Angiotensin II Type 1a Receptor Research Articles

Myogenic Vasoconstriction Requires Canonical Gq/11 Signaling of the Angiotensin II Type 1 Receptor.

BackgroundBlood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) arterial tone. However, many of the molecular determinants of this response are unknown. We previously found that mice with targeted disruption of the gene encoding the angiotensin II type 1a receptor (AT1AR) (Agtr1a), the major murine angiotensin II type 1 receptor (AT1R) isoform, showed reduced myogenic tone; however, uncontrolled genetic events (in this case, gene ablation) can lead to phenotypes that are difficult or impossible to interpret.Methods and ResultsWe tested the mechanosensitive function of AT1R using tamoxifen‐inducible smooth muscle‐specific AT1aR knockout (smooth muscle‐Agtr1a −/−) mice and studied downstream signaling cascades mediated by Gq/11 and/or β‐arrestins. FR900359, Sar1Ile4Ile8‐angiotensin II (SII), TRV120027 and TRV120055 were used as selective Gq/11 inhibitor and biased agonists to activate noncanonical β‐arrestin and canonical Gq/11 signaling of the AT1R, respectively. Myogenic and Ang II‐induced constrictions were diminished in the perfused renal vasculature, mesenteric and cerebral arteries of smooth muscle‐Agtr1a −/− mice. Similar effects were observed in arteries of global mutant Agtr1a −/− but not Agtr1b −/− mice. FR900359 decreased myogenic tone and angiotensin II‐induced constrictions whereas selective biased targeting of AT1R‐β‐arrestin signaling pathways had no effects.ConclusionsThis study demonstrates that myogenic arterial constriction requires Gq/11‐dependent signaling pathways of mechanoactivated AT1R but not G protein‐independent, noncanonical pathways in smooth muscle cells.

2D-QSAR Modeling of Quinazolinone Derivatives as Angiotensin II Type 1a Receptor Blockers

Angiotensin receptor blockers (ARBs) are a group of drugs primarily used in the treatment of cardiovascular disease. Multiple quantitative structural activity relationship (QSAR) models established for prediction of angiotensin II type 1a (AT-1a) receptor blocking activity of quinazolinone derivatives to investigate the structural attributes that have significant correlation with biological activity. The genetic algorithm (GA) approach was used to generate a highly predictive models using easily interpretable Py, OEstate and Padel descriptors. OECD principles have been followed to develop statistically robust QSAR models (R2tr = 0.8055 – 0.8625) with good external predictivity (CCCex = 0.7528-0.8450). The multiple QSAR models successfully identified that increase in surface area of negatively charge carbon atom within four bonds from N atom, presence of tetrazole substituents and sp3 N atoms governs the AT-1a receptor blocking activity. The validated QSAR models of present study might be helpful for evaluation AT-1a receptor blocking activity to identify novel hits.

Abstract P148: Angiotensin II Type 1a Receptor Deletion In Renal Proximal Tubular Cells Does Not Attenuate Atherosclerosis In Hypercholesterolemic Mice

Objective: AngII (Angiotensin II) acts through AT1aR (AngII type 1a receptor) to promote atherosclerosis. Despite the consistency that global inhibition of AT1aR markedly attenuates atherosclerosis, it remains undefined in which cell types that AT1aR contributes to atherosclerosis formation. Since renal AngII is associated with increased atherosclerosis in hypercholesterolemic mice, and AT1aR is abundant on PTCs (proximal tubular cells), the present study investigated the effects of AT1aR deletion in PTCs on atherosclerosis in hypercholesterolemic mice. Approach and Results: First, both male and female LDL receptor -/- mice were fed a Western diet and infused with either vehicle or losartan for 12 weeks. Losartan led to more profound increases of plasma renin concentrations and greater reduction of systolic blood pressure in female than in male mice, whereas atherosclerosis was attenuated by losartan equivalently in both sexes. Secondary, we determined whether the effects of losartan were attributed to inhibition of AT1aR on PTCs. To generate PTC-specific AT1aR -/- mice, AT1aR floxed mice were bred with mice expressing Cre under the control of the N-myc downstream regulated gene 1. These mice were injected with 150 mg/kg/day of tamoxifen for 5 days to activate Cre at the age of 4 - 6 weeks. RNAscope detected AT1aR mRNA in both PTCs and glomeruli; mRNA of AT1aR was absent in PTCs, but not glomeruli, of PTC-specific AT1aR -/- mice. Two weeks after completion of tamoxifen injection, these mice in LDL receptor -/- background were fed Western diet for 12 weeks. Deletion of AT1aR in PTCs did not affect plasma renin concentrations, blood pressure and atherosclerosis in either sex. Finally, to investigate the impacts of PCT-specific AT1aR deletion under the conditions with enhanced AngII stimulation, AngII was infused subcutaneously for 12 weeks. There were no differences in both blood pressure and atherosclerosis between genotypes. Conclusion: Although pharmacological inhibition of AT1aR reduced atherosclerosis profoundly in both sexes, PTC-specific AT1aR deletion did not affect atherosclerosis in hypercholesterolemic mice. Our future study will determine whether the presence of AT1aR in both PTCs and glomeruli are needed to promote atherosclerosis.

Activation of the renin-angiotensin system in high fructose-induced metabolic syndrome.

High fructose intake induces hyperglycemia and hypertension. However, the mechanism by which fructose induces metabolic syndrome is largely unknown. We hypothesized that high fructose intake induces activation of the renin-angiotensin system (RAS), resulting in hypertension and metabolic syndrome. We provided 11-week-old Sprague–Dawley rats with drinking water, with or without 20% fructose, for two weeks. We measured serum renin, angiotensin II (Ang II), and aldosterone (Aldo) using ELISA kits. The expression of RAS genes was determined by quantitative reverse transcription polymerase chain reaction. High fructose intake increased body weight and water retention, regardless of food intake or urine volume. After two weeks, fructose intake induced glucose intolerance and hypertension. High fructose intake increased serum renin, Ang II, triglyceride, and cholesterol levels, but not Aldo levels. High fructose intake increased the expression of angiotensinogen in the liver; angiotensin-converting enzyme in the lungs; and renin, angiotensin II type 1a receptor (AT1aR), and angiotensin II type 1b receptor (AT1bR) in the kidneys. However, expression of AT1aR and AT1bR in the adrenal glands did not increase in rats given fructose. Taken together, these results indicate that high fructose intake induces activation of RAS, resulting in hypertension and metabolic syndrome.

Differential effects of membrane sphingomyelin and cholesterol on agonist-induced bitter taste receptor T2R14 signaling.

Membrane lipids regulate the structure and function of G protein-coupled receptors (GPCRs). Previously we have shown that membrane cholesterol regulates the signaling of two human bitter taste receptors (T2Rs), T2R4 and T2R14. Another major plasma membrane lipid known to influence the function of membrane proteins including GPCRs is sphingomyelin. The role of sphingomyelin in T2R function is unexplored thus far. In this work, we examined the significance of sphingomyelin in T2R14 signaling. Results suggest that unavailability of membrane sphingomyelin did not affect the agonist-promoted T2R14 Ca2+ signaling in heterologous expression system and also in primary airway smooth muscle cells (HASM cells). In addition, T2R14 mediated downstream AMPK activation was also unaffected in sphingomyelin-depleted condition; however, cholesterol depletion impaired the T2R14-mediated AMPK activation. Angiotensin II type1A receptor (AT1R) expressed in HASM cells and signals through Ca2+ and AMPK was used as a control. Results suggest that similar to T2R14, membrane sphingomyelin depletion did not affect AT1R signaling. However, membrane cholesterol depletion impaired AT1R mediated Ca2+ signaling and AMPK activation. Interestingly, amino acid sequence analysis revealed the presence of putative sphingolipid binding motif in both T2R14 and AT1R suggesting that the presence of a motif alone might not be suggestive of sphingomyelin sensitivity. In conclusion, these results demonstrate that in contrast to membrane cholesterol, sphingomyelin does not affect the agonist-induced T2R14 signaling, however it may play a role in other aspects of T2R14 function.

Abstract P2036: Effects of Angiotensin II Type 1 A Receptor (AT1aR) on Renal and Urinary Biomarkers of Acute Kidney Injury in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension

Renin angiotensin system (RAS) plays a key role in the pathogenesis and progression of hypertension and renal injury. Angiotensin-II (Ang-II) type 1 receptor (AT1R) mediates the actions of Ang II to increase blood pressure, fluid retention, and aldosterone secretion. Ang II is involved in cell injury, vascular remodeling, and inflammation by releasing inflammatory and pro-inflammatory mediators. The effects of Ang II could be counteracted through its catabolism into the vasodilator peptide Ang (1-7) by angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Diabetes and high blood pressure are the major causes of chronic kidney disease which could lead to end stage renal disease. Our previous studies demonstrated that shedding of renal ACE2 and NEP in the urine of db / db diabetic mice could act as biomarkers for diabetic kidney disease. The aim of the study is to test the hypothesis that there is increased renal ACE2 and NEP shedding in the urine of two-kidney one clip (2K1C) model of renovascular hypertension. In addition, we investigated the effects of deleting AT1aR on these RAS components and on kidney injury molecule-1 (KIM-1). Renovascular hypertension was induced in AT1aR knock out (KO) and WT mice by 2K1C surgery. Blood pressure (BP) measurement by radio-telemetry revealed significant increase of 46.1± 3.6 mmHg in 2K1C mice compared to control mice (p<0.001). Deleting AT1aR significantly decreased the BP in 2K1C mice compared to 2K1C WT mice (69.86 ± 0.19 vs. 151.69 ± 12.22 mmHg, p<0.001). Renal expression and activity of ACE2 and NEP were significantly decreased in the clipped kidney of WT and AT1KO group (p<0.05). In contrast to diabetes, there was no detectable urinary ACE2 and NEP expression and activity in 2KIC mice. In addition, urinary expression of KIM-1 was increased in the clipped kidney. Although deleting AT1R attenuated albuminuria and hypertension, it did not altered urinary expression of KIM-1. These results suggest that the decreased renal NEP and ACE2 in the clipped kidney of 2K1C model may thus worsen kidney injury via impairment of Ang (1-7) formation. In conclusion, urinary KIM-1 could serve as an early indicator of non-diabetic acute kidney injury. Urinary ACE2 and NEP could be used as biomarker of diabetic kidney disease.

Abstract P2026: Involvement of Subfornical Organ Insulin Receptor Signaling in the Development of Angiotensin-II Hypertension

Neurocardiovascular dysfunction due to angiotensin-II (Ang-II) signaling within the brain has emerged as a key mechanism in hypertension development. Interestingly, the hormone insulin is also closely associated with multiple forms of hypertension. Emerging evidence suggests interactions between insulin receptor and Ang-II type 1a receptor (AT 1a R) signaling, although the extent to which this contributes to hypertension development remains unclear. Based on this, we hypothesized that brain insulin receptor signaling is involved in Ang-II hypertension. Male C57Bl/6J mice were instrumented with intracerebroventricular cannula (ICV) and radiotelemeters for conscious cardiovascular measurements. Subsequently, administration of Ang-II (600 ng/kg/min) over 2 wk resulted in the development of hypertension in control animals; daily ICV insulin receptor antagonism with the pharmacological agent S961 (3.5 nM) prevented hypertension development (e.g. day 14 mean arterial pressure 118±6 vs 110±6 mmHg, ICV vehicle vs ICV S961, p=0.05). Given the well-known role for the subfornical organ, a forebrain region that lacks a blood-brain-barrier, in this model of hypertension, we next focused our attention on this nucleus. Insulin receptor immunohistochemistry in AT 1a R eGFP reporter mice (GENSAT NZ44) revealed that insulin receptors were present on 69% of AT 1a R cells (42 of 62, n=3) in the SFO. Acute insulin administration (i.p. 5U) was also used to explore SFO insulin receptor-associated intracellular signaling including phosphorylation of AKT [pAkt; phosphatidylinositol 3-kinase (PI3K) signaling] and ERK1/2 [pERK; mitogen-activated protein kinase (MAPK) signaling]. Analysis of over 4000 SFO cells indicated that insulin resulted in activation of the PI3K pathway as demonstrated by an increase in the percentage of cells with medium to high pAKT expression (5±1 vs 14±1% per section, vehicle vs insulin, n=3-4, 5-10 sections/animal). In contrast, acute insulin administration did not influence SFO pERK. Collectively, these findings indicate that brain insulin receptor signaling is necessary for the development of Ang-II hypertension, and that this response may be mediated by interactions between Ang-II and insulin and/or PI3K signaling in the SFO.

Investigation of Insulin Receptor‐Associated Neuronal Phenotypes in the Subfornical Organ of the Brain

The subfornical organ (SFO), a forebrain nucleus that lacks a blood‐brain‐barrier and integrates circulating factors with downstream neural circuits, is strongly implicated in cardiovascular and metabolic physiology. Despite remarkable progress in unraveling the function of the SFO in cardiometabolic control, the underlying cellular phenotypes and signaling mechanisms remain unclear. The hormone insulin acts within the central nervous system to influence cardiometabolic regulation, and alterations in brain insulin signaling have been implicated in metabolic syndrome‐associated conditions including obesity, diabetes and hypertension. We have recently demonstrated that insulin receptors are widely expressed throughout the rostral to caudal extent of the SFO. Building upon this, we investigated insulin receptor‐associated cellular phenotypes and intracellular signaling pathways in the SFO. Immunohistochemistry in male C57Bl/6 mice (n=4) for insulin receptors and the neuronal marker NeuN revealed that SFO insulin receptors are predominately expressed on neurons (80 ± 3% of insulin receptor positive cells). To dissect the neuronal phenotype upon which SFO insulin receptors are expressed, we next utilized a GABA reporter mouse strain (GAD67‐mCherry; n=3). Immunohistochemistry revealed that approximately one third of all SFO insulin receptor positive cells were localized to GABAergic neurons. On the other hand, double immunohistochemistry for insulin receptors and the excitatory neuronal marker Ca 2+ /calmodulin‐dependent protein kinase II (CamKII) demonstrated abundant insulin receptor expression on SFO glutamatergic neurons (77 ± 3% insulin receptor/CAMKII co‐localization, n=3). Importantly, the SFO is critical in mediating angiotensin‐II responses, and within the SFO angiotensin‐II type1a receptor (AT1aR) expressing neurons are exclusively glutamatergic. Therefore, we also examined potential co‐localization of insulin receptors and AT1aR in the SFO using an AT1aR eGFP reporter strain (GENSAT NZ44). Interestingly, insulin receptors were found to be highly expressed on SFO AT1aR‐expressing cells (69 ± 4% insulin receptor/AT1aR co‐localization, n=3). Brain insulin receptors act through two major intracellular signal cascades: mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3K) pathways. Therefore, we further examined SFO insulin receptor‐associated downstream signaling using immunohistochemistry. Relative to vehicle controls, acute insulin administration (i.p. 5U) resulted in activation of the PI3K pathway, as indicated by phosphorylation of AKT (100 ± 8 vs. 159 ± 7, density/area, saline vs. insulin, p<0.05, n=3–4). SFO phosphorylated ERK1/2, a marker for MAPK pathway activation, was not changed with insulin stimulation (100 ± 21 vs. 114 ± 9, density/area, saline vs. insulin, p=0.5, n=3–4). Collectively, these findings indicate that in the SFO: 1) Insulin receptors are abundantly expressed in inhibitory and excitatory neurons; 2) Insulin may interact with angiotensin‐II; and 3) Insulin appears to predominately utilize PI3K intracellular signaling in the SFO.Support or Funding InformationNIH R01DK117007 & R01HL141393This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Renal Fibrosis and Glomerular Injury by Sacubitril/Valsartan, a Combination Angiotensin Receptor Blocker and Neprilysin Inhibitor, in a Salt-Sensitive Hypertensive Model Using Angiotensin 1 Receptor Knockout Mice: The Contribution of Non-Angiotensin Blocking Effects to Renal Protection

Introduction: “Aldosterone breakthrough,” which is observed in patients receiving long term treatment with angiotensin blockade, is strongly associated with the increased risk of a declining glomerular filtration rate through the profibrotic actions of aldosterone. Sacubitril/valsartan is a newly created combination medicine (the angiotensin receptor blocker valsartan and the neprilysin-inhibitor sacubitril). Therefore, sacubitril/valsartan should have additional organ-protective actions besides the angiotensin blockade. Methods: In this study, we examined the renal protective effect of sacubitril/valsartan in a salt-sensitive hypertension model using angiotensin II type 1a receptor (AT1aR) knockout mice. An oral administration of 1% NaCl solution with sacubitril/valsartan (30 or 60 mg/kg/day) or valsartan (15 or 30 mg/kg/day) alone beginning 7 days before administration of aldosterone was examined in an aldosterone infusion AT1R knockout mouse model as an aldosterone breakthrough model. Results / Conclusion: A significant decrease in Blood Pressure (BP) was observed in the sacubitril/valsartan group compared to the valsartan group under low and high doses. In addition, the pathological analysis of the kidney for glomerular fibrosis by Sirius red staining and for injury by PAS staining demonstrated significant reductions accompanied by a significant reduction in TGF-β in the sacubitril/valsartan group compared to the valsartan group. Overall, sacubitril/valsartan, which has the dual actions of the AT1R blockade and neprilysin inhibition, may have additional clinical values for the treatment of hypertensive patients with aldosterone breakthrough.

The expressional disorder of the renal RAS mediates nephrotic syndrome of male rat offspring induced by prenatal ethanol exposure

This study aimed to prove that prenatal ethanol exposure (PEE) can induce nephrotic syndrome in male rat offspring and to explore the underlying intrauterine programming mechanisms. Pregnant Wistar rats were intragastrically administered ethanol (4 g/kg d) from gestational day (GD) 9 to GD 20, and the male fetuses were delivered by cesarean section at GD20 and the male adult offspring were euthanized at postnatal week (PW) 24. In vitro, the primary metanephric mesenchyme cells were treated with ethanol at concentrations of 15–60 mM. The results indicated that the kidneys of adult offspring in the PEE group exhibited glomerulosclerosis as well as interstitial fibrosis. The levels of serum creatinine and urine protein were elevated; the serum total cholesterol level was increased and the serum albumin concentration was reduced. In the fetal kidney, developmental retardation was presented in the PEE group via pathological examinations, accompanied by the expressional inhibition of the glial-cell-line-derived neurotrophic factor/c-ret tyrosine kinase receptor (GDNF/c-ret) signaling pathway. Although serum angiotensin II (Ang II) level and the gene expression of renal angiotensin-converting enzyme (ACE) were increased in the PEE group, the expression of renal angiotensin II type 2 receptor (AT2R) was significantly inhibited, accompanied by a reduction in the H3K27ac level on the AT2R gene promoter. In the non-classical renin-angiotensin system (RAS), the expression of renal angiotensin converting enzyme 2 (ACE2) and Mas receptor (MasR) were inhibited in the PEE group. The above changes of the classical and non-classical RAS all sustained from utero to adulthood. In vitro, ethanol elevated the gene expression of ACE and angiotensin II type 1a receptor (AT1aR) whereas it reduced the expression of AT2R, ACE2, and MasR, accompanied by a reduction in the H3K27ac level on AT2R gene promoter. Taken together, these results suggested that PEE can induce fetal kidney developmental retardation and adult nephrotic syndrome, and direct regulation of ethanol to the renal RAS was involved in the mechanism of nephrotic syndrome induced by PEE.

Renoprotective effect of the xanthine oxidoreductase inhibitor Topiroxostat under decreased angiotensin II type 1a receptor expression

The aim of this study was to confirm the renoprotective effect of xanthine oxidoreductase (XOR) inhibitor, topiroxostat, compared with another XOR inhibitor, febuxostat, under decreased angiotensin II type 1a (AT1a) receptor expression in the model of renal injury caused by adenine. To evaluate the degree of tubular damage using urinary liver-type fatty acid-binding protein (L-FABP) under decreased AT1a expression, we used AT1a receptor knockdown hetero and human L-FABP chromosomal transgenic (Tg) mice (AT1a+/-L-FABP+/-). Male AT1a+/-L-FABP+/- mice were divided into two groups: the adenine diet group (n = 40) was given a diet containing only 0.2% w/w adenine, and the normal diet group (n = 5) was given a normal diet. When renal dysfunction was confirmed in the adenine diet group 4 weeks after starting the diet, the adenine diet group was further divided into five groups. The adenine diet group (n = 8) was continuously given only the adenine diet. Each group receiving high-dose (3mg/kg) or low-dose (1mg/kg) topiroxostat (Topiroxostat-H group, n = 8, Topiroxostat-L group, n = 8) or febuxostat (Febuxostat-H group, n = 8, Febuxostat-L group, n = 8) was given the adenine diet including the drug for another 4 weeks. The levels of renal XOR, renal dysfunction, urinary L-FABP, tubulointerstitial damage, hypoxia, and oxidative stress were decreased or attenuated after treatment with topiroxostat or febuxostat compared with the adenine diet group. Furthermore, antioxidant capacity was maintained owing to these treatments.In conclusion, topiroxostat and febuxostat attenuated renal damage under decreased AT1a expression in the adenine-induced renal injury model.

Perivascular Adipose Tissue Angiotensin II Type 1 Receptor Promotes Vascular Inflammation and Aneurysm Formation.

Perivascular adipose tissue exhibits characteristics of active local inflammation, which contributes to the development of atherosclerotic disease as a complication of obesity/metabolic syndrome. However, the precise role of perivascular adipose tissue in the progression of abdominal aortic aneurysm remains unclear. To test the hypothesis that genetic deletion of angiotensin II type 1a (AT1a) receptor in perivascular visceral adipose tissue (VAT) can attenuate aortic aneurysm formation in apolipoprotein E-deficient (ApoE-/-) mice, we performed adipose tissue transplantation experiments by using an angiotensin II-induced aneurysm murine model, in which we transplanted VAT from ApoE-/- or ApoE-/- AT1a-/- donor mice onto the abdominal aorta of ApoE-/- recipient mice. Compared with ApoE-/- VAT transplantation, ApoE-/- AT1a-/- VAT transplantation markedly attenuated aortic aneurysm formation, macrophage infiltration, and gelatinolytic activity in the abdominal aorta. AT1a receptor activation led to the polarization of macrophages in perivascular VAT toward the proinflammatory phenotype. Moreover, osteopontin expression and gelatinolytic activity were considerably lower in ApoE-/- AT1a-/- perivascular VAT than in ApoE-/- perivascular VAT, and angiotensin II-induced osteopontin secretion from adipocytes was eliminated after deletion of AT1a receptor in adipocytes. Notably, induction of macrophage migration by conditioned medium from angiotensin II-stimulated wild-type adipocytes was suppressed by treatment with an osteopontin-neutralizing antibody, and ApoE-/- OPN-/- VAT transplantation more potently attenuated aortic aneurysm formation than ApoE-/- VAT transplantation. Our findings indicate a previously unrecognized effect of AT1a receptor in perivascular VAT on the pathogenesis of abdominal aortic aneurysm.

Angiotensin II synergizes with BAFF to promote atheroprotective regulatory B cells

Angiotensin II (AngII) promotes hypertension, atherogenesis, vascular aneurysm and impairs post-ischemic cardiac remodeling through concerted roles on vascular cells, monocytes and T lymphocytes. However, the role of AngII in B lymphocyte responses is largely unexplored. Here, we show that chronic B cell depletion (Baffr deficiency) significantly reduces atherosclerosis in Apoe−/− mice infused with AngII. While adoptive transfer of B cells in Apoe−/−/Baffr−/− mice reversed atheroprotection in the absence of AngII, infusion of AngII in B cell replenished Apoe−/−/Baffr−/− mice unexpectedly prevented the progression of atherosclerosis. Atheroprotection observed in these mice was associated with a significant increase in regulatory CD1dhiCD5+ B cells, which produced high levels of interleukin (IL)-10 (B10 cells). Replenishment of Apoe−/−/Baffr−/− mice with Il10−/− B cells reversed AngII-induced B cell-dependent atheroprotection, thus highlighting a protective role of IL-10+ regulatory B cells in this setting. Transfer of AngII type 1A receptor deficient (Agtr1a−/−) B cells into Apoe−/−/Baffr−/− mice substantially reduced the production of IL-10 by B cells and prevented the AngII-dependent atheroprotective B cell phenotype. Consistent with the in vivo data, AngII synergized with BAFF to induce IL-10 production by B cells in vitro via AngII type 1A receptor. Our data demonstrate a previously unknown synergy between AngII and BAFF in inducing IL-10 production by B cells, resulting in atheroprotection.

Angiotensin II Type 1a Receptor Knockdown in Subfornical Organ Attenuates Central Expression of Inflammatory Mediators and Ameliorates Peripheral Manifestations of Heart Failure in Rats After Myocardial Infarction

Myocardial infarction (MI)‐induced heart failure (HF) is characterized by increased proinflammatory cytokines (PICs) and renin‐angiotensin system (RAS) activity in the periphery and in the brain, contributing to sympathetic excitation and the progression of HF. Although circulating angiotensin II (ANGII) and PICs can both up‐regulate inflammation and RAS activity in cardiovascular regions of the brain, their interactions within the brain in HF remains unclear. Receptors for ANGII and PICs are highly expressed in the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood–brain barrier and senses circulating humoral factors. We previously reported (FASEB J., 30:1b719, 2016) that knockdown of the tumor necrosis factor‐α (TNF‐α) receptor 1 (TNFR1) in the SFO of HF rats reduced the expression of RAS and inflammatory components in the SFO and downstream in the hypothalamic paraventricular nucleus (PVN), with improvements in indices of sympathetic drive and hemodynamic function. The present study examined the effect of knocking down the ANGII type 1a receptor (AT1aR) in the SFO of HF rats. Rats received SFO microinjections of an adeno‐associated viral vector carrying AT1aR shRNA or a scrambled shRNA and green fluorescent protein (GFP). One week later, transfection potential and knockdown efficiency were examined in some rats receiving shRNAs; other rats underwent coronary ligation to induce HF confirmed by echocardiography, or a sham operation. One week after SFO microinjections of AT1aR shRNA or scrambled shRNA, GFP fluorescence was visualized in the SFO but not in other brain areas. Rats receiving AT1aR shRNA also had significantly reduced AT1aR mRNA in the SFO compared with rats receiving scrambled shRNA. Four weeks after MI, HF+scrambled shRNA rats had increased mRNA for AT1aR, TNF‐α, TNFR1, interleukin (IL)‐1β, cyclooxygenase (COX)‐2, c‐fos (a marker of neuronal excitation), GFAP (an astrocyte marker) and CD68 (a microglial marker) in SFO and in PVN, along with increased plasma norepinephrine levels and lung/body weight and right ventricle/body weight ratios, compared with SHAM+scrambled shRNA rats. All of these abnormalities were ameliorated in the HF+AT1aR shRNA rats, that had reduced mRNA for AT1aR. Notably, SHAM+AT1aR shRNA rats also had reduced AT1aR mRNA in the SFO, but did not exhibit alterations in expression of the above mediators in the SFO and PVN, compared with SHAM+ scrambled shRNA rats. The results support the view that circulating ANGII, acting upon AT1aR in the SFO, upregulates the inflammatory response in the SFO with downstream effects in the PVN. Taken together with our previous study of the effects of TNFR1 knockdown in SFO, these results suggest that the SFO is a site at which reciprocal facilitatory interactions occur between RAS and PICs, both contributing to sympathetic excitation in HF, and that interrupting either may have beneficial effects. Astrocytes or microglia that express receptors for both ANGII and PICs may play a role in mediating these interactions.Support or Funding InformationSupported by NIH RO1 HL‐073986 and Department of Veterans Affairs.

Losartan reduces ensuing chronic kidney disease and mortality after acute kidney injury.

Acute kidney injury (AKI) is an important risk factor for incident chronic kidney disease (CKD). Clinical studies disclose that ensuing CKD progresses after functional recovery from AKI, but the underlying mechanisms remain illusive. Using a murine model representing AKI-CKD continuum, we show angiotensin II type 1a (AT1a) receptor signaling as one of the underlying mechanisms. Male adult CD-1 mice presented severe AKI with 20% mortality within 2 weeks after right nephrectomy and left renal ischemia-reperfusion injury. Despite functional recovery, focal tubular atrophy, interstitial cell infiltration and fibrosis, upregulation of genes encoding angiotensinogen and AT1a receptor were shown in kidneys 4 weeks after AKI. Thereafter mice manifested increase of blood pressure, albuminuria and azotemia progressively. Drinking water with or without losartan or hydralazine was administered to mice from 4 weeks after AKI. Increase of mortality, blood pressure, albuminuria, azotemia and kidney fibrosis was noted in mice with vehicle administration during the 5-month experimental period. On the contrary, these parameters in mice with losartan administration were reduced to the levels shown in control group. Hydralazine did not provide similar beneficial effect though blood pressure was controlled. These findings demonstrate that losartan can reduce ensuing CKD and mortality after functional recovery from AKI.

Role of angiotensin II type 1a receptor in renal injury induced by deoxycorticosterone acetate-salt hypertension.

The aim of this study was to investigate the in vivo role of angiotensin II type 1a (AT1a) receptor in renal damage as a result of hypertension by using transgenic mice with AT1a receptor gene disruption. Transgenic mice that express human liver-type fatty acid binding protein (L-FABP) with or without disruption of the AT1a receptor gene (L-FABP+/− AT1a−/−, and L-FABP+/− AT1a+/+, respectively) were used with urinary L-FABP as an indicator of tubulointerstitial damage. Those female mice were administered subcutaneously deoxycorticosterone acetate (DOCA)–salt tablets plus drinking water that contained 1% saline for 28 d after uninephrectomy. In L-FABP+/− AT1a+/+ mice that received DOCA-salt treatment, hypertension was induced and slight expansion of glomerular area, glomerular sclerosis, and tubulointerstitial damage were observed. In L-FABP+/− AT1a−/− mice that received DOCA-salt treatment, hypertension was similarly induced and the degree of glomerular damage was significantly more severe than in L-FABP+/− AT1a+/+-DOCA mice. Urinary L-FABP levels were significantly higher in L-FABP+/− AT1a−/−-DOCA mice compared with those in L-FABP+/− AT1a+/+-DOCA mice. Hydralazine treatment significantly attenuated renal damage that was found in L-FABP+/− AT1a−/−-DOCA mice along with a reduction in blood pressure. In summary, activation of the AT1a receptor may contribute to maintenance of the glomerular structure against hypertensive renal damage.—Hisamichi, M., Kamijo-Ikemori, A., Sugaya, T., Ichikawa, D., Natsuki, T., Hoshino, S., Kimura, K., Shibagaki, Y. Role of angiotensin II type 1a receptor in renal injury induced by deoxycorticosterone acetate–salt hypertension.

Effect of Chronic Mild Stress on AT1 Receptor Messenger RNA Expression in the Brain and Kidney of Rats.

The purpose of the study was to determine whether exposure to chronic mild stress (CMS) affects expression of angiotensin II Type 1a receptor (AT1aR) messenger RNA (mRNA) in the brain and kidney. Male Sprague-Dawley rats were divided into an unchallenged control group, which remained at rest, and an experimental group, exposed to CMS produced by a series of unexpected, disturbing stimuli applied at random over a period of 4 weeks. After sacrificing the animals, samples of the septal/accumbal and hypothalamic/thalamic diencephalon, brain medulla, cerebellum, and the renal medulla were harvested for determination of AT1aR mRNA. Expression of AT1a receptor mRNA was significantly greater in the rats in the CMS condition than in the controls (septal/accumbal diencephalon: 1.689 [0.205] versus 0.027 [0.004], hypothalamic/thalamic diencephalon: 1.239 [0.101] versus 0.003 [0.001], brain medulla: 2.694 [0.295] versus 0.028 [0.003], cerebellum: 0.013 [0.002] versus 0.005 [0.001; p < .001 for all comparisons], and renal medulla: 409.92 [46.92] versus 208.06 [30.56; p < .01]). There was a significant positive correlation between AT1a mRNA expression in the septal/accumbal diencephalon and brain medulla (p < .025). The results provide evidence that CMS significantly enhances expression of the AT1aR gene in the brain and kidney and indicate that changes in expression of AT1aR mRNA in different brain regions during CMS may be causally related. It is suggested that the up-regulation of AT1a receptors by chronic stress may potentiate negative effects of angiotensin II in pathologies associated with activation of the renin-angiotensin system.

Abstract WMP109: Activation of the Brain Renin-Angiotensin System (RAS) Causes Selective Cerebrovascular Dysfunction

Hypertension is a leading risk factor for cerebrovascular disease. Although many changes during hypertension may be mediated by the RAS, most hypertensive patients have normal or reduced levels of circulating renin, suggesting that local tissue RAS play a key role. Surprisingly, little is known regarding the impact of the brain RAS on the cerebral vasculature. We tested the hypothesis that activation of the central RAS using deoxycorticosterone (DOCA)-salt (which simultaneously suppresses the peripheral RAS) alters vascular function. Male C57Bl/6 mice treated with DOCA were given both tap water and water with 0.9% salt for 3 weeks, followed by measurements of vessel reactivity. Mean arterial pressure was elevated modestly after DOCA-salt (79±2 vs 95±3 mmHg; P&lt;0.01). In DOCA-salt treated mice, endothelium-dependent dilation of isolated middle cerebral arteries was almost abolished compared to shams, whereas dilation in mesenteric arteries was normal. This striking effect in brain extended to small parenchymal arterioles. Endothelium-independent vasodilation was similar in all groups. Analysis of mRNA revealed that expression of renal renin was markedly reduced by DOCA-salt while expression of RAS components (eg, AGT, ACE) were increased in both brain and cerebral arteries. In NZ44 reporter mice that express GFP driven by the angiotensin II type 1A receptor (AT1AR) promoter, DOCA-salt significantly increased AT1R-GFP protein expression in cerebral arteries. In cerebral arterioles in vivo, local inhibition of AT1R, mineralocorticoid receptors (MR), or Rho kinase (including ROCK2) reversed endothelial dysfunction in DOCA-salt treated mice. These findings suggest for the first time that activation of the brain and the cerebrovascular RAS profoundly, but selectively, impairs vascular function in brain. Our findings identify AT1R, MR and Rho kinase as key contributors to vascular dysfunction in brain in a clinically relevant model of hypertension.

A single mutation in helix 8 enhances the angiotensin II type 1a receptor transport and signaling

The amphipathic helix 8 in the membrane-proximal C-terminus is a structurally conserved feature of class A seven transmembrane-spanning G protein-coupled receptors (GPCRs). Mutations of this helical motif often cause receptor misfolding, defective cell surface transport and dysfunction. Surprisingly, we demonstrated here that a single point mutation at Lys308 in helix 8 markedly enhanced the steady-state surface density of the angiotensin II type 1a receptor (AT1aR). Consistent with the enhanced cell surface expression, Lys308 mutation significantly augmented AT1aR-mediated mitogen-activated protein kinase ERK1/2 activation, inositol phosphate production, and vascular smooth muscle cell migration. This mutation also increased the overall expression of AT1aR without altering receptor degradation. More interestingly, Lys308 mutation abolished AT1aR interaction with β-COP, a component of COPI transport vesicles, and impaired AT1aR responsiveness to the inhibition of Rab6 GTPase involved in the Golgi-to-ER retrograde pathway. Furthermore, these functions of Lys308 were largely dependent on its positively charged property. These data reveal previously unappreciated functions of helix 8 and novel mechanisms governing the cell surface transport and function of AT1aR.

Fibroblast Angiotensin II Type 1a Receptors Contribute to Angiotensin II-Induced Medial Hyperplasia in the Ascending Aorta.

Angiotensin II (Ang II) infusion causes aortic medial thickening via stimulation of angiotensin II type 1a (AT1a) receptors. The purpose of this study was to determine the cellular loci of AT1a receptors that mediate this Ang II-induced aortic pathology. Saline or Ang II was infused into AT1a receptor floxed mice expressing Cre under control of cell-specific promoters. Initially, AT1a receptors were depleted in aortic smooth muscle cell and endothelium by expressing Cre under control of SM22 and Tie2 promoters, respectively. Deletion of AT1a receptors in either cell type had no effect on Ang II-induced medial thickening. To determine whether this effect was related to neural stimulation, AT1a receptors were depleted using an enolase 2-driven Cre. Depletion of AT1a receptors in neural cells attenuated Ang II-induced medial thickening of the ascending, but not descending aorta. Lineage tracking studies, using ROSA26-LacZ, demonstrated that enolase 2 was also expressed in adventitial cells adjacent to the region of attenuated thickening. To determine whether adventitial fibroblasts contributed to this attenuation, AT1a receptors in fibroblasts were depleted using S100A4 driven Cre. Similar to enolase 2-Cre, Ang II-induced medial thickening was attenuated in the ascending, but not the descending aorta. Lineage tracking demonstrated an increase of S100A4-LacZ positive cells in the media of the ascending region during Ang II infusion. AT1a receptor depletion in fibroblasts attenuates Ang II-induced medial hyperplasia in the ascending aorta.