in both groups. However, the increase of MPs was numerically higher in a subgroup of endothelial MPs in SHR after 12 wk compared to controls (AnV negative and S-endoglin (CD105) positive, median 34333 vs 10328 MPs/μl plasma, p=0.08). Subgroups of circulating MPs show a distinct pattern in the SHR. It remains to be determined whether these vesicles reflect early end-organ damage in HTN and have the potential to guide treatment.
Abstract P299: Thymosin Beta15 is a Novel Precursor of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP)
Authors: Liao Tang-Dong , Romero Cesar , Kumar Nitin , Rhaleb Nour-Eddine , Carretero Oscar
Published: June 2021

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with anti-inflammatory and antifibrotic properties that is released from its precursor thymosin β4 (Tβ4). At present, Tβ4 is the only identified Ac-SDKP precursor with no other candidates reported. Our search of National Center for Biotechnology Information (NCBI) database by Basic Local Alignment Search Tool (BLAST) revealed that Tβ15 has similar N-terminal amino acid structure as Tβ4. We hypothesize that in addition to Tβ4, Tβ15 may also be a precursor of Ac-SDKP. To test our hypothesis we used wild-type (WT) and Tβ4 global knockout mice (Tβ4KO). We detected the presence of Ac-SDKP in various organs (pmol/mg protein) in both WT and Tβ4KO mice, including lymph node (459±35
vs
234±37), thymus (241±36
vs
179±24), heart (95±14
vs
71±10), kidney (90±19
vs
56±11), testis (55±16
vs
55±17), spleen (57±10
vs
16±1) and liver (49±16
vs
18±2). We also measured urinary Ac-SDKP execration before and after angiotensin converting enzyme inhibitor (ACEi) treatment. In both group of mice we determined the basal Ac-SDKP excretion (ng/24hrs) and that was significantly increased with ACEi treatment in both WT (36±8
vs
117±17) and Tβ4KO (22±3
vs
36±3), with the increase being lower in Tβ4KO mice. Immunohistochemistry analysis of Tβ15 in the kidney revealed positive cells located in the thick ascending limb, distal tubule, macula densa and collecting duct in both groups of mice. Interestingly, we found that in Tβ4KO mice thymus weight was higher than in WT mice, while there was no difference between the groups with regard to heart and kidney weight. We concluded that Tβ15 is a potential novel precursor of Ac-SDKP.
Abstract P301: Renal Expression of Collectrin (TMEM27) is Downregulated During Angiotensin II-induced Hypertension
Authors: Cechova Sylvia , Chu Pei-Lun , Gigliotti Joseph , Le Thu H
Published: June 2021

Collectrin (
Tmem27
) is transmembrane glycoprotein with homology to ACE-2, but lacks any catalytic domain. It plays a key role as a chaperone of amino acid transporters, and is abundantly expressed in the kidney in the proximal tubules and collecting duct. Deletion of collectrin in the mouse results in hypertension (HTN) at baseline and augmented salt-sensitivity that are associated with decreased renal nitric oxide and increased superoxide levels. During high salt diet, renal expression of collectrin is upregulated, suggesting an adaptive homeostatic response to salt loading. Here, we queried whether the expression of collectrin is regulated by angiotensin II (Ang II). Wild-type 129S6 mice were made hypertensive with Ang II osmotic minipump @ 600 ng/kg/min x 2 weeks, and were compared to age-matched untreated WT 129 mice. Shown in
Fig. 1
, renal mRNA expression of collectrin is significantly reduced after 2 weeks of Ang II (Panel A). Immunostaining shows collectrin protein level is also significantly diminished to near undetectable level (Panel B). We show for the first time that Ang II regulates the expression of collectrin, suggesting that the action of Ang II on blood pressure may be mediated, in part, through the downregulation of collectrin. Further studies are needed to determine the effect of AT
1
and AT
2
receptor signaling on renal expression of collectrin during Ang II-HTN in vivo.
Abstract P302: A Positive Correlation Between High Levels of Inflammatory Markers and Angiotensin II in Obese Appalachian Females
Authors: Chaudhry Hibba , Nichols Alexandra , Nawab Athar , Lakhani Vishal , Srikanthan Krithika , Sodhi Komal
Published: June 2021

Introduction/Background:
Obesity increases levels of certain biomarkers related to metabolic syndrome (leptin and TNF-α) in Appalachian women. It also leads to vascular dysfunction and atherosclerosis, which is assessed by levels of inflammatory markers angiotensin II (Ang II), a powerful vasoconstrictor, and serum adiponectin. We previously demonstrated a marked increase in levels of inflammatory markers in humans and mice with hypertension, obesity, and diabetes, and hypothesize that a high-BMI subject would present with increased levels of angiotensin II, leptin, and TNF-α, as well as decreased adiponectin.

Methods:
Serum adiponectin, Ang II, leptin, and TNF-α were assayed in female Appalachian subjects. Linear regression analysis was used to analyze the relationship between BMI, leptin, adiponectin, and Ang II. Nonlinear regression was used to determine the odds ratio and confidence intervals. The effect on murine pre-adipocytes was also measured.

Results:
Lipidomic analysis revealed a significant increase in Ang II among high-BMI females (50-72) compared to lower-BMI subjects (32-45) (p<0.05). Treatment of murine pre-adipocytes with Ang II decreased serum adiponectin levels and increased adipogenesis by 70% (p<0.05), implicating both Ang II and oxidative stress as factors in the pathogenesis of BMI-related disease. Serum leptin and TNF-α were significantly increased in high-BMI subjects (p<0.05) compared to lower-BMI subjects, while adiponectin levels were decreased (p<0.05) in high-BMI subjects compared to lower-BMI subjects.

Discussion/Conclusions:
Increased BMI in Appalachian females correlates with an increase in Ang II level, serum TNF-α, and leptin expression, and a decrease in serum adiponectin. This represents a novel mechanism by which high-BMI females with controlled blood pressure remain sensitive to the development of atherogenesis, vascular dysfunction, and metabolic syndrome.
Abstract P303: Cilostazol Attenuates Angii-induced Cardiac Fibrosis in Mice
Authors: Okuyama Yuka , Uchida Haruhito A , Umebayashi Ryoko , Kakio Yuki , Takeuchi Hidemi , Okuyama Michihiro , Wada Jun
Published: June 2021

Background:
Cilostazol, a phosphodiesterase-3 inhibitor, plays vasoprotective roles such as an improvement of endothelial function, a vasodilatation and a suppression of proliferation of vascular smooth muscle cells. The aim of study was to investigate a cardioprotective effect of cilostazol.

Method: Male apolipoprotein E deficient mice (8-12 weeks old) were fed with either normal chow diet or cilostazol-containing (0.1% wt/v) diet. After 1 week of cilostazol administration, mice were infused subcutaneously with either angiotensin II (AngII, 1,000 ng/kg/min, n = 16 - 19) or saline (n = 5 - 6) by osmotic minipumps for 4 weeks.

Results:
AngII equivalently increased systolic blood pressure, irrespective of cilostazol administration. Cilostazol had no effect on serum cholesterol concentrations, triglycerides, high-density lipoprotein-cholesterol, body weights, heart rates, and systolic blood pressures. AngII increased heart weight but was attenuated by cilostazol administration (6.7±0.8 to 6.0±0.7 mg/gBW, p < 0.05). Cilostazol prevented both perivascular and interstitial cardiac fibrosis induced by AngII (p < 0.05, each). Quantitative real-time PCR revealed that mRNA expressions of
Ctgf
,
Collagen I
,
Collagen III
,
Tgf-
,
Hgf
and
Spp-1
increased by AngII infusion but were attenuated by cilostazol administration (p < 0.05). Immunohistochemical analysis demonstrated that AngII administration enhanced OPN expression in heart but was suppressed by cilostazol administration. Further, to investigate the mechanism, human cardiac myocytes were cultured and stimulated with AngII (1x10
-7
M). Co-treatment of Cilostazol (1x10
-7
to 1x10
-5
M) attenuated AngII-induced increase of
Spp-1
gene expression in dose-dependent manner. This effect was mimic by a treatment with forskolin, which was diminished by co-treatment with H-89.

Conclusion:
Cilostazol attenuated AngII-induced cardiac fibrosis in vivo. Cilostazol attenuated AngII-induced increment of
Spp-1
gene expression through cAMP-PKA dependent pathway.
Abstract P306: Inflammatory Biomarkers Participate in Physiopathology of Hypertensive Crisis
Authors: Vilela-Martin Jose F , Oliveira-Andrade Days , Cosenso-Martin Luciana N , Gregorio Michele L , Godoy Moacir F , Yugar-Toledo Juan C , Souza Doroteia R
Published: June 2021

Background:
Recent evidence suggests the existence of an underlying inflammation process to vascular disease associated with chronic high blood pressure (BP), playing an important role in the pathophysiology of hypertension (HT). However, few studies show the participation of the inflammatory process in the pathophysiology of acute BP elevation.

Objectives:
To identify clinical and metabolic profile of presentation of hypertensive crisis (HC) divided into hypertensive urgency (HUrg) and emergency (HEmerg); 2 - To assess the involvement of inflammatory cytokines: IL-1β, IL-6, IL-8, IL-18, TNF-α and anti-inflammatory IL-10 in subjects with HC.

Methods:
We studied 274 individuals: 74 normotensive (NT), 74 controlled hypertensive (CHT), 50 HUrg and 78 HEmerg. Serum levels of cytokines were made by MULTIPLEX and ELISA techniques. Analysis of variance was used to compare the groups, with significant p-value <0.05.

Results:
The diastolic BP and heart rate were greater in the HC group (120mmHg and 85bpm, respectively) compared to the CHT group (75mmHg and 68 bpm, respectively; p<0.05). Individuals with HEmerg were older. Glycaemia was significantly higher in the HEmerg group (113mg/dL) in comparison to NT and CHT (91mg/dL and 98mg/dL, respectively; p<0.05). HDL-c was significantly lower in HEmerg group compared to the NT group (p=0.0088). Potassium levels were lower in HEmerg group compared to the other groups NT, CHT and HUrg (p=0.0118, p=0.036 and p=0.036; respectively). All measured cytokines were significantly greater in subjects who had HC compared to NT and CHT groups. Logistic regression showed that IL-1β, IL-6, IL-8 and IL-18 were predictors for HC development with odds ratios of: 4.29 (2.01 - 9.19), 11.02 (3.98 - 30.45), 22.56 (7.99 - 63.64) and 3.85 (1.85 - 8.02), respectively.

Conclusions:
The levels of inflammatory cytokines (IL-1β, IL-6, IL-8 and IL-18) and anti-inflammatory (IL-10) are higher in HUrg and HEmerg groups compared to NT and CHT groups. This suggests the involvement of inflammatory cytokines in the pathogenesis of acute hypertensive event.
Abstract P312: A Novel Selective Melanocortin-4 Receptor Agonist Attenuates Bradycardia and Hyperglycemia in Diabetic Rats
Authors: do Carmo Jussara M , da Silva Alexandre A , Hall John E
Published: June 2021

We previously demonstrated that functional brain melanocortin 3/4 receptors (MC3/4R) are required for leptin to exert its chronic cardiovascular and antidiabetic actions. To determine if chronic treatment with a selective MC4R agonist reduces blood glucose and prevents bradycardia in diabetic rats, we used a novel compound with high affinity to MC4R. Male 12-week-old Sprague-Dawley rats (n=5/group) were instrumented with telemetry probes for determination of mean arterial pressure (MAP) and heart rate (HR) 24-hrs/day and an intracerebroventricular (ICV) cannula was placed in the brain lateral ventricle for continuous infusion of the selective MC4R agonist PL6214 (2.5 μg/hr) or MTII (a non-selective MC3/4R agonist, 10 ng/hr) via osmotic minipump. Induction of diabetes caused hyperphagia (20±1 to 32±2 g), hyperglycemia (89±3 to 494±44 mg/dl) and bradycardia (-47 bpm). Chronic infusion of PL6214 for 11 days transiently reduced food intake which returned to diabetic values by day 6 after starting the infusions, whereas chronic infusion of MTII caused a reduction in food intake lasting only 3-4 days. PL6214 reduced blood glucose by 63% on day 2 and 16% by day 11 of infusion, and prevented further bradycardia induced by diabetes (-35±14 bpm on the last day of infusion). Chronic MTII infusion reduced blood glucose by 31% on day 2 and by day 6 glucose levels had already returned to values observed before treatment was started. MTII infusion did not attenuate the bradycardia (-99±13 bpm on the last day of infusion). Diabetes did not alter MAP, while the MC4R agonist increased MAP by 5±1 mmHg compared to control values. These results indicate that selective activation of MC4R agonist attenuates bradycardia and hyperglycemia in type 1 diabetes, and may provide a new strategy for treatment of diabetes. (P20GM104357, NHLBI-PO1HL51971, AHA-SDG5680016 and Palatin Technologies).
Abstract P313: Alterations in Notch Signaling in Diabetic Coronary Microvascular Smooth Muscle and Endothelial Cells
Authors: McCallinhart Patricia , Lilly Brenda , Trask Aaron J
Published: June 2021

Notch signaling between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is crucial during normal vascular development and homeostasis and may contribute to vasculopathies. Notch signaling is known to regulate VSMC phenotypic mechanisms, including proliferation and differentiation. We previously reported differences in VSCM Notch3 and EC Jagged1 mRNA expression, as well as a de-differentiated VSMC phenotype, in diabetic coronary resistance microvascular (CRM) cells as a potential mechanism for the inward remodeling observed in those vessels. The current study tested the hypothesis that VSMC-EC dependent Notch signaling accounts for phenotypic changes in VSMCs that may promote adverse remodeling observed in diabetic CRMs. Low-passage primary coronary VMSCs were isolated from normal (n=6) and type 2 diabetic db/db mice (n=4) and were either cultured alone or co-cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively. Notch2 expression was significantly attenuated in diabetic CRM VSMCs in both mono- and co-culture conditions (0.53±0.12 vs. 1.00±0.06,
p
<0.01 and 0.59±0.09 vs. 0.94±0.04,
p
<0.01, respectively). There were no significant differences in Hes1, and Hrt3 was increased in only normal VSMCs that were co-cultured with ECs. We further found that proliferation inhibitor p53 expression was significantly lower in diabetic CRM VSMCs in when co-cultured with ECs (0.78±0.15 vs. 1.34±0.13,
p
<0.01), and Smad3 was significantly reduced in diabetic co-cultured CRM VSMCs (0.90±0.02 vs. 1.14±0.06,
p
<0.05). Collectively, these data implicate that Notch signaling through p53 and Smad3 may account for phenotypic alterations of diabetic CRM VSMCs.
Abstract P314: Urinary Renin and Kidney RAS Activation in Mice with Diabetic Kidney Disease
Authors: Rein Johannes , Valles Patricia , Wysocki Jan , Ye Minghao , Afkarian Mariam , Batlle Daniel
Published: June 2021

RAS is overactive in kidneys from patients with diabetic nephropathy (DN), but circulating plasma renin activity (PRA) is usually low. This is known as the renin-paradox. We evaluated juxtaglomerular (JGA), tubular and urinary renin, as a potential source of local RAS activation, to gain some understanding of this paradox.

Mice with STZ induced diabetes were used which had mild albuminuria and glomerular mesangial expansion consistent with early DN. Renin expression in the JGA and in the collecting tubule (CT) was evaluated by immunohistochemistry. IF was used to localize renin within CT cells. Proximal tubular renin was evaluated by RT-real time PCR in microdissected proximal tubules (PT). Urinary renin and Ang II were measured by ELISA.

Urinary Ang II was increased (37.8±11.4 vs. 99.0±21.6 pg/mg creat, p<0.05) reflecting an active kidney RAS. Urinary renin was also increased in STZ-treated as compared to controls (Table). In microdissected PTs there were no significant differences in renin mRNA between control and STZ-mice. By immunostaining, renin was localized to principal cells in the CT and the number of renin stained CTs was higher in STZ than in control mice. In sharp contrast, renin staining of the JGA of STZ-mice was significantly reduced as compared to controls.

We conclude that in DN renin expression in the JGA, the physiologic site of renin secretion into the circulation is suppressed, whereas in the CT it is increased. Activation of the kidney RAS, as inferred from increased urinary Ang II, likely occurs as a result of renin of tubular origin rather than from JGA renin. Since PT renin is not increased, the CT may provide the source of tubular renin for RAS activation.
Abstract P316: Angiotensin-(1-7) Contributes to the Insulin-sensitizing Effects of Renin-angiotensin System Blockade in High Fat Fed Mice
Authors: Arnold Amy C
Published: June 2021

Angiotensin-converting enzyme (ACE) inhibitors reduce body weight, lower blood pressure, and improve glucose homeostasis in animal models of the cardiometabolic syndrome. These effects are generally attributed to a reduction in angiotensin (Ang) II formation; however, these therapies also increase circulating levels of Ang-(1-7), a peptide with direct anti-hypertensive and insulin-sensitizing effects. In this study, it was hypothesized that endogenous Ang-(1-7) generation contributes to the beneficial cardiometabolic effects of ACE inhibition. To test this hypothesis, diet-induced obesity was produced in adult male C57BL/6J mice by placing them on a 60% high fat diet for 11 weeks. The Ang-(1-7) mas receptor antagonist A779 (400 ng/kg/min) or saline was given after 8 weeks of the high fat diet by subcutaneous osmotic mini-pumps. Immediately following mini-pump implantation, mice received water containing the ACE inhibitor captopril (50 mg/L) or plain tap water. Hyperinsulinemic (4 mU/kg/min) euglycemic clamps were performed in conscious, unrestrained vehicle (n=6), captopril (n=6), or captopril plus A779 (n=13) mice at the end of the 3-week treatment period. Blood pressure was measured via an indwelling carotid artery catheter connected to a transducer on the morning of the clamp. Captopril reduced body weight (28±2 vs. 41±2 g vehicle; p=0.001), lowered blood pressure (systolic: 109±6 vs.144±7 mmHg vehicle; p=0.003), and improved whole-body insulin sensitivity (steady-state glucose infusion rate: 31±4 vs. 16±2 mg/kg/min; p=0.008) in high-fat fed mice.
Mas
receptor antagonism with A779 attenuated the improvement in insulin sensitivity produced by captopril in high fat fed mice (23±2 mg/kg/min; p=0.042). There was no effect of A779 on the weight loss (32±2 g) or blood pressure lowering effects (111±7 mmHg) of captopril. These findings suggest that the improvement in insulin sensitivity produced by ACE inhibition is at least partly mediated by Ang-(1-7) pathways, and provide new insight into potential mechanisms involved in renin-angiotensin system blockade.
Abstract P320: Proliferationof Endogenous T-regs Improves the Pathophysiology Associated with Placental Ischemia of Pregnancy
Authors: Ibrahim Tarek , Przybyl Lukasz , Harmon Ashlyn C , Connelius Denise C , Cunningham Mark W , Amaral Lorena M , Faulkner Jessica L , Dechend Ralf , LaMarca Babbette
Published: June 2021

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with pro-inflammatory cytokines and decreased regulatory immune mechanisms such as Tregs, IL-10 and IL-4. We believe this decrease in immune regulatory mechanisms leads to an uncontrolled proinflammatory response which contributes to most of the pathophysiology associated with PE. The Reduced Uterine Perfusion Pressure, RUPP, rat model of induced placental ischemia exhibits similar characteristics as women with PE including high blood pressure, elevated pro-inflammatory cytokines and cells and decreased Tregs, IL-4 and IL-10. Therefore, we hypothesized that stimulating Tregs by administration of a superagonist (SA) would increase the Treg profile in the RUPP rats which could reduce pro-inflammatory cytokines and blood pressure. The RUPP procedure was performed at gestation day 14 (GD14); SA was administered intraperitoneally at GD15, GD18 carotid catheters inserted, and GD19 MAP and pup weight, serum and tissues were collected. MAP in NP rats was 98.6 mmHg ± 4.71, 122.2 mmHg ± 1.84 in RUPPs which was improved to 110.789mmHg ± 1.23 in RUPP+SA. Circulating FoxP3+ Treg cells were 5.987% ± 1.69% of total T-cells population in NP, 0.77% ± 0.49% in RUPP rats but increased to 11.218% ± 2.9% in RUPP+SA; Circulating IL-6 levels were 41.008 ± 4.79 pg/mL in NP, 108.26 ± 25.99 pg/mL in RUPP, and 40.37 ± 4.49 pg/mL in RUPP+SA, Administration of the SA to the NP rats did not affect IL-6 Levels in comparison to NP at 36.79 ± 3.9 pg/mL. Plasma IL-10 Levels were at 58.399 ± 9.527 pg/mL in NP rats, these levels were significantly decreased in the RUPP rats, 26.298 ± 4.33 pg/mL, and treatment of the RUPPs with the SA significantly increased plasma IL-10 levels to 51.5486 ± 3.329 pg/mL. When the SA was given to NP rats, the levels for IL-10 were significantly higher compared to any of the other groups at 85.5207 ± 9.067 pg/mL. Placental Pre-pro Endothelin-1 (PPET-1) was increased 44.42 ± 0.269 fold in RUPP compared to NP 1 ± 0.255, but was decreased to 18.78 ± 0.48 in RUPP+SA. These data suggest an important role for up-regulating Treg cells to enhance the immune regulatory interactions and lower the hypertension without causing further reduction in fetal weight in response to placental ischemia during pregnancy.
Abstract P323: Arginine Vasopressin and Indoleamine 2,3 Dioxygenase: The Early Immunovascular Interface in Preeclampsia
Authors: Santillan Donna A , Scroggins Sabrina M , Devor Eric J , Hunter Stephen K , Grobe Justin L , Sigmund Curt D , Santillan Mark K
Published: June 2021

Preeclampsia (PreE), a hypertensive disease of pregnancy, causes maternal and fetal health complications. Abnormal placental development/angiogenesis and poor immunoregulation (involving T cells, Indoleamine 2, 3 Dioxygenase (IDO), and dendritic cells) are central to the development of PreE. IDO, produced by regulatory immune cells, degrades tryptophan to arrest inflammatory T cell proliferation and induces T regulatory cell development. Our data and others show decreased IDO placental expression from human preeclamptic pregnancies. We published that deletion of IDO (IDO KO) in pregnant mice results in pathognomonic glomerular endotheliosis, proteinuria, and intrauterine growth restriction, hallmark features of PreE. Our group also demonstrated that, plasma copeptin, a stable bio-marker of vasopressin (AVP) secretion, is elevated early in human PreE pregnancies; and AVP infusion into wild-type C57BL/6J dams phenocopies human PreE, including increased inflammatory T cells and highly activated dendritic cells. Here, we test our hypothesis that AVP (via copeptin measurement) is elevated in the IDO KO mouse model of PreE and that IDO activity is decreased in the AVP mouse model of PreE. Copeptin, measured by ELISA, was elevated in both the placenta (2.4±0.2 vs. 1.7±0.2 pg/mg, p=0.03) and maternal serum (2.1±0.2 vs. 1.2±0.4 pg/mg, p=0.03) from IDO KO pregnancies compared to wild-type at gestational day (GD) 18. In our chronic infusion of AVP model of PreE (24 ng/hour), GD 18 colorimetric IDO activity was decreased by 22% in the maternal kidney (N=10 per group) and by 27% in the amniotic fluid (saline N=8 vs. AVP N=12) of AVP-infused dams in comparison to controls. Collectively, these data demonstrate an inverse relationship between IDO activity and copeptin expression in PreE pregnancies. As both IDO and AVP sit at the crossroads between vascular and immune dysfunction, these data suggest that the IDO-AVP interaction may contribute to the maternal and fetal renal phenotype observed in preeclampsia.
Abstract P325: Enhanced Afferent Arteriole Dilatation in Dahl Salt-sensitive Rats (dahlss): Role of Connecting Tubule Glomerular Feedback (ctgf)
Authors: Wang Hong , Romero Cesar A , Janic Branislava , Peterson Edwards , Carretero Oscar A
Published: June 2021

Afferent arteriole (Af-Art) resistance is modulated by 2 intrinsic nephron feedbacks: the vasoconstrictor tubuloglomerular feedback (TGF) and the vasodilator CTGF. TGF is mediated by NKCC2 channel in the macula densa and blocked by furosemide; and CTGF is mediated by ENaC in the connecting tubule and blocked by benzamil. Previously we measured CTGF indirectly, by differences between TGF response with and without CTGF blocker benzamil. Thus, using this indirect measurement we reported that Dahl SS have greater CTGF than Dahl salt-resistant rats (Dahl SR). We have recently developed a new method to measure CTGF more directly and we found that when we simultaneously blocked TGF with furosemide and CTGF with benzamil, the increasing tubular perfusion caused Af-Art constriction (TGF-like) that is mediated by the NHE. W
e hypothesize that in vivo during simultaneous inhibition of NKCC2 and the NHE, CTGF causes an Af-Art dilatation revealed by an increase
in
stop-flow pressure (P

SF
) and that is greater in Dahl SS than in Dahl SR in a high salt diet.

In the presence of furosemide alone, increasing nephron perfusion did not change the P
SF
in neither Dahl SS nor Dahl SR. When we blocked both, NKCC2 with furosemide and NHE with DMA, increase in tubular flow caused Af-Art dilation that was demonstrated by an increase in P
SF
.

This increase was greater in Dahl SS (5.1±0.4 mmHg) than in Dahl SR (2.9±0.3 mmHg;
P
< 0.01), (Fig).We confirm that CTGF causes this vasodilation, since benzamil completely blocked this effect. We conclude that during inhibition of NKCC2 and NHE in the nephron CTGF (Af-Art dilatation) is enhanced in Dahl SS as compared to Dahl SR.
Abstract P329: Age- and Sex-differences in Salt Sensitivity and Renal Function in a Rat Model of Autosomal Recessive Polycystic Kidney Disease
Authors: Jin Chunhua , Mrug Michal , Yoder Bradley K , Kesterson Robert A , Inscho Edward W , Pollock David M
Published: June 2021

Background:
Most forms of hypertension are influenced by gender and salt sensitivity, however, the interplay of these two factors and their functional consequences in polycystic kidney disease (PKD)-associated hypertension are poorly understood. Thus, we tested the hypothesis that age and male sex would pre-dispose animals with PKD to hypertension and risk of renal dysfunction.

Methods:
We used the
Pkhd1
pck
rat model of autosomal recessive PKD to study sex differences in blood pressure in response to a high salt diet (HSD). Two month- and eight month-old male and female
Pkhd1
pck
rats were surgically implanted with telemetry transmitters and allowed to recover for at least one week before obtaining baseline mean arterial pressure (MAP). Rats were maintained on either HSD (4% NaCl) or normal rat chow (0.49% NaCl) for 3 weeks. At the end of the study, rats were placed in metabolic cages and a 24 hr urine sample was collected before measuring GFR (transdermal sinistrin clearance).

Results:
In the 2-month old
Pkhd1
pck
rats, blood pressures were in a normal range and there were no differences between males and females. Furthermore, 3 weeks on a HSD had no effect on 24 hr MAP. GFR was similar between male and female rats on either diet. However, urinary protein excretion was significantly higher in HSD fed rats compared to normal rat chow in both male (83±26 vs. 17±4 mg/day, respectively, p<0.05) and female rats (106±25 vs. 6±2 mg/day, p<0.05). In 8-month old rats, again there were no sex differences between animals on normal rat chow. However, MAP increased progressively in male rats after 3 weeks of HSD feeding, a change that was significantly greater than females (Δ39±6 vs Δ15±5 mmHg, p<0.05). The blood pressure increase in male rats was associated with higher urinary protein excretion compared to female rats (755±118 vs. 390±48 mg/day, p<0.05). HSD significantly reduced GFR in male, but not female rats (0.48±0.13 vs. 1.24±0.05 mL/min/100 g bwt, respectively, p<0.05). GFR was similar between older male and female rats on normal salt diet.

Summary:
Our studies demonstrate that male
Pkhd1
pck
rats with advanced cystic kidney disease are more vulnerable to salt sensitive hypertension and renal injury than age-matched females.
Abstract P330: Role of Na-H Exchanger Regulatory Factor 1 (NHERF1) In Salt-sensitive Hypertension and Inflammation in Aging
Authors: Khundmiri Syed J
Published: June 2021

Aging animals develop salt-sensitive hypertension due, in part, to desensitization of dopamine receptors and sensitization of AT1R in renal proximal tubules. We demonstrated old FBN rats develop salt-dependent hypertension. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 may contribute to salt reabsorption in aging. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed 1% or 8% NaCl diet for one week and, urinary volume, sodium, potassium, and chloride and expression of Na-K ATPase α1 subunit (NKA), NHERF1, and D1R were measured. Feeding 8% NaCl increased 24 h urine volume compared to rats fed 1% NaCl diet in 1m (13.6±0.98 vs 29.2±3.8 mL) and 4m (15.3±1.5 vs 39.2±2.4) but not in 24 m rats (20.7±4.7 vs 15.9±2.4). 24 h urine Na increased 7 fold in 4 m old rats fed an 8% NaCl diet but only 3 fold in 24m old rats. To our surprise, NHERF1 expression increased with age in FBN rats (0.21±.03 (4m) vs 0.71±0.07 (24m) AU ratio NHERF1:actin). In F344 rats NHERF1 expression decreased with age (0.86±0.14 vs 0.39±0.05). Increased NHERF1 expression with age in FBN rats was associated with decreased D1R expression (0.8±0.16 vs 0.4±0.04) while in F344 rats D1R expression increased with age (0.6±0.1 vs 0.9±0.1). FBN rats are salt-sensitive while F344 are salt-resistant. F344 rats develop kidney inflammation with aging. We determined TNFα in kidney homogenates from FBN and F344 rats. TNFα increased with age in both F344 (4.04±0.15 vs 6.9±0.42) and FBN rats (2.1±0.3 vs 4.05±0.31 ng/mg protein). To determine if lack of NHERF1 is responsible for salt wasting, we measured urine volume and Na in 18 m old WT and NHERF1 KO mice. The urine volume in WT mice (2.7±0.8 (1% NaCl) vs 5.6±1.2 mL/24 h (8% NaCl)) were significantly lower than KO mice (3.6±0.57(1% NaCl) vs 7.5±0.88 mL/24 h (8% NaCl)). Similar results were observed in Na excretion. D1R expression increased in NHERF1 KO mice as compared to WT mice. TNFα increased significantly in NHERF1 KO mice kidneys as compared to WT mice. We conclude that increased NHERF1 expression as seen in FBN rats results in salt retention while lack of NHERF1 as seen F344 rats and NHERF1 KO mice increases renal inflammation. Further studies are required to delineate the two roles of NHERF1.
Abstract P333: The Impact of Sodium on Blood Pressure and Arterial Stiffness is Moderated by Indices of Obesity in People of African Ancestry.
Authors: Muzi Maseko
Published: June 2021

Background:
Obesity is on the rise worldwide and like Na
+
it is associated with blood pressure (BP) and target organ changes. Our study population has a high incidence of obesity (67%) and a dietary sodium intake that is slightly above the recommended threshold. Previous studies conducted in this population have shown no relationship between Na
+
and both BP and arterial stiffness. With the high incidence of obesity in this population, it is possible that the indices of obesity blunt this relationship. Therefore in this study we investigate whether the relationship between Na
+
and both BP and PWV is moderated by the indices of obesity.

Methods:
We recruited 1219 South Africans of African ancestry and measure 24-h ambulatory on 796 participants and 597 had complete 24-hour urine collection. Anthropometric measurements were taken and a standard questionnaire was issued to determine lifestyle habits and history of medication. To assess arterial stiffness we used applanation tonometry to measure pulse wave velocity (PWV).

Results:
After correcting for covariates, there was an association between Na
+
and PB in participants with normal BMI but not in obese participants. Similarly there was a relationship between Na
+
and PWV (p=0.0447) in individuals with normal BMI only. When waist circumference was used as an index of obesity, gender disparities were observed in the relationship between Na
+
and PWV. There was no relationship between Na
+
and PWV irrespective the waist circumference in males but a multivariate regression analyses showed a relationship between Na
+
and PWV in all women (p=0.0142) and in women with a normal waist circumference (p=0.0006).

Conclusion:
In a population with a high incidence of obesity, the relationship between Na
+
and both BP and PWV is modified by the indices of obesity.