bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 043: A Gwas Prioritized Candidate Gene,Nr2f2, Attenuates Salt-sensitive Hypertension and Associated Renal Function
Authors: Kumarasamy Sivarajan , Waghulde Harshal , Mell Blair , Joe Bina
Published: March 2022

A haplotype-based re-analysis of the GWAS study by the Wellcome Trust Case Control consortium prioritized the transcription factor, Nuclear receptor2, family2 (NR2F2) also known as Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) as a gene associated with essential hypertension in humans. This gene is also prioritized as a differentially expressed positional BP QTL candidate gene on rat chromosome 1. To further examine Nr2f2 as a determinant of blood pressure, a mutant Dahl salt-sensitive (S) rat model was generated using the zinc-finger nuclease (ZFN) technology. As a result of this ZFN mediated disruption, a 15-bp deletion occurred within exon 2 of the Nr2f2 locus, resulting in the loss of 5 amino acids from the hinge region of the Nr2f2 protein. Systolic BP of the homozygous Nr2f2mutant rats was lower than that of the S rats (179±3 vs. 197±5 mmHg; p<0.001) as measured by the tail-cuff method and (165±2 vs. 201±3 mmHg; p<0.001) as monitored by the radio-telemetry method. Nr2f2mutant rats demonstrated lower proteinuria compared to S rats (87.97±7.07 vs. 122.63±7.07mg/day; p<0.05). Since Nr2f2 is reported to negatively regulate renin promoter activity, we tested renin activity in the Nr2f2mutant rats. However, there was no significant difference in either the plasma renin activity or renal renin protein expression when compared with S rats. Renal histological analysis showed that Nr2f2mutant rats have decreased collagen compared with the S rats. Furthermore, Nr2f2mutant rats showed significantly decreased TGF-β protein levels, when compared with S rat kidney (p<0.05). These data, taken together with our recently published data on the improved cardiac and vascular function in the Nr2f2mutant rats (Nat Commun. 2015 Feb 17;6:6252), lend support to Nr2f2 as a determinant of blood pressure and associated renal function. While the observed improvement in renal function could be a secondary consequence of lower hypertension, our data, combined with the important mechanistic finding that the enhanced transcription factor-transcription factor interaction of Nr2f2 with Fog2 (Friend of GATA4), suggest that the hinge region of Nr2f2 is important for regulating blood pressure and associated cardiac and renal functions.
Abstract 046: Deep Sequencing in mpkCCD Collecting Duct Cells Identifies Transcriptional Response to Vasopressin
Authors: Sandoval Pablo , Lee Jae Wook , Claxton J'Neka , Hoffert Jason D , Knepper Mark A
Published: March 2022

Blood pressure regulation depends in part on control of ion and water transport in the renal collecting duct by vasopressin and other hormones. To identify genes that are transcriptionally regulated by vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq (n=9) and ChIP-seq for RNA polymerase II (Pol-II, n=3) after 24-hr treatment with vasopressin analog dDAVP or vehicle (Illumina HiSeq2000 sequencer). RNA-seq analysis showed significant changes in 33 transcripts out of 8393 quantified (Volcano plot thresholds: p<0.05 (Wilcoxon) and log2[ratio] outside of 99% confidence interval (CI) for vehicle-vs-vehicle controls). More transcripts increased (26) than decreased (7). The largest increases were seen for the transcriptional coactivator
Cited1
(log2[dDAVP/Vehicle]= 5.2) and the water channel
Aqp2
(log2[dDAVP/Vehicle]= 4.2). The ChIP-seq analysis showed significantly increased RNA-Pol-II binding in 209 genes versus only 9 with decreases. Plotting RNA-seq data versus ChIP-seq data for 3883 genes revealed 43 genes that exceeded the 95% CI for control-vs-control measurements for both variables, i.e., are transcriptionally regulated. The transcriptionally regulated genes included those involved in control of Na+/Cl- transport and/or blood pressure, viz.
Fxyd4
(aldosterone-regulated channel-inducing factor),
Tsc22d3
(the aldosterone-regulated leucine-zipper protein GILZ),
Atp1b1
(Na+-K+-ATPase beta subunit) and
Sik1
(salt-inducible kinase 1). Among transcriptionally upregulated genes were several transcription factors (
Elf3
,
Nfkbiz
,
Nr4a1
,
Myc
) that form the core of a proposed vasopressin/cAMP-activated transcriptional network. Conclusions: 1) Vasopressin signaling effects on transcription are biased with more genes increased than decreased. 2) Among the genes that are transcriptionally regulated in response to vasopressin are several that have been implicated in regulation of salt and water transport in the collecting duct.
Abstract 049: Preeclampsia in the Dahl Salt Sensitive Rat is Associated with Increased Uterine Artery Resistance and Reduced Placental Microvascular Density
Authors: Gillis Ellen E , Chade Alejandro R , Spradley Frank T , Garrett Michael R , Sasser Jennifer M
Published: March 2022

We showed that the Dahl salt sensitive (Dahl S) rat is a spontaneous model of preeclampsia superimposed on chronic kidney disease and hypertension. This model exhibits intrauterine growth restriction, decreased pup weight, increased fetal death, and placental hypoxia; however, the underlying mechanisms are unknown. We hypothesized that a pathological remodeling and rarefaction in the maternal placental bed drives the development of preeclampsia in this model. Pregnant Dahl S and Sprague-Dawley (SD) rats were used in this study. Uterine artery resistance index (UARI) was calculated via Doppler ultrasound (Vevo 770) on day 18 of pregnancy (UARI=(peak systolic flow velocity - end diastolic flow velocity)/ peak systolic flow velocity). On day 20 of pregnancy, placentas were perfused through the uterine circulation with a silicon-polymer contrast (Microfil MV122). Individual placentas were then excised, scanned using a micro-CT scanner (SkyScan 1076), and 3D reconstructed for analysis and quantification of the placental vasculature on the maternal side. Microvascular density was calculated for vessels of diameters in the 0-500 μm ranges. UARI was higher in the Dahl S compared to the SD (0.71±0.02 vs 0.51±0.02, n=4-12, p<0.05). Density of placental microvessels in the 200-500 μm range was significantly decreased in placentas from Dahl S rats, and we observed a strong trend towards a decrease in density of microvessels in the 0-200 μm range (n=3-5, Figure). These results suggest that the impaired fetal growth and placental hypoxia in the Dahl S rat may be mediated by insufficient placental vascularization and reduced blood flow to the feto-placental unit.
Abstract 059: Loss of AT1 Receptors in the Podocyte Does Not Protect Against Renal Injury
Authors: Johnson Stacy A , White Tiffani , Griffiths Robert , Gurley Susan B , Coffman Thomas
Published: March 2022

Chronic kidney disease (CKD) arising from hypertension or other etiologies often progresses to ESRD despite treatment. One of the mainstays of treatment for CKD is blockade of the renin-angiotensin system (RAS). RAS blockers - angiotensin converting enzyme inhibitors (ACE-Is) and angiotensin receptor blockers (ARBs) - have the unique ability to decrease urine albumin excretion independent of systemic blood pressure. Despite their clinical effectiveness, RAS blockers do not completely stop CKD progression, thus there exists a need for new therapies. The association between proteinuria and an over-active RAS is well described and is mediated by angiotensin II (ang II) via the AT1 angiotensin receptor. This suggests that ang II, via AT1 receptors, promotes proteinuria and that this may be an important pathway in the pathogenesis of CKD. Yet, the mechanism and cellular site of action where ang II causes proteinuria have not been clearly demonstrated. To investigate the role of AT1 in podocytes we used cell-specific gene targeting to generate mice lacking all AT1 receptors in the podocyte (PodKOs). PodKOs were then crossed with mice expressing a renin transgene (RenTg) to generate PodKO-RenTg mice. RenTg mice are hypertensive and provide an effective genetic model in which to study RAS over-activation. We hypothesized that AT1 signaling is a critical mediator of albuminuria in hypertensive kidney disease and that actions of AT1 receptors in the podocyte mediate kidney injury. PodKO mice develop normally and there is no difference in kidney weight between Cre- and Cre+ mice (6.9 ± 0.5 mg/g BW vs 7.0 ± 0.3 mg/g BW). At baseline, there was no significant difference in albumin excretion between 24 week old RenTg and PodKO-RenTg mice (210 ± 70.9 μg vs 228.7 ± 37.6 μg). When the mice were subjected to uninephrectomy to induce CKD, both RenTg and PodKO-RenTg mice developed similar degrees of albuminuria (279.9 ± 105.8 μg vs 366.8 ± 105.9 μg). However, kidneys from PodKO-RenTg mice had higher levels of glomerulosclerosis and mesangial expansion. Taken together, these data suggest that AT1 receptors in podocytes do not promote albuminuria and may have protective effects.
Abstract 074: An Obligatory Role for B Cells in the Development of Angiotensin II-dependent Hypertension
Authors: Chan Christopher , Sobey Chris , Lieu Maggie , Ferens Dorota , Kett Michelle , Diep Henry , Kim Hyun Ah , Krishnan Shalini , Salimova Ekaterina , Vinh Antony , Samuel Chrishan , Tipping Peter , Kyaw Tin Soe , Toh Ban-Hock , Bobik Alexander , Drummond Grant
Published: March 2022

Background:
Clinical hypertension is associated with raised serum antibody levels. However, no studies have examined whether B cells and antibodies play causative roles in the pathogenesis of hypertension. We investigated whether experimental hypertension is similarly associated with elevated IgG production, and if B cell/antibody deficiency affords protection against hypertension and associated vascular remodeling.

Methods and Results:
Ang II-dependent hypertension in mice was associated with B cell activation/maturation as revealed by a: (1) a 25% increase in the proportion of splenic B cells that expressed the activation marker CD86; (2) an 80% increase in numbers of splenic plasma cells, (3) a 500% increase in serum IgG levels; and (4) marked accumulation of IgG deposits in the aortic adventitia. Indicative of a causative role for B cells, B cell activating factor-receptor-deficient (BAFF-R-/-) mice, which lack mature B cells, displayed blunted hypertensive responses to chronic angiotensin II infusion (Δ30±4 mmHg) compared to wild-type (WT) mice (Δ41±5 mmHg). Importantly, adoptive transfer of B cells into BAFF-R-/- mice restored the hypertensive response. BAFF-R-/- mice showed no evidence of Ang II-induced increases in aortic IgG accumulation. They also had 80% fewer F4/80+ macrophages, 60% fewer CD4+ T cells, and 70% reduction in TGF-β expression and collagen in their aortas compared to Ang II-treated wild-type mice. Ang II doubled aortic pulse-wave velocity, a measure of vessel stiffening, whereas BAFF-R-/- mice were largely protected (70% reduced) from this effect. Finally, pharmacological depletion of B cells with an anti-CD20 antibody blunted Ang II-induced hypertension (Δ37±9 mmHg vs Δ57±6 mmHg).

Conclusions:
We provide the first evidence that B cells are activated during development of hypertension and contribute to vessel remodeling and elevated blood pressure. These findings suggest B cells and/or the IgG antibodies they produce could be targeted for anti-hypertensive therapy.
Abstract 091: High Density Adrenal MRI Detects Clinically Relevant Aldosterone-producing Adenomas with Higher Precision than Computer Tomography
Authors: Raber Inbar , Isom Robert , Louie John , Vasanawala Shreyas , Bhalla Vivek
Published: March 2022

Background:
Primary hyperaldosteronism is the most common cause of secondary hypertension, and part of the noninvasive diagnostic studies used to confirm the diagnosis include biochemical and computed tomography (CT) of the adrenal glands. If no adenomas are seen on imaging, invasive adrenal vein sampling, to diagnose unilateral disease amenable to surgery, is limited to cases with high pre-test probability. We developed a dedicated MRI protocol and investigated whether surgically-treatable unilateral aldosterone-producing adenomas missed by CT could be detected using this protocol.

Methods:
We developed a dedicated MRI protocol of the adrenal glands using thin single shot T2-weighted scans, noncontrast MR angiography, reduced field of view diffusion, and dynamic contrast enhancement through the kidneys and adrenal glands with gadolinium (or ferumoxytol if eGFR < 30 mL/min/1.73m2). We then enrolled patients with biochemical evidence of primary hyperaldosteronism without evidence of adrenal lesions by CT and performed MRI.

Results:
Five subjects with primary hyperaldosteronism and negative CT underwent MRI. Each had adrenal nodules by MRI. In three cases, a single, unilateral adrenal nodule was detected and followed up with ipsilateral lateralization of aldosterone/cortisol production by adrenal vein sampling. These patients underwent laparoscopic adrenalectomy with histologic diagnosis of an aldosterone-producing adenoma with significant improvement in blood pressure and/or reduction in antihypertensive medications. In the other two cases, adrenal nodules were found but adrenal vein sampling detected no lateralization of aldosterone/cortisol ratio.

Conclusions:
As proof of concept, MRI of the adrenal glands may be a superior imaging modality to diagnose subtypes of primary hyperaldosteronism. With identification of adrenal lesions, this may capture additional cases of secondary hypertension than are amenable to surgical cure improving outcomes and quality of life for patients with severe hypertension. Moreover, it may imply that some cases of primary hyperaldosteronism ascribed to bilateral adrenal hyperplasia may indeed be due to aldosterone-producing adenomas undetectable by CT.
Abstract 094: Gain-of-function EPHX2 Variant rs41507953 Is Associated With Acute Kidney Injury Following Cardiac Surgery
Authors: Shuey Megan M , Billings Frederic T , Wei Shouzuo , Milne Ginger L , Brown Nancy J
Published: March 2022

Acute kidney injury (AKI) affects 20-30% of patients following cardiac surgery and predicts increased mortality. Murine models suggest that epoxyeicosatrienoic acids (EETs) protect against renal ischemia/reperfusion injury, a contributor to AKI following cardiac surgery. Soluble epoxide hydrolase (sEH), encoded by
EPHX2,
hydrolyzes EETs to less active DHETs.

We tested the hypothesis that a gain-of-function
EPHX2
variant, rs41507953, is associated with AKI following cardiac surgery.

We first studied 371 cardiac surgery patients (67.3% male, mean age 65.6±12.9years and BMI 28.5±6.17kg/m
2
) enrolled in a clinical trial in which DNA was collected. Ninety-eight patients (26.4%) developed AKI, defined by AKIN criteria. Rs41507953 genotypes were in Hardy-Weinberg equilibrium (AA:AG:GG=297:68:6). There was a significant association between the gain-of-function “G” allele and AKI, p=0.006. Adjusting for known risk factors for AKI including estimated glomerular filtration rate (eGFR), age, sex, race, history of diabetes, BMI, and use of cardiopulmonary bypass the rs41507953 “G” allele remained independently associated with higher rates of AKI [OR=2.09 (95%CI 1.132-3.848, p=0.018). We replicated this association between the rs41507953 “G” allele and AKI in another cohort of 800 cardiac surgery patients.

To assess the association between the rs41507953 “G” allele and sEH activity we measured 9,10 or 12,13-dihydroxyoctadecanoic acid/9,10 or 12,13-epoxyoctadecanoic acid (DiHOME/EpOME) ratios, measures of sEH activity, in plasma collected from 26 AA and 5 AG individuals in the original cohort. sEH activity was highest in plasma collected post-protamine administration. The 12,13 DiHOME/EpOME and the total DiHOME/EpOME ratios post-protamine were significantly greater in the AG vs. the AA genotype group, 11.66±12.91 vs. 2.32±3.03 (p=0.002) and 28.35±47.79 vs. 4.03±7.96 (p=0.014), respectively.

An
EPHX2
variant with increased sEH activity is associated with AKI following cardiac surgery. Pharmacologic inhibition of the sEH enzyme might protect patients from AKI following cardiac surgery.
Abstract 102: Renal Mechanisms of Blood Pressure Homeostasis and Salt Sensitivity by Collectrin
Authors: Chu Pei-Lun , Gigliotti Joseph C , Cechova Sylvia , Chan Fang , Ralph Donna L , Franceschini Nora , McDonough Alicia A , Le Thu H
Published: March 2022

Collectrin (
Tmem27
), an ACE-2 homologue and a chaperone of amino acid transporters, is expressed in the endothelium and renal epithelia, including the proximal tubules and collecting duct. We previously reported that collectrin-deficient (KO) mice exhibit hypertension (HTN) at baseline and augmented salt-sensitivity associated with impaired endothelial-dependent relaxation and a rightward shift of the pressure-natriuresis relationship. To determine the effect of renal vs. extra-renal collectrin on blood pressure (BP) regulation, we performed renal cross-transplantation studies (at the Duke O’Brien Center), then treated with normal salt (NSD) and high salt (HSD) diets, in the following groups (n = 3-5/group): 1) wild-type (WT) mice with WT kidney (WT WT), 2) collectrin KO mice with WT kidney (WT KO), and 3) WT mice with collectrin KO kidney (KO WT). Collectrin KO WT group displayed a trend towards higher baseline systolic BP (SBP, mmHg); however, under HSD, collectrin KO WT had significantly higher SBP (WT WT 148.9, WT KO 151.6, KO WT 168.6, p = 0.037).

To determine the renal endothelial and epithelial contribution of collectrin in BP regulation, we assessed renal blood flow (RBF) by contrast-enhanced ultrasound and renal sodium transporters and channels by immunoblotting. KO mice displayed significantly reduced total, cortical and medullary RBF under both NSD and HSD (n ≥ 11 each, p ≤ 0.01). Moreover, at baseline, compared to WT mice (n = 4), KO mice (n = 5) displayed significantly elevated abundance of NHE3 (p = 0.002), activated NCC (NCC-pS71) (p = 0.004) and ENaC alpha (p = 0.03).

We next conducted a candidate gene association study in the HyperGen cohort (1,270 white participants, 50% men), using intronic SNPs in the
ACE2
,
TMEM27
and
CA5BP1
genes on the X chromosome. Only 2 SNPs, rs6629114 and rs41492646, both of
TMEM27
and in perfect linkage disequilibrium (r
2
=1.0), were associated with diastolic BP (DBP). Meta-analysis of men and women showed this was statistically significant, (p=0.04 for both), with stronger effects in men (p=0.028).

In conclusion, deletion of collectrin alters renal hemodynamics and epithelial sodium handling to favor sodium reabsorption. Collectrin is associated with DBP in human subjects.
Abstract 104: Targeting the Alms1 Gene in Rats Causes Hypertension, Obesity and Enhanced NKCC2 Trafficking
Authors: Jaykumar Ankita Bachhawat , Caceres Paulo , Henson Emily , Ares Gustavo , Ortiz Pablo
Published: March 2022

Enhanced NaCl reabsorption by the thick ascending limb (TAL) is associated with salt sensitive hypertension in rodents and humans. NaCl absorption by the TAL depends on the apical Na/K/2Cl cotransporter - NKCC2. NKCC2 activity is regulated in part by protein-protein interactions with its carboxyl terminus that controls its trafficking to the apical membrane. We hypothesized that the proteins binding to this region of NKCC2 may be involved in NKCC2 regulation and trafficking. To identify new TAL proteins that bind NKCC2, we performed a proteomics screen between the NKCC2 carboxyl terminus and TAL proteins. We identified ALMS1 (Alstrom syndrome 1) as a specific interacting partner for this NKCC2 region and confirmed that ALMS1 is expressed in TALs. Little is known about ALMS1 in renal function. Mutation of this gene causes severe metabolic syndrome in humans and is associated with hypertension. To study ALMS1 function we obtained ALMS1 knockout (KO) rats in collaboration with the rat genome editing consortium. We found that ALMS1 KO rats fed a normal salt diet have higher systolic blood pressure (151± 5 mmHg) compared to wild type littermates (125± 4 mmHg, p< 0.02). We also observed an increase in body weight in ALMS1 KO at all ages (WT: 344± 4 g vs KO: 429±11 g, at 11 weeks, p<0.05). ALMS1 KO rats showed large intra-abdominal and sub-cutaneous fat deposits. We then obtained TALs and measured surface and total NKCC2 expression. In TALs from ALMS1 KO, the percentage of total NKCC2 at the surface was higher compared to WT (27±4 vs 14±2 %, p<0.05, n=6). Total NKCC2 expression was not significantly different between ALMS1 KO and WT rats. We conclude that ALMS1 is important for blood pressure regulation. The mechanism for hypertension in ALMS1 KO is unclear but may involve an increase in NKCC2 trafficking to the apical membrane and activity. ALMS1 has been associated with poor kidney function, hypertension, and type 2 diabetes in humans. Understanding the function of ALMS1 could develop new avenues for research into these diseases.
Abstract 121: Gstm1 Deletion Exaggerates Hypertension, Oxidative Stress, and Kidney Pathology in Angiotensin II Hypertension Independent of Nox2 and Nox4
Authors: Cechova Sylvia , Chan Fang , Ruiz Phillip , Le Thu H
Published: March 2022

Gstm1 gene encodes an enzyme that belongs to a superfamily of Glutathione-S-transferases that metabolize a broad range of reactive oxygen species. In the African American Study of Kidney Disease Trial (AASK), we reported that participants with decreased expression of GSTM1 as carriers of the GSTM1 null allele have accelerated kidney disease progression. To establish cause and effect, we generated Gstm1-/- (KO) mice and determined their response in angiotensin II (AngII)-induced hypertension (at 1000 ng/kg/min for 4 weeks). By radiotelemetry, Gstm1 KO mice (n=6) displayed a modest but significantly higher baseline systolic blood pressure (SBP, mmHg) compared to their wild type (WT) littermates (n=6): KO 135.8 ± 1.4; WT: 129.9 ± 1.2; P = 0.01. AngII augmented the difference in SBP between WT and KO mice (KO 175.4 ± 6.2; WT 158.0 ± 2.6, p = 0.04). There were no differences in albumin/creatinine ratio (μg/mg) either at baseline (KO 29.1± 4.1; WT 27.3 ± 2.9) or after 4 weeks of Ang II (KO 4657 ± 631.6; WT 4697.7 ± 533.8). By quantitative real-time PCR, the renal expressions of Nox2 and Nox4 genes were almost identical and not statistically different between WT and KO mice. However, by luminescence, Gstm1 KO mice had significantly increased levels of superoxide radicals: [counts/min/1 mg of dry tissue] KO 128 ± 12 vs. WT 42 ± 8.5; P < 0.0001. Furthermore, there was significantly worse kidney pathology in Gstm1 KO mice (Figure 1), including focal segmental glomerulosclerosis (FSGS), fibrosis, chronic inflammation and epithelial cell reactivity. In conclusion, loss of GSTM1 enzyme increases Ang II induced hypertension, oxidative stress and kidney injury independent of Nox2 or Nox4.
Abstract 126: Systemic Administration of (Pyr1)-Apelin-13 at Late Pregnancy Reduces Blood Pressure, Proteinuria, and Improves Autonomic Function in Preeclamptic Rats
Authors: Yamaleyeva Liliya M , Brosnihan K. Bridget , McGee Carolynne , Shi Sara , Varagic Jasmina , Bader Michael , Dechend Ralf , Shaltout Hossam A
Published: March 2022

Preeclampsia is associated with maternal perinatal morbidity and mortality and a high risk of premature birth and intrauterine growth restriction. The apelin system is a novel pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. We have previously reported that total apelin content is lower in human preeclamptic chorionic villi. In this study, we determined whether (Pyr1)-apelin-13 improves hypertension, proteinuria, fetal characteristics, uteroplacental hemodynamics, and the autonomic function in preeclamptic rats (TgA, female transgenic for human angiotensinogen mated to male transgenic for human renin). (Pyr1)-apelin-13 (2 mg/kg/day) (n=7) or saline (n=5) was infused in TgA via osmotic minipumps starting at day 13 of gestation, when blood pressure begins to increase in these animals. Pregnant SD (n=6) rats were used as controls. At the 20th day of pregnancy, TgA rats had higher MAP (138±6 vs. 79±3 mmHg in SD, p<0.001) which was reduced by (Pyr1)-apelin treatment to 119±2 mmHg vs. TgA, p<0.006. TgA rats also had impaired heart rate variability measured as root of mean successive differences (rMSSD) compared with SD (2.7±0.4 vs. 3.8±0.3 ms in SD, p<0.05). Apelin treatment normalized rMSSD to 3.6±0.3, p<0.05. Similarly, baroreflex sensitivity measured in the sequence domain was lower in TgA (0.7±0.1 vs. 2.6±0.5 ms/mmHg in SD, p<0.01) and normalized with (Pyr1)-apelin-13 to 2.0 ± 0.4 ms/mmHg, p<0.05. Proteinuria was greater in TgA (53±9 vs. 10±2 mg/kg/day, p<0.001), and normalized by (Pyr1)-apelin-13 (18±6, p<0.05). Pup (3.0±0.1 vs. 3.7±0.1 g, p<0.01) and placental weight (0.41±0.01 vs. 0.45±0.01 g, p<0.01), and pup number (10.7±1.1 vs. 14.0±0.8, p<0.01) were lower in TgA vs. SD; however they were not changed by (Pyr1)-apelin-13. Uterine artery peak systolic velocity was not different between SD and TgA, but increased with (Pyr1)-apelin-13 treatment (179.5±16.7 vs. 122.6±16.7 ml/min, p<0.05) with no change in resistance index. In conclusion, our findings suggest that (Pyr1)-apelin-13 may be beneficial for the treatment of preeclampsia due to its hemodynamic and renoprotective effects. We also report for the first time that these changes may involve central control of the cardiovascular system.
Abstract 128: Role of Hypoxia-dependent Autophagy in the Trophoblast of BPH/5, a Mouse Model of Pre-eclampsia (PE)
Authors: Valbuena-Diez Ana Cristina , Sones Jenny , Butler Scott , Davisson Robin
Published: March 2022

Autophagy, a mechanism of cell survival, plays an important role in normal placentation and protects the fetus during stress. Previously, we showed that autophagy was increased in fetoplacental units (FPUs) from pregnant BPH/5 mice, a model that develops a PE-like syndrome including late-gestational hypertension, proteinuria, abnormal placentation and poor feto-placental outcomes. We also showed that increased autophagy led to reduced invasion capacity of BPH/5 trophoblasts. Evidence suggests that hypoxia increase autophagy, so we measured the invasion capacity of trophoblasts isolated from C57 mice in normoxic/hypoxic conditions. Trophoblasts cultured in hypoxia exhibited defective invasion, effect partially blocked by autophagy inhibitor chloroquine diphosphate (CQ; 1.7-fold decrease, n=4 p<0.005 hypoxia vs. normoxia; 1.35-fold increase p<0.5 CQ vs. hypoxia). We also tested the effect of conditioned media from BPH/5 or C57 placental explants on the invasion capacity of JAR cells, a human choriocarcinoma trophoblast cell line. Conditioned media from BPH5 explants decreased JAR invasion, but only for 24h after placental collection (1.8-fold decrease, n=4 p<0.005 vs. C571.33-fold p<0.05 vs. BPH/5). However, when the explants were kept in hypoxia, JAR invasion capacity decreased following treatment with BPH5 explants conditioned media, even after 48h; effect partially inhibited by CQ (1.6-fold decrease, n=4 p<0.005 vs. CTR; 1.7-fold, n=4 p<0.05 vs. BPH/5). As autophagy plays a key role in the invasion of trophoblasts and thus remodeling of uterine arteries, we performed a tube formation assay with endothelial cells treated with conditioned media from BPH/5 or C57 placental explants. Tube formation decreased after treatment with conditioned media from BPH/5 explants (1.7-fold decrease, n=3 p<0.05). Co-culture of endothelial and trophoblast cells under hypoxia resulted in less efficient tube formation. Treatment with CQ partially blocked this effect (1.55-fold decrease, n=3 p<0.05 vs normoxia; 1.2-fold, n=3 p<0.05 vs hypoxia). These data demonstrate that invasion capacity and tube formation are defective in trophoblasts from BPH/5 mice, suggesting that hypoxia-dependent autophagy may play a causal role in PE in the BPH/5 model.
Abstract 131: Endothelial Cell Mineralocorticoid Receptor: A Critical Player in Aortic Stiffness and Cardiac Diastolic Dysfunction in Female Mice
Authors: Jia Guanghong , Habibi Javad , Aroor Annayya R. , Sun Zhe , Ma Lixin , DeMarco Vincent G. , Martinez-Lemus Luis A , Meininger Gerald A , Jaffe Iris Z , Sowers James R
Published: March 2022

Enhanced activation of mineralocorticoid receptors (MRs) impairs insulin metabolic signaling, increases oxidative stress, and induces maladaptive immune responses with associated CV abnormalities. Emerging information suggests that obesity and insulin resistance predict CV stiffness in females. However, the specific role of endothelial cell MR (ECMR) has not been explored. Accordingly, we hypothesized that ECMR signaling modulated by a western diet (WD) impairs insulin signaling and increases inflammation, fibrosis and CV stiffness in females. Four week-old ECMR knockout (ECMR
-/-
) and wild-type female mice were fed a mouse chow or WD containing fat (46%), sucrose (17.5%), and high fructose (17.5%) for 16 weeks. WD prompted MR to bind the hormone response element (nGnACAnnnTGTnCn) on the site of ENaC promoter and induced an increase in ENaC expression that was associated with increased aortic and EC stiffness as determined by in vivo pulse wave velocity and ex vivo atomic force microscopy techniques, respectively The elevated aortic stiffness was accompanied by increased expression of cytokines IL-17, MCP-1 and M1 markers CD 86, and CD11c. ENaC expression was reduced in the ECMR
-/-
vasculature with a decrease in WD-increase in aortic and EC stiffness.. ECMR
-/-
also improved aortic vasorelaxation to Ach, SNP (10
-9
-10
-4
mol/L), and insulin (0.1- 300 ng/ml), which were impaired by WD. Additionally, ECMR
-/-
restored WD-induced cardiac diastolic dysfunction assessed by cardiac MRI and echocardiography. Diastolic dysfunction was related to cardiomyocyte hypertrophy, oxidative stress, and fibrosis and occurred with enhanced activation of S6 kinase-1, Erk 1/2, serine phosphorylation of IRS-1, inactivation of PI3K-AKT-eNOS signaling pathways and the pro-fibrotic TGF-β1/ Smad signaling pathway and increased macrophage pro-inflammatory polarization. ECMR
-/-
markedly attenuated the cardiac functional and changes signaling induced by WD. These findings suggest that increased ECMR signaling and associated ENaC activation plays a key role in WD induced insulin metabolic sinaling impairment, adaptive pro-inflammatory responses, macrophage polarization and associated aortic stiffness and cardiac diastolic dysfunction in females.
Abstract 135: Activated Intrarenal Renin Angiotensin System Contributes to the Development of Chronic Kidney Disease in Primary Aldosteronism
Authors: Iwakura Yoshitsugu , Morimoto Ryo , Kikuchi Kumi , Ono Yoshikiyo , Kudo Masataka , Ito Sadayoshi , Satoh Fumitoshi
Published: March 2022

Background:
Patients with primary aldosteronism (PA) have higher prevalence of albuminuria than those with essential hypertension (EH). We have shown that about 15% in PA patients had chronic kidney disease (CKD). Hence, the active screening of PA is important to prevent CKD. Urinary angiotensinogen (UAGT) has been reported as the earliest predictor of nephropathy in patients with type 1 diabetes (DM). UAGT indicates the intrarenal renin angiotensin systems (RAS) activation which plays an important role in the development of CKD. This study aimed to confirm the existence of activated intrarenal RAS by measuring UAGT in patients with PA.

Methods:
Consecutive 58 patients of aldosteronoma (APA) participated. No patients had CKD defined as eGFR <60 ml/min/1.73m
2
or urinary albumin (UA) ≥30 mg/g cre and DM defined as HbA1c ≥6.5% or taking treatments. As control subjects, 59 EH patients were included. All APA patients were treated by sufficient dose of mineralocorticoid receptor (MR) antagonists (MRA) until the day of adrenalectomy. Urine samples were collected on 10g salt diet between baseline and after MRA treatment. UAGT (ng/g cre) was measured and the value was transformed by logarithm.

Results:
Age, sex distribution, blood pressure, duration of hypertension, eGFR, and UA were similar between two groups at baseline. However, UAGT was significantly higher in APA than in EH (Fig). In multivariate analysis, only plasma aldosterone was significantly related with UAGT in APA (t=3.572, p=0.0007). UAGT was significantly decreased after MRA treatment (Fig).

Conclusion:
Intrarenal RAS is activated by aldosterone/MR in patients of PA, which might contribute to the development of CKD.
Abstract 136: Mineralocorticoid Receptor Activation In Macrophages Mediates High Fat/high Sucrose Induced Vascular Stiffness in Female Mice
Authors: Manrique Camila , Sowers James , Aroor Annayya , Jia Guanghong , Habibi Javad , Mortensen Richard M , Lastra Guido
Published: March 2022

Enhanced mineralocorticoid receptor (MR) activation promotes vascular dysfunction and remodeling. We studied the role of MR activation in perivascular adipose tissue (PVAT) macrophages in the development of high-fat/high-fructose diet (“Western Diet”, WD) induced vascular stiffness in female mice.

Myeloid MR KO mice (MyMRKO) were fed a WD for 8 weeks (n=3). MyMRKO female mice (n=2) were infused with aldosterone (Aldo) for 3 weeks. Aortic stiffness was assessed in vivo by pulse wave velocity and ex vivo by atomic force microscopy. Vascular reactivity was studied in aortic rings.

MyMRKO mice were protected from Aldo and WD induced aortic stiffness and had greater endothelial-dependent and independent vasodilation compared to littermates (Fig. 1, 2).

Immunohistochemistry suggested decreased peri-aortic fibrosis and macrophage infiltration in Aldo infused MyMRKO (Fig.1).

We conclude that MR KO in myeloid cells protects against WD and Aldo-induced vascular stiffness in female mice. Our data support a role for MR activation in PVAT macrophages in the pathogenesis of WD-induced vascular dysfunction.
Abstract 139: Renal Afferent Nerve Sensitivity and the Pathophysiology of Salt-sensitive Hypertension
Authors: Wainford Richard D , Walsh Kathryn R
Published: March 2022

Aim:
Altered renal afferent nerve responsiveness contributes to hypertension in the Spontaneously Hypertensive rat. We hypothesized that increased salt-intake differentially impacts the renal afferent nerve sensitivity in salt-resistant vs salt-sensitive rats.

Methods:
Groups of naïve Sprague-Dawley (SD), Dahl Salt-Resistant (DSR), Dahl Salt-Sensitive (DSS) or SD rats receiving a s.c. norepinephrine (NE:600ng/min) infusion were fed a 0.4% (NS) or 8% NaCl (HS) diet for 14 (SD) or 21 days (DSR & DSS). Following HS intake MAP and plasma NE content were determined. Via a renal pelvis preparation afferent nerve activity was assessed as NE-evoked (6250 pmol) substance P (SP) release (N=5/gp).

Results:
Salt-resistant phenotypes (SD & DSR) remain normotensive and exhibit HS-evoked suppression of plasma NE and increased renal afferent nerve release of substance P. In contrast in salt-sensitive phenotypes (DSS and NE infused SD) a high salt diet evoked hypertension, increased plasma NE and abolished sodium evoked increased responsiveness of the renal afferent release of substance P.

Significance symbols:
*p<0.05 vs. resp. NS gp; τ p<0.05 vs. resp. SD Naïve gp. value.

Conclusion:
These data support a role of the afferent renal nerves in blood pressure regulation during high salt-intake. Increased responsiveness of the renal afferent nerves contributes to the maintenance of normotension in a salt-resistant rat phenotype. Conversely, our data suggest that in salt-sensitive phenotypes excess release of NE triggers impaired activation of the renal afferent nerves, a factor we postulate contributes to the development of salt-sensitive hypertension.
Abstract 141: G-Protein Coupled Receptors GPR37 and GPR37L1 Regulate Sodium Reabsorption in Renal Proximal Tubule Cells
Authors: Zheng Xiaoxu , Armando Ines , Jose Pedro A , Konkalmatt Prasad
Published: March 2022

GPR37 and GPR37L1 are closely related G-protein coupled receptors that are expressed mainly in brain glial cells and muscle-myenteric nerve layers in the GI tract. GPR37L1 transgenic mice have decreased systolic blood pressure (SBP) whereas GPR37L1 KO mice have increased SBP. However, there are no studies reporting kidney expression of GPR37 or GPR37L1 or their role in renal blood pressure regulation. Immunostaining and immunoblotting showed that GPR37 and GPR37L1 are expressed in the apical membrane of proximal tubules of the mouse kidney; RT-PCR on proximal tubule and collecting duct cells obtained by laser capture micro-dissection of mouse kidney sections, confirmed these findings. In addition, chronic high salt diet increased the renal expression of prosaposin, a precursor for saposin C, a natural ligand for GPR37 and GPR37L1. Infusion of prosaptide, a synthetic ligand for GPR37 and GPR37L1, decreased SBP in mice by 10 mm Hg. To determine the roles of GPR37 and GPR37L1 in renal Na+ transport, we over-expressed separately the two proteins in human renal proximal tubule (RPT) cells (n=3). Intracellular Na+ was increased in GPR37- or GPR37L1-transfected RPT cells (3.1 ± 0.8 and 3.2 ± 0.6 fold respectively, P<0.001) compared with mock-transfected cells. Immunoblot analyses showed increased phosphorylation of Erk1/2 (1.33 ± 0.03 and 1.52 ± 0.06 fold, P<0.05), and ribosomal S6 protein (1.29 ± 0.03.5 and 1.39 ± 0.08 fold, P<0.01) in RPT cells over-expressing GPR37 or GPR37L1, respectively. Na+,K+-ATPase expression was decreased by 29% ± 3.5 (P<0.05) in GPR37L1 transfected RPT cells, but was unaltered in GPR37 transfected RPT cells. Taken together, these results show that both GPR37 and GPR37L1 are expressed in RPT cells and signal via the Erk1/2 pathway. GPR37L1 increases intracellular Na+ in RPT cells by decreasing the exit of Na+ due to a decrease in Na+,K+-ATPase expression and activity at the basolateral membrane while GPR37 increases intracellular Na+, by a mechanism independent of Na+,K+-ATPase. These results indicate that GPR37 and GPR37L1 may play a role in Na+ reabsorption in RPT cells and may be novel targets to designing drugs to treat patients with hypertension.
Abstract MP03: Relationships between Serum Soluble (Pro)Renin Receptor Levels and Endocrine or Metabolic Factors in Patients with Essential Hypertension
Authors: Kimura Shihori , Morimoto Satoshi , Yamashita Kaoru , Takano Noriyoshi , Mizuguchi Yuki , Seki Yasufumi , Niiyama Michita , Yoshida Naohiro , Bokuda Kanako , Yatabe Midori , Yatabe Junichi , Watanabe Daisuke , Ando Takashi , Ichihara Atsuhiro
Published: March 2022

Background:
(Pro)renin receptor[(P)RR] is a single trans-membrane receptor that binds to both renin and its precursor prorenin to activate tissue renin-angiotensin system (RAS). (P)RR is cleaved by furin to generate soluble (pro)renin receptor [s(P)RR], which is secreted into the extracellular space. Blood s(P)RR level is a candidate biomarker reflecting the status of the tissue RAS, and we have reported that serum s(P)RR levels are associated with renal function. Endocrine or metabolic factors contribute to progression of endorgan damages in hypertensive patients. Therefore, in the present study, we investigated the relationships between serum s(P)RR levels and endocrine or metabolic factors in patients with essential hypertension.

Methods:
We measured serum s(P)RR levels and assessed the relationships between serum s(P)RR levels and background factors including endocrine or metabolic factors such as age, body mass index (BMI), abdominal circumference, hemoglobin, uric acid, HbA1c, LDL-cholesterol (C), HDL-C, triglyceride (TG), high-sensitivity (hs) CRP, plasma renin activity, aldosterone concentration, noradrenaline, adrenaline, thyroid stimulating hormone, free T3, and free T4 in essential hypertensive patients.

Results:
A total of 302 patients (138 males) were enrolled. The mean value for age was 57±12 y.o., BMI was 24.9±5.1 Kg/m2, blood pressure was 140±16/85±12 mmHg, hemoglobin was 14.0±1.3 g/dl, estimated glomerular filtration rate (eGFR) was 75.2±20.6 ml/min/1.73 m2, HbA1c was 5.7±0.7%, and serum s(P)RR level was 21.0±5.8 ng/ml. Serum s(P)RR levels were significantly positively correlated with age (r=0.209, p<0.0005), abdominal circumference (r=0.177, p<0.05), HbA1c (r=0.219, p<0.001), TG (r=0.180, p<0.01), hs CRP (r=0.162, p<0.05), free T3 (r=0.521, p<0.01), and free T4 (r=0.523, p<0.005), and were significantly negatively correlated with hemoglobin (r=-0.243, p<0.05), eGFR (r=-0.272, p<0.0001), and HDL-C (r=-0.199, p<0.005).

Conclusion:
In patients with essential hypertension, serum s(P)RR levels were shown to be associated with endocrine or metabolic factors in addition to renal function, suggesting serum s(P)RR level as a useful biomarker that reflects the status of endocrine or metabolic disorders.
Abstract MP06: Mitochondrial Dynamics and Vascular Effects: Role of OPA1 in Hypertension
Authors: Nguyen Phuc Minh Chau , Grenier Celine , Lelievre Eric , Grimaud Linda , Vessieres Emilie , Sarzi Emmanuelle , Bonneau Dominique , Reynier Pascal , Fassot Celine , Lenaers Guy , Henrion Daniel , Loufrani Laurent
Published: March 2022

Background:
Defects in mitochondrial dynamics have been associated with various disorders, including cardiovascular diseases. OPA1 is essential for mitochondrial inner membrane fusion and mutation in OPA1 is associated with autosomal dominant optic atrophy. Since OPA1 has been reported to be associated with cell apoptosis, cell proliferation, mitochondrial ATP synthesis, calcium homeostasis and reactive oxygen species (ROS) production, we investigated its role in vascular function and/or structure in physiological and pathological condition like hypertension.

Methods and Results:
We used the heterozygous Opa1 mouse model carrying the recurrent Opa1 delTTAG mutation and OPA1 silencing in artery smooth muscle and endothelial cells. Electron microscopy revealed altered mitochondrial cristae structure in vascular smooth muscle cells and endothelial cells of Opa1+/- mice, 6 month-old Opa1+/- mice had a normal baseline blood pressure and vascular function (contraction and dilation). A chronic treatment with L-NAME induced hypertension in mice. Systolic blood pressure was significantly greater in Opa1+/- than in wild type (WT) mice. This was associated with a stronger reduction in endothelium-dependent relaxation to acetylcholine of resistance arteries in Opa1+/- than in WT animal. On the other hand, hypertension-induced wall hypertrophy in the aorta was absent in Opa1+/- in association with reduced proliferation and increased apoptosis of vascular cells.

Conclusions:
Thus mitochondria alteration due to OPA1 down-regulation did not affect baseline blood pressure and vascular tone but induced an excessive elevation in blood pressure in hypertension. These results suggest for the first time that OPA1 may play an important role in protection of the vasculature in pathological conditions such as hypertension.
Abstract MP14: A K572Q Mutation in Gamma-adducin Is Responsible for the Impaired Myogenic Response and Autoregulation of Renal and Cerebral Blood Flow in FHH Rats
Authors: Fan Fan , Geurts Aron M , Pabbidi Mallikarjuna R , Ge Ying , Harder David R , Roman Richard J
Published: March 2022

The FHH rat is a genetic model of hypertension induced CKD, but the genes and pathways involved are unknown. We recently reported that the myogenic response of the renal and cerebral arteries is impaired in FHH rats and it was restored in a FHH.1BN congenic strain in which a small region of Chr. 1 containing 15 genes, including gamma-Adducin (Add3), from BN rats was transferred into the FHH background. We further identified a K572Q mutation in Add3 in FHH versus FHH.1BN rat. The present study examined the contribution of Add3 to the impaired myogenic reactivity using Add3 transgenic and KO rats. The diameter of the middle cerebral artery (MCA) decreased by 20-30% in SD, FHH.1BN and FHH.Add3 transgenic rats when perfusion pressure was increased from 40 to 140 mmHg. In contrast, the MCA of FHH and SD.Add3 knockout rats did not constrict. The myogenic response of the MCA is also impaired in MNS rats that share the same mutation in Add3 as in FHH rat and this phenotype was complemented in a F1 cross of FHH and MNS strain. Autoregulation of CBF was impaired in FHH rats and rose by 99 ± 7% when MAP was increased from 100 to 190 mmHg and was restored in FHH.Add3 and FHH.1BN rats. Similarly, the diameter of Af-art of FHH, MNS and a F1 cross of FHH and MNS rat increased in response to increase in perfusion pressure but decreased in FHH.1BN rats that have wild type Add3. FHH rats exhibited impaired autoregulation of RBF in comparison with FHH.1BN rats. Pgc estimated from the stopflow pressure increased by 20 mmHg in FHH rats when RPP was increased from 100 to 140 mmHg versus only 4 mmHg in the congenic strain. FHH rats developed severe renal injury and proteinuria rose from 37 ± 2 to 260 ± 32 mg/day as they aged from 12 to 21 weeks, but rose by a significant lesser extent in FHH.1BN and FHH.Add3 rats. BK current in VSMC isolated from the MCA was 5-fold higher in FHH vs. FHH.1BN rats. Administration of IBTX normalized the elevated BK channel current and restored the myogenic response in FHH rats but it had little effect in FHH.1BN. These results indicate that the K572Q mutation in Add3 plays a causal role in the impaired myogenic response and autoregulation of renal and cerebral circulation in FHH rats and may contribute to the development of renal and cerebral end organ damage with aging and after the onset of hypertension.
Abstract P001: Comparative Study on Steroidgenic Activity in Aldosterone-Producing Adenoma With ATPase or CACNA1D Gene Mutations in Japanese Patients With Primary Aldosteronism
Authors: Kitamoto Takumi , Suematsu Sachiko , Matsuzawa Yoko , Saito Jun , Omura Masao , Nishikawa Tetsuo
Published: March 2022

Object:
Our aim is to clarify the regulatory mechanism of aldosterone synthesis in patients with aldosterone-producing adenomas (APA) harboring ATPase or CACNA1D gene mutations.

Design and patients:
We subjected 108 patients with unilateral APA, and tested somatic mutations by using each APA tissue. ATPase and CACNA1D genes were analyzed among 33 APAs without KCNJ5 gene mutations. We also evaluated pathological findings of steroidgenic enzymes and isolated cells prepared from 2 ATP2B3- and 1 CACNA1D-mutated APAs were incubated with various stimulants for clarifying steroidgenic activity.

Results:
There were 1, 2, and 2 cases whose APAs possessed ATP1A1, ATP2B3, and CACNA1D mutations, respectively. Compared with the wild-type group without any somatic mutations of KCNJ5, ATPase or CACNA1D, the patients with ATPase mutations showed severe phenotype of hyperaldosteronemia even with smaller-sized tumors, although the CACNA1D-mutated APA patients showed similar characteristics. Pathological findings clearly demonstrated that the ATPase-mutated APA was mainly composed of compact eosinophilic tumor cells, while the CACNA1D-mutated APA mainly did of clear tumor cells with relatively weak 3βHSD2 immunoreactivity. In vitro incubation study with isolated APA cells demonstrated that aldosterone production of ATP2B3 mutated APA cells was more responsive to (Bt)2cAMP than that of the other types of cells (almost 2-fold in the wild group and the CACNA1D-mutated cells vs. 4-fold increase in the ATPase-mutated cells). On the other hand, CACNA1D-mutated APA cells showed greater responsiveness to ACTH compared with the other types of cells (almost 2-fold in the wild group and ATPase-mutated cells vs. 4-fold increase in the CACNA1D-mutated cells).

Conclusion:
Responsiveness of aldosterone production stimulated by ACTH or cyclic AMP differed in each case with different cell types. The mutation of ATPase seems to promote accelerated intracellular Ca signaling systems, of which activation may be quantitatively differed in the case of CACNA1D mutation. Thus, our data suggested that the regulatory effect of ACTH on aldosterone synthesis might vary according to the basal intracellular conditions, such as upregulation of Ca signaling induced by each mutation.