Exploring type I angiotensin (AT1) receptor functions through gene targeting.

The renin-angiotensin system (RAS) modulates a diverse set of physiological processes including development, blood pressure, renal function and inflammation. The principal effector molecule of this system, angiotensin II, mediates most of these actions. The classically recognized functions of the RAS are triggered via the type 1 (AT(1)) class of angiotensin receptors. Pharmacological blockade of the AT(1) receptor lowers blood pressure and slows the progression of cardiovascular and renal diseases. Gene-targeting technology provides an experimental approach for precisely dissecting the physiological functions of the RAS. Here, we review how gene-targeting experiments have elucidated AT(1) receptor functions.

Deficiency of COX-1 causes natriuresis and enhanced sensitivity to ACE inhibition.

BACKGROUND: Prostanoid products of the cyclo-oxygenase (COX) pathway of arachidonic acid metabolism modulate blood pressure (BP) and sodium homeostasis. Conventional non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit both COX isoforms (COX-1 and -2), cause sodium retention, exacerbate hypertension, and interfere with the efficacy of certain anti-hypertensive agents such as angiotensin-converting enzyme (ACE) inhibitors. While a new class of NSAIDs that specifically inhibit COX-2 is now widely used, the relative contribution of the individual COX isoforms to these untoward effects is not clear. METHODS: To address this question, we studied mice with targeted disruption of the COX-1 (Ptgs1) gene. Blood pressure, renin mRNA expression, and aldosterone were measured while dietary sodium was varied. To study interactions with the renin-angiotensin system, ACE inhibitors were administered and mice with combined deficiency of COX-1 and the angiotensin II subtype 1A (AT1A) receptor were generated. RESULTS: On a regular diet, BP in COX-1-/- mice was near normal. However, during low salt feeding, BP values were reduced in COX-1-/- compared to +/+ animals, and this reduction in BP was associated with abnormal natriuresis despite appropriate stimulation of renin and aldosterone. Compared to COX-1+/+ mice, the actions of ACE inhibition were markedly accentuated in COX-1-/- mice. Sodium sensitivity and BP lowering also were enhanced in mice with combined deficiency of COX-1 and AT1A receptor. CONCLUSIONS: The absence of COX-1 is associated with sodium loss and enhanced sensitivity to ACE inhibition, suggesting that COX-1 inhibition does not cause hypertension and abnormal sodium handling associated with NSAID use.

Deficiency of 5-lipoxygenase accelerates renal allograft rejection in mice.

Acute renal allograft rejection is associated with alterations in renal arachidonic acid metabolism, including enhanced synthesis of leukotrienes (LTs). LTs, the products of the 5-lipoxygenase (5-LO) pathway, are potent lipid mediators with a broad range of biologic activities. Previous studies, using pharmacological agents to inhibit LT synthesis or activity, have implicated these eicosanoids in transplant rejection. To further investigate the role of LTs in acute graft rejection, we transplanted kidneys from CByD2F1 mice into fully allogeneic 129 mice that carry a targeted mutation in the 5lo gene. Unexpectedly, allograft rejection was significantly accelerated in 5-LO-deficient mice compared with wild-type animals. Despite the marked reduction in graft survival, the 5lo mutation had no effect on the hemodynamics or morphology of the allografts. Although LTB4 levels were reduced, renal thromboxane B2 production and cytokine expression were not altered in 5-LO-deficient allograft recipients. These findings suggest that, along with their proinflammatory actions, metabolites of 5-LO can act to enhance allograft survival.

Receptors for prostaglandin E(2) that regulate cellular immune responses in the mouse.

Production of prostaglandin E(2) (PGE(2)) is enhanced during inflammation, and this lipid mediator can dramatically modulate immune responses. There are four receptors for PGE(2) (EP1-EP4) with unique patterns of expression and different coupling to intracellular signaling pathways. To identify the EP receptors that regulate cellular immune responses, we used mouse lines in which the genes encoding each of the four EP receptors were disrupted by gene targeting. Using the mixed lymphocyte response (MLR) as a model cellular immune response, we confirmed that PGE(2) has potent antiproliferative effects on wild-type responder cells. The absence of either the EP1 or EP3 receptors did not alter the inhibitory response to PGE(2) in the MLR. In contrast, when responder cells lacked the EP2 receptor, PGE(2) had little effect on proliferation. Modest resistance to PGE(2) was also observed in EP4-/- responder cells. Reconstitution experiments suggest that EP2 receptors primarily inhibit the MLR through direct actions on T cells. Furthermore, PGE(2) modulates macrophage function by activating the EP4 receptor and thereby inhibiting cytokine release. Thus, PGE(2) regulates cellular immune responses through distinct EP receptors on different immune cell populations: EP2 receptors directly inhibit T cell proliferation while EP2 and EP4 receptors regulate antigen presenting cells functions.

Enhanced central response to dehydration in mice lacking angiotensin AT(1a) receptors.

The objective was to determine the central nervous system (CNS) responses to dehydration (c-Fos and vasopressin mRNA) in mice lacking the ANG AT(1a) receptor [ANG AT(1a) knockout (KO)]. Control and AT(1a) KO mice were dehydrated for 24 or 48 h. Baseline plasma vasopressin (VP) was not different between the groups; however, the response to dehydration was attenuated in AT(1a) KO (24 +/- 11 vs. 10.6 +/- 2.7 pg/ml). Dehydration produced similar increases in plasma osmolality and depletion of posterior pituitary VP content. Neuronal activation was observed as increases in c-Fos protein and VP mRNA. The supraoptic responses were not different between groups. In the paraventricular nucleus (PVN), c-Fos-positive neurons (57.4 +/- 10.7 vs. 98.4 +/- 7.4 c-Fos cells/PVN, control vs. AT(1a) KO) and VP mRNA levels (1.0 +/- 0.1 vs. 1.4 +/- 0.1 microCi, control vs. AT(1a) KO) were increased with greater responses in AT(1a) KO. A comparison of 1- to 2-day water deprivation showed that plasma VP, brain c-Fos, and VP mRNA returned toward control on day 2, although plasma osmolality remained high. Data demonstrate that AT(1a) KO mice show a dichotomous response to dehydration, reduced for plasma VP and enhanced for PVN c-Fos protein and VP mRNA. The results illustrate the importance of ANG AT(1a) receptors in the regulation of osmotic and endocrine balance.

Activation of the murine EP3 receptor for PGE2 inhibits cAMP production and promotes platelet aggregation.

The importance of arachidonic acid metabolites (termed eicosanoids), particularly those derived from the COX-1 and COX-2 pathways (termed prostanoids), in platelet homeostasis has long been recognized. Thromboxane is a potent agonist, whereas prostacyclin is an inhibitor of platelet aggregation. In contrast, the effect of prostaglandin E2 (PGE2) on platelet aggregation varies significantly depending on its concentration. Low concentrations of PGE2 enhance platelet aggregation, whereas high PGE2 levels inhibit aggregation. The mechanism for this dual action of PGE2 is not clear. This study shows that among the four PGE2 receptors (EP1-EP4), activation of EP3 is sufficient to mediate the proaggregatory actions of low PGE2 concentration. In contrast, the prostacyclin receptor (IP) mediates the inhibitory effect of higher PGE2 concentrations. Furthermore, the relative activation of these two receptors, EP3 and IP, regulates the intracellular level of cAMP and in this way conditions the response of the platelet to aggregating agents. Consistent with these findings, loss of the EP3 receptor in a model of venous inflammation protects against formation of intravascular clots. Our results suggest that local production of PGE2 during an inflammatory process can modulate ensuing platelet responses.

The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure.

The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1-/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1-/- mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor-deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.

Role of EP(2) and EP(3) PGE(2) receptors in control of murine renal hemodynamics.

The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE(2) E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE(2). Specifically, we determined the extent to which the EP(2) and EP(3) receptor subtypes mediate the actions of PGE(2) on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP(2) or EP(3) (-/-) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP(2) receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP(2)-/- mice [RBF EP(2)-/-: 5.3 +/- 0.8 ml. min(-1). 100 g kidney wt(-1); renal vascular resistance (RVR) 19.7 +/- 3.6 mmHg. ml(-1). min. g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 +/- 0.5 ml. min(-1). 100 g kidney wt(-1); RVR +/+: 25.4 +/- 4.9 mmHg. ml(-1). min. 100 g kidney wt(-1)). This was also the case for the peak RBF increase after local PGE(2) (500 ng) injection into the renal artery (EP(2)-/-: 116 +/- 4 vs. +/+: 112 +/- 2% baseline RBF). In contrast, we found that the absence of EP(3) receptors in EP(3)-/- mice caused a significant increase (43%) in basal RBF (7.9 +/- 0.8 ml. min(-1). g kidney wt(-1), P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 +/- 1.4 mmHg. ml(-1). min. g kidney wt(-1), P < 0.05 vs. +/+). Local administration of 500 ng of PGE(2) into the renal artery caused more pronounced renal vasodilation in EP(3)-/- mice (128 +/- 2% of basal RBF, P < 0.05 vs. +/+). We conclude that EP(3 )receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP(3) receptors are capable of buffering PGE(2)-mediated renal vasodilation.

Circadian rhythms of activity and drinking in mice lacking angiotensin II 1A receptors.

Angiotensin II (ANG II) type 1 receptors are found in the mouse suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, but their significance for circadian timekeeping is unknown. We examined circadian rhythms of wheel running and drinking in angiotensin AT(1a) receptor knockout (KO) mice. Mean daily running and drinking activity were elevated in KO mice under a light-dark (LD) cycle and in constant dark (DD). These increases were confined to the usual active (dark) period, thus, the 'amplitude' of running and drinking rhythms was higher in KO mice. The phase of entrainment to LD (measured by the onset of the daily active period) did not differ between groups, either in LD or on the first day of DD ('unmasked' phase). KO mice showed a modestly shorter free-running period (tau) in DD. The direction and magnitude of phase shifts to light pulses at two circadian times (CTs) in DD did not differ between groups. Core functions of the circadian system appear intact following AT(1a) receptor KO. The modestly shorter tau and increased rhythm amplitude in KO mice may be secondary to an effect of the mutation on the level of running and drinking activity.

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection.

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.

What can knockout mice contribute to an understanding of hypertension?

The generation of knockout mice using homologous recombination in embryonic stem cells is a powerful tool for physiologic investigations. This experimental approach has provided unique insights into the study of hypertension. Studies using knockout mice have shed new light on blood pressure regulatory mechanisms, molecular mechanisms of end-organ injury, and genetic mechanisms for hypertension. With the development of more accessible approaches for carrying out sophisticated manipulation of the mouse genome, there will be continuing utility of this technique for future studies of hypertension.

Insights into the functions of type 1 (AT1) angiotensin II receptors provided by gene targeting.

The renin-angiotensin system (RAS) has a wide range of actions in biological processes ranging from development and reproduction to cardiovascular and renal functions. Most of these actions are mediated by the octapeptide hormone angiotensin II. The identified family of angiotensin II receptors is divided into two pharmacological classes: type 1 (AT1) and type 2 (AT2). The classically recognized actions of the RAS are primarily mediated by the AT1 subtype of angiotensin receptors, and these receptors are the targets of a new class of anti-hypertensive agents. In recent years, our understanding of the physiological functions of AT1 receptors has been advanced through the use of gene-targeting technology. In this review, we will summarize the emerging picture of AT1 receptor functions that has been provided by gene-targeting experiments.

Divergent functions of angiotensin II receptor isoforms in the brain.

The renin-angiotensin system (RAS) plays a critical role in cardiovascular and fluid homeostasis. The major biologically active peptide of the RAS is angiotensin II, which acts through G protein-coupled receptors of two pharmacological classes, AT(1) and AT(2). AT(1) receptors, expressed in brain and peripheral tissues, mediate most classically recognized actions of the RAS, including blood pressure homeostasis and regulation of drinking and water balance. In rodents, two highly homologous AT(1) receptor isoforms, termed AT(1A) and AT(1B) receptors, are expressed at different levels in major forebrain cardiovascular and fluid regulatory centers, with AT(1A) expression generally exceeding AT(1B) expression, but the relative contributions of these receptor subtypes to central angiotensin II responses are not known. We used gene targeting in combination with a unique system for maintaining catheters in the cerebral ventricles of conscious mice to test whether there are differential roles for AT(1A) and AT(1B) receptors in responses elicited by angiotensin II in the brain. Here we show that the blood pressure increase elicited by centrally administered angiotensin II can be selectively ascribed to the AT(1A) receptor. However, the drinking response requires the presence of AT(1B) receptors. To our knowledge, this is the first demonstration of a primary and nonredundant physiological function for AT(1B) receptors.

H2-DMalpha(-/-) mice show the importance of major histocompatibility complex-bound peptide in cardiac allograft rejection.

The role played by antigenic peptides bound to major histocompatibility complex (MHC) molecules is evaluated with H2-DMalpha(-/)- mice. These mice have predominantly class II-associated invariant chain peptide (CLIP)-, not antigenic peptide-bound, MHC class II. H2-DMalpha(-/)- donor heart grafts survived three times longer than wild-type grafts and slightly longer than I-A(beta)(b)-(/)- grafts. Proliferative T cell response was absent, and cytolytic response was reduced against the H2-DMalpha(-/)- grafts in vivo. Residual cytolytic T cell and antibody responses against intact MHC class I lead to eventual rejection. Removal of both H2-DMalpha and beta2-microglobulin (beta2m) in cardiac grafts lead to greater (8-10 times) graft survival, whereas removal of beta2m alone did not have any effect. These results demonstrate the significance of peptide rather than just allogeneic MHC, in eliciting graft rejection.

Cardiovascular responses to the isoprostanes iPF(2alpha)-III and iPE(2)-III are mediated via the thromboxane A(2) receptor in vivo.

BACKGROUND: Isoprostanes (iPs) are free radical-catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. Several iPs exert biological effects in vitro and may contribute to the functional consequences of oxidant stress. For example, iPF(2alpha)-III (8-iso PGF(2alpha)) and iPE(2)-III modulate platelet function and vascular tone. Although these effects are blocked by antagonists of the receptor (TP) for the cyclooxygenase product thromboxane A(2), it has been speculated that the iPs may activate a receptor related to, but distinct from, the TP. METHODS AND RESULTS: Transgenic mice (TPOEs) were generated in which the TP-beta isoform was under the control of the preproendothelin promoter. They overexpressed TP-beta in the vasculature but not in platelets and exhibited an exaggerated pressor response to infused iPF(2alpha)-III compared with wild-type mice. This was blocked by TP antagonism. The platelet response to the iP was unaltered in TPOEs compared with wild-type mice. By contrast, both the pressor response to iPF(2alpha)-III and its effects on platelet function were abolished in mice lacking the TP gene. This was also true of the effects of infused iPE(2)-III on mean arterial pressure and platelet aggregation. CONCLUSIONS: Both iPF(2alpha)-III and iPE(2)-III exert their effects on platelet function and vascular tone in vivo by acting as incidental ligands at membrane TPs rather than via a distinct iP receptor. Activation of TPs by iPs may be of importance in syndromes in which cyclooxygenase activation and oxidant stress coincide, such as in atherosclerosis and reperfusion after tissue ischemia.

A role for leukotrienes in cyclosporine nephrotoxicity.

BACKGROUND: Nephrotoxicity associated with cyclosporine A (CsA) administration is characterized by marked renal vasoconstriction, interstitial fibrosis, and arteriolar hypertrophy. While the molecular mechanisms of CsA toxicity are not well characterized, previous studies have demonstrated that altered arachidonic acid (AA) metabolism plays a role its pathogenesis. Using a rat renal transplant model, the purpose of this study was to examine the effects of CsA on the 5-lipoxygenase (5-LO) pathway of AA metabolism. METHODS: The PVG (RT1c) strain of rats underwent kidney transplantation, and recipients of nonrejecting kidney transplants were treated with either 50 mg/kg/day CsA or vehicle (N = 24). To determine the physiologic significance of increased leukotriene (LT) production, the peptidoleukotriene receptor antagonist SKF 106203 was administered to CsA-treated animals for six days. RESULTS: CsA caused a substantial reduction in glomerular filtration rate (GFR) in the transplanted rats compared with the vehicle-treated controls (1.5 +/- 0.6 vs. 4.1 +/- 0.8 mL/min/kg, P < 0.05). The reduction in renal function was associated with enhanced urinary excretion of the peptidoleukotriene metabolites LTE4 (1431 +/- 207 vs. 953 +/- 125 pg/24 h, P < 0.05) and N-acetyl-LTE4 (4411 +/- 848 vs. 463 +/- 70 pg/24 h, P < 0.001). LT receptor blockade had a significant protective effect on renal transplant function in CsA-treated animals (GFR, 4.8 +/- 1.1 vs. 1.7 +/- 0.9 mL/min/kg, P < 0.05), such that CsA-treated animals that received SKF106203 maintained GFR at levels similar to controls that never received CsA (4.1 +/- 0.8 mL/min/kg). Peptidoleukotriene receptor blockade also prevented the histomorphological abnormalities caused by CsA, including tubular vacuolization. CONCLUSIONS: These studies identify a critical role for LTs in the pathophysiology of CsA nephrotoxicity and suggest that LT antagonists may be useful in preventing CsA-associated kidney toxicity.

Altered intragraft immune responses and improved renal function in MHC class II-deficient mouse kidney allografts.

BACKGROUND: During renal allograft rejection, expression of MHC class II antigens is up-regulated on the parenchymal cells of the kidney. This up-regulation of MHC class II proteins may stimulate the intragraft alloimmune response by promoting their recognition by recipient CD4+ T cells. In previous studies, absence of donor MHC class II antigens did not affect skin graft survival, but resulted in prolonged survival of cardiac allografts. METHODS: To further explore the role of MHC class II antigens in kidney graft rejection, we performed vascularized kidney transplants using donor kidneys from A(beta)b-deficient mice that lack MHC class II expression. RESULTS: At 4 weeks after transplant, GFR was substantially depressed in control allografts (2.18+/-0.46 ml/min/kg) compared to nonrejecting isografts (7.98+/-1.62 ml/min/kg; P<0.01), but significantly higher in class II- allografts (4.38+/-0.60 ml/min/kg; P<0.05). Despite the improvement in renal function, class II- allograft demonstrated histologic features of acute rejection, not unlike control allografts. However, morphometric analysis at 1 week after transplantation demonstrated significantly fewer CD4+ T cells infiltrating class II- allografts (12.8+/-1.2 cells/mm2) compared to controls (25.5+/-2.6 cells/mm2; P=0.0007). Finally, the intragraft profile of cytokines was altered in class II- allografts, with significantly reduced expression of Th2 cytokine mRNA compared to controls. CONCLUSIONS: These results support a role of MHC class II antigens in the kidney regulating immune cells within the graft. Further, effector pathways triggered by class II antigens promote renal injury during rejection.