bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 276: Angiotensin AT1 Receptor Blockade Reverses Age-Related Changes in Myocardial Energy Metabolism and Increases Mitochondrial Biogenesis
Authors: Abadir Peter M , Akki Ashwin , Carey Robert , Gupta Ashish , Chacko Vadappuram , Weiss Robert , Walston Jeremy
Published: July 2021

Aging and mitochondrial function have been closely linked. We recently reported the identification of a mitochondrial angiotensin system. We hypothesized that angiotensin AT1 receptor blockade would increase energy production and mitochondrial biogenesis and reduce oxidative stress in aging hearts. We used Magnetic resonance spectroscopy to measure cardiac energy metabolism and function in young (20 wks old), aged (150 wks old) mice at baseline and after 4 weeks of losartan (50 mg/kg/day). For mitobiogenesis, qPCR was used to calculate CytB (mitochondrial gene)/GAPDH (nuclear gene) ratio and to measure mito-survival genes Sirt1, Sirt3, Nampt, and PGC-1α. Cardiomyocyte mitochondria from young, aged and treated mice were examined with electron microscopy. The expression of nitrotyrosine was quantified by immunohistochemistry. Older animals hearts (n=9) exhibited increase in LV mass (103±9 mg versus 120±8 mg, young (n=8) versus old (n=9), P<0.002). The mean cardiac PCr/ATP was reduced in older animals (1.5±0.2) than that of young mice (2.0±0.3, P<0.0004). Losartan abolished the LV mass increase in older animals (109±11 mg vs 101±7 mg, young versus old, P<0.1) and improved the impaired energy metabolism of the older hearts increasing the PCr/ATP ratios towards those observed in younger animals (1.94±0.01 vs 1.87±0.4, control versus old, P<0.7). Losartan increased EF in older animals (56±5% vs 63±5%, old versus old treated, P<0.01). Losartan increased mitobiogenesis in the hearts of treated young and old mice (3.8+2.5 folds, P<0.02 and 4.3+ 0.9 folds, P<0.0001). Mito-survival genes in the heart were not increased but PGC-1α was up-regulated by 2.8+1.6-fold, P<0.05 and 7+ 1.9-fold, P<0.001 in young and old treated mice. Electron micrograph analysis revealed that aging was associated with swollen cardiac mitochondria and disrupted cristae, which were reversed by Losartan. Losartan in older animals significantly reduced oxidative damage as evidenced by less Nitrotyrosine staining score in cardiomyocytes (2.5±0.5 vs. 1.3±0.4, old versus old treated, P<0.0009). Our results indicate that Losartan in aging increased mitobiogenesis, reduced oxidative stress, improved energy production and restored cardiac function to the healthy young adult level.
Abstract 29: An Orally Active Epoxyeicosatrienoic Acid (eet) Analog Attenuates Kidney Injury in Hypertensive Dahl Salt Sensitive Rat by Reducing Oxidative Stress, Inflammation and Endoplasmic Reticulum Stress
Authors: Khan Md Abdul H , Narayanan Praveena , Falck John R , Imig John D
Published: July 2021

Renal injury is associated with salt-sensitive hypertension. The Dahl salt-sensitive hypertensive rat (Dahl SS) is a model of salt-sensitive hypertension and the related kidney injury. The kidney injury in Dahl SS hypertension is associated with oxidative stress, inflammation and ER stress. We have recently developed orally active epoxyecosatrienoic acid (EET) analogs that provide organ protection in hypertension and other pathologies through multiple signaling pathways. We hypothesized that these EET analogs with their anti-inflammatory, anti-oxidant and anti-ER stress effects will protect the kidney in Dahl SS hypertension. Dahl SS rats received high salt diet and treated either with an EET analog (10 mg/kg/d) or vehicle (0.05% EtOH+0.1% PEG 400) in drinking water for 14 days. Blood pressure (SBP) was measured by tail-cuff plethysmography and urine, plasma and tissue samples were collected at the end of the treatment protocol. Urinary creatinine, albumin, nephrin, and renal tissue TBARS content were measured using ELISA or colorimetric assays. Renal expression of oxidative, inflammatory and ER stress marker genes were examined using RT-PCR. Histological analysis was done to assess renal injury. In Dahl SS rats, EET analog treatment did not affect the SBP compared to vehicle (183±6 vs. 185±12 mmHg). Interestingly, the EET analog treatment in Dahl SS reduced albumin-creatinine ratio (49±17 vs. 12±3) and nephrinuria (11±1 vs. 4±1 mg/d). EET analog treatment also reduced glomerular injury, intra-tubular cast formation and kidney fibrosis by 30-60% in Dahl SS rats compared to vehicle. In Dahl SS rats, EET analog treatment caused a 40% reduction in kidney TBARS content compared to vehicle. Moreover, the EET analog treatment in Dahl SS rats resulted 2-3 fold attenuation in the renal expression of oxidative (
p47PHOX
), inflammation (
IL-6, TGF-β
), and ER stress (
CHOP, GADD34
) genes. These data, demonstrate that a novel orally active EET analog provides kidney protection in Dahl SS hypertension by reducing oxidative stress, inflammation and ER stress, and this kidney protection was independent of blood pressure reduction.
Abstract 329: Oral Administration of New Calcium Channel Antagonist Reverses Right Ventricular Dysfunction in Rats With Pulmonary Arterial Hypertension
Authors: Pereira Sharlene L , Alencar Allan K , Kummerle Arthur E , Ferraz Emanuelle B , Nascimento José , Fraga Carlos A , Barreiro Eliezer J , Sudo Roberto T , Zapata-Sudo Gisele
Published: July 2021

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, endothelial injury and vascular remodeling, right ventricular hypertrophy and increased right ventricular systolic pressure. This work investigated the effects of LASSBio-1289, a new calcium channel antagonist, in rats with PAH induced by monocrotaline (MCT).

METHODS:
Male Wistar rats (200 - 250 g) received a single MCT i.p. injection (60 mg/kg) for induction of PAH. The experimental groups were: control, MCT, MCT + vehicle, MCT + LASSBio-1289 (50 mg/kg p.o.). Animals were treated either with vehicle or LASSBio-1289 for 14 days after the onset of disease. The following parameters were analyzed: right ventricular systolic pressure (RVSP), RV contractility (dp/dtmax), RV relaxation (dp/dtmin)and RV weight / left ventricle + septum weight (RV/LV+S). Echocardiography was performed to determine RV area and wall thickness. The protocols used in the present study were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro.

Results:
RVSP (mmHg) increased from 25.3 ± 1.9 (control) to 49.0 ± 6.2 (P < 0.05) and 52.7 ± 6.2 (P < 0.05) in MCT and MCT + vehicle groups, respectively. When MCT-injected rats were treated with LASSBio-1289, RVSP was decreased to 36.4 ± 2.0 mm Hg (P < 0.05). RV/LV+S increased from 0.24 ± 0.02 to 0.56 ± 0.04 (P < 0.05) and 0.49 ± 0.03 in the MCT and MCT + vehicle groups (P < 0.05) and reduced to 0.30 ± 0.03 (P < 0.05) after treatment with LASSBio-1289. The dp/dtmax (mmHg/s) was 1179 ± 137, 2180 ± 238, 2482 ± 371 and 1134 ± 131 for control, MCT, MCT + vehicle and MCT + LASSBio-1289 groups. In addition to this, the dp/dtmin (mmHg/s) increased from -702 ± 83 to -1359 ± 120, -1720 ± 243 in the MCT and MCT + vehicle groups. In contrast, treatment with LASSBio-1289 reduced this parameter to -701 ± 122 (P < 0.05). RV area (mm2) was increased from 8.2 ± 0.5 to 17.8 ± 1.4 and 18.7 ± 1.0 in the PAH groups. RV area was reduced in LASSBio-1289-treated rats to 9.4 ± 0.9 (P < 0.05). PAH increased the RV wall thickness (cm) from 0.089 ± 0.002 to 0.125 ± 0.006 (P < 0.05) and was reduced to 0.098 ± 0.006 after treatment with LASSBio-1289.

Conclusions:
LASSBio-1289 effectively reversed right ventricular dysfunction and hypertrophy in rats with monocrotaline-induced PAH.
Abstract 346: Mice Overexpressing Renin: a New Model of Progressive Chronic Kidney Disease
Authors: Huby Anne-Cecile , Abed Ahmed , Kavvadas Panos , Alfieri Carlo , Rastaldi Maria-Pia , Dussaule Jean-Claude , Chatziantoniou Christos , Chadjichristos Christos
Published: July 2021

Background:
Hypertension-induced chronic kidney disease in mouse models is quite fast and consequently away from the human pathology. There is an increasing need for a mouse model that can be used to delineate the pathogenic process leading to progressive renal disease.

Aim:
Our objective was dual: to investigate whether mice overexpressing renin ectopically at constant and high levels by genetic clamping (RenTg) could mimic kinetics and the physiopathological characteristics of hypertension-induced CKD and to identify cellular and/or molecular events characterizing the different steps of the progression of CKD.

Results:
We found that RenTg mice are hypertensive (123±7 vs to 90±2 mm Hg for the wt age-matched animals, p<0.05) and slightly albuminuric (22.1±5.3 vs. 5.2±0.4 g/mol, p<0.01) as early as 3 month old. At this age, the expressions of adhesion markers such as vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 are 4-5 fold increased in the renal cortical vasculature indicating the beginning of endothelial dysfunction. Five month-old RenTg mice show perivascular and periglomerular infiltrations of macrophages and their GFR is starting to decrease(-10%). At 8 months, the renal cortex of RenTg mice is altered by leukocyte invasion, decreased expression of nephrin (a protein controlling filtration barrier), increased expression of KIM-1 (a protein typical of tubular cell stress) and of several pro-fibrotic agents of the TGFbeta family, and establishment of fibrotic lesions. At the age of 12 months, RenTg mice display several lesions of renal structure typical of hypertensive renal disease (such as glomerular ischemia, glomerulo- and nephroangio-sclerosis, mesangial expansion, tubular dilation), important proteinuria (138±20 g/mol) and a 55% fall of GFR.

Conclusions:
The RenTg strain develops progressively with age CKD. In this model, endothelial dysfunction is an early event preceding the structural and fibrotic alterations which ultimately lead to the development of CKD. This model can provide a useful tool allowing to gain new insights into the mechanisms of chronic renal failure and to identify new targets for arresting and/or reversing the development of CKD
Abstract 352: Anti-aging Gene Klotho Deficiency Causes Hypertension and Vascular Dysfunction via Upregulation of Mammalian Target of Rapamycin (mTOR)
Authors: Song Ying , Sun Zhongjie
Published: July 2021

Klotho is a recently discovered anti-aging gene. Genetic mutation of klotho expedites the aging process and shortens the lifespan while overexpression of klotho slows down the aging process and extends the lifespan by 20%. Interestingly, blood pressure (BP) was elevated significantly and vasodilatory responses to acetylcholine and sodium nitroprusside were impaired in klotho heterozygeous (+/-) mice, suggesting that klotho deficiency causes hypertension and vascular dysfunction. It is noted that klotho deficiency is associated with upregulation of mTOR expression and NADPH oxidase activity and downregulation of Mn-SOD expression in aortas and kidneys. Inhibition of mTOR by rapamycin abolished the upregulation of NADPH oxidase activity and O
2
-
production and the downregulation of Mn-SOD expression and decreased BP to the control levels. Inhibition of mTOR also abolished vascular endothelial dysfunction and macrophage infiltration in kidneys in klotho (+/-) mice. The upregulation of NADPH oxidase activity and downregulation of Mn-SOD may be involved in klotho deficiency-induced hypertension which can be decreased significantly by apocynin (NADPH oxidase inhibitor) or Tempol (O
2
-
scavenger). These results demonstrate, for the first time, that klotho is essential in the maintenance of normal blood pressure. Klotho deficiency-induced hypertension and vascular dysfunction are mediated by upregulation of mTOR. This study also reveals a previously unidentified role of mTOR in the regulation of NADPH oxidase and MnSOD. (Supported by HL105302 and HL102074).
Abstract 358: Signature microRNA Expression Pattern of Essential Hypertension in a Portuguese Population
Authors: Pinho Maria J , Vaz-da-Silva Manuel , Soares-da- Silva Patricio
Published: July 2021

Hypertension is a complex, multifactorial disease, and its development is determined by a combination of genetic susceptibility and environmental factors. microRNAs have been implicated in numerous biologic processes. Moreover, several studies have demonstrated associations between diseases and specific microRNAs, especially in the cardiovascular system. The present study we assessed the microRNA system in a Portuguese population of hypertensive subjects and explored the potential of microRNAs as biomarkers for essential hypertension. Using Agilent Human miRNA Microarrays, Release 16.0 in combination with a one-color based hybridization protocol and bioinformatic analysis, we determined the microRNA signature of peripheral blood mononuclear cells (PBMCs) from a total of 66 hypertensive subjects (divided into three classes: group 1, medicated HTA I and II; group 2, medicated HTA III; group 3, non-hypertensive with cardiac disease) and 13 non-hypertensive subjects. Bioinformatic analysis revealed 4 miRNAs (miR-4306, miR-146a, miR-22 and miR-146b-5pn) as upregulated not yet related to essential hypertension, and one (miR-186) previously described in myocardial infarction. Endothelial-specific miR-126, nephrosclerosis marker miR-192 and miR-98 (previously implicated in NO and ANP signaling) were found to be upregulated exclusively in group 1 comparing to control. Determining functions and identifying the specific targets of miR-4306, miR-146a, miR-22 and miR-146b-5pn, could determine their value as therapeutic targets for essential hypertension. Supported by FCT (PIC/IC/83204/2007)
Abstract 365: Differential Regulation of NOX2, NOX4 and SOD Expression in the Heart and Kidneys by PPAR-α During Angiotensin II Hypertension
Authors: Wilson Justin L , Sankavaram Kavitha , Duan Rong , Allard Joanne S , Lee Dexter L
Published: July 2021

Previous reports suggest peroxisome proliferator-activated receptor-α (PPAR-α) agonist attenuates hypertension by suppressing oxidative stress and increasing superoxide dismutase (SOD) activity. We tested the hypothesis that the absence of PPAR-α would increase NADPH oxidase (NOX)2, NOX4 and decrease SOD expression in mice heart and kidneys during Ang II hypertension. Male (10 - 12 weeks old) PPAR-α knockout (KO) mice and their wild-type (WT) controls were implanted with biotelemetry devices and infused with a slow pressor dose of Ang II (400 ng/kg/min) for 12 days. Separate groups of KO + Ang II and WT + Ang II mice were given the NOX inhibitor, apocynin (1 g/L) or PPAR-α agonist, fenofibrate (145 mg/kg/day). Mean arterial pressure (MAP) was similar between KO (119 ± 2 mmHg) and WT (122 ± 2 mmHg) during the control period. On day 12 of Ang II, MAP was significantly higher in KO than WT, 161 ± 5 and 145 ± 4 mmHg, respectively. Fenofibrate significantly reduced MAP in WT + Ang II mice (134 ± 5 mmHg) and apocynin reduced MAP in KO + Ang II and WT + Ang II mice. Heart and kidney NOX2 expression were significantly increased (61 ± 5% and 33 ± 5 %, respectively) in KO + Ang II when compared to WT + Ang II mice. Apocynin treatment significantly reduced heart and kidney NOX2 expression in KO + Ang II mice. Heart NOX4 expression was significantly decreased (28 ± 3%) in WT + Ang II mice when compared to WT controls. No significant changes were observed in KO + Ang II heart NOX4 expression. Kidney NOX4 expression was significantly decreased in both Ang II-treated KO (77 ± 5%) and WT (50 ± 5%) mice when compared to their respective controls. WT + Ang II SOD expression in the heart was significantly lower than WT controls. Ang II did not lower heart SOD expression in KO mice. SOD expression in the kidneys was significantly decreased in KO + Ang II and WT + Ang II when compared to their respective controls. Fenofibrate significantly increased kidney SOD expression in WT + Ang II mice above WT controls. Fenofibrate did not significantly increase kidney SOD expression in KO + Ang II mice above KO controls. Our results suggest that increased heart and kidney NOX2 expression, along with decreased SOD expression in PPAR-α KO mice contributes to significant increases in MAP during Ang II hypertension.
Abstract 37: Deletion of Notch3 Gene Impairs Contractility of Renal Resistance Vessels
Authors: Helle Frank , Kavvadas Panagiotis , Hultström Michael , Iversen Bjarne , Chatziantoniou Christos
Published: July 2021

Background:
Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. In previous studies we showed that mice lacking Notch3 display deficient renal autoregulation.

Objective:
Our aim was to study the mechanisms involved in the Notch3-mediated control of renal vascular response.

Methods and Results:
To this end, renal afferent arterioles were isolated from Notch3-/- and wild type littermates and stimulated with ANG II. Contractions and intracellular Ca2+ concentrations were blunted in Notch3-/- vessels in a dose-dependent manner (diameter decrease: 15±3 vs. 38±5%, p<0.01; change of fura 2 ratio: 0.18±0.02 vs. 0.39±0.05, p<0.001, at 10-7Ang II for Notch3-/- and wt vessels, respectively). Differential transcriptomic analysis of 47 genes known to participate in vasoreactivity indicated an important downregultion of the cacna1h gene expressing the α1H subunit of the T-type Ca2+ channel in the renal vessels of Notch3-/- mice. This finding was confirmed by real-time qPCR and western blotting. In subsequent experiments, addition of EGTA (Ca2+ chelating agent), nifedipine (L-type channel blocker) or mibefradil (T-type channel blocker) blunted as expected the response to ANG II in wild-type vessels. In sharp contrast, these agents did not affect vessel responsiveness in Notch3-/- indicating dysfunctional extracellular Ca2+-entry. Abolishing stored Ca2+ with thapsigargine, reduced Ca2+ responses equally in both strains, signifying intact Ca2+-mobilization in Notch3-/- vessels. In contrast to renal resistance vessels, pre-capillary muscle arterioles of Notch3-/- mice reacted normally to ANG II, suggesting a focal role of Notch3 in the renal vasculature.

Conclusions:
Notch3-/- mice display deficient renal vascular reactivity because of blunted expression and function of calcium channels. Consequently, intact Notch3 expression is necessary for proper regulation of renal vascular tone in the kidney, whereas dysfunction of Notch3 can have important physiopathological consequences by impairing regulation of renal hemodynamics
Abstract 370: Upregulation of Renal D5 Dopamine Receptor Ameliorates Hypertension in D3 Deficient Mice
Authors: Wang Xiaoyan , Escano Crisanto S , Asico Laureano , Jones John E , Barte Alan , Jose Pedro A , Armando Ines
Published: July 2021

D
3
dopamine receptor (D
3
R) deficient mice have renin-dependent hypertension but the hypertension is mild and is not associated with oxidative stress. In order to determine if any compensatory mechanism in the kidney is involved in the regulation of blood pressure with disruption of D
3
R, we measured the renal protein expression of dopamine receptors in D
3
R homozygous (D
3
-/-) and heterozygous (D
3
+/-) knockout mice and their wild type (D
3
+/+) littermates. D
5
dopamine receptor (D
5
R) (169±23%, reported as % of D
3
+/+, n=5/group) expression was increased but D
4
dopamine receptors protein expression (59±8%) was decreased, while no significant changes were found with D
1
and D
2
dopamine receptors. Immunocytochemistry showed a stronger renal staining of D
5
R but without a change in renal tubule cell distribution in D
3
-/- relative to D
3
+/+ mice. D
5
R abundance was also increased in D
3
+/- (205±30%, n=5/group) relative to D
3
+/+ mice, while D
1
R abundance was similar between D
3
+/- and D
3
+/+ mice. The increase in D
5
R expression was abolished while blood pressure was increased further in D
3
-/- mice fed a high salt diet. Treatment of the D
1
-like (including D
1
and D
5
receptors) antagonist,
SCH23390
, increased the blood pressure to a greater extent in anesthetized D
3
-/- mice than in D
3
+/+ mice (n=4/group), suggesting that the upregulation of D
5
R may modulate the hypertension in mice caused by the disruption of D
3
R. Since dopamine inhibits the NADPH oxidase-induced production of reactive oxygen species (ROS) via the D
5
R, we also measured the protein expression of NOXs in the kidney and isoprostane in the urine. No NADPH oxidase subunit was increased in D
3
-/- and D
3
+/- mice relative to D
3
+/+ mice fed a normal or salt high salt diet, and urinary isoprostane excretion was also similar in D
3
-/- and D
3
+/+ mice. Our findings suggest that the upregulation of D
5
R may minimize the hypertension and prevent oxidative stress in D
3
-/- mice.
Abstract 371: Nitrosonifedipine, a Photodegradation Product of Nifedipine, Prevents the Progression of Diabetic Nephropathy in Type II Diabetic Mice
Authors: Fujii Shoko , Ishizawa Keisuke , Sakurada Takumi , Yamano Noriko , Izawa-Ishizawa Yuki , Imanishi Masaki , Nuno Asami , Suzuki Yuta , Kihira Yoshitaka , Ikeda Yasumasa , Tomita Shuhei , Tsuchiya Koichiro , Tamaki Toshiaki
Published: July 2021

Diabetic nephropathy (DN) is a major microvascular complication of diabetes involving oxidative stress and endothelial damage. Nitrosonifedipine (NO-NIF) is converted from nifedipine under the light illumination. The ability of NO-NIF to block calcium channels is quite weak compared with that of nifedipine. Recently, we have demonstrated that NO-NIF reacts with unsaturated fatty acid leading to generate NO-NIF radical, which acquires radical scavenging activity. Also we have reported that NO-NIF reduces cytotoxicity of tumor necrosis factor-α (TNF-α) in cultured human glomerular endothelial cells. However, it is unclear the effects of NO-NIF on the pathogenesis related with oxidative stress, such as DN. In this study, we investigated whether NO-NIF treatment improves DN by using KKAy mice developing type II diabetes. Urinary albumin (UA) and total urinary protein (TUP) were elevated in KKAy mice (16 weeks old) compared with C57BL/6 mice, which were significantly decreased by NO-NIF treatment (30 mg/kg/day, i.p., 4 weeks) (UA; 1415.2 ± 312.5 vs 609.6 ± 151.5 μg/24hr, p<0.05, TUP; 73.1 ± 11.2 vs 44.8 ± 9.4 mg/24hr, p<0.05). Mesangial expansion was estimated by PAS staining. Glomerular diameter (GD) and glomerular tuft area (GTA) were extended in KKAy mice compared with C57BL/6 mice, which were significantly decreased by NO-NIF treatment (GD; 81.4 ± 2.6 vs 65.7 ± 1.9 μm, p<0.05, GTA; 4198.8 ± 254.6 vs 3051.9 ± 140.4 μm2, p<0.05). NO-NIF significantly suppressed urinary 8-hydroxy-20-deoxyguanosine (144.1 ± 21.4 vs 75.2 ± 11.1 ng/24hr, p<0.05) in KKAy mice. Also NO-NIF reduced the elevation of reactive oxygen species in kidney of KKAy mice detected by DHE staining. However NO-NIF had no influence on SOD activity in kidney. NO-NIF significantly inhibited the mRNA expression of TNF-α in kidney of KKAy mice (2.9 ± 0.1 vs 1.9 ± 0.1 fold increase, p<0.05). ICAM-1 protein expression was increased in glomeruli of KKAy mice, which was decreased by NO-NIF treatment. On the other hand, NO-NIF had no effect on glucose tolerance and systolic blood pressure (111.1 ± 2.3 vs 108.3 ± 3.3 mmHg, N.S.) in KKAy mice. These findings suggest that NO-NIF prevents the progression of DN via the attenuation of oxidative stress independent of antihypertensive or antihyperglycemic action.
Abstract 372: Role of Redox-Sensitive Calcium Channel TRPM2 and Oxidative Stress in Vascular Smooth Muscle Cell Differentiation to an Osteogenic Phenotype: Implications in Hypertension.
Authors: Montezano Augusto C , Yusuf Hiba , Callera Glaucia E , Touyz Rhian M
Published: July 2021

Vascular smooth muscle cell (VSMC) transformation to an osteoblast-like phenotype is a major factor contributing to vascular calcification, often associated with chronic kidney disease and hypertension. Exact molecular mechanisms underlying VSMC transformation remain unclear but intracellular calcium and ROS have been implicated. Whether these factors are interlinked is unknown. Here, we tested the hypothesis that ROS and redox-sensitive calcium channels (TRPM2) induce an osteogenic phenotype transition in VSMCs from WKY and SHRSP rats. Cultured VSMCs from WKY and SHRSP rats were exposed to calcification medium (CaM) (Ca2+ 1.8 mmol/L, PO4 2.0 mmol/L) for 10 days in the presence/absence of tempol (superoxide dismutase mimetic), and N-(p-amylcinnamoyl)anthranilic acid (ACA, TRPM2 inhibitor). Osteocalcin (OC), BMP-2, BMP-7, TRPM2 and TRPM2-S expression, as well as p47 phox translocation (cytosol:membrane), were determined by immunoblotting. ROS generation was evaluated by chemioluminescence. ROS production (Ctl: 30 AU/ug protein; CaM: 60 AU/ug protein, p<0.05) and p47 translocation (Ctl: 0.7 AU; CaM: 1.1AU, p<0.05) were increased by the CaM in VSMCs from WKY. CaM-induced increase in OC (Ctl: 5 AU; CaM: 13 AU) and BMP-2 (Ctl: 0.60 AU; CaM: 0.75AU) (p<0.05), followed by a decrease in BMP-7 (Ctl: 1.12 AU; CaM: 0.7 AU) (p<0.05), expression in VSMCs from WKY; an effect that was inhibited by tempol and ACA. In SHRSP, the increase in OC (Ctl: 1.25 AU; CaM: 2 AU) and BMP-2 (Ctl: 0.6 AU; CaM: 0.85 AU, p<0.05) expression induced by the CaM was also blocked by tempol. TRPM2 expression was higher in SHRSP (1.40 AU) than in WKY (1.05 AU) VSMCs (p<0.05). However, TRPM2-S expression, an intracellular inhibitor of TRPM2, was decreased in SHRSP compared to WKY VSMCs (SHRSP: 0.9 AU; WKY: 1.5 AU; p<0.05), and it was further decreased by the CaM (0.6 AU). In conclusion, ROS, through TRPM-2 sensitive mechanisms, seems to play an important role in VSMC transformation to an osteogenic, a phenomenon that may be exacerbated in hypertension.
Abstract 384: Nov/ccn3: A Potential Antagonist of Angiotensin Ii Effects in Kidney Disease
Authors: Chadjichristos Christos , Marchal Pierre-Olivier , Cuinet Aurelie , Pakradouni Jihane , Toubas Julie , Kazazian Chantal , Calmel Claire , Leblais Veronique , Chatziantoniou Christos , Martinerie Céle
Published: July 2021

Progression of chronic kidney disease (CKD) represents a constant challenge for the identification of new therapeutic targets. The NOV/CCN3 matricellular protein, belonging to the CCN (CYR61/CCN1, CTGF/CCN2, NOV/CCN3) family, could represent a new endogenous inhibitor of the fibrotic process that plays a crucial role in tissue damage and regression of renal function. However, the relation between NOV and renal disease remains unclear. Our objective was to study whether alterations in renal NOV expression are involved in the progression of CKD. NOV mRNA expression was significantly induced by 2.5-3 fold at 12-16 weeks in renin-transgenic mice (RenTg), a well established model of AngII-mediated nephropathy, through the Angiotensin receptor 1 (AT
1
R) compared to normotensive control animals (n=7 mice per group, p<0.05). NOV immunodetection revealed an up-regulation mainly in vascular smooth muscle cells (VSMC) in the renal cortex of RenTg mice. Interestingly, NOV expression was inversely correlated to known fibrotic markers such as Connective tissue growth factor, Transforming growth factor and collagen I, during the progression of renal disease in RenTg mice. NOV up-regulation at mRNA and protein levels (∼ 2.5 fold p<0.05) by AngII through AT
1
R was also observed in VSMC cultures. Furthermore, infection of these cells with an adenovirus recombinant for NOV strongly repressed in a dose-dependent manner the expression of the Angiotensin Receptor 1 reaching 72 and 78% (p<0.05) at mRNA and protein levels respectively. Treatment with exogenous NOV gave similar results. Conversely, knocking down NOV by small interfering RNA increased Angiotensin Receptor 1 by ∼3.5 to 4.5 fold and by ∼7.5 fold (p<0.05) at mRNA and protein levels respectively. These results indicate that NOV is involved in an AngII- AT
1
R regulatory loop. In subsequent studies the NOV-induced downregulation of AT
1
R was abrogated by integrin β1 specific siRNAs. Thus, NOV can attenuate the deleterious profibrotic effects of AngII during the progression of CKD and may provide a potential therapeutic option against this pathology.
Abstract 391: Modeling of the Angiotensin Peptide Processing Network
Authors: Schwacke John H , Spainhour John Christian , Arthur John M , Janech Michael G , Velez Juan Carlos Q
Published: July 2021

New insights into the intrarenal renin-angiotensin system (RAS) have modified the traditional view of the system. Nonetheless, the complexity of this network of angiotensin (Ang) peptides and peptidases is not completely understood. We hypothesized that a computational systems biology approach, applied to peptidomic data, could elucidate the network of enzymatic conversions and the resulting net stimulatory signal. To that effect, we built and refined a Bayesian network model and a dynamic systems model of the Ang peptide fragmentation utilizing a database of MALDI-TOF mass spectra from experiments conducted in mouse podocytes exposed to exogenous Ang substrates, augmented by new experiments guided by findings from the archive. A model-building process suggested novel steps, three of which were confirmed in vitro. When 1 μM Ang 2-10 was added to cell media, 102 ± 17 nM Ang 2-7 was detected at 4 hours, decreasing to 30 ± 12 nM in the presence of a neprilysin (NEP) inhibitor, supporting the model-predicted cleavage of Ang 2-10 by NEP. In addition, using Ang 1-9 as substrate, we observed Ang 2-9 production (15.3 ± 3% total Ang ion current at 2 hours) that decreased to 4.6 ± 0.5% during co-treatment with an aminopeptidase A (APA) inhibitor, supporting the model-predicted cleavage of Ang 1-9 by APA. Similarly, incubation of Ang 1-7 showed evidence of Ang 2-7 (28.1 ± 2.2% total Ang ion current at 2 hours) with undetectable amounts in cell-free controls. These data concur with the well-recognized robust degrading capacity of NEP and APA localized in glomerular podocytes. The performance of our model improved after inclusion of the newly discovered steps by Deviance Information Criterion. Although the products from these novel reactions are not known to be bioactive, the cleavages shunt Ang substrates away from formation of known bioactive peptides. In conclusion, our results demonstrate that systems biology methods are effective in identifying novel steps in the Ang peptide processing network, expanding our understanding of the RAS. Besides, the study illustrates the potential for mining archives of peptidomic data. Further exploration of the Ang network using this strategy will likely improve the study of the intricacies of the RAS, with potential clinical impact.
Abstract 394: Chronic Knockdown of the NTS AT1R Increases Circulating Inflammatory-Endothelial Progenitor Cells Ratio and Exacerbates Hypertension in the SHR
Authors: Shan Zhiying , Zubcevic Jasenka , Shi Peng , Jun Joo Yun , Dong Ying , Murça Tatiane M , Lamont Gwyneth J , Qi Yanfei , Li Qiuhong , Katovich Michael J , Sumners Colin , Raizada Mohan K
Published: July 2021

AT1R expression is increased in the nucleus of solitary tract (NTS) of many animal models of hypertension. However, the impact of its chronic regulation on cardiovascular control is not well understood. In this study, we investigated the hypothesis that NTS AT1R is involved in peripheral inflammatory status and hypertension-linked cardiovascular pathophysiology. AAV2- mediated expression of a shRNA targeted the AT1R in the NTS has been used to test the hypothesis. First,the efficacy of the AAV2-AR1R-shRNA was established with the use of neuronal cells in primary culture. Transduction of these cultures by AAV2-AT1R-shRNA resulted in a 72% decrease in AT1R mRNA compared with the AAV2- Sc-shRNA transduced control. This was accompanied by complete attenuation of AngII-induced increase in ERK1/2 phosphorylation and neuronal action potential frequency (control: 2.2±0.4 vs. AT1R-shRNA: 0.5±0.3 hz). We then microinjected AAV2-AT1R-shRNA or control vector, AAV2-Scrambled (Sc)-shRNA into the NTS of 8-week-old SHR and WKY rats. Nine weeks following microinjection, AAV2-AT1R-shRNA group of SHRs showed a 29 mmHg increase in mean arterial pressure (MAP) (control: 154.2±4.7; shRNA: 183.0±10.2 mmHg), insignificant effects on the MAP of WKY rats was observed. The increase in MAP in the SHR by AAV2-AT1R-shRNA was associated with a 42% decrease in baroreflex bradycardia (control: 0.57±0.02; shRNA: 0.33±0.06 ms/mmHg), profound cardiac hypertrophy, and significant increase in the plasma norepinephrine (control: 6.36±0.39, shRNA: 13.0±4.13 ng/mL). In addition, AAV2-AT1R-shRNA rats showed significant increases in water intake (control: 32.5±1.5; shRNA: 12.5±2.5 ml/16h) and urine output (control: 1.6 ±0.5; shRNA: 9.3±2.5 ml/16h). Furthermore, AAV2-AT1R-shRNA treated SHR exhibited 74% decrease in circulating endothelial progenitor cells (EPC, CD90+, CD4/5/8-), and ∼300% increases in inflammatory cells (IC) including CD4/8+, T lymphocytes (CD45/3+) and macrophages (CD68+) . As a result, EPC/IC ratio was decreased by 8∼15 fold in AT1R-shRNA treated SHR. These observations suggest that increased AT1R in the NTS of SHR may present a counter-hypertensive mechanism involving inflammatory/angiogenic cells.
Abstract 396: Inhibition of Prolyl Hydroxylase Domain-containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor
Authors: Ichiki Toshihiro
Published: July 2021

Background:
Prolyl hydroxylase domain-containing protein (PHD) mediates hydroxylation of hypoxia-inducible factor (HIF)-1α and thereby induces proteasomal degradation of HIF-1α. Inhibition of PHD by hypoxia or hypoxia mimetics such as cobalt chloride (CoCl2) stabilizes HIF-1 and increases the expression of target genes such as vascular endothelial growth factor (VEGF). Although hypoxia activates the systemic renin angiotensin system (RAS), the role of PHD in regulating RAS remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin (Ang) II type 1 receptor (AT1R) and its signaling.

Methods and Results:
Hypoxia (1% O2), CoCl2 (100-300 μmol/L), and dimethyloxalylglycine (0.25-1.0 mmol/L), all known to inhibit PHD, reduced AT1R expression by 37.7±7.6, 39.6±8.4-69.7±9.9, and 13.4±6.1-25.2±7.0%, respectively (p<0.01) in cultured vascular smooth muscle cell. The same stimuli increased the expression of nuclear HIF-1α and VEGF (p<0.05), suggesting that PHD activity is inhibited. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT1R expression by 55.3±6.0% (p<0.01). CoCl2 decreased AT1R mRNA through transcriptional and posttranscriptional mechanisms (p<0.01 and <0.05, respectively). CoCl2 and PHD2 knockdown diminished Ang II-induced ERK phosphorylation (P<0.01). Over-expression of the constitutively active HIF-1α did not impact the AT1R gene promoter activity. Oral administration of CoCl2 (14 mg/kg/day) to C57BL/6J mice receiving Ang II infusion (490 ng/kg/min) for 4 weeks significantly reduced the expression of AT1R in the aorta by 60.9±11.3% (p<0.05) and attenuated coronary perivascular fibrosis by 85% (p<0.01) without affecting blood pressure. However, CoCl2 did not affect Ang II-induced renal interstitial fibrosis.

Conclusion:
PHD inhibition downregulates AT1R expression independently of HIF-1α, reduces the cellular response to Ang II, and attenuates profibrotic effect of Ang II on the coronary arteries. PHD inhibition may be beneficial for the treatment of cardiovascular diseases, in which activation of RAS plays a critical role.
Abstract 400: A Nonpeptide Angiotensin (Ang) II Type 2 Receptor (AT2R) Agonist Improves Diabetic Nephropathy via Inhibition of Renal Inflammation
Authors: Matavelli Luis , Siragy Helmy M
Published: July 2021

We explored the hypothesis that direct AT2R stimulation improves albuminuria in diabetes by reducing renal inflammation and improving oxidative stress. Sham and DM Sprague-Dawley rats were treated for 4 weeks with vehicle (V) or AT2R agonist Compound 21 (C21; 0.3 mg/kg/d). C21 was infused systemically by osmotic minipump. Diabetes was induced by streptozotocin (65 mg/kg IP). At the end of study, we monitored BP, 24h urine collection for measurements of urinary albumin to creatinine ratio (UACR), and renal interstitial fluid (RIF) levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), cGMP, and 8-isoprostane (ISO). Data are shown as mean±SE. There were no significant differences in BP between different treatments. UACR (μg/mg), compared to Control+V (14±2), increased significantly in DM+V (60±3, p<0.001) and did not change in Control+C21 (16±2). Compared to DM+V, UACR decreased significantly by 20% in DM+C21 (50±3, P<0.04). RIF TNF-α and IL-6 (pg/min), compared to Control+V (0.040±0.001 and 0.613±0.012, respectively), increased in DM+V (0.046±0.001 and 0.652±0.005, P<0.01) and did not change in Control+C21 (0.041±0.001 and 0.622±0.008). Compared to DM+V, RIF TNF-α and IL-6 decreased in DM+C21 (0.041±0.001 and 0.616±0.007, P<0.01). RIF NO (μmol/min) and cGMP (fmol/min), compared to Control+V (7.0±0.3 and 3.8±0.4), decreased in DM+V (4.3±0.5 and 1.7±0.2, P<0.001) and did not change in Control+C21 (6.6±0.3 and 3.7±0.5). RIF NO and cGMP increased in DM+C21 (6.2±0.8 and 2.7±0.4, P<0.04) compared to DM+V. RIF ISO (pmol/min), compared to Control+V (0.135±0.005), increased in DM+V (0.158±0.007, P<0.02) and did not change in Control+C21 (0.134±0.010). Compared to DM+V, RIF ISO significantly decreased in DM+C21 (0.135±0.006, P<0.03). We concluded that direct AT2R stimulation by the nonpeptide agonist C21 improves diabetic nephropathy through the reduction of renal inflammatory factors and improvement of oxidative stress.
Abstract 412: Electrophysiological Properties of Kidney-related Neurons in the Rostral Ventrolateral Medulla
Authors: Gao Hong , Krantz Amanda , Derbenev Andrei V
Published: July 2021

Presympathetic neurons in the rostral ventrolateral medulla (RVLM) play an important integrative role in the neuronal network mediating cardiovascular regulation. In this study, we employed a retrograde, transsynaptic pseudorabies viral label (PRV-152), a PRV construct which expresses enhanced green fluorescent protein (EGFP), to identify kidney-related neurons in the RVLM. Whole-cell, patch-clamp recordings were made from 86 kidney-related neurons in the RVLM in transverse brainstem slices from 4-7-week-old rats. Under control conditions, we identified two populations of PRV-labeled RVLM neurons based on their electrophysiological properties. The resting membrane potential of large kidney-related neurons was -48 ± 2 mV and the input resistance was 205 ± 28 MΩ. The resting membrane potential of small kidney-related neurons was -51 ± 2 mV and the input resistance was 430 ± 36 MΩ. Spontaneous firing was observed in 2 out of 15 large cells with frequency of firing 0.008 and 0.05 Hz and 7 out of 16 small cells with average of firing 0.4 ± 0.2 Hz. Both spontaneous and miniature inhibitory and excitatory postsynaptic currents (i.e., sEPSCs, sIPSCs and mEPSCs, mIPSCs) were observed in small and large neurons. Tonic inhibitory and excitatory currents were identified in large kidney-related neurons of the RVLM. Application of the type A γ-aminobutyric acid (GABA
A
) receptor-linked Cl
-
channel blocker, bicuculline methiodide (30 μM), blocked sIPSCs and revealed a robust tonic inhibitory current with an average amplitude of 145.3 ± 30 pA. The cocktail of potent N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate ionotropic glutamate receptor antagonist, AP-5 (50 μM) and CNQX (10 μM), blocked sEPSCs and revealed a two-component tonic excitatory current mediated by NMDA and AMPA/kainate ionotropic glutamate receptors with overall amplitude of 65.2 ± 11.3 pA. These data demonstrate the synaptic complexity involved in the regulation of kidney-related RVLM neurons.
Abstract 440: Female Spontaneously Hypertensive Rats are More Dependent on Nitric Oxide in Modulating Blood Pressure and Renal Injury Than Males
Authors: Elmarakby Ahmed A , Pye Chelsey , Brinson Krystal , Yamamoto Tatsuo , Sullivan Jennifer C
Published: July 2021

We recently demonstrated that female spontaneously hypertensive rats (SHR) have higher nitric oxide (NO) bioavailability in the kidney compared to males. Therefore, we hypothesize that female SHR are more dependent on the NO synthase (NOS) system to modulate their blood pressure, and as a result will have a greater rise in blood presure and renal injury in response to chronic NOS inhibition compared to males. Mean arterial pressure in SHR was very sensitive to NOS inhibition, with both sexes exhibiting a significant increase in blood pressure to the non-specific NOS inhibitor N
G
-nitro-L-arginine methyl ester (L-NAME) at a dose of 2 mg/kg/day. However, there was not a sex difference in the percent increase in mean arterial pressure. Treatment of female and male SHR with 7 mg/kg/day L-NAME for 2 weeks significantly increased mean arterial pressure and indices of renal injury in both females and males; the percent increases in mean arterial pressure and injury were greater in females during the last three days of L-NAME treatment (% increase in mean arterial pressure was 44± 1.3 in females
vs.
35± 2.9 in males, P<0.05). L-NAME treatment also increased indices of inflammation and oxidative stress as urinary monocyte chemoattractant protein-1 (MCP-1) and thiobarbituric acid reactive substances (TBARs) excretion levels were increased by L-NAME; however, the increases were greater in females. Renal cortical macrophage and T cell infiltrations and soluble intracellular adhesion molecule-1 (sICAM-1) were also elevated following L-NAME treatment (cortical sICAM-1 levels increased from 4.7± 0.2 to 9± 0.1 ng/mg in males
vs.
4.6± 0.1 to 7.9± 0.3 ng/mg in females, P<0.05). Although renal cortical macrophage infiltration was greater in female SHR
vs.
males following L-NAME treatment, the increase in cortical T cell infiltration and sICAM-1 were greater in males. These data suggest that the NO system plays an important role in modulating blood pressure, renal injury and inflammation in SHR, with females being more dependent on the NOS system than males.
Abstract 46: Peripheral Inflammation Prior to rhe Onset of Hypertension and Proteinuria Characterizes the BPH/5 Mouse Model of Preeclampsia
Authors: Lob Heinrich E , Sones Jennifer L , Song Jiunn , Davisson Robin L
Published: July 2021

Evidence suggests that the immune system plays an important role in the pathogenesis of preeclampsia (PE), a hypertensive disease of pregnancy. Increased levels of circulating pro-inflammatory Th1 polarized T cells have been observed in PE patients. However, because it is not possible to diagnose PE until after its onset at ∼20 weeks of pregnancy, it is not known whether overactivity of the immune system is a cause or an effect of this disease. The BPH/5 mouse strain spontaneously develops the hallmarks of PE, including late-gestational (∼e14-16) hypertension and proteinuria, making it a valuable model system for studying early-pregnancy factors that may play a causal role in PE. Here we utilized flow cytometry to measure inflammatory cells and tested the hypothesis that BPH/5 mice exhibit T cell activation prior to the development of PE. First, similar to humans, there are increased circulating pro-inflammatory T cells at the hypertensive late-gestation stage (e18.5) in BPH/5 mice compared to C57Bl/6 controls (11 ± 0.2 x 10
5
vs 1.5 ± 0.4 x 10
5
CD3
+
cells, n=5; p<0.05). Aortas of BPH/5 mice at this time-point also show increased CD3
+
T cells compared to C57 (7.2 ± 1.2 x 10
3
vs 1.1 ± 0.4 x 10
3
CD3
+
cells, n=5; p<0.05). Next, to investigate if peripheral inflammation is increased before the onset of PE in this model, we measured circulating and aortic pro-inflammatory T cells at e10.5. BPH/5 mice showed a significant increase in both circulating (7.8 ± 0.2 x 10
5
vs 2.2 ± 0.2 x 10
5
CD3
+
cells, n=5; p<0.05) and aortic (3.9 ± 0.9 x 10
3
vs 0.8 ± 0.2 x 10
3
CD3
+
cells, n=5; p<0.05) T cells compared to controls. These data demonstrate that BPH/5 mice, similar to humans, show peripheral inflammation during PE. However, more importantly, our data show that activation of the immune system occurs prior to the onset of hypertension and proteinuria in this model, suggesting that peripheral inflammation may play a causal role in this devastating disease of pregnancy.
Abstract 463: Cardiometabolic Risk Factors and Vitamin D 25OH levels in Puerto Rican Adolescents
Authors: Suarez-Rivera Marta P , Bruckman Wilmarie , García Ana , Bonilla-Felix Melvin
Published: July 2021

Purpose:
Overweight, obesity, hypertension (HTN), insulin resistance, and dyslipidemia have been recognized as cardiometabolic risk factors in adults. Early markers of cardiovascular disease in children are lacking. Low vitamin D (VITD) levels are associated with higher blood pressure, obesity and insulin resistance. We explored the association between traditional cardiometabolic risk factors and VITD levels with HTN status in adolescents. DESIGN

METHODS:
Cross-sectional pilot study in students from grades 9th - 12th in Puerto Rico. Fasting blood samples to estimate serum lipids, fasting glucose and vitamin D25OH levels were obtained, with anthropometric and blood pressure measurements. Variables described as mean [standard deviation (sd)] or frequencies and percentages as appropriate. Bivariate analysis using chi-square test; statistical significance was set at p<0.05.

Results:
78 adolescents, mean age: 16 years were studied. Mean BMI: 23.8 (range 17-39); 19.2% (15/78) were overweight and 16.7% (13/78) were obese. Pre-HTN observed in 7.7% (6/78), with 1 HTN. Hypercholesterolemia (≥170 mg/dl): 16.9% 13/77), low HDL levels (≤ 40 mg/dl): 67.5% 23/77), and hypertriglyceridemia (≥ 110 mg/dl): 15.6% (12/77). VITD levels were low (< 30ng/dL) in 31% 24/77. Obese subjects were more likely to have insufficient VITD levels when compared with the non-obese, (61.5% vs 25%; p=0.02).At least 1 cardiometabolic risk factor was found in 69% (54/78); 33% (26/78) had 2 or more.

Conclusion:
Obesity is associated with low VITD levels and preHTN. Cardiometabolic risk factors are highly prevalent in our population. Interventions to prevent cardiovascular disease in Puerto Rican adolescents are required.