Renin Transgene Research Articles

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.

Rats transgenic for human renin and human angiotensinogen as a model for gestational hypertension.

Animal models of gestational hypertension are problematic. A novel mouse model was described earlier. The dams in that study were transgenic for human angiotensinogen and the sires for human renin; human renin was expressed in and produced by the placenta. This model was adapted to the rat, which has greater utility in terms of chronic instrumentation and physiologic measurements. Female rats transgenic for human angiotensinogen were mated with rats transgenic for human renin. Telemetry BP increased on day 5 of pregnancy from 110/80 mmHg to as high as 180/140 mmHg, while heart rate increased slightly. The renin transgene was expressed in the placenta, which resulted in increased human plasma renin concentration from 0 to 937 +/- 800 ng angiotensin I ml/h; the values returned to 0 after delivery. Female rats transgenic for human renin that were mated with male rats transgenic for human angiotensinogen in contrast exhibited a decrease in BP. In these rats, human angiotensinogen in plasma remained undetectable. Double transgenic offspring of these transgenic rats developed hypertension and end-organ damage, regardless of the source of the transgenes. The conclusion is that transgenic rats that bear human renin and angiotensinogen genes make an attractive model for gestational hypertension. The rat model will have greater utility than the mouse model.

Gene Therapy for Hypertension: Recombinant Adeno-Associated Virus Vector Delivery of Angiotensin Type 1 Receptor Antisense in Hypertensive Double Transgenic Mice

P204 Introduction: The goal of gene therapy for hypertension is to produce prolonged reductions of high blood pressure with a single administration of a transgene. We have developed gene therapy using adeno-associated virus (AAV)antisense (AS) as a vector because it is safe, stable and effective. However, because large quantities of AAV are not yet available, our previous studies were in baby rats or by brain injections to achieve an effective concentration of AAV. Since mice are one-tenth the size of rats, systemic injection of AAV-AS achieves higher concentrations than in rats. To test systemic injection in an adult hypertensive model, this study uses double transgenic (dt) mice, with human renin (hR) and human AGT (hAGT) transgenes. In these mice, plasma Ang II levels are elevated and blood pressure increased (∼156 mmHg), compared to controls (∼ 100 mmHg). Methods: dt mice with established baseline BP of >156 mmHg (n=5) were injected (100 μl) with a single dose of 4x10 10 infectious particles of rAAV-AT 1 R-AS. Blood pressure recordings by the tailcuff method were made once per week for 6 months. Results: One week after intracardiac injection of rAAV-AT 1 R-AS, blood pressure decreased by 35-50 mmHg (p<0.01,compared to baseline). The normalized blood pressure persisted for the full length of the study, 28 weeks. Individual mice were sacrificed at 14-28 weeks and tissues taken for detection of rAAV-AT 1 R- AS, At both time periods, the neo r transgene was present in lung, kidney, liver, heart, adrenal gland and fat. The rAAV was not detected in the brain. Renal arterioles (n=3) showed a reduced (50%)contractile response to doses of Ang II, compared to controls (n=3). Conclusion: The results demonstrated that a single systemic delivery of rAAV-AT 1 R-AS in adult, hypertensive mice produces a profound decrease in blood pressure for at least 6 months. The prolonged effect is due to the continuous expression of the AT 1 -R antisense transgene inhibiting AT 1 receptors.

Gene Therapy for Hypertension: Recombinant Adeno-Associated Virus Vector Delivery of Angiotensin Type 1 Receptor Antisense in Hypertensive Double Transgenic Mice

P204 Introduction: The goal of gene therapy for hypertension is to produce prolonged reductions of high blood pressure with a single administration of a transgene. We have developed gene therapy using adeno-associated virus (AAV)antisense (AS) as a vector because it is safe, stable and effective. However, because large quantities of AAV are not yet available, our previous studies were in baby rats or by brain injections to achieve an effective concentration of AAV. Since mice are one-tenth the size of rats, systemic injection of AAV-AS achieves higher concentrations than in rats. To test systemic injection in an adult hypertensive model, this study uses double transgenic (dt) mice, with human renin (hR) and human AGT (hAGT) transgenes. In these mice, plasma Ang II levels are elevated and blood pressure increased (∼156 mmHg), compared to controls (∼ 100 mmHg). Methods: dt mice with established baseline BP of >156 mmHg (n=5) were injected (100 μl) with a single dose of 4x1010 infectious particles of rAAV-AT1R-AS. Blood pressure recordings by the tailcuff method were made once per week for 6 months. Results: One week after intracardiac injection of rAAV-AT1R-AS, blood pressure decreased by 35-50 mmHg (p<0.01,compared to baseline). The normalized blood pressure persisted for the full length of the study, 28 weeks. Individual mice were sacrificed at 14-28 weeks and tissues taken for detection of rAAV-AT1R- AS, At both time periods, the neor transgene was present in lung, kidney, liver, heart, adrenal gland and fat. The rAAV was not detected in the brain. Renal arterioles (n=3) showed a reduced (50%)contractile response to doses of Ang II, compared to controls (n=3). Conclusion: The results demonstrated that a single systemic delivery of rAAV-AT1R-AS in adult, hypertensive mice produces a profound decrease in blood pressure for at least 6 months. The prolonged effect is due to the continuous expression of the AT1-R antisense transgene inhibiting AT1 receptors.

Angiotensin Paradoxically Upregulates a Human Renin Transgene Containing a 900bp Promoter by a Pressure-Independent Mechanism

P196 Renin gene expression is negatively regulated by angiotensin II (AII) and blood pressure (BP). In transgenic mice containing the entire human renin (HREN) gene with only 900 bp of promoter sequence, pressor AII infusion paradoxically increases renal HREN mRNA. Lack of upregulation during subpressor AII suggests that this increase might be mediated by increased BP. To test this hypothesis, the AII-induced increase in BP was prevented by coadministration of the vasodilator, hydralazine (H). AII and H were infused at 1000 ng/kg/min and 15 mg/kg/day, respectively, by osmotic minipump. Isotonic saline was infused as vehicle (V). BP was measured daily by tail cuff, after a 7-day training period and a 5-day baseline period. Renal renin mRNA expression was measured by ribonuclease protection assay (normalized to expression of actin). BP during the last 3 days of a 5 day infusion of AII (n=5) was increased by an average of 37 ± 4 mmHg. There was no significant change in BP in mice infused with V (n=4) or with AII+H (n=4). The level of endogenous mouse renin mRNA was appropriately decreased by about 60% in both AII and AII+H mice relative to V mice. In contrast, HREN mRNA increased almost 2-fold (1.9 ± 0.3) in AII mice compared to V mice. In addition, the normalization of BP in AII+H mice did not prevent the increase in HREN mRNA (2.1 ± 0.3 fold). Thus, renal HREN in transgenic mice containing the entire HREN gene with 900 bp of promoter sequence is increased by pressor AII even when the BP increase is ameliorated by H, suggesting that the upregulation may be due to a direct effect of AII. Moreover, the finding from our previous study of a lack of upregulation during subpressor AII infusion may indicate that this action of AII is dose-dependent. We are currently performing similar studies in transgenic mice containing a well regulated HREN transgene to further elucidate the molecular mechanisms of renin gene regulation and to better understand the defect existing in the present model.

Raised blood pressure, not renin-angiotensin systems, causes cardiac fibrosis in TGR m(Ren2)27 rats.

Elevated systemic arterial blood pressure is associated with left ventricular hypertrophy and fibrosis. It has been suggested that both circulating and local myocardial renin-angiotensin systems play a role in mediating these responses. Here we describe the natural history of ventricular hypertrophy and fibrosis in the transgenic (mRen2)27 rat--a monogenetic model--which has a high tissue expression of the murine renin transgene, and suffers severe hypertension. We further explored the relative contribution of both hypertensive burden and circulating and tissue renin-angiotensin systems to the fibrotic process. The transgenic rats were treated from 28 days old with (1) a hypotensive dose of the ACE inhibitor ramipril which inhibited both tissue and circulating ACE activity, (2) the calcium antagonist amlodipine, or (3) a non-hypotensive dose of ramipril which inhibited about 60% of tissue ACE activity with little effect on circulating ACE. Normotensive Sprague-Dawley rats were used as controls. The transgenics developed left ventricular hypertrophy along with perivascular and interstitial fibrosis which became progressively worse up to 24 weeks of age. Both the high dose of ramipril and amlodipine prevented the hypertrophy and fibrosis, whereas tissue ACE inhibition without lowering blood pressure had no effect, and actually led to a worsening of the fibrosis by 24 weeks. These results suggest that the development of left ventricular hypertrophy and fibrosis in the transgenic (mRen2)27 rat are regulated by blood pressure and not activity of the renin-angiotensin systems and that progression of fibrosis at 24 weeks involves a mechanism unrelated to local renin-angiotensin activity.

Human renin mRNA expression and renin activity in transgenic mice.

The renin-angiotensin system (RAS) plays a very important role in the regulation of blood pressure and electrolyte homeostasis in mammals. It has also been hypothesized to regulate local tissue and organ blood supply in an autocrine/paracrine manner. To further study RAS physiology, transgenic mice carrying the human renin gene were produced by microinjection and were screened by PCR. The human renin transgene was found, by RT-PCR, to be expressed in the heart, kidney, and lung of transgenic mice, but not in the liver or skeletal muscle. The mean levels of renin activity in various tissues were determined by radioimmunoassay. The mean level of renin activity in the plasma and the heart/body weight ratio of the transgenic mice (n=7) were also found to be significantly higher (p< 0.05) than those of the control mice (n= 8). However, no significant differences were seen in the mean levels of renin activities in the kidney and heart between transgenic and control mice. Thus, the transcription of human renin gene in transgenic mice is regulated in a tissue-specific manner. This transgenic model will be useful in clarifying the regulatory mechanisms of the renin gene and the relationship between this gene and diseases.

Aminopeptidase A inhibitors as potential central antihypertensive agents.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT(1)) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT(1) receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Different effects of antihypertensive agents on cardiac and vascular hypertrophy in the transgenic rat line TGR(mRen2)27

The hypertensive transgenic rat model TGR(mRen2)27 has been used to investigate the development of cardiac and vascular hypertrophy in response to two different drug regimes. Cardiac hypertrophy was shown to be related to age and gender with the copy number of mouse renin transgenes having an additive effect. A similar observation was noted for hypertrophy in the vasculature, which was assessed using flow cytometry cell cycle DNA analysis of aortic vascular smooth muscle cells. Chronic treatment from weaning with equihypotensive doses of perindopril (2 mg/kg/day) or hydralazine and hydrochlorothiazide (4 mg/day of each) prevented the development of cardiac hypertrophy. Perindopril treatment also effectively prevented the development of vascular hypertrophy; however, treatment with hydralazine and hydrochlorothiazide was not as effective despite equivalent blood pressure reduction. These studies have demonstrated the presence of marked vascular and cardiac hypertrophy in the hypertensive transgenic TGR(mRen2)27 model of hypertension. Furthermore, these results provide new evidence to support the role of a locally activated renin angiotensin system in the blood vessel wall, which is involved in the pathogenesis of vascular hypertrophy in this transgenic rat model.

Appropriate regulation of renin and blood pressure in 45-kb human renin/human angiotensinogen transgenic mice.

The renin-angiotensin system is normally subject to servo control mechanisms that suppress plasma renin levels in response to increased blood pressure and increase plasma renin levels when blood pressure falls. In most species, renin is rate limiting, and angiotensinogen circulates at a concentration close to the Km, so varying the concentration of either can affect the rate of angiotensin formation. However, only the plasma renin level responds to changes in blood pressure and sodium balance to maintain blood pressure homeostasis. Therefore, the high plasma human renin levels and the hypertension of mice and rats containing both human renin and angiotensinogen transgenes indicate inappropriate regulation of renin and blood pressure. These anomalies led us to develop new lines of transgenic mice with a longer human renin gene fragment (45 kb) than earlier lines (13 to 15 kb). Unlike their predecessors, the 45-kb hREN mice secrete human renin only from the kidneys, and both the human and mouse renins respond appropriately to physiological stimuli. To determine whether blood pressure is also regulated appropriately, we crossed these new 45-kb hREN mice with mice containing the human angiotensinogen gene. All doubly transgenic mice were normotensive like their singly transgenic and nontransgenic littermates. Moreover, among doubly transgenic mice, both human and mouse plasma renin concentrations were suppressed relative to the singly transgenic 45-kb hREN mice. These findings demonstrate the importance of appropriate cell and tissue specificity of gene expression in constructing transgenic models and affirm the pivotal role played by renal renin secretion in blood pressure control.

Angiotensin-(1–7) immunoreactivity in the hypothalamus of the (mRen-2d)27 transgenic rat

The distribution of angiotensin-(1–7) immunoreactive neurons was compared to those of vasopressin-(VP) and oxytocin-(OT) immunoreactive (IR) neurons in the hypothalamus of adult (mRen-2d)27 transgenic hypertensive and Sprague–Dawley rats. In both strains, angiotensin (Ang)-(1–7)-IR cells were found in the supraoptic nucleus (SON), and in the anterior (ap-), medial (mp-), and lateral (lp-) parvocellular, and posterior magnocellular (pm-) subdivisions of the paraventricular (PVN) nucleus. Three-dimensional reconstructions showed that cells immunoreactive to Ang-(1–7) and VP were specifically co-distributed in the SON and in the pmPVN. Double-labeling neurons for both peptides revealed that both Ang-(1–7) and VP were colocalized in a subpopulation of neurons in the pmPVN and SON. In combination with previous studies, our results suggest that Ang-(1–7) and VP are colocalized, co-released and may have a combined action at a common target. In addition, the introduction of the mouse submandibular renin (mRen-2d) transgene into Sprague–Dawley rats does not appear to have altered the fundamental organization of hypothalamic peptide systems involved in fluid homeostasis.

High human renin hypertension in transgenic rats.

We developed a model of spontaneously high human renin hypertension in the rat by producing two transgenic strains, one for human angiotensinogen with the endogenous promoter and one for human renin with the endogenous promoter. Neither transgenic strain was hypertensive. These strains were then crossed, producing a double transgenic strain. The double transgenic rats, both males and females, developed severe hypertension (mean systolic pressure, 200 mm Hg) and died after a mean of 55 days if untreated. The rats had a human plasma renin concentration of 269 +/- 381 (+/-SD) ng angiotensin I (Ang I)/mL per hour, plasma renin activity of 177 +/- 176 ng Ang I/mL per hour, rat angiotensinogen concentration of 1.49 +/- 1 microgram Ang I/mL, and human angiotensinogen concentration of 78 +/- 39 micrograms Ang I/mL (n = 49). Control rats had plasma renin activity of 3.7 +/- 3.9 ng Ang I/mL per hour and rat angiotensinogen of 1.32 +/- 0.16 micrograms Ang I/mL. Angiotensinogen transgene expression by RNase protection assay was ubiquitously present but most prominent in liver. Renin transgene expression was high in kidney but absent in liver. The rats featured severe cardiac hypertrophy, with increased cross section of cardiomyocytes but little myocardial fibrosis. The kidneys showed atrophic tubules, thickened vessel walls, and increased interstitium. Both the angiotensin-converting enzyme inhibitor lisinopril and the specific human renin inhibitor remikiren lowered blood pressure to normal values. Double transgenic mice have been developed that exhibit features quite similar to those described here; their gene expressions are similar. The specificity of rodent and human renin is similarly documented. Although many elegant physiological studies can now be done in mice, rats nevertheless offer flexibility, particularly in terms of detailed cardiac and renal physiology and pharmacology. We conclude that this double transgenic strain will facilitate simultaneous investigation of genetic and pathophysiological aspects of renin-induced hypertension. The fact that human renin can be studied in the rat is a unique feature of this model.

Lifelong angiotensin-converting enzyme inhibition, pressure natriuresis, and renin-angiotensin system gene expression in transgenic (mRen-2)27 rats.

The transgenic rat (TGR) (mRen-2)27 is said to have low circulating active renin values in plasma and little or no renin gene expression in the kidney. Nevertheless, intrarenal angiotensin II-related effects appear to be responsible for the rightward shift in pressure-natriuresis curves of TGR. To clarify the role of the intrarenal renin-angiotensin system in modulating TGR pressure-natriuresis, TGR were given lifelong lisinopril by treating TGR and their mothers before conception. Rat and mouse renin, AT1 receptor, and angiotensinogen gene expression in the kidneys were studied with in situ hybridization. Neural and endocrine regulatory differences between TGR and Sprague-Dawley Hannover (SDH) rats were eliminated by renal denervation and infusion of vasopressin, aldosterone, 17-OH corticosterone, and norepinephrine. TGR with lisinopril had blood pressures similar to SDH. In TGR with lisinopril, the pressure-natriuresis curve was shifted leftward but not quite to the values observed in SDH given lisinopril. The histology of lisinopril-treated TGR was indistinguishable from normal SDH. Lisinopril increased rat renin and angiotensinogen gene expression both in SDH and TGR, but it did not influence mouse renin gene expression in TGR. Discontinuing lisinopril increased blood pressure in TGR and shifted the pressure-natriuresis relationship rightward. Thus, the components of the endogenous renin-angiotensin system and the mouse renin transgene were present and expressed in kidneys of TGR. The rat gene components responded to lisinopril as expected, but the mouse renin transgene expression was not influenced. Lisinopril normalized TGR blood pressure; however, a detectable leftward shift in pressure-natriuresis remained. These studies underscore the role of angiotensin-mediated effects of the mouse renin transgene in terms of shifting pressure-natriuresis in TGR.

Chromosomal mapping of human angiotensinogen gene and human renin gene by fluorescence in situ hybridization (FISH) in transgenic mice.

We attempted to apply FISH to chromosomal mapping of the human angiotensinogen (hAG) and human renin (hRN) transgenes which are carried by the parental strains of the Tsukuba hypertensive mouse. We report here that the hAG gene is mapped in the C2 region of Chr 19 and the hRN gene in the A1 region of Chr 6.

Infertility in a transgenic rat due to impairment of cytoplasmic elimination and sperm release from the Sertoli cells.

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2 [CP-2]), none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.

Infertility in a Transgenic Rat Due to Impairment of Cytoplasmic Elimination and Sperm Release from the Sertoli Cells

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2[CP-2], none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.