AT2 Receptor Knockout Mice Research Articles

Understanding Angiotensin II Type 1 Receptor Signaling in Vascular Pathophysiology.

Angiotensin II is the most important endocrine ligand in the renin–angiotensin system (RAS), contributing to the development of several cardiovascular diseases including hypertension.1 Angiotensin II mediates its signal transduction and functions via the angiotensin II receptors.2 Historically, the presence of 2 subtypes of angiotensin II receptors were pharmacologically recognized based on the sensitivity to the first orally active nonpeptide angiotensin II receptor antagonist, losartan. The losartan-sensitive receptor was termed AT1 (angiotensin II receptor type 1) receptor. It was assumed to be a heterotrimeric GPCR (G protein–coupled receptor) because it generates inositol triphosphate and diacylglycerol, leading to intracellular Ca2+ elevation and protein kinase C activation, respectively. Most known physiological and pathophysiological functions of angiotensin II, including stimulation of vasoconstriction and salt and water reabsorption, are mediated through the AT1 receptor. The losartan-insensitive receptor was termed AT2 receptor, whereas its G protein coupling remains unclear.1,3,4 In 1991, 2 research groups in the United States independently isolated cDNA (termed AGTR1) encoding the mammalian AT1 receptor.5,6 Subsequently, rat AT2 receptor cDNA (AGTR2) was cloned in 1993.7,8 This pioneer work revealed complete amino acid sequences of the angiotensin II receptor subtypes belonging to the 7-transmembrane GPCR superfamily. In the early 1990s, several studies reported that AT1 receptor elicits tyrosine phosphorylation of multiple proteins and activation of MAPK (mitogen-activated protein kinase; eg, p42/p44 MAPK)/ERK (extracellular signal–regulated kinase; eg, ERK1/2) in various cell types including vascular smooth muscle cells (VSMC). The early 1990s also saw the establishment of the concept that angiotensin II via the AT1 receptor has a direct action on cardiac myocytes, fibroblasts, and VSMCs causing hypertrophic and fibrotic cardiovascular remodeling.9,10 The cardiovascular remodeling caused by angiotensin II seemed to be …

Chronic angiotensin AT2R activation prevents high-fat diet-induced adiposity and obesity in female mice independent of estrogen

ObjectiveObesity is a known risk factor for various metabolic disorders and cardiovascular diseases. Recently we demonstrated that female angiotensin AT2 receptor (AT2R) knockout mice on high-fat diet (HFD) had higher body weight and adiposity with a parallel reduction in estrogen (17,β-estradiol/E2). The present study investigated whether the anti-adiposity effects of the AT2R are estrogen-dependent in female mice. MethodsFemale C57BL/6 ovary-intact (Ovi) mice were treated with an AT2R agonist (C21, 0.3mg/kg, daily i.p.). Ovariectomized (Ovx) mice, supplemented with E2 (5μg/day, pellets implanted subcutaneously), were treated with an AT2R agonist (C21, 0.3mg/kg, daily i.p.) or vehicle. After 4-days of pre-treatment with C21, Ovi and Ovx mice were placed on either normal diet (ND) or HFD while the C21 treatment continued for the next 10days. For a long-term study, Ovi mice were placed on HFD and treated with C21 for 12weeks. ResultsOvi mice fed the HFD had increased parametrial white adipose tissue (pWAT) weight, plasma free fatty acid and triglycerides compared to Ovi mice on ND. Ovariectomy alone caused similar changes in these parameters which were further increased by HFD-feeding. C21 treatment attenuated these HFD-induced changes in Ovi as well as Ovx mice. HFD also, increased the liver/body-weight ratio and decreased the liver expression of the β-oxidation enzyme, carnitine palmitoyltransferase 1 (CPT1-A). C21 treatment attenuated these changes as well. The long-term C21 treatment of Ovi mice lowered the HFD-induced body weight gain, increase in pWAT weight, parametrial adipocyte size and hyperinsulinemia induced by HFD. Finally, HFD drastically reduced urinary estrogen and the beneficial metabolic changes in response to C21-treatment occurred without significantly increasing urinary estrogen. ConclusionWe suggest that the pharmacological activation of AT2R by the agonist C21 reduces adiposity and body weight gain independent of estrogen in female mice. Improvement in fatty acid metabolism is a potential mechanism by which the AT2R exerts anti-adiposity effects.

Abstract 465: AT 2 Receptor Signaling Plays An Important Role In Conversion Of From White To Beige Fat

Background: We have previously reported that treatment with angiotensin II type-2 (AT 2 ) receptor agonist (compound 21; C21) ameliorated insulin resistance with the improvement of adipocyte dysfunction in obese type 2 diabetic mouse model, KKAy. Until quite recently, adipose tissue has been divided into two groups that have directory-opposed capacities; white adipose tissue is best known for its role in energy store, while brown adipose tissue functions for energy expenditure (thermogenesis). In addition, new clusters of adipocyte with thermogenic capability have been discovered in white adipose tissue, which are distinct origins from brown adipose tissue. These adipocytes have been named ‘beige’. However, the role of AT 2 receptor signaling in white-to-beige fat conversion is not known. Methods: KKAy mice were subjected to i.p. injection of C21 (10ug/kg/day) for 2 weeks. After the treatment, the expression of thermogenic genes (Ucp1, Cidea, Pgc1α) in adipose tissue were determined with real-time RT-PCR. Next, we examined the difference in conversion from white to beige fat between wild-type (WT) mice and AT 2 receptor knockout (AT 2 KO) mice. Eight-week-old mice were fed a high-fat and high-cholesterol diet (HFCD) (10% coconut oil + 1.25% cholesterol) for 2 weeks. To examine the effect of exercise on fat conversion, 9-week-old mice were forced swim test (2 minх3 times/day) for 7days. After the swim test, mice were sacrificed for examinations. Results: In KKAy mice, treatment of C21 increased the expression of thermogenic genes in subcutaneous fat, but not in visceral fat. Adipose tissue weight was not changed by treatment of C21. In AT 2 KO mice fed HFCD for 2 weeks, visceral fat was greater than that in WT mice, but subcutaneous fat was not. The expression of thermogenic genes in subcutaneous fat was lower in AT 2 KO mice than in WT mice. In both mice, swim exercise increased the expression of thermogenic genes; however, the up-regulation of these genes in AT 2 KO mice did not reach the level of that in WT mice. Conclusion: These results suggest that AT 2 receptor signaling has an important role in white-to-beige fat conversion, and therefore AT 2 receptor signaling might be a potential therapeutic target for lipid metabolic disorders.

Angiotensin AT2 receptors and the baroreflex control of renal sympathetic nerve activity

Angiotensin AT2 receptors and the baroreflex control of renal sympathetic nerve activity

Effect of Shenxinning decoction on ventricular remodeling in AT1 receptor-knockout mice with chronic renal insufficiency.

Objective:To observe the efficacy of Shenxinning Decoction (SXND) in ventricular remodeling in AT1 receptor-knockout (AT1-KO) mice with chronic renal insufficiency (CRI).Materials and Methods:AT1-KO mice modeled with subtotal (5/6) nephrectomy were intervened with SXND for 12 weeks. Subsequently, blood urea nitrogen (BUN), serum creatinine (SCr), brain natriuretic peptide (BNP), echocardiography (left ventricular end-diastolic diameter, LVDD; left ventricular end-systolic diameter, LVDS; fractional shortening, FS; and ejection fraction, EF), collagen types I and III in the heart and kidney, myocardial mitochondria, and cardiac transforming growth factor-β1 (TGF-β1) of the AT1-KO mice were compared with the same model with nephrectomy only and untreated with SXND.Results:AT1-KO mice did not affect the process of CRI but it could significantly affect cardiac remodeling process. SXND decreased to some extent the AT1-KO mice's BUN, SCr, BNP, and cardiac LVDD, LVDS, and BNP, improved FS and EF, lowered the expression of collagen type I and III in heart and kidney, increased the quantity of mitochondria and ameliorated their structure, and down-regulated the expression of TGF-β1.Conclusion:SXND may antagonize the renin–angiotensin system (RAS) and decrease uremia toxins, thereby ameliorating ventricular remodeling in CRI. Furthermore, SXND has a mechanism correlated with the improvement of myocardial energy metabolism and the down-regulation of TGF-β1.

Possible Role of Angiotensin-Converting Enzyme 2 and Activation of Angiotensin II Type 2 Receptor by Angiotensin-(1–7) in Improvement of Vascular Remodeling by Angiotensin II Type 1 Receptor Blockade

Cross talk between the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis and the ACE2/Ang-(1-7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Furthermore, possible stimulation of the Ang II type 2 (AT2) receptor by Ang-(1-7) has been highlighted as a new pathway. Therefore, we examined the possibility of whether the ACE2/Ang-(1-7)/Mas axis and Ang-(1-7)/AT2 receptor axis are involved in the inhibitory effects of AT1 receptor blockers on vascular remodeling. Wild-type, Mas-knockout, and AT2 receptor knockout mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. Some mice were treated with azilsartan, an AT1 receptor blocker, or Ang-(1-7). Neointimal formation 2 weeks after cuff placement was more marked in Mas-knockout mice compared with wild-type mice. Treatment with azilsartan or Ang-(1-7) attenuated neointimal area, vascular smooth muscle cell proliferation, increases in the mRNA levels of monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-1β, and superoxide anion production in the injured artery; however, these inhibitory effects of azilsartan and Ang-(1-7) were less marked in Mas-knockout mice. Administration of azilsartan or Ang-(1-7) attenuated the decrease in ACE2 mRNA and increased AT2 receptor mRNA but did not affect AT1 receptor mRNA or the decrease in Mas mRNA. The inhibitory effect of Ang-(1-7) on neointimal formation was less marked in AT2 receptor knockout mice compared with wild-type mice. These results suggest that blockade of the AT1 receptor by azilsartan could enhance the activities of the ACE2/Ang-(1-7)/Mas axis and ACE2/Ang-(1-7)/AT2 receptor axis, thereby inhibiting neointimal formation.

Abstract 162: Possible Roles of Crosstalk Between Angiotensin-converting Enzyme 2/Angiotensin (1-7)/Mas Axis and Angiotensin (1-7)/angiotensin II Type 2 Receptor Axis in Vascular Remodeling

Objectives: Crosstalk between the angiotensin (Ang)-converting enzyme (ACE)/Ang II/ Ang II type 1 (AT 1 ) receptor axis and ACE2/Ang (1-7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Moreover, possible stimulation of Ang II type 2 (AT 2 ) receptor by Ang-(1-7) has been highlighted as new axis; however, pathophysiological significance of this possibility has to be elucidated in more detail. Therefore, we examined the possibility whether ACE2/Ang (1-7)/Mas axis and Ang (1-7)/AT 2 receptor axis is involved the inhibitory effects of AT 1 receptor blocker (ARB) on vascular remodeling. Methods: Ten-week-old male C57BL/6J (wild type: WT), Mas knockout (MasKO), and AT 2 receptor knockout (AT 2 KO) mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. After cuff placement, some mice were treated with azilsartan (0.3 mg/kg/day), an ARB, or Ang (1-7) (0.5 mg/kg per day). Two weeks after operation, femoral arteries were removed, fixed and stained by Elastica van Gieson staining for evaluating neointimal formation. Results: Neointima formation induced by cuff placement was further increased in MasKO mice compared with WT mice. Treatment with azilsartan or Ang-(1-7) decreased neointima area and vascular smooth muscle cell proliferation, and attenuated the increases in the mRNA level of MCP-1, TNF-α and IL-1β in the injured artery in WT mice without influencing blood pressure. These inhibitory effects of azilsartan or Ang-(1-7) were less in Mas KO mice. One week after cuff placement, the mRNAs of ACE2 and Mas in the injured artery were markedly decreased in WT mice. Administration of azilsartan or Ang-(1-7) attenuated the decrease in ACE2 mRNA, but not affect the decrease in Mas mRNA. Neointima formation in AT 2 KO mice was more exaggerated compared with that in WT mice. Interestingly, we observed that inhibitory effect of Ang-(1-7) on neointima formation was less in AT 2 KO mice compared with WT mice. Conclusion: These results suggest that blockade of AT 1 receptor by azilsartan could enhance the activities of the ACE2/Ang (1-7)/Mas axis and ACE2/Ang-(1-7)/AT 2 receptor axis, thereby inhibiting the neointima formation.

Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)

Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.

Commercially Available Angiotensin II At2 Receptor Antibodies Are Nonspecific

Commercially available angiotensin II AT2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available AT2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and AT2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or AT2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of AT2 receptor binding or gene expression. We conclude that none of these commercially available AT2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study AT2 receptor expression.

The Pharmacological Effects of Novokinin; a Designed Peptide Agonist of the Angiotensin AT<sab>2</sab> Receptor

Novokinin (RPLKPW) was designed based on ovokinin (FRADHPFL), a vasorelaxing peptide derived from ovalbumin. Novokinin relaxed a mesenteric artery isolated from the spontaneously hypertensive rat (SHR) at 10(-5) M, and reduced SHR blood pressure at a dose of 0.1 mg/kg (po.) emulsified in 30% egg yolk. Novokinin exhibited an affinity for the AT2 receptor Ki=7x10(-6) M, and its antihypertensive and vasorelaxing activities were blocked by PD123319, an AT2 receptor antagonist. The hypotensive effect of novokinin in normotensive mice was not observed in the AT2 receptor-knockout mice. Its antihypertensive and vasorelaxing activities in SHR were also blocked by CAY-10441, an antagonist of the IP receptor for prostaglandin I2 PGI2 suggesting that these activities are mediated by the AT2 receptor, followed by the prostaglandin I2-IP receptor pathway. Novokinin suppressed food intake after icv. or po. administration in mice. The anorexigenic activity was not observed in the AT2 receptor- knockout mice, but was observed in the AT 1 receptor-knockout mice. The anorexigenic activities of novokinin and angiotensin II were blocked by PD123319, and ONO-AE3-208, an antagonist of the EP4 receptor suggesting that the anorexigenic activities of the AT2 agonists are mediated by the PGE 2-EP4 receptor pathway downstream of the AT2 receptor. Novokinin given icv. in mice antagonized the antinociceptive effect of morphine. The antiopiod activites of novokinin and angiotensin II were are blocked by PD123319, and by ONO-AE3-240, an antagonist of the EP3 receptor, suggesting that the antiopioid activities of AT2 agonists is mediated by the PGE2-EP3 receptor downstream of the AT2 receptor.

Compound 21 Induces Vasorelaxation via an Endothelium- and Angiotensin II Type 2 Receptor-Independent Mechanism

Angiotensin II type 2 (AT(2)) receptor stimulation has been linked to vasodilation. Yet, AT(2) receptor-independent hypertension and hypotension (or no effect on blood pressure) have been observed in vivo after application of the AT(2) receptor agonist compound 21 (C21). We, therefore, studied its effects in vitro, using preparations known to display AT(2) receptor-mediated responses. Hearts of Wistar rats, spontaneously hypertensive rats (SHRs), C57Bl/6 mice, and AT(2) receptor knockout mice were perfused according to Langendorff. Mesenteric and iliac arteries of these animals, as well as coronary microarteries from human donor hearts, were mounted in Mulvany myographs. In the coronary vascular bed of Wistar rats, C57Bl/6 mice, and AT(2) receptor knockout mice, C21 induced constriction followed by dilation. SHR hearts displayed enhanced constriction and no dilation. Irbesartan (angiotensin II type 1 receptor blocker) abolished the constriction and enhanced or (in SHRs) reintroduced dilation, and PD123319 (AT(2) receptor blocker) did not block the latter. C21 relaxed preconstricted vessels of all species, and this did not depend on angiotensin II receptors, the endothelium, or the NO-guanylyl cyclase-cGMP pathway. C21 constricted SHR iliac arteries but none of the other vessels, and irbesartan prevented this. C21 shifted the concentration-response curves to U46619 (thromboxane A(2) analog) and phenylephrine (α-adrenoceptor agonist) but not ionomycine (calcium ionophore) to the right. In conclusion, C21 did not cause AT(2) receptor-mediated vasodilation. Yet, it did induce vasodilation by blocking calcium transport into the cell and constriction via angiotensin II type 1 receptor stimulation. The latter effect is enhanced in SHRs. These data may explain the varying effects of C21 on blood pressure in vivo.

Cerebral Microvascular Inflammation in DOCA Salt-Induced Hypertension: Role of Angiotensin II and Mitochondrial Superoxide

Angiotensin II-mediated hypertension (HTN) is accompanied by a pro-inflammatory and pro-thrombotic state in the cerebral microvasculature. Whether comparable phenotypic changes are elicited in other models of HTN remains unclear. Using wild-type mice with deoxycorticosterone acetate (DOCA) salt-induced HTN and intravital microscopy, we observed significant increases in the adhesion of both leukocytes and platelets in cerebral venules, compared with uninephrectomized control mice, without an accompanying increase in blood-brain barrier permeability. The cell-cell interactions in hypertensive mice were more pronounced after ischemic stroke, but no difference in infarct size was detected. The blood cell recruitment was largely prevented in the following groups of DOCA salt mice: losartan (angiotensin II AT1 receptor blocker) treated, AT1 receptor knockout mice, tempol (a membrane-permeable oxygen radical scavenger) treated, and mito-TEMPO (a mitochondria-targeted antioxidant) treated. A similar pattern of protection was noted in mice subjected to ischemic stroke. The blunted cell recruitment responses were not accompanied by reductions in blood pressure (BP). These findings implicate mitochondria-derived oxygen radicals and angiotensin II in the cerebral inflammation associated with DOCA salt HTN and suggests that BP per se is not a critical determinant of the phenotypic changes that accompany HTN, even after ischemic stroke.

Podocytes of AT2 Receptor Knockout Mice Are Protected from Angiotensin II-Mediated RAGE Induction

Background/Aims: The interaction of ‘advanced glycation end products’ (AGEs) and their receptor ‘RAGE’ plays an important role in diabetic nephropathy. We have previously found that in cultured differentiated podocytes, angiotensin II (ANG II) induces RAGE expression via an AT2 receptor-mediated pathway. Methods: To further confirm our results in an in vivo study, AT2 receptor knockout mice (AT2^–/–) and wild-type mice were infused with ANG II by osmotic minipumps for 14 days. Results: As shown by immunohistochemistry, ANG II treatment of wild-type animals (C57BL6) allowed a significantly increased RAGE expression in renal podocytes in comparison to sham-operated C57BL6 mice. In contrast, RAGE expression in podocytes of ANG II-treated knockout mice (AT2^–/–) was only moderately higher than in control animals, but significantly lower than in ANG II-treated wild-type mice. For the AGE species NΕ-carboxymethyllysine, a similar immunohistochemical staining pattern was found. There was no significant change in glomerular AT1a receptor expression. However, no difference in RAGE mRNA expression could be found between ANG II-infused wild-type and AT2^–/– animals by real-time PCR using whole kidney mRNA, presumably due to the low abundance of podocyte mRNA in these preparations. No effects were seen on glomerular apoptosis. Conclusion: These data support the fact that ANG II-mediated RAGE induction in podocytes occurs via AT2 receptors. The present findings may suggest that not all ANG II-mediated changes in diabetic nephropathy can be treated with AT1 receptor blockers.

Renal vasoconstrictor and pressor responses to angiotensin IV in mice are AT1a-receptor mediated

Angiotensin (Ang) IV was reported to induce renal vasoconstriction or vasodilation in rats via AT1 or AT4 receptors, respectively, whereby the latter one has been identified to be the insulin-regulated aminopeptidase (IRAP). We investigated the effects of Ang IV on mean arterial pressure (MAP) and renal cortical blood flow (CBF) in AT1a, AT1b, AT2 receptor and IRAP knockout (-/-) mice and their corresponding wild-type littermates. Ang II, known as a renal vasoconstrictor in mice, was used as a reference. MAP was recorded via a femoral catheter and CBF was measured using a light amplification by stimulated emission of radiation (LASER) Doppler probe; cortical vascular resistance (CVR) was calculated as MAP divided by CBF. Baseline MAP, CBF and CVR in AT1a (-/-) mice were significantly lower than wild-type mice. AT2 (-/-) mice had a significantly higher baseline MAP, but similar CBF. In wild-type mice, Ang IV and Ang II induced dose-dependent pressor and renal vasoconstrictor responses, which were antagonized by the AT1 receptor blocker candesartan. These responses were almost completely absent in AT1a (-/-) mice, but were enhanced in AT2 (-/-) mice; responses in AT1b (-/-) and IRAP (-/-) mice were comparable to those in corresponding wild-type mice. Ang IV mediates pressure and renal vasoconstrictor effects in mice via AT1a receptors, whereas IRAP/AT4 is not involved.

Angiotensin II type 2 receptor deficiency aggravates renal injury and reduces survival in chronic kidney disease in mice

Angiotensin II (Ang II) activates at least two receptors, AT1 and AT2, with the majority of its effects-such as vasoconstriction, inflammation, and matrix deposition-mediated by the AT1 receptor. It is thought that the AT2 receptor counteracts these processes; however, recent studies have found proinflammatory and hypertrophic effects of this receptor subtype. To identify the physiological roles of the AT2 receptor in chronic kidney disease, we performed renal ablation in AT2 receptor knockout and wild-type mice. Renal injury caused a greater impairment of renal function, glomerular injury, albuminuria, and mortality in the knockout mice than in the wild-type mice. There was increased fibronectin expression and inflammation in the knockout mice, as shown by augmented monocyte/macrophage infiltration and higher chemokine monocyte chemotactic protein-1 (MCP-1) and RANTES expression in the remnant kidney. The higher mortality and renal morbidity of the knockout mice was not due to differences in systemic blood pressure, glomerular volume, AT1 receptor, renin, or endothelial nitric oxide synthase expression. Whether activation of the AT2 receptor will have therapeutic benefit in chronic kidney disease will require further study.

The angiotensin II type 2 (AT2) receptor: an enigmatic seven transmembrane receptor

Angiotensin II (AngII) interacts with two receptor subtypes, AT1 and AT2, belonging to the seven transmembrane receptor superfamily. Pharmacological investigations initially suggested that AT2 receptors antagonize AT1 effects. Data from AT2 receptor transgenic and knock-out mice have not been entirely consistent with this interpretation. At the cellular level, a clear mechanistic model of AT2 transduction and signalling has yet to emerge. The AT2 receptor displays the hallmark motifs and signature residues of a G protein-coupled receptor (GPCR), but fails to demonstrate most of the classic features of GPCR signalling. In recent years, unbiased screens for AT2-interacting proteins have identified novel partner proteins involved in AT2 signalling, providing new insight into the mechanisms of AT2 action. A growing body of evidence suggests that the AT2 receptor is constitutively active (i.e. signals without AngII). This review critically evaluates controversies surrounding physiological functions and signalling mechanisms of the AT2 receptor, primarily in a cardiovascular context. Recent advances in the field are highlighted and findings challenging the concept that the AT2 receptor is a conventional angiotensin receptor are considered.

Renal cortical cyclooxygenase 2 expression is differentially regulated by angiotensin II AT 1 and AT 2 receptors

Macula densa cyclooxygenase 2 (COX-2)-derived prostaglandins serve as important modulators of the renin-angiotensin system, and cross-talk exists between these two systems. Cortical COX-2 induction by angiotensin-converting enzyme (ACE) inhibitors or AT(1) receptor blockers (ARBs) suggests that angiotensin II may inhibit cortical COX-2 by stimulating the AT(1) receptor pathway. In the present studies we determined that chronic infusion of either hypertensive or nonhypertensive concentrations of angiotensin II attenuated cortical COX-2. Angiotensin II infusion reversed cortical COX-2 elevation induced by ACE inhibitors. However, we found that angiotensin II infusion further stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT(2) receptor-mediated pathway when the AT(1) receptor was inhibited. Both WT and AT(2) receptor knockout mice were treated for 7 days with either ACE inhibitors or ARBs. Cortical COX-2 increased to similar levels in response to ACE inhibition in both knockout and WT mice. In WT mice ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation was attenuated by the AT(2) receptor antagonist PD123319. In the knockout mice ARBs led to significantly less cortical COX-2 elevation, which was not attenuated by PD123319. PCR confirmed AT(1a) and AT(2) receptor expression in the cultured macula densa cell line MMDD1. Angiotensin II inhibited MMDD1 COX-2, and CGP42112A, an AT(2) receptor agonist, stimulated MMDD1 COX-2. In summary, these results demonstrate that macula densa COX-2 expression is oppositely regulated by AT(1) and AT(2) receptors and suggest that AT(2) receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate AT(1)-mediated responses.

Anti-fibrogenic function of angiotensin II type 2 receptor in CCl 4-induced liver fibrosis

The renin-angiotensin system (RAS) contributes to fibrogenesis in a variety of organs. We recently showed that a lack of angiotensin (Ang) II type 1 (AT1) receptor activity reduces liver fibrosis. In this study, we investigated whether the Ang II type 2 (AT2) receptor is implicated in the development of liver fibrosis. A comparison was made between AT2-receptor knockout (AT2KO) and wild type (WT) mice after 4 weeks of treatment with carbon tetrachloride (CCl 4). Fibrosis was assessed by Azan–Mallory staining and hepatic hydroxyproline (HP) content. The expression of fibrogenic mRNA was measured by real-time quantitative reverse-transcription polymerase chain reaction (PCR). Liver fibrosis evaluated by regular histological analyses and immunohistochemical α-SMA staining was observed in both groups of mice. The extent of fibrosis was greatest in the AT2KO mice. Fibrosis was associated with increases in hepatic HP content and mRNA expression for TGF-β1 and α-SMA, as well as an increase in hepatic TBARS. These findings suggest that CCl 4 induces oxidative stress which leads to activation of hepatic stellate cells (HSCs). These changes were considerably more pronounced in the AT2KO mice than the WT mice. Taken together, we conclude that AT2 signal has anti-fibrogenic and/or cytoprotective effects on oxidative stress-induced liver fibrosis. We therefore suggest that RAS-associated liver fibrogenesis may be determined by the balance between AT1 and AT2 signals.

ROLE OF ANGIOTENSIN II IN MODULATING FLOW‐DEPENDENT BICARBONATE TRANSPORT IN MOUSE PROXIMAL TUBULES

We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ and HCO3− absorption and that flow-dependent modulation of proximal tubule bicarbonate reabsorption is due to changes in both NHE3 and H-ATPase activity within the luminal cell membrane. Since Angiotension II is one of the important autocrine hormones that regulate Na+ and HCO3− transport in the proximal tubule, we examined flow-dependent transport in AT1 receptor knockout mice as well as the effect of blockers of AT1, NHE3 and H-ATPase. Mouse proximal tubule S2 segments were microperfused in vitro at flow rates of 5 and 20nl/min in the absence and presence of losartan, EIPA or bafilomycin in the lumen. HCO3− (JHCO3) absorption was significantly lower under both flow rates in AT1 knockout compared to the wild-type mice (see table). Table 1. Flow-dependent Bicarbonate Absorption The fractional increases in JHCO3 in the AT1 KO, the losartan treated, and the wild type mice were 100%, 130% and 94%, the absolute changes were 42.74, 57.76 and 58.5pmole/min/mm and were all comparable. When the components of JHCO3 were assessed using inhibitors, it was found that the fractional increases in JHCO3 were reduced 49% by EIPA plus losartan or by bafilomycin plus losartan in the wild-type mice. We conclude that angiotensin II is not critical to the signal cascade that mediates flow-dependent proton secretion, and that flow impacts transport by both NHE3 and the H-ATPase comparably.

Insights into angiotensin II receptor function through AT2 receptor knockout mice.

Angiotensin II signals via at least two receptors termed AT1 and AT2. The function of the AT1 receptor is well defined, while that of the AT2 receptor is still shrouded in uncertainty. AT2 gene-deficient (-/-) mice have been helpful in unravelling the function of the AT2 receptor. We have studied AT2-/- and AT2+/+ mice with classical physiological techniques developed for the rat. We found that although AT2-/- mice have normal glomerular filtration rate, the pressure-natriuresis relationship in these mice, compared with AT2+/+ mice, is shifted rightward. Moreover, medullary blood flow fails to increase with increased perfusion pressure while the AT1 receptor expression in the kidneys is increased. We used telemetry and found that AT2-/- mice have about 10 mmHg higher blood pressures than AT2+/+ mice and that their circadian rhythm is disturbed. Moreover, their baroreflexes, as measured by spectral analyses, differs from AT2+/+ controls. The cardiac function of AT2-/- mice is remarkably preserved and the differences are subtle. However, if the mice are given l-NAME hypertension, they exhibit an end-systolic pressure-volume relationship that reveals decreased contractility and probable increased vascular stiffness. Furthermore, the hearts of AT2-/- mice hypertrophy more in response to l-NAME than those of AT2+/+ mice and perivascular fibrosis is increased. DOCA-salt treatment also shows a more rightward pressure-natriuresis relationship in AT2-/- compared with AT2+/+ mice. The renal iNOS expression is increased with DOCA-salt treatment. Our findings support the notion that the AT2 receptor signals antiproliferative and antifibrotic effects and that its presence results in lower blood pressures and lesser responses to secondary forms of hypertension. Technical advances that have allowed us to adapt methods for the rat to the much smaller mouse have facilitated our studies.