Angiotensin Type 2 Receptor Activation Research Articles

β-Arrestin2 Deficiency in the Subfornical Organ Alters Fluid Intake and Blood Pressure Regulation

The activation of the angiotensin type 1 receptor (AT1R) in the brain through canonical Gαq-mediated signaling results in dipsogenic and pressor responses to angiotensin II (ANG). Non-canonical or β-arrestin-mediated signaling of AT1R activation is hypothesized to counterbalance maladaptive G protein signaling during disease. Recently, we found that global genetic deletion of β-arrestin2 ( Arrb2) resulted in higher 0.15 M saline intake at baseline and an exacerbated increase in blood pressure (BP) in response to deoxycorticosterone acetate-salt treatment, suggesting a protective role for ARRB2 during hypertension. We hypothesized that ARRB2 activation specifically within the subfornical organ (SFO) contributes to fluid intake and BP regulation. Male and female Arrb2FLOX mice received intracerebroventricular (ICV) injection of adeno-associated virus (AAV)-CRE to induce deletion of Arrb2 from the SFO. Infection with ICV AAV-CRE primarily targeted the SFO with few off-targets. Mice receiving ICV-AAV-Cre-GFP are denoted Arrb2SFO-KO, and littermate controls receiving ICV-AAV-GFP are denoted Arrb2WT. First, fluid intake was evaluated using the two-bottle choice paradigm, where mice were presented with two bottles, one containing water and one containing 0.15M NaCl. At baseline, Arrb2SFO-KO mice exhibited a significant increase in saline intake compared to controls ( Arrb2WT=1.7±0.9 and Arrb2SFO-KO 3.0±1.4 mL/day p<0.05; n=13 and 18, respectively). This resulted in a saline preference, meaning mice preferred saline over water by more than 50% by volume. Second, we challenged the animals to water- and sodium-depleted conditions to elevate levels of endogenous ANG. After 18h of water and sodium depletion, mice were subjected to the same two-bottle choice paradigm and fluid intake was recorded for 4 hours. Preliminarily, we found Arrb2SFO-KO mice to exhibit higher 0.15M saline intake in both water replete and depleted conditions ( Arrb2WT=0.39±0.13 vs. Arrb2SFO-KO=0.82±0.80 mL/day; n=7-17 p=0.15 and Arrb2WT=1.66±0.95 vs. Arrb2SFO-KO=1.93±1.07 mL/day; n=9-12 p=0.55). BP was evaluated in response to ICV-ANG infusion (1 μg in 5 min) in awake animals. Arrb2SFO-KO mice had higher BP within the first 15 minutes in response to ICV-ANG infusion ( Arrb2WT=1566±85 vs. Arrb2SFO-KO=1726±61 mmHg*min; n=11 p<0.05). Collectively, selective Arrb2 deletion in the SFO increased saline intake and exacerbated the pressor response to ICV-ANG. Overall, these data indicate that ARRB2 in the SFO is protective against sodium ingestion and increases in BP. Stimulation of the AT1R/β-arrestin axis in the brain may represent a novel strategy to treat hypertension. AHA to NMM, NIH to CDS and JLG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of the brain renin-angiotensin system in Parkinson´s disease.

The renin-angiotensin system (RAS) was classically considered a circulating hormonal system that regulates blood pressure. However, different tissues and organs, including the brain, have a local paracrine RAS. Mutual regulation between the dopaminergic system and RAS has been observed in several tissues. Dysregulation of these interactions leads to renal and cardiovascular diseases, as well as progression of dopaminergic neuron degeneration in a major brain center of dopamine/angiotensin interaction such as the nigrostriatal system. A decrease in the dopaminergic function induces upregulation of the angiotensin type-1 (AT1) receptor activity, leading to recovery of dopamine levels. However, AT1 receptor overactivity in dopaminergic neurons and microglial cells upregulates the cellular NADPH-oxidase-superoxide axis and Ca2+ release, which mediate several key events in oxidative stress, neuroinflammation, and α-synuclein aggregation, involved in Parkinson's disease (PD) pathogenesis. An intraneuronal antioxidative/anti-inflammatory RAS counteracts the effects of the pro-oxidative AT1 receptor overactivity. Consistent with this, an imbalance in RAS activity towards the pro-oxidative/pro-inflammatory AT1 receptor axis has been observed in the substantia nigra and striatum of several animal models of high vulnerability to dopaminergic degeneration. Interestingly, autoantibodies against angiotensin-converting enzyme 2 and AT1 receptors are increased in PD models and PD patients and contribute to blood-brain barrier (BBB) dysregulation and nigrostriatal pro-inflammatory RAS upregulation. Therapeutic strategies addressed to the modulation of brain RAS, by AT1 receptor blockers (ARBs) and/or activation of the antioxidative axis (AT2, Mas receptors), may be neuroprotective for individuals with a high risk of developing PD or in prodromal stages of PD to reduce progression of the disease.

Restoring Angiotensin Type 2 Receptor Function Reverses PFOS-Induced Vascular Hyper-Reactivity and Hypertension in Pregnancy.

Perfluorooctane sulfonic acid (PFOS) exposure during pregnancy induces hypertension with decreased vasodilatory angiotensin type-2 receptor (AT2R) expression and impaired vascular reactivity and fetal weights. We hypothesized that AT2R activation restores the AT1R/AT2R balance and reverses gestational hypertension by improving vascular mechanisms. Pregnant Sprague-Dawley rats were exposed to PFOS through drinking water (50 μg/mL) from gestation day (GD) 4-20. Controls received drinking water with no detectable PFOS. Control and PFOS-exposed rats were treated with AT2R agonist Compound 21 (C21; 0.3 mg/kg/day, SC) from GD 15-20. In PFOS dams, blood pressure was higher, blood flow in the uterine artery was reduced, and C21 reversed these to control levels. C21 mitigated the heightened contraction response to Ang II and enhanced endothelium-dependent vasorelaxation in uterine arteries of PFOS dams. The observed vascular effects of C21 were correlated with reduced AT1R levels and increased AT2R and eNOS protein levels. C21 also increased plasma bradykinin production in PFOS dams and attenuated the fetoplacental growth restriction. These data suggest that C21 improves the PFOS-induced maternal vascular dysfunction and blood flow to the fetoplacental unit, providing preclinical evidence to support that AT2R activation may be an important target for preventing or treating PFOS-induced adverse maternal and fetal outcomes.

Abstract 039: B-arrestin Defficiency In The Subfornical Organ Alters Blood Pressure Regulation

The activation of the angiotensin type 1 receptor (AT 1 R) in the brain through the canonical Gαq-mediated signaling results in dipsogenic and pressor responses to angiotensin (AngII). Non-canonical or β-arrestin-mediated signaling of AT 1 R activation is hypothesized to counterbalance maladaptive G protein signaling during disease. Recently, we found that global genetic deletion of β-arrestin2 (ARRB2) resulted in an exacerbated increases in blood pressure (BP) in response to deoxycorticosterone acetate-salt treatment, suggesting a protective role for ARRB2 in hypertension. We hypothesized that ARRB2 activation specifically within the brain contributes to BP regulation. First, global male and female ARRB2-deficient (ARRB2-KO) mice were used to evaluate acute BP responses to intracerebroventricular (ICV) infusion of AngII (1μg). Although no differences at baseline BP were observed, ARRB2-KO mice exhibited greater maximal BP after AngII (144±17 vs. 177±24 mmHg; n=14 and 19, p<0.05). Next, we examined the role of ARRB2 within the brain via ICV injection of adeno-associated virus (AAV)-CRE into ARRB2 FLOX mice. Infection with ICV AAV-CRE primarily targeted the subfornical organ with minimal off-targets. In preliminary studies, these mice exhibited higher BP in response to AngII (179±5 vs. 187±6 mmHg; n=4 p=0.09). Thus, deletion of ARRB2 within the subfornical organ exacerbates the pressor response to Ang II. Finally, we evaluated BP responses to ICV infusion of a β-arrestin biased agonist specific for the AT 1 R (TRV120027 0.5 ug/h) in spontaneously hypertensive BPH/2J and normotensive BPN/3J mice. BPH/2J mice exhibited significantly higher baseline BP compared to BPN/3J (124±5 vs. 153±3 mmHg; n=4 p<0.05), and greater reductions in BP with ICV infusion of TRV120027 (-4±3 vs. -9±1 mmHg; n=4 p<0.05), suggesting that the beneficial BP-lowering effects of β-arrestin activation are more robust during hypertension. Overall, these data indicate that ARRB2 in the subfornical organ is protective against increases in BP. Stimulation of the AT 1 R β-arrestin axis in the brain may represent a novel strategy to treat hypertension.

Renin-angiotensin system in the central nervous system: focus on Huntington's disease.

Renin-angiotensin system in the central nervous system: focus on Huntington's disease.

Angiotensin type 2 receptor antagonism as a new target to manage gout.

There is a growing search for therapeutic targets in the treatment of gout. The present study aimed to evaluate the analgesic and anti-inflammatory potential of angiotensin type 2 receptor (AT2R) antagonism in an acute gout attack mouse model. Male wild-type (WT) C57BL/6 mice either with the AT2R antagonist, PD123319 (10pmol/joint), or with vehicle injections, or AT2R KO mice, received intra-articular (IA) injection of monosodium urate (MSU) crystals (100µg/joint), that induce the acute gout attack, and were tested for mechanical allodynia, thermal hyperalgesia, spontaneous nociception and ankle edema development at several times after the injections. To test an involvement of AT2R in joint pain, mice received an IA administration of angiotensin II (0.05-5nmol/joint) with or without PD123319, and were also evaluated for pain and edema development. Ankle joint tissue samples from mice undergoing the above treatments were assessed for myeloperoxidase activity, IL-1β release, mRNA expression analyses and nitrite/nitrate levels, 4h after injections. AT2R antagonism has robust antinociceptive effects on mechanical allodynia (44% reduction) and spontaneous nociception (56%), as well as anti-inflammatory effects preventing edema formation (45%), reducing myeloperoxidase activity (54%) and IL-1β levels (32%). Additionally, Agtr2tm1a mutant mice have largely reduced painful signs of gout. Angiotensin II administration causes pain and inflammation, which was prevented by AT2R antagonism, as observed in mechanical allodynia 4h (100%), spontaneous nociception (46%), cold nociceptive response (54%), edema formation (83%), myeloperoxidase activity (48%), and IL-1β levels (89%). PD123319 treatment also reduces NO concentrations (74%) and AT2R mRNA levels in comparison with MSU untreated mice. Our findings show that AT2R activation contributes to acute pain in experimental mouse models of gout. Therefore, the antagonism of AT2R may be a potential therapeutic option to manage gout arthritis.

AT1R‐mediated neurodegeneration is part of the etiology of hypertension‐associated cognitive impairment

Hypertension has now been closely associated to cognitive impairment, due to its contribution to cerebrovascular dysfunction. To investigate whether there is neuronal mechanism, hypertension was induced in C57BL/6j male mice (6 months) by DOCA‐salt treatment (1.5 mg/g, s.c., 28 days). Firstly, behavioral study, which contains object location behavioral task, novel object recognition, Barnes maze, and nesting score test, was performed to assess cognitive function, meanwhile, blood pressure (BP) was monitored by tail‐cuff device. In NT (non‐transgenic) mice, DOCA+1%NaCl increases BP significantly, before vs. after DOCA (systolic BP): 114.8 ±0.94 vs. 133.7 ±3.3 mmHg. In whole, compared to sham controls, the treated NT mice performed worse in all four behavioral tests, indicating cognition decline after DOCA‐salt treatment. Later, brain tissues were collected to evaluate possible degenerative alterations in neurons by transmission electron microscope (TEM) imaging, Golgi‐Cox staining, fluorescence‐activated cell sorting (FACS) and qRT‐PCR. It was shown via the TEM that DOCA‐salt hypertension induces mitochondrial damage and morphological changes in prefrontal cortical neurons, meanwhile, Golgi‐Cox staining revealed decreased numbers of dendritic spines, suggesting the onset of neurodegeneration in DOCA‐salt hypertension. In both prefrontal cortex and hippocampus, the mRNA levels of BDNF (brain‐derived neurotrophic factor) and PI4KIIIβ (phosphatidylinositol 4‐kinase IIIβ) were found to be significantly lower in hypertensive NT mice. Using FACS, neurons were sorted from those regions, then analyzed via qRT‐PCR, which identified that the downregulated expression of PI4KIIIβ takes place in neurons. Using laser speckle contrast imaging system, we detected only decreased functional hyperemia, but no impairment in basal cerebral blood flow of the hypertensive mice, suggesting that the neurodegeneration is not caused by cerebral ischemia. We previously demonstrated that neuronal angiotensin type 1 receptors (AT1R) plays a pivotal role in the maintenance of neurogenic hypertension, and here we hypothesized that activation of AT1R could also exacerbate hypertension‐induced reduction in neuronal function. Particularly, in the hippocampal CA1 of adult mice, treatment of angiotensin II (Ang‐II, 500 nM, bath application) induces a significant inhibition in the induction of long‐term potentiation (LTP), while pre‐treatment with losartan (5 μM, 15 min), the AT1R antagonist, can efficiently block this Ang‐II‐induced inhibition, supporting the involvement of neuronal AT1R. Indeed, in mice with selective deletion of neuronal AT1aR, DOCA‐salt‐induced neurodegeneration was attenuated, as evidenced by significantly higher mRNA levels of cortical BDNF and PI4KIIIβ, compared with the NT. In summary, our data suggest that neuron‐expressing AT1R participates in the development of hypertension‐associated cognitive impairment, independently of vascular AT1R.

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK—but not ERK, PI3K, and p38—signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.

Renoprotective Effect of KLF2 on Glomerular Endothelial Dysfunction in Hypertensive Nephropathy.

Kruppel-like factor 2 (KLF2) regulates endothelial cell metabolism; endothelial dysfunction is associated with hypertension and is a predictor of atherosclerosis development and cardiovascular events. Here, we investigated the role of KLF2 in hypertensive nephropathy by regulating KLF2 expression in human primary glomerular endothelial cells (hPGECs) and evaluating this expression in the kidney tissues of a 5/6 nephrectomy mouse model as well as patients with hypertension. Hypertension-mimicking devices and KLF2 siRNA were used to downregulate KLF2 expression, while the expression of KLF2 was upregulated by administering simvastatin. After 4 mmHg of pressure was applied on hPGECs for 48 h, KLF2 mRNA expression decreased, while alpha-smooth muscle actin (αSMA) mRNA expression increased. Apoptosis and fibrosis rates were increased under pressure, and these phenomena were aggravated following KLF2 knockdown, but were alleviated after simvastatin treatment; additionally, these changes were observed in angiotensin II, angiotensin type-1 receptor (AT1R) mRNA, and interleukin-18 (IL-18), but not in angiotensin type-2 receptor mRNA. Reduced expression of KLF2 in glomerular endothelial cells due to hypertension was found in both 5/6 nephrectomy mice and patients with hypertensive nephropathy. Thus, our study demonstrates that the pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, and are associated with IL-18.

Sympathetic modulation by antihypertensive drugs.

Sympathetic modulation by antihypertensive drugs.

Angiotensin Type 2 Receptors: Painful, or Not?

Pain in response to various types of acute injury can be a protective stimulus to prevent the organism from using the injured part and allow tissue repair and healing. On the other hand, neuropathic pain, defined as ‘pain caused by a lesion or disease of the somatosensory nervous system’, is a debilitating pathology. The TRPA1 neurons in the Dorsal Root Ganglion (DRG) respond to reactive oxygen species (ROS) and induce pain. In acute nerve injury and inflammation, macrophages infiltrating the site of injury undergo an oxidative burst, and generate ROS that promote tissue repair and induce pain via TRPA1. The latter discourages using the injured limb, with a lack of movement helping wound healing. In chronic inflammation caused by diabetes, cancer etc., ROS levels increase systemically and modulate TRPA1 neuronal functions and cause debilitating neuropathic pain. It is important to distinguish between drug targets that elicit protective vs. debilitating pain when developing effective drugs for neuropathic pain. In this context, the connection of the Angiotensin type 2 receptor (AT2R) to neuropathic pain presents an interesting dilemma. Several lines of evidence show that AT2R activation promotes anti-inflammatory and anti-nociceptive signaling, tissue repair, and suppresses ROS in chronic inflammatory models. Conversely, some studies suggest that AT2R antagonists are anti-nociceptive and therefore AT2R is a drug target for neuropathic pain. However, AT2R expression in nociceptive neurons is lacking, indicating that neuronal AT2R is not involved in neuropathic pain. It is also important to consider that Novartis terminated their phase II clinical trial (EMPHENE) to validate that AT2R antagonist EMA401 mitigates post-herpetic neuralgia. This trial, conducted in Australia, United Kingdom, and a number of European and Asian countries in 2019, was discontinued due to pre-clinical drug toxicity data. Moreover, early data from the trial did not show statistically significant positive outcomes. These facts suggest that may AT2R not be the proper drug target for neuropathic pain in humans and its inhibition can be harmful.

β-Arrestin-Biased Agonist Targeting the Brain AT1R (Angiotensin II Type 1 Receptor) Increases Aversion to Saline and Lowers Blood Pressure in Deoxycorticosterone Acetate-Salt Hypertension.

Activation of central AT1Rs (angiotensin type 1 receptors) is required for the increased blood pressure, polydipsia, and salt intake in deoxycorticosterone acetate (DOCA)-salt hypertension. TRV120027 (TRV027) is an AT1R-biased agonist that selectively acts through β-arrestin. We hypothesized that intracerebroventricular administration of TRV027 would ameliorate the effects of DOCA-salt. In a neuronal cell line, TRV027 induced AT1aR internalization through dynamin and clathrin-mediated endocytosis. We next evaluated the effect of chronic intracerebroventricular infusion of TRV027 on fluid intake. We measured the relative intake of water versus various saline solutions using a 2-bottle choice paradigm in mice subjected to DOCA with a concomitant intracerebroventricular infusion of either vehicle, TRV027, or losartan. Sham mice received intracerebroventricular vehicle without DOCA. TRV027 potentiated DOCA-induced water intake in the presence or absence of saline. TRV027 and losartan both increased the aversion for saline-an effect particularly pronounced for highly aversive saline solutions. Intracerebroventricular Ang (angiotensin) II, but not TRV027, increased water and saline intake in the absence of DOCA. In a separate cohort, blood pressure responses to acute intracerebroventricular injection of vehicle, TRV, or losartan were measured by radiotelemetry in mice with established DOCA-salt hypertension. Central administration of intracerebroventricular TRV027 or losartan each caused a significant and similar reduction of blood pressure and heart rate. We conclude that administration of TRV027 a selective β-arrestin biased agonist directly into the brain increases aversion to saline and lowers blood pressure in a model of salt-sensitive hypertension. These data suggest that selective activation of AT1R β-arrestin pathways may be exploitable therapeutically.

The COVID-19 Code – Cracked – WHO can Save The World (?)!

The first step in COVID-19 pathogenesis is the viral spike protein priming by Trans Membrane Peptide Receptor Serine S2 (TMPRSS2). TMPRSS2 promotes viral entry, cell to cell transmission, evasion of host immune response, and Angiotensin-Converting Enzyme 2 (ACE2) downregulation. Androgen through Androgen Receptor (AR) increases TMPRSS2 gene expression. Blocking AR may prevent viral entry and other TMPRSS2 mediated actions. ACE2 acts as an entry point for COVID-19 and as the counter regulator in Renin-Angiotensin-Aldosterone System (RAAS). RAAS maintains homeostasis of blood pressure, salt and water, inflammation, and immune response – through its two arms called “killer” and “protective pathways.” The balance between these two pathways determines life or death in disease states. ACE2 converts Angiotensin II to Angiotensin (1-7), which through Mas receptors mediates antiinflammatory, immune-modulatory, and anti-fibrotic actions. Angiotensin II also acts on Angiotensin type 2 Receptor (AT2R) to produce similar actions, called a "protective pathway." Further, Angiotensin II acts through its primary Angiotensin type 1 Receptor (AT1R), causing inflammatory, cytokine storm, and profibrotic response – called "Killer pathway." In COVID, down-regulated ACE2 leads to unabated Angiotensin II/AT1R – "Killer pathway" – actions producing a vicious cycle of "hyper-inflammatory state," resulting in ALI, ARDS, and death. AT1R activation further stimulates the secretion of aldosterone, which through Mineralocorticoid Receptor (MR), augments AT1R mediated 'killer pathway”. None of the COVID guideline drugs modulate this pathogenic mechanism. We examine the first time in history the scientific rationale for combined AR/AT1R/MR blockade for COVID-19 treatment and prevention.

Modulation of Angiotensin Converting Enzyme 2 Activity by Ubiquitination

The major roles on the regulation of cardio‐vascular activity of angiotensin II (angII), the main effector hormone of the renin angiotensin system (RAS), were firmly established. These effects are mediated in large part by activation of angiotensin type 1 receptor (AT1R). Angiotensin converting enzyme 2 (ACE2) catalyzes the conversion of angII to angiotensin1‐7 (ang1‐7), which exerts opposite effects to AT1R activation, presumably through MAS1 receptor. Our group previously demonstrated that angII enhanced AT1R/ACE2 interactions which leads to enzyme ubiquitination followed by its degradation into lysosomes. The levels of protein ubiquitination are controlled by an enzymatic cascade involving ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). Ubiquitination can be reversed under the actions of deubiquitinating enzymes (DUBs). In this study, we aimed to characterize the mechanisms modulating ACE2 ubiquitination. Treatment with angII (100 nM for 4h) of ACE2‐transfected HEK293T cells induced a significant decrease in the enzymatic activity (72.21±12.9 % from control n=8), which was reflected in a decrease of its expression levels by 64.45±7.3 (n=3) after 18h. Pretreatment with the effective concentration of PYR‐41, a cell permeable inhibitor of E1 (50 μM for 2h), fully reversed the effects of angII (100.67±23.2% from control, n=9). In contrast, pretreatment with PR‐619, a broad‐spectrum reversible inhibitor of cysteine‐reactive DUBs (20μM, 6h), did not change the effects of angII on ACE2 activity and expression levels (60.93±9.21% from control, n=9). Furthermore, our preliminary proteomic experiments indicated that the expression levels of several E3 ligases were significantly augmented in hypothalami from DOCA‐salt hypertensive mice, including NEDD4 (Neural precursor cell‐Expressed Developmentally Downregulated gene 4). Accordingly, overexpression of NEDD4 in ACE2‐transfected HEK293T cells significantly reduced basal ACE2 activity to 56±7.5 % from control (n=12) and its expression levels (48.97±2.45 %, n=2). Remarkably, angII effects on ACE2 activity and expression levels were eliminated in cells transfected with a catalytically inactive NEDD4 dominant negative mutant (125 ± 5.25%, n=12, and 94.51±15.56, n=2, respectively from control), indicating that angII requires NEDD4 activity to attenuate ACE2 actions. Similarly, angII treatment was unable to change the activity of an ACE2 ubiquitination‐deficient mutant. This mutant also displayed enhanced ACE2 activity in basal conditions (195±4.5%, n=12). In summary, NEDD4 modulates the levels of ACE2 ubiquitination, whereas PYR‐41 prevented the effects of angII on ACE2, indicating that ubiquitination may represent a novel target for the treatment of cardio‐vascular diseases.Support or Funding InformationThis work was supported in part by a Bridge Grant from Howard University to CMF, National Institute of General Medical Sciences P30 GM106392 to JJG and Veterans Affairs Merit Award (BX004294) and LSUHSC‐NO Research Enhancement Program to EL.

Renal Nerve Deafferentation Attenuates the Fall in GFR during Intravenous Infusion of Furosemide in Anesthetized Rats

Introduction: Furosemide reduces the glomerular filtration rate (GFR) and increases the renal vascular resistance (RVR) despite inhibiting tubuloglomerular feedback but increases proximal tubule pressure, renin release, and renal nerve activity. Objective: This study tested the hypothesis that the fall in GFR with furosemide is due to volume depletion or activation of angiotensin type 1 (AT1) receptors or renal nerves. Methods: Furosemide was infused for 60 min at 1.0 mg·kg⁻¹·h⁻¹ in groups of 5–8 anesthetized rats. Additional groups received intravenous volume replacement to prevent fluid and Na⁺ losses or volume replacement plus losartan or plus sham denervation or plus renal denervation or renal nerve deafferentation. Results: At 60 min of infusion, furosemide alone reduced the GFR (–37 ± 4%; p < 0.01). This fall was not prevented by volume replacement or pretreatment with losartan, although losartan moderated the increase in RVR with furosemide (+44 ± 3 vs. +82 ± 7%; p < 0.01). Whereas the GFR fell after furosemide in rats after sham procedure (–31 ± 2%), it was not changed significantly after prior renal deafferentation. Proximal tubule pressure increased significantly but returned towards baseline over 60 min of furosemide, while urine output remained elevated, and GFR and renal blood flow depressed. Conclusions: The fall in GFR over 60 min of furosemide infusion is independent of volume depletion or activation of AT1 receptors but is largely dependent on renal afferent nerves.

Interference With Endothelial PPAR (Peroxisome Proliferator-Activated Receptor)-γ Causes Accelerated Cerebral Vascular Dysfunction in Response to Endogenous Renin-Angiotensin System Activation.

Low-salt diet is beneficial in salt-sensitive hypertension but may provoke cardiovascular risk in patients with heart failure, diabetes mellitus, or other cardiovascular abnormalities because of endogenous renin-angiotensin system activation. PPAR (peroxisome proliferator-activated receptor)-γ is a transcription factor which promotes an antioxidant pathway in the endothelium. We studied transgenic mice expressing a dominant-negative mutation in PPAR-γ selectively in the endothelium (E-V290M) to test the hypothesis that endothelial PPAR-γ plays a protective role in response to low salt-mediated renin-angiotensin system activation. Plasma renin and Ang II (angiotensin II) were significantly and equally increased in all mice fed low salt for 6 weeks. Vasorelaxation to acetylcholine was not affected in basilar artery from E-V290M at baseline but was significantly and selectively impaired in E-V290M after low salt. Unlike basilar artery, low salt was not sufficient to induce vascular dysfunction in carotid artery or aorta. Endothelial dysfunction in the basilar artery from E-V290M mice fed low salt was attenuated by scavengers of superoxide, inhibitors of NADPH oxidase, or blockade of the Ang II AT1 (angiotensin type-1) receptor. Simultaneous AT1 and AT2 receptor blockade revealed that the restoration of endothelial function after AT1 receptor blockade was not a consequence of AT2 receptor activation. We conclude that interference with PPAR-γ in the endothelium produces endothelial dysfunction in the cerebral circulation in response to low salt-mediated activation of the endogenous renin-angiotensin system, mediated at least in part, through AT1 receptor activation and perturbed redox homeostasis. Moreover, our data suggest that the cerebral circulation may be particularly sensitive to inhibition of PPAR-γ activity and renin-angiotensin system activation.

Angiotensin AT2 Receptor Stimulation is Protective in Lipopolysaccharide‐Induced Inflammation and Renal Injury

Recently angiotensin type‐2 receptor (AT2R) activation has been shown to exert anti‐inflammatory and reno‐protective response against chronic injury. Our prior in vitro studies using the proximal tubule epithelial cells (PTECs) and macrophages revealed that prior activation of angiotensin‐II type 2 receptor (AT2R) reduces pro‐inflammatory cytokine production in response to lipopolysaccharide (LPS). However, the in vivo effects of AT2R stimulation on LPS‐mediated inflammatory renal injury are not known. The present study tests the hypothesis that AT2R activation attenuates inflammation and preserves renal structure and function in the early stages of LPS‐mediated renal injury. Mice were pretreated with vehicle or AT2R agonist C21 (300 μg/kg, i.p.) prior to LPS (5 mg/kg, i.p) challenge. Pretreatment with C21 significantly prevented the LPS‐induced increase in the levels of renal pro‐inflammatory cytokines (TNF‐α, IL‐6), TLR4 expression, and markers of renal injury (blood urea nitrogen, plasma creatinine and albuminuria). Moreover, C21 treatment also protected against LPS‐induced tubular vacuolization and tubular degeneration. Renal and circulating anti‐inflammatory IL‐10 levels were higher with C21 pretreatment at time of LPS challenge. In vitro studies in HK‐2 cells (human PTECs) demonstrate that AT2R activation directly stimulates IL‐10 production. Further, conditioned‐media derived from the C21‐treated HK‐2 cells reduced LPS‐induced TNF‐α production via IL‐10 originating from HK‐2 cells. Our findings suggest that prior activation of AT2R prevents LPS‐mediated inflammatory renal injury, possibly via an increase in renal IL‐10 production.Support or Funding InformationThis study was supported by NIH R01 grant DK61578This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carotid body and subfornical organ AT1R‐mediated sympathoexcitation following repetitive hypoxia requires intrarenal ischemia in rats

Recurrent hypoxemia induced by acute intermittent hypoxia (AIH) elevates sympathetic nerve activity (SNA); a pre‐clinical neural manifestation before the onset of resistant hypertension. Blocking angiotensin type 1 receptors (AT1R) in the rodent brain prevents arterial pressure (AP) elevation after chronic intermittent hypoxia (CIH), but becomes ineffective once the vascular pathology establishes. Hence, understanding how angiotensin II (AngII) recruits neural circuits for enhancing sympathetic tone is important. We produce AIH‐induced elevations in SNA and chemoreceptor‐mediated reflex sensitivity, while AP remains unchanged (i.e. a ‘pre‐hypertensive’ phase). The sympathetic effects of AIH were alleviated by pre‐treating animals with losartan that target AT1R. We demonstrate that CB denervation reduces the elevated sympathetic tone by 56% following AIH, whereas subfornical organ (SFO) inhibition by GABAA receptor agonist, isoguvacine, reduces the effect by 65%. Combined CB ablation and SFO inhibition abrogates the enhanced SNA after AIH. Extracellular single unit recording demonstrates that 43% of SFO neurons (26/60) recorded increases firing rate in response to AngII and hypoxia. Type I SFO neurons (n=16) immediately responds to both AngII and hypoxia upon the onset of the stimulus. Type II SFO neurons (n=10) produce a delayed response that manifests 228 ± 22 and 233 ± 23 secs after AngII and hypoxia respectively. Type III neurons (n=9) respond specifically to hypoxia, while type IV (n=16) neurons are unresponsive to both stimuli, and type V (n=9) neurons display attenuated activity to both AngII and hypoxia. To confirm that peripheral RAS activity increases during hypoxia, we hypothesised that renal perfusion is reduced during hypoxia that leads to an elevation in plasma renin. We use laser speckle imaging and doppler flowmetry to demonstrate that hypoxia causes intense renal ischemia. A comparable ischemic effect can be pharmacologically induced by α1‐adrenergic receptor‐mediated vasoconstriction in the kidney using phenylephrine. Intermittent injections of phenylephrine augments SNA, and this effect is abolished by pre‐treating the animals with a renin‐inhibitor, aliskiren. To confirm that renal ischemia is the pathophysiological cause for evoking increased SNA in AIH, aliskiren was injected prior to AIH and this prevented the increase in SNA. Together, our results suggest that renal ischemia induced by hypoxia leads to an activation of AT1Rs in the CB/SFO pathways to subsequently enhance sympathetic efferent activity. It appears that the kidney‐brain connection is crucial for the induction of increased SNA during AIH, and possibly for the generation of elevated AP in CIH.Support or Funding InformationNational Health and Medical Research Council of Australia Fellowship [PMP; 1024489], [NHMRC Project Grants: 1065485, 1082215] and the Heart Research Institute. S.B.G.A. is supported by the National Health and Medical Research Council of Australia (Grant GNT1052674) and the University of Sydney. S.J.K. is supported by an Australian Postgraduate Award [APA SC0042] from the University of Sydney and a Heart Research Institute scholarship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P288: Systemic Administration of an Angiotensin Type-2 Receptor Agonist Decreases Renal Regulatory T Cells

There is increasing evidence supporting a critical role of the immune system in the development of hypertension. Our lab has previously reported sex differences in the renal T cell profile in both Spontaneously Hypertensive Rats (SHR) and Angiotensin II (Ang II) models of hypertension, with females having more anti-inflammatory regulatory T cells (Tregs) than males. Ang II has a well-defined role in the activation of pro-inflammatory T cells in hypertension via the angiotensin type-1 receptor (AT1R). Less is known about the role of the angiotensin type-2 receptor (AT2R) in the regulation of immune cells, although the AT2R has been shown to be cardioprotective and AT2R expression is greater in females than males. Based on the potential anti-hypertensive role of AT2Rs, we hypothesized that administration of an AT2R agonist, Compound 21 (C21), would increase renal Tregs, and this increase would be greater in females due to greater AT2R expression. Male and female SHR (10 weeks of age, n=3-4) were implanted with telemetry units for continuous monitoring of mean arterial pressure (MAP). Following 10 days of recovery, baseline MAP was recorded for 5 days. Rats were then divided into the following treatment groups: surgical controls, low dose C21 (150 ng/kg/min, sc by osmotic minipump), high dose C21 (300 ng/kg/min, sc by osmotic minipump). Kidneys were harvested after 2 weeks of treatment and flow cytometry was performed on whole kidney homogenates. MAP was not altered by C21 treatment in males (137±4 vs 134±4 vs 134±4 mmHg; n.s.) or females (128±2 vs 136±5 vs 134±4 mmHg; n.s.). Interestingly, despite having no effect on MAP, there was a significant decrease in renal CD3 + CD4 + FoxP3 + Tregs in females following both low and high doses of C21 (data expressed as % CD3 + CD4 + cells: 6±0.6 vs 3±0.6 vs 3.5±1.3 %, respectively; p=0.02). Tregs decrease in males following the high dose of C21 only (data expressed as % CD3 + CD4 + cells: 3.3±0.3 vs 3.3±0.5 vs 1.7±0.7 %, respectively; p=0.05). Total CD3 + T cells, CD3 + CD4 + T cells, and Th17 cells were not altered by C21 treatment. In conclusion, AT2R activation suppresses renal Tregs, and females are more sensitive than males. These data suggest a novel role for AT2R regulation in the kidney in hypertension.

Local renin-angiotensin system regulates the differentiation of mesenchymal stem cells into insulin-producing cells through angiotensin type 2 receptor

Differentiation of stem cells into insulin-producing cells (IPCs) suitable for therapeutic transplantation offers a desperately needed approach for the diabetic patients. Elucidation of the molecular mechanisms during the differentiation of mesenchymal stem cells (MSCs) into IPCs assists the successful production of IPCs and provides an important insight into the improvement of the role of MSCs as a therapeutic tool for diabetes mellitus (DM). The present study aimed to investigate the role of local renin-angiotensin system (RAS) on MSCs differentiation into IPCs by measuring the expression of local RAS in MSCs during the differentiation into IPCs and assessing the effect of angiotensin type 1 receptor (AT1R) blocker and angiotensin type 2 receptor (AT2R) blocker on the differentiation process. Our data showed that the differentiation of MSCs into IPCs was associated with an increase in cellular angiotensinogen, angiotensin-converting enzyme (ACE), renin, and AT2R expression and undetectable expression of AT1R. The net effect was an increase in cellular angiotensin II (Ang II) during the differentiation process. AT1R blockade allowed the differentiation of MSCs into IPCs, whereas AT2R blockade alone and blockade of both AT1R and AT2R inhibited the differentiation of MSCs into IPCs. Our data demonstrated an important role of local RAS in the regulation of MSCs differentiation into IPCs and that Ang II mainly orchestrates this role through AT2R activation.