bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 237: Structural Analysis of Ang Peptide Specificity in Binding to ACE, ACE2, Neprilysin, and PRCP
Authors: Prokop Jeremy W , Araujo Fabiano C , Watanabie Ingrid K , Caserini Dulce E , Milsted Amy
Published: July 2021

A combination of known biochemistry and physiology data on angiotensin converting enzyme (ACE), ACE2, neprilysin and prolylcarboxypeptidase (PRCP) with structural biochemistry and bioinformatics reveals several key amino acids contributing to the orientation of Ang peptides in the active site of the various enzymes. A total of four structures are known for the ACE N-terminus, twelve for the ACE C-terminus, eleven for ACE2, seven for neprilysin, and one for PRCP. Of all the known structures, none include angiotensin peptides bound into an active site. Autodock experiments reveal possible binding conformations of Ang I, Ang II, Ang-(1-9), Ang-(1-7), or Ang III into the active site of each enzyme. These autodock experiments were validated using combinations of disease associated natural variants, known mutagenesis data, conservation of amino acids in multiple species and inhibitor based structural data, all revealing amino acids of high functionality that interact with Ang peptides. Aromatic amino acids (Phe, Tyr, and Trp) tend to dictate the cleavage site of the Ang peptides through interactions with amino acid 4 (Tyr), 6 (His) or 8 (Phe) of angiotensin. The ACE C-terminus and N-terminus have three conserved aromatic amino acids 10 Å or less from the catalytic Zn, ACE2 has two 11 Å or less from the catalytic Zn, neprilysin has a Phe 8.4Å from the catalytic Zn, and PRCP has five aromatic amino acids 12Å or less from the active site. A high density of serine and threonine amino acids is found in the active sites of some enzymes when compared with total serines and threonines (ACE N-term=39%, ACE C-term=46%, ACE2=32%, neprilysin=15%, PRCP=10%), suggesting a strong likelihood of a hydrogen bonding network with angiotensin peptides in the active site, aiding in proper orientation for cleavage. Positions of serines and threonines (along with Lys, Arg, Val, Tyr, Phe, and Met) differ in the two active sites of ACE and are in contact with docked Ang I. This study shows enzyme differences and similarities in amino acids contacting the Ang peptides. Further, our results suggest that it may be possible to target multiple enzymes with the same (or a single) treatment in hypertensive patients, minimizing additive side effects of multidrug treatments.
Abstract 243: Structure and Sequence Analysis of AT1, AT2, and MAS: Insights into Differences and Similarities in Binding and Activation by Angiotensin Molecules
Authors: Prokop Jeremy W , Santos Robson A , Milsted Amy
Published: July 2021

The renin-angiotensin system is involved in multiple conditions ranging from cardiovascular disorders to cancer. The components of the pathway are targets for disease treatments including ACE inhibitors, renin inhibitors and AT1 blockers. However, very little is understood about the molecular mechanisms by which G-protein coupled receptors (GPCRs) are activated by angiotensin peptides. This study addresses three known receptors of the pathway: AT1, AT2, and MAS. Combining biochemical and amino acid variation data with multiple species sequence alignments, structural models, and docking site predictions allows for visualization of how angiotensin peptides may bind and activate the three receptors. It also addresses conserved and variant mechanisms among receptors. Models of each receptor align with a root mean squared deviation of less than 2.1Å with sequence conservation of 14-30% when comparing to another GPCR (rhodopsin), revealing amino acids required to maintain the seven helixes of the structure. This study reveals that MAS possibly differs in binding to angiotensin peptides, favoring a binding to Ang-(1-7) and not Ang II. Consensus amino acids 512 (Lys) and 621 (His), believed to interact with Phe 8 of Ang II are not conserved in MAS but amino acids (118, 233, 231, 268, 419, 719, and 725) predicted to interact with amino acids 1-7 of either Ang II or Ang-(1-7) are conserved. Analysis of MAS related proteins shown to be activated by Ang peptides reveals possible amino acids (114, 120, 318, 342, 426, 526, 527, and 720) that may contribute to homo or heterodimer formations with other membrane bound proteins, a possible mechanism of activation by the MAS receptor family. Finally a new model of Ang II binding and activation of AT1 and AT2 is proposed that correlates data from site directed mutagenesis and photolabled binding. The initial binding occurs between amino acids 512 and 621of either AT1 or AT2 with Phe 8 of Ang II. Then the Phe of Ang II shifts position via conserved aromatic amino acids to the final photolabled position relative to either AT1 (725) or AT2 (336). Our new model of Ang II-receptor interactions can be verified in future experiments to allow for a clear understanding of angiotensin peptide receptors and their interactions with other molecules.
Abstract 255: The Kidney and Heart are Hemogenic Organs During Embryonic Life.
Authors: Hu Yan , Li Minghong , Gomez R. Ariel , Sequeira Lopez Maria Luisa S
Published: July 2021

During early embryonic life, the heart starts to beat before an effective circulation is established, and the kidney starts to form its vasculature before it connects to the general circulation. We and others have shown a close lineage relationship between endothelial cells (ECs) and hematopoietic cells. In fact, during embryonic development erythroblasts bud from the endothelium of developing vessels, a process we termed hemovasculogenesis. Those studies suggested the possibility that embryonic organs may have hemogenic potential. To test this hypothesis, we performed lineage studies and colony forming unit (CFC) assays to trace the fate of hematopoietic stem cells (HSCs), erythroid cells, and ECs in heart and kidney from embryonic mice. Using ER-GFPcre mice that express both GFP and cre under control of the erythropoietin receptor locus in the erythroid cells, we identified hematopoietic progenitors (Hb+Nanog+) within nascent vessels in the early embryonic kidney and heart. Using EC-SCL-Cre-ERT transgenic mice that specifically express tamoxifen inducible Cre in EC progenitors, we found both circulating and non-circulating cells from the EC lineage in the early embryonic heart and kidney. CFC assays using HSC-SCL-Cre-ERT; mTmG mice (which express GFP in the cells from the HSC lineage upon tamoxifen induction) showed that both the embryonic kidney and heart possess HSCs. Further, transplantation studies of pre-vascular embryonic kidneys from EC-SCL-Cre-ERT;R26R mice under the kidney capsule of WT adult mice showed blood cells derived from the embryonic kidney suggesting that the embryonic kidney also possesses HSCs that originate in situ. These studies indicate that the embryonic kidney and heart function as hematopoietic organs during early embryogenesis. In addition to solve an important scientific controversy in our understanding of lineage/fate relationships in the developing embryo, these findings are relevant for tissue repair/regeneration and may help explain why under pathological circumstances, hematopoiesis occurs in extramedullary organs.
Abstract 256: Rbp-j Controls the Fate of the Kidney Vasculature
Authors: Lin Eugene , Sequeira Lopez Maria Luisa S , Gomez Roberto A
Published: July 2021

Proper assembly of the renal vasculature is essential for post-natal life, and alterations to the renal vasculature are at the root of many types of cardiovascular disease. However, the mechanisms underlying the establishment, assembly and maintenance of the renal blood vessels are poorly understood. We have identified a population of renal stromal cells (marked by their expression of the transcription factor Foxd1) that differentiate to form the mural cells of the kidney arterial tree (excluding endothelial cells) and the glomerular mesangium. We previously demonstrated that RBP-J, the final transcriptional effector of the Notch signaling pathway, controls the phenotype of renin cells which are also derived from the Foxd1 lineage. We therefore hypothesized that RBP-J regulates the differentiation of stromal cells into the mural cells of the kidney arterioles. To answer this question, we deleted RBP-J in the metanephric stromal precursor cells, and found that mutant mice displayed striking kidney abnormalities in early life. Staining for vascular markers showed a significant decrease in the number of arteries and arterioles. Vessel walls were thinner due to a decrease in both the size and number of smooth muscle cells. We also noted a near absence of renin cells, supporting our earlier findings regarding the key role of RBP-J in establishing the differentiated renin cell endowment. These findings were accompanied by delayed nephrogenesis and other renal abnormalities including tubular dilation. In addition, mutant kidneys lacked Foxd1-lineage cells within the glomeruli, resulting in a depletion of mesangial cells and glomerular aneurysms. Thus, we conclude that RBP-J in Foxd1+ stromal cells plays a key role in the development of the kidney vasculature, and regulates the fate of cells that compose the arterial tree and the glomerular tuft.
Abstract 258: Angiotensin 1-7 Attenuates Endothelin-1-Induced Endothelial Cell Inflammation and Growth Through Nitric Oxide Production and Activation of Mas and EndothelinB Receptors
Authors: Yusuf Hiba , Montezano Augusto C , Callera Glaucia E , Nguyen Dinh Cat Aurille , Santos Robson A , Castro Carlos H , Touyz Rhian M
Published: July 2021

In pulmonary hypertension, where the endothelin system plays a major role, the vasoprotective axis of the rennin angiotensin system (ACE2-Ang (1-7)-Mas) seems to be protective. However, the exact mechanisms are still elusive and whether Ang 1-7 counterbalancing effects are beyond interactions with Ang II system is unknown. In this study, we assessed whether Ang 1-7 influences/interacts with the ET-1 system in endothelial cells. Cultured human microvascular endothelial cells (HMEC) were studied. HMEC were stimulated with ET-1 (10-7 mol/L) in the absence and presence of Ang 1-7 (10-7 mol/L), BQ788 (an ETBR antagonist), BQ 123 (an ETAR antagonist) and A779 (Mas receptor inhibitor) (10-6 mol/L). Expression of pro-inflammatory mediator (VCAM-1), cell growth marker (PCNA), Mas, ETBR expression and eNOS activation was determined by immunoblotting. ET-1 significantly increased expression of VCAM-1 (138.90% vs control, p<0.05) and PCNA (125% vs control, p<0.05). Ang 1-7 alone did not modulate pro-inflammatory and growth mediators, but significantly inhibited the effects of ET-1 on VCAM-1 (95.55%) and PCNA (103.83%) expression, an effect mediated by Mas receptor activation (after A779: VCAM-1: 226.15%; PCNA: 120% vs control, p<0.05). Ang 1-7 increased NO production (Ctl: 7.5 vs Ang 1-7: 20 RFU/ug of protein, by microfluorescence). Inhibition of Ang 1-7-induced NO production by L-NAME, inhibited Ang 1-7-mediated effects on ET-1-induced VCAM-1 (160%) and PCNA (125%), p<0.05. Ang 1-7 significantly increased expression of ETB receptors (175.63% vs control, p<0.05), an effect attenuated by A779. Ang 1-7 (166.94% vs control, p<0.05) and ET-1 (146.04% vs control, p<0.05) increased eNOS phosphorylation in HMEC. Blockade of Mas and ETB receptor inhibited Ang 1-7 and ET-1 effects on eNOS activation. BQ123, but not BQ788, blocked ET-1-stimulated inflammation/growth in HMEC (VCAM-1: 75%, PCNA: 100%, p<0.05). In conclusion, Ang 1-7 negatively modulates proinflammatory and mitogenic actions of ET-1, through crosstalk between Mas and ETB receptors, and increase in NO production. These data highlight some molecular mechanisms whereby Ang 1-7 may exert beneficial effects in pulmonary hypertension and suggests a novel mechanism for Ang 1-7 signalling in HMEC.
Abstract 265: Chronic Treatment with Nebivolol Attenuates the Vascular Changes in the 2-Kidney, 1-Clip Model of Renovascular Hypertension
Authors: Ceron Carla S , Rizzi Elen , Guimarães Danielle A , Martins-Oliveira Alisson , Gerlach Raquel F , Tanus-Santos José
Published: July 2021

Nebivolol (Nebi) is a β-1 receptor antagonist with vasodilator and antioxidant properties. Matrix metalloproteinases (MMPs) are enzymes involved in cardiovascular remodeling, and MMP upregulation has been associated with increased reactive oxygen species (ROS). Because vascular remodeling is associated with increased ROS and MMPs, it is possible that Nebi reverses the increased MMP levels and vascular remodeling associated with 2K-1C hypertension through its antioxidant activity. Hypertension was induced in male Wistar rats by clipping the left renal artery. Six weeks after surgery, hypertensive and sham rats were treated with Nebi (10 mg/kg/day), metoprolol (Meto; 20 mg/kg/day) or vehicle for four weeks. Systolic blood pressure (SBP) was monitored weekly by tail-cuff plethysmography. Structural changes of the aortic wall were studied in hematoxylin/eosin sections. MMPs levels and activity were determined by zymography and in situ zymography. NADPH oxidase activity and ROS production were evaluated by luminescence and dihydroethidium. Similar reductions in SBP were found with both Meto or Nebi treatments (156 ± 8 mmHg and 151 ± 9 mmHg, respectively, versus 206 ± 7 mmHg in hypertensive controls; both P<0.05). However, only Nebi (all P<0.05) reversed aortic hypertrophy (aortic cross sectional area x 10
4
= 89±5, 68±3, and 80±8 μm
2
, respectively, in the 2K1C, 2K1C+Nebi, and 2K1C+Meto groups), the increases in aortic MMP-2 levels (0.18±0.04, 0.07±0.02, and 0.12±0.06 arbitrary units; AU, respectively in the 2K1C, 2K1C+Nebi, and 2K1C+Meto groups), in aortic MMP activity (21071±700, 16217±818, and 18848±1396, respectively in the 2K1C, 2K1C+Nebi, and 2K1C+Meto groups), in aortic NADPH oxidase activity (253887±13712, 143765±15642, and 232465±14352 AU, respectively in the 2K1C, 2K1C+Nebi, and 2K1C+Meto groups) and in aortic ROS levels (8057±800, 4400±480, and 6230±1190 AU, respectively, in the 2K1C, 2K1C+Nebi, and 2K1C+Meto groups). No significant differences were found in the Sham groups. Our results suggest that lower vascular NADPH oxidase may explain, at least in part, the attenuation of oxidative stress and vascular remodeling associated with Nebi.
Abstract 268: Angiotensin II Up-regulates Serum and Glucocorticoid Inducible Kinase 1 in Remodeled Blood Vessels
Authors: Stewart-Hutto Tarianna , Francis Sharon
Published: July 2021

Angiotensin II (Ang II) is a potent vasoconstrictor that activates vascular smooth muscle and in excess amounts is an important contributing factor in the development of hypertension. However, the downstream signaling pathways mediating the effects of Ang II in the vasculature is not fully known. The present study examines the regulation of serum- and glucocorticoid inducible kinase (SGK1) a serine/threonine kinase that has been implicated in hyperglycemia- and salt-induced hypertension. We hypothesized that SGK1 is up-regulated in pathologically remodeled blood vessels in the context of Ang II-induced hypertension and by Ang II in vascular smooth muscle cells
in vitro
. Therefore, we examined SGK1 protein expression in human aortic smooth muscle cells (HASM) stimulated with increasing doses of Ang II (0-100nM) in vitro. Our results demonstrated a dose-dependent increase in SGK1 protein expression. SGK1 expression was increased approximately 10-fold following 60 minutes of stimulation with 100nM Ang II. Next, we examined SGK1 expression in the vasculature in vivo in a mouse model of Ang II-induced hypertension. Based on immunohistochemistry, our data indicated that SGK1 was up-regulated in the medial layer of the aorta in mice infused with 0.7mg/kg/day Ang II, a dose that significantly increases blood pressure. Overall, these results indicate that Ang II up-regulates SGK1 in both smooth muscle cells and blood vessels. Our results also suggest that SGK1 may be responsible for the increase in blood pressure and remodeling of the blood vessels.
Abstract 313: Protective Effects of Iron-Restricted Food against Diabetic Nephropathy in db/db Mice
Authors: Enomoto Hideaki , Ikeda Yasumasa , Tajima Soichiro , Izawa-Ishizawa Yuki , Kihira Yoshitaka , Ishizawa Keisuke , Tomita Shuhei , Tsuchiya Koichiro , Tamaki Toshiaki
Published: July 2021

Background:
Iron, an essential trace metal for organisms, catalyzes highly toxic hydroxyl radical via Fenton/Haber-Weiss reaction. We recently have clarified that iron chelation prevents the development of adipocyte hypertrophy through the reduction of oxidative stress in diabetic obese mice. It is suggested that iron reduction potentiates to be beneficial effects against diabetic complications. In the present study, we investigated the protective action of iron restriction against the progression of diabetic nephropathy.

METHODS:
We employed and divided male db/db mice of 8 weeks old age, a model of diabetic nephropathy, into 2 groups, normal diet group (Fe 100mg/kg food; ND) and low iron diet group (Fe 10mg/kg food; LID). After 8 weeks treatment, the mice were used for analysis.

Results:
LID group showed the decreased renal iron content (7.5±3.0 μg/g tissue vs 5.7±0.3 μg/g tissue, ND vs LID, p<0.05), serum iron concentration (213±15 μg/dL vs 157±14 μg/dL, ND vs LID, p<0.05) and hemoglobin level (13.5±0.7g/dL vs 10.4±0.5g/dL, ND vs LID, p<0.01). Urinary albumin excretion was significantly decreased in LID group compared to ND group (2.6±0.4mg/g·Cre vs 1.4±0.3mg/g·Cre, ND vs LID, p<0.05). In histological analysis, mesangial proliferation was ameliorated in db/db mice with LID (39% vs 28%, ND vs LID, p<0.05). LID reduced the deposition of collagen IV, fibronectin and desmin by 61%, 60%, and 42%, respectively, in glomeruli of db/db mice. Superoxide production, p22phox and NOX4 expression were also diminished in kidney of LID group.

Conclusion:
Iron restriction is suggested to prevent the development of diabetic nephropathy through the suppression of oxidative stress.
Abstract 315: Supression of Aldosterone and Progesterone in Preeclampsia: Role of Marinobufagenin: A Translational Approach
Authors: Uddin Mohammad N , Horvat Darijana , Allen Steven R , Jones Richard O , Zawieja David C , Kuehl Thomas J
Published: July 2021

Preeclampsia (preE) is a hypertensive disorder of pregnancy. In an animal model of preE, we have shown that the urinary excretion of marinobufagenin (MBG) is elevated prior to the development of hypertension and proteinuria. Several research groups provided evidence for reduced aldosterone (Aldo) and progesterone (Prog) availability in preE. This study focuses on the alterations in Aldo & Prog in a rat model of preE,
in vitro
study and in preE patients. Three groups of animals were studied: normal pregnant (NP, n = 9); pregnant rats that received weekly injections of desoxycorticosterone acetate and saline drinking water (PDS; preE rat model, n = 9); and NP rats injected with MBG (7.65 μg/kg/d, NPM, n = 8). The plasma and urinary levels of Aldo were assayed in all groups. To see the effect of MBG in the secretion of Aldo, adrenal cells were treated with DMSO (vehicle), 0.1, 1, 10 or 100 nM MBG. The levels of Aldo & Prog in the culture media of MBG-treated adrenal cells and in cell lysates were measured by ELISA. We recruited 17 PreE and 23 normally pregnant patients at Scott & White hospital. Aldo, Prog and MBG levels were assayed in plasma samples by ELISA. Both plasma and urinary levels of Aldo (NP: 578.5 ± 152.4, PDS: 379.5 ± 89.6, NPM: 348.7 ± 91.3 pg/mL for plasma; NP: 86.7± 10.7, PDS: 52.0 ± 5.2, NPM: 58.9 ± 6 for urine, p<0.05 for each case) & Prog (NP: 31.5 ± 2.9, PDS: 18.6 ± 2.1, NPM: 16.5 ± 91.3 ng/mL for plasma; NP: 6.5 ± 0.5, PDS: 3.5 ± 0.4, NPM: 3.2 ± 0.5 for urine, p<0.05 for each case) were significantly lower in PDS and NPM rats compared to NP. MBG ≥ 1 nM significantly decreased the secretion of Aldo in MBG- treated adrenal cells when compared to the vehicle-treated cells. The mean plasma Aldo of 398.6 ± 115.8 pg/mL and Prog of 46.2 ± 9.5 ng/ml in PreE patients were significantly suppressed (p<0.05) from 548.4 ± 137.5 pg/mL and 82.5 ± 13.6 ng/l, respectively, in NP women. On the contrary, plasma MBG was significantly higher (p<0.05) in preE compared to NP (preE: 59 ± 17 & NP: 12 ± 2 pg/mL). These data suggest that Aldo & Prog are paradoxically concealed in PreE. Moreover, it has been suggested that MBG is involved in the downregulation of Aldo & Prog expression in preE rat model and in
in vitro
adrenal cells. This study provides new evidence that MBG plays a key role in the suppression of Aldo & Prog in PreE.
Abstract 316: Effects of Inhibiting of Inducible Nitric Oxide Synthase in the Pathophysiology of Experimental Preeclampsia
Authors: Amaral Lorena M , Palei Ana Carolina T , Pinheiro Lucas C , Sertorio Jonas T , Guimaraes Danielle A , Portela Rafael L , Tanus-Santos Jose Eduardo
Published: July 2021

The pathophysiology of preeclampsia (PE) is not entirely known. However, increased oxidative stress possibly leading to impaired nitric oxide activity has been implicated in the critical condition. Increased oxidative stress with increased levels of highly reactive species including superoxide may generate peroxynitrite. We examined the role of inducible nitric oxide synthase (iNOS) and oxidative stress in the reduced uterine perfusion pressure (RUPP) preeclampsia experimental model.

METHODS:
RUPP was induced in wistar rats. Pregnant rats in the RUPP group had their aortic artery clipped at day 14 of gestation. After a midline incision, a silver clip (0.203 mm) was placed around the aorta above the iliac bifurcation; silver clips (0.100 mm) were also placed on branches of both the right and left ovarian arteries that supply the uterus. Sham-operated (pregnant control rats) and RUPP rats were treated with oral vehicle or 1 mg/kg/day 1400W (iNOS inhibitor) for 5 days. Mean arterial pressure (MAP) and plasma levels of thiobarbituric acid-reactive species (TBARS) and total radical-trapping antioxidant potential (TRAP) were measured determined. Aortic iNOS expression (Western blotting) and reactive oxygen species (ROS; assessed by fluorescence microscopy with dihydroethidium-DHE) were measured. We found increased mean arterial pressure in RUPP compared with pregnant control rats (MAP= 128±1 vs. 100±1.8 mmHg, respectively; P<0.05) and 1400W exerted antihypertensive effects (MAP= 114±2 vs.128±1 mmHg in RUPP treated and untreated rats, respectively; P<0.05). Higher reactive oxygen species (ROS) concentrations were found in RUPP compared with pregnant control rats (7.1±0.5 vs. 5.1±0.5 arbitrary units (A.U.), respectively; P<0.05) and 1400W decreased ROS production to 5.8±0.02 A.U. in RUPP treated rats, P<0.05. In addition, 1400W attenuated iNOS expression in RUPP rats (0.29±0.02 vs. 0.55±0.8 A.U. in RUPP treated and untreated rats, respectively; P<0.01) and had no effects on plasma TBARS and TRAP levels. Our results suggest that 1400w exerts antihypertensive effects in the RUPP model and suppresses ROS formation. Supported by FAPESP,Cnpq.
Abstract 319: Potential Lifetime Cost-Effectiveness of Catheter-Based Renal Sympathetic Denervation in Patients with Resistant Hypertension
Authors: Dorenkamp Marc , Bonaventura Klaus , Leber Alexander W , Schulze Julia , Sohns Christian , Boldt Leif-Hendrik , Haverkamp Wilhelm , Frei Ulrich , Roser Mattias
Published: July 2021

Aims
- Recent studies have demonstrated the safety and efficacy of catheter-based renal sympathetic denervation (RD) for the treatment of resistant hypertension. We aimed to determine the cost-effectiveness of this approach separately for men and women of different ages.

Methods and results
- A Markov state-transition model accounting for costs, survival, quality of life, and incremental cost-effectiveness was developed to compare RD with best medical therapy (BMT). The model ran from age 30 to 100 years or death, with a Markov cycle length of 1 year. All patients entered the model with elevated systolic blood pressure (SBP, 180 mmHg), but free from prior cardiovascular disease (CVD) events. Patients could remain in this disease-free state or could have one of the following primary CVD events: myocardial infarction, angina, stroke or heart failure. After an event, patients transitioned into chronic disease states and/or could suffer from secondary CVD events. In the base case scenario, RD resulted in a sustained SBP reduction of 20 mmHg and in a corresponding relative risk reduction of adverse CVD events and death. The cost analysis was conducted from a health care payer perspective. Effectiveness was expressed as quality-adjusted life-years (QALY) gained. Costs and effects were discounted at 3% per annum, respectively. Deterministic and probabilistic sensitivity analyses were conducted to evaluate the robustness of the results. As compared with BMT, RD gained 0.99 QALYs in men and 0.91 QALYs in women 60 years of age at an additional cost of $3,265 and $2,594, respectively. As the incremental cost-effectiveness ratio increased with patient age, RD consistently yielded more QALYs at lower costs in lower age groups. Considering a willingness to pay threshold of $50,000/QALY, there was a 95% probability that RD would remain cost-effective up to an age of 82 and 80 years in men and women, respectively. Cost-effectiveness was influenced most by the extent of SBP lowering by RD, the rate of RD non-responders, and the costs associated with the RD procedure.

Conclusion
- RD offers a cost-effective treatment option for the prevention of CVD events in patients with resistant hypertension. An earlier intervention produces better cost-effectiveness ratios.
Abstract 321: Combination Dri With At1R Blockade Does Not Confer Additional Benefit to Improvement in Myocardial Tissue Hypertrophy and Fibrosis
Authors: Whaley-Connell Adam T , Habibi Javad , Rehmer Nathan , Ardhanari Sivakumar , Chockalingham Anand , Hayden Melvin R , Pulakat Lakshmi , Krueger Caroline , Ferrario Carlos M , DeMarco Vincent G , Sowers James R
Published: July 2021

Data support the notion that direct renin inhibition and angiotensin type 1 receptor (AT
1
R) blockade improve myocardial hypertrophy and fibrosis. Even with contemporary use of AT
1
R blockade and renin inhibition, the burden of heart failure remains high. Thereby, we sought to determine if combination of direct renin inhibition with AT
1
R blockade
in vivo
, through greater reductions in systolic blood pressure (SBP) and aldosterone would attenuate left ventricular (LV) hypertrophy and interstitial fibrosis to a greater extent than either intervention alone. We utilized the transgenic Ren2 rat which manifests increased expression of murine renin which, in turn, results in increased tissue RAS activity, aldosterone secretion and elevated SBP. Ren2 rats were treated with renin inhibition (aliskiren), AT
1
R blockade (valsartan), the combination (aliskiren+valsartan), or vehicle for 21 days. Compared to Sprague-Dawley controls, Ren2 rats displayed increased SBP, serum aldosterone levels, LV and cardiomyocyte hypertrophy, interstitial fibrosis and ultrastructural remodeling. These biochemical and functional alterations were accompanied by increases in LV tissue NADPH oxidase subunit Nox2 and 3-nitrotyrosine (3-NT) content along with increases in mammalian target of rapamycin (mTOR) and reductions in protein kinase B phosphorylation. Combination therapy contributed to greater reductions in SBP and serum aldosterone but did not result in greater improvement in metabolic signaling or markers of oxidative stress, fibrosis or hypertrophy beyond either intervention alone. Thereby, our data suggest that the greater impact of combination therapy on reductions in aldosterone does not translate into greater reductions in myocardial fibrosis or hypertrophy in this transgenic model of tissue renin overexpression.
Abstract 322: Imbalance Between MMP-2 and TIMP-4 Associated With Oxidative Stress Contributes to Temporal Renovascular Hypertension-Induced Cardiac Hypertrophy
Authors: Rizzi Elen , Ceron Carla S , Guimaraes Danielle A , Prado Cibele M , Rossi Marcos A , Gerlach Raquel F , Tanus-Santos Jose E
Published: July 2021

Imbalanced matrix metalloproteinase (MMP) activity is associated with left ventricular hypertrophy (LVH). Increased reactive oxygen species (ROS) formation enhances MMP activity and decrease tissue inhibitor of MMP (TIMP) activity. We examined the temporal relationship between ROS, MMP-2, and TIMP-4 levels in the heart during the progression of LVH in two-kidney, one-clip (2K1C) hypertension. Sham or 2K1C hypertensive rats were studied after 15, 30, and 75 days of hypertension, and the results are expressed as % of values found in corresponding sham animals at each time point. Systolic blood pressure increased with time in 2K1C rats: 167±3, 201±1, and 203±2 mmHg after 15, 30, and 75 days of hypertension (all P<0.05). Hypertension induced LVH in a time-dependent manner (P<0.05). Collagen deposition increased by 250±23%, 213±20% and 235±16% in 2K1C rats, respectively, after 15, 30, and 75 days of hypertension; all P<0.05
vs.
respective sham). Cardiac MMP-2 levels increased from 13±1 (sham) to 19±2, 17±1, and 20±2 arbitrary units (AU) after 15, 30, and 75 days of hypertension, respectively (all P<0.05
vs.
sham). Cardiac TIMP-4 levels increased by 325±68% (P<0.05) only after 75 days of hypertension. Cardiac gelatinolytic activity increased by 129±4%, 150±6%, and 152±6% after 15, 30, and 75 of hypertension, respectively (all P<0.05
vs.
sham). Cardiac ROS levels increased by 137±5%, 154±15%, and 130 ±10% after 15, 30, and 75 days of hypertension, respectively (all P<0.05
vs.
sham). These results indicate that LVH in is an early process associated with imbalanced MMP-2 and TIMP-4 levels in renovascular hypertension, thus resulting in increased gelatinolytic activity and fibrosis, which may be due to oxidative stress.
Abstract 327: Apelin-13 Increases Myocardial Progenitor Cells and Improves Myocardial Remodeling of Post-myocardial Infarction.
Authors: Li lanfang , Zeng Heng , Chen Jian-Xiong
Published: July 2021

ABSTRACT:
Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and has beneficial effects against hypertension and myocardial ischemia/reperfusion injury. Little is known about the role of apelin in the homing of vascular progenitor cells (PCs) and cardiac remodeling post-myocardial infarction (MI). The present study investigates whether apelin affects PCs homing to the infarcted myocardium thereby mediating cardiac remodeling post-MI. Mice were infarcted by coronary artery ligation and apelin-13 (1 mg/kg.d) was injected for three days prior to MI and for either 24 hours or 14 days post MI. Homing of vascular progenitor cell (CD133
+
/c-kit
+
/Sca1
+
, CD133
+
/SDF-1α
+
and CD133
+
/CXCR4
+
) into the ischemic area were examined at 24 hours and 14 days post-MI. Myocardial Akt, eNOS, VEGF, Jagged1, Notch3, SDF-1α and CXCR4 expression were assessed. Functional analyses were performed at day 14 after MI. Mice receiving apelin-13 treatment demonstrated upregulation of SDF-1α/CXCR4 expression and dramatically increased the number of CD133
+
/c-kit
+
/Sca1
+
, CD133
+
/SDF-1α
+
and c-kit
+
/CXCR4
+
cells in the infarcted hearts. Apelin-13 also significantly increased Akt and eNOS phosphorylation and upregulated VEGF, Jagged1, Notch3 expression in the ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Further, treatment with apelin-13 promoted myocardial angiogenesis, attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 14 days post-MI mice. Apelin-13 increases angiogenesis and improves cardiac remodeling by a mechanism involving upregulation of SDF-1α/CXCR4 and homing of vascular progenitor cells.
Abstract 339: Splice Acceptor Site Mutation in Cullin 3 is Causing Pseudohypoaldosteronism Type II.
Authors: Friesema Edith C , Verkerk Annemieke J , Koole Rita H , van der Lubbe Nils , van Rooij Jeroen , Kraaij Robert , Uitterlinden Andre G , Sijbrands Eric J , van den Meiracker Anton H
Published: July 2021

A rare Mendelian form of hypertension is Pseudohypoaldosteronism type II (PHAII), also referred to as familial hyperkalemic hypertension (FHHt, OMIM
145260
). Mutations in
WNK1
and
WNK4
, and the two recently identified genes
KLHL3
and
CUL3
have been reported as causes of this monogenic form of arterial hypertension. Mutations in
WNK1
and
WNK4
result in over-activity of the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule. In a Turkish family (parents and 5 children) one daughter was suspected of PHAII. This daughter has been diagnosed at age 24 years with severe hypertension (200/130 mm Hg), hyperkalemia (6.5 mmol/l; reference range 3.5-5.1), and normal renal function. Besides a short stature, no other phenotypical abnormalities were identified. The patient’s hypertension and hyperkalemia completely normalized with hydrochlorothiazide, 50 mg daily. All other family members are healthy. Sanger sequencing of
WNK1
and
WNK4
did not reveal any mutation. A
de novo
mutation in the daughter is the most likely cause of her disease. Full exome sequencing (Nimblegen SeqCapEZV2, Illumina HiSeq2000) of the seven family members identified 34,733 unique variants in our patient. By comparing the recent mutations reported by Boyden et al, we found an identical
de novo
mutation in
CUL3
, a splice acceptor site mutation in intron 8. RT-PCR analysis of RNA extracted from fresh blood showed two cDNA species in the patient, corresponding to a normal sized
CUL3
fragment and an abrogated variant. Sanger sequencing of this shorter form revealed a loss of all nucleotides of exon 9 excluding the first nucleotide and including the first nucleotide of exon 10. This results in an in-frame 57 amino acid deletion abrogating the function of CUL3. After 1 week withdrawal from diuretic medication, we found in the patient’s urinary exosomes, vesicles derived from renal epithelial cells, an increase in the abundance of phosphorylated (P)-NCC (active form) compared to urinary exosomes extracted from urine when using thiazide diuretics. This
de novo
splice acceptor site mutation found in a second patient with PHAII confirmed
CUL3
as a cause of PHAII. This patient also showed up-regulation of P-NCC as reported in
WNK1
and
WNK4
mutations pointing to the same mechanism of FHHt.
Abstract 356: A Role for Renal Proximal Tubule Sodium Bicarbonate Cotransporter SLC4A5 in Salt-Sensitivity of Blood Pressure
Authors: Carlson Julia M , Gildea John J , McGrath Helen E , Felder Robin A
Published: July 2021

SLC4A5 is a sodium-bicarbonate co-transporter involved with sodium homeostasis. Based on unpublished data, two SLC4A5 single nucleotide polymorphisms (SNPs rs1017783 and rs7571842) have been highly associated with an individual’s salt-sensitivity status. Since the renal proximal tubule (RPT) regulates a large percentage of renal sodium transport, we investigated whether SLC4A5 was present in this nephron segment. Using confocal immunofluorescence microscopy, we found expression of SLC4A5 in human RPT cell plasma membrane and intracellular membrane vesicles. We then examined the physiologic implications of the SLC4A5 SNPs in human RPT cells. Using immunoblotting and RT-PCR, we found no significant differences in basal SLC4A5 expression in RPT cells between individuals that are homozygous variant at both SNPs and individuals that are wild-type (WT) for both alleles. Stimulation of the dopaminergic system with 1μM fenoldopam, or the renin-angiotensin system with 10 nM angiotensin II or 10 nM angiotensin III (n=18 per treatment) over 3 and 24 hours did not significantly alter SLC4A5 protein or 24 hour mRNA expression. These data indicate that SLC4A5 is not directly regulated by either the renal dopaminergic or renin-angiotensin system. However, 24 hour stimulation with the sodium ionophore monensin (MON, 1μM) significantly increased overall mRNA expression of SLC4A5 by 182±0.098% over vehicle (VEH) (ΔCq VEH=0.283±0.035; n=18, p<0.001). There was also a significant increase in SLC4A5 mRNA in three cell lines homozygous variant for both alleles compared to three WT cell lines following MON treatment at both 3 hours (138±0.10%; ΔCq WT MON = 0.5±0.052; n=9, p<0.05) and 24 hours (161±0.11%; ΔCq WT MON = 0.39±0.066; n=9, p<0.02). Three but not 24 hour stimulation with MON also significantly increased overall expression of SLC4A5 protein (137±0.00041%; RFU VEH=0.0030±0.00022; n=18, p<0.01). MON, by allowing salt to enter a cell, may be activating an enhancer that leads to increased transcription of SLC4A5 mRNA that is more effective in homozygous variant cell lines. These novel observations demonstrate that SNPs located in a non-promoter DNA intron are associated with enhanced promoter activity that is regulated by altered intracellular sodium.
Abstract 375: Knockdown of (Pro)renin Receptor and Wnt3a Improves Diabetic Albuminuria Via (Pro)renin Receptor-TGFβ1-Wnt3a Signaling Pathways
Authors: Huang Jiqian , Pitkin William B , Siragy Helmy M
Published: July 2021

Recently we demonstrated involvement of (pro)renin receptor (PRR) in renal inflammation of diabetic rats. We hypothesized that in presence of hyperglycemia, PRR upregulates renal Wnt3a and the extracellular matrix component fibronectin (FN) via TGFβ1 signaling pathway, leading to development of albuminuria. Rats underwent unilateral nephrectomy of right kidneys. Diabetes (DM) was induced by IP streptozocin 65 mg/kg. Lentiviral particles constructed with shRNA of PRR or Wnt3a were injected under the capsule of left kidneys of diabetic rats (n=6 each group) respectively. Control shRNA lentiviral particles were also delivered to the kidneys of normal (n=6) and diabetic (n=6) rats. Studies were conducted for 4 weeks. At the end of study, we monitored renal expressions of PRR, TGFβ1, Wnt3a and FN by quantitative RT-PCR and Western blotting, and 24h urine albumin to creatinine ratio (UACR). Compared to control, DM rats had increases in renal mRNA and protein expressions of PRR by 65% and 40% (p<0.01), TGFβ1 by 57% and 46% (p<0.05) and Wnt3a by 205% and 50% (p<0.01) respectively. Renal mRNAs and proteins of PRR and Wnt3a, and UACR in diabetic rats were significantly reduced by corresponding PRR- or Wnt3a-shRNA (20.14% and 46.77%, p<0.001; 37.80% and 24.09%, p<0.01; 40.00% and 55.29%, p<0.01) respectively. There were no changes in cardiac or hepatic expressions of PRR and Wnt3a in response to shRNAs treatments. PRR shRNA significantly reduced TGFβ1 and Wnt3a mRNA and protein expressions (67% and 30%, p<0.01 and 24.61% and 66.83%, p<0.01) respectively. In DM rats, TGFβI receptor inhibition by SD208 decreased Wnt3a mRNA and protein expression (46% and 37%, p<0.01). FN mRNA expression increased by 79% (p<0.01) in the kidneys of DM rats. The increase in FN mRNA expression was attenuated by PRR shRNA and TGFβ receptor blockade by 73% (p<0.01) and 97% (p<0.01) in DM rats. Both PRR and Wnt3a shRNAs attenuated the DM-induced glomerular hypertrophy. We conclude that in hyperglycemic state, PRR upregulates renal Wnt3a and FN via TGFβ1 signaling pathway. Blockade of PRR-Wnt3a signaling pathway improves diabetic albuminuria.
Abstract 385: TRPA1 Mediates Angiotensinogen Augmentation in Renal Proximal Tubular Cells
Authors: Satou Ryousuke , Zsombok Andrea , Derbenev Andrei V
Published: July 2021

Overactivity of the intrarenal renin-angiotensin system contributes to the development of hypertension and renal injury. In this process, regulation of angiotensinogen (AGT) in renal proximal tubular cells (RPTC) is a key factor. Oxidative stress stimulates AGT expression accompanied by activation of p38 MAPK in RPTC. Transient receptor potential A1 (TRPA1) is known to be a sensor activated by a variety of noxious stimuli including hydrogen peroxide. Activated TRPA1 induces calcium influx across the plasma membrane leading to activation of AGT-inducible signal transducers. However, the presence and function of TRPA1 in the kidney have not been delineated. Therefore, this study was performed to demonstrate expression of intrarenal TRPA1 and its role in AGT augmentation in RPTC. Expression of TRPA1 in mouse kidney and cultured mouse RPTC were determined by RT-PCR, in situ hybridization, western blot analysis and immunocytochemistry. The RPTC were treated with 100 μM H2O2 for 1 hr with or without 10μM HC030031, a TRPA1 specific antagonist. EGTA was used to the culture medium for depletion of extracellular calcium. Thereafter, AGT expression levels were evaluated by real-time RT-PCR, and TRPA1 expression levels and p38 MAPK activity were determined by western blot analyses. TRPA1 expression was observed in tubules of renal cortex, and the cultured PRTC expressed TRPA1 mRNA and protein. H2O2 increased AGT expression (1.54 ± 0.08, ratio to control) in RPTC. The AGT augmentation was suppressed by HC030031 (1.19 ± 0.05, ratio to control). Calcium depletion also resulted in attenuation of the AGT augmentation induced by H2O2. Although H2O2 induced phosphorylation of p38 MAPK, HC030031 did not inhibit the p38 MAPK activation. TRPA1 expression was increased by H2O2 under the experimental condition (1.90 ± 0.06, ratio to control). These results suggest that TRPA1 is expressed in RPTC, which contributes to H2O2-induced AGT augmentation via calcium-dependent but p38 MAPK-independent pathways. Furthermore, increase in TRPA1 expression by H2O2 likely facilitates the stimulation of AGT expression. Therefore, TRPA1 may play an important role in the progression of oxidative stress-associated renal injury and hypertension via the stimulation of AGT expression in RPTC.
Abstract 387: Individual Biochemical Characterization of the Renin-Angiotensin-System by RAS-Fingerprinting in Healthy Volunteers Treated with RAS-Blockers
Authors: Poglitsch Marko , Schwager Cornelia , Domenig Oliver , van Oyen Dunja , Schuster Manfred , Loibner Hans
Published: July 2021

The Renin-Angiotensin-System (RAS) is critically involved in the regulation of important physiological functions including blood pressure and fluid balance. It is constituted by multiple enzymes giving rise to a meshwork of effector peptides, which mediate their functions through binding to specific receptor molecules. Therefore, the modification of angiotensin peptide levels represents a common strategy for the treatment of RAS-associated diseases and is frequently achieved by the pharmacologic regulation of enzymes taking part in angiotensin metabolism. We developed a highly sensitive method, which allows the simultaneous absolute quantification of up to 10 different angiotensin peptides in human plasma and whole blood (RAS-Fingerprinting). The measurement of RAS-Fingerprints provides unique insights into the physiology of the human RAS and therefore represents a powerful tool for the patient specific evaluation of this physiologically important peptide hormone system. Beside the precise quantification of angiotensin peptides in plasma and whole blood, RAS-Fingerprints analyze the biochemical hardware of the RAS at a previously unachieved level of detail and accuracy. During the development and validation of our LC-MS/MS based method, comprehensive datasets showing multiple applications for RAS-Fingerprinting have been generated. Investigation of healthy volunteers revealed that a patient specific state of the RAS exists, which is defined by a unique composition of RAS enzyme activities that might have crucial effects on the outcome of RAS targeted therapies in individual patients. RAS-Fingerprinting in healthy volunteers treated with an Angiotensin-Receptor-Blockers, ACE-Inhibitor or Renin-Inhibitor revealed that the human RAS reacts to different drugs in a very dynamic and specific way. Therefore, RAS-Fingerprinting could contribute to the development of innovative therapeutic approaches affecting the RAS. The implementation of RAS-Fingerprinting into clinical studies would substantially enhance our understanding of the human RAS and could lead to the development of personalized treatment schemes in the management of RAS-associated diseases in the near future.
Abstract 42: AT1 Angiotensin Receptors in Principal Cells of the Collecting Duct Have Minimal Impact on Blood Pressure and the Development of Hypertension.
Authors: Chen Daian , Stegbauer Johannes , Sparks Matthew A. , Kohan Donald , Gurley Susan B. , Coffman Thomas M.
Published: July 2021

The main actions of the renin angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT
1
R). The major murine AT
1
R isoform, AT
1A
is highly expressed in epithelial cells throughout the nephron, including the collecting duct (CD). The CD consists of two cell types, principal (PC) and intercalated cells (IC) with distinct functions. Activation of AT
1
R in PC stimulates solute reabsorption in the CD by increasing the activity of epithelial sodium channels. To examine the role of the AT
1A
R in the PC to BP regulation and the development of hypertension
in vivo
, we generated inbred 129SvEv mice with cell-specific deletion of AT
1A
R in PC (PC-KO, n=6).
AQP2-Cre
transgenic mouse line was used to excise the floxed
Agtr1a
allele in PC. mTmG reporter mouse line was used to confirm specific expression of
AQP2-Cre
transgene in PC, and found the pattern of expression to be predominantly in medullary CD. Using RT-PCR, mRNA levels for AT
1A
R were reduced by ≈50% in the inner medulla of PC-KO mice (
P
<0.05), but unaffected in cortex. Baseline BP measured by radiotelemetry was similar between PC-KO (122±1.6 mm Hg) and controls (122±1.6 mm Hg). During one week of feeding a low salt (<0.002% NaCl) diet, MAP fell significantly (P<0.05) and to a similar extent in both groups, (PC-KO: 116±1 mm Hg; Controls: 117±2 mm Hg). High salt (6% NaCl) diet increased BP (P<0.01), but was not different between groups (PC-KO: 129±2 mm Hg; Controls: 131±2 mm Hg). Finally, we induced hypertension with chronic infusion of Ang II (1000 ng/kg/min) by osmotic mini-pumps. During the initial phase (days 1-8), there was a modest but significant attenuation of hypertension in PC-KO (167±7 mm Hg) compared to controls (178±3 mm Hg, P<0.001). However, by day 9 levels of BP were indistinguishable between groups and there was no difference in BP in the later phase (days 9-14) of hypertension (PC-KO: 175±12 mm Hg; Controls: 177±7 mm Hg). Heart to body weight ratio after Ang II infusion was not different between groups. In summary, AT
1A
R in PC of the medullary CD have little influence on BP, adaptation to changes in dietary salt intake or development of hypertension. We suggest that other mediators, such as aldosterone, may have a more important role to influence sodium handling in this nephron segment.