bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP42: T Cell Deletion Normalizes Sex Differences Of Blood Pressure But Not Renal Damage In Dahl Salt-sensitive Rats
Authors: Abais-Battad Justine M , Lund Hayley , Dasinger John Henry , Fehrenbach Daniel J , Mattson David L
Published: September 2020

The immune system has a clear role in the development of hypertension and renal damage in male Dahl salt-sensitive (SS) rats, but far less is known about this phenotype in females, especially in regard to the contribution of immune-related mechanisms. The current study examined the hypertensive and kidney injury phenotype in response to a 3 week high salt challenge (HS, 4.0% NaCl, AIN-76A) in female versus male SS rats (n>10/group). Measured via radiotelemetry, there was no difference in low salt (LS, 0.4% NaCl) mean arterial pressure (MAP) between females and males (129±2 vs 124±2 mmHg, respectively). However, after 3 weeks of HS, females had a significant attenuation in the development of hypertension compared to males (161±3 vs 177±7 mmHg). This coincided with significantly less renal damage, evident by markedly reduced albuminuria (105±16 vs 183±16 mg/day) and medullary protein cast formation (7.0±0.7 vs 14.6±1.1%) in the females. Assessed via flow cytometry, there were fewer CD45+ total leukocytes (48% reduction), CD3+ T cells (51%), CD45R+ B cells (73%), and CD11b/c+ monocytes/macrophages (47%) in female versus male kidneys. To interrogate the contribution of adaptive immunity, specifically T cells, to the development of this sex difference, the same parameters were investigated in female and male SS
CD247-/-
rats which lack CD3+ T cells. The absence of functional T cells significantly reduced blood pressure in both females (147±6 vs 161±3 mmHg; SS
CD247-/-
vs SS) and males (157±8 vs 177±7 mmHg) after 3 weeks of HS, with no statistical difference between female and male SS
CD247-/-
rats. While a reduction in albuminuria (females: 37±12 vs 105±16 mg/day; males: 88±13 vs 183±16 mg/day, SS
CD247-/-
vs SS) and medullary protein cast formation (females: 1.8±0.4 vs 7.0±0.7%; males: 9.1±0.7 vs 14.6±1.1%) was observed in SS
CD247-/-
versus SS rats regardless of sex, there was greater protection from these measures of kidney damage in SS
CD247-/-
female versus SS
CD247-/-
males. Together, our data indicate that female SS rats are significantly protected from high salt-induced hypertension and renal disease compared to males rats. The deletion of T cells normalized high salt blood pressure between male and female rats but sex differences in renal damage still persisted.
Abstract MP43: The Minor Allele Of A Single Nucleotide Polymorphism In
SH2B3
Promotes Hypertension And Renal Dysfunction In Mice
Authors: Alexander Matthew R , Dale Bethany L , Hank Samuel , Himmel Lauren , Smart Charles D , Chen Yuhan , Prabakaran Nitin , Tirado Brian , Levy Daniel , Madhur Meenakshi S
Published: September 2020

SH2B3, also known as LNK, is an adaptor protein that negatively regulates growth factor and cytokine signaling. The minor allele of a single nucleotide polymorphism in
SH2B3
(rs3184504) encodes a tryptophan (Trp) at amino acid 262 as opposed to arginine (Arg) and is strongly associated with hypertension in genome-wide association studies. Whether this variant is causal and how it impacts hypertension development and end-organ damage is unknown. We used CRISPR-Cas9 to engineer mice homozygous for the major and minor alleles of this
SH2B3
polymorphism, resulting in Arg/Arg and Trp/Trp mice, respectively. Trp/Trp mice exhibited increased systolic blood pressure (SBP) by radiotelemetry during weeks 3-4 of angiotensin II (Ang II) infusion compared to Arg/Arg mice (nighttime SBP 168 vs 158 mm Hg, respectively). Renal dysfunction was also exacerbated in Ang II-treated Trp/Trp compared to Arg/Arg mice, as evidenced by significantly increased urinary albumin/creatinine ratio (0.41 vs 0.17), renal perivascular fibrosis (fibrosis score 2.0 vs 1.0), and renal macrophages (8,341 vs 6,413 per kidney). In addition, renal CD8
+
T cells from Ang II-treated Trp/Trp mice produced significantly more IFNγ compared to Arg/Arg controls (median fluorescence intensity 20,455 vs 14,134), and
ex vivo
stimulated splenic CD8
+
T cells from Trp/Trp compared to Arg/Arg mice made 2.7-fold more IFNγ. To determine a mechanism for increased T cell IFNγ production, dendritic cells and naïve T cells from Trp/Trp and Arg/Arg mice were co-cultured in different combinations. The greatest increase in GM-CSF-induced IFNγ production occurred when both dendritic cells and T cells came from Trp/Trp mice (3.4-fold greater than both cell types from Arg/Arg mice). This effect appears to be due to loss of SH2B3-mediated suppression of GM-CSF signaling, as overexpression of Trp-encoding SH2B3 in HEK cells exhibited significantly less repression of GM-CSF-induced Stat5 activation compared to Arg-encoding SH2B3. Taken together, these findings suggest that the Trp encoding allele of rs3184504 is a causal variant promoting blood pressure elevation and renal dysfunction, at least in part through loss of SH2B3-mediated repression of T cell IFNγ production.
Abstract P001: Diurnal Dysfunction Of The Renal Endothelin System Is Associated With Obesity-induced Hypertension In Rats
Authors: Soliman Reham H , Jin Chunhua , Taylor Crystal M , Pollock David M
Published: September 2020

Loss of endothelin (ET) control of Na
+
reabsorption results in salt-sensitive hypertension. Diet-induced obesity such as via high fat (HF) diet can produce salt-sensitive hypertension. The goal of this study was to determine if obesity produced by HF diet impairs the renal ET system. Male SD rats were fed either HF (45 Kcal% fat) or normal fat (10 Kcal% fat, NF) diet at 6 weeks of age and for 8 weeks duration. Telemetry transmitters were implanted for systolic blood pressure (SBP) and activity monitoring. During the last 2 weeks of the diet, HF and NF rats were sub-divided into 2 groups to receive an additional high salt (4% NaCl, HS) diet or continue on normal salt (0.3% NaCl, NS) diet. Urine was collected and food and water intakes monitored in 12-hr increments the final 2 days. All groups maintained a diurnal BP pattern. However, rats on HF had significantly higher SBP compared to NF (146 ± 1 vs 138 ± 2 mmHg during the active period, p=0.003, n= 12/group). 12-hr urine volume or Na
+
excretion values were similar between groups. However, urinary ET-1, a measure of intrarenal ET-1 production, was significantly lower in the HF compared to NF controls (0.92 ± 0.15 vs 1.52 ± 0.09 pg/12hrs, p= 0.0007, n= 12/group). Relative mRNA expression of ET-1 in the renal cortex collected at ZT12 was significantly lower in the HF vs. NF (0.8 ± 0.1 vs 1.3 ± 0.2, p= 0.008, n= 6/group). A similar reduction of ET-1 mRNA was observed in the OM of HF rats. However, there was no change in ET-1 mRNA in the IM of HF rats compared to NF (0.8 ± 0.1 vs 1.0 ± 0.2, p= 0.28, n= 8/group). HS diet led to a further increase in SBP in HF vs the HF/NS group (152 ± 2 vs 147 ± 1 mmHg, p=0.04, n= 8/group) while addition of HS to the NF group showed no significant increase in SBP (145 ± 2 vs 140 ± 3 mmHg, p=0.37 n= 6/group). The NF/HS group had a significant increase in active-time ET-1 excretion compared to NF/NS (4.0 ± 0.4 vs 1.5 ± 0.2 pg/12hrs, p<0.0001, n= 6/group). ET-1 excretion was elevated in the HF/HS vs HF/NS rats (2.6 ± 0.3 vs 1.1 ± 0.1 pg/12hrs, p= 0.02, n= 8/group), but was significantly lower than NF/HS rats (4.0 ± 0.4 pg/12hrs p=0.009). These findings demonstrate that HF diets impair the renal ET-1 system and are consistent with our hypothesis that loss of renal ET-1 contributes to salt-sensitive hypertension in diet-induced obesity.
Abstract P005: GRK4 Inhibitors Suppress Breast Cancer Cell Growth And Potentiate The Inhibitory Effect Of Dopaminergic D
1
Receptor Agonist SKF38393
Authors: Yue Wei , Wang Jiping , Gildea John J , Xu Peng , Marshall Stephen , Schiermeyer Katherine , Felder Robin A
Published: September 2020

The renal dopaminergic D
1
receptor (D
1
R) regulates sodium excretion which is terminated by phosphorylation by G protein-coupled receptor kinases 4 (GRK4). GRK4 gene variants are associated with increased GRK4 activity and reduced sodium excretion resulting in hypertension. Breast cancer incidence is higher in hypertensive women. We found that GRK4 is a potential molecule linking these two diseases. We hypothesized that GRK4 inhibitors would be beneficial to patients with hypertension and breast cancer. Three potential GRK4 inhibitors (compounds A, B, and C) were tested for their effect on the growth of breast cancer cells MDA-MB-468, MCF-7, and benign mammary epithelial cells MCF-10A controls. These cell lines had high, low, and no GRK4 expression respectively. Growth of MDA-MB-468 and MCF-7 cells was effectively inhibited by compound C with IC
50
16.8±1.9 nM (n=2). MCF-10A cells were relatively resistant to compound C with IC
50
49.3±4.0 nM (n=3) that is significantly higher than the cancer cells (p<0.001). Compound B was the least effective inhibitor in all three cell lines (IC
50
was 1.5-2.3 μM). Growth inhibition of compound A was similar in MCF-7 and MCF-10A cells but less effective in MDA-MB-468 cells indicating GRK4 inhibition may not be the only target for growth inhibition of this compound. It has been reported that D
1
R agonists inhibit growth of breast cancer cells. We hypothesized that blockade of GRK4 would increase sensitivity of breast cancer cells to the inhibitory effect of a D
1
R agonist. In MDA-MB-468 cells, SKF38393 (SKF) at 20 μM caused a 36% reduction in cell number (from 276.7±0.47E4 to 177.7±4.33E4). Compound C alone reduced cell number by 37% (172.4±0.04E4, 5 nM) and 51% (136.3±7.87E4, 10 nM) respectively. Combination treatment induced more reduction in cell number, 63% (100.4±5.54E4, 5 nM) and 84% (44.1±12.7E4, 10 nM). Similarly, Compound A also enhanced the inhibitory effect of SKF. A left-shift of the SKF dose-response curve in GKR4 knock-down MDA-MB-468 cells confirmed that inhibition of GRK4 increases sensitivity of breast cancer cells to SKF. Our preliminary results suggest that targeting GRK4 with compound C and a dopaminergic agonist could be a novel strategy for breast cancer therapy especially for the patients with hypertension.
Abstract P006: Purinergic Receptor Activation Protects Glomerular Microvasculature From Increased Mechanical Stress In Angiotensin Ii-induced Hypertension: A Modeling Study
Authors: Richfield Owen , Cortez Ricardo , FRANCO Martha , NAVAR L Gabriel G
Published: September 2020

Angiotensin II (Ang II)-induced hypertension increases afferent and efferent arteriole resistances via the actions of Ang II on the AT1 receptor. In addition to the increased interstitial levels of Ang II, the increased arterial pressure increases interstitial ATP concentrations which act on the purinergic receptors P2X1 and P2X7, to constrict the AA, preventing increases in plasma flow and single nephron GFR (SNGFR). Blockade of the P2 receptors also mitigates the effects of Ang II, thus increasing blood flow and SNGFR, but the resulting increases in mechanical stresses (shear stress and circumferential hoop stress) on the glomerular microvasculature have not been quantified. A mathematical microvascular hemodynamic glomerular model was developed to simulate blood flow and plasma filtration at each of 320 capillary segments in an anatomically-accurate rat glomerular capillary network topology. Afferent and efferent arteriole resistances and network hydraulic conductivity were adjusted to match glomerular hemodynamic data for control, Ang II-induced hypertension and P2X1-blocked conditions (Franco, Martha, et al.
Amer. J. Physiology-Renal
313.1 (2017): F9-F19). Ang II infusion increased both afferent and efferent resistances, reducing blood flow while slightly raising glomerular pressure. Blockade of the purinergic receptors reduced both afferent and efferent resistances, maintaining glomerular pressure at hypertensive levels but increasing blood flow significantly, increasing shear stress from 24.9 dynes/cm
2
in hypertensive conditions to 71.3 dynes/cm
2
after purinergic blockade. Because glomerular pressure was maintained, hoop stress barely changed from 69.5 kPa in hypertensive conditions to 70.9 kPa after purinergic blockade. Purinergic blockade also increased hydraulic conductivity and filtering surface area, increasing SNGFR. In hypertension, purinergic stimulation does not prevent the transmission of increased arterial pressure to the glomerular capillaries to reduce capillary hoop stress. However, activation of the purinergic system protects the glomerular microvasculature from increases in shear stress caused by a marked increase in blood flow that would occur in the absence of purinergic stimulation.
Abstract P014: 1,3-butanediol Induces Weight Loss But No Antihypertensive Effects In Male S.SHR(11) Rats, An Accelerated Model Of Renal Injury
Authors: Ishimwe Jeanne , Garrett Michael R , Sasser Jennifer M
Published: September 2020

The ketone body precursor 1,3-Butanediol (BD) has been reported to lower blood pressure in male Dahl Salt-sensitive rats fed a 2% NaCl diet and female S.SHR(11) rats on a 0.3% NaCl diet. Treatment in female S.SHR(11) was also associated with weight loss and suppression of renal injury. The present study tested whether BD attenuates hypertension and renal injury in male S.SHR(11) rats. The S.SHR(11) rat is a congenic strain generated from genetic modification of the Dahl S rat. Previously, we demonstrated that the S.SHR(11) rat fed a 0.3% NaCl diet exhibits earlier onset of kidney injury, a greater decline in kidney function compared to the Dahl S rat despite having similar blood pressure, thus providing a better model to test potential therapeutic interventions. Rats (7 -18 weeks old) were divided into two groups: the treatment group that received BD (20%) for 10 weeks via drinking water and controls on ad libitum water and both maintained on a low-salt rodent chow (Teklad 7034, 0.3% NaCl; n=5-6/group). Systolic blood pressure was measured after 9 weeks by tail-cuff plethysmography. After 10 weeks of BD treatment, 24h urine was collected and tissues were harvested. Treated rats were smaller than controls (330.2 ± 14.2 vs. 449.8 ± 22.9 g, p= 0.001). Urine excretion rate was lower in treated rats (11.3± 1.1 vs. 19.6 ± 3.8 ml/day, p=0.047) whereas kidney weight normalized to body weight was higher in this group (0.95 ± 0.04 vs. 0.77 ± 0.03 %, p=0.011). BD did not lower systolic blood pressure (157.0 ±14.0 vs. 163.9 ±14.6 mmHg, p=0.740). Proteinuria was diminished in the treated group, although not significant (135 ± 51 vs. 311 ± 67 mg/day, p= 0.064). Renal fibrosis (7.6 ± 0.61 vs. 9.4 ± 0.95 %, p=0.139), plasma creatinine (0.46 ±0.03 vs. 0.49 ± 0.01 mg/dL, p=0.466), and creatinine clearance normalized to kidney weight (0.57 ±0.05 vs. 0.67 ± 0.02 ml/min, p= 0.138) were not changed by treatment. Blood urea nitrogen was similar between groups (30.7 ± 2.57 vs. 27.4 ± 2.25mg/dL, p= 0.367). The current data demonstrate that BD does not mitigate hypertension nor improve renal function in male S.SHR(11) rats suggesting potential sex differences in the antihypertensive effects of BD. However, weight loss which has been associated with ketogenic interventions such as BD was still observed in this study.
Abstract P016: Sex Differential Effects Of Hyperoxia And Thioredoxin Reductase-1 Inhibition On The Kidney Endothelin-1 System
Authors: De Miguel Carmen , George Aleena , Biswal Sara N , Kraus Abigayle , Dunigan Katelyn , Tipple Trent
Published: September 2020

The vasoactive peptide endothelin-1 (ET-1) is critical in lung and kidney injury. Notably, renal damageand hyperoxia-induced lung disease are more prevalent in males than females. Aurothioglucose (ATG),an inhibitor of thioredoxin reductase-1, attenuates hyperoxia-induced lung injury in mice; however, theeffects of hyperoxia and/or ATG treatment on the kidney ET-1 system remain unknown. Wehypothesized that hyperoxia would activate the renal ET-1 system and that ATG treatment wouldattenuate this activation. Male and female adult C57Bl/6 mice received a single injection of saline orATG (25mg/Kg, i.p.) and were exposed to room air (RA) or >90% O2 for 72 hours. Kidney and spleen werecollected for assessment of the ET-1 system by RT-PCR and glomerular morphology and immune cellinfiltration were evaluated by histology and immunohistochemistry, respectively. In male mice,hyperoxia reduced the cortical expression of ET-1 (RA vs. hyperoxia: 1 ± 0.05 vs. 0.34 ± 0.04, p<0.05;n=3-4/group) and ameliorated the expression of ETA receptor. In RA males, treatment with ATGsignificantly halved the expression of ET-1 and ETB receptor (saline vs. ATG, ET-1: 1 ± 0.05 vs. 0.45 ± 0.07,p<0.05; n=3-4/group; ETB receptor: 1 ± 0.09 vs. 0.37 ± 0.10, p<0.05; n=3-4/group), but had no effect inhyperoxic males. Contrarily, hyperoxic females demonstrated a 3-fold upregulation of corticalETB receptor expression, which was significantly prevented by ATG (saline vs. ATG: 2.80 ± 0.37 vs. 0.95 ±0.27, p<0.05; n=3-4/group). ATG treatment in hyperoxic females also decreased the cortical expressionof ET-1 and ETA receptor. No changes in cortical inflammation or glomerular morphology were observed.Further preliminary results showed that hyperoxia led to a 4-fold increase in splenic ET-1 expression insaline-treated mice, and a 7-fold increase in mice treated with ATG. These results demonstrate sexdifferences in the effects of hyperoxia and ATG treatment in the renal ET-1 system and highlight ATG asa possible therapeutic target to attenuate hyperoxia-induced kidney damage. Funded by NIHK01HL145324 and UAB Diabetes Research Center Pilot Project grant to CDM.
Abstract P017: Increased Sodium Excretion And Urinary Output During Acute Human Sleep Deprivation
Authors: Yang Huan , Vazquez Michael , Chatterton Bryan , Mullington Janet
Published: September 2020

Introduction:
Insufficient sleep contributes to an increased risk (e.g. ~60%) of hypertension. While BP is influenced by renal regulation of sodium and water, the underlying mechanism involved the modulation of renin-angiotensin-aldosterone-system (RAAS) by sleep deprivation is unknown. This project investigated the RAAS regulation of water and salt balance (e.g. urine output, sodium excretion) during prolonged sleep deprivation in humans.

Methods:
Twenty-six subjects (35±1 yrs; 9 women) completed a highly controlled 7-day in hospital study. They were randomly assigned to an 88-h total sleep deprivation (TSD; N=17) condition or an 8-h/night sleep control (SC; N=9) condition. Twenty-four-hour urinary output was collected from baseline (Day 2) to recovery (Day 7). Water and sodium intake were controlled and data were collected. The 24-h sodium excretion value (mmol) was calculated by multiplying the concentrations of sodium in the urine by the urinary volume. Frequent blood samples were collected throughout the experimental period in this study.

Results:
There was a significant interaction effect of TSD on sodium excretion and urinary output (condition x day, p<0.05). Both TSD and SC groups showed continuous increases in sodium excretion during experimental period compared to baseline (p<0.05). The increases in TSD were significantly higher compared to SC (condition x day, p<0.05). In addition, there was a dose dependent effect of TSD on sodium excretion (i.e. p<0.05 for 48-h TSD compared to 24-h TSD). Diet and fluid intake were controlled and there was no interaction effect for TSD on 24-h water intake or oral sodium intake (condition x day, p>0.05). Urinary output significantly increased from baseline, to 24-h and 72-h (p<0.05) during TSD, but remained at baseline levels throughout days in SC group. Preliminary plasma renin activity analyses (assays ongoing) showed a decreasing statistical trend during TSD compared to baseline (p=0.058).

Conclusion:
Prolonged TSD significantly increases sodium excretion and urinary output from the body compared to control group, despite similar water and sodium intake levels between two groups. In addition, the increases of sodium excretion are also dose dependent during TSD.
Abstract P019: Renal Mitochondrial Bioenergetics In Salt-sensitive Hypertension Is Affected By ANP
Authors: Sultanova Regina , Domondon Mark , Nikiforova Anna , Schibalski Ryan , Ilatovskaya Daria
Published: September 2020

There are clinical data suggesting that low levels of Atrial Natriuretic Peptide (ANP) aggravate susceptibility to salt-sensitive (SS) hypertension. ANP is known to affect mitochondria in many tissues, however, little is known about the effects of ANP on renal mitochondrial function. According to our earlier studies, Dahl SS rats lacking ANP exhibit increased blood pressure and pronounced kidney injury. We hypothesized that in SS hypertension ANP deficiency affects renal mitochondrial bioenergetics and contributes to renal function impairment. SS hypertension was induced in male SS
NPPA-/-
(
Nppa
knockout in Dahl SS background, KO) and SS
WT
(wild type, WT) rats by a high salt (HS) 4% NaCl diet administered for 21 days. Age-matched control animals were fed a normal (NS) 0.4% NaCl salt diet. A combination of
in vivo
studies, molecular biology and tests of mitochondria isolated from renal cortex (seahorse respiration and spectrofluorimetry assays using TMRM, Amplex Red and MCLA) were used to probe the role ANP in mitochondrial function. Data was analyzed with ANOVA followed by Holm-Sidak post hoc. We report a significant decrease in mitochondrial membrane potential in the SS
NPPA-/-
rats vs SS
WT
(25 ± 4% decrease in KO vs WT on NS, and a 16 ± 5% decrease in KO on HS). Furthermore, mitochondrial H
2
O
2
(57.7 ± 7.4 (WT) vs 57.6 ± 1.3 au (KO), p<0.0001) and superoxide (36.6± 1.8 au (WT) vs 67.9 ± 5.2 au (KO), p<0.0001) levels were increased in the KO on a HS diet. Next, we antioxidant capacity was elevated in the SS
NPPA-/-
rats on HS diet in comparison with SS
WT
, and increased SOD2 levels were observed in the KO animals. Furthermore, SS
NPPA-/-
rats exhibit higher MCU (mitochondrial calcium uniporter) activity than SS
WT
, implying an ANP-dependent effect on mitochondrial calcium influx. Seahorse assay revealed dramatic elevation of the basal, ATP-linked, maximal and spare oxygen consumption rate (OCR) in the cortical mitochondria of the knockout rats on HS, while in HS fed SS
WT
rats these OCR parameters were, as expected, reduced (vs NS). Therefore, deficiency of circulating ANP leads to significant changes of renal mitochondrial bioenergetics, potentially via effects on mitochondrial calcium uptake, which in turn affects respiratory chain activity leading to oxidative stress.
Abstract P023: Relationship Between TNF-α And SBP In The Baseline Cohort Of The Dietary Approaches To Stop Hypertension (DASH)-Sodium Trial
Authors: Farhan Khalid , Drugge Elizabeth D , Zhou Hong , Graham Lesley , Hao Shoujin , Ferreri Nicholas R
Published: September 2020

Hypertension (HTN) is a leading global noncommunicable cause of mortality and dietary salt is the most common modifiable risk factor for HTN. Evidence suggests that tumor necrosis factor alpha (TNF-α) modulates mechanisms that may contribute to salt-sensitive hypertension (SSHT). We conducted a secondary analysis of the baseline cohort of the DASH-Sodium trial, to explore the relationship between TNF-α and systolic blood pressure (SBP) in subjects categorized by race, sex, and baseline blood pressure.

Urinary TNF-α levels adjusted for creatinine (pg/mg) were measured in (91%) (374 of 412) subjects ingesting high salt (150 mmol) and control diet for 2 weeks prior to randomization. Descriptive analyses were used to determine the sample prevalence of demographic, laboratory, and outcome variables, and bivariate analyses were used to check for highly correlated variables. Robust multiple linear regression was used to evaluate the association of TNF-α and SBP with and without covariates, p < 0.05.

SBP, urinary sodium and potassium adjusted for creatinine, and 24-hour urine volume were positively associated with TNF-α, whereas African American, male, and increasing waist circumference exhibited negative associations with TNF-α, p < 0.05. Predicted TNF-α increased from 22.7 (19.3,26.8) to 33.2 (26.9,41.0) pg/mg with increasing SBP (100 to 180 mm Hg). TNF-α was 25.4 (23.7, 27.1) pg/mg for AA vs. 28.9 (27.2, 30.8) pg/mg for non-AA, and 24.2 (22.6, 25.9) for males vs. (27.3, 30.7) pg/mg for females, respectively. TNF-α increased from 22.3 (20.3, 24.5) to 46.8 (36.1, 60.7) pg/mg as urinary potassium increased from 10 to 100 mg/g, and from 22.7 (20.3, 25.5) to 33.1 (29.0, 37.8) pg/mg as urinary sodium increased from 10 to 250 mg/g. There was a statistically significant interaction between urinary sodium and potassium: TNFα increased to a greater extent with increasing urinary potassium at a lower level of urinary sodium (-1 SD),16.7 (14.0, 20.2) to 38.1 (31.3, 47.3) pg/mg compared to that at higher urinary sodium levels (+1SD), 24.4 (21.0, 28.4) to 35.2 (30.9, 40.1) pg/mg.

These results suggest that TNF-α may regulate ion transport mechanisms that contribute to differences in potassium handling and changes in sodium and potassium excretion observed in SSHT.
Abstract P024: Proximal Tubule Specific Dpp4 Deletion Slows Kidney Disease Progression In Western Diet-fed Obese Mice
Authors: Nistala Ravi , Ren Jing , An Jianzhong , Strawn Tammy , Rawat Suryanshi , Patel Priyanka , Whaley-connell Adam
Published: September 2020

Background:
Obesity is a major risk factor for Chronic Kidney Disease progression to End Stage Renal Disease and/or Dialysis. Increased absorption of fats and/or sugars from Western Diet (WD), likely leads to kidney tubular injury in obesity. We observed that global dipeptidyl peptidase 4 (DPP4) deletion as well as use of inhibitors in WD-fed mice results in decreased kidney injury which, in turn was associated with a decrease in proximal tubule DPP4. Therefore, we hypothesized that proximal tubule (PT) DPP4 activation leads to injury and progression of kidney disease.

Methods:
PT DPP4-KO and WT littermates were fed a WD starting 4-6 wks of age and continued for 2 years. FITC-sinestrin-based GFR and albuminuria were monitored periodically. Tissue histology was performed at select intervals. GeLC-MS was used to separate kidney peptides and Scaffold 4/iPathwayguide used to analyze the Proteomics data.

Results:
WD-fed WT mice gained 150-200% weight of chow-fed [CD] mice and had a greater decline in GFR than CD-fed animals over 2 years (50% vs. 20%, p<0.05). WD-fed PT-DPP4-KO mice had a lesser decline (~35%) when compared to WT mice. This was true for both male and female mice. Concomitantly, there was an increase in albuminuria in WD-fed WT mice that was mitigated in PT-DPP4-KO mice. PAS/PSR stained sections showed worsening fibrosis, tubular dilatation and glomerulomegaly, tubular vacuolization in WD-fed WT mice that was mitigated in PT-DPP4-KO mice. Oil Red O staining showed increased fat accumulation in glomeruli and tubules of WD-fed WT mice that was mitigated in KO mice. Proteomics analysis followed by immunoblots showed that WD-feeding led to an increase in cell adhesion proteins and ribosomal machinery that was significantly suppressed by KO. In addition, there was a shift towards reduction in gluconeogenesis and improved fatty acid oxidation in KO mice.

Conclusion:
Obesity without diabetes can lead to chronic decline in GFR in both male and female mice. DPP4 inhibition may slow the decline if started early in the course of developing obesity and/or metabolic syndrome.
Abstract P029: Aspartate And Potassium Aspartate May Contribute To A Mechanism By Which Saccharina Japonica Extract Attenuates Hypertension In 2-kidney, 1-clip Renovascular Hypertensive Rats.
Authors: Segawa Yukiko , Maruyama Saki , Hashimoto Hiroko , Osera Tomoko , Kurihara Nobutaka
Published: September 2020

Introduction:
Saccharina japonica (SJ) is one of brown algae. The extract of SJ (SJE) is often used for soup stock in Japanese cuisine. We recently observed the decreases in blood pressure (BP) by intake of SJ or SJE diet in 2-kidney, 1-clip renovascular hypertensive (2K1C) rats. SJ is rich in aspartate (Asp), which is the umami taste of amino acid. The intake of Asp was reported to reduce BP in Dahl salt sensitive rats. As well, the potassium Asp (AspK) intake was reported to improve BP in hypertensive patients. 70% (70 of 100) of amino acids such as Asp are eluted into the SJE from SJ. Therefore, we hypothesized that Asp in SJE contributes to the mechanism of BP reduced by dietary SJE in 2K1C rats. In this study, we observed BP in 2K1C rats fed a diet containing Asp or AspK and investigated whether those diets alleviate hypertension to the same extent of the diet containing SJE.

Methods:
Male Sprague-Dawley rats (6 wks) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, the rats fed a control diet (C), a diet containing Asp, AspK or SJE for 6 weeks. The content of SJE was set to be the same as that in our previous study. The amount of Asp in diet was set to be the same as that in our previous study which showed the antihypertensive effect of SJ. The amount of AspK in diet was set to be equimolar to Asp. The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the procedure, mean arterial BP (MAP) was measured in each rat under anesthesia.

Results:
Six weeks after the surgery, 2K1C-C significantly increased in SBP compared with SHAM-C (126±2 vs 159±4 mmHg, p<0.05). However, 2K1C-Asp (138±3), -AspK (142±4), and -SJE (135±4) decreased compared with 2K1C-C (p<0.05) respectively. MAP was significantly higher in 2K1C-C than in SHAM-C (162±3 vs 131±5, p<0.05). It showed a significant decrease in 2K1C-Asp (142±3), -AspK (148±3) and -SJE (144±4), compared with 2K1C-C (p<0.05), respectively. There were no significant differences among 2K1C-Asp, -AspK and -SJE, in either SBP or MAP.

Conclusions:
Asp and AspK may attenuate hypertension in 2K1C renovascular hypertension as much as SJE which contains similar quantities of Asp. It is possible that Asp and/or AspK eluted into SJE contributes to the mechanism of alleviating hypertension by SJE in 2K1C.
Abstract P031: Reduced Augmentation Of The Intrarenal Renin-angiotensin System (ras), Lower Systolic Blood Pressure, And Preserved Renal Function In Female Compared To Male Rats With Unilateral Renal Artery Stenosis
Authors: Bell Annie L , Shao Weijian A , Katsurada Akemi , Sato Ryosuke , NAVAR L Gabriel G
Published: September 2020

Despite growing evidence of sex differences in the progression of hypertension, there are no guidelines that differentiate treatment between men and women. Intrarenal renin-angiotensin system (RAS) activation and tissue injury in 2-kidney, 1-clip (2K1C) hypertensive rats have been characterized in previous studies of male but not female rats.

To evaluate possible sex differences in response to renovascular hypertension, urinary angiotensinogen (uAGT) excretion, systolic blood pressure (BP), urinary protein excretion, and renal function were assessed in female rats.Female (n=8) and male (n=6) rats underwent placement of a 0.2 mm clip on the left renal artery to simulate unilateral renal artery stenosis. BP was measured by tail-cuff plethysmography, and clearance studies were conducted in anesthetized rats to assess renal function. Urine protein concentration was determined by pyrogallol red method. uAGT was measured by ELISA as an index of intrarenal RAS activity.

Systolic BP increased from 120±1 to 176±8 mmHg, and urinary protein excretion reached 20.2±5.6 mg/day in female rats. Although uAGT excretion increased from 13.2±7.7 ng/day to 74.1±29.9 ng/day in female rats, male rats had a significantly higher uAGT excretion of 1572.6±750 ng/day. Nonclipped kidneys exhibited more uAGT excretion compared to clipped kidneys, consistent with previous findings in males. Although 2K1C female rats demonstrate significantly lower renal function than sham females, they show more preserved renal function than male rats. Female rats also demonstrate significantly lower increases in systolic BP and urinary protein excretion compared to male rats.

The data support substantial sex-dependent differences in renal responses to unilateral renal artery stenosis. The results show substantial increases in systolic BP, uAGT, and urinary protein excretion and decreased renal function after renal artery clipping in females, but the magnitude of the changes is markedly lower than in males. Nonclipped kidneys of both sexes exhibit greater uAGT excretion than clipped kidneys. Notably, females show less augmentation of the intrarenal RAS compared to male rats in renovascular hypertension.
Abstract P038: Renoprotective Effect Of SGLT1/2 Inhibitor In Rat Renal Congestion Model
Authors: Hirose Takuo , Matsuki Takuma , Takahashi Chika , Yamakoshi Seiko , Oba-Yabana Ikuko , Endo Akari , Anan Go , Kato Toshiko , Tajima Ryo , Seki Takayuki , Nakayama Shingo , Nakamura Hannah , Tani Junichi , Mori Takefumi
Published: September 2020

Renal venous congestion by increased central venous pressure in congestive heart failure is responsible for renal dysfunction. We have created a novel rat renal congestion model presenting increased renal interstitial hydrostatic pressure and vasa recta expansion, which leads to renal congestion-mediated fibrosis. Current clinical trials suggest the renoprotective effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors in heart failure patients; however, the underlying mechanisms are still discussing. Thus, we investigated the effect of phlorizin, a commercially available SGLT1/2 inhibitor, in our renal congestion model. All animal procedures were performed in accordance with the policies and guidelines of the “Position of the American Heart Association on Research Animal Use” and were approved by the Tohoku Medical and Pharmaceutical University Animal Experiment Committee (registration number: A18019-a, A19039-cn and A20005-cn). The inferior vena cava between the renal veins was ligated, which induced congestion only in the left kidney. Phlorizin (400 mg/kg body weight) or saline were injected subcutaneously daily from 1 day before the operation. Both the right control kidney and left congested kidney were collected 3 days after the surgery. Increased kidney weight and renal fibrosis were observed in the congested kidneys. Phlorizin attenuated the increased kidney weight and suppressed renal fibrosis staining. Molecules related to kidney injury (Kim1) and fibrosis (Fn1 and αSma) were also suppressed by Phlorizin in the congested kidney. Low-vacuum electron microscopy revealed the vasa recta dilatation in the congested kidney. This morphological change was alleviated by Phlorizin. These results suggest that SGLT1/2 inhibitor could suppress renal congestion-mediated fibrosis. This mechanism could be a potential therapeutic target for renoprotection against heart failure.
Abstract P039: The Endogenous Ouabain And Changes In Blood Pressure And Sodium Excretion Following An Acute Saline Load In Essential Hypertension
Authors: Lanzani Chiara Livia , Grim Clarence E , Simonini Marco , Citterio Lorena , Messaggio Elisabetta , Zagato Laura , Hamlyn John , Manunta Paolo
Published: September 2020

Among ~25% of patients with essential hypertension (EH), circulating endogenous ouabain (EO) and aldosterone (Aldo) are typically coelevated and their BP is especially salt-sensitive (SS). We probed for exaggerated natriuresis in a large cohort (712) of hypertensive patients that underwent an acute 2hr (T
120
) load with saline (0.9%/2 L).

Results:
Using a quartiles analysis of sodium excretion, the 4th quartile subgroups showed higher SBP and DBP at T
120
, and at 2 hrs recovery (T
240
). Fractional sodium excretion (FE Na) was significantly higher during loading and recovery in the 4
th
quartile subgroup. Plasma Aldo was suppressed after saline in all subgroups as expected. SS patients showed an increased EO. In the 4
th
quartiles EO was elevated at baseline and after saline (see figure), p
Manova
> 0.001 and the urinary Na/K ratio output was higher. Further, increases in plasma Na
+
were greater in the 4
th
quartile group (1.91 ± 0.15 vs 0.83 ± 0.13 mM, p<0.001). These data suggest circulating EO is a potential mediator of the exaggerated natriuresis in SS EH, and that this phenomenon is reminiscent of an “aldosterone escape”.
Abstract P065: A Novel Risk Score For The Progression Of Carotid Plaque In The Japanese Community: The Suita Study
Authors: Kokubo Yoshihiro , Watanabe Makoto , Higashiyama Aya , Nakao Yoko , Honda-Kohmo Kyoko , Miyamoto Yoshihiro
Published: September 2020

Purpose:
We have first reported that the new progression of carotid artery plaque (Prog-PQ) is a risk factor for incident cardiovascular disease (CVD), but the Prog-PQ risk score has not been established.

Methods:
We studied 4,724 Japanese (mean age 59.7 years) without CVD, whose intima-media thickness (IMT) was measured with carotid ultrasonography on both sides of the entire carotid artery areas as a baseline survey (April 1994 to August 2001). We defined carotid plaque as a maximum IMT of the common carotid artery >1.1mm. We excluded subjects with carotid plaque at baseline (n=1,044), lost to follow-up (n=167), and missing data (n=2). For 3,511 subjects, carotid ultrasonographic follow-ups were performed every two years until March 2016. Blood pressures (BPs) were used the average of two measurements recorded more than 1 min apart. Cox proportional hazard ratios were analyzed after adjusting for cardiovascular risk factors. We used the area under the receiver operating characteristic curve to evaluate the model's discrimination.

Results:
During 38,454 person-years of follow-up, 1,771 instances of the Prog-PQ occurred. Compared to subjects with systolic BP (SBP) <120 mmHg, the adjusted hazard ratios (95% confidence intervals, CI) of the Prog-PQ were 1.18 (1.04-1.33), 1.35 (1.11-1.63), and 1.65 (1.23-2.23) in subjects with SBP=120-139 mmHg, 140-159 mmHg, and ≥160 mmHg, respectively. We developed a scoring system for each risk factor as follows: 0, 2, 4, 6, 9, and 11 points for age 30's to 80's, respectively; -1 point for women; 0, 1, and 2 points for SBP<120, 120-159, and ≥160mmHg, respectively; 0, 1, 2, and 3 points for total cholesterol<160, 160-239, 240-279, and ≥280mg/dL, respectively; 2, 0, and -1 points for HDL cholesterol<35, 35-59, and ≥60mg/dL, respectively; 1 point for overweight or current smoking; and 2 points for diabetes (C-statistic 0.647; 95% CI, 0.629-0.665). Individuals with 2 or 10 points had 27% or 62% observed probability of the Prog-PQ in 10 years, respectively.

Conclusion:
High SBP was a significant risk factor for the Prog-PQ. We have developed a 10-year risk score for the Prog-PQ, which can efficiently predict atherosclerosis in routine clinic/health examinations without carotid ultrasonography.
Abstract P086: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, And Hypertension
Authors: Wu Jing , Fang Shi , Donato Silva Sebastiao , Hu Chunyan , Otanwa Adokole J , Agbor Larry N , Nair Anand R , Lu Ko-Ting , Sigmund Curt D
Published: September 2020

Mutations in
CULLIN3
gene (causing in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT), which is driven by a combination of renal tubular and vascular mechanisms. We have previously shown that disruption of Cullin3 (CUL3) in vascular smooth muscle impairs nitric oxide (NO) signaling and vasodilation through decreased cGMP bioavailability, strongly supporting a role of vascular CUL3 in blood pressure (BP) regulation. To test the importance of endothelial Cul3
in vivo
, we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE
ERT2
mice. Four weeks after tamoxifen, the resultant mice (E-Cul3Δ9) developed nocturnal HT (Night time peak systolic BP, E-Cul3Δ9: 135±3 vs Control: 124±3 mmHg) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s). No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting there was no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg
2
) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%) and cerebral resistance basilar artery (41% vs 77%). No difference in smooth muscle function was observed. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS, and NO production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). Thus, CUL3 mutations in the endothelium contribute to human HT in part through decreased NO bioavailability, endothelial dysfunction and secondary sympathoexcitation.
Abstract P112: Elevated Blood Pressure In Conventionalized Germ-free Rats Is Coupled With Upregulation Of Kynurenic Pathway Metabolites And Central Immune Responses
Authors: Yang Tao , Chakraborty Saroj , Saha Piu , Mell Blair , Cheng Xi , Yeo Ji-Youn , Mei Xue , Zhou Guannan , Mandal Juthika , Golonka Rachel , Yeoh Beng San , Putluri Vasanta , Piyarathna Danthasinghe Waduge Badrajee , Putluri Nagireddy , McCarthy Cameron , Wenceslau Camilla F , Sreekumar Arun , Vijay-Kumar Matam , Joe Bina
Published: September 2020

Background:
Recent evidence supports that metabolic dysfunction underlies hypertension. Injection of kynurenate, a metabolite of tryptophan pathway, into the paraventricular nucleus of the hypothalamus (PVN) lowers blood pressure (BP). Intestinal absorption and metabolism of tryptophan are impacted by gut microbiota. Since gut-brain axis contributes to gut dysbiosis-inducd hypertension, we hypothesized that gut microbiota modulates the levels of kynurenic pathway metabolites that have central impact on BP regulation.

Methods:
We, for the first time, used 7 weeks old male Germ-free (GF) Spague Dawley (SD) rats (n=5) and GF rats co-housed with conventional SD rats for 10 days (GFC) (n=6). BP was measured by tail-cuff. Serum metabolites were quantified by 6495 triple quandrople mass spectrometryand data was normalized using isotoplic labelled compounds. The nucleus of the solitary tract (NTS), the principal sensory nucleus for peripheral changes, and the PVN, a relay center projecting sympathetic output based on the integrated afferent inputs from brain regions including NTS, were analyzed by microarray hybridization for mRNA expression.

Results:
Compared to the GF rats, GFC rats had significantly higher systolic (139 mmHg vs 115 mmHg,
p
<0.05), diastolic BP (96 mmHg vs 79 mmHg,
p
<0.05), and serum levels of kynurenic acid (-9.76 vs -10.21,
p
<0.05) and 3-hydroxy kynurenine (-6.49 vs -7.34,
p
<0.01). Coupled with these increases in kynurenic pathway metabolites, microarray analyses demonstrated increased immune responses (e.g.
Cd74, Il1b, Cxcl1, Mmp14
) in the PVN (gene ontology analysis,
p
<0.001) and increased cell differentiation and synaptic plasticity (e.g.
Sox11, Tp53, Cdk6, Hoxb4, Foxo4, Cyr61
) in the NTS (gene ontology analysis,
p
<0.01).

Conclusion:
Colonization of gut microbiota in GF rats induced increased cell differentiation and synaptic plasticity in the NTS and immune responses in the PVN, indicating the restructured sensory neurons of the NTS and enhanced sympathetic output from the PVN. These are in line with increased levels of kynurenic acid and 3-hydroxy kynurenine, and BP, respectively, suggesting that BP regulation by the gut-brain axis may be mediated by kynurenic pathway.
Abstract P114: Foxo4 Regulates Vascular Smooth Muscle Soluble Guanylyl Cyclase Expression Expression
Authors: Galley Joseph C , Miller Megan , Hahn Scott , Sanker Subramaniam , Straub Adam C
Published: September 2020

Soluble guanylyl cyclase (sGC) modulator drugs may offer a new approach to attenuate hypertension by enhancing vasorelaxation through production of cGMP. Loss of sGC leads to impaired vasorelaxation and hypertension, however the mechanisms that regulate sGC transcription are not fully understood. We recently published that Forkhead box subclass O (FoxO) transcription factor inhibition led to decreased sGC mRNA and protein expression and subsequent loss of downstream enzymatic sGC function and vasoreactivity in the aorta. We conducted luciferase experiments in HEK cells which identified several regions which were sensitive to FoxO inhibition and may be responsible for the regulation of sGCβ protein expression (n=4).The objective of this study was to identify which FoxO transcription factor(s) regulates sGC transcription in vascular aortic smooth muscle cells (SMCs) and at what location on the promoter. FoxO1 shRNA produced a 3-fold increase in sGCα and sGCβ mRNA expression (n=3) and a 27% elevation in sGCβ protein expression (n=6). FoxO3a shRNA produced a similar 2-fold increase in sGCα and sGCβ mRNA (n=3) and a 41% increase in sGCβ protein expression (n=6). FoxO4 shRNA, however, produced approximately 55% decrease in sGCα and sGCβ mRNA (n=3) and a similar 52% decrease in sGCβ protein expression (n=6). FoxO1 and FoxO3a shRNA treatment caused no significant change in pVASP expression at baseline or following stimulation with the nitric oxide donor molecule DEA NONOate (n=3). FoxO4 shRNA treatment resulted in a 73% decrease in pVASP expression at baseline and blunted the DEA NONOate-mediated increase in phosphorylation by 33% (n=3). Preliminary ChIP experiments in human aortic SMCs and have identified 4 sites of enriched Foxo4 binding on the human sGCβ promoter consistent with locations previously identified as sensitive to FoxO inhibitor. These data indicate FoxO4 plays a critical role in the maintenance of sGC transcription, protein expression, and downstream enzymatic function of sGC in vascular smooth muscle. Additionally, our data demonstrate that human sGCβ promoter expression requires functional FoxO activity and FoxO4 promoter binding occurs in promoter regions of human smooth muscle found to be important for promoter-luciferase expression.