bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 158: Sex Differences in Response to Chronic Administration of Immunosuppressive Agents in Spontaneously Hypertensive Rats
Authors: Moulana Mohadetheh , Lima Roberta , Zhang Huimin , Mathbout Mohammad , Reckelhoff Jane F
Published: March 2022

Hypertension in male spontaneously hypertensive rats (SHR) is thought to be mediated in part by enhanced immune function. Tacrolimus (FK-506) and Mycophenolate Mofetil (MMF); potent immunosuppressive agents are widely used for the prevention of graft rejection in organ transplantation. Tacrolimus inhibits proliferation of T cells, whereas MMF inhibits proliferation of T cells, B cells and monocytes. However, the impact of the drug on the cardiovascular system of different sexes is unknown. The present study tested the hypothesis that there are sex differences in response to FK-506 and/or MMF in SHR. Young male (YM, age 3 mos; n= 6) and young female (YF, aged 3 mos; n= 6) SHR received tacrolimus (0.25 mg/kg/day i.p. for 14 d) and (YM, age 3 mos; n= 6) and young female (YF, aged 3 mos; n= 6) SHR received MMF (20 mg/kg/day i.p.) for 14 d. Rats were implanted with radiotelemeters and mean arterial pressure (MAP) was measured chronically. Tacrolimus increased MAP in males (Control: 143 ±3 mmHg vs. Tacrolimus-treated: 163 ±4 mmHg, p=S, p<0.05), but had no effect in females (Control: 132 ±3 mmHg vs. Tacrolimus-treated: 133 ±2 mmHg, p=NS). In contrast, Mycophenolate Mofetil significantly reduced MAP in both males (Control: 153 ±2 mmHg vs. MMF-treated: 140 ± 2 mmHg; p <0.05), and females (Control: 128 ±2 mmHg vs. MMF-treated: 114 ±2 mm Hg, p<0.05). These data suggest that immunosupproessive drugs may have different effects in men and women.
Abstract 16: Adoptive Transfer of T Lymphocytes Prevents Angiotensin II-induced Vascular Stiffness in rag-1 Knockout Mice.
Authors: Mian Muhammad Oneeb Rehman , Barhoumi Tlili , Briet Marie , Ene Adriana Cristina , Paradis Pierre , Schiffrin Ernesto L
Published: March 2022

Background:
Both innate and adoptive immune systems are involved in the pathogenesis of hypertension and vascular damage. T lymphocytes participate in the low-grade inflammatory response that contributes to vascular injury in cardiovascular disease, including hypertension. Angiotensin (Ang) II-induced hypertension and endothelial dysfunction are blunted in rag1 knockout (rag1-/-) mice, which are deficient in T and B lymphocytes, and restored with adoptive transfer of T but not B lymphocytes. We hypothesized that adoptive transfer of T lymphocytes from C57Bl/6 mice (WT) will exacerbate Ang II-induced vascular damage in rag1-/- mice.

METHODS:
Eleven-week old male rag1-/- mice were injected i.v. with PBS or 10 x 106 pan T lymphocytes, and 2 weeks later implanted with a dummy pump (control) or infused with Ang II (490 ng/kg/min, s.c.) for 14 days (n=5-8). Systolic (SBP) and diastolic blood pressure (SBP) were measured by telemetry. Endothelial function and vessel structure were assessed in second order mesenteric arteries by pressurized myography. Successful adoptive transfer was confirmed at time of sacrifice by determining numbers of T cells in the spleen by flow cytometry.

Results:
Ang II induced a similar 40 mmHg SBP rise in rag-1-/- mice injected with PBS or T cells, but DBP rise was greater for T cell-injected mice (24 mmHg) than for PBS injected mice (9 mmHg). Ang II impaired vasodilatory responses to acetylcholine in T cell-injected mice (54.0±5.9%) but not in PBS injected mice (82.1±2.9%). Ang II treatment induced hypertrophic remodeling in PBS injected mice, but not in T cell-injected mice (media-to-lumen ratio: 4.1±0.3 from 2.7±0.3 vs. 3.3±0.2 from 2.6±0.1, media cross-sectional area (μm2): 5757±565 from 4382±580 vs. 4811±281 from 4350±254). Ang II increased vascular stiffness, indicated by a leftward shift of the small artery stress/strain relationship, in PBS injected mice but to a lower extent in T cell-injected mice.

Conclusions:
These findings suggest that T lymphocytes have a protective role in Ang II-induced vascular stiffness.
Abstract 164: CYP17A1 Variant is Associated with Blood Pressure Reactivity to a Mental but not a Physical Challenge in Male Adolescents and May Be an Early Marker of Hypertension Risk
Authors: Van Woudenberg Mariel , Melka Melkaye G , Bernard Manon , Syme Catriona , Abrahamowicz Michal , Leonard Gabriel , Perron Michel , Richer Louis , Veillette Suzanna , Gaudet Daniel , Paus Tomas , Pausova Zdenka
Published: March 2022

Individuals who exhibit exaggerated blood pressure (BP) reactivity to physical and mental challenges at a young age are at increased risk of developing hypertension in adulthood. Hypertension is a complex genetic trait that is more prevalent in men than women during reproductive age. CYP17A1 is one of the best-established gene loci of hypertension. It encodes a key enzyme in the steroidogenic pathway that produces mineralocorticoids, glucocorticoids, androgens, and estrogens; as such, it may influence BP reactivity in a sex-specific manner. Here, we investigated whether CYP17A1 is associated with BP reactivity to physical and mental challenges in adolescence. In 285 male and 311 female adolescents (age 12-18 years), we measured systolic BP (SBP) and diastolic BP (DBP) beat-by-beat during a 52-minute protocol, which included a physical (10-min standing) and mental (2-min math test) challenge and we genotyped 8 tagging single nucleotide polymorphisms (SNPs) that covered the entire region of CYP17A1. Genotype-phenotype association tests were performed separately in males and females, while adjusting for age, height and initial BP. Our results showed that 6 SNPs were associated with higher SBP and DBP reactivity (p=0.003-0.0001), but only in response to mental stress and only in boys. At
rs619824
, minor allele homozygotes differed from major allele homozygotes by 8.7 mm Hg of SBP (p=3x10-4) and 4.8 mm Hg of DBP (p=1x10-4) in boys, and the two groups varied only by 2.0 mm Hg of SBP (p=0.31) and 0.9 mm Hg of DBP (p=0.42) in girls. These results suggest that CYP17A1 may contribute to the development of hypertension through the regulation of the stress response and that, at least in adolescence, this effect appears to be present in males but not in females.
Abstract 172: Toll-like Receptor-4 Mediates Augmented Contractility In Diabetic Resistance Vessels
Authors: Carrillo-Sepulveda Maria Alicia , Szasz Theodora , Nunes Kenia P , Webb R Clinton
Published: March 2022

Hyperglycemia, the hallmark of diabetes, plays a central role in the pathogenesis of diabetic vascular dysfunction; however the underlying mechanisms are not entirely understood. We previously reported that inhibition of Toll-Like Receptor- 4 (TLR4), a key receptor of the innate immune system, improves vascular function in spontaneously hypertensive rats (SHR) revealing a novel role of TLR4 in the vascular system. In our current study, we hypothesized that upregulation of TLR4 in the vasculature following hyperglycemia leads to augmented vascular contractility. Third order mesenteric arteries from streptozotocin (STZ)-induced diabetic Sprague-Dawley rats (glucose 279 ± 11.6mg/dL) and their respective controls (98 ± 8.03mg/dL ), were incubated with either neutralizing antibody to TLR4 (1ug/mL) or IgG (1ug/mL) for 30 min and contractile responses to phenylephrine (PE) were evaluated ex vivo. Vessels from STZ rats exhibited greater maximal contractile force to PE compared to control (Emax 38.99 ± 1.45 vs. 23.98 ± 0.73 (mN), p<0.05, respectively). Anti-TLR4 incubation attenuated this response to PE in vessels from STZ rats (29.71 ± 1.69mN) but had no effect in control vessels (21.03 ± 1.02mN). To test whether hyperglycemia modulates vascular TLR4, mesenteric vascular smooth muscle cell (MVSM) cultures were stimulated with high glucose (HG 25mM) or normal glucose (NG 5mM) for 48 hours. HG increased protein expression of TLR4 in MVSM compared to MVSM stimulated with NG (2.74±0.34 vs 1.38±0.20, p<0.05). Concurrently, HG caused activation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) (5.04±0.83 vs 3.80±0.90, p<0.05), increased levels of PCNA, a marker of proliferation, (1.34±0.11 vs 0.35±0.04, p<0.05), and decreased levels of calponin, a marker of contractile phenotype, (1.45±0.14 vs 3.62±0.40, p<0.05). Levels of total intracellular reactive oxygen species (ROS) were robustly augmented under HG condition (2.2 fold of the normal glucose). Our results suggest that hyperglycemia upregulates vascular TLR4 leading to augmented vascular contractility, potentially due to activation of ERK1/2, increased ROS and MSMCs proliferation.
Abstract 187: A Postnatal Diet Rich in Fat and Sucrose Leads to the Differential Alterations in Renal Function and Metabolic Health in Male Control and Intrauterine Growth Restricted Offspring
Authors: Intapad Suttira , Brown Andrew D , Tull F L , Dasinger John H , Ojeda Norma B , Alexander Barbara T
Published: March 2022

We previously reported that reduced uterine perfusion induced at day 14 of gestation in the rat programs a marked increase in mean arterial pressure (MAP) in male intrauterine growth restricted (IUGR) offspring compared to male control with no impact on renal function or metabolic health. Recent studies suggest that the degree of mismatch between the pre- and postnatal environments can alter the programming of adult health and disease. This study tested the hypothesis that IUGR followed by exposure to a diet rich in fat and sucrose would exacerbate programmed increases in blood pressure, alter renal function and impair metabolic health in male IUGR offspring. Control and IUGR offspring were maintained on regular rodent chow (RC) or switched to high fat/high sucrose (HF/HS) diet at delivery and maintained on the RC or HF/HS diet for 6 months (control RC: n=12, IUGR RC: n=9, control HF/HS: n=13, IUGR HF/HS n=12). MAP, measured in conscious, chronically instrumented via arterial catheter, was significantly increased by postnatal exposure to a HF/HS diet in both control and IUGR offspring (149.7±3 versus 150.6±3 mmHg, control HF/HS versus IUGR HF/HS, respectively); thus, normalizing the significant difference in MAP observed in control versus IUGR offspring on RC. Glomerular filtration rate (GFR) adjusted per kidney weight did not differ in control RC or IUGR RC. However, it was significantly reduced in IUGR HF/HS relative to control HF/HS (1.9±0.5 vs. 0.3±0.1 mg/min/gram kidney;
P
<0.05, control HF/HS versus IUGR HF/HS, respectively) indicating that despite no difference in MAP in the HF/HS groups, a mismatch of pre- and post-natal diet altered renal function in IUGR offspring. Represented by area under the curve (AUC), an impaired oral glucose tolerance test was observed in control HF/HS relative to all other groups (
P
<0.05); AUC did not differ in IUGR HF/HS relative to offspring on RC (control RC: 436.6±15 versus IUGR RC: 464.0±27, control HF/HS: 552.2±23, and IUGR HF/HS 469.7±23). Thus, male IUGR offspring were protected from impaired metabolic health following postnatal exposure to HF/HS diet. These findings indicate that male control and IUGR offspring exhibit a differential response to postnatal exposure to a HF/HS diet on renal function and metabolic health.
Abstract 200: Time Course of Macrophages Infiltration and Apha 2 Adrenergic Autoreceptor Impairment in DOCA-salt Hypertensive Rats
Authors: Thang Loc V , Galligan James J
Published: March 2022

The development of DOCA-salt hypertension displayed a phasic phenomenon: pre-hypertension, phase 1, phase 2 and established hypertension. We have reported that in established DOCA-salt hypertension rats there were in increased number of pro-inflammatory macrophages (Mϕ) in the mesenteric arteries (MA) adventitia, and the α2-adrenergic autoreceptor (α2AR) regulating norepinephrine (NE) release from sympathetic nerves supplying arteries is impaired. DOCA-salt rats treated with apocynin, an NADPH-oxidase inhibitor, exhibited reduced inflammation and are protected against established hypertension. The proposed studies tested the hypothesis that as blood pressure increased in DOCA-salt rats, Mϕ infiltrated into the adventitia of MA. Mϕ released O2- that disrupted α2AR function causing increased NE release and further increased in blood pressure. Using immunohistochemistry, we detected the number of Mϕ in MA adventitia of phase 1, 2 and established hypertension was 4-5 higher than control (p<0.05). Similarly, the level of O2-, detected with relative ratio of DOCA-Sham mean fluorescence intensity of dihydroethidium, in DOCA-salt (2-2.5) peaked higher than control (1.0) during the same time period (p<0.05). However, using amperometry we found that the normalized NE current in the presence of Idazoxan, an α2AR antagonist, was significantly difference between DOCA and SHAM MA only during Phase 2 and established hypertension (p<0.05). The data suggests in DOCA-salt hypertension development, as the blood pressure increases to Phase 1 hypertension Mϕ is initially recruited to the MA adventitia where they release O2-. Consequently, over a few days period the O2- disrupts the α2AR function causing increased NE release and further increased in blood pressure.
Abstract 202: Temporal Evaluation of Baroreflex Dysfunction: Role in Target Organ Damage
Authors: Jorge Luciana , Paulini Janaina , Mostarda Cristiano , Moreira Edson D , irigoyen Maria Claudia
Published: March 2022

Introduction:
Baroreceptors regulate moment-to-moment blood pressure (BP) variations, but their long-term effect on the cardiovascular system and target organ damage remains unclear.

Purpose:
to investigate the temporal mechanisms enrolled in the relationship between baroreceptor reflex dysfunction and end-organ damage in different pathophysiological conditions as hypertension, heart failure and myocardial infarction.

Method:
Male Wistar rats were divided into 5 groups: control (n = 9),SAD1(n=8,24h), SAD2(n=8,28 days), SAD3 (n=8, 90 days) and SAD4 (n=7, 6 months). Sinoaortic denervation (SAD) was used as a model of arterial baroreflex deficit. BP, HR were analysed using a data acquisition system (Codas, 2kHz). Cardiac output and tissues blood flow were evaluated using colored microspheres.

Result:
BP and the HR was incresed in SAD after 24h (C:106 ± 0.6
vs
. SAD1: 142 ± 2 mmHg; C: 355 ± 7
vs
. SAD1: 453 ± 10 bpm), but it were normalizaed in chronic SAD. Moreover, it was observed a gradual reduction over time in cardiac output (C: 111 ± 7, SAD1:81±5, SAD2:85 ± 6, SAD3:69±5 SAD4:35±2 mL/min) accompanied by an increase in total peripheral resistance (C:0.97±0.07, SAD1:1.74±0.05, SAD2:1.23±0.07, SAD3:1.60±0.09, SAD4:3.00±0.05 mL/min/mmHg) in SAD groups. Those alterations resulted in a decreased renal blood flow (C:3.47±0.21, SAD1:1.37±0.19, SAD2:1.49±0.18, SAD3:1.12±0.13, SAD4:0.42±0.16 mL/min/g) , cardiac blood flow (C:2.91±0.71, SAD1:2.07±0.19, SAD2:1.58±0.28, SAD3:0.92±0.13, SAD4:0.34±0.16 mL/min/g) and muscle blood flow (C:0.24±0.02, SAD1:0.08±0.005, SAD2:0.10±0.02, SAD3:0.05±0.01, SAD4:0.05±0.007 mL/min/g) in SAD groups.

Conclusion:
The acute total baroreflex dysfuntion incresed BP and HR and reduced blood flow and cardiac output, these changes are associated with the body’s attempt to maintain homeostasis. Howerer, the temporal evaluation showed a progressive reduction in CO and regional blood flow associated with an increase in RPT to maintain blood pressure levels. These adjustements may play an important role in target organ-damage in different pathological conditions, independently of BP levels.
Abstract 205: Tissue Transglutaminase as a Novel Pathogenic Factor and Therapeutic Target for Preeclampsia
Authors: Liu Chen , Wang Wei , Parchim Nicholas , Irani Roxanna , Ning Chen , Sun Kaiqi , Blackwell Sean , Kellems Rodney , Xia Yang
Published: March 2022

Preeclampsia (PE) is a prevalent life-threatening complication of pregnancy featuring hypertension, placental abnormality, and renal damage. The condition is associated with the presence of agonistic autoantibodies (AT1-AA) capable of activating the major angiotensin receptor, AT1R, and believed to contribute to disease pathogenesis. Tissue transglutaminase (tTG), a prominent member of a family of enzymes that crosslink proteins by catalyzing the formation of inter- or intra-molecular ε-(γ-glutamyl)-
lysine
isopeptide bonds, is known to interact with G protein-coupled receptors and contribute to hypertension. Thus, we hypothesize that AT1-AA may function via tTG to crosslink AT1R and contribute to pathophyhsiology of PE. To test this hypothesis, using immunoprecipation with anti-tTG antibody, we successfully pulled down more AT1R from human trophoblast (HTR) cells treated with IgG from PE women than IgG from normotensive (NT) women, thereby providing initial evidence for the promotional effect of AT1-AA on tTG and AT1R interaction. Subsequently, we measured tTG and its modification on AT1R in both NT and PE placentas. We found increased levels of tissue transglutaminase (∼2 folds), ε-(γ-glutamyl)-
lysine
isopeptides (∼2 folds), and AT1R with ε-(γ-glutamyl)-
lysine
isopeptide bonds at the microvillous membrane of PE placentas, indicating that tTG-induced AT1 receptor modification is elevated in PE placenta. Finally, in an effort to determine the role of elevated tTG in AT1-AA-induced PE in vivo, we treated PE-IgG injected pregnant mice with cystamine, a potent tTG inhibitor. Compared with PE-IgG injected mice, less AT1 receptor with ε-(γ-glutamyl)-
lysine
isopeptide bonds was found in the placental labyrinth zone of PE-IgG injected mice treated with cystamine. Moreover, cystamine treatment significantly attenuated key features associated with PE including hypertension (from 159.5±5.6 to 132.6±2.7 mm Hg) and proteinuria (from 106.5±37.8 to 38.5±6.9 ng albumin/mg creatine) in the PE-IgG-injected pregnant mice as well. Taken together, here we report a previously unrecognized role of tTG in the pathogenesis of PE by crosslinking AT1Rs and suggest a novel therapeutics for the disease.
Abstract 208: TLR3-Induced Placental miR-210 Down-Regulates the STAT6/IL-4 Pathway
Authors: Chatterjee Piyali , Kopriva Shelley E , Chiasson Valorie L , Mitchell Brett M
Published: March 2022

Several clinical studies have reported differential expression of microRNAs (miRs) in placentas from women with preeclampsia (PE) compared to normotensive women. Whether or not miRs play a role in the etiology of PE is unknown. Given the important role of maternal immune system activation and inflammation in PE, we developed a mouse model of PE via activation of Toll-like receptor 3 (TLR3) which exhibit hypertension, endothelial dysfunction, and proteinuria only when pregnant. miR-210 is increased in placentas of women with PE, but whether TLR3 activation in pregnant mice and human cytotrophoblasts (CTBs) increases miR-210 and the effects on miR-210 targets related to inflammation are unknown. miR-210 expression was up-regulated in placentas of poly I:C-treated control mice (P-PIC: 4.9 fold, p<0.05 vs. controls) similar to that in women with PE. Both HIF-1α and NF-κB p50 are known to bind to the promoter of miR-210 and induce its expression. HIF-1α (1.5 fold, p<0.05 vs. controls) and NF-κB p50 (2.7 fold, p<0.05 vs. controls) expression were increased significantly in placentas of P-PIC mice. Target identification algorithms and gene ontology predicted STAT6 as a target of miR-210 related to inflammation and STAT6 was decreased significantly in placentas of P-PIC mice. Increased production of the anti-inflammatory cytokine IL-4, which is regulated by STAT6, plays a critical role during normotensive pregnancy but failed to increase in P-PIC mice. Pregnant TLR3 KO mice treated with poly I:C, which did not develop hypertension, did not have increased placental expression of HIF-1α, NF-κB p50, and miR-210 or decreased expression of STAT6 and IL-4. To determine the placental etiology, human CTBs were treated with poly I:C and HIF-1α, NF-κB p50, and miR-210 expression were increased significantly while STAT6 and IL-4 expression decreased significantly. Inhibition of miR-210 in CTBs using anti-miR-210 significantly increased STAT6 and IL-4 expression and overexpression of miR-210 decreased STAT6 and IL-4 expression in CTBs. Taken together our data suggest that TLR3 activation induces placental miR-210 via HIF-1α and NF-κB p50 which in turn decreases STAT6 and IL-4 expression and may contribute to the development of PE.
Abstract 210: Urinary Renin as a New Marker of Preeclampsia?
Authors: Verdonk Koen , Visser Willy , van den Meiracker Anton H , Danser A.H.Jan
Published: March 2022

Urinary renin-angiotensin system (RAS) components have been suggested to reflect intrarenal RAS activity. Urinary renin is elevated in diabetes, thus explaining the effectiveness of RAS blockade in diabetic nephropathy despite the low-to-normal plasma renin levels in this condition. Here we investigated urinary renin levels in preeclampsia (PE), a disease characterized by hypertension and proteinuria. We speculated that urinary renin levels might be of additional value on top of the elevated sFlt-1/PlGF ratio in PE. Renin, prorenin, creatinine (Cr), total protein, s-Flt-1 and PlGF were determined in plasma (15 PE, 6 non-PE) and/or 24 hour-urine samples (39 PE, 27 non-PE) of pregnant women. Gestational age was 33±1 in PE vs. 37±0 weeks in non-PE. The sFlt-1/PlGF ratio (523 (30-49996) vs. 10 (2.9-32.5)) (geometric mean and range) was higher in PE vs. non-PE. Plasma renin and prorenin levels in PE were 42 (range 11-181) and 190 (95-274) ng/L, versus 41 (16-80) ng/L and 129 (87-160) ng/L in non-PE. Plasma total protein levels in PE and non-PE were 50 (21-68) and 61 (58-66) g/L, resp., versus 1.0 (0.3-13) and 0.2 (0.1-1.5) g/L in urine. Plasma Cr levels in PE and non-PE were 61 (39-110) and 61 (47-83) μmol/L, resp., versus 31 (2.6-241) and 11 (3.4-56) mmol/L in urine. Urinary renin levels were ≈3 times higher in PE than in non-PE (3.2 (0.1-410) vs. 1.4 (0.3-13.0) ng/L), and 5-10 times higher than expected if filtration of plasma renin paralleled filtration of plasma protein. In contrast, urinary prorenin levels were 0.4 (0.1-84) and 0.1 (0.1-0.9) ng/L in PE and non-PE, i.e., as high as expected on the basis of plasma protein filtration. In line with these observations, urinary total protein and Cr correlated with urinary prorenin (r=0.38, P<0.05 and r=0.71, P<0.01, resp.), but not with urinary renin. In conclusion, urinary prorenin levels run in parallel with urinary total protein and Cr levels, and are as high as can be expected based upon protein filtration in the kidney. In contrast, urinary renin levels are 5-10 fold higher than expected based on filtration, and do not correlate with urinary protein or Cr. Urinary renin may therefore reflect an activation of the renal RAS in PE, and could serve as a new marker of the severity of this disease, in particular its renal consequences.
Abstract 216: Mechanism of Pressure-Natriuresis: Evidence That Na+/K+-ATPase is a Renal Extracellular Cyclic Guanosine 3’,5’-Monophosphate Receptor
Authors: Gherghe Cristina , Kemp Brandon A , Howell Nancy L , Carey Robert M
Published: March 2022

Pressure-natriuresis (P-N) is the main compensatory mechanism by which the kidney excretes sodium (Na+) in response to an increase in blood pressure. Extracellular renal interstitial (RI) cyclic guanosine 3’,5’-monophosphate (cGMP) modulates P-N by binding to an unidentified receptor on the basolateral membrane of renal proximal tubule cells and activating the Src tyrosine kinase signaling cascade. In this study, we hypothesized that the cGMP receptor is the extracellular domain of Na+/K+-ATPase (NKA). In uninephrectomized Sprague Dawley rats, the renal perfusion pressure (RPP) was increased from 110 to 140 mmHg by ligating the infrarenal aorta, celiac, and mesenteric arteries, in a standard P-N model. In response to increased RPP, urinary Na+ excretion (UNaV) increased from 0.35±0.065 to 2.19±0.31 umol/min (P<0.0001; N=14). Rostafuroxin, a digitoxigenin derivative that specifically displaces ouabain binding from NKA without inhibiting NKA pump activity, significantly decreased UNaV in a dose-dependent manner, from 2.19±0.31 to 1.38±0.18 (P<0.05; N=8) and to 0.34±0.080 umol/min (P<0.0001; N=6), at RI infusion rates of 2.4 and 12 ng/kg/min, respectively. In contrast, at higher infusion rates, of 60 and 120 ng/kg/min, rostafuroxin increased UNaV to 2.49±0.45 (P=0.60; N=8) and to 3.68±0.49 umol/min (P<0.05; N=8), respectively. Thus, rostafuroxin is a pharmacologic agent with NKA antagonist/agonist activity, depending on dose. To show that cGMP-induced natriuresis depends upon NKA binding, we microinfused cGMP directly into the RI compartment of uninephrectomized Sprague Dawley rats without changing the RPP. We compared UNaV in the second hour period of a 2-hour RI microinfusion of vehicle (0.66±0.086 umol/min; N=5), to rostafuroxin 12 ng/kg/min (0.69±0.11 umol/min; P=0.81; N=6), cGMP 72 ng/kg/min (3.41±0.57 umol/min; P<0.005; N=7), or cGMP together with rostafuroxin (0.64±0.21 umol/min; P=0.93; N=6). The rostafuroxin dose that abolished P-N also nullified the natriuretic response to cGMP. In summary, RI microinfusion of rostafuroxin blocked both pressure- and cGMP-induced natriuresis. These data provide strong evidence that the extracellular domain of NKA serves as a receptor for RI cGMP, which mediates P-N.
Abstract 22: Ablation of Leptin Receptors in the Brain Subfornical Organ Attenuates Leptin-induced Increases in Renal Sympathetic Nerve Activity (RSNA) But Spares Its Effect on Food Intake and Body Weight
Authors: Young Colin N , Morgan Donald A , Mark Allyn L , Davisson Robin L
Published: March 2022

The adipocyte-derived hormone leptin acts within the central nervous system to decrease food intake and body weight and to increase sympathetic nerve activity to the kidney. Although most studies have focused on hypothalamic brain regions, recent findings have identified the long form of the leptin receptor (ObR) in the circumventricular subfornical organ (SFO), a forebrain region devoid of a blood-brain barrier. Here we tested the hypothesis that ObR receptors in the SFO are functionally involved in the decreases in food intake and body weight and increases in RSNA due to leptin. To delete ObR receptors selectively in the SFO, an adenovirus encoding Cre-recombinase (Cre; n=4) was targeted to this brain region in ObR
flox
mice. LacZ served as a control (n=4). After 7 days, systemic leptin (i.p. 30μg bi-daily) was administered over 3 days. Leptin caused a significant decrease in body weight and food intake in control animals. Interestingly, ablation of the ObR receptor in the SFO did not attenuate the leptin-induced decreases in either body weight (peak decrease: Δ-2.2±0.1 vs. Δ-2.4±0.2 g; LacZ vs. Cre, p>0.05) or food intake. Similar findings were obtained with daily intracerebroventricular (ICV) administration of leptin. To examine a role for SFO-ObR in cardiovascular-sympathetic regulation, ObR
flox
mice underwent SFO-targeted adenoviral delivery of Cre or LacZ as above. Following recovery, mice were instrumented for anesthetized recordings of RSNA and leptin was administered systemically (i.v. 120μg) or ICV (2μg). Both systemic and centrally administered leptin caused robust increases in RSNA over 4 hours in control mice. This response was completely abolished in mice with SFO-targeted deletion of ObR (e.g., i.v. leptin % RSNA 4hr: 116±16 vs. -20±9 v*s/min; LacZ vs. Cre, p<0.05, n=5/group). These findings suggest that ObR receptors in the SFO play a critical role in leptin-induced renal sympathoexcitation, but not in the body weight and food intake effects of leptin. These findings emphasize two important concepts: 1) A distributed brain network as opposed to a hypothalamic-centric concept of leptin action; and 2) Selectivity of leptin action mediated by distinct brain regions, including the circumventricular organs.

HL063887,
HL084207
Abstract 221: Renoprotective Effects of SGLT2 Inhibitor, Luseogliflozin, on the Progression of Diabetic Nephropathy in T2DN Rats
Authors: Kojima Naoki , Williams Jan M , Takahashi Teisuke , Miyata Noriyuki , Roman Richard J
Published: March 2022

This study examined the effect of long term control of hyperglycemia with a new selective SGLT2 inhibitor, luseogliflozin (TS-071), given alone and in combination with lisinopril on the development of renal injury in a rat model of type 2 diabetic nephropathy (T2DN). Chronic treatment of 14-month old T2DN rats with luseogliflozin (10 mg/Kg/day) reduced plasma glucose levels from 285.7±17.7 mg/dL to 161.7±5.5 mg/dL and HbA1c from 8.9±0.4% to 6.4±0.2% in 17-month old T2DN rats but it had no effect on mean arterial pressure (MAP). Chronic treatment with lisinopril (10 mg/Kg/day) markedly reduced MAP (113.0±4.6 mmHg) compared with 17-month old T2DN rats (140.0±4.1 mmHg) but it had no effect on plasma glucose levels. Combination therapy with luseogliflozin and lisinopril significantly reduced MAP (92.9±4.0 mmHg) more than that seen in lisinopril treated rats. The kidneys of T2DN rats exhibited marked focal glomerulosclerosis with a glomerular injury score of 3.1±0.1 on a 0-4 scale and renal cortical fibrosis of 27.9±0.9%. Control of hyperglycemia with luseogliflozin reduced the glomerular injury score (2.6±0.1) and the renal cortical fibrosis (20.8±0.8%). Reducing blood pressure with lisinopril also reduced the glomerular injury score (2.5±0.1) but not the renal cortical fibrosis (29.0±1.5%). Combination therapy with luseogliflozin and lisinopril markedly reduced the glomerular injury score (2.0±0.1) and the renal cortical fibrosis (15.6±1.0%) more than either drug alone. GFR decreased from 0.44±0.04 to 0.29±0.03 mL/min/g- kwt and RBF decreased from 2.7±0.2 to 2.2±0.2 mL/min/g-kwt in T2DN rats over the course of this study. Chronic treatment of luseogliflozin or lisinopril prevented the decrease of GFR (0.44±0.05 and 0.54±0.05 mL/min/g-kwt) and RBF (3.2±0.2 and 6.7±1.1 mL/min/g-kwt). These results suggest that strict control of blood glucose with the luseogliflozin slows the progression of diabetic nephropathy and combination therapy with luseogliflozin and lisinopril is more effective at lowering blood pressure and is more renoprotective than administration of lisinopril alone.
Abstract 228: Transcriptional Networks Involved in Differentiation of Renal Progenitor Cells to the Juxtaglomerular Cell Fate
Authors: Gomez Jose A , Chien Jennifer , Jameson Samantha , Jayawardena Tilanthi , Perez-Marco Romelia , Yang Yanqiang , Hodgkinson Conrad , Capel Blanche , Dzau Victor J , Mirotsou Maria
Published: March 2022

Background:
The molecular processes regulating the development of renin producing juxtaglomerular (JG) cells are not fully understood. We have previously shown that LXRα and cAMP treatment can induce mesenchymal stromal cells (MSCs) from different sources to differentiate into renin expressing JG-like cells. Here, we investigated the LXRα/cAMP induced transcriptional networks involved in the commitment of progenitor cells to the JG lineage.

METHODS:
Adult renal CD44+ MSCs were isolated from C57BL/6 Ren1c YFP mice. MSCs were differentiated into YFP+ renin expressing cells by treatment with LXRα ligand (T0901317) or cAMP. The Affymetrix Mouse 430 2.0 array was used for profiling. Data analysis was focused on transcriptional and epigenetic regulators and performed by using Dchip, David or Toppgene.

Results:
Differentiated YFP+ cells from either cAMP or LXRα treatment expressed high levels of renin and renin regulators such as Hoxd8 and Cebp. Untreated MSCs showed expression of metanephric markers such as Foxd1, Sox11 and Eya. Treatment with cAMP resulted in differential expression of 108 transcripts in YFP+ cells as compared to control MSC. The majority of these genes (82) were down-regulated and involved in chromatin acetylation (23 out of 82, P<2E-5) or embryonic and hematopoietic development (36 out of 82 respectively, P<2E-5). Mesenchymal markers (i.e. Sox11, FC= -3) and repressors of differentiation (i.e. Id2, FC= -2) were also decreased significantly. Similarly, MSC activation by LXRα resulted in differential expression of 98 transcripts, 67 of which were decreased compared to baseline. As with cAMP, the majority of genes were involved in differentiation and chromatin remodeling (i.e Tbx3, Klfs and Mll); still, the overlap of genes between the two treatments was minimal (24 genes) suggesting that cAMP and LXRα might act complementary to each other.

Conclusion:
Our data indicate that cAMP and LXRα, rather than directly define JG cell fate, modulate the epigenetic status of MSCs and prevent differentiation to other lineages. Systems biology approaches are under way to evaluate the importance of the transcriptional networks identified. We anticipate that these results will provide novel insights into the mechanisms regulating JG cell fate.
Abstract 233: Evidence for an Interaction Between Systemic and Renal Angiotensinogen in the Control of Arterial Pressure
Authors: Ramkumar Nirupama , Kohan Donald
Published: March 2022

Both the liver and proximal tubule make angiotensinogen (AGT), and overexpression of AGT in either region increases blood pressure (BP). To determine the relative contributions of renal and systemic AGT in modulating BP, mice were used with AGT overexpression in kidney (K), liver (L) or both sites (KL). Male transgenic and wildtype (WT) controls aged 24 weeks underwent BP recording via telemetry and metabolic cage studies on normal (0.25%) and high (3.2%) sodium diets. Mean BP (MAP) was higher in K and KL vs L and WT mice fed normal salt, while MAP in L mice was higher than controls. High salt intake did not alter MAP in WT, K or KL mice but increased it in L mice. Plasma AGT levels, plasma renin concentration, urine Na excretion and urine volume were similar between groups regardless of sodium intake. Urinary AGT (ng/day) on normal sodium intake was: WT: 5±1, L: 4±1, K: 20±5 and KL: 34±9. High salt increased urine AGT in all groups (WT: 19±4, L: 42±8, K: 290±75, KL: 407± 72). High salt did not alter plasma AGT levels but reduced plasma renin concentration in all groups. Taken together, these data show a correlation between urinary AGT and BP. Mice with renal AGT overexpression may be maximally hypertensive on a normal sodium diet since renal AGT is already high. Mice with liver AGT overexpression have mild hypertension on a normal sodium diet that is exacerbated by high sodium intake; this is associated with a significant increase in urinary AGT as compared to WT animals. Thus, systemic AGT may have the potential to regulate renal AGT and this effect may be a key determinant of BP.
Abstract 238: Nuclear Renin-Angiotensin System in Renal Proximal Tubule Cells
Authors: Alzayadneh Ebaa M , Brosnihan K. Bridget , Diz Debra I
Published: March 2022

Compelling evidence for an intracellular renin-angiotensin system (RAS) is evident in multiple tissues including the heart, vasculature, brain and kidney. Indeed, the full complement of angiotensin receptors is localized on renal cortical nuclei; however, elucidation of the intracellular pathways that contribute to the expression of Ang II or Ang-(1-7) is incomplete. The current study sought to identify a proximal tubule epithelial cell model to facilitate our understanding of the regulation and function of this intracellular system. Utilizing the rat-derived NRK52E epithelial cells, immunofluorescent staining and western blots revealed nuclear expression of angiotensinogen (Aogen), renin and the prorenin receptor (PRR). Further characterization of Aogen expression utilizing an antibody against the protein region distal to Ang I ([des-Ang I]-Aogen) revealed a single 60 kDa band evident in the nuclear and the cytosolic fractions of the cells. A second antibody to the Ang I sequence of Aogen revealed expression in the cytosol and collected cell media, but not the nuclear fraction. The large molecular form of renin in the nuclear fraction suggests the predominance of prorenin in the nucleus. Basal renin activity in the nuclear fraction as measured by Ang I generation was significantly inhibited by the specific renin inhibitor aliskiren [770 ± 46 vs. 129 ± 14 pg Ang I/mg protein, n=3; p<0.01]. Conversion of the proform to active renin (total renin) by trypsin treatment of the nuclear fraction increased renin activity 2-fold [1933 ± 230 pg Ang I/mg; n=3; p<0.01]; aliskiren significantly reduced total renin activity by 92% [167 ± 24 pg Ang I/mg, n=3; p<0.01]. Utilizing specific radioimmunoassays, we detected both Ang II and Ang-(1-7) at concentrations of 50 ± 24 and 51 ± 39 pg/mg protein, respectively (n=3) in nuclear extracts of NRK cells. Collectively, these data suggest that processing of Aogen from either prorenin or renin may occur on the nucleus forming [des-Ang I]-Aogen and the active peptides Ang II and Ang-(1-7). We conclude that a nuclear prorenin/renin-Aogen pathway may constitute one source of peptide ligands for intracellular angiotensin receptors.
Abstract 240: Blood Pressure, Renal Function and Hemodynamic Responses to Systemic Ang1-7 in Rats Infused Chronically With Ang Ii: Does the Ang1-7 Natriuretic Effect Depend on Renal Hemodynamics?
Authors: Kuczeriszka Marta , Seth Dale M , Prieto Minolfa C , Navar Gabriel L
Published: March 2022

Angiotensin II (AngII) is the primary effector of the renin-angiotensin system (RAS) eliciting hypertensinogenic effects. The final levels of AngII depend on AngII synthesis by angiotensin-converting enzyme (ACE) and its degradation to Ang1-7 by carboxypeptidase (ACE2) and to AngIII by aminopeptidase A. Ang 1-7 exerts natriuretic effects and counteracts some of the actions of increased AngII. The downregulation of ACE2 protein in the kidneys of hypertensive rats suggests that reduced Ang1-7 may play a role in the overall blood pressure, renal hemodynamic and excretory responses to chronic AngII infusion. In the present study, we examined the effects of systemic Ang1-7 infusions on blood pressure, renal hemodynamics and urinary excretion in male Sprague-Dawley rats subjected to chronic AngII infusion (80ng/min) for 14 days (N=8) and in control (N=6) rats. We examined if the natriuretic activity is associated with renal hemodynamic changes and if it is modified by RAS activation in the AngII infused rats. In surgically prepared rats, Ang1-7 was infused systemically at a dose of 110 fmol/min. Systemic Ang1-7 infusion increased MABP slightly at 60-120 min of infusion in both control and AngII infused rats (114±3 to 123±4 and 140±6 to 148 ±7 mmHg; p<0.05). RBF significantly decreased in both control and AngII infused rats (7.5±0.9 to 6.4±1 and 9.5 ±1.3 to 8.4±1; p<0.05). Ang1-7 infusion also decreased MBF in control rats, but not in AngII infused rats (124±22 to 113±19 PU, p<0.05). Acute Ang1-7 infusion significantly increased urine flow predominantly in rats that received AngII infusion (5.2±0.4 to 15.6±3.5 μl/min; p<0.05). Sodium excretion was enhanced in control rats (0.14±0.08 to 0.94±0.3 μmol/min; p<0.05) as well as in those chronically infused with AngII (0.04±0.02 to 0.42±0.1; p<0.05). These changes were observed despite the decreases in RBF and MBF. The results indicate that the decreased Ang1-7 levels observed in hypertensive rats chronically infused with AngII do not contribute to the alterations in renal hemodynamics. However, the natriuretic actions of Ang1-7 may exert opposing effects on tubular transport.
Abstract 242: The pH Influences Angiotensin-(1-7) Forming Enzyme Activities in the Kidney
Authors: Grobe Nadja , Weir Nathan M , Morris Mariana , Elased Khalid M
Published: March 2022

The kidney plays a pivotal role in acid-base balance. Hydrogen and bicarbonate titration through the nephron produces ranges in urinary pH from 4.5 to 8. All of the components of the renin angiotensin system (RAS) are expressed in the kidney and intrarenal angiotensin II (Ang II) and Ang-(1-7) are formed by multiple independent enzymatic pathways. Most enzymes function optimally at the pH of their native environment. Since small changes in pH can have significant effects on enzyme activities, we investigated the influence of pH on renal Ang II processing to Ang-(1-7). Wild type (WT) and angiotensin converting enzyme 2 (ACE2) knock out (KO) mouse kidney homogenates (20-30 μg protein) were incubated for 30 min at 37°C in 50 mM citrate, phosphate or glycine buffer (pH 4-10) containing 50 μM substrate. First, activity was analyzed using the fluorogenic substrate MCA-Ala-Pro-Lys(Dnp)-OH. A second approach, matrix assisted laser desorption ionization mass spectrometry ((MALDI MS), allowed for the use of the natural substrate, Ang II. In the WT, the fluorogenic assay system detected activity at pH 4, 5, 5.5, 6, 7, 8, 9, and 10 with minimal differences. Using MALDI MS, renal enzymatic Ang-(1-7) formation was observed at a pH range between 4 and 9 with two peaks at pH 5.5 and 8. At pH < 7, conversion of the fluorogenic substrate as well as Ang-(1-7) formation was also detected in KO mice. The activity at pH < 7 was not blocked in KO and WT by ACE2 inhibitor MLN-4760 but was quenched in only WT at pH ≥ 7. The combined prolyl carboxypeptidase (PCP)/prolyl endopeptidase (PEP) inhibitor [Z-pro-prolinal (ZPP)] reduced Ang-(1-7) formation in WT and KO kidney homogenates at pH < 7. Results suggest enzymes responsible for renal Ang-(1-7) formation shift with changes in pH. ACE2 independent, pH dependent formation of Ang-(1-7) from Ang II has not been described before and underscores the usefulness of the MALDI MS approach.
Abstract 251: Regulator of G Protein Signaling 5 is a Novel Peroxisome Proliferator-Activated Receptor (PPAR)-gamma and PPAR-delta Target Gene and Regulator of Resistance Vessel Tone
Authors: Ketsawatsomkron Pimonrat , Keen Henry L , Weatherford Eric T , Liu Xuebo , Faraci Frank M , Sigmund Curt D
Published: March 2022

PPARγ is a ligand-activated transcription factor expressed in the vasculature. Targeted expression of dominant negative (DN) PPARγ to vascular muscle of transgenic mice (S-P467L) caused hypertension and aortic dysfunction. Recently, we demonstrated a robust increase in myogenic tone in S-P467L resistance artery and a reduced large conductance Ca
2+
-activated K
+
(BKCa) current in S-P467L smooth muscle cells (SMC). Inhibition of protein kinase C (PKC) corrected enhanced myogenic constriction and impaired BKCa channel function. Here, we tested the hypothesis that altered expression of PPARγ target genes in S-P467L resistance artery contributes to increased vessel tone. Gene expression profiling in mesenteric artery revealed a marked loss of the regulator of G protein signaling 5 (RGS5) in S-P467L compared to non-transgenic (NT) (fold change compared to NT; 0.18±0.03, p<0.05). Knock down of RGS5 by siRNA in SMC cultures enhanced PKC activity. Consistent with the function of RGS5 as a negative regulator of angiotensin (Ang) II signaling, S-P467L mesenteric artery exhibited an increased Ang II-induced constriction (at 3 nM: 31.4±4% in S-P467L vs 12.9±2% in NT, p<0.05). Pre-incubation of the artery for 30 minutes with the AT1 receptor (AT1R) antagonist losartan completely abolished Ang II-induced vasoconstriction. Neither AT1aR nor AT1bR mRNA expression was different between the groups, implicating a post-receptor mechanism in response to Ang II. Using a sequence-based model, we identified potential PPAR response elements (PPREs) close to the RGS5 promoter. Electrophoretic mobility shift assays showed that both PPARγ and PPARδ can bind at one of the identified PPREs, suggesting that RGS5 might be a direct target of both PPARs. Whereas a significant induction of RGS5 expression was observed in normal mesenteric artery following PPARγ (3.3±0.4 fold) or PPARδ (3.5±0.3 fold) agonist treatment, these inductions were completely lost in S-P467L. We conclude that PPARγ is required for normal resistance vessel tone and have identified RGS5 as a functionally important PPAR target gene.
Abstract 252: Impact of Endothelial Microparticles on Endothelial Cell Signaling and Endothelium-dependent Vasorelaxation
Authors: Burger Dylan , Nguyen Dinh Cat Auralie , He Ying , Montezano Augusto C , Touyz Rhian M
Published: March 2022

Microparticles (MPs), fragments of membrane shed from stressed/damaged cells, are found in the plasma of healthy individuals with levels increased in vascular disease. However, whether MPs themselves contribute to endothelial dysfunction and damage is unclear. We examined the effects of endothelial MPs (eMPs) on cultured endothelial cells (ECs)
in vitro
and on the function of isolated mesenteric arteries
ex vivo
. eMPs were isolated from the media of cultured mouse aortic ECs and quantified by flow cytometry. ECs were treated with eMPs (10
5
/ml) and effects on kinase signaling pathways (Akt, c-Src, ERK1/2, p38 MAPK), superoxide anion generation (dihydroethidium) and NO production (diaminofluorescin) were examined. eMPs increased phosphorylation of ERK1/2 at 5, 15, and 30 minutes post-treatment (P<0.05) and increased phosphorylation of c-Src at 2, 4, and 8 hours post-treatment (P<0.05). Phosphorylation of p38 MAPK and Akt were not altered by eMP exposure. eMPs increased superoxide anion production (208% of control, P<0.05) and decreased ionomycin-induced NO production (39% of control, P<0.05) in ECs after 4 hours. Additionally, the sensitivity to acetylcholine was impaired in MP-treated vessels (pD2: 5.6±0.2) compared to untreated vessels (pD2: 6.7±0.1, P<0.01), assessed in 2nd order mesenteric arteries using wire myography. Finally, we explored mechanisms by which MPs achieve their effects. Co-treatment with an epidermal growth factor receptor inhibitor (AG1478, 10 μM), but not a platelet-derived growth factor receptor inhibitor (Tyrphostin AG1296, 10 μM), blocked microparticle-mediated effects on superoxide generation, and NO production in ECs. In summary, we demonstrate that eMPs impair vasorelaxation
ex vivo
and activate kinase signaling pathways, promote superoxide production and inhibit endothelial NO production
in vitro
. These effects may be mediated, at least in part, through EGFR.
Abstract 254: Treatment With Anti-TLR4 Antibody Lowers Blood Pressure and Improves Erectile Function in Doca-salt Rats
Authors: Nunes Kenia Pedrosa , Bonfim Gisele F , Szasz Theodora , Carvalho Maria Helena , Webb R Clinton
Published: March 2022

Inflammation and endothelial dysfunction play important roles in hypertension, disease associated with erectile dysfunction (ED). Growing evidence suggests that the immune system through activation of toll-like receptors (TLRs), such as TLR4, may contribute to this process. We hypothesized that chronic treatment with anti-TLR4 antibody improves erectile function in DOCA-salt hypertensive rats. Sprague-Dawley normotensive rats (Sham) and DOCA-salt (deoxycorticosterone acetate 200mg/kg, 6 weeks) hypertensive rats were treated with antibody anti-TLR4 (1ug/day, i.p.) or vehicle in the last 3 weeks. Erectile function was evaluated in vivo through measurements of changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation (EFS) of the major pelvic ganglion (MPG). Functional responses of cavernosal tissue were also performed. Anti-TLR4 treatment improved erectile function and significantly lowered blood pressure in DOCA-salt rats (Fig. *p<0.05 DOCA treated vs. untreated). DOCA-salt rats exhibited increased maximal contractile response to phenylephrine (1.53±0.2 vs. 0.92±0.12mN) and EFS (1.36±0.14 vs. 0.88±0.11mN, respectively) compared to Sham, which was attenuated (20±1.3% to PE and 26.5±2% to EFS) by anti-TLR4 treatment. Furthermore, this treatment enhanced nitrergic relaxation (16Hz) in penile tissue from DOCA-salt rats (33±3% untreated vs. 52±4% treated, p<0.05). In conclusion, these results suggest that TLR4 may mediate hypertension-associated ED.