Transgenic mice expressing an intracellular fluorescent fusion of angiotensin II demonstrate renal thrombotic microangiopathy and elevated blood pressure.

We have generated transgenic mice that express angiotensin II (ANG II) fused downstream of enhanced cyan fluorescent protein, expression of which is regulated by the mouse metallothionein promoter. The fusion protein, which lacks a secretory signal, is retained intracellularly. In the present study, RT-PCR, immunoblot analyses, whole-animal fluorescent imaging, and fluorescent microscopy of murine embryonic fibroblasts confirm expression of the fusion protein in vivo and in vitro. The transgene is expressed in all tissues tested (including brain, heart, kidney, liver, lung, and testes), and radioimmunoassay of plasma samples obtained from transgenic mice indicate no increase in circulating ANG II over wild-type levels, consistent with intracellular retention of the transgene product. Kidneys from transgenic and corresponding wild-type littermates were histologically evaluated, and abnormalities in transgenic mice consistent with thrombotic microangiopathy were observed; microthrombosis was frequently observed within the glomerular capillaries and small vessels. In addition, systolic and diastolic blood pressures, measured by telemetry (n = 8 for each group), were significantly higher in transgenic mice compared with wild-type littermates. Blood pressure of line A male transgenic mice was 125 + or - 1.7 over 97 + or - 1.6 compared with 109 + or - 1.7 over 83 + or - 1.4 mmHg in wild-type littermates (systolic over diastolic). In summary, overexpression of an intracellular fluorescent fusion protein of ANG II correlates with elevated blood pressure and kidney pathology. This transgenic model may be useful to further explore the intracellular renin-angiotensin system and its implication in abnormal kidney function and hypertension.

RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.

Angiotensinogen (ANG) is the sole substrate of the renin-angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury.

Transgenic mice for studies of the renin-angiotensin system in hypertension.

Hypertension is a polygenic and multi-factorial disorder that is extremely prevalent in western societies, and thus has received a great deal of attention by the research community. The renin-angiotensin system has a strong impact on the control of blood pressure both in the short- and long-term, making it one of the most extensively studied physiological systems. Nevertheless, despite decades of research, the specific mechanisms implicated in its action on blood pressure and electrolyte balance, as well as its integration with other cardiovascular pathways remains incomplete. The production of transgenic models either over-expressing or knocking-out specific components of the renin-angiotensin system has given us a better understanding of its role in the pathogenesis of hypertension. Moreover, our attention has recently been refocused on local tissue renin-angiotensin systems and their physiological effect on blood pressure and end-organ damage. Herein, we will review studies using genetic manipulation of animals to determine the role of the endocrine and tissue renin-angiotensin system in hypertension. We will also discuss some untraditional approaches to target the renin-angiotensin system in the kidney.

Macrophage-specific expression of human lipoprotein lipase accelerates atherosclerosis in transgenic apolipoprotein e knockout mice but not in C57BL/6 mice.

Transgenic mice with macrophage-specific expression of human (hu) lipoprotein lipase (LPL) were generated to determine the contribution of macrophage LPL to atherogenesis. Macrophage specificity was accomplished with the scavenger receptor A promoter. Complete characterization demonstrated that macrophages from these mice expressed huLPL mRNA and secreted enzymatically active huLPL protein. Expression of huLPL was macrophage specific, because total RNA isolated from heart, thymus, lung, liver, muscle, and adipose tissues was devoid of huLPL mRNA. Macrophage-specific expression of huLPL did not exacerbate lesions in aortas of C57BL/6 mice even after 32 weeks on an atherosclerotic diet. However, when expressed in apolipoprotein E knockout background, the extent of occlusion in the aortic sinus region of male huLPL+ mice increased 51% (n=9 to 11, P<0.002) compared with huLPL- mice after they had been fed a Western diet for 8 weeks. The proatherogenic effect of macrophage LPL was confirmed in serial sections of the aorta obtained after mice had been fed a Western diet for 3 weeks. By immunohistochemical analysis, huLPL protein was detected in the lesions of huLPL+ mice but not in huLPL- mice. Our results establish that macrophage LPL accelerates atherosclerosis in male apolipoprotein E knockout mice.

Genetic manipulation of the renin-angiotensin system in the kidney.

Over the past 5 years, genetic manipulation has revolutionized the way we examine physiological processes by providing a targeted specificity that was not possible previously. The application of transgenesis and gene targeting has been applied to numerous physiological pathways; and both will remain important tools as we reach the completion of the human genome project and begin to assess the function of newly identified genes. The renin-angiotensin system (RAS) has been the target of numerous transgenic and gene targeting studies designed to help uncover its role in cardiovascular regulation and organ development. Each gene of the system has now been both over-expressed and knocked out. It will be discussed as to how new advances in tissue-specific gene targeting by both over-expression and gene ablation can be used as powerful tools to dissect the role of the RAS in individual tissues.

NF-Y antagonizes renin enhancer function by blocking stimulatory transcription factors.

We previously reported that the promoter proximal portion of the mouse renin enhancer contains a binding site for NF-Y (Ea) that overlaps with a positive regulatory element (Eb). In the context of the renin enhancer, NF-Y acts to oppose enhancer activity. We tested the hypothesis that NF-Y acts as a negative regulator by physically blocking the binding of transcription factors to element-b (Eb). Increasing the spacing between the NF-Y binding site (Ea) and Eb by 2, 5, or 10 nucleotides increased activity of the enhancer to the same extent as mutations abolishing NF-Y binding. The increase in transcription caused by increasing the spacing between Ea and Eb was not due to a shift of NF-Y from a negative regulator to a positive regulator because there was no loss of activity when Ea was also mutated. Oligonucleotides containing the normal or increased spacing mutants still allowed the binding of both NF-Y to Ea and transcription factors to Eb. In fact, we present evidence that both NF-Y and the Eb-binding factor(s) can each bind together on the same oligonucleotide containing either a 5- or 10-bp spacing between Ea and Eb. Our data strongly suggest that the mechanism by which NF-Y opposes renin enhancer activity is to sterically block the binding of factors to Eb.

Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression.

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin expressing cells, express high levels of renin mRNA from their endogenous Ren-1(c) gene. We have previously identified a 242-base pair enhancer (coordinates -2866 to -2625 relative to the CAP site) upstream of the mouse Ren-1(c) gene. This enhancer, in combination with the proximal promoter (-117 to +6), activates transcription nearly 2 orders of magnitude in an orientation independent fashion. To further delimit sequences necessary for transcriptional activation, renin promoter-luciferase reporter gene constructs containing selected regions of the Ren-1(c) enhancer were analyzed after transfection into As4.1 cells. These results demonstrate that several regions are required for full enhancer activity. Sequences from -2699 to -2672, which are critical for the enhancer activity, contain a cyclic AMP responsive element (CRE) and an E-box. Electrophoretic mobility shift assays demonstrated that transcription factors CREB/CREM and USF1/USF2 in As4.1 cell nuclear extracts bind to oligonucleotides containing the Ren-1(c) CRE and E-box, respectively. These two elements are capable of synergistically activating transcription from the Ren-1(c) promoter. Moreover, mutation of either the CRE or E-box results in almost complete loss of enhancer activity, suggesting the critical roles these two elements play in regulating mouse Ren-1(c) gene expression. Although the Ren-1(c) gene contains a CRE, its expression is not induced by cAMP in As4.1 cells. This appears to reflect constitutive activation of protein kinase A in As4.1 cells since treatment with the protein kinase A inhibitor, H-89, caused a significant reduction in Ren-1(c) gene expression and this reduction is mediated through the CRE at -2699 to -2688.

Elevated blood pressure in transgenic mice with brain-specific expression of human angiotensinogen driven by the glial fibrillary acidic protein promoter.

In addition to the circulatory renin (REN)-angiotensin system (RAS), a tissue RAS having an important role in cardiovascular function also exists in the central nervous system. In the brain, angiotensinogen (AGT) is expressed in astrocytes and in some neurons important to cardiovascular control, but its functional role remains undefined. We generated a transgenic mouse encoding the human AGT (hAGT) gene under the control of the human glial fibrillary acidic protein (GFAP) promoter to experimentally dissect the role of brain versus systemically derived AGT. This promoter targets expression of transgene products to astrocytes, the most abundant cell type expressing AGT in brain. All transgenic lines exhibited hAGT mRNA expression in brain, with variable expression in other tissues. In one line examined in detail, transgene expression was high in brain and low in tissues outside the central nervous system, and the level of plasma hAGT was not elevated over baseline. In the brain, hAGT protein was mainly localized in astrocytes, but was present in neurons in the subfornical organ. Intracerebroventricular (ICV) injection of human REN (hREN) in conscious unrestrained mice elicited a pressor response, which was abolished by ICV preinjection of losartan. Double-transgenic mice expressing the hREN gene and the GFAP-hAGT transgene exhibited a 15-mm Hg increase in blood pressure and an increased preference for salt. Blood pressure in the hREN/GFAP-hAGT mice was lowered after ICV, but not intravenous losartan. These studies suggest that AGT synthesis in the brain has an important role in the regulation of blood pressure and electrolyte balance.

Genetic manipulation of the renin-angiotensin system: targeted expression of the renin-angiotensin system in the kidney.

The renin-angiotensin system is a classic endocrine system that also exists within individual tissues. All components of the renin-angiotensin system (RAS) are expressed in the kidney suggesting the potential for local production and action of angiotensin II. Although the importance of the kidney in hypertension is unequivocal, our understanding of the functional significance of intrarenal production of angiotensin II remains incomplete. Using genetic manipulation of the mouse genome we generated a novel transgenic model expressing human angiotensinogen specifically in the proximal tubule cells of the kidney. Herein I describe the generation and physiologic characterization of this model, and discuss the implications of our findings in terms of the genetics of human hypertension. The experimental results presented support the hypothesis that a tissue-specific RAS cascade exists in the kidney of this transgenic model, and that this system may play an important role in blood pressure (BP) and renal homeostasis in this model.

Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine beta-synthase-deficient mice.

Hyperhomocysteinemia is associated with increased risk for cardiovascular events, but it is not certain whether it is a mediator of vascular dysfunction or a marker for another risk factor. Homocysteine levels are regulated by folate bioavailability and also by the methyl donor S-adenosylmethionine (SAM) and its metabolite S-adenosylhomocysteine (SAH). We tested the hypotheses that endothelial dysfunction occurs in hyperhomocysteinemic mice in the absence of folate deficiency and that levels of SAM and SAH are altered in mice with dysfunction. Heterozygous cystathionine beta-synthase-deficient (CBS(+/-)) and wild-type (CBS(+/+)) mice were fed a folate-replete, methionine-enriched diet. Plasma levels of total homocysteine were elevated in CBS(+/-) mice compared with CBS(+/+) mice after 7 weeks (27.1+/-5.2 versus 8.8+/-1.1 micromol/L; P<0.001) and 15 weeks (23.9+/-3.0 versus 13.0+/-2.3 micromol/L; P<0.01). After 15 weeks, but not 7 weeks, relaxation of aortic rings to acetylcholine was selectively impaired by 35% (P<0.05) and thrombomodulin anticoagulant activity was decreased by 20% (P<0.05) in CBS(+/-) mice. Plasma levels of folate did not differ between groups. Levels of SAH were elevated approximately 2-fold in liver and brain of CBS(+/-) mice, and correlations were observed between plasma total homocysteine and SAH in liver (r=0.54; P<0.001) and brain (r=0.67; P<0.001). These results indicate that endothelial dysfunction occurs in hyperhomocysteinemic mice even in the absence of folate deficiency. Endothelial dysfunction in CBS(+/-) mice was associated with increased tissue levels of SAH, which suggests that altered SAM-dependent methylation may contribute to vascular dysfunction in hyperhomocysteinemia.

Physiological insights from genetic manipulation of the renin-angiotensin system.

The renin-angiotensin system is one of the most widely studied endocrine systems. It has an important role in the regulation of normal homeostasis, and disturbances in this system may be important in numerous pathological states. This review will focus on the major insights and important questions raised from gene targeting of this system.

Paradoxical regulation of short promoter human renin transgene by angiotensin ii.

We previously reported the generation of transgenic mice containing the entire human renin gene with a 900-bp promoter. To determine whether all the required elements for angiotensin II-mediated suppression of human renin are present in these mice, angiotensin II was chronically infused by means of osmotic minipump at both low and high doses, 200 and 1000 ng/kg per minute, respectively. Blood pressure was measured by tail-cuff, and kidney renin mRNA levels were quantitated using ribonuclease protection assays. Blood pressure was unchanged in mice receiving either vehicle or low-dose angiotensin II infusion but was increased by approximately 40 mm Hg with the higher dose of angiotensin II. Mouse renin mRNA decreased by >60% during both pressor and nonpressor angiotensin II infusion. Human renin mRNA was not suppressed by nonpressor angiotensin II and was paradoxically increased 1.9-fold by pressor angiotensin II. The lack of upregulation during nonpressor angiotensin II suggested that the increase might be pressure-mediated. To test this, the angiotensin II-induced increase in blood pressure was prevented by coadministration of the vasodilator, hydralazine (15 mg/kg per day). Hydralazine alone decreased blood pressure (-27+/-3 mm Hg) and increased mouse renin mRNA 2.4-fold. Human renin mRNA was unresponsive to this vasodilator-induced fall in pressure and despite the normalization of blood pressure by hydralazine, high-dose angiotensin II still caused a 2.1-fold increase in human renin mRNA. Thus, the first 900 bp of the human renin promoter does not contain all the elements required for appropriate angiotensin II-mediated suppression of human renin mRNA.

Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.

We previously reported a novel transgenic model expressing human angiotensinogen from the kidney androgen-regulated protein promoter, and demonstrated sexually dimorphic expression. Herein, we investigated the hormonal regulation of this transgene. Testosterone increased transgene expression in female mice in a dose- and time-dependent manner and was not detectable 3-days after treatment was halted. High doses of estrogen were required to induce the transgene. Expression of transgene mRNA decreased after castration of male transgenic mice. As in females, however, transgene expression could be induced after administration of testosterone. Flutamide, an androgen receptor antagonist, dose dependently blocked transgene expression in males and blunted the induction caused by testosterone in females. Neither testosterone nor estrogen altered the proximal tubule cell-specific expression of the transgene. The data suggest that the level of transgene expression in this model can be controlled temporally and in magnitude by manipulating the levels of androgen. The fortuitous androgen regulation of this transgene can be used as a molecular "on-off" switch to control transgene expression and potentially manipulate blood pressure levels in this model.

Genetic evidence that lethality in angiotensinogen-deficient mice is due to loss of systemic but not renal angiotensinogen.

Angiotensinogen (AGT)-deficient mice die shortly after birth presumably due to renal dysfunction caused by the presence of severe vascular and tubular lesions in the kidney. Because AGT is expressed in renal proximal tubule cells, we hypothesized that its loss may be the primary mediator of the lethal phenotype. We generated two models to test this hypothesis by breeding transgenic mice expressing human renin with mice expressing human AGT (hAGT) either systemically or kidney-specifically. We then bred double transgenic mice with AGT+/- mice, intercrossed the compound heterozygotes, and examined the offspring. We previously reported that the presence of the human renin and systemically expressed hAGT transgene complemented the lethality observed in AGT-/- mice. On the contrary, we show herein that the presence of the human renin and kidney-specific hAGT transgene cannot rescue lethality in AGT-/- mice. An analysis of newborns indicated that AGT-/- mice were born in normal numbers, and collection of dead 10-day old pups revealed an enrichment in AGT-/-. Importantly, we demonstrated that angiotensinogen protein and functional angiotensin II was generated in the kidney, and the kidney-specific transgene was temporally expressed during renal development similar to the endogenous AGT gene. These data strongly support the notion that the loss of systemic AGT, but not intrarenal AGT, is responsible for death in the AGT-/- mouse model. Taken together with our previous studies, we conclude that the intrarenal renin-angiotensin system located in the proximal tubule plays an important role in blood pressure regulation and may cause hypertension if overexpressed, but may not be required for continued development of the kidney after birth.

Munc18c regulates insulin-stimulated glut4 translocation to the transverse tubules in skeletal muscle.

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.

Retinoic acid-mediated activation of the mouse renin enhancer.

Previous studies demonstrate that the mouse renin gene is regulated by a complex enhancer of transcription located 2.6 kilobases upstream of the transcription start site which is under both positive and negative influence. We demonstrate herein that a positive regulatory element (Eb) is repeated 10 bp upstream (Ec), and both are required for baseline activity of the enhancer. The Eb and Ec core sequences are identical to the consensus sequence for the nuclear hormone receptor superfamily of transcription factors, and transcriptional activity of constructs containing the enhancer is increased after treatment with retinoic acid. Maximal induction requires both Eb and Ec. Expression of endogenous renin and a renin-promoter controlled transgene in As4.1 cells, and kidney renin mRNA in C57BL/6J mice was induced after retinoid treatment. Gel mobility supershift analysis revealed the binding of RARalpha and RXRalpha to oligonucleotides containing both Eb and Ec. Reverse transcriptase-polymerase chain reaction analysis revealed that As4.1 cells express both receptor isoforms, along with RARgamma, but do not express RARbeta, RXRbeta, or RXRgamma. Co-transfection of an expression vector encoding wild-type RARalpha increased enhancer activity, whereas a dominant negative mutant of RARalpha significantly attenuated retinoic acid-induced activity of the enhancer. These results demonstrate the importance of the Eb and Ec motifs in controlling baseline activity of the renin enhancer, and suggest the potential importance of retinoids in regulating renin expression.

Differential requirement for SLP-76 domains in T cell development and function.

The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.

Genetic Manipulation of the Renin-Angiotensin System Using Cre-loxP-Recombinase.

The advent of gene-targeting technology in embryonic stem cells has provided an important tool for the dissection of complex biological systems by allowing investigators to generate germ line mutations in selected genes. Since the introduction of this technology in the early 1980s, hundreds of genes have been targeted for systemic deletion (knocked out), including each gene of the renin-angiotensin system (RAS). Although the technique is very powerful, there are weaknesses that limit its usefulness for studying the RAS. For example, systemic deletion of several of the RAS genes leads to a phenotype, of varying severity depending on the gene in question, in which the mice suc5cumb to severe renal lesions and ultimately die before the age of weaning. This is observed in angiotensinogen (Agt-/-), angiotensin-converting enzyme (ACE-/-), and combined angiotensin receptor subtype 1A and 1B (At1a-/-, At1b-/-) deficient "knockout" mice (1-5). Mice deficient in At1a, but wild type at the At1b locus, are phenotypically normal on mixed genetic backgrounds, but exhibit the same renal lesions and reduced mortality when bred onto "pure" genetic backgrounds, suggesting that renal morphology in response to Ang-II may be under some complex genetic control (6). Presumably, Ang-II is required during the early neonatal period for the continued development of the kidney, and the mice die if they are unable to either generate or utilize An-II during this period.

Identification of three human renin mRNA isoforms from alternative tissue-specific transcriptional initiation.

We have reported that mice transgenic for 140- and 160-kb P1 phage artificial chromosomes (PACs) containing the human renin gene express the gene in a highly tissue-restricted and regulated manner. Herein, we demonstrate that the transgene is also expressed appropriately throughout development. In the course of this investigation, we identified the existence of three transcriptional isoforms of human renin mRNA derived from the utilization of alternative transcription start sites. The first isoform is the kidney-specific isoform, which utilizes the classic renin promoter. The second is a brain-specific isoform, which when previously identified in rats and mice was due to a transcription initiation site within intron A. However, the start site in the human gene resides approximately 1,325 bp upstream of the classic promoter and encodes a new exon 1 (termed exon 1b) that splices directly to exon 2. The third isoform is lung specific and is due to transcriptional initiation 79 bp directly upstream of exon 2, fusing additional DNA within intron A (termed exon 1c) directly to exon 2 without splicing. Importantly, the alternative first exons observed in the PAC transgenic mice were identical to those used to transcribe renin in human fetal kidney, brain, and lung, suggesting these sites are bona fide isoforms of human renin mRNA and not artifacts of transgenesis. Moreover, the subtle differences in tissue-specific transcriptional initiation observed in the renin gene of rats and humans can be faithfully and accurately emulated in a transgenic model.