Robert Di Nicolantonio

Angiotensinogen and angiotensin-converting enzyme gene copy number and angiotensin and bradykinin peptide levels in mice.

Objective To test the hypothesis that changes in gene expression that may accompany angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) gene polymorphism cause alteration in angiotensin and bradykinin peptide levels. Design Mice with one or two genes for AGT and ACE allow assessment of the effects of modest alteration in AGT and ACE gene expression on angiotensin and bradykinin peptide levels. Methods Angiotensin and bradykinin peptides were measured in the blood, kidney, heart, lung, adrenal, brain, and aorta of mice that were either wild-type (+/+), heterozygous (+/−) or null (−/−) for either the AGT or ACE gene. Results Angiotensin I and angiotensin II were not detectable in blood or tissues of AGT −/− mice, which had increased bradykinin levels in kidney and lung. ACE −/− mice had markedly reduced angiotensin II levels and increased bradykinin levels in blood and tissues. However, despite reduced AGT and ACE gene expression, angiotensin and bradykinin peptide levels in AGT and ACE +/− mice were no different from the levels in wild-type mice. Conclusion Although the AGT and ACE genes are fundamental determinants of angiotensin and bradykinin peptide levels, compensatory mechanisms attenuate the effect of modest change in AGT and ACE gene expression on the levels of these peptides. Identification of these compensatory mechanisms may provide new candidate genes for investigation in humans.

Preferred salt levels and salt taste acuity in human subjects after ingestion of untasted salt

We have examined whether salt loading alters the salt preference or salt taste acuity of nine human subjects on continuous low salt diet. Subjects were randomly assigned to either untasted salt tablets (120 mmol/day) or placebo over a 2-week period at the end of which salt preference and salt recognition thresholds were measured. Subjects then received the alternate substance for another 2 weeks and the measurements were repeated. While urinary Na+ and Cl- were significantly increased while on salt tablets, urinary volume, K+, urea and creatinine concentrations, blood pressure, body weight, salivary and plasma electrolyte concentrations were unchanged. Plasma renin and aldosterone levels were reduced while on salt tablets but not significantly. Salt tablets caused a significant increase in sodium recognition threshold but a significant decrease in salt addition to unsalted tomato juice and in ideal salt level assessed by presalted (150 mmol/l) tomato juice. Thus, an increase in untasted dietary salt may reduce salt preference in human subjects, a finding opposite to that with an increased, tasted salt intake over a similar period.

Osmoregulatory fluid intake but not hypovolemic thirst is intact in mice lacking angiotensin

Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.

Altered age-dependent modulation of tissue renin messenger RNA levels in the spontaneously hypertensive rat

Objective To evaluate the possible role of tissue renin overproduction in the pathogenesis and time course of hypertension development in the spontaneously hypertensive rat (SHR) of the Okamoto strain by measuring tissue renin gene expression at different stages of hypertension development Design Measurements of kidney, adrenal gland and brain stem renin messenger RNA( mRNA) levels were performed in SHR aged 4, 12, 20 and 36 weeks and in age-matched normotensive Wistar-Kyoto (WKY) rats Methods A fully quantitative, competitive reverse transcriptase polymerase chain reaction method was used to measure tissue renin mRNA levels. Plasma renin concentrations were measured by radioimmunoassay Results Compared with age-matched WKY rats, renin mRNA levels in the adrenal gland of SHR were significantly higher at ages 4,12 and 36 weeks. Brain stem renin mRNA levels were significantly higher in SHR than in WKY rats at ages 4, 12 and 20 weeks. In contrast, renal renin mRNA levels were consistently lower in SHR at all ages, as were plasma renin concentrations Conclusions The present study indicates an important role for renin gene expression both in adrenal and in brain stem tissues in the pathogenesis of hypertension in SHR. Adrenal and brain renin-angiotensin systems may interact with each other synergistically in the development and maintenance of hypertension in SHR. Suppressed renal renin gene expression could then be an indirect consequence of the amplified renin-angiotensin system in the adrenal gland and in the brain or a baroreceptor-mediated consequence of its hypertension

RELATIVE CONTRIBUTION OF THE PRENATAL VERSUS POSTNATAL PERIOD ON DEVELOPMENT OF HYPERTENSION AND GROWTH RATE OF THE SPONTANEOUSLY HYPERTENSIVE RAT

1 To determine the relative roles of the prenatal and postnatal (preweaning) environment on the development of blood pressure and growth rate in the spontaneously hypertensive rat (SHR) of the Okamoto strain, we used combined embryo transfer and cross‐fostering techniques between SHR and normotensive Wistar‐Kyoto (WKY) rats to produce offspring whose development was examined during the first 20 weeks of life. 2 We measured litter sizes, bodyweights and tail‐cuff blood pressures in offspring at 4, 8, 12 and 20 weeks of age. We also recorded heart, kidney and adrenal weights at 20 weeks of age, when the study concluded. 3 We found that both the in utero and postnatal environments provided by the SHR mother could significantly affect WKY rat offspring growth rates, but blood pressure was unaffected in this strain. In SHR offspring, the SHR maternal in utero and suckling period both contributed to the rate of blood pressure development in the SHR, but not the final blood pressure of offspring at 20 weeks of age. This effect was greater for male than female offspring. Organ weights were largely unaffected by the perinatal environment in either strain. 4 We conclude that although the SHR maternal in utero and immediate postnatal environment both contribute to the rate of blood pressure development in the SHR, they do not appear to contribute to the final blood pressure of offspring at maturity. The SHR maternal environment also alters growth rate that may, in turn, underlie these effects on SHR blood pressure development, particularly in males.

TRANSITORY REDUCTION IN ANGIOTENSIN AT2 RECEPTOR EXPRESSION LEVELS IN POSTINFARCT REMODELLING IN RAT MYOCARDIUM

1. Myocardial infarction (MI) poses a significant risk for sudden cardiac death. The effectiveness of angiotensin‐converting enzyme (ACE) inhibitors and AT1 receptor blockade in attenuating unfavourable post‐MI outcomes indicates an important role for angiotensin (Ang) II signalling in the post‐MI remodelling process.

Altered nuclear protein binding to the first intron of the renin gene of the spontaneously hypertensive rat.

We and others have reported elevated levels of renin mRNA in extrarenal tissues of the spontaneously hypertensive rat (SHR) of the Okamoto strain. We hypothesise that this is due to mutations we have found in putative, cis-regulatory regions in the first intron of its renin gene. Here we report two G-A mutations at position +502 and +934 of the first intron of the SHR renin gene, when compared to normotensive Wistar Kyoto (WKY) and Sprague Dawley (SD) rats. These mutations fall within consensus sequences for the well described E2A and peroxisome proliferator-activated receptor (PPAR) transcription factors. We used electrophoretic mobility shift assays to determine if these mutations alter the pattern or affinity of nuclear protein binding to oligonucleotides homologous to these regions of the renin gene. Both mutations significantly altered the intensity and pattern of nuclear protein binding to oligonucleotides homologous with the renin gene regions bearing these putative transcription factor binding sites. Thus these mutations have the potential to alter the type and/or affinity of transcriptional factors for the SHR renin gene in vivo, and result in renin overproduction at an extra-renal tissue site subserving blood pressure control.

Central angiotensin converting enzyme blockade and thirst.

The role of endogenous brain angiotensin II (AII) in various thirst states was examined in the rat using the angiotensin converting enzyme inhibitor, captopril. Intracerebroventricular (ICV) captopril (7 micrograms) significantly attenuated the dipsogenic response to centrally administered angiotensin I (AI) (200 ng) for up to 2 hours. The same dose of captopril significantly potentiated the dipsogenic response to ICV AII (100 ng) but failed to alter the dipsogenic response to ICV carbachol (200 pmoles). Central pretreatment with captopril (7 micrograms), for 30 minutes, failed to alter markedly the cumulative water intake of 24 hour water deprived rats. However, a small, significant 8% decrease in water intake was noted in ICV captopril treated rats 60 minutes following the return of water. The same dose of captopril, administered intraperitoneally, significantly potentiated the cumulative water intake of 24 hour water deprived rats. Central pretreatment with captopril (7 micrograms), for 30 minutes, failed to alter the cumulative water intake of rats treated intraperitoneally with hypertonic saline (0.75 M given at a dose of 1% of the body weight). From these studies it would appear that central angiotensin converting enzyme plays only a minor role in thirst induced by water deprivation.

Fetal versus maternal determinants of the reduced fetal and placental growth in spontaneously hypertensive rats

Objective Epidemiological studies indicate that a reduced birth weight and enlarged placenta increase the likelihood of human cardiovascular disease later in life. The relative importance of fetal versus maternal factors in these phenomena is not known. To assess the relative role of genotypic versus environmental factors in this effect, we examined whether the altered fetal and placental growth rates and amniotic fluid volume of spontaneously hypertensive rat (SHR) fetuses of the Okamoto strain, are modified by gestation in normotensive Wistar–Kyoto (WKY) rat mothers and vice versa. Design One-day-old SHR embryos were gestated for 16 days in either SHR or WKY recipients. Similarly, 1-day-old WKY rat embryos were gestated for 16 days in either SHR or WKY surrogates. At 16 days, fetal and placental weights were recorded. Paternal and maternal donor and recipient blood pressures, maternal body weight and average litter size within the four groups were also studied. Methods One cell SHR and WKY embryos were harvested from timed matings and transferred to psuedopregnant mothers of the same or opposite strain. Timed matings required routine vaginal smears for the detection of proestrus and the presence of sperm following overnight matings. Harvested embryos were temporarily maintained in culture medium in a 37°C incubator until injection into the oviduct of recipients. Blood pressures were meaured using indirect, tail-cuff plethysmography and a computerized data acquisition system. Results SHR fetal and placental weights at 16 days gestation were significantly lower than WKY fetal and placental weights, irrespective of maternal strain. At 16 days of gestation, the fetal and placental weights of SHR fetuses gestated in WKY rat surrogate mothers (0.21 ± 0.01 g and 0.19 ± 0.01 g, respectively) were not significantly different from those of SHR gestated in a surrogate SHR mother (0.21 ± 0.01 g and 0.18 ± 0.01 g, respectively). Similarly, the fetal and placental weights of WKY fetuses gestated in a WKY rat (0.27 ± 0.01 g and 0.25 ± 0.01 g, respectively) were unaltered by gestation in a SHR recipient (0.25 ± 0.01 g and 0.23 ± 0.01 g, respectively). The amniotic fluid volumes of SHR gestated in WKY rats and those of WKY fetuses gestated in SHR were not significantly different to each other (0.37 ± 0.01 ml versus 0.38 ± 0.01 ml, respectively) and were intermediate between the values for SHR and WKY fetuses gestated in a mother of the same strain (0.34 ± 0.01 ml versus 0.44 ± 0.02 ml, respectively). Conclusion The SHR fetus exhibited reduced growth rate and placental size irrespective of maternal surrogate strain, suggesting that these measures are likely to be determined by the fetus or the placenta and, presumably, are independent of maternal blood pressure or altered electrolyte and hormonal milieu.

Blood pressure, salt appetite and mortality of genetically hypertensive and normotensive rats maintained on high and low salt diets from weaning.

1. Blood pressure, bodyweight, saline preference and mortality rate were examined in spontaneously hypertensive rats (SHR) of the Okamoto strain and normotensive control Wistar‐Kyoto (WKY) rats maintained on low (0.1% NaCl w/w), control (0.8% w/w) and high (3% w/w) salt diets from weaning until 6 months of age.