bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 383: Candesartan, in a Dose-Related Manner, Upregulates the Protective Axis of the Renal Renin Angiotensin System and Prevents Progression of Diabetic Nephropathy in db/db Mice.
Authors: Callera Glaucia E , Zimermann Danielle , Correa Jose , Gutsol Alexey , He Ying , Yogi Alvaro , Nguyen Dinh Cat Aurelie , Briones Ana , Zimpelmann Joe , Burns Kevin , Touyz Rhian
Published: March 2022

The renin-angiotensin system (RAS) plays a pivotal role in the pathophysiology of cardiovascular and kidney disease. Protection against kidney injury has been evidenced by high dose angiotensin II (Ang II) type 1 receptor (AT1R) blockers. In this study we sought to determine whether a high dose of candesartan has renoprotective actions associated with the modulation of RAS component expression in type 2 diabetic db/db mice. Systolic blood pressure (SBP), urinary albumin excretion (albumin/creatinine, ACR), and protein expression/activity of RAS components were assessed in db/db and control non-diabetic db/+ mice infused with the AT1R blocker candesartan (1, 5, 25, 75 mg/kg/day, sc, 4 weeks). Vehicle treated db/db mice exhibited increased albuminuria (38.9 ± 11.9 ACR) compared with db/+ mice (6.77 ± 1.7 ACR) with no differences in SBP between the two groups (110 ± 9 vs 116 ± 4 mmHg). Candesartan (1 mg/kg/day) had no effect on SBP, although albuminuria was decreased in db/db mice (8.8 ± 3.0 ACR). Candesartan 5 mg/kg/day reduced SBP only in db/+ mice (92 ± 8 mmHg). Diabetic mice receiving 5mg/kg/day showed less albuminuria compared with vehicle treated db/db mice (21.4 ± 6 ACR). Candesartan 25mg/kg/day reduced SBP in both db/db and db/+ mice (77 ± 5 and 76 ± 4 mmHg) and decreased albuminuria in db/db mice (14.95 ± 6.4 ACR). Reduction in SBP (80 ± 7 mmHg) with no improvement of albuminuria (55.9 ± 6 mmHg) was observed in diabetic mice receiving 75 mg/kg/day candesartan. Histological findings showed that candesartan at low dose, but not at high dose, prevented tubular damage in db/db mice. Candesartan, 1-25 mg/kg/day, increased renal expression of AT2R in db/db mice. Expression of ACE2 and MAS receptor was increased only by candesartan (1 mg) in db/db mice. ACE activity was reduced in plasma and kidney cortex of db/db mice, without effect of candesartan. ACE2 activity was increased by candesartan in plasma (5 mg/kg/day) and kidney cortex (1 mg/kg/day) from db/db mice. These results indicate that low to intermediate doses of candesartan reduce the magnitude of albuminuria and tubular damage in diabetic nephropathy. These effects were not evidenced at a higher dose of candesartan. Such processes may be due to activation of the protective axis (ACE2/MAS/AT2R) of the renal RAS.
Abstract 388: Identification of FOXO1 as a Major Transcriptional Mediator of Angiotensin-(1-7) Signaling in Human Endothelium
Authors: Verano-Braga Thiago , Schwäle Veit , Sylvester Marc , Silva Danielle G , Peluso Antonio A , Etelvino Gisele M , Roepstorff Peter , Santos Robson A
Published: March 2022

Angiotensin-(1-7) has been shown to induce vasodilation, positive regulation of insulin, anti-proliferative and anti-tumorigenic activities via Mas receptor activation. However, little is known about the Ang-(1-7) signal transduction. The aim of this study was to identify proteins involved in the Ang-(1-7) signaling using mass spectrometry-based time-resolved quantitative phosphoproteome of human aortic endothelial cells (HAEC) treated with Ang-(1-7). HAEC were incubated with 10-7 M of Ang-(1-7) for 3, 5 and 20 min. After cell harvesting and protein digestion with tripsin, peptides were labeled (iTRAQ). Enriched phosphopeptides were separated by reversed-phase liquid chromatography. An LTQ Orbitrap XL instrument was used for mass-espectrometry analysis. We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Among these proteins forkhead box protein O1 (FOXO1) was dephosphorylated at Ser256. Following S256 dephosphorylation, FOXO1 becomes active and then translocates to the nucleus where it can increase transcription of genes involved in apoptosis, cell arrestment, glucose metabolism, oxidative protection and tumor suppression. Indeed, additional studies in HAEC showed that following 3 minutes of Ang-(1-7) treatment, FOXO1 was accumulated in the HAEC nucleus which is in accordance with the downphosphorylation of S256. Furthermore, we observed that FOXO1 accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7), supporting the antitumorigenic activity of this angiotensin peptide. Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, we performed for the first time a large-scale MS-based phosphoproteomics approach to interrogate the Ang-(1-7)/Mas signaling in human endothelial cells. Our data open new possibilities for the understanding of the mechanisms underlying Mas activation in the human endothelium.
Abstract 39: The Macula Densa PGE2 Activates Adult Renal CD44+ Mesenchymal Stromal Cell (MSC) Migration and Differentiation to Renin Producing cells via EP4 Receptor and Wnt Canonical Signaling
Authors: Yang Yanqiang , Gomez Jose , Herrera Marcela , Repenning Peter , Zhang Zhiping , Payne Alan , Pratt Richard , Koller Beverly , Coffman Thomas , Mirotsou Maria , Dzau Victor J
Published: March 2022

Background:
We have previously shown that CD44+ MSCs exist in the adult kidney and that under chronic sodium deprivation these cells migrate to the juxtaglomerular (JG) area and differentiate to new renin expressing cells. The mechanisms mediating these biological processes remain elusive. Renin expression and release by JG cells is initiated at the Macula densa (MD) primarily by the release of PGE2. Interestingly, renal MSC expresses the PGE2 receptor EP4. Here, we hypothesized that MD-derived PGE2 acting via the EP4 is responsible for the migration of renal MSCs and their differentiation to renin expressing cells.

Methods:
MSCs were isolated by FACS from the kidney of C57BL/6 adult mice and co-cultured in a transwell system with the MD cell line MMDD1 at normal or low NaCl medium. For the in vivo study, wild type and EP4 knockout mice were fed a low salt diet and given furosemide to induce JG hyperplasia. Renin, CD44 and B-catenin stabilization were detected by immunochemistry staining and Western blot analysis.

Results:
Low salt stimulation of MMDD1 cells significantly increased MSC migration in transwell assays (P<0.05). These effects were abrogated by addition of a COX2 inhibitor (NS398) to MMDD1 cells or an EP4 receptor antagonist (AH23848) to the MSCs. Treatment of MSCs with a PGI2 receptor antagonist (CAY 10441) had no effect on this migratory response. Furthermore, direct addition of PGE2, but not PGI2 to MSCs increased cell migration as compared to controls (P<0.05). PGE2 did not affect proliferation of MSCs but did induce renin expression, suggesting that PGE2 plays an important role in promoting the migration and differentiation of renal MSCs. PGE2 treatment also resulted in increased beta catenin stabilization and activation of canonical Wnt signaling; a pathway that plays an important role in progenitor cell migration and differentiation. Finally, the absence of the EP4 receptor in mice attenuated expansion, migration of renal CD44+ cells and JG hyperplasia induced by sodium deprivation.

Conclusion:
The MD/ PGE2/ EP4 axis plays a key role in the activation of MSCs and their differentiation to renin producing cells. This process could potentially be an important physiologic mechanism of maintaining blood pressure under conditions of sodium deprivation.
Abstract 392: Sexual Dichotomy in Blood Pressure Regulation: Is Ang (1-7) a Key Player?
Authors: Sullivan Jennifer C , Zimmerman Margaret , Harris Ryan A
Published: March 2022

The prevalence of cardiovascular disease is greater in men than in women, and although the mechanism responsible is unknown, sex differences in Angiotensin (Ang) peptides have been suggested to contribute. In fact, evidence in rats indicates Ang (1-7), a vasodilatory peptide, is higher in females when compared to males. In humans, the flow-mediated dilation (FMD) test represents a bioassay of NO-dependent vasodilation and is a predictor of future cardiovascular disease. This study sought to test the hypotheses that 1) Ang (1-7) is higher in women compared to men, and 2) determine if relationships exist among concentrations of Ang (1-7), FMD, and blood pressure. A total of 29 subjects participated in this study; FMD was obtained on 14 (7 men; 7 women). Following an overnight fast, blood pressure and FMD were performed and blood samples were collected. Systolic blood pressure was greater in men (121±5 mm Hg) compared to women (101±3 mm Hg; p=0.01); however, no difference in diastolic blood pressure was observed (66±4 vs. 63±2 mm Hg, respectively). Plasma concentrations of Ang (1-7) were significantly higher in women compared to men (43±5 vs. 32±4 pg/mL; p=0.04). FMD was positively associated with Ang (1-7) independent of sex (r=0.592; p=0.02); however, the relationship between Ang (1-7) and diastolic blood pressure was evident only in women (women r=-0.717; p=0.07 and men r=0.186; p=0.69). These data support a sexual dimorphism in Ang (1-7) in humans, similar to what we have observed previously in our animal model. For the first time in humans, we have also observed a sex dependent relationship between Ang (1-7) and diastolic blood pressure supporting the hypotheses that Ang (1-7) may play an important role in BP regulation and maintenance of endothelial function particularly in women. These data may provide insight into the cardiovascular disease risk disparity between sexes.
Abstract 404: The Use of Renin and Aldosterone Profiling in the Treatment of Complex Hypertension
Authors: Haddad Danny , Cohen Debbie
Published: March 2022

Uncontrolled hypertension (HTN) remains highly prevalent and contributes to the risk for increased CVD and stroke. The purpose of this study is to determine if renin/aldosterone profiling in patients new to the complex HTN clinic would aid in identification of certain subsets of HTN and lead to improved BP control.

Methods:
We conducted a retrospective study of all new patients seen at the Penn HTN clinic by a single HTN specialist between 2005-2010. We examined all adult patients that were screened with plasma renin (ng/ml) and serum aldosterone (ng/dl) levels. Patients with known causes of secondary HTN such as pheochromocytoma were excluded. All data was reviewed from the first visit and for all subsequent visits for the first year. The patients were classified as follows: normal, salt sensitive (renin < 1, aldo <15), hyperaldosteronism (renin < 1, aldo ≥ 15) and high renin (renin ≥ 10). Medications were then tailored to the individual profile.

Results:
We identified 158 patients with a mean age of 52.9 ± 1.25 years, 47% (74 of 158) males; 53% (84 of 158) females; 75% (117 of 158) were white; 21% (33 of 158) black and 4 % (6 of 158) other. The mean SBP/DBP at baseline for the entire cohort was 149/87 ± 1.85/1.17 mmHg and was decreased by a mean of 20.3/8.6 ± 1.88/1.2 at 1 year (p=0.000). There was no change in the overall number of medications at baseline and at one year despite improved BP control ( p = 0.770).

Conclusion:
Renin/aldosterone profiling in subjects with severe HTN helps the physician tailor therapy resulting in improved BP control without an overall increase in the number of medications needed. Most patients did require at least 3 meds to achieve good BP control.
Abstract 41: A Novel Role of the Na+/H+ Exchanger 3 (NHE3) in Angiotensin II-induced Hypertension in Wild-type and NHE3-deficient Mice
Authors: Li Xiao C , Schull Gary E , Miguel-Qin Elisa , Zhuo Jia L
Published: March 2022

The renal mechanisms underlying angiotensin II (ANG II)-induced hypertension remain incompletely understood. We reasoned that the Na
+
/H
+
exchanger 3 (NHE3) in proximal tubules of the kidney may play a key role. In the present study, we used NHE3-deficient mice (

Nhe3
-/-
)

to test the hypothesis that NHE3 is required for maintaining long-term blood pressure responses to ANG II. Three groups (n=8 each) of wild-type (

Nhe3
+/+

) and

Nhe3
-/-

mice were treated with vehicle, ANG II (40 ng/min, i.p.,
via
minipump), or ANG II plus losartan (20 mg/kg/day, p.o.) for 2 weeks. Basal and weekly systolic blood pressure (SBP) and urinary water, sodium (Na
+
), potassium (K
+
) and chloride (Cl
-
) excretory responses were measured by the tail-cuff and metabolic cage methods. Under basal conditions,

Nhe3
-/-

mice grew normally (

Nhe3
+/+

: 23±0.3 g vs.

Nhe3
-/-

: 23±0.7 g, n.s.), but had a significantly lower SBP level (

Nhe3
+/+

: 120±3 mmHg vs.

Nhe3
-/-

: 106±3 mmHg,
p
<0.01), 24 h urine excretion (

Nhe3
+/+

1.23±0.15 vs.

Nhe3
-/-

: 0.82±0.10 ml,
p
<0.05), urinary Na
+
excretion (

Nhe3
+/+

: 232.1±10.3 vs.

Nhe3
-/-

: 34.8±3.6 μmol/24 h,
p
<0.01), and urinary K
+
excretion (

Nhe3
+/+

: 343.8±19.4 vs.

Nhe3
-/-

: 194.2±19.8 μmol/24 h,
p
<0.01). Basal plasma ANG II (

Nhe3
+/+

: 313.4±19.9 vs.

Nhe3
-/-

: 397.7±17.2 pg/ml,
p
<0.05) and aldosterone levels (

Nhe3
+/+

: 505.6±26.3 vs.

Nhe3
-/-

: 802.8±17.3 pg/ml,
p
<0.01) were significantly elevated in

Nhe3
-/-

mice. Kidney ANG II levels were not statistically different between

Nhe3
+/+

and

Nhe3
-/-

mice. In

Nhe3
+/+

mice, ANG II infusion markedly increased SBP in a time-dependent manner (
p
<0.01). However, the SBP response to ANG II was markedly attenuated in

Nhe3
-/-

mice despite plasma ANG II and aldosterone levels were further elevated by ANG II infusion (

Nhe3
+/+

: 165±4 mmHg vs.

Nhe3
-/-

: 119±4 mmHg,
p
<0.01 at 2 weeks). 24 h urinary K
+
and Cl
-
excretory responses were increased by ANG II in

Nhe3
-/-

mice without altering 24 h urinary Na
+
excretion. Concurrent losartan treatment normalized SBP responses to ANG II in

Nhe3
+/+

mice, but markedly increased the mortality in

Nhe3
-/-
mice

. We concluded that NHE3 in proximal tubules of the kidney, along with NHE3 in intestines, is required for maintaining long-term blood pressure responses to ANG II and body salt and fluid homeostasis in mice.
Abstract 415: Brain Epidermal Growth Factor Receptor and c-Src Tyrosine Kinase Contribute to Sympathetic Excitation Induced by Systemically Administered Aldosterone in Rats
Authors: Zhang Zhi-Hua , Yu Yang , Wei Shun-Guang , Felder Robert B
Published: March 2022

Introduction:
Epidermal growth factor receptor (EGFR) and c-Src tyrosine kinase mediate multiple functions of aldosterone and angiotensin II in peripheral cardiovascular tissues by regulating mitogen-activated protein kinase (MAPK) signaling. Hypothesis: EGFR and c-Src activity in cardiovascular regions of the brain contribute to sympatho-excitatory responses induced by circulating aldosterone.

METHODS:
In anesthetized rats, we recorded renal sympathetic nerve activity (RSNA), mean blood pressure (MBP) and heart rate (HR) at baseline and for 4 hours during intravenous infusion of aldosterone (30 μg/hr), with or without concomitant intracerebroventricular (ICV) infusion of the EGFR inhibitor AG1478 (1 nmol/hr), the mineralocorticoid receptor (MR) antagonist RU28318 (10 μg/hr) or the p44/42 MAPK inhibitor PD98059 (5 μg/hr), or pretreatment with a bolus ICV injection of the c-Src inhibitor SU6656 (1 nmol). In some rats, hypothalamic paraventricular nucleus (PVN) tissues were collected to examine the expression of phosphorylated p44/42 MAPK.

Results:
Aldosterone infusion significantly (*p<0.05 vs baseline) increased RSNA (25.3 ± 4.6 %*), MBP (9.8 ± 2.2 mmHg*) and HR (28.2 ± 6.2 bpm*), as well as the PVN expression of phosphorylated p44/42 MAPK (86%, p<0.05), as compared with vehicle. Concomitant ICV infusion of the EGFR inhibitor AG1478 significantly (†p<0.05 vs aldosterone alone) diminished the phosphorylation of p44/42 MAPK (28 %†) in PVN and the increases in RSNA (3.6 ± 1.5 %†), MBP (3.2 ± 1.3 mmHg†) and HR (5.3 ± 5.1 bpm†) induced by aldosterone. ICV pretreatment with the c-Src inhibitor SU6656 had similar significant (p<0.05) inhibitory effects on the RSNA, MBP and HR responses to aldosterone. Concomitant ICV infusion of either the MR antagonist RU28318 or the p44/42 MAPK inhibitor PD98059 prevented the aldosterone-induced sympathetic excitation.

Conclusion:
Central inhibition of EGFR and c-Src significantly reduced the brain MR- and MAPK-dependent sympathetic excitation elicited by systemically administered aldosterone. Brain EGFR and c-Src are potential therapeutic targets for cardiovascular disease states characterized by high circulating levels of aldosterone.
Abstract 430: Race Dependent Effects of C Reactive Protein on the Vasomotor Modulators ET1 and NO in HUVECs
Authors: Grimm Heather , Ling Chenyi , Kretzschmar Jan , Park Joon Y , Brown Michael D
Published: March 2022

Background:
Chronic inflammation and elevated levels of C reactive protein (CRP) can lead to reduced bioavailability of endothelium relaxing factors such as Nitric Oxide (NO) and increased production of vasoconstrictive pro-inflammatory agents such as endothelin-1 (ET1). African Americans (AA) have impaired vasodilatory capabilities evident through reduced NO bioavailability. The purpose of this study was to investigate the potential differential racial effect of CRP on ET1 and NO.

METHODS:
AA and Caucasian (C) Human Umbilical Vein Endothelial Cells (HUVECs) were cultured and treated with 0, 25, 50, and 100μg/mL CRP. The secretion of ET1 and NO in HUVEC culture medium supernatant was assessed by ELISA and a modified Griess assay, respectively.

Results:
At baseline (0μg/mL CRP) there were no significant differences between AA and C in ET1 (457.5 ± 44.8 vs 420.6 ± 34.8 pg/mL) or NO (8.0 ± 1.4 vs 7.4 ± 1.2 μmol/L). Differences (p≤.05) were found at 50μg/mL CRP between ET1 and NO between C and AA HUVECs (ET1: 566.7 ± 17.7 vs 595.8 ± 73.6 pg/mL) (NO: 0 ± 0 vs 1.1 ± 1.0 μmol/L). There was a significant increase in ET1 in the AA HUVECs from 0μg/mL (457.5 ± 44.8 pg/mL) to 25μg/mL CRP (546.8 ± 39.8 pg/mL) while NO decreased significantly in AA HUVECs from 0μg/mL (8.0 ± 1.4 μmol/L) to 25μg/mL (0.5 ± 0.2 μmol/L), 50μg/mL (1.1 ± 1.0 μmol/L), and 100μg/mL CRP (0.1 ± 0.1 μmol/L). C HUVECs showed significant changes in ET1 between 0μg/mL (420.6 ± 34.8 pg/mL) and 50μg/mL CRP (566.7 ± 17.7 pg/mL) and in NO from 0μg/mL CRP (7.4 ± 1.2 μmol/L) to 25μg/mL (0.8 ± 0.5 μmol/L), 50μg/mL (0 ± 0 μmol/L), and 100μg/mL (0.9 ± 0.6 μmol/L). Without the influence of race, significant increases in ET1 production occurred from 0μg/mL CRP (439.0 ± 39.8 pg/mL) to 25μg/mL (512.0 ± 38.9 pg/mL) (p ≤ 0.001) and 50μg/mL CRP (581.2 ± 45.6 pg/mL) and significant decreases in NO (p ≤ 0.001) from 0μg/mL CRP (7.7 ± 1.3 μmol/L) to 25μg/mL CRP (0.6 ± 0.4 μmol/L), 50μg/mL CRP (0.5 ± 0.5 μmol/L), and 100 μg/mL CRP (0.5 ± 0.3 μmol/L).

Conclusions:
Our data show that differential CRP induced ET1 and NO production does not appear to contribute to the difference in hypertension prevalence between AA and C. Further investigation is needed to determine the cause of the racial differences in Hypertension between African Americans and Caucasians.
Abstract 441: Central Infusion of Angiotensin(1-7) Attenuates Aldosterone/Salt-Induced Increases in Blood Pressure in Ovariectomized Female Rats
Authors: Xue Baojian , Zhang Zhongming , Guo Fang , Hay Meredith , Johnson Alan K
Published: March 2022

In comparison to male rodents, females are protected against angiotensin II (ANG II)- and aldosterone (Aldo)-induced hypertension. However, the mechanism underlying this protective effect is not well understood. ANG(1-7) is formed from ANG II by angiotensin converting enzyme 2 (ACE2) and plays an anti-hypertensive effect in the CNS. Recent studies from our laboratory demonstrate that central blockade ANG(1-7) augments Aldo-induced hypertension in intact female rats. The present study further determined whether central infusion of ANG(1-7) will attenuate Aldo/salt-induced increase in blood pressure (BP) in ovariectomized (OVX) female rats. Systemic infusion of Aldo (0.75 μg/h, 4 weeks) into OVX female rats with 1% salt as their sole drinking fluid resulted in a significant increase in BP (Δ26.9±2.9 mmHg). Central infusion of ANG(1-7) significantly attenuated this Aldo/salt pressor effect (Δ11.9±2.8 mmHg). RT-PCR analysis revealed that mRNA expression of renin (from 3.6 to1.1-fold) and ACE1 (from 2.6 to 0.9-fold) were significantly reduced in Aldo/salt treated rats with central infusion of ANG(1-7) when compared with central vehicle infusion. In contrast, mRNA expression of ACE2 (from 1.0 to 1.4-fold), AT
2
R (from 0.4 to 1.2-fold) and estrogen receptor alpha (ERα, from 0.9 to 1.7-fold) were significantly increased in the lamina terminalis. Taken together these results suggest that a central antihypertensive arm of the brain renin-angiotensin system (ACE2/ANG(1-7)/Mas) may play an important compensatory role in the development of Aldo/salt induced hypertension in females. (HL-14388, HL-98207, DK-66086, and MH-80241)
Abstract 444: Proximal Tubule Angiotensin AT2 Receptors Mediate Anti-inflammatory Response via Interleukin-10: Role in Renoprotection in Obese Rats.
Authors: Dhande Isha , Ali Quaisar , Hussain Tahir
Published: March 2022

The angiotensin AT2 receptor (AT2R) has been shown to lower inflammation in the kidney. However the role of the anti-inflammatory cytokine IL-10 in AT2R mediated attenuation of inflammation has not been elucidated. We hypothesized that AT2R activation is renoprotective by directly increasing the levels of anti-inflammatory cytokine IL-10 in the kidney via nitric oxide (NO) signaling. For in vitro studies, the human proximal tubule epithelial cell-line (HK-2) was treated with lipopolysaccharide (LPS, 1 μg/ml) and/or AT2R agonist C21 (1μM) for 24 hrs and media cytokine levels were assessed. Compared to control, LPS treatment significantly increased TNF-α (1.25 ± 0.22 vs 46.27 ± 3.22 pg/ml), IL-6 (1.43 ± 0.06 vs 5.204 ± 0.41 ng/ml) and IL-10 (5.11 ± 0.46 vs 13.35 ± 0.74 pg/ml) levels. C21 treatment with LPS lowered the levels of both, TNF-α and IL-6 by 65% but caused 55% increase in IL-10 levels. C21 treatment alone significantly increased IL-10 levels (5.11 ± 0.46 vs 18.37 ± 0.87 pg/ml) while there was no effect on TNF-α or IL-6 levels. The anti-inflammatory effect of C21 was abolished in the presence of neutralizing IL-10 antibody (1 μg/ml). Also, pre-incubation with NO synthase inhibitor L-NAME (1 mM) prevented the C21-mediated decrease in IL-6 levels and increase in IL-10 levels in activated cells. For in vivo studies, 5 wk old lean and obese Zucker rats (OZR) were treated for 2 wks with C21 (300 μg/kg/day, i.p) and/or AT2R antagonist (PD123319, 50 μg/kg/min, s.c. infusion). Compared to LZR, OZR had higher levels (pg/mg protein) of renal TNF-α (1.42 ± 0.14 vs 2.78 ± 0.42) and IL-6 (6.59 ± 0.77 vs 11.71 ± 0.42). C21 treatment decreased levels of TNF-α by 75% and IL-6 by 60% while PD had no effect. Conversely, PD treatment lowered the renal IL-10 levels in OZR (8.5 ± 0.49 vs 3.69 ± 0.65 pg/mg protein) while C21 had no effect. Renal morphometry revealed increased mesangial matrix expansion score in OZR (0.27 ± 0.08 vs 1.3 ± 0.12) which was worsened by PD (1.97 ± 0.16) and improved by C21 treatment (0.8 ± 0.13). Our findings suggest that proximal tubule AT2R activation is anti-inflammatory by increasing IL-10 production which is largely NO-dependent and thus offers renoprotection by preventing early inflammation-induced renal injury in obesity.
Abstract 452: Impact of Aryl Hydrocarbon Receptor Agonist, Leflunomide, on Components of Stem Cells in the Setting of Renal Ischemia-Reperfusion Injury
Authors: Baban Babak , Liu Jun Yao , Mozaffari Mahmood S
Published: March 2022

The aryl hydrocarbon receptor (AHR) has emerged as a regulator of bone marrow-derived stem cells (BMDSCs). Further, mobilization and homing of BMDSCs into the ischemic-reperfused kidney contribute importantly to the repair and recovery of the injured tissue. We tested the hypothesis that the recently described non-dioxin AHR agonist, leflunomide, mobilizes BMDSCs into the ischemic-reperfused kidney in association with reduction in cell death. Accordingly, male mice were treated with leflunomide (40 mg/kg; i.p.). Thereafter, the right kidney of each animal was subjected to ischemia (45 min) followed by reperfusion (4 hr) while the left kidney served as sham control; vehicle-treated animals served as controls. Peripheral blood and renal cells prepared from kidneys of experimental groups were subjected to flow cytometry-based assays using markers for endothelial progenitor cells (EPCs; Sca1
+
/CD31
+
), mesenchymal stem cells (MSCs; Sca1
+
/CD11b
-
) and hematopoietic stem cells (HSCs; Sca1
+
/CD11b
+
). Compared to the vehicle-treated mice, peripheral blood of leflunomide-treated mice showed significantly higher levels of MSCs and HSCs with the effect more marked for the latter. On the other hand, the ischemic-reperfused, but not sham, kidneys of leflunomide-treated animals displayed significant and similar increase in the level of each component of BMDSCs compared to their vehicle-treated counterparts. Further, leflunomide-treated mice displayed preservation of mitochondrial membrane potential as well as decreased apoptosis and necrosis compared to vehicle-treated ischemic-reperfused or sham kidneys. Collectively, the results indicate that AHR stimulation may represent a novel renoprotective mechanism likely involving mobilization and recruitment of components of Sca1+ stem cells into the damaged kidney.
Abstract 455: Decreased Experimental Hypertension and Inflammation in the Absence of Prolylendopeptidase Activity
Authors: Ong Frank S , Blackwell Wendell-Lamar B , Shah Kandarp H , Fuchs Sebastien , Shen Xiao Z , Bernstein Kenneth E
Published: March 2022

Objective:
Previously, we showed that inhibition of prolyloligopeptidase eliminated the differences in macrophage TNFα expression and BP response in mice lacking N-domain activity of ACE. Thus, functional studies of BP and inflammatory response in a model lacking prolylendopeptidase activity are performed.

Results:
After a 14 day high-dose AngII infusion (980 ng/kg-min), POP-KO(n=12) BP was 130±3 mmHg and 147±4 mmHg for POP-WT(n=12). After 14 days of low-dose AngII infusion(490 ng/kg-min), POP-KO(n=8) BP was 126±4 mmHg and 139±3 mmHg for POP-WT(n=8). To investigate differences in the inflammatory response, we examined peritoneal macrophage cytokine expression in response to AngII. The percentage of F4/80+/TNFαhigh cells vs. total F4/80+ was 17%±4.7% for POP-KO (n=8) and 49%±2.8% for POP-WT (n=8)[p<0.005]. The percentage of F4/80+/IL-6high cells vs. total F4/80+ was 15%±3.2% for POP-KO(n=8) and 39%±4.8% for POP-WT(n=8)[p<0.01]. To investigate whether there were baseline differences, TNFalpha secretion was measured in POP-KO(8%) vs. POP-WT(16%) without AngII stimulation. Baseline bone marrow analysis by flow shows 48% CD11b+Ly6ghi cells in POP-KO (n=6) vs. 39% in POP-WT (n=4). Moreover, F4/80hiLy6cintermediate cells were 1.19% in the POP-KO (n=6) vs. 1.75% in POP-WT(n=4).

Conclusion:
Inflammation is an emerging player in hypertension and prolylendopeptidase inhibition may play a role in reducing that inflammation. In the POP-KO mouse model, baseline differences in quantity and quality of inflammatory cells may contribute to the phenotypic differences found
in vivo
.
Abstract 460: Impaired Glucose Tolerance is Associated With an Exaggerated Renin-Angiotensin-Aldosterone System Response to Hydrochlorothiazide in Subjects With Metabolic Syndrome
Authors: Byrne Loretta , Luther James M
Published: March 2022

Renin-angiotensin-aldosterone system (RAAS) activation worsens insulin resistance, and aldosterone impairs insulin secretion via a direct beta-cell effect in mice. To test the hypothesis that the endogenous RAAS contributes to impaired insulin secretion and contributes to impaired glucose tolerance in humans, we assessed glucose homeostasis in 37 subjects with metabolic syndrome (18 Female; 29 white/7 African American; age 45.6±1.8 years). After washout of anti-hypertensive medications, glucose tolerance was characterized as normal glucose tolerance (NGT, n=21), impaired glucose tolerance (IGT, n=11), or type 2 diabetes (T2DM, n=5). Baseline body mass index, body fat percentage, age, plasma renin activity (PRA) and aldosterone were similar among glucose tolerance groups.

We then provided hydrochlorothiazide 12.5mg daily for one month and conducted hyperglycemic clamps on a controlled diet (Na 160, K 80 mEq/d). Potassium was supplemented to similarly maintain serum K
+
≥3.8 (3.90±0.2 NGT, 3.82±0.2 IGT, and 3.86±0.2 mEq/L T2DM). During HCTZ, plasma aldosterone and PRA increased to a greater extent in subjects with IGT compared to NGT or T2DM groups (
Figures a, b
). Plasma aldosterone-renin ratio was similar among groups. Insulin sensitivity index was similar among groups (5.63±2.8 NGT, 7.27±5.4 IGT, and 4.13±3.7 in T2DM;
P
=0.29). The acute insulin response (AIR) to glucose was significantly impaired in IGT and T2DM subjects (
figure c
). The AIR correlated inversely with aldosterone (
figure d
) but not PRA.

HCTZ treatment activates the RAAS to a greater extent in subjects with IGT, and the aldosterone response specifically correlates with impaired insulin secretion.
Abstract 462: The Effect of Body Mass Index on Blood Pressure Varies by Race among Children Referred for Treatment of Obesity
Authors: Hannon Tamara , Li Zhuokai , Eckert George , Gupta Sandeep , Tu Wanzhu , Pratt J Howard
Published: March 2022

Black school-aged children tend to have greater body mass index (BMI) and higher BP than their age-matched white peers. But few studies have considered racial differences in the magnitude of the effect of increasing BMI on BP among equally obese black and white children. We used a novel analytic technique to examine the influence of age and BMI on BP in children seen at a tertiary care center- based obesity clinic (POWER, Riley Hospital for Children, Indianapolis, IN). The study sample included 821 obese children (306 males, 515 females, 362 blacks, and 459 whites). The mean age of the study subjects was 11.72 ± 3.48 years, the mean BMI was 36.22 ± 8.51 kg/m2, and the mean systolic and diastolic BP were 109.36 ± 16.10 and 69.99 ± 10.48 mmHg, respectively. In comparison, blacks and whites were similar in age (11.89 vs 11.58; p=0.197); while black patients had higher mean BMI (37.32 vs 35.34 kg/m2; p=0.0010), and higher systolic BP% than whites (58.71 vs 50.72 mmHg; p=0.00062). Semiparametric regression models showed that while age and BMI were associated with systolic BP% in both race groups, black children had significantly higher BP% values as compared with white children of the same age and BMI (Fig 1). In conclusion, among children referred for treatment of obesity, there are race-dependent differences in the effect of BMI on blood pressure. Black children are at a significantly greater risk for having elevated BP as compared with their white peers of similar age and severity of obesity. Further research is needed to better understand this population-specific intensification of the adiposity effect on BP, as this has implications for clinical care and treatment of hypertension in black children.
Abstract 470: Treatment of Orthostatic Hypotension with the Combination Ergotamine and Caffeine
Authors: Shibao Cyndya , Ramirez Claudia E , Okamoto Luis E , Arnold Amy C , Gamboa Alfredo , Muppa Prasuna , Raj Satish R , Diedrich Andre , Robertson David , Biaggioni Italo
Published: March 2022

Orthostatic hypotension is the most disabling symptom of autonomic failure. Severely affected patients often require medication to counteract the blood pressure fall and to improve pre-syncopal symptoms on standing. Isolated clinical observations have suggested that the combination ergotamine and caffeine can be useful in the treatment of orthostatic hypotension. The aim of this study was to examine the effect of the combination ergotamine and caffeine on blood pressure and pre-syncopal symptoms on standing in a group of patients with autonomic failure. A total of 12 patients participated in this study. Eight were men and the average age was 64±10 years. The combination ergotamine and caffeine increased seated SBP by 19 mm Hg compared with placebo (95% CI: -2 to 40 mm Hg, P=0.07) (Figure A) and reduced the orthostatic symptom score by 9 points (95% CI: 0.9 to 16, P=0.03), compared with baseline (Figure B). In conclusion, the combination ergotamine and caffeine could be an alternative therapy for orthostatic hypotension in patients with autonomic failure.
Abstract 471: Trpv1 Ablation Leads to Elevated Blood Pressure at Night but Not Day Time in Mice Fed a Western Diet
Authors: Zhong Beihua , Wang Donna H
Published: March 2022

Obesity is associated with an increase in renal sympathetic nerve activity (RSNA) and impairment in baroreflex (BR) that raises cardiovascular morbidity and mortality. This study tests the hypothesis that ablation of the transient receptor potential vanilloid type 1 (TRPV1) channels exacerbates impairment of BR in mice fed a western diet (WD) and leads to distinct diurnal and nocturnal blood pressure patterns. TRPV1-null mutant (TRPV1
-/-
) or wild type (WT) mice were given a WD (42% kcal from fat) or normal diet (CON) for 4 months. Perfusion of capsaicin (CAP, 10
-6
M
), a selective TRPV1 agonist, into the left renal pelvis increased ipsilateral afferent renal nerve activity (ARNA) in WT but not TRPV1
-/-
mice, with a smaller magnitude in WT-WD compared to WT-CON (P<0.05). The sensitivity of RSNA and heart rate (HR) responses to BR was assessed during changes in mean arterial pressure (MAP) induced by IV infusion of sodium nitroprusside (SNP)- or phenylephrine (PE). The sensitivity of RSNA and HR responses to BR was reduced to the same degree in TRPV1-/-CON and WT-WD and further decreased in TRPV1-/-WD (SNP infusion-RSNA: WT-CON183 ±37.7; TRPV1-/-CON 114.5 ± 15.3; WT-WD 92.1 ± 33.3; TRPV1-/-WD 53.9 ± 26.6%; P<0.05; SNP infusion-HR: WT-CON 47.4 ± 7.6; TRPV1-/-CON 38.6 ± 5.3; WT-WD 36.6 ± 3.0; TRPV1-/-WD 28.2 ± 3.9 beats/min; P<0.05; PE infusion-RSNA: WT-CON -93.9 ±6.4; TRPV1-/-CON -74.3 ± 9.2; WT-WD -66.1 ± 9.0; TRPV1-/-WD -40.6 ± 10.2%; P<0.05; PE infusion-HR: WT-CON -35.5 ± 6.5; TRPV1-/-CON -29.0 ± 3.2.; WT-WD -27.6± 2.9; TRPV1-/-WD -20.2± 3.3 beats/min; P<0.05). Urinary norepinephrine levels at day and night times were increased in WT-WD and TRPV1-/-WD, with further elevated levels at night time in TRPV1-/-WD (
P
<0.05). Urinary albumin and plasma creatinine levels were increased in WT-WD and TRPV1-/-WD, with further elevated urinary albumin levels in TRPV1-/-WD (
P
<0.05). Telemetry MAP was not different at the day time between all groups, but increased at the night time in TRPV1-/-WD compared to WT-CON, TRPV1-/-CON, and WT-WD (P<0.05). Thus, TRPV1 ablation exacerbates impairment of BR and BR control of RSNA in the face of WD intake, and leads to elevated nocturnal but not diurnal blood pressure possibly attributed to further enhanced sympathetic drives at night time.
Abstract 479: A novel Interaction Between G protein-Coupled Receptor Kinase 4 and Sorting Nexin 5 in the Regulation of Dopamine D1 Receptor Phosphorylation
Authors: Villar Van Anthony M , Armando Ines , Jurgens Julie A , Evans Sarah , Ennis Riley C , Yu Peiying , Jose Pedro A , Jones John E.
Published: March 2022

A dysfunction of the renal dopaminergic system is implicated in the pathogenesis of essential hypertension. Independent of renal nerves, the kidney synthesizes dopamine to regulate salt and water homeostasis by inhibiting ion transport in the nephron. The dopamine D
1
receptor (D
1
R) plays a pivotal role in regulating salt metabolism and blood pressure. The regulatory mechanisms or proteins involved in D
1
R trafficking are not completely understood, although our group has reported how G protein-coupled receptor kinase 4 (GRK4) promotes agonist-activated D
1
R desensitization and plasma membrane localization. We have recently identified the sorting nexin 5 (SNX5)_a protein involved in protein trafficking and a member of the mammalian retromer_as a novel binding partner of D
1
R using a yeast two-hybrid screen and have endeavored to elucidate how GRK4 and SNX5 cooperatively interact and regulate the D
1
R. Using a series of biological, biophysical and immunological approaches, we showed that GRK4 and SNX5 colocalize with the D
1
R and that these proteins co-segregate in lipid rafts in human renal proximal tubule cells (hRPTCs). Moreover, endogenous SNX5 co-immunoprecipitated with GRK4, but not with the β-arrestins, another class of proteins involved in D
1
R desensitization; treatment with the D1-like dopamine receptor agonist fenoldopam (1 uM, 15 min) enhanced this process 3-fold (n=3/group). We next determined the functional relevance of this interaction by silencing SNX5 and then evaluating the extent of agonist-activated D
1
R phosphorylation. In control cells that were transfected with non-silencing siRNA, fenoldopam increased D
1
R phosphorylation at 10, 20, and 30 min (137%, 140%, and 148%, respectively, compared to basal; P<0.5 vs. 0 min, n=3/group). Interestingly, SNX5 depletion resulted in 160% increase in phosphorylated D
1
R at the basal state and 145%, 170%, 178% and 200% increase after 5, 10, 20 and 30 min of agonist treatment (P<0.5 vs. 0 min, n=3/group). These indicate that SNX5 restricts the GRK4 from phosphorylating the receptor and may help explain why GRK4 is not involved in early phase of D
1
R desensitization. These data highlight the importance of SNX5 and its interaction with GRK4 in regulating D
1
R function in the control of blood pressure.
Abstract 484: Role of GRK4 Variants in Renal Dopamine D3 Receptor Expression and Function
Authors: Pinho Maria J , Villar Van A , Zhang Yanrong , Soares-da- Silva Patricio , Jose Pedro A
Published: March 2022

In the kidney, dopamine promotes natriuresis by inhibiting both proximal and distal tubule NaCl reabsorption through its action on renal dopamine receptors, namely dopamine D
3
receptor (DRD3). DRD3 promotes natriuresis, especially during salt-replete states. The involvement of DRD3 dysfunction in the etiology of hypertension has been demonstrated through pharmacologic and genetic interventions. While its gene locus at 3q13.3 has been linked to hypertension, the signal transduction pathway of DRD3 has not been fully ascertained. The physiological effects of the GRK4 sequence variants on DRD3 function
in vitro
remain to be determined, therefore, the present study aimed to elucidate whether GRK4 sequence variants (142V and 486V) promote DRD3 hyper-phosphorylation that results in receptor dysfunction. For this purpose, Chinese hamster ovary cells heterologously expressing GRK4γ variants (A142V and A486V) were co-transfected with human DRD3. Stimulation with the highly selective D3R agonist PD128907 resulted in an increase in receptor phosphorylation in CHO cells expressing GRK4-A142 and GRK4γ-A486V variants. PD128907 treatment increased the phosphorylation of p44/42 MAP kinases and their downstream target p90RSK n both GRK4γ variants and wild type. Contrasting, S6 ribosomal protein showed a decrease in phosphorylation upon D3R activation. The abundance of active-Gα
o
was decreased in wild type and GRK4γ-A486V after stimulation with PD128907, but not in CHO cells expressing GRK4-A142. In conclusion, these results indicate that these GRK4 variants phosphorylate and possible desensitize DRD3, suggesting an impairment of its function. Supported by grant PIC/IC/83204/2007.
Abstract 485: Transglutaminase 2 is a Regulator of Angiotensin II-induced ERK1/2 Activation in Vascular Smooth Muscle Cells.
Authors: Rautureau Yohann , Paradis Pierre , Schiffrin Ernesto L
Published: March 2022

Background:
In essential hypertension, angiotensin (Ang) II induces arterial remodeling that is dependent on the activation of ERK1/2 pathway in vascular smooth muscle cells (VSMC). Arterial remodeling is also dependent on transglutaminase 2 (TG2), a protein with an emerging role in vascular pathophysiology. Beside its effect on extracellular matrix, TG2 is a multifunctional protein with GTPase activity and as such, could potentially be a signaling molecule. We hypothesized that TG2 mediates Ang II-induced ERK1/2 activation in VSMC during arterial remodeling.

Methods and Results:
To determine the role of TG2 in Ang II type 1 receptor (AT
1
R) signaling, His
6
tagged-TG2 was overexpressed in the human embryonic kidney (HEK) 293 cell line stably overexpressing HA tagged-AT
1
R. Protein and phosphorylation levels were determined by Western blot. When stimulated for 2-60 min, TG2 overexpression potentiated 100 nM Ang II-induced ERK1/2 phosphorylation compared to control cells overexpressing green fluorescent protein (GFP,
P
<0.05, n=4). Maximal potentiation was observed after 2 min of stimulation at which time TG2 transfected cells showed an increase of 64±31% compared to GFP transfected cells. The role of TG2 in Ang II-induced phosphorylation of ERK1/2 was studied by TG2 siRNA knockdown in mouse vascular smooth muscle cell line (MOVAS). TG2 siRNA decreased TG2 expression by 83±5% compared to cells treated with control siRNA (n=4). Transfection of TG2 siRNA did not significantly change cell viability when compared to ctrl siRNA (n=3). Ten min stimulation with 1 and 100 nM Ang II increased ERK1/2 phosphorylation by 38±18% and 60±29%, respectively, and this was prevented by TG2 knockdown (
P
<0.01). Using western blots, HA-AT
1
R dimer/monomer ratio was increased by co-expression of His
6
-TG2 compared to HA-AT1R expressed alone.

Conclusion:
These results suggest that TG2 mediates ERK1/2 activation by Ang II through a mechanism involving TG2-induced AT
1
R dimerization. Overall, our results demonstrate that TG2 is involved in the activation of ERK1/2 in response to Ang II and could participate in arterial remodeling induced by Ang II.
Abstract 491: Activation of Voltage-dependent L-type Calcium Channels and the Rho Kinase Pathway Are Involved in S1P-induced Vasoconstriction in Rat Juxtamedullary Afferent Arterioles
Authors: Guan Zhengrong , Singletary Sean T , Cook Anthony K , Inscho Edward W
Published: March 2022

Sphingosine-1-phosphate (S1P) is an important regulator of resistance vessels and is implicated in pathological processes including diabetic nephropathy and acute kidney injury. Previous studies show that S1P causes marked vasoconstriction in preglomerular but not postglomerular microvessels, however, the intracellular signaling pathways underlying S1P-mediated vasoconstriction are undefined. We postulated that S1P-mediated afferent arteriolar (AA) vasoconstriction involves activation of voltage-dependent L-type Ca
2+
channels (L-VDCC) and the rho kinase pathway. Studies were conducted
in vitro
using the blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (n=6) evoked concentration-dependent AA vasoconstriction. Control diameter averaged 13.8±0.9 μm and declined to 95±2, 85±4, 75±6, 60±5 and 46±5% of control diameter in response to S1P (from 10
-9
to 10
-5
M), respectively. Superfusion with nifedipine (10
-6
M, n=6), a L-VDCC inhibitor, increased basal diameter by 39±18% (p<0.05) and significantly attenuated the S1P-induced vasoconstriction. S1P reduced AA diameter to 99±1, 98±1, 90±3, 79±3 and 52±5% of control (p<0.05 vs. S1P alone at 10
-8
to 10
-6
M) during nifedipine treatment. Superfusion with the rho kinase inhibitor, Y27632 (10
-5
M, n=6), increased diameter by 60±12% and inhibited S1P-induced vasoconstriction. AA diameter averaged 103±1, 101±1, 93±3, 72±9 and 56±10% of control in response to increasing concentrations of S1P (p<0.05 vs. S1P alone at 10
-9
to 10
-7
M). In contrast, S1P-induced vasoconstriction was unaltered by Ca
2+
store depletion using the Ca
2+
-ATPase inhibitors, thapsigargin (Tg) and cyclopiazonic acid (CPA). Although Tg (10
-6
M, n=4) increased diameter by 38±16%, the S1P-induced vasoconstrictor profile was not different from that without Tg. AA declined to 99±3, 95±5, 85±10, 60±13 and 32±8% (p>0.05), respectively. Similar results were obtained with CPA (10
-5
M, n=6). S1P reduced diameter to 97±2, 93±2, 75±5, 53±6 and 36±5% of the control (p>0.05), respectively, suggesting that mobilization of intracellular Ca
2+
store is not required for S1P-induced vasoconstriction. Overall, these data indicate that both L-VDCC and rho kinase pathways contribute to S1P-mediated AA vasoconstriction.
Abstract 497: H2O2 in Fumarase Insufficiencies in Dahl Salt-Sensitive Rats.
Authors: Liu Yong , Liang Mingyu
Published: March 2022

We have shown previously that renal insufficiencies of fumarase, an enzyme in the tricarboxylic acid cycle, appear to contribute to salt-induced hypertension in Dahl salt-sensitive (SS) rats. The present study examined the molecular mechanism underlying fumarase insufficiencies in SS rats. Protein abundance and enzymatic activity of fumarase was significantly lower in the renal medulla of SS rats compared to salt-insensitive Sprague-Dawley rats. We screened several factors including TGFβ1, TNFα, and H
2
O
2
for their effects on fumarase expression. These factors were selected for screening because they had been reported to be elevated in the kidneys of the SS rat and they represent a pro-fibrotic, pro-inflammatory, and oxidative milieu present in the kidneys of the SS rat. Cultured human renal epithelial cells were treated with TGFβ1 (3 ng/ml), TNFα (25 ng/ml), or H
2
O
2
(500 μM) for 24 hours. H
2
O
2
substantially reduced fumarase mRNA levels (normalized by 18S rRNA) to less than 10% of control. TNFα tended to suppress fumarase expression, but the effect did not reach statistical significance. TGFβ1 did not have significant effects on fumarase mRNA levels. The greater than 90% loss of fumarase following the H
2
O
2
treatment was accompanied by significant cell death. To determine if the down-regulation of fumarase was simply a consequence of the cytotoxicity of H
2
O
2
, we treated HRE cells with 100 μM of H
2
O
2
for 48 hours. The lower dose of H
2
O
2
did not cause significant cell death. Yet, fumarase mRNA was still down-regulated to 79.6% ± 4.7% of control (n=4, P<0.05 vs. control). Moreover, we repeated the treatment in cells cultured in the Dulbecco’s Modified Eagle Medium (DMEM) instead of the regular epithelial growth medium. HRE cells cultured in DMEM were highly resistant to the cytotoxicity of H
2
O
2
. H
2
O
2
(500 μM) did not cause any significant cell death in HRE cultured in DMEM, yet it again significantly suppressed fumarase expression to 77.1% ± 6.6% of control (n=7-8, P<0.05 vs. control). The result suggests that elevated levels of H
2
O
2
in the renal medulla might contribute to fumarase insufficiencies in SS rats.