bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 495: A Novel Test for Low Salt Sensitivity: Angiotensin type-II Receptor Recruitment After Dopamine-1 Receptor Stimulation in Urine-Derived Renal Proximal Tubule Cells
Authors: Gildea John J , Keene Staci A , Lahiff Dylan T , Van Sciver Robert E , Schoeffel Cynthia D , Carey Robert M , Felder Robin A
Published: July 2021

Salt-sensitivity of blood pressure is an inappropriate increase in blood pressure following high salt intake. Subjects in our clinical study were typed according to their salt-sensitivity status into 3 categories: High-Salt-Sensitive (HSS; ≥ 7 mmHg increase in mean arterial pressure (MAP) on a high salt diet of 300 mEq of sodium, 17% prevalence), Low-Salt-Sensitive (LSS:, who paradoxically showed a ≥ 7 mmHg increase in MAP on a low salt diet of 10 mEq of sodium, 11% prevalence), and Salt-Resistant (SR, individuals who showed no significant increase in blood pressure on either diet, 72% prevalence). We previously demonstrated that LSS subjects show increased recruitment of the natriuretic dopamine-1 receptor (D1R) to the plasma membrane following a salt stimulation as compared to HSS subjects. Stimulation of the D1R in RPTC with fenoldopam (dopaminergic agonist) results in recruitment of the natriuretic angiotensin type-2 receptor (AT2R) to the cell surface. We hypothesized that LSS individuals may also demonstrate an enhanced AT2R RPTC membrane recruitment compared to HSS individuals when challenged with fenoldopam. In order to gain access to fresh RPTC from each subject, we isolated exfoliated RPTC from randomly voided urine from SR, LSS, and HSS subjects from our clinical study. We measured three subjects from each category with a minimum of three voids for each subject. We counted individual cells as independent events using both the confocal microscope (n=245) and the flow cytometer (n=5344). We found an inverse correlation between AT2R recruitment and the degree of salt-sensitivity of blood pressure. Fenoldopam stimulated AT2R recruitment as measured by confocal microscopy (y = -0.0047x + 0.4966, R2 = 0.2488, P<0.0001) and flow cytometry (y =-0.057x + 1.5645, R2=0.2912, P=0.0185). Flow cytometry provided a more sensitive diagnostic for LSS than HSS subjects. AT2R recruitment was more predictive of LSS than HSS. AT2R recruitment may be used as a rapid method to test for LSS individuals who need to be identified and encouraged to increase their sodium intake in order to avoid paradoxical hypertension.
Abstract 505: Specific Activation of the Ang all/AT1R-Jak2-Rho Kinase Pathway in Salt Sensitive but not in Salt Independent Hypertension: Clinical and Pathophysiologically Implications
Authors: Chadipiralla Kiranmia , Juncos Ramiro , Zhou Ming-sheng , Raij Leopoldo
Published: July 2021

In hypertension aortic (Ao) stiffness is a main determinant of increased systolic (SBP) and pulse- pressure (PP). Brachial and Ao SBP and PP often differ; Ao pressures predict cardiovascular events more accurately than brachial pressures. It is surmised that increased pressure-workload is cause and effect of Ao stiffening but the mechanisms involved are poorly understood. Rho Kinase is a master regulator of vascular tone and remodeling: In endothelium Rho kinase decreases eNOS expression and NO production. In VSMC Ang II/ AT1R specifically activates Rho Kinase via phosphorylation of Jak2 /Arhgef1 which phosphorylates (inhibits) myosin light chain phosphatase (MYPT1) thereby promoting vasoconstriction. Groups (n= 6) of Dahl SS (DS) rats fed either 0.5% or 4% NaCl diets for 10 weeks were matched with SHR of similar age (6 and 16weeks old) and SBP (147+8 & 211+7). Hypertensive DS rats, but not SHR showed increased Ao weight/length (17%, P<0.05) and left ventricle weight/ body weight ratios (16%, P<0.05). Compared to normotensive DS, Ao AT1Rs in hypertensive DS rose 230% accompanied by increased pJak2/Jak2 (3.5 fold) and pMYPT1/MYPT1 (3.95 fold) all p<0.05. Switch of hypertensive DS to a 0.5 % NaCl diet for 4 additional weeks did not reduce either SBP or the activated Ang l1/Rho Kinase pathways. The ARB Candesartan prevented Angll/AT1-Jak2-Rho Kinase activation in hypertensive DS fed 4% NaCl diet and normalized SBP, LVH and Ao weight/length in hypertensive DS switched to a 0,5% NaCl diet. Hypertensive SHR did not show Ao upregulation of AT1Rs or activation of the Angll/Jak2-Rho Kinase pathway. These studies show for the first time that in low renin salt sensitive hypertension there is specific Ao activation of the Ang II/Jak2-Rho Kinase pathway that is independent of the severity of SBP.

Clinically the variable end-organ disease observed in individuals with similar severity of hypertension may be linked, at least in part, to genetically conditioned differences in Angll/AT1R, Jak-2 activation in response to high dietary salt. These novel studies extend and complement previous studies by us and others implicating abnormal bioactivity of NO and aldosterone in salt sensitive hypertension and unravel a new and intricate pathway in which Rho Kinase plays a pivotal role.
Abstract 509: Essential Role of Vascular Smooth Muscle Bmal1 in Diurnal Variations of Contraction and Blood Pressure
Authors: Su Wen , Xie Zhongwen , Guo Zhenheng , Gong Ming C
Published: July 2021

Bmal1 is an obligatory core clock gene that is ubiquitously expressed but has been demonstrated to have tissue specific functions. However, the vascular smooth muscle specific function of bmal1 is unknown. We generated a smooth muscle specific bmal1 knockout mouse model (SM-bmal1-ko) and investigated the role of bmal1 in vascular smooth muscle contraction and blood pressure regulation. Isometric contractions were measured in isolated right renal artery and 2
nd
order branch of mesenteric artery helical strips. Blood pressure was monitored in conscious free-moving mice using radiotelemetry. We demonstrated that bmal1 was selectively deleted in smooth muscle enriched tissues like mesenteric arteries. Moreover, the diurnal variations of bmal1 target genes per1/2 were abolished in mesenteric arteries. The isometric contractions in response to alpha1 agonist phenylephrine and to 5-HT were significantly diminished in vascular helical strips isolated from SM-bmal1-ko mice compared to that from control flox mice. The contractile diurnal variations detected in the renal arteries isolated from control flox mice were significantly diminished in samples isolated from SM-bmal1-ko mice. Moreover,
in vivo
, the diurnal variations in the instantaneous pressor responses to intravenous phenylephrine injection were significantly diminished in SM-bmal1-ko mice compared to control flox mice. Twenty four hour mean arterial blood pressure was significantly decreased under 12:12 light:dark, constant light or constant dark conditions. Importantly, the amplitude of blood pressure diurnal variations was significantly diminished in SM-bmal1-ko mice. Importantly, neither the level nor the diurnal variations of locomotor activity was affected by bmal1 deletion. This indicates that the central SCN clock function is not affected in the SM-bmal1-ko mice and the blood pressure alterations in SM-bmal1-ko mice is not a consequence of changed locomotor activity. Taken together, our results demonstrate an essential role of bmal1 in the diurnal variations of vascular smooth muscle contraction and blood pressure.
Abstract 516: Knockdown of the Hypertension Associated Gene NOSTRIN Alters Glomerular Barrier Function in Zebrafish (Danio rerio)
Authors: Kirsch Torsten , Kaufeld Jessica , Korstanje Ron , Hentschel Dirk , Haller Hermann , Schiffer Mario
Published: July 2021

The bioavailability of nitric oxide (NO) has been associated with the development and progression of vascular and renal disease. NOSTRIN (for eNOS Traffic Inducer) has primarily been recognized as one important regulator of eNOS, the prime source of NO in the cardiovascular system, with a possible role in the pathogenesis of pre-eclampsia and the development of increased intrahepatic resistance in liver disease.

Here, we identified NOSTRIN in the center of a QTL-overlap region in rat and human trait loci that are associated with hypertension. Glomerular NOSTRIN expression is detectable in podocytes in human and rat glomeruli and podocytic NOSTRIN expression is diminished in hypertensive kidney disease. We show that knockdown of NOSTRIN alters the glomerular filtration barrier function in larval zebrafish, inducing proteinuria and leading to ultrastructural morphological changes on the endothelial as well as epithelial side and the GBM of the glomerular capillary loop. We also demonstrate that NOSTRIN interacts with proteins associated with the podocyte slit membrane. We conclude that NOSTRIN expression is an important factor for the integrity of the glomerular filtration barrier. Disease related alteration of NOSTRIN expression may not only affect the vascular endothelium and therefore contribute to endothelial cell dysfunction but may also contribute to the development of podocyte disease and proteinuria.
Abstract 520: Thromboxane Receptors in Smooth Muscle Promote Hypertension, Vascular Remodeling and Sudden Death
Authors: Sparks Matthew A , Makhanova Natalia , Griffiths Robert C , Snouwaert John N , Koller Beverly , Coffman Thomas M
Published: July 2021

The prostanoid thromboxane (TxA2) is a potent vasoconstrictor and platelet aggregant that has been implicated in the pathogenesis of cardiovascular diseases including hypertension. Actions of thromboxane (TP) receptors in platelets and the vasculature have both been implicated in cardiovascular pathogenesis. To distinguish the contributions of vascular TP receptors in isolation, we generated mice with cell-specific deletion of TP receptors in smooth muscle cells (TP-SMKOs) using Cre/Loxp technology. We used the KISM22α-Cre transgenic mouse line, with Cre recombinase “knocked-in” to the Sm22α gene locus, to excise the conditional Tp receptor allele specifically in smooth muscle. mRNA for the TP-receptor was easily detected in aortae from control mice, but not from TP-SMKOs (P<0.005). Similarly, TP receptor mRNA expression in mesenteric arteries, with intact endothelium and adventitia, was decreased by ≈80% in TP-SMKOs (P=0.05). In TP-SMKOs, acute vasoconstrictor responses to the TP agonist
U46619
were dramatically attenuated by ≈60% in both the peripheral and renal circulations (P<0.05), whereas acute vascular responses to angiotensin II were unaffected. Infusion of high-dose
U46619
caused circulatory collapse and death in a majority of control mice, but TP-SMKOs were completely protected from
U46619
-induced sudden death (P<0.05). Baseline blood pressures measured by radiotelemetry were similar in TP-SMKOs (111±1 mmHg) and Controls (114±1 mmHg; P=NS). However, the absence of TP receptors in vascular smooth muscle cells caused significant attenuation of angiotensin II-induced hypertension (controls: 159±2 mm Hg; TP-SMKO: 145±8 mm Hg, P<0.05) and diminished aortic medial hypertrophy in TP-SMKOs (59±4 μm) vs. Controls (79±7 μm; P<0.05). Thus, vascular TP receptors play a major role in shock, angiotensin II-induced hypertension, and vascular remodeling.
Abstract 537: Marked Attenuation of Age-Related Increases in Aortic Pulse Wave Velocity in Critical Lower Limb Ischemia
Authors: Norton Gavin R , Brand Martin , Woodiwiss Angela J , Michel Frederic , Booysen Hendrik L , Majane Olebogeng H , Maseko Muzi J , Veller Martin G
Published: July 2021

Although an increased aortic pulse wave velocity (PWV) may be employed to predict cardiovascular risk, the extent to which this may be limited by decreases in PWV in peripheral arterial disease (PAD) is uncertain. Applanation tonometry (SphygmoCor software) and vascular B-mode ultrasound were employed to assess carotid-femoral PWV and carotid intima-media thickness (IMT) respectively in 1030 randomly selected healthy adults, and in 217 patients with chronic critical lower limb ischemia (CLI). Patients with CLI had a marked attenuation of age-related increases in aortic PWV (slope of the multivariate adjusted age-log PWV relationship: CLI group=0.23±0.07, Controls=0.54±0.03, p<0.0001 for comparison of slopes) and multivariate adjusted aortic PWV (m/sec)(CLI group=4.48±0.20, Controls=6.77±0.08, p<0.0001). As compared to the community sample carotid IMT was greater in patients with CLI (p<0.0001). Carotid IMT was correlated with aortic PWV in the community sample (r=0.50, p<0.0001), but not in patients with CLI (r=0.06, p=0.37). In a multivariate model with both carotid IMT and aortic PWV in the same model, both an increased IMT (standardized β-coefficient=0.18±0.04, p<0.0001) and a reduced PWV (standardized β-coefficient=-0.23±0.03, p<0.0001) were independently associated with CLI. In conclusion, a marked decline in age-related increases in aortic PWV occurs in CLI and a reduced aortic PWV is associated with CLI independent of the extent of atherosclerosis, as indexed by carotid IMT. Thus, the presence of PAD may limit the sensitivity of the use of an increased aortic PWV as a risk predictor and a decrease in aortic PWV may herald the onset of advanced PAD.
Abstract 55: Involvement of Matrix Metalloproteinase-2 During the Progression of Renal Disease in Diabetic Dahl Salt-sensitive Rats
Authors: Slaughter Tiffani , Paige Adrienne , Fan Fan , Kyle Patrick , Geurts Aron M , Jacob Howard J , Roman Richard J , Williams Jan M
Published: July 2021

Recently, our laboratory reported that Dahl salt-sensitive (SS) rats develop a form of renal disease following induction of diabetes with streptozotocin (STZ) that is similar to patients with diabetic nephropathy (DN). The progression of renal injury in this model was associated with increased levels of matrix metalloproteinase-2 (MMP-2) in the renal cortex. The present study examined the role of MMP-2 during the progression of diabetes-induced renal injury by comparing the development of proteinuria and renal injury following the induction of type I diabetes in SS rats and in a MMP-2
Zinc finger
KO strain of SS rats with an 8 base pair frame-shift deletion (1433-1440) in exon 7 (MMP-2 ZN KO strain). The glomerular expression of MMP-2 protein was non-detectable in the MMP-2 ZN KO strain compared to SS rats fed a low salt (LS) diet. We next performed studies using 9 week-old SS and MMP-2 ZN KO rats treated with either vehicle or (2) STZ, 50 mg/kg (i.p.) to induce diabetes and fed the rats a low salt (LS) diet containing 0.4% NaCl to minimize the development of hypertension. At 18 weeks of age, protein excretion increased to 303±39 mg/day in STZ-treated SS rats (n=7) versus 112±12 mg/day in vehicle treated rats. Protein excretion only increased to 150±23 mg/day in the STZ-treated MMP-2 ZN KO strain (n=7). Blood pressure was not significantly altered and averaged 109±4 mmHg in vehicle and STZ-treated SS rats versus 108±4 mmHg in the STZ-treated MMP2-ZN KO animals. STZ-treated SS rats exhibited marked glomerular injury and extensive renal fibrosis. The degree of glomerulosclerosis and renal interstitial fibrosis was significantly reduced in the kidneys of the MMP-2 ZN KO strain. These data indicate that the progression of diabetes-induced renal injury in STZ-SS rats is associated with upregulation of the expression and activity of MMP-2 and that KO of MMP-2 gene function markedly reduces the development of proteinuria and renal injury in this model of type I diabetic nephropathy. These results also suggest that selective MMP inhibitors may be useful to prevent the development and/or progression of chronic kidney disease in the millions of patients suffering from diabetes.
Abstract 610: A Pepstatin-Sensitive Aspartyl Peptidase as an Early Urinary Marker of Renal Injury in the Diabetic Hypertensive mRen2.Lewis Female Rat
Authors: Americo Anna , Pirro Nancy , Yamaleyeva Lilliya , Lindsey Sarah , Chappell Mark
Published: July 2021

We previously reported that induction of diabetes in estrogen-depleted, hypertensive mRen2.Lewis (mRen2) female rats leads to significant albuminuria, proteinuria and tubulointerstial oxidative stress at 28 days. The current study assessed the time course for the development of proteinuria and peptidase activities in the urine of the diabetic hypertensive mRen2 to begin to establish potential biomarkers of renal injury in this model. Heterozygous mRen2 underwent ovariectomy and diabetes was induced by a single administration of streptozotocin (65 mg/kg; STZ) at 15 weeks of age. Urinary samples were taken prior to (day 0) and 5 and 28 days following STZ. Aspartyl peptidases were assayed with the quenched fluorescent substrate Mca-GWPILFFRLW(Dnp)R in the presence or absence of the inhibitor pepstatin. Enzyme activities were expressed as nmols Mca per 30 min per mg creatinine. Pepstatin-sensitive activity [IC50 ∼ 0.4 nM] was detectable in the pre-diabetic urines [26 ± 9; n=8]; however, the diabetic rats exhibited a significant increase in urinary activity at days 5 and 28 [184 ± 25 and 204 ± 53, respectively; p<0.05 vs. day 0]. In contrast, pepstatin-insensitive activity was markedly lower and decreased between days 0 and 5 [22 ± 2 vs. 12 ± 4; p<0.05], but was unchanged at day 28 [34 ± 12]. Proteinuria was similar at days 0 and day 5, but significantly increased by day 28. Aspartyl peptidases in the kidney and urine that are sensitive to pepstatin include cathepsins D and E, as well as the enzyme napsin. However, immunoblots of concentrated urine samples using selective cathepsin and napsin antibodies failed to match protein expression to the increased activities in the diabetic rats. Finally, we assessed peptidase activity in isolated cortical slices from diabetic mRen2 to determine the extent of enzyme release. Aspartyl peptidase activity was readily detectable in the perfusate of the isolated kidney [6 ± 0.3 pmols Mca per min release, n=3] and completely inhibited by pepstatin suggesting that the urinary activity is of renal origin. We conclude that a pepstatin-sensitive aspartyl peptidase, potentially distinct from cathepsin D/E and napsin may constitute an early marker of renal injury in the diabetic hypertensive mRen2 strain.
Abstract 627: Antisense Inhibition Of Angiotensinogen Reduces BP In Normotensive Sprague-Dawley Rats And Effectively Eliminates RAS-dependent BP Control In Hypertensive SHR Rats
Authors: Mullick Adam E , Brown Heather L , Graham Mark J , Crooke Rosanne M
Published: July 2021

Uncontrolled hypertension is an important contributor to stroke, myocardial infarction, heart failure and renal failure. Despite the armamentarium of antihypertensive therapeutics, there still exists a need for a novel agent effective in individuals with Resistant Hypertension (RHTN). Second generation antisense oligonucleotides (ASOs) are a novel therapeutic class of inhibitors that cause RNase H-mediated target mRNA degradation in organs such as the liver and kidneys. Experiments were conducted to determine the efficacy of ASO treatment targeting angiotensinogen (Agt). Normotensive Sprague-Dawley rats were given 10 - 80 mg/kg/wk of an Agt ASO for 4 weeks via subcutaneous injections. Dose-responsive reductions of liver Agt mRNA (20 - 96%), kidney Agt mRNA (40 - 75%) and plasma Agt (25 - 90%) were observed. BP, measured by tail-cuff volume-pressure recordings, was dose-dependently reduced by Agt ASO treatments as well. At the maximal ASO dose SBP, MAP and DBP were reduced by 39, 43 and 45 mm Hg, respectively. Studies comparing Agt ASO to captopril treatments were conducted in SHR rats. Agt ASO administered at 50, 75 or 100 mg/kg/wk for 2 weeks resulted in dose-dependent reductions of BP of 35 - 57 mm Hg (SBP), 32 - 52 mm Hg (MAP) and 31 - 49 mm Hg (DBP). Addition of 50 mg/kg/day of captopril did not result in further BP reductions in rats receiving 100 mg/kg/wk Agt ASO, suggesting maximal inhibition of RAS signaling was already achieved via Agt ASO treatment in this group. However, captopril treatment in the 50 and 75 mg/kg/wk Agt ASO treated groups did result in further reductions of 17 & 27 mm Hg (SBP), 17 & 25 mm Hg (MAP) and 17 & 24 mm Hg (DBP). These data demonstrate robust and predictive reductions of BP with long term Agt ASO treatment in normo- or hypertensive rats. Additionally, complete inhibition of RAS-dependent BP control could be achieved within 2 wks of ASO treatment. Finally, additive BP lowering was achieved with the combination treatment of Agt ASO and captopril, suggesting improvements in efficacy are possible when ASOs are added to existing RAS inhibitor therapeutics. Such improvements could be desirable in RHTN individuals and/or individuals not at their BP goal with existing RAS inhibitors.
Abstract 646: A Critical Role of CD8+ T cells in the Genesis of Renal Dysfunction in Hypertension
Authors: Chen Wei , Kirabo Annet , Thabet Salim R , Trott Daniel W , Zhang Feng , Wu Jing , Arendshorst William J , Goldstein Anna , Bikineyeva Alfiya , Dikalov Sergey , Madhur Meena S , Titze Jens M , Harrison David G
Published: July 2021

T cells have been implicated in the genesis of hypertension however the mechanisms and subtype of T cells involved are still poorly understood. Moreover, the manner by which T cells of various subtypes affect renal function remains poorly defined. Recently, we found that CD8
+
T cells play a critical role in the blood pressure elevation caused by angiotensin II. We sought to determine if CD8
+
T cells have a role in the renal responses to angiotensin II. In metabolic chamber studies, we found that wild-type mice excrete 80% of a saline load (10% of body weight) in 4 hours, and that this is decreased in response to angiotensin II to 60 ± 5% (p < 0.05) in WT mice but not in CD8
-/-
mice. In contrast, CD4
-/-
mice demonstrate a markedly exaggerated anti-natriuretic and anti-diuretic response to angiotensin II (p < 0.001). We also found that chronic angiotensin II infusion increases superoxide production in the renal cortex of WT and CD4
-/-
mice, but not in CD8
-/-
mice. In addition, we found that angiotensin II infusion caused significant hypertrophy of the renal arterioles (< 25 μm diameter) as reflected by an increase in the media/lumen ratio in wild type and CD4
-/-
mice, (1.5 to 4.5 and 2.9 to 5.3 respectively, p < 0.05 for each), but to a lesser extent in CD8
-/-
mice (1.8 to 3.3). We also observed that the number of arterioles 0-25 micron in diameter decreased from 16.5 and 15.5 to 11.8 and to 11.4/mm2 in wild type and CD4
-/-
mice respectively in response to angiotensin II, but was not changed in CD8
-/-
mice. Our study therefore shows a previously unrecognized role for CD8
+
T cells in modulating renal sodium and volume retention, renal cortical superoxide production, vascular hypertrophy and rarefaction. Thus, CD8
+
T cells are an important intermediate in the actions of angiotensin II on the kidney, and a potential therapeutic target.
Abstract 648: Heme-oxygenase-1 Gene Expression Ameliorates Cardiac Dysfunction Following Myocardial Infarction In C57 Mice By Recruitment Of Angiogenic Factors And Angiogenesis In C57 Mice
Authors: Sacerdoti David , Monu Sumit R , Pesce Paola , Peterson Stephen J , Sodhi Komal , Abraham Nader G
Published: July 2021

Rational:
Angiogenesis is essential in order to increase blood circulation in infarcted tissue of MI (Myocardial infarction). Increased Heme-Oxygenase (HO)-1 gene expression increases angiogenic proteins, e.g. VEGF, bFGF, EGF, angiopoietin and adiponectin.

Objective:
To investigate whether increased levels of HO-1, after the occurrence of a MI, improves angiogenesis and capillary formation in ischemic myocardium, thereby improving cardiac function.

METHODS:
Experimental MI was induced by LAD (Left anterior descending artery) ligation. C57BL6 mice were divided into 4 groups: Sham; MI; 5 days after MI treated with the HO-1 inducer, cobalt protoporphyrinIX (CoPP); and, CoPP in the presence of the HO activity inhibitor, Stannous Mesoporphyrin (SnMP). HO-1 downstream signaling proteins were determined including VEGF, CD31 and adiponectin. Echocardiography was performed weekly for 4 weeks after surgery.

Results:
5 days after MI, CoPP treatment significantly increased VEGF (p<0.05 vs.MI), CD31 (p<0.05 vs.MI), and adiponectin levels (p<0.05 vs.MI). These findings were associated with a significant increase in capillary formation and blood flow in CoPP-treated animals (p<0.05 vs.MI). Echocardiography showed that left ventricle dilatation, measured as end diastolic area (EDA), was significantly reduced in CoPP- treated animals compared to MI groups (EDA: MI: 0.216±0.02cm2; MI+CoPP: 0.172±0.03 cm2; (-13%) p<0.01). This was associated with a significant decrease in apoptosis and fibrosis (P<0.05). These beneficial effects were reversed by SnMP administration.

Conclusion:
HO-1 improved cardiac function and enhanced angiogenesis via the recruitment of pro-angiogenic factors.
Abstract 74: Heme Oxygenase-PPARα Induction of FGF21 in Hepatocytes Recruits pAMPK/pAKT and Attenuates Insulin Resistance in Obese Mice
Authors: Hinds Terry D , Abraham Nader G
Published: July 2021

The peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that is known to regulate glucose and lipid homeostasis and increases expression of the hormone fibroblast growth factor 21 (FGF21), which is secreted to blood, increases phosphorylation of AMPK and AKT in adipocytes and has been purported to stimulate adiponectin synthesis. The heme oxygenase (HO) system is functionally important for reducing ROS and inflammation whose induction has been shown to increase serum adiponectin levels and affect lowering of blood glucose and fatty acids. This study was designed to examine the hypothesis that a HO-1-PPARα crosstalk could elevate hepatic FGF21 thus leading to enhanced adiponectin secretion and abatement of metabolic imbalance. Preliminary experiments were conducted in human hepatocellular carcinoma cells (Hep G2) cultured in the absence or presence of the HO-1 inducer, cobalt protoporphyrin (CoPP, 2μmoles/l). A 2.0 fold induction of HO-1 in Hep G2 cells by CoPP (p<0.05) increased expression of FGF21 1.5 fold (n=3, p=0.0119 vs. vehicle control) without affecting PPARα expression. Additional experiments in obese (ob/ob) mice treated with CoPP (5mg/kg/day) resulted in 2 fold (p<0.05) increase of FGF21 mRNA expression in liver as compared to mice treated with the vehicle. These observations were complemented by blood glucose measurements, which showed significant attenuation in obese mice treated with CoPP (vehicle: 285.0±24 vs. CoPP: 160.0±27.3, p<0.05, n= 5), along with enhancement of adiponectin levels in CoPP treated obese mice (p<0.05). Taken together these results demonstrate that HO-1 could increase serum adiponectin levels and insulin sensitivity by elevating hepatic FGF21 levels. Our overall hypothesis is that the HO-1 can stimulate PPARα activity with resultant activation of hepatic FGF21 secretion and that this HO-PPARα-FGF21 axis could work in concert to modulate the development of insulin resistance and diabetes.
Abstract 83: Intracerebroventricular Administration of Autologous Bone Marrow-derived Cells Attenuate Slow Pressor Angiotensin II Initiated Hypertension in Rats
Authors: Yamazato Masanobu , Ishida Akio , Yamazato Yoriko , Sakima Atsushi , Ohya Yusuke
Published: July 2021

Objectives:
Subcutaneous infusion of non-pressor doses of angiotensin II (Ang II) gradually produces hypertension in part via central mechanism. Bone marrow-derived cells (BMC) have an ability to secrete paracrine factors leading to improvements in tissue injury. We hypothesized that implantation of autologous BMCs in the brain would attenuate Ang II initiated neurogenic hypertension via secreted paracrine factors.

METHODS:
Sprague-Dawley rats were divided into following 4 groups: rats received vehicle infusion with intracerebroventricular (icv) administration of medium (V/M group, n=5); rats received vehicle infusion with icv administration of DiI labeled BMCs (V/B group, n=3); rats received Ang II infusion (150 ng/kg/min) with icv administration of medium (A/M group, n=4); rats received Ang II infusion with icv administration of BMCs (A/B group, n=5). Three weeks following initiation of the infusion, rats had surgery to implant arterial and venous catheters. Resting mean arterial pressure (MAP) of rats was recorded in conscious unrestrained state and resting sympathetic tone was evaluated with a peak depressor response produced by hexamethonium (C6) injection.

Results:
Systolic blood pressure (SBP) measured by tailcuff method was similar among the groups, only A/M group had a trend to increase in SBP. Resting MAPs of V/M, V/B, A/M and A/B group were 125±2, 116±2, 174±7, 134±8 mmHg, respectively. Ang II infusion significantly increased MAP in A/M group, however in A/B group, icv administration of BMCs attenuated the Ang II mediated increase in MAP. Heart rate was similar among the groups. The peak depressor responses to C6 injection in V/M, V/B, A/M and A/B group were 38±3, 46±6, 77±5, 48±1 mmHg, respectively. Ang II infusion significantly increased the peak depressor response to C6 and icv administration of BMCs attenuated the increase in peak depressor response. DiI positive cells were distributed to the choroid plexus, surface of ventricle, and subventricular zone but not to the subfornical organ.

Conclusion:
Icv administration of autologous BMCs attenuate slow pressor Ang II initiated hypertension via attenuating the Ang II mediated sympathetic activation in rats. Paracrine factors secreted from the BMCs may be involved in this process.
Abstract 92: Hvcn1 Contributes to Superoxide Production in the Medullary Thick Ascending Limb
Authors: O'Connor Paul M , Sun Jing-Ping
Published: July 2021

We have previously reported that H
+
efflux stimulates superoxide (O
2
-
) production in the medullary thick ascending limb of the loop of Henle (mTAL) in rats by activation of an as yet unidentified H
+
transport pathway. As HVCN1, a voltage gated H
+
channel associated with NADPH oxidase in immune cells, is also expressed in mTAL, we hypothesized that HVCN1 is responsible for H
+
efflux induced production of O
2
-
in mTAL. In order to test this hypothesis we utilized HVCN1
-/-
mice and wild type litter mates obtained from KOMP. To confirm that HVCN1 was functionally knocked out in HVCN1
-/-
mice we stimulated the respiratory burst using PMA (100uM) in peritoneal macrophages extracted from HVCN1
-/-
and WT mice and determined maximal O
2
-
production using L-012 luminescence. Maximal O
2
-
production during the respiratory burst was significantly lower in macrophages from HVCN1
-/-
mice (23±4AU; n=6) compared to WT mice (92±3AU; n=5; p<0.05), consistent with loss of HVCN1 function. mTAL were isolated from the inner stripe of the outer-medulla by micro-dissection and loaded for 60 min with the O
2
-
sensitive dye dihydroethidium (DHE). Live mTAL were then imaged on a heated chamber attached to a fluorescent microscope and the ratio of ethidium (Eth) to DHE quantified using metafluor imaging software as an index of O
2
-
production. O
2
-
producing H
+
currents were isolated in 0 Na
+
, 100uM BaCl
2
media and cellular H
+
efflux stimulated using an NH
4
Cl (20mM) prepulse. Following removal of NH
4
Cl from the bath there was a significant increase in O
2
-
production in mTAL from WT mice (Delta slope Eth/DHE 15.9±3.6 AU/sec; p<0.02 (n=5)) but not HVCN1
-/-
mice (delta slope 4.7±9.6; P=0.64 (n=6)). The response of WT mice was significantly greater than that of HVCN1
-/-
mice (P<0.001). We conclude that HVCN1 is required for maximal O
2
-
production in mTAL in response to H
+
efflux. These data suggest that HVCN1 may be a novel target to prevent renal oxidative stress.
Abstract 100: Mechanisms That Mediate the Cardioprotective Activities of Resveratrol in Pressure Overload-Induced Heart Failure
Authors: Supowit Scott , Gupta Prakash , DiPette Donald
Published: July 2021

We previously demonstrated that the potent antioxidant, resveratrol, significantly attenuates the adverse cardiac remodeling and dysfunction in pressure overload heart failure induced by transverse aortic constriction (TAC) in C57/BL6 mice. The purpose of this study was to identify the mechanisms that mediate the cardioprotective activities of resveratrol in this setting. Male C57BL6 mice (26-28 g) were subjected to either sham or TAC surgery. One group of TAC mice was given daily resveratrol treatment (oral gavage, 100 mg/kg/body weight for 28 days starting on day 2 after surgery. At the end of the protocol (28 days), the hearts were removed for analysis of markers of oxidative stress, inflammation, and apoptosis. Immunohistological staining for left ventricular (LV) 4-hydroxy-2-nonenal, a marker of lipid peroxidation was significantly elevated in the TAC mice (18.4±3.0 IOD/μm
2
) compared to sham mice (3.4±1.0). Resveratrol treatment significantly attenuated this increase (14.4±2.0). In contrast, LV content of the antioxidant glutathione was significantly decreased in the TAC mice (5.3±0.2 nM/mg protein) compared to the sham mice (8.5±0.2). Resveratrol treatment significantly increased glutathione (7.6±0.3) to near sham levels. Likewise, LV activity of sodium oxide dismutase 2 was markedly reduced in TAC mice (7.3±0.9 U/mg protein) compared to sham mice (10.7±1.3) and was significantly increased by resveratrol treatment (7.6±0.3). LV macrophage and mast cell infiltration was markedly increased in the TAC mice (14.8±0.1 and 12.6±0.0 %) compared to sham mice (1.7±0.1 and 2.6±0.1). This increase was significantly attenuated by resveratrol administration (10.4±0.1 and 8.0±0.1). Apoptotic cell death, as determined by the TUNEL assay, was also increased in the TAC mice (0.13±0.01 % cells) compared to sham mice (0.01±0.01). This increase was significantly attenuated in the resveratrol treated group (0.06±0.01). A similar pattern was observed when activated LV caspase-3, a second marker of apoptosis, was measured. In conclusion, these data indicate that the cardioprotective effects of resveratrol in pressure overload-induced heart failure are mediated through the inhibition of oxidative stress, inflammation, and apoptotic cell death.
Abstract 192: Activation of Kidney Renin-angiotensin System (RAS) and Renoprotective Effects of Direct Renin Inhibition (DRI) in Glomerulonephritis
Authors: Miyata Kayoko , Satou Ryousuke , Sato Akemi , Seth Dale , Davis Allison , Urushihara Maki , Kobori Hiroyuki , Mitchell Kenneth D. , Navar L. Gabriel
Published: July 2021

The development of glomerulonephritis causes glomerular injury and reduced renal function and is associated with increased renin release. The aims of this study were to demonstrate activation of intrarenal RAS in glomerulonephritis and determine the effects of DRI with aliskiren on progression of glomerulonephritis associated renal injury induced by administration of an anti-Thy1.1 antibody. We divided rats into 3 groups; control group, anti-Thy1.1 glomerulonephritis group and anti-Thy1.1 glomerulonephritis treated with aliskiren (30 mg/kg/day) group. In the anti-Thy1.1 rats, protein excretion and micro albumin excretion levels increased markedly after anti-Thy1.1 antibody injection. The increases in protein (94.7 ± 13.0 mg/day) and micro albumin (7.5 ± 2.6 mg/day) excretions were attenuated by aliskiren treatment (43.6 ± 4.5 mg/day of protein and 2.6 ± 0.7 mg/day of micro albumin). The glomerular expansion score was significantly higher in the anti-Thy1.1 group compared to the control group. The anti-Thy1.1 group also showed elevation of intrarenal TGF-β and PAI-1 mRNA levels compared with control rats. The progression of glomerular expansion and elevation of these mRNA levels was prevented by aliskiren. Importantly, the anti-Thy1.1 group had markedly increased urinary angiotensinogen (AGT) excretion at days 3 (3102.9 ± 683.8 ng/day), but these changes were markedly suppressed by aliskiren treatment (441.3 ± 104.8 ng/day). AGT protein expression in the kidney was significantly lower in the aliskiren treatment group compared with anti-Thy1.1 group (western blot, 0.8 ± 0.04 and immunohistochemistry, 0.7 ± 0.11 relative to anti-Thy1.1 group). Furthermore, plasma renin activity (PRA) and plasma angiotensin (Ang) II levels were significantly increased in the anti-Thy1.1 group and aliskiren treatment prevented the elevation of PRA and plasma Ang II. Anti-Thy1.1 antibody also elicited left ventricular hypertrophy and fibrosis, which were not ameliorated by aliskiren. These results demonstrate that DRI prevents or reduces intrarenal RAS activation and glomerular injury in progressive glomerulonephritis. Accordingly, DRI may be an effective approach to treat glomerulonephritis as well as other intrarenal RAS associated kidney diseases.
Abstract 202: Activation of Cyp4a12-20-HETE Synthase Increases Blood Pressure and Promotes Vascular Hypertrophy and Cardiac Dysfunction
Authors: Joseph Gregory , Ding Yan , Garcia Victor , Steadman Elisabeth , Capdevila Jorge , Falck John R , Peterson Stephen , Schwartzman Michal L
Published: July 2021

20-Hydroxyeicosatetraenoic acid (20-HETE) is a microcirculatory cytochrome P450-derived eicosanoid shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promotes hypertension. We developed a mouse model (Cyp4a12tg) in which the expression of Cyp4a12, the sole 20-HETE synthase in mouse, is under the control of the doxycycline (DOX) promoter. Administration of DOX to Cyp4a12tg mice increased blood pressure (131±3 vs 100±2 mmHg, p<0.05), which was prevented by co-treatment with the 20-HETE antagonist, 20-HEDGE (96±3 mmHg, p<0.05). Media-to-lumen ratio and medial cross sectional area of renal microvessels from DOX-treated Cyp4a12tg mice significantly increased compared to untreated (M/L, 0.15±0.01 vs 0.07±0.01; mCSAx103 10.8±0.92 vs. 6.4±0.48 mm2); these increases were abolished by co-treatment with 20-HEDGE. Cardiac output and heart rate were unchanged, whereas %EF and %FS were reduced and LV volume and diameter at systole increased in DOX-treated Cyp4a12tg. Total peripheral resistance (TPR) was significantly increased in DOX-treated Cyp4a12tg mice (8.00±0.39 vs 6.77±0.36 mmHg/ml/min, p<0.05); co-treatment with 20-HEDGE decreased (p<0.0%) DOX-induced TPR in Cyp4a12tg mice (7.45±0.22 mmHg/ml/min). These results indicate that activation of Cyp4a12-20-HETE synthase causes hypertension, microvascular remodeling, and cardiac dysfunction. The results also suggest that 20-HETE promotes hypertension by increasing TPR. However, the mechanisms underlying Cyp4a12-20-HETE-driven microvascular remodeling and cardiac dysfunction are yet to be explored.
Abstract 210: Stem Cell Conditioned Culture Media Attenuated Albumin-induced Epithelial-mesenchymal Transition in Renal Tubular Cells
Authors: Hu Junping , Han Weiqing , Zhu Qing , Li Pin-Lan , Li Ningjun
Published: July 2021

Mesenchymal stem cells (MSCs) have been shown to be a promising therapy for many different diseases. Stem cell conditioned culture media (SCM) exhibit similar beneficial effects as MSCs. Albuminuria-induced epithelial-mesenchymal transition (EMT) plays an important role in progressive renal tubulointerstitial fibrosis in chronic renal disease. The present study tested the hypothesis that SCM inhibit albumin-induced EMT in cultured renal tubular cells. SCM were obtained by culturing rat adult MSCs for 3 days. Cultured renal proximal tubular cells were incubated with rat albumin (20μg/ml) and treated with SCM or control culture media. Our results showed that 48 h albumin incubation stimulated EMT in renal proximal tubular cells as shown by significant decrease in the protein levels of epithelial marker E-cadherin from 2.30 ± 0.27 to 0.87 ± 0.11 (
P
< 0.05) and increase in the protein levels of mesenchymal marker fibroblast-specific protein 1 (FSP-1) (2.18±0.33 folds,
P
< 0.05). SCM treatment significantly inhibited these albumin-induced changes in E-cadherin and FSP-1 by 2.33±0.17 and 1.95±0.23 folds (
P
< 0.05), respectively. Meanwhile, albumin increased the mRNA levels of pro-inflammatory factor monocyte chemoattractant protein-1 (MCP)-1 by nearly 30 folds compared with control. SCM almost abolished the increase of MCP-1 induced by albumin. Furthermore, Western blot results displayed that albumin rapidly decreased the cytosolic levels and increased the nuclear levels of NF-κB, indicating a translocation of NF-κB; immunofluorescence microscopy also demonstrated that albumin induced NF-κB translocation from the cytosol into nucleus. SCM blocked the translocation of NF-κB into nucleus. These results suggest that SCM attenuated albumin-induced EMT in renal tubular cells via inhibiting NF-κB activation and inflammation, which may serve as a new therapeutic approach for chronic kidney diseases. (Supported by NIH grant HL89563 and HL106042)
Abstract 211: Variation of NEDD4L is Associated with Hypertension in Chinese Hans Chronic Kidney Disease Patients
Authors: Wang Cheng , Zhang Jun , Li Cuicui , Gong Wenyu , Lou Tanqi
Published: July 2021

Background:
Neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L) is a candidate gene for hypertension, and carriers of an intact NEDD4L C2-domain,encoded by the NEDD4L rs4149601 (G/A) GG genotype, together with the C-allele of the NEDD4L rs2288774 (C/T) polymorphism have been found be associated with hypertension both in African Americans and whites. However, there is no data on the relationship between polymorphism of NEDD4L rs4149601 and rs2288774 and hypertension in Chinese chronic kidney disease (CKD) patients. The purpose of the current study was to investigate the relationship between the variation of NEDD4L rs4149601, rs2288774 and hypertension in CKD patients.

Methods:
A total of 546 Chines Hans CKD patients were enrolled in our study. The SNPs were genotyped using PCR-based techniques. All patients underwent ambulatory blood pressure monitoring, and clinical data were also collected. Multivariate logistic regression analysis was used to identify the relationship between polymorphisms and hypertension.

Results:
506 patients carried GG/GA genotype and 30 carried AA genotype. Rs4149601 AA genotype carriers had significantly higher rate of hypertension (68.3% vs 46.2%, P = 0.022) than GG/GA genotype carriers by Chi-squared test. AA genotype carriers also had a higher day-time and bedtime systolic blood pressure (142±16 vs 135±23, P=0.036; 137±18 vs 127±13, P=0.022, respectively) when compared with GG/GA genotype carriers. AA genotype [OR= 3.08, 95% CI (1.06-9.80)], lowever eGFR [OR=0.98, 95% CI (0.97-0.99)], older age [OR=1.03, 95% CI (1.01-1.05)] were independently associated with hypertension in CKD patients by multivariate logistic regression. However, No difference was found in blood pressure with rs2288774 TT/TC/CC genotypes, and no difference was found in the incidence of hypertension among patients with three genotypes.

Conclusions:
Our results suggested 4149601AA genotype of NEDD4L may be associated with hypertension in CKD patients, and further genetic and functional studies are required to understand its role in the manifestation of hypertension in Chinese CKD patients.
Abstract 220: Exercise Training Attenuates Hypertension and Insulin Resistance in Old Fructose-fed Ovariectomized Rats
Authors: Machi Jacqueline F , Bernardes Nathalia , Dias Daniele S , Moraes-Silva Ivana C , Dos Santos Fernando , Sartori Michelle , Wichi Rogério B , De Angelis Kátia , Irigoyen. Maria Cláudia C
Published: July 2021

Menopause and aging are associated with a gradual increase in systolic blood pressure. Poor eating habits through food containing fructose, have also been related to alterations in the cardiovascular system; however, there are few studies available in the literature relating exercise training (ET), menopause, aging and metabolic syndrome. The aim of the present study was to investigate the effects of ET in ovarian hormone deprivation and/or fructose consumption in old Wistar rats. Female Wistar rats (24 months old) were ovariectomized, fed with fructose (100g/L in drinking water) or normal water, and submitted to an ET protocol in treadmill (1 h/day; 5 days/wk for 8 wk, ~ 40-60% of maximum test) or kept sedentary. The groups were: ovariectomized (O, n= 8), ovariectomized trained (OT, n= 8), ovariectomized fructose (OF, n= 8) and ovariectomized fructose trained (OFT, n= 8). Glycemia, triglycerides, adipose tissue, insulin resistance, physical capacity and blood pressure (BP) were evaluated at the end of the protocol. While the glycemia values remained within the normal range (O:90.1±7.; OT:85.2±2; OF:86±2; OFT:80.6±3 mg/dl) and the triglycerides values were increased in OF group (OF:182±20 vs. O:140.3±9; OT:143.5±7;; OFT:140 ±12 mg/dl). The physical capacity was increased in both OFT (1.8±0.1 Km/h) and OF (1.6±0.07 Km/h) when compared with sedentary peers (O:1.1±0.05; OF: 1.1±0.05 Km/h). The metabolic results showed that ET decreased the adipose tissue in (OT: 6.7±0.9 and OFT: 5.28±0.6 vs. OF: 10.07±0.6 g), and insulin resistance (OT:4.8±0.2 and OFT: 5.0±0.2 vs. OF: 3.2±0.6 mg/dl/%). No differences were observed in O group (O: 6.5±0.8g and 4.5±0.2 mg/dl/min). Finally, ET attenuated the increase in mean BP in both OT and OFT rats (O:119±2; OT 110±2; OF:119±2; OFT: 107±1 mmHg). The same behavior was observed in systolic BP (O:142±2; OT 127±2; OF:143±2; OFT: 129±1 mmHg) and diastolic BP (O:100±2; OT 92±2; OF:98±2; OFT: 89±1 mmHg). In conclusion, low to moderate aerobic ET can attenuate the deleterious effects of ovarian hormone deprivation, and/or fructose consumption, especially the blood pressure increase in old female Wistar rats.
Abstract 230: Evaluation of Hyperglycemia-Driven Alterations on Vascular Endothelial Function
Authors: Hoffmann Brian R , Prisco Anthony R , Wang Jingli , Suboc Tisha M , Widlansky Michael E , Greene Andrew S
Published: July 2021

Type 2 diabetes mellitus (T2DM) afflicts ~25 million people in the United States and hyperglycemia is a major causative factor contributing to vascular dysfunction among this population. This study evaluated the hypothesis that hyperglycemia-induced changes in endothelial glycoproteome leads to alterations of important homeostatic signaling pathways that result in vascular dysfunction. Utilizing targeted glycoprotein enrichment coupled with tandem mass spectrometry for protein identification and quantification, the cell surface glycoproteins were assessed from rat microvascular endothelial cells (RMVECs) cultured in ‘normal’ glucose (5 mM) or ‘high’ glucose (25 mM). Altogether, 273 N-glycosylation modified proteins were identified; 65 proteins uniquely N-glycosylated and 22 proteins significantly increased in N-glycosylation (p<0.05) from ‘high’ glucose RMVECs. Additionally, 499 O-glycoproteins were identified; 78 proteins uniquely O-glycosylated and 65 proteins significantly increased in O-glycoslyation (p<0.05) from ‘high’ glucose RMVECs. The type-1A angiotensin II receptor (AT1; p=0.029), along with numerous cell adhesion molecules, had increased N-glycosylation in ‘high’ glucose RMVECs. Numerous ion channels important for vascular function and the 5-HT2A serotonin receptor (p=5.3e-28), recently shown to contribute to vascular dysfunction in T2DM, had elevated O-glycosylation in ‘high’ glucose RMVECs. In order to evaluate these targets, functional assays were developed to measure endothelial and vascular function. Immobilization assays in a parallel plate flow chamber (PPFC) indicated that ‘high’ glucose significantly increased RMVEC adhesion to gelatin substrate under laminar flow (p<0.05). In addition, incubation of RMVECs under ‘high’ glucose conditions increased the adherence of healthy platelets to RMVECs, suggesting the importance of adhesion protein glycosylation in endothelial dysfunction. Finally, studies in human subcutaneous arterioles demonstrated that acute exposure to ‘high’ glucose impaired acetylcholine-induced vasodilation. Utilizing these assays, differentially hyperglycemia-regulated targets will be functionally evaluated for potential therapeutic intervention.