Jean E Robillard

Chronic hypertension and altered baroreflex responses in transgenic mice containing the human renin and human angiotensinogen genes.

We have generated a transgenic model consisting of both the human renin and human angiotensinogen genes to study further the role played by the renin-angiotensin system in regulating arterial pressure. Transgenic mice containing either gene alone were normotensive, whereas mice containing both genes were chronically hypertensive. Plasma renin activity and plasma angiotensin II levels were both markedly elevated in the double transgenic mice compared with either single transgenic or nontransgenic controls. The elevation in blood pressure caused by the human transgenes was independent of the genotype at the endogenous renin locus and was equal in mice homozygous for the Ren-1c allele or in mice containing one copy each of Ren-1c, Ren-1d, or Ren-2. Chronic overproduction of angiotensin II in the double transgenic mice resulted in a resetting of the baroreflex control of heart rate to a higher pressure without significantly changing the gain or sensitivity of the reflex. Moreover, this change was not due to the effects of elevated pressure itself since angiotensin-converting enzyme inhibition had minimal effects on the baroreflex in spontaneously hypertensive BPH-2 control mice, which exhibit non-renin-dependent hypertension. This double transgenic model should provide an excellent tool for further studies on the mechanisms of hypertension initiated by the renin-angiotensin system.

Developmental Aspects of Renal Tubular Reabsorption of Water in the Lamb Fetus

Summary: The effects of intrauterine maturation on the tubular reabsorption of water by the fetal kidney was determined in 44 chronically catheterized fetal lambs divided in three groups. Fourteen fetuses were studied between 101 and 119 days, 13 between 120 and 130 days, and 17 between 131 and 142 days. The urinary flow rate increased (P < 0.01) when fetuses <120 days were compared to fetuses between 120-130 days of gestation. A 35% increase in Uosm was found in fetuses over 130 days and was paralleled by significant increases in (U/P) iothalamate and (U/P) osm ratios. An increase in (U/P) urea ratio in fetuses over 130 days correlated with an increase in (U/P) iothalamate (r = 0.87). The (U/P) ratios for Na+ and Cl− were not correlated with fetal age and did not increase during gestation. The percent of filtered water excreted (V/GFR) was high before 120 days of gestation (26.8 ± 3.04%) and remained elevated in fetuses of 120-130 days (28.7 ± 2.53%). After 130 days of gestation, it decreased significantly to 18.3 ± 2.56% (P < 0.05). Similarly, CH2O/GFR values were, respectively, 12.02 ± 1.53 and 13.18 ± 1.46 ml/min/100 ml GFR in groups of fetuses of less than 120 days and between 120-130 days; after 130 days CH2O/GFR decreased significantly to 5.82 ± 1.97 ml/min/ 100 ml GFR (P < 0.02). Cosm/GFR remained unchanged in all three groups of fetuses at 14.76 ± 2.57, 15.49 ± 1.90, and 12.51 ± 1.34 ml/min/100 ml GFR, respectively. The simultaneous significant decreases in V/GFR and CH2O/GFR without changes in Cosm/GFR in fetuses over 130 days suggest that the increase in Uosm in near-term fetuses represents an increase in free water reabsorption by the fetal kidney.Speculation: The increase in free water reabsorption by the near-term fetal kidney may represent either an increase in end-organ responsiveness to arginine vasopressin or maturation of mechanisms involved in the buildup of the osmotic gradient in the fetal medulla or both.

The Dynamics of Vasopressin Release and Blood Volume Regulation During Fetal Hemorrhage in the Lamb Fetus

Summary: Experiments were performed on 11 long-term fetal lamb preparations (103–138 days of gestation) to investigate the sensitivity and relative responsiveness of the fetal volume receptors in modulating fetal plasma arginine vasopressin (pAVP) secretion and plasma renin activity (PRA) secretion during fetal hypovolemia and after fetal blood volume replacement. During fetal hemorrhage there were significant decreases (P < 0.05) in fetal hematocrit (34.7 ± 2.58 to 27.0 ± 1.64%), plasma proteins (3.14 ± 0.15 to 2.78 ± 0.19 g/100 ml), mean arterial blood pressure (MABP) (58.1 ± 2.59 to 52.2 ± 2.60 mmHg) and fetal arterial pH (7.38 ± 0.01 to 7.35 ± 0.01). A significant increase in fetal pAVP concentration from 0.73 ± 0.21 to 34.9 ± 10.04 μU/ml (P < 0.01) and fetal PRA from 4.78 ± 2.22 to 40.4 ± 18.31 ng/ml/hr (P < 0.05) was demonstrated at the peak of fetal hemorrhage. Two hr after correction of the fetoplacental blood volume, these values were back to base line levels. No change in either maternal pAVP or PRA was seen during fetal hemorrhage. When individual values for log pAVP and log PRA were plotted as a function of percent of fetoplacental blood volume removed the correlation coefficients were 0.82 and 0.60, respectively. A multiple regression analysis showed a high correlation of log pAVP and log PRA with the volume of blood removed and a low partial correlation with the fetal MABP. This suggests the decrease in fetal MABP was not the primary factor explaining the increase in pAVP and PRA during fetoplacental blood volume depletion. The data indicate that the fetal volume receptors for control of arginine vasopressin secretion are fully functional in the last trimester of gestation and suggests that fetal pAVP and PRA are released as an exponential function of the percent of fetoplacental blood volume depletion. Finally, an isosmotic water shift from the fetal interstitial space to the fetal vascular space is described during fetal hemorrhage.Speculation: It is suggested that a change in the equilibrium between the forces regulating fluid movement through the fetal capillary membranes, in accordance with Starlings principle, activates isosmotic water fluxes from the fetal interstitial space to the fetal vascular compartment counteracting the effects of fetal blood volume depletion. Therefore, the role of arginine vasopressin (AVP) release during fetal hemorrhage, if any, will be to act as a pressor substance helping to maintain fetal blood pressure. No major effect of AVP on placental membranes was demonstrated in vivo.

Effect of Cortisol on Gene Expression of the Renin-Angiotensin System in Fetal Sheep

ABSTRACT: Components of the renin-angiotensin system have been found in a variety of tissues during fetal and postnatal life and appear to be developmentally regulated. We postulated that hormonal changes associated with parturition participate in the regulation of renin, angiotensinogen (Ao) and angiotensin type 1 receptor (AT1) gene expression. Cortisol, which increases rapidly in fetal blood before delivery, has been shown to influence the maturation of various systems in the developing fetus. To test the hypothesis that an increase in cortisol regulates fetal renin, Ao, and AT1 mRNA gene expression, we used Northern blot analysis to study the effects of an intraperitoneal infusion of cortisol (3 mg/h, 1 mL/h) for 48 h on the expression of these genes in twin ovine fetuses (n = 10 pairs) at 130-d gestation (term 145 d); one twin in each pair served as a saline-treated control (0.9% NaCl, 1 mL/h). Plasma cortisol levels were significantly higher in cortisol-treated fetuses (113 ± 23 nmol/dL) than in twin controls (4.6 ± 0.8 nmol/dL). Cortisol infusion significantly decreased AT1 receptor mRNA levels in kidney and liver by 24 ± 7% and 27 ± 8%, respectively, when compared with controls (p < 0.05), whereas in contrast, increased mRNA levels (p < 0.05) in heart right atrium (91 ± 23%) and ventricle (59 ± 20%). Renin mRNA levels decreased in renal cortex by 77 ± 13% (p < 0.05) in cortisol-treated animals compared with controls. Hepatic Ao mRNA levels decreased by 15 ± 5% in response to cortisol (p < 0.05), whereas no significant effect was seen on renal Ao gene expression. These findings demonstrate that cortisol exhibits tissue specific positive and negative regulation of renin, Ao and AT1 receptor gene expression during fetal life and may act as an important modulator of the renin-angiotensin system during parturition.

Ontogeny of Single Glomerular Perfusion Rate in Fetal and Newborn Lambs

Summary: The developmental pattern of regional glomerular density and glomerular perfusion rate (GPR) was studied in 20 chronically catheterized fetal lambs between 106 and 140 days of gestation (term, 145 days) and in six newborn lambs between 3 and 19 days of age. The present study demonstrates for the first time in lambs that the nephrogenic zone disappears around 130 days of gestation and that the total glomerular counts per kidney in fetuses over 130 days (468296 ± 41173 glomeruli per kidney) is not significantly different than in newborn lambs (433704 ± 21553). Glomerular density, determined in four cortical zones (zone I being the outermost portion of the cortex) did not show any significant changes during fetal life; however, significant decreases in glomerular density were observed in each cortical zone after birth. The relative distribution of glomeruli during fetal life decreased in the outer portion (zones I and II) and increased in the inner portion (zones III and IV) of the cortex as fetuses matured and approached term. After birth, this difference became even more prominent; the outer cortical fraction (zone I) decreased from 49.6 ± 2.9% in fetuses of less than 120 days to 37.8 ± 1.5% (P < 0.05) in newborn lambs, whereas the fraction found in zone III increased from 14.9 ± 1.3% to 20.8 ± 0.7% (P < 0.05). Small but significant increases in glomerular filtration rate (GFR) (P < 0.01) and total renal blood flow (P < 0.05) were observed during fetal life: GFR and total renal blood flow increased, respectively, from 1.84 ± 0.11 and 37 ± 2 ml/min in fetuses <120 days to 3.05 ± 0.2 and 46 ± 4 ml/min in fetuses >130 days gestation. During the same period, filtration fraction (FF) did not increase significantly whereas a significant (P < 0.01) 58% increase in FF was observed after birth: FF increased from 10.16 ± 0.74% in fetuses >130 days gestation to 16.12 ± 1.66% in newborn lambs. Renal vascular resistance decreased from 1.03 ± 0.08 mm Hgml-1 min-1 in fetuses >130 days to 0.51 ± 0.05 mm Hgml-1min-1 (P < 0.01) in newborn lambs. Glomerular perfusion rate (GPR), computed for each cortical zone did not change significantly during the last trimester of gestation. After birth, GPR increased almost three times in zones I and II (from 69.2 ± 8.9 and 53.6 ± 4.2 nl/min in fetuses >130 days to 206.9 ± 16.8 and 161.3 ± 20.7 nl/min in newborn lambs, respectively), doubled in zone III (from 55.6 ± 10 nl/min in fetuses >130 days to 119.5 ± 8.9 nl/min in newborn lambs) and remained unchanged in zone IV when compared to fetal GPR values. After birth, the increase in GFR correlated closely with the GPR increase in zone I (r = 0.87) and zone II (r = 0.87), suggesting that the developmental pattern of GFR after birth may depend on the increase in GPR in the outer zones of the renal cortex.Speculation: The addition of new nephron units may be an important determinant of glomerular filtration rate (GFR) in fetuses of less than 130 days of gestation. In fetuses over 130 days of gestation, the addition of new nephron units is no longer a factor to explain the increase in fetal GFR but other factors such as increases in surface area for filtration, effective filtration pressure and capillar filtration coefficient may then play an important role. After birth, one can speculate that a decrease in glomerular vascular resistance is a major determinant in the postnatal increase in glomerular perfusion rate and GFR.

Effect of antenatal glucocorticoids on sympathetic nerve activity at birth in preterm sheep

Renal sympathetic nerve activity (RSNA) increases rapidly after delivery of term fetal sheep and parallels the rise in heart rate (HR) and arterial pressure. To examine the RSNA response at birth in immature lambs, experiments were performed in chronically instrumented preterm fetal sheep (118- to 125-day gestation, term 145 days) before and after delivery by cesarean section. HR remained unchanged from fetal values at 1 and 4 h after birth, whereas mean arterial blood pressure (MABP) decreased significantly ( P < 0.05) by 4 h after delivery. RSNA significantly decreased after premature birth in all animals studied ( n = 6), achieving only 39 ± 17% of fetal RSNA ( P< 0.05; all results are mean ± SE). Because cardiovascular function after premature birth is improved by the use of antenatal corticosteroids, we also tested the hypothesis that corticosteroid administration would evoke a more pronounced sympathetic response in prematurely delivered lambs ( n = 7, 118- to 125-day gestation). After maternal administration of dexamethasone (5 mg im, 48 and 24 h before delivery), RSNA increased after birth in six of seven fetuses to 166 ± 32% of the fetal RSNA value. Dexamethasone treatment also decreased the sensitivity of baroreflex-mediated changes in HR in response to increases in MABP. Because the sympathetic response at birth is depressed in preterm compared with term lambs, we performed an additional study ( n = 8) to determine if immature sheep are capable of mounting a sympathetic response to cold. In utero cooling produced rapid and sustained increases in MABP (20 ± 4%), HR (26 ± 6%), and RSNA (282 ± 72%) (all P < 0.05), consistent with a generalized sympathoexcitation. These results suggest that sympathoexcitation is absent after premature delivery despite the presence of functional descending autonomic pathways. Furthermore, exogenous corticosteroids appear to have a maturational effect on the sympathetic response at birth, which may be one mechanism by which maternal steroid administration improves postnatal cardiovascular homeostasis.Renal sympathetic nerve activity (RSNA) increases rapidly after delivery of term fetal sheep and parallels the rise in heart rate (HR) and arterial pressure. To examine the RSNA response at birth in immature lambs, experiments were performed in chronically instrumented preterm fetal sheep (118- to 125-day gestation, term 145 days) before and after delivery by cesarean section. HR remained unchanged from fetal values at 1 and 4 h after birth, whereas mean arterial blood pressure (MABP) decreased significantly (P < 0.05) by 4 h after delivery. RSNA significantly decreased after premature birth in all animals studied (n = 6), achieving only 39 +/- 17% of fetal RSNA (P < 0.05; all results are mean +/- SE). Because cardiovascular function after premature birth is improved by the use of antenatal corticosteroids, we also tested the hypothesis that corticosteroid administration would evoke a more pronounced sympathetic response in prematurely delivered lambs (n = 7, 118- to 125-day gestation). After maternal administration of dexamethasone (5 mg i.m., 48 and 24 h before delivery), RSNA increased after birth in six of seven fetuses to 166 +/- 32% of the fetal RSNA value. Dexamethasone treatment also decreased the sensitivity of baroreflex-mediated changes in HR in response to increases in MABP. Because the sympathetic response at birth is depressed in preterm compared with term lambs, we performed an additional study (n = 8) to determine if immature sheep are capable of mounting a sympathetic response to cold. In utero cooling produced rapid and sustained increases in MABP (20 +/- 4%), HR (26 +/- 6%), and RSNA (282 +/- 72%) (all P < 0.05), consistent with a generalized sympathoexcitation. These results suggest that sympathoexcitation is absent after premature delivery despite the presence of functional descending autonomic pathways. Furthermore, exogenous corticosteroids appear to have a maturational effect on the sympathetic response at birth, which may be one mechanism by which maternal steroid administration improves postnatal cardiovascular homeostasis.

Differential gene expression and regulation of renal angiotensin II receptor subtypes (AT1 and AT2) during fetal life in sheep.

ABSTRACT: Previous studies have shown that angiotensin II subtype 2 (AT2) receptors appear early during renal embryonic development. Factors involved in the regulation of AT2 receptors during renal development, however, have not been investigated. The present study was designed 1) to characterize the ontogeny of renal AT2 gene expression during the last half of gestation in fetal sheep and newborn lambs, 2) to compare changes in AT1 and AT2 gene expression during renal development, 3) to determine the influence of AII in modulating renal AT1 and AT2 gene expression during fetal life, and 4) to characterize the role of cortisol in modulating renal AT2 gene expression during the last trimester of gestation in fetal sheep. To perform these studies, we first isolated and cloned a polymerase chain reaction product that has 92 and 90% homology with the cDNA encoding the human and rat AT2 receptors, respectively. Using this sheep AT2 cDNA probe, we demonstrated that the sheep AT2 gene was encoded in a single locus. In addition, we showed that renal AT2 mRNA expression was high early during fetal life (60–90-d gestation) and decreased rapidly thereafter. In contrast, the expression of renal AT1 receptor gene was low at 60-d gestation and increased during the last trimester of gestation. We found that a continuous i.v. infusion (I mL/h) of AII (9.5 nM/h) for 24 h, which raised plasma AII levels from 84 ± 9 pg/mL to 210 ± 21 pg/mL, decreased the expression of both renal AT1 and AT2 genes in third trimester fetal sheep. On the other hand, we observed that cortisol, known to decrease AT1 gene expression in the fetus, had no effect on AT2 gene expression. In summary, this study demonstrates that AII, but not glucocorticoids, contributes to the regulation of renal AT2 gene expression during development and that there is differential regulation of AT1 and AT2 receptors.

Renal Hemodynamics and Functional Changes during the Transition from Fetal to Newborn Life in Sheep

ABSTRACT: The effects of delivery on renal function and renal hemodynamics were studied in conscious and chronically instrumented fetal sheep. Each fetus was studied 1 h before delivery and 1, 4, and 24 h following delivery by cesarean section. Delivery was not associated with significant changes in plasma renin activity, plasma angiotensin II, plasma aldosterone, and plasma arginine vasopressin concentrations when determined 1 h after birth. On the other hand, the transition from fetal to newborn life was accompanied by significant increases in plasma epinephrine and norepinephrine concentrations. No significant changes in renal blood flow velocity or in renal vascular resistance were observed during the transition from fetal to newborn life; percent changes in renal blood flow velocity and renal vascular resistance values were respectively 15.4 ± 11 and - 2.4 ± 1.0% at 1 h, 4.0 ± 8.0 and 5.8 ± 9.1% at 4 h, and 3.2 ± 8.0 and 9.7 ± 13% at 24 h. No significant changes in urinary flow rate, urine osmolality, free water clearance, and osmolar clearance were observed in the first 24 h following delivery. On the other hand, glomerular filtration rate increased 3-fold from 3.3 ± 0.4 ml/min in fetuses to 10.1 ± 1.2 ml/min in newborn lambs at 24 h of age. This rise in glomerular filtration rate was associated with significant decreases in urinary sodium excretion (UNaV) (from 36 ± 7 to 13 ± 3 μEq/min) and fractional excretion of sodium (FENa) (from 7.6 ± 0.9 to 1.1 ± 0.3%). In summary, present results demonstrate that transition from fetal to newborn life is associated with a rapid rise in glomerular filtration rate and an important decrease in urinary sodium excretion and fractional excretion of sodium. These changes seem to be independent of changes in renal blood flow velocity, renal vascular resistance, and blood pressure.

Maturational changes in the fetal glomerular filtration rate

Few data are available documenting the evolution of glomerular filtration rate (GFR) during gestation. In order to investigate this aspect, 33 fetal sheep have been studied, with the use of an intrauterine fetal preparation. It appears that the actual GFR (AGFR) in milliliters per minute increased during the last third of gestation without a significant increase in GFR expressed as milliliters per minute per kilogram of fetal weight (GFR-FW) and as milliliters per minute per gram of kidney weight (GFR-KW). It is suggested that a parallel and constant increase in AGFR and in total fetal weight or kidney weight might explain the absence of variation if GFR-FW or GFR-KW during the last third of gestation.

Mechanisms regulating renal sodium excretion during development.

The present review focuses on the ontogeny of mechanisms involved in renal sodium excretion during renal maturation. The effect of birth on renal excretion of sodium and the role played by the different tubular segments in the regulation of sodium excretion during maturation are discussed. The influence of circulating catecholamines and renal sympathetic innervation in regulating sodium excretion during renal development is reviewed. The effects of aldosterone, atrial natriuretic factor, and prostaglandins on sodium regulation during renal maturation are discussed. Special emphasis is given to the potential role of glucocorticoids in modulating sodium excretion early in life.