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Angiotensin and Bradykinin Peptides in the TGR(mRen-2)27 Rat

Originally publishedhttps://doi.org/10.1161/01.HYP.25.5.1014Hypertension. 1995;25:1014–1020

    Abstract

    Abstract The transgenic TGR(mRen-2)27 rat, in which the Ren-2 mouse renin gene is transfected into the genome of the Sprague-Dawley rat, develops severe hypertension at a young age that responds to inhibitors of angiotensin-converting enzyme and to antagonists of the type 1 angiotensin II (Ang II) receptor. Despite this evidence that the hypertension is Ang II dependent, TGR(mRen-2)27 rats have suppressed renal renin and renin mRNA content, and there is controversy concerning the plasma levels of renin and Ang II in these rats. We investigated the effect of the transgene on circulating and tissue levels of angiotensin and bradykinin peptides in 6-week-old male homozygous TGR-(mRen-2)27 rats. Systolic blood pressure of TGR(mRen-2)27 rats was 212±4 mm Hg (mean±SEM, n=25) compared with 108±2 mm Hg (n=29) for age- and sex-matched Sprague-Dawley rats. Compared with control rats, TGR(mRen-2)27 rats had increased plasma levels of active renin (4.5-fold), prorenin (300-fold), and Ang II (fourfold) as well as tissue levels of Ang II (twofold to fourfold in kidney, adrenal, heart, aorta, brown adipose tissue, and lung and 18-fold in brain). Plasma angiotensinogen levels were reduced to 73% of control, and plasma aldosterone levels were increased fourfold. Plasma angiotensin-converting enzyme was reduced to 64% of control. Compared with control rats, TGR-(mRen-2)27 rats had increased bradykinin levels in brown adipose tissue (1.9-fold) and lung (1.6-fold). The elevated circulating and tissue levels of Ang II in TGR(mRen-2)27 rats provide direct support for an Ang II–dependent mechanism of hypertension in these rats.

    The transgenic TGR(mRen-2)27 rat, in which the Ren-2 mouse renin gene is transfected into the genome of the Sprague-Dawley (SD) rat, develops severe hypertension at a young age. The hypertension responds to inhibitors of angiotensin-converting enzyme (ACE) and antagonists of the type 1 (AT1) angiotensin II (Ang II) receptor,1234 suggesting an Ang II dependence of the hypertension. The transgene is expressed at high levels in the adrenal and also in the thymus, small intestine, testis, ovary, coagulation gland, kidney, brain, lung, blood vessels, pituitary, and thyroid.1256 TGR(mRen-2)27 rats have suppressed renal renin and renin mRNA contents,1267 and the major form of circulating renin is derived from the adrenally expressed transgene.789 TGR(mRen-2)27 rats have increased plasma aldosterone levels and urinary aldosterone secretion.1810

    Although TGR(mRen-2)27 rats have markedly increased plasma prorenin levels, initial studies reported that these rats have suppressed plasma levels of renin and immunoreactive angiotensin peptides,1711 leading to the hypothesis that the hypertension is due to increased angiotensin peptide formation in a specific tissue compartment.1211 We investigated this hypothesis by measuring angiotensin peptide levels in plasma, kidney, adrenal, heart, aorta, brown adipose tissue, lung, and brain of control male SD rats and male homozygous TGR(mRen-2)27 rats. We also measured tissue levels of bradykinin-(1-9) [BK-(1-9)] and its metabolite BK-(1-7) to determine the effects of the expression of the renin transgene on the activity of the bradykinin system.

    Methods

    Animals

    In June 1993, we received from Drs Detlev and Ursula Ganten two families of homozygous TGR(mRen-2)27 rats, each comprising two males and two females from generation F30. The homozygous TGR(mRen-2)27 strain was maintained with between-family matings, and the TGR(mRen-2)27 rats used in this experiment were from generation F32. TGR(mRen-2)27 breeding stock were administered lisinopril in their drinking water (10 mg/L) to control hypertension and ensure survival. Rats were fed a diet of GR 2+ pellets (Clarke King & Co) and received tap water to drink. These studies were performed in accordance with the guidelines of the Animal Experimentation Ethics Committees of St Vincent’s Hospital and Melbourne University, Victoria, Australia.

    To minimize the influence of lisinopril, TGR(mRen-2)27 dams were withdrawn from lisinopril 2 weeks before giving birth, and the pups were fostered to normal SD dams at birth. Control SD pups were also fostered to normal SD dams at birth. These experiments were performed with 25 male homozygous TGR(mRen-2)27 rats and 29 male control SD rats. Systolic blood pressures were measured by the tail-cuff method (model PE-300 programmed electro-sphygmomanometer, Narco Bio-Systems, Inc) at 38 to 40 days of age. At the age of 40 to 44 days, rats were killed by decapitation between noon and 2 pm without prior anesthetic.

    Extraction and Radioimmunoassay of Angiotensin Peptides From Plasma

    Plasma levels of Ang-(1-7), Ang II, and Ang I were measured as described previously.12 Briefly, trunk blood (1 to 2 mL) was rapidly collected into tubes containing 0.33 mL inhibitor solution (220 μmol/L pepstatin, 76 mmol/L 1,10-phenanthroline, 190 mmol/L EDTA, 3 g/L neomycin sulfate, 3% dimethyl sulfoxide, and 3% ethanol in water) at 4°C. The renin inhibitor JF1 (0.17 mL of 3 mmol/L) was immediately mixed with the blood. JF1 has the sequence N-acetyl-His-Pro-Phe-Val-Leu-(R)-Leu-Leu-Phe-NH2, where –(R)– is a reduced bond; this peptide was synthesized at St Vincent’s Institute of Medical Research (Victoria, Australia) and is based on the series of rat renin inhibitors described by Hui et al.13 JF1 produces 50% inhibition of rat renin at approximately 0.2 μmol/L and greater than 95% inhibition of rat and mouse renin at 10 μmol/L. Blood from two rats was pooled to obtain sufficient sample for analysis. The blood was centrifuged and the plasma (1 to 2 mL) immediately extracted with Sep-Pak C18 cartridges (Waters Chromatography Division, Millipore). Angiotensin peptides were acetylated and piperidine treated before high-performance liquid chromatography (HPLC), and assay of HPLC fractions was performed by N-terminal–directed radioimmunoassay.1214 Data were corrected for recovery, as reported elsewhere.12

    Extraction and Radioimmunoassay of Angiotensin and Bradykinin Peptides From Blood and Tissues

    Kidney, adrenals, heart (cardiac ventricles), aorta, periaortic brown adipose tissue with associated connective tissue, lung, and brain (comprising brain stem, hypothalamus, thalamus, septum, and midbrain) were rapidly removed, weighed, and immediately homogenized in 4 mol/L guanidine thiocyanate and 1% (vol/vol) trifluoroacetic acid in water and processed as described previously15 before acetylation and piperidine treatment, HPLC, and measurement of angiotensin and bradykinin peptides by N-terminal–directed radioimmunoassay.1215 Data were corrected for recovery, as reported elsewhere.1215 The time delay between decapitation and homogenization for adrenals was 60 seconds and for kidney was 90 seconds; heart and lung were homogenized within 120 seconds, aorta and brown adipose tissue were homogenized within 150 seconds, and brain was homogenized within 180 seconds. For adrenals, lung, aorta, brown adipose tissue, and brain, homogenates from two rats were pooled to obtain sufficient sample for analysis.

    Measurement of ACE, Renin, Angiotensinogen, and Aldosterone in Plasma

    Trunk blood for measurement of ACE, renin, angiotensinogen, and aldosterone was collected into heparinized tubes on ice and centrifuged and the plasma rapidly frozen on dry ice and stored at −30°C. The plasma concentrations of ACE enzymatic activity and angiotensinogen were measured as described previously.16 ACE enzymatic activity was measured with the use of 3-(2-furylacryloyl)-l-phenylalanyl-glycyl-glycine (FAPGG) as substrate.17 Plasma aldosterone was measured by radioimmunoassay (Coat-a-Count Direct RIA, Diagnostic Products Corp).

    Renin was measured by radioimmunoassay of Ang I generated during incubation of samples with nephrectomized rat plasma at 37°C, pH 7.4. Renin and prorenin activities are expressed as picomoles Ang I generated per milliliter per hour of incubation in the presence of excess angiotensinogen. Experiments performed with hog renin as reference standard (National Standards Laboratory, Holly Hill, London, UK) showed that 1 pmol Ang I/mL per hour corresponds to 0.015×10−3 Goldblatt units/mL. For the measurement of active renin, 20 μL thawed plasma was mixed with 12.5 μL nephrectomized plasma, 30 μL inhibitor mixture (25 mmol/L N-ethylmaleimide, 20 mmol/L disodium EDTA, and 100 mmol/L benzamidine) and 37.5 μL diluent (100 mmol/L sodium phosphate, pH 7.4, 1 mg/mL bovine serum albumin, and 1 mmol/L disodium EDTA). The final angiotensinogen concentration was 0.9 μmol/L. Samples were incubated for 30 minutes at 37°C and then placed on ice, and 0.1 mL diluted Ang I antibody and 0.25 mL 125I-labeled Ang I were added for radioimmunoassay of the Ang I generated. After incubation at 4°C for 18 to 24 hours, dextran-coated charcoal was added to the tubes, which were centrifuged, the supernatant was separated, and both the supernatant and charcoal pellet were counted in a gamma counter. A parallel series of tubes kept at 4°C instead of incubation at 37°C showed negligible generation of Ang I. For measurement of total renin, 40 μL plasma was incubated with 20 μL trypsin (6 mg/mL) for 10 minutes at 4°C, and the reaction was terminated by addition of 20 μL soybean trypsin inhibitor (8 mg/mL). The trypsin-treated plasma was assayed for renin activity as described above. Plasma prorenin levels were calculated by subtraction of active renin from the total plasma renin determined after trypsin activation.

    In separate experiments, we assessed whether mouse prorenin was activated during collection of trunk blood from TGR(mRen-2)27 rats. Six male homozygous TGR(mRen-2)27 rats aged 12 to 15 weeks and withdrawn from lisinopril for 2 to 3 weeks had carotid arterial cannulas inserted while under sodium pentobarbital anesthesia. On the following day, the cannula of conscious, unrestrained rats was flushed with approximately 0.1 mL heparinized (20 IU/mL) 0.15 mol/L sodium chloride, and 0.5 mL blood was collected from the cannula, after which the rats were immediately decapitated, and trunk blood was collected. Each blood sample was initially collected into a heparinized tube on ice, and 0.2 to 0.5 mL blood was added to a tube containing a dried mixture of protease inhibitors (0.75 μmol N-ethylmaleimide, 0.6 μmol disodium EDTA, and 3 μmol benzamidine). The blood was centrifuged and the plasma rapidly frozen on dry ice. The cannula plasmas and trunk plasmas, each collected in the presence or absence of inhibitors, were assayed for active renin, and plasma samples without inhibitors were assayed for total renin.

    Statistical Analysis

    Data are presented as mean±SEM. Comparisons between control SD and TGR(mRen-2)27 rats were made using the unpaired Student’s t test. Comparisons within each rat were made by paired Student’s t test. All probability values were two-tailed; a value of P<.05 was considered statistically significant. When more than half of the samples comprising a mean had values below the minimum detectable, the sample mean is shown as less than the minimum detectable. Where values were below the minimum detectable, they were set at half the minimum detectable for statistical calculations. Logarithmic transformation of data was performed where appropriate to obtain similar variances among groups.

    Results

    Six-week-old male homozygous TGR(mRen-2)27 rats had the same body weight as normotensive SD rats but had increased heart, kidney, and adrenal weights and ratios of heart weight to body weight, kidney weight to body weight, and adrenal weight to body weight (Table 1). TGR(mRen-2)27 rats were markedly hypertensive, with increased plasma levels of renin (4.5-fold), prorenin (300-fold), and aldosterone (fourfold). Plasma angiotensinogen levels were reduced to 73% of control, and plasma ACE levels were 64% of control.

    To determine whether inadvertent activation of prorenin occurred during collection of trunk blood, we compared the levels of renin in blood collected from the cannula of a separate group of conscious cannulated TGR(mRen-2)27 rats, with the levels in trunk blood collected from the same rats. Cannula and trunk blood samples were collected in either the presence or absence of protease inhibitors. Renin levels in trunk plasma were 31% to 35% higher than the renin levels of cannula plasma (cannula plasma renin without inhibitors, 7.4±0.7 pmol Ang I/mL per hour, n=6; trunk plasma renin without inhibitors, 10.0±1.4, P<.02 by paired t test; cannula plasma renin with inhibitors, 7.0±0.9; trunk plasma renin with inhibitors, 9.2±1.4, P<.02). The collection of blood into protease inhibitors did not influence the measured renin level. Prorenin levels in cannula and trunk plasma were similar (cannula plasma prorenin, 451±44 pmol Ang I/mL per hour, n=6; trunk plasma prorenin, 441±42).

    Compared with control SD rats, TGR(mRen-2)27 rats had increased plasma levels of Ang-(1-7) (twofold), Ang II (fourfold), and Ang I (3.3-fold). TGR(mRen-2)27 rats also had increased tissue levels of Ang II of twofold to fourfold in kidney, adrenal, heart, aorta, brown adipose tissue, and lung and 18-fold in brain (Table 2). Compared with control SD rats, Ang I levels in brown adipose tissue and lung were increased in TGR(mRen-2)27 rats, but the low tissue Ang I levels in adrenal, heart, aorta, and brain did not show a statistically significant increase. By contrast, renal Ang I levels in TGR(mRen-2)27 rats were only 35% of levels in control SD rats. Thus, the kidney showed an eightfold higher ratio of Ang II to Ang I than control SD rats. Plasma also showed a small but statistically significant increase in Ang II–Ang I ratio in TGR(mRen-2)27 rats, but for all other tissues, the Ang II–Ang I ratios were similar for TGR(mRen-2)27 and SD rats.

    Although tissue bradykinin peptide levels tended to be higher in TGR(mRen-2)27 rats than in control rats for all tissues studied, the increase was statistically significant for only brown adipose tissue (1.9-fold) and lung (1.6-fold) (Table 3). For all tissues, the ratio of BK-(1-7) to BK-(1-9) was similar in TGR(mRen-2)27 and control SD rats.

    Discussion

    The TGR(mRen-2)27 rat was originally characterized as an Ang II–dependent form of hypertension with low circulating levels of renin and angiotensin peptides.1711 In contrast to previous reports, we found that 6-week-old male homozygous TGR(mRen-2)27 rats have elevated circulating renin and angiotensin levels in association with increased tissue levels of angiotensin peptides. Tissue bradykinin peptide levels were also elevated in brown adipose tissue and lung of TGR(mRen-2)27 rats.

    Several factors may have contributed to the differences between the present results and previous findings of reduced plasma renin and angiotensin levels in TGR-(mRen-2)27 rats. First, we studied male homozygous TGR(mRen-2)27 rats to achieve maximal transgene expression.18 An advantage of homozygous TGR(mRen-2)27 rats is that the data obtained are directly comparable to those of previous studies and avoid confounding factors related to breeding the transgene into different genetic backgrounds.19 Second, we used 6-week-old rats with minimal prior exposure to ACE inhibitor. If allowed to live longer than 6 weeks without antihypertensive treatment, the rats rapidly die.19 In previous reports,12345678910112021 the nature of exposure of TGR(mRen-2)27 rats to ACE inhibitor in utero and during suckling is not documented; however, exposure to ACE inhibitor during this period may slow the rate of development of hypertension in these rats. Third, we paid particular attention to the method of blood collection for measurement of plasma levels of renin and angiotensin peptides to minimize stress-related increases in renin secretion.

    Given the fact that TGR(mRen-2)27 rats had higher plasma renin levels than control SD rats, we were concerned that activation of prorenin may have occurred during collection of trunk blood from these rats. In a comparison of renin levels in trunk blood and blood obtained from arterial cannulas of conscious animals, we found that trunk blood renin levels were 31% to 35% higher than cannula blood levels. However, this small increase in renin levels in trunk blood contributed little to the 4.5-fold higher plasma renin levels in TGR(mRen-2)27 rats. Support for the higher plasma renin levels in TGR(mRen-2)27 rats is the lower plasma angiotensinogen levels in these rats.

    Mullins and Mullins19 have noted that the original transgenic founder was not generated on an inbred background, so there is no true genetic control for the model; the Hannover SD strain is probably the best alternative. Although our control SD rats were not the Hannover SD strain, our plasma renin (and other) data for 6-week-old male SD rats were very similar to data we have previously obtained for 6-week-old male Donryu rats (unpublished data from this laboratory, 1994). Thus, we believe that our data for SD rats are representative of normotensive rats. In agreement with our own data, Tokita et al8 found increased plasma renin and Ang II levels in TGR(mRen-2)27 rats compared with Harlan SD rats and SD rats of the Hannover strain. Moreover, the plasma renin levels reported by these authors for the Hannover SD rats (approximately 5 pmol Ang I/mL per hour) are very similar to the levels measured in SD rats in this laboratory. The higher renin levels in control SD rats measured in previous studies suggest that renin secretion may have been stimulated by the use of ether anesthesia during blood collection.11122 Tokita et al8 found that light ether anesthesia causes a 2- to 10-fold increase in plasma renin levels in both Harlan and Hannover strains of SD rats, and these authors suggest that plasma renin levels of the TGR(mRen-2)27 rats may not respond to ether anesthesia, given the fact that extrarenal tissues are the main source of plasma renin in these rats. Consequently, stress may affect comparisons of plasma renin or angiotensin levels between TGR(mRen-2)27 and normotensive SD rats.

    In addition to the original report of reduced plasma levels of immunoreactive angiotensin peptides in TGR(mRen-2)27 rats,1 several other laboratories have reported plasma angiotensin peptide levels in these rats.35820 Hilgers et al5 measured plasma Ang II levels in conscious rats with arterial cannulas and found lower Ang II levels in female heterozygous TGR(mRen-2)27 rats than in female SD controls. However, the plasma Ang II levels measured in control rats were high (98±23 fmol/mL), suggesting that these rats had elevated renin secretion. Two other studies reported that plasma Ang II levels in trunk blood of decapitated female heterozygous TGR(mRen-2)27 rats are either similar to3 or increased20 compared with levels in normotensive SD rats. In agreement with our own findings, Tokita et al8 found increased plasma Ang II levels in trunk blood of decapitated male TGR(mRen-2)27 rats. The high plasma Ang I levels and low Ang II–Ang I ratios reported for control SD rats by some laboratories may relate to a failure to collect blood into appropriate inhibitors of renin, thus allowing continued generation of Ang I during sample processing.1320 For the present studies, blood was collected into a potent renin inhibitor.

    The angiotensin peptide levels of kidney of TGR(mRen-2)27 rats are of particular interest, given that the pressure-diuresis-natriuresis relationship is shifted to higher pressures in TGR(mRen-2)27 rats; this is associated with enhanced tubular reabsorption of sodium,423 which is consistent with their higher plasma aldosterone levels. Lo et al4 have proposed that the TGR(mRen-2)27 rat may develop sodium retention. We found renal Ang II levels to be increased threefold in TGR(mRen-2)27 rats, and renal Ang I levels were decreased to one third of the levels of control SD rats. It is well established that renin gene expression is suppressed in the TGR(mRen-2)27 kidney.267 This suppression of renin gene expression may be a consequence of the hypertension and also the high renal Ang II levels observed in the present study. What was the mechanism of the high renal Ang II levels in TGR(mRen-2)27 kidney? We propose that the high Ang II levels in TGR(mRen-2)27 kidney were due to the same processes operating in many of the other tissues of the TGR(mRen-2)27 rat, ie, the delivery of higher renin levels to tissues via the blood.

    We12 and others24 have shown that Ang II levels in extrarenal tissues of normal animals are higher than can be accounted for by the plasma content of tissues and are consistent with Ang II formation by tissues. Moreover, the marked suppression of tissue Ang II and renin levels after nephrectomy1224 indicates that kidney-derived renin is the predominant mechanism of Ang II formation in extrarenal tissue, caused by the tissue uptake of renin from plasma. By a similar mechanism, renal uptake of circulating renin may account for Ang II formation in the TGR(mRen-2)27 kidney. In previous studies of the effects of ACE inhibitors and AT1 antagonists in SD rats, we have shown that increased renin secretion from the kidney occurs without a commensurate increase in renal angiotensin peptide levels (References 1616 and 2525 and unpublished observations, 1994). We proposed that this failure of renal angiotensin peptide levels to increase is a consequence of local exhaustion of angiotensinogen because the kidney, as the source of circulating renin, necessarily has very high renin levels within the renal tissue.1625 However, in the TGR(mRen-2)27 rat, the kidney is not the source of the high circulating levels of renin. Consequently, local renal angiotensinogen levels would not be exhausted, and renal Ang II levels are able to increase in proportion to the increase in plasma renin.

    Why were Ang I levels suppressed in the TGR(mRen-2)27 kidney compared with control SD kidney? Whereas brown adipose tissue and lung of TGR(mRen-2)27 rats showed an increase in Ang I levels, most tissues (adrenal, heart, aorta, and brain) had low Ang I levels that were not increased. This may be due to efficient conversion and/or degradation of Ang I within tissues. Studies of the regional production of angiotensin peptides emphasize the high degree of compartmentalization of Ang I and Ang II production.26 For the kidney, Ang I formation by locally produced renin may occur in a different compartment than Ang I formed by plasma renin. Thus, the reduced renal Ang I levels in TGR(mRen-2)27 rats may be a reflection of reduced renin secretion by the TGR(mRen-2)27 kidney.

    Compared with control SD rats, TGR(mRen-2)27 rats had increased Ang II levels in all tissues studied. Our findings are in agreement with those of Hilgers et al,5 who found increased release of angiotensin peptides from isolated perfused hindquarters of TGR(mRen-2)27 rats. For plasma, kidney, adrenal, heart, aorta, brown adipose tissue, and lung of TGR(mRen-2)27 rats, the twofold to fourfold increase in Ang II levels was similar to the 4.5-fold increase in plasma renin levels. By contrast, the brain of TGR(mRen-2)27 rats showed an 18-fold increase in Ang II levels. In agreement with our finding, Senanayake et al20 found increased levels of Ang II and Ang I in hypothalamus and medulla oblongata of 10-week-old female heterozygous TGR(mRen-2)27 rats. The study of Senanayake et al is significant for another reason; these authors have previously reported levels of greater than 1000 fmol/g for Ang II in hypothalami of control SD rats,27 whereas they now report hypothalamic Ang II levels of less than 10 fmol/g in control SD rats,20 in close agreement with estimates from this laboratory.28 We have previously noted that these Ang II levels in control SD brain are of very low abundance for a neurotransmitter.28 Moreover, the absence of angiotensinogen and its mRNA in neurons2930 is against a neurotransmitter role for Ang II in the brain. It will be of interest to discover the cellular location of the much higher Ang II levels in TGR(mRen-2)27 rat brain.

    Although the adrenal of TGR(mRen-2)27 rats shows the highest levels of transgene expression126 and is a major source of circulating renin and prorenin,78 the increase in adrenal Ang II levels was only 2.6-fold. We have observed much greater increases (eightfold) in adrenal Ang II levels in rats administered the AT1 antagonist losartan (unpublished data from this laboratory, 1994). Thus, there was no apparent correlation between the levels of transgene expression in each tissue and the increase in tissue levels of Ang II. It is not possible to infer from the present data which tissues or organs play a major role in the pathogenesis of hypertension in TGR(mRen-2)27 rats. As we have mentioned previously, the kidney plays an essential role, and the increased renal Ang II levels may be critical to this role. The marked 18-fold increase in brain Ang II levels is certainly suggestive of a role for the central nervous system in the pathogenesis of hypertension in these rats, but an obvious mediator, the sympathoadrenal system, is not notably active in TGR(mRen-2)27 rats.4

    We found increased plasma aldosterone levels in TGR(mRen-2)27 rats, in agreement with previous studies.1810 Whether these increased plasma aldosterone levels are consequent to the increased circulating or adrenal Ang II levels is unknown. We also found that TGR(mRen-2)27 rats have larger adrenals and an increased ratio of adrenal weight to body weight. Although we did not measure plasma corticotropin or glucocorticoid levels in these experiments, previous studies found increased urinary glucocorticoid excretion in TGR(mRen-2)27 rats.10 Sander et al10 found that heterozygous TGR(mRen-2)27 rats have normal plasma corticotropin levels, although these rats show an increased glucocorticoid responsiveness to corticotropin. Springate et al21 have previously noted an increased ratio of kidney weight to body weight in TGR(mRen-2)27 rats. The increased adrenal and kidney weights of TGR(mRen-2)27 rats may be due to trophic effects of high circulating and tissue levels of Ang II.

    In the present study, only brown adipose tissue and lung showed an increase in bradykinin peptide levels in TGR(mRen-2)27 rats. Little is known of the regulation of circulating and tissue levels of bradykinin. Although mineralocorticoids stimulate renal kallikrein gene expression,31 the fourfold increase in plasma aldosterone levels in TGR(mRen-2)27 rats had little effect on renal bradykinin levels. Madeddu et al32 reported that blockade of the bradykinin type 2 receptor with Hoe 140 potentiates the pressor effects of Ang II, indicating an interaction between Ang II and bradykinin. However, the present data suggest that the renin-angiotensin system is not an important regulator of the bradykinin system.

    Moriguchi et al3 found no difference between plasma ACE levels of SD and TGR(mRen-2)27 rats when using Hip-His-Leu as substrate. By contrast, we found reduced ACE levels in TGR(mRen-2)27 rats when FAPGG was used as substrate. The measurement of plasma ACE activity is clearly substrate dependent,333435 which may be due to different substrates having differential metabolism by non-ACE enzymes. The studies of Nussberger et al34 and Delacrétaz et al36 show that the plasma Ang II–Ang I ratio is a reliable index of ACE activity. The TGR(mRen-2)27 rats showed a significant increase in Ang II–Ang I ratio in plasma, indicating that true ACE activity is probably increased in plasma of TGR(mRen-2)27 rats.

    Although we have shown that TGR(mRen-2)27 rats have higher renin, Ang II, and aldosterone levels, many aspects of the mechanism of the hypertension in these rats remain to be defined. Long-term infusion of pressor doses of Ang II increases the vascular media-lumen ratio, media thickness, and media cross-sectional area.37 However, TGR(mRen-2)27 rats have increased media-lumen ratio and media thickness without an increase in media cross-sectional area.38 Thus, Ang II infusion causes vascular hypertrophy, whereas the TGR(mRen-2)27 rat demonstrates vascular remodeling without hypertrophy or hyperplasia. These differences between long-term Ang II infusion and the TGR(mRen-2)27 rat suggest that the effects of the transgene on vascular structure are not solely due to an increase in Ang II formation.

    Table 1. Measurements in 6-Week-Old Normotensive Male Sprague-Dawley and Homozygous TGR(mRen-2)27 Rats

    ParameterSprague-DawleyTGR(mRen-2)27P
    Systolic blood pressure, mm Hg108 ±2212±4<.001
    Body weight, g156 ±4147±2NS
    Heart weight, mg558 ±16653±12<.001
    Heart weight–body weight ratio, mg/g3.8±0.14.4±0.1<.001
    Left kidney weight, mg659±21748±13<.01
    Left kidney weight–body weight ratio, mg/g4.49±0.085.05±0.05<.001
    Adrenal weight, mg35±143±1<.001
    Adrenal weight–body weight ratio, mg/g0.23±0.010.30±0.01<.001
    Plasma renin, (pmol Ang I/mL)/h8±136±1<.001
    Plasma prorenin, (pmol Ang I/mL)/h1.4±0.6433±31<.001
    Plasma angiotensinogen, pmol/mL542±22398±11<.001
    Plasma ACE, U/L291 ±18187±2<.001
    Plasma aldosterone, pmol/L254 ±991047±315<.01

    ACE indicates angiotensin-converting enzyme. Data are mean±SEM. For blood pressure and body weight, n=29 for control rats and n=25 for TGR(mRen-2)27 rats. For heart and kidney weights and ratios of heart weight to body weight and kidney weight to body weight, n=12. For adrenal weights and ratio of adrenal weight to body weight, n=24. For plasma renin, angiotensinogen, ACE, and aldosterone, n=9. Heart refers to cardiac ventricles.

    Table 2. Circulating and Tissue Levels of Angiotensin Peptides in 6-Week-Old Normotensive Male Sprague-Dawley and Homozygous TGR(mRen-2)27 Rats

    PeptideSprague-DawleyTGR(mRen-2)27P
    Plasma
    Ang-(1-7)6.3±0.812.8 ±1.3<.001
    Ang II31±3125 ±9<.001
    Ang I23±278±5<.001
    Ang II–Ang I ratio1.3±0.11.6±0.1<.05
    Kidney
    Ang II155±10474±40<.001
    Ang I57±720 ±2<.001
    Ang II–Ang I ratio2.9±0.324 ±2<.001
    Adrenal
    Ang II2225±1625891 ±344<.001
    Ang I<20<20
    Ang II–Ang I ratioNDND
    Heart
    Ang II15±328±3<.001
    Ang I5 ±27±1
    Ang II–Ang I ratio2.4±0.84.0±0.5
    Aorta
    Ang II290±28583±45<.001
    Ang I<410±3
    Ang II–Ang I ratioNDND
    Brown adipose tissue
    Ang II157±11486 ±46<.001
    Ang I<58.4 ±1.4<.05
    Ang II–Ang I ratioND71±12
    Lung
    Ang II174±8474±28<.001
    Ang I<26.3±1.0<.02
    Ang II–Ang I ratioND99±17
    Brain
    Ang II14±2260±11<.001
    Ang I<2.54±1
    Ang II–Ang I ratioNDND

    Ang indicates angiotensin, expressed as fmol/mL plasma or fmol/g tissue; blank spaces, NS. Data are mean±SEM. For plasma, n=10 for control rats and n=8 for TGR(mRen-2)27 rats. For tissues of control and TGR(mRen-2)27 rats, n=10. Heart refers to cardiac ventricles.

    Table 3. Tissue Levels of Bradykinin Peptides in 6-Week-Old Normotensive Male Sprague-Dawley and Homozygous TGR(mRen-2)27 Rats

    PeptideSprague-DawleyTGR(mRen-2)27P
    Kidney
    BK-(1-7)104±11126 ±12
    BK-(1-9)97±12144±23
    BK-(1-7)/BK-(1-9) ratio1.1±0.11.0±0.1
    Adrenal
    BK-(1-7)236 ±39277±29
    BK-(1-9)215±41230±34
    BK-(1-7)/BK-(1-9) ratio1.1±0.11.3±0.1
    Heart
    BK-(1-7)50±757±8
    BK-(1-9)39±846±6
    BK-(1-7)/BK-(1-9) ratio1.4±0.11.2±0.1
    Aorta
    BK-(1-7)99±15119±24
    BK-(1-9)85±6121 ±36
    BK-(1-7)/BK-(1-9) ratio1.2±0.21.1±0.1
    Brown adipose tissue
    BK-(1-7)117±15220 ±34<.01
    BK-(1-9)95±15183 ±28<.02
    BK-(1-7)/BK-(1-9) ratio1.3±0.11.3±0.1
    Lung
    BK-(1-7)68±1294±8
    BK-(1-9)51 ±683±10<.02
    BK-(1-7)/BK-(1-9) ratio1.3 ±0.21.2±0.2
    Brain
    BK-(1-7)38±445±8
    BK-(1-9)15±224±4
    BK-(1-7)/BK-(1-9) ratio2.5 ±0.22.1±0.3

    BK indicates bradykinin, expressed as fmol/g; blank spaces, NS. Data are mean±SEM, n=10. Heart refers to cardiac ventricles.

    This study was funded by grants from the National Health and Medical Research Council of Australia. We are grateful to Thaddeus P. Gorski for performing the assays for plasma ACE.

    Footnotes

    Correspondence to Dr D.J. Campbell, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy 3065, Australia.

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