bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 342: RNA-seq Profiling of Individually Microdissected Renal Tubule Segments
Authors: Lee Jae Wook , Chou C.-L. , Miller R. Lance , Knepper Mark A
Published: July 2021

Control of renal tubule NaCl transport plays an important role in blood pressure regulation. In the past, transport studies and biochemical assays have been conducted in microdissected renal tubule segments to discover mechanisms of transport regulation. Here, we extend the use of microdissected renal tubules to transcriptomic analysis using the RNA-seq method with small sample amplification. The feasibility of the approach was tested in microdissected cortical thick ascending limbs (cTAL) isolated by hand dissection from collagenase-incubated rat renal cortex. After lysis of isolated cTAL cells, poly(A)-targeted reverse transcription and library preparation were performed (
Nature Protocols
5:516-535). Paired-end sequencing of the cDNA library was done using an Illumina HiSeq 2000 sequencer. The generated 50-bp reads were aligned to the UCSC rat reference genome (
Bioinformatics
25, 1754-60). Reads per kilobase exon model normalized by million mapped reads (RPKM) were calculated. In an initial sample of 5 mm cTALs, 451,694,588 total reads were obtained of which 235,735,784 reads (52%) were mapped and properly paired. In this experiment, 5410 RefSeq genes were identified. To assess accuracy, we surveyed G-protein coupled receptors (GPCRs) and transport proteins that were identified. Among the GPCRs known to be expressed in cTAL (
Avpr2, Pth1r, Gcgr, Calcr, Casr
, and
Ptger3
) only the calcitonin receptor was not found. Among the transporter proteins generally accepted to be expressed in cTAL (
Slc12a1, Kcnj1, Clcnkb, Cldn16, Slc9a3, Slc2a4
and
Atp1a1
) only
Slc9a3
was not found. In contrast, transporter proteins characteristic of neighboring tubule segments were not found. In addition, gene-set analysis showed overrepresentation of mitochondrial genes among transcripts with high RPKM . When longer lengths of cTAL were used, the number of mapped transcripts increased, but not linearly (15 mm, 8455 genes; 25 mm, 12,110 genes). Experiments with a second segment, medullary TALs, gave a profile very similar to that of the cTAL. These studies establish the feasibility of RNA-seq-based transcriptomic profiling in microdissected renal tubule segments and ‘open the door’ for profiling of gene expression along the entire renal tubule.
Abstract 36: Deficiency in the Production of 20-HETE Contributes to Impaired Myogenic and TGF Responses in the Afferent Arteriole of Dahl S Rats
Authors: Ge Ying , Fan Fan , Murphy Sydney R , Williams Jan Michael , Liu Ruisheng , Falck John , Roman Richard J.
Published: July 2021

Previous studies have indicated that a deficiency in the formation of 20-HETE in the proximal tubule and thick ascending limb of Henle in Dahl S rats increases sodium reabsorption and contributes to the development of hypertension. The present study examined whether the lack of 20-HETE production in the renal vasculature contributes to the progression of renal injury by altering the myogenic or tubuloglomerular feedback (TGF) response of the afferent arteriole (Af-Art). The production of 20-HETE was significantly lower by 54% in renal microvessels isolated from the kidneys of Dahl S rats versus that seen than in SS.5BN consomic strain in which chromosome 5 from the Brown Norway (BN) rat containing the CYP4A genes responsible for the formation of 20-HETE was transferred into the Dahl S genetic background. The luminal diameter of the Af-Art decreased by 14.7± 1.5% (from 20.5 ± 0.7 to 17.5 ± 0.8 μm, n=6) in SS.5BN rats whereas the diameter of the Af-Art remained unaltered in Dahl S rats (from 20.1 ± 0.6 to 21.7 ± 0.6 μm, n=7) when the perfusion pressure was increased from 60 mmHg to 120 mmHg. In other experiments, adenosine (1 μM) reduced the diameter of the Af-Art in the SS.5BN rats by 15±0.7% (from 20.1 ±0.4 to 17.1 ± 0.9 μm, n=3) whereas the Af-Art of Dahl S rats was unaltered. However, administration of a 20-HETE synthesis inhibitor, HET0016 (1 μM, n=6), or a selective 20-HETE antagonist, 6, 15-20-HEDE (10 μM, n=6) completely blocked the myogenic and adenosine responses in the Af-Art of SS.5BN rats but it had no effect in Dahl S rats. Administration of a 20-HETE agonist, 5, 14-20-HEDE (1 μM) restored the myogenic response (from 20.7 ± 0.7 to 17.6 ± 0.6 μm, n=7) and vasoconstrictor response to adenosine in the Af-Art of Dahl S rats. These studies confirm the key role of 20-HETE in modulating the responsiveness of the Af-Art and indicate that a deficiency in the formation of 20-HETE in renal microvessels contributes to the marked susceptibility of Dahl S rats to develop hypertension induced renal injury.
Abstract 361: Characterization of a 1,45 Mbp Blood Pressure QTL in Rat Chromosome 13.
Authors: Moreno Quinn Carol P , Yang Chun , Liu Pengyuan , Kurth Terry , Zappa Allison , Xiao Bing , Lazar Jozef , Jacob Howard J , Liang Mingyu , Cowley Allen W
Published: July 2021

We previously showed that transfer of a 13.3 Mb segment of the Brown Norway (BN) rat chromosome 13 (from position 65.9 to 79.21 Mb) into the Dahl salt-sensitive (SS) background attenuates the development of hypertension. In order to narrow the region and identify the causative genes and sequence variants, we created and phenotyped 13 overlapping congenic strains spanning the candidate region. We performed sequence analysis and gene expression analysis by rtPCR and whole-genome RNA-seq on kidneys of the congenic carrying the 13.3 Mb BN segment and the SS rat. Seven strains carrying segments of BN chromosome 13 spanning the region from 73.59 to 75.04 Mb (1.45 Mbp region) were protected from the development of hypertension by at least 20 mmHg. Proteinuria was also markedly reduced in these strains. In contrast, blood pressure was high in the six strains carrying the SS allele in this 1.45 Mb segment. NextGen sequencing of this 1.45 Mbp candidate region, which contained five genes, revealed 2,198 sequence variants between SS and BN. Two of these were nonsynonymous SNPs in
Astn1
and one variant was located on
miR488
. Initial expression analysis of the genes within this region suggests that
Astn1
and
Rfwd2
are differentially expressed between SS and the protected congenic strain after high salt; Pappa2 was differentially expressed between SS and the congenic carrying the BN allele at both high and low-salt diet. In summary, we have narrowed a region for salt-sensitive hypertension to 1.55 Mbps, containing five candidate genes, each of which may contribute in some way to salt-sensitive hypertension.
Abstract 366: Nox1-Derived Superoxide Anion Mediates Stretch-induced Phenotypic Switching in Smooth Muscle Cells
Authors: Rodriguez Andres I , Pagano Patrick J
Published: July 2021

Hemodynamic forces in the vessel wall have profound influences on function, structure and pathology of vascular cells. The predominant mechanical force influencing vascular smooth muscle cells (VSMC) structure and signaling is cyclic stretch (CS). Aberrant CS can direct VSMC to a synthetic phenotype (SP) characterized by increased VSMC migration, loss of contractility and matrix production. Some evidence suggests that CS is capable of inducing reactive oxygen species (ROS); however, the mechanisms by which stretch-derived ROS can induce a VSMC SP are poorly understood. Here we postulate that the NADPH oxidase isoform Nox1, which is an important source of ROS in vascular disease, is involved in a CS-mediated SP. In VSMC subjected to CS (10%, 1Hz) there was an increase in Nox1 mRNA levels (3.9 ± 0.14 fold of increase after 6 hr of CS), Nox1 protein levels (1.44 ± 0.04 fold increase after 24 hr CS) and Nox1-derived superoxide production (CS: 13.82±1.34 vs static: 6.61±0.67 pmol of superoxide/min/mg protein after 24 hr). Using our recently-developed specific peptide inhibitor of Nox1 (NoxA1ds), CS-induced superoxide production abated to baseline levels (6.61±0.67, 13.82±1.34 and 5.06±0.76 pmol of superoxide/min/mg protein; for static, CS and CS+NoxA1ds respectively). Furthermore, osteopontin protein levels, a VSMC SP marker, were increased by CS for 24 hrs (1.25±0.04 fold higher than static) and abolished by NoxA1ds pretreatment (0.96±0.19 fold of static). On the other hand, calponin protein levels, a VSMC contractile phenotype marker, were decreased by CS and restored by pretreatment with NoxA1ds (0.67±0.05- and 0.93±0.12-fold change from static respectively). Finally, we demonstrate that stretch-induced migration of smooth muscle cells is promoted by Nox1-derived superoxide (46.36±1.47, 56.16±1.74 and 40.98±2.46 % of scratch wound closure for static, CS and CS+NoxA1ds treatments respectively). These results suggest that Nox1-derived superoxide mediates a synthetic phenotype in vascular smooth muscle under CS, and identify Nox1 as a potential therapeutic target for the treatment of diseases such as atherosclerosis and hypertension which are characterized by VMSC SP.
Abstract 374: Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-AUpregulates Renal (Pro)Renin Receptor Gene Expression in Npr1 Mutant Mice
Authors: Periyasamy Ramu , Das Subhankar , Nguyen Vickie , Pandey Kailash N
Published: July 2021

Mice carrying targeted-disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (
Npr1
) exhibits hypertension, cardiac, and kidney hypertrophy, and congestive heart failure. The objective of the present study was to determine the role of (pro)renin receptor (P)RR in the kidneys of
Npr1
gene-disrupted (-/-; 0-copy), and wild-type (+/+; 2-copy) mice. Disruption of
Npr1
gene leads to increased systolic blood pressure by 36mmHg in 0-copy mice (138.4 ± 3.6) as compared with wild-type mice (102.2 ± 3.2 mm Hg). The renal (P)RR protein and mRNA levels were increased by 3-fold (4.8 ± 0.7 vs.1.3±0.4) and 3.5-fold (5.0 ± 0.5 vs.1.9 ± 0.3), respectively, in 0-copy mice compared with 2-copy control mice. To identify the Angiotensin II-independent mechanism of (P)RR, angiotensin converting enzyme (ACE) inhibitor, captopril and angiotensin II receptor-1 (AT1R) antagonist, losartan were administered to all
Npr1
mice genotypes. The protein expression of ACE (3.9 ± 0.6 vs.1.9 ± 0.4; 2-fold) and AT1R (4.5 ± 0.4 vs.1.7; 2.5-fold) were decreased in 0-copy treated mice compared with 0-copy control mice, whereas the (P)RR expression was not altered by both inhibitors. Further, expression of phospho-extracellular regulated kinases (p-Erk1/2, 5.3 ± 0.7 vs 1.6 ± 0.5; 3-fold) and phospho-p38 (p-p38, 8.0 ± 0.8 vs. 2.1 ± 0.7; 4-fold) were increased in 0-copy mice as compared with 2-copy control mice. Treatment with Erk1/2 inhibitor U0126 and p38 inhibitor SB203580 attenuated the levels of p-Erk1/2 (6.8 ± 0.5 ± vs.1.1 ± 0.3; 6-fold) and p-p38 (4.7 ± 0.4 vs. 1.6 ± 0.5; 3-fold) in 0-copy treated mice as compared with 0-copy control mice. Captopril and losartan did not alter the expression levels of p-Erk1/2 and p-p38 in 0-copy mice as compared with 0-copy treated mice. The present results suggests that absence of NPRA/cGMP signaling causes increased expression of (P)RR and leads to MAPKs activation in the
Npr1
null mutant mice.
Abstract 376: The Expression of (pro)renin Receptor is Upregulated in the Kidney by High Salt Diet and No Inhibition
Authors: Rong Rong , Ito Osamu , Mori Nobuyoshi , Tamura Yuma , Sakuyama Akihiro , Takahashi Kazuhiro , Totsune Kazuhito , Kohzuki Masahiro
Published: July 2021

The (pro)renin receptor ((P)RR)-bound (pro)renin not only causes the generation of angiotensin II via the increased enzymatic activity, it also activates the receptor’s own intracellular signaling pathways up-regulating the expression of the profibrotic proteins. To clarify the regulation of (P)RR expression, the present study examined the effects of high salt diet and nitric oxide synthase (NOS) inhibition on the (P)RR expression in the kidney. The nephron segments were isolated from male Sprague-Dawley (SD) rats by microdissection and bulk isolation technique, and the (P)RR mRNA and protein expressions were examined by using reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. In adiition, 5 week-old, male SD rats were randomly divided into 3 groups: a control group, a high salt diet (HS) group and a Nω-Nitro-L-arginine (L-NAME) group, and each group was treated with vehicle, high salt diet (8%, NaCl), or L-NAME (600mg/ml in drinking water), respectively. After 4 weeks, the (P)RR expression in the kidney was compared among these groups. The (P)RR mRNA was expressed in the glomerulus (Glm), the proximal convoluted and straight tubule, the cortical and medullary thick ascending limb (TAL) and collecting duct. The (P)RR protein as well as mRNA was expressed widely in the nephron segments; the preglomerular arteriole, the Glm, the proximal tubules (PT), the medullary TAL (mTAL) and inner medullary collecting duct (IMCD). Compared with the control group, the (P)RR protein levels significantly increased in the kidney cortex of both HS group and L-NAME group by 96% (p<0.01) and 506% (p<0.01) and in the inner medulla of L-NAME group by 148% (p<0.05), but did not significantly change in the outer medulla of HS group or L-NAME group. HS increased the (P)RR protein levels in the Glm and PT by 48% (p<0.05) and 39% (p<0.01), but did not affect them in other nephron segments. These results indicated that (P)RR is expressed widely in the nephron segments and that HS and NOS inhibition upregulate the (P)RR expression in the kidney, suggesting roles of (P)RR in hypertensive kidney disorder.
Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell
Authors: Castellanos Rivera Ruth M , Pentz Ellen S. , Gross Kenneth W. , Medrano Silvia , Yu Jing , Sequeira-Lopez Maria Luisa S. , Gomez R. Ariel
Published: July 2021

RBP-J
, the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition,
RBP-J
regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that
RBP-J
acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether
RBP-J
directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its
RBP-J-
binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking
RBP-J
(cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice (
RBP-J
fl/fl
; Ren1d
+/cre
; R26R
+/-
) in which cells lacking
RBP-J
simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells
in vivo
did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that
RBP-J
directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.
Abstract 399: The Circadian Rhythm of Plasma Angiotensinogen in Healthy Volunteers
Authors: Kaifu Kumiko , Kobori Hiroyuki , Nishijima Yoko , Nishiyama Akira , Kohno Masakazu
Published: July 2021

Background:
We have previously reported that urinary angiotensinogen (AGT) excretion did not have a circadian rhythm and could be a novel biomarker for the activity of the renin-angiotensin system (RAS) in kidney. However, there have been few reports investigating the circadian rhythm of plasma AGT in human body. Thus, this study was performed to examine the circadian rhythm in plasma AGT in human.

METHODS:
Evaluating RAS in clinical practice is generally performed in a recumbent position after a 30-minute stabilization period. However, to determine the necessity of recumbent position, we first compared plasma AGT concentrations measured right after waking up and after a 5-minute sitting rest. Next, we examined the circadian rhythm of plasma AGT in 43 healthy volunteers who had shown no abnormalities in the medical examinations in 2011. Plasma AGT was measured at three time points (9 a.m., 1 p.m., and 4 p.m.) in the above volunteers. Blood was collected by a micro hematocrit capillary tube with heparin, frozen for storage after centrifugation, and thawed for the measurement of plasma AGT using an ELISA kit.

Results:
There was no significant difference between the plasma AGT values of the two measuring methods (P = 0.1202, n = 5). Based on the result, we performed blood sampling after a 5-minute sitting rest in the volunteers consisting of 17 men and 26 women. Average blood pressure was 116.3/75.1 mmHg at 9 a.m., 116.3/71.9 mmHg at 1 p.m., and 115.5/70.1 mmHg at 4 p.m.; average pulse rate was 78.7/min at 9 a.m., 77.1/min at 1 p.m., and 73.3/min at 4 p.m. Blood pressure and pulse rate did not change throughout the day. Average plasma AGT was 20.4 ± 6.0 ng/ml at 9 a.m., 20.7 ± 5.0 ng/ml at 1 p.m., and 19.8 ± 6.4 ng/ml at 4 p.m. Plasma AGT did not show a circadian rhythm (P = 0.3803).

Conclusion:
We found in this study that plasma AGT did not have a circadian rhythm. We also found that plasma AGT was not affected by daily life actions. Thus, future patients may not be required to rest nor wait for certain time points before measuring plasma AGT. We also have to unveil the normal AGT levels and the influence on the levels by diseases. As we think that plasma AGT and ratio of urinary AGT to plasma AGT can be a new surrogate marker of hypertension and kidney diseases, we further need to go into this research area.
Abstract 402: Specified Proximal Tubule Segments Augmented Angiotensinogen mRNA and Protein Expressions in Diabetic Kidneys in Rats and Humans
Authors: Kamiyama Masumi , Farragut Kristina , Garner Michelle , Sofue Tadashi , Hara Taiga , Morikawa Takashi , Konishi Yoshio , Imanishi Masahito , Nishiyama Akira , Kobori Hiroyuki
Published: July 2021

Others and we showed the increased levels of intrarenal angiotensinogen (AGT) in diabetic humans, mice, and rats. However, the precise localization of the augmented AGT mRNA and protein in proximal tubule segments of kidneys in diabetes remains to be established. To investigate the detailed localization of AGT in 3 proximal tubule segments of the kidneys in diabetes, the Otsuka Long-Evans Tokushima fatty (OLETF) rat (15 weeks) and the age-matched genetic control, the Long-Evans Tokushima Otsuka (LETO) rat were used. Kidney tissues were also prepared from OLETF rats that were administered with angiotensin II type 1 receptor blocker, olmesartan (0.02% in chow) or a combination of vasodilator agents (0.03% hydralazine, 0.006% reserpine, and 0.012% hydrochlorothiazide in chow; HRH). Moreover, biopsied samples of human kidney cortex were obtained by fine-needle aspiration to confirm the below-mentioned results of the rat localization study. Using 3-μm sections of zinc-saturated formalin-fixed paraffin-embedded slides, we examined the co-localization of AGT mRNA or protein with segment-specific protein markers (Segment (S)1-specific anti-sodium glucose cotransporter 2 antibody, S2-specific anti-carbonic anhydrase IV antibody, and S3-specific anti-ecto-adenosine triphosphatase antibody) by double staining using fluorescence in situ hybridization and/or immunofluorescence. AGT mRNA expression was not recognized much in S1 segments of either OLETF or LETO rat kidneys. In S3 segments, the AGT mRNA expression area was augmented in the OLETF rats than in the LETO rats (2.31 ± 0.02-fold; P = 0.0029). AGT protein expression areas in S1 and S3 segments were also increased (8.50 ± 2.78-fold; P = 0.0476, and 3.71 ± 0.83-fold; P = 0.0317, respectively). Chronic treatment with olmesartan, but not with HRH, ameliorated the augmented AGT mRNA and protein expression areas. AGT mRNA and protein expression areas in S2 segments were unchanged in OLETF rats compared with LETO rats. Biopsied samples of human kidney cortex showed the same results as for rat AGT mRNA and protein expression in the S1 and S2 segments. These data suggest that the augmented AGT mRNA in S3 and AGT protein in S1 and S3 play an important role in the development and the progression of diabetic nephropathy.
Abstract 403: Renal Denervation Reveals Renal Sympathetic Activation in Adult Rats Exposed to Early Life Stress
Authors: Loria Analia S , Brands Michael W , Pollock David M , Pollock Jennifer S
Published: July 2021

We previously reported that maternal separation (MS), a model of early life stress, does not modify baseline blood pressure in adult rats, but increases sensitivity to hypertensive stimuli. Under baseline conditions, adult male rats exposed to MS have significantly reduced glomerular filtration rate (GFR). Acute phenylephrine-induced reductions in renal blood flow is significantly attenuated in rats exposed to MS compared to control rats. Furthermore, norephinephrine (NE) content was increased in renal cortex of MS rats compared to control rats (p<0.05). These data indicate that MS induces increased renal sympathetic outflow. Thus, we hypothesized that renal denervation will normalize GFR in rats exposed to MS. Male WKY rat pups were separated from their mothers for 3 hrs/day during the morning hours from day 2 to 14 of life. Male non-separated littermates served as control rats. Experiments were performed in 300-320 g adult rats. Denervation (DnX) was performed mechanically stripping all visible renal nerves followed by topical phenol (10%) on the renal artery. Control-sham, MS-sham, control-DnX, and MS-DnX rats were instrumented with catheters in the femoral vein and abdominal aorta. Rats were placed in metabolic cages, connected to swivels, and allowed to recover for 4-5 days. Sodium intake was clamped at 2.8 mEq/day in both groups by combining sodium deficient diet and 24 hr/day 0.9% iv saline infusion (20 ml/day). GFR was determined by plasma clearance of [125I]iothalamate in the conscious state. During baseline conditions, MAP was not different between control-sham and MS-sham rats (99±4 vs 97±2 mmHg, respectively). MAP was reduced in both control-DnX and MS-DnX rats (91±2 mmHg and 83±3 mmHg, p<0.05, respectively) compared with the respective sham group. The reduction in MAP tended to be greater in MS than in control rats (-9±1 and -14±2 mmHg, p=0.074). DnX did not modify GFR in control rats (sham: 3.1±0.1 ml/min vs DnX: 3.5±0.4 ml/min). However, DnX significantly increased GFR in rats exposed to MS (sham: 2.4±0.2 ml/min vs DnX: 3.8±0.4 ml/min, p<0.05). These data support our hypothesis that MS induces increased renal sympathetic tone to reduce GFR in MS male rats, and may contribute to the exacerbated response to hypertensive stimuli observed in MS rats.
Abstract 433: Serum Chloride is an Independent and Robust Predictor of Mortality Than Serum Sodium, Potassium or Bicarbonate in Hypertensive Patients
Authors: McCallum Linsay , Jeemon Panniyammakkal , Walters Matthew , Muir Scott , Dominiczak Anna F , McInnes Gordon T , Padmanabhan Sandosh
Published: July 2021

Objectives:
Extremes of serum Na
+
, K
+
and HCO
3
have been associated with poorer outcomes. The role of serum Cl
-
has been largely considered to be linked to serum Na
+
and HCO
3
levels.

Design and Methods:
We studied the association of serum and other serum electrolytes in relation to mortality outcomes in 13,415 hypertensive patients attending the Glasgow Blood Pressure Clinic
(GBPC)
with serum Cl
-
assessed at the registration visit. Cox proportional hazards (Cox-PH) models were used for survival analysis. To determine whether Cl
-
effects are independent of low serum sodium, we performed the analysis in subjects with Na
+
>135 and stratified by Na
+
and Cl
-
levels.

Results:
The total time at risk was 197,101 person years during which 3,373 all-cause deaths occurred. Compared to quintile 1 (Cl
-
<=100), each higher quintile of Cl
-
was associated with lower mortality (H.R.[95%C.I.]0.82[0.74-0.90];0.79[0.71-0.87];0.79[0.70-0.89];0.80[0.68-0.93]) after adjustment for all conventional risk factors, other serum electrolytes and diuretic use (Figure-1). In the subset with Na
+
>135, each unit increase in Cl
-
was associated with a 3% reduction in mortality(H.R =0.97 CI:0.96-0.98). Finally subjects with Na
+
>135&Cl
-
>100(reference) had the best survival and those with Na
+
<135 and Cl
-
<100 the poorest survival. The group with normal Na
+
>135 and Cl
-
<100 had significantly higher mortality than the reference group (1.21[1.11-1.31]).

Conclusion:
Serum Cl
-
independently predicts mortality outcomes in the hypertensive population irrespective of serum Na
+
, K
+
and HCO
3
levels.
Abstract 446: Deletion of Il-6 Prevents Development of Cardiac Damage and Dysfunction in Chronic Ang Ii-salt-induced Hypertension
Authors: González Germán E , Rhaleb Nour-Eddine , Nakagawa Pablo , Liu Yun-He , Carretero Oscar A
Published: July 2021

IL-6 knockout (KO) mice were reported to spontaneously develop cardiac dysfunction and fibrosis. These KO mice also develop less hypertension when fed high salt and infused with angiotensin II (Ang II). We tested the hypothesis that in IL-6-KO mice the attenuated hypertension in response to Ang II-salt is due to the development of cardiac dysfunction. Male C57Bl/6J and IL-6-/- mice (B6.129S6-

Il6
tm1Kopf

) were implanted with telemetry devices for blood pressure measurements, infused with vehicle (V) or Ang II (90 ng/min/mouse subcutaneously) and feed a high salt diet (4% salt diet, HS) for 8 weeks (W). We studied 4 experimental groups: 1) C57BL/6J + V (n=9); 2) IL6-KO + V (n=9); 3) C57BL/6J + Ang II (n=8) and 4) IL6-KO + Ang II (n=6). Blood pressure and echocardiography data were collected before starting the HS diet and Ang II infusion (baseline) and 8 weeks after HS alone or combined with Ang II.

Results
(Mean±SEM)

Conclusion:
Our results do not support our hypothesis and shows that the lack of IL-6 does not affect development of hypertension or cardiac hypertrophy but rather prevents cardiac dysfunction, LV dilation, myocardial inflammation and fibrosis in Ang II-salt-induced hypertension, suggesting that IL-6 plays an important role in cardiac dysfunction associated with hypertension.
Abstract 45: Pathological Role of LIGHT, a New TNF-alpha Superfamily Member, in Preeclampsia via Activation of HVEM and LTβR
Authors: Wang Wei , Irani Roxanna A , Liu Haixiong , Zhang Yujing , Tao Lijian , Kellems Rodney E , Xia Yang
Published: July 2021

Preeclampsia (PE) is a life-threatening hypertensive disorder of pregnancy that is a leading cause of maternal and neonatal mortality and morbidity. The current treatment for PE is limited because underlying pathophysiologic mechanisms remain undefined. PE is, however, recognized as an autoimmune disease, which is associated with a series of inflammatory cytokines. Using sensitive ELISA arrays, we discovered a pronounced inflammatory response was stimulated in circulation of PE patients, including elevated levels of LIGHT, a novel TNF superfamily member, which is involved in pathogenesis of series of autoimmune diseases. Next, we confirmed that LIGHT is significantly increased in the circulation and placenta women with PE. In order to determine the role of LIGHT in pathophyisology of PE, we injected recombinant LIGHT into pregnant mice and non-pregnant mice. We demonstrated that LIGHT directly induces major clinical PE features including hypertension (161±3mmHg vs. control 122±3mmHg, p<0.05) and proteinuria (57±2.7μg/mg vs. control 22±2.5μg albumin/mg creatinine, p<0.05) in the pregnant mice. In contrast,LIGHT only induced hypertension (149±2mmHg vs. control 122±8mmHg, p<0.05) but not proteinuria in non-pregnant mice, indicating LIGHT has a previously unrecognized role in pathophysiology of PE and its detrimental effects on kidney injury is pregnancy-dependent. Mechanistically, using neutralizing antibodies for LIGHT receptors, we found that LIGHT transmembrane receptors, HVEM and LTβR, are required for LIGHT-induced hypertension and proteinuria in the pregnant mice by inducing sFlt-1 production, impaired placental angiogenesis and endothelium dysfunction in pregnant mice. Overall, our studies provide both human and mouse evidence that elevated LIGHT contributes to pathophysiology of PE via both HVEM and LTβR signaling and immediately suggest a novel therapeutic possibility.
Abstract 494: P21 Plays a Protective Role Against Renal Ischemia Reperfusion Injury and is an Essential Factor for Ischemic Preconditioning
Authors: Nishioka Satoshi , Nakano Daisuke , Kitada Kento , Ohosaki Hiroyuki , Sofue Tadashi , Nishiyama Akira , Kohno Masakazu
Published: July 2021

Background:
We previously reported that various pathological conditions including high blood pressure increase p21 expression in the kidney; however, the functional importance of renal p21 up-regulation has not been clarified yet. In the present study, we evaluated the role of p21 in acute kidney injury, a life-threatening disease that can occur independently of the pathological background of patients (whether renal p21 is up-regulated or not).

Methods and Results:
The mice lacking functional p21 (p21-KO, n=9) and its wild-type control (WT, n=7) underwent a 45-min renal ischemia followed by a 24-h reperfusion (I/R). I/R significantly increased both mRNA expression and nuclear immunoreactivity of p21 in the kidney of WT compared with sham surgery (p21/β-actin, 1.28±0.23 vs. 0.57±0.15, respectively, P<0.05). I/R injury analyzed by blood urea nitrogen (BUN) and kidney histological changes were exacerbated in p21-KO mice (BUN: WT; 103.8±4.6 mg/dL, p21-KO; 127.7±5.2 mg/dL, P<0.05). The results suggest that p21 plays a protective role against I/R injury. Therefore, we next examined whether p21 is also associated with the protective effect of ischemic preconditioning (IPC), which is an established method of attenuating the I/R injury. IPC (4 sets of a 5-min ischemia and a 5-min reperfusion) clearly improved the I/R injury in WT (BUN: sham; 87.7±22.0 mg/dL, IPC; 39.0±2.3 mg/dL, n=3 and n=7, respectively, P<0.05), whereas there was no difference in the I/R injury in p21-KO mice (BUN: sham; 136.5±13.6 mg/dL, IPC; 127.9±6.9 mg/dL, n=5 and n=8, respectively). IPC increased the renal expression of p21 prior to I/R compared with sham surgery (p21/β-actin: 1.07±0.08 vs. 0.26±0.05 fold, respectively, P<0.05).

Conclusion:
Renal p21 plays a protective role against I/R injury and is necessary for the beneficial effect of renal IPC.
Abstract 507: G Proteins are Required for Lipoxygenase EDHF Activity of Rabbit Arterial Smooth Muscle Cells
Authors: Gauthier Kathryn M , Falck J. R , Campbell William B
Published: July 2021

Arachidonic acid 15-lipoxygenase (15-LO) metabolites function as endothelium-derived hyperpolarizing factors in rabbit and human arteries. In rabbit arteries, LO metabolites mediate nitric-oxide and prostaglandin-independent relaxations to acetylcholine and AA. Previously, we characterized 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) as a major vasoactive 15-LO metabolite in rabbit arteries. 11,12,15-THETA requires a specific structure for vascular activity. 11(R),12(S),15(S)-THETA causes concentration-related relaxation whereas 11(R),12(R),15(S)-THETA is without activity. The specific structure requirement suggests a role for a receptor. Therefore, we examined the role of G proteins in 11(R),12(S),15(S)-THETA vascular activity. Western immunoblot verified protein expression of Gαs, Gαi and a Gαo in rabbit endothelial and smooth muscle cells. 11(R),12(S),15(S)-THETA increased GTPγ35S binding to rabbit arterial membranes 280±25% while 11(R),12(S),15(S)-THETA was without effect. In cell-attached patches of rabbit smooth muscle, 11(R),12(S),15(S)-THETA (100 nM) increased mean open time of apamin-sensitive, calcium-activated, small conductance potassium (SK) channels from 0.0001±0.0001 to 0.0015±0.0006. In inside-out patches, 11(R),12(S),15(S)-THETA did not increase channel opening (0.0001±0.0001) unless GTP was present (0.0051±0.0023). In the presence of GTP, an antibody against Gαs and a Gαs inhibitory peptide inhibited 11(R),12(S),15(S)-THETA SK channel activation (0.0007±0.0005, 0.0013±0.0012, respectively) whereas an antibody against Gαi was without effect (0.0042±0.0018). A cell-permeant, penetratin-linked Gαs inhibitory peptide also inhibited 11(R),12(S),15(S)-THETA SK channel activation in cell-attached patches (0.0005±0.0002) and blocked 11(R),12(S),15(S)-THETA relaxations in rabbit aorta (max relaxations = 74±6%, 23±7% for control and permeant peptide, respectively). These studies indicate that 11,12,15-THETA-induced SK channel activation and vascular relaxation are mediated by a Gs-coupled mechanism and that 11,12,15-THETA acts via a stereo-specific G protein coupled receptor/binding site.
Abstract 508: TNFα Downregulates DNA Methylation and Increases Pulmonary Arterial Smooth Muscle Cell (PASMC) Proliferation
Authors: Crosswhite Patrick , Sun Zhongjie
Published: July 2021

Background:
Chronic cold exposure triggers robust TNFα production in the lungs and pulmonary arteries (PAs) and causes PA remodeling in rats. A hallmark of the PA remodeling is the over-proliferation of PASMCs. The purpose of this experiment was to investigate potential mechanisms of TNF-alpha induced PASMC proliferation.

Methods and Results:
PASMCs were isolated from 8-week-old male Sprague Dawley rats and treated with 0, 20 or 200 ng/ml TNFα for 24 or 48 hours. After treatment, cell number, superoxide production, histone acetylation and DNA methylation were assessed. TNFα treatment significantly increased the number of PASMCs, indicating excessive proliferation. TNFα also increased NADPH oxidase activity and superoxide production compared with untreated controls. TNFα treatment however did not affect histone acetylation at either dose. Interestingly, DNA methylation was significantly decreased by TNFα treatment compared to controls. Unexpectedly, the activity of DNA methyltransferase-1, the major enzyme for DNA methylation, was not altered by TNFα treatment. Further investigation using QRT-RT-PCR revealed that TNFα upregulated several factors including Gadd45α that regulate DNA de-methylation.

Conclusions:
Our results show that TNFα treatment induced cell proliferation and increased superoxide production in PASMCs. TNFα decreased DNA methylation and upregulated mediators of de-methylation in PASMCs. These findings suggest that TNF-alpha induced PASMCs proliferation may involve the DNA methylation mechanism. (Supported by NIH HL077490 and AHA 11PRE7830040).
Abstract 514: Caspase-1 Activation Contributes to Angiotensin-II-induced Vascular Dysfunction and Hypertension
Authors: Cau Stefany B , Silva Marcondes A , Coimbra Terezila M , Alves-Filho José , Zamboni Dario S , Tostes Rita C
Published: July 2021

Caspase-1 (casp-1) regulates key steps in microbial and sterile inflammatory responses. Hypertension is associated with inflammation of the vascular wall, but it is not known whether casp-1 mediates this phenomenon. We hypothesized that casp-1 contributes to vascular dysfunction in angiotensin II (AngII)-induced hypertension.

Male C57BL/6 wild type (WT) and casp-1 knockout (casp-1
-/-
) mice were infused with AngII (90 ng/min, 14 days, osmotic mini-pumps). Control mice were sham-operated. Systolic blood pressure (SBP, mmHg) was significantly increased in AngII-WT mice (162±7.9
vs.
138±2.9 WT-sham, p<0.05), however this elevation was less in AngII-casp-1
-/-
mice (148±13
vs.
140±6.2 casp-1
-/-
-sham, p>0.05). AngII-WT mice exhibited cardiac hypertrophy (mg/g: 10±0.36 vs. 4.9±0.19 WT-sham, p<0.05), which was not observed in AngII-casp-1
-/-
mice (mg/g: 7.6±0.70
vs.
5.6±0.68 casp-1
-/-
-sham, p>0.05). Severe endothelial dysfunction [acetylcholine (ACh)-dependent dilation, second-order mesenteric arteries] was observed in AngII-WT mice, but this was attenuated in AngII-casp-1
-/-
mice (inserted Figure). Vascular superoxide anion production (dihydroethidium fluorescence, arbitrary units) augmented in AngII-WT (6.2±0.60
vs.
4.9±0.48 WT-sham, p<0.05), but not in AngII-casp-1
-/-
(4.9±0.48
vs.
4.0±0.64 casp-1
-/-
-sham, p>0.05) mice. Preliminary data also show that AngII
in vivo
and
in vitro
induces casp-1 activation (western blotting for cleaved casp-1). These data provide evidence that activation of casp-1 plays a role in vascular dysfunction associated with AngII-hypertension.
Abstract 532: New Agonist of A2a Receptor Reduces Ventricular and Vascular Dysfunction in Monocrotaline-induced Pulmonary Arterial Hypertension in Rats
Authors: Alencar Allan K , Pereira Sharlene L , Kummerle Arthur E , Langraf Sharon S , Caruso-Neves Celso , Baptista Emanuelle F , Nascimento José , Barreiro Eliezer J , Fraga Carlos A , Sudo Roberto T , Zapata-Sudo Gisele
Published: July 2021

Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance with subsequent remodeling and right ventricular hypertrophy. Vascular reactivity and ventricular function were investigated in rats with monocrotaline-induced PAH and treated with a new N-acylhydrazone derivative named as LASSBio-1359.

METHODS:
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Male Wistar rats received a single i.p. injection of monocrotaline (MCT) (60 mg/kg) for PAH induction and were randomly divided in groups which were treated with: saline, vehicle and LASSBio-1359 (50 mg/kg p.o.). After 14 days of treatment, some parameters were evaluated: pulmonary acceleration time (PAT); right ventricular systolic pressure (RVSP); vascular reactivity to acetylcholine; expression of iNOS in pulmonary tissue; wall thickness of pulmonary artery (PAWT).

Results:
PAT (ms) was increased from 26.2 ± 2.8 to 41.3 ± 3.9 in PAH group treated with vehicle (n=8, p<0.05) and was reduced to 24.2 ± 1.7 when PAH group was treated with LASSBio-1359. RVSP (mmHg) increased from 26.0 ± 2.0 to 55.2 ± 2.3 in PAH group (p<0.05) but was similar to control after treatment with LASSBio-1359 (31.8 ± 2.3 mm Hg). Ratio of right ventricle and body weight (mg/g) was 0.66 ± 0.02, 1.63 ± 0.16 and 0.87 ± 0.10 for control, vehicle- and LASSBio-1359-treated PAH groups, respectively. PAH promoted ventricular dysfunction which was reduced by LASSBio-1359. The pulmonary artery maximum relaxation (%) was 57.3 ± 5.5, 43.6 ± 1.2 and 61.4 ± 8.4 for control, vehicle and LASSBio-1359-treated groups indicating that PAH promoted endothelium injury which was recovered by LASSBio-1359. iNOS expression in pulmonary tissue was increased from 0.48 ± 1.31 to 0.98 ± 3.14 in PAH group and reduced to 0.53 ± 1.83 in rats treated with LASSBio-1359. The PAWT (%) were increased from 74.1 ± 1.3 to 90.2 ± 2.7 in PAH group (p<0.05) but was 74.4 ± 1.3 when treated with LASSBio-1359. This compound showed an in vitro vasodilatory activity mediated by activation of adenosinergic A2A receptor.

Conclusion:
LASSBio-1359 reduced ventricular and vascular dysfunction in monocrotaline-induced PAH in rats indicating a possible new alternative to treat PAH.
Abstract 6: Heterozygous TGFβ1 Zinc-Finger Knockout Dahl S Rats Reveal Protection Against High-Salt Induced Renal Injury
Authors: Chen Chun Cheng Andy , Geurts Aron , Jacob Howard J , Fan Fan , Roman Richard J
Published: July 2021

We used a zinc-finger nuclease strategy to create heterozygous TGFβ1 knockout rats (TGFβ1
+/-
) on a Dahl SS/Jr genetic background with a 22 base-pair frame shift mutation between nucleotides 191-212 which introduced a premature stop codon at amino acid 34. Intercrossing TGFβ1
+/-
rats did not produce homozygous knockout rats, indicating that the mutation is embryonic lethal. Wildtype (WT) littermates and TGFβ1
+/-
rats were fed either a 0.4% (normal salt, NS) or 8% NaCl (high salt, HS) diet for 5 weeks. When fed a NS diet, WT and TGFβ1
+/-
exhibit similar renal cortical TGFβ1 expression (1.00±0.12 vs 1.05±0.05, arbitrary units), urinary TGFβ1 excretion (3.9±1.2 vs 5.3±0.4, μg/day), proteinuria (43±5 vs 36±4, mg/day), and minimal glomerular injury and tubulointerstitial fibrosis (TIF). 5 weeks of HS increased renal cortical TGFβ1 protein expression to a greater extent in WT versus TGFβ1
+/-
(1.89±0.14 vs 1.52±0.09, arbitrary units) and TGFβ1 levels in urine increased to a greater extent in WT (41±10 μg/day) versus TGFβ1
+/-
rats (18±4 μg/day) fed a HS diet for 1 week. Systolic blood pressure (SBP), measured by tail-cuff, was similar in WT (161±6 mmHg) and TGFβ1
+/-
(162±7 mmHg) fed a NS diet and increased to the same extent in both WT (235±2 mmHg) and TGFβ1
+/-
(239±4 mmHg) fed a HS diet for 5 weeks. Urinary protein excretion increased to a greater extent in WT versus TGFβ1
+/-
(463±28 vs 313±36 mg/day) fed a HS diet for 5 weeks. Glomerular injury and renal cortical interstititial fibrosis were markedly reduced in TGFβ1
+/-
versus WT after 5 weeks on a HS diet. Similarly, TIF in the renal medulla was less in TGFβ1
+/-
compared with WT. These findings suggest that loss of one copy of the TGFβ1 gene blunts the increase in renal TGFβ1 protein in Dahl S rats fed a HS diet and slows the progression of proteinuria, glomerulosclerosis, and renal interstitial fibrosis independent of changes in blood pressure.
Abstract 604: Differences In The Prevalence And Control Of Hypertension Between Mercury Sphygmomanometer And Omron (hem_907xl): Nhanes 2009-2010
Authors: Yoon Sarah , Nwankwo Tatiana , Carroll Margaret , Ostchega Yechiam
Published: July 2021

Objectives -
Precise, reliable blood pressure (BP) measurement, whether in clinical practice or in epidemiological research, is essential for diagnosis and data interpretation. The study objectives were to compare differences in the prevalence and control of hypertension among adults aged 18 years and older using two standard devices: the mercury sphygmomanometer and the Omron Digital Blood Pressure Monitor (HEM_907XL).

Methods -
5,185 individuals aged 18 years and older participated in the National Health and Nutrition Examination Survey 2009-2010 BP methodology study. Hypertension was defined as a mean systolic blood pressure (SBP) ≥ 140 mmHg or diastolic blood pressure (DBP) ≥ 90 mmHg, or currently taking BP medication. Mean BP was the average of up to three readings for each device. Controlled Hypertension among hypertensives was defined as SBP <140 mmHg and DBP < 90 mmHg.

Results -
Overall the age-adjusted prevalence of hypertension among adults was higher by mercury measurement (27.7%) than by Omron (26.4%,
p
<0.05). There were significant differences in hypertension prevalence between the two devices among men (mercury: 28.9% vs. Omron: 26.5%,
p <
0.05) and Hispanics (mercury: 25.1% vs. Omron: 22.8%,
p <
0.05). The overall hypertension control rate among hypertensives was significantly higher using Omron (64.6%) vs. mercury measurement (57.5%,
p
<0.05). In subgroup analyses, rates of hypertension control among hypertensives were systematically higher using Omron: for those 40-59 years (mercury: 60.0%; Omron: 67.9%); for those 60 years and older (mercury: 59.7%; Omron: 65.4%); for men (mercury: 54.9%; Omron: 63.9%); women (mercury: 61.5%; Omron: 66.1%,); non-Hispanic whites (mercury: 60.9%; Omron: 68.4%); non-Hispanic blacks(mercury: 48.8%; Omron: 54.3%); and Hispanics (mercury: 34.6%; Omron: 44.8%), (
p
<0.05 for all groups).

Conclusion -
Hypertension prevalence measured by mercury was significantly higher than that measured by the Omron device. Lower Omron readings resulted in apparently higher estimates for the rate of controlled hypertension.
Abstract 617: Impact Of Pterostilbene On Blood Pressure and Other Metabolic Parameters In Adults
Authors: Riche Daniel M , Deschamp David , Griswold Michael E , McEwen Corey L , Riche Krista D , Sherman Justin J , Wofford Marion R
Published: July 2021

Objective:
Pterostilbene is a polyphenol that is chemically related to resveratrol and commonly found in berries, such as blueberries. The purpose of this trial was to evaluate the effect of pterostilbene on metabolic parameters.

METHODS:
The trial was a prospective, randomized, double-blind, placebo-controlled study of patients with a total cholesterol ≥200 mg/dL and/or LDL ≥100 mg/dL. Patients were included if they were ≥18 years old and on either no cholesterol therapy or cholesterol medication at a stable dose for at least 2 months prior to baseline laboratory. Patients were excluded if they had significant hepatic, renal or GI tract disease or current overt cardiovascular disease; were receiving thiazolidinediones or fibric acids; were women who were pregnant or of reproductive potential. Eighty subjects were divided equally into one of four groups: (1) pterostilbene 125 mg twice daily; (2) pterostilbene 50 mg twice daily; (3) pterostilbene 50 mg + grape extract (GE) 100 mg twice daily; (4) matching placebo twice daily for 6-8 weeks. Patients received identical counseling on lifestyle intervention. Metabolic endpoints included blood pressure, body weight, and lipids. Linear mixed models were used to examine changes in metabolic parameters over time within treatment groups and compare changes over time across groups. Models were adjusted for age, sex and race.

Results:
The majority of patients completed the study (73/80; 91%). The average age was 54 years. The majority of patients were female (57/80; 71%), Caucasian (56/80; 70%), and had HTN (44/80; 55%). Both systolic (-7.8 mmHg; p<0.01) and diastolic blood pressure (-7.3 mmHg; p<0.001) were reduced with high dose pterostilbene. The only change in lipids was an increase in LDL with pterostilbene monotherapy (24.9 mg/dL; p<0.001) which was not seen with GE combination (p=0.47). Presence of a baseline cholesterol medication appeared to attenuate LDL effects. Patients not on cholesterol medication (n=51) exhibited minor weight loss with pterostilbene (-0.59 kg/m2; p=0.014).

Conclusion:
Pterostilbene reduces blood pressure in adults. Future studies should evaluate high dose pterostilbene with GE in a hypertensive population. Clinicaltrials.gov identifier NCT 01267227.