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Previous studies have shown that angiotensin I1 subtype 2 (AT,) receptors appear early during renal embryonic development. Factors involved in the regulation of AT, receptors during renal development, however, have not been investigated. The present study was designed 1 ) to characterize the ontogeny of renal AT, gene expression during the last half of gestation in fetal sheep and newborn lambs, 2) to compare changes in AT, and AT, gene expression during renal development, 3) to determine the influence of A11 in modulating renal AT, and AT, gene expression during fetal life, and 4) to characterize the role of cortisol in modulating renal AT, 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 AT, receptors, respectively. Using this sheep AT, cDNA probe, we demonstrated that the sheep AT, gene was encoded in a single locus. In addition, we showed that renal AT, mRNA expression was high early during fetal life (60-90-d gestation) and decreased rapidly thereafter. In contrast, the expression of renal AT, receptor gene was low at 60-d gestation and increased during the last trimester of gestation. We found that a continuous i.v. infusion (1 mL/h) of A11 (9.5 nM/h) for 24 h, which raised plasma A11 levels from 84 t-9 pg/mL to 210 i-21 pg/mL, decreased the expression of both renal AT, and AT, genes in third trimester fetal sheep. On the other hand, we observed that cortisol, known to decrease AT, gene expression in the fetus, had no effect on AT, gene expression. In summary, this study demonstrates that AII, but not glucocorticoids, contributes to the regulation of renal AT, gene expression during development and that there is differential regulation of AT, and AT, receptors. (2), and the expression of renin, angiotensinogen, and angiotensinconverting enzyme genes appear to be developmentally regulated (3-6).It has been suggested that A11 is implicated in the regulation of renal function (1) and renal growth (7) during development.The biologic effects of A11 are mediated by two distinct specific receptors (AT, and AT,) located in the plasma membrane of different tissues (8). Studies in rats (9, 10) and sheep (11) have shown that the expression of kidney AT, receptor mRNA is developmentally regulated. In the sheep, renal AT, mRNA expression is elevated during the last trimester of gestation and decreases during the second postnatal week (1 1).In the rat, the expression of renal AT, receptor mRNA is also higher in immature than in adult animals (12). Discrete expression of AT, receptor has been observed as early as 2 d of postnatal age in rat immature glomeruli (10).Both in situ hybridization and autoradiographic studies have also shown that AT, receptors are present in the fetal mesenchyme (13), in the mesonephros before its involution (14), in

Factors regulating the expression of the angiotensin I1 subtype 1 (AT,) receptor during fetal life have not been investigated previously. The present study was designed 1) to characterize the ontogeny of AT, receptor gene expression in the kidney of fetal and newborn sheep and 2) to determine the influence of both glucocorticoids and renal nerves in modulating AT, gene expression during fetal life and during the transition from fetal to newborn life. We first isolated and cloned a PCR product that has 98 and 94% homology with the cDNA encoding the bovine and pig AT, receptors, respectively, and 99 and 98% homology with the corresponding deduced protein sequences. Probing with this cDNA, we demonstrated that renal AT, mRNA expression did not change significantly during the last trimester of gestation in fetal sheep or immediately after birth but decreased significantly 10 d after birth. We also demonstrated that renal denervation in the fetus had no effect on renal AT, gene expression in 24-h-old newborn lambs. On the other hand, we observed in 130-d twin fetuses that a continuous intraperitoneal infusion (1 mL/h) of cortisol (3 mg/h or 6.2 p,mol/h) for 48 h in one of the twins increased the fetal plasma cortisol concentration from 32.0 + 7.1 to 1126 r 231 nmol/L and produced a significant decrease ( p < 0.005) in renal AT, gene expression compared with the control twin receiving an intraperitoneal infusion of 0.9% NaCl. In summary, this study demonstrates that renal AT, gene expression is elevated during fetal life and decreases after birth. It is also shown that glucocorticoids, but not renal nerves, contribute to the regulation of renal AT, gene expression during development. (Pediatr Res 36: 755-762, 1994)Abbreviations RAS, renin-angiotensin system AII, angiotensin I1 AT,, angiotensin I1 subtype 1 receptor AT,, angiotensin I1 subtype 2 receptor PCR, polymerase chain reaction RSNA, renal sympathetic nerve activity PRA, plasma renin activity rRNA, ribosomal RNA It has become apparent that the physiologic role of the RAS changes during embryonic and fetal maturation (1-4). Early during development, the RAS exerts a major influence o n cellular growth and organ differentiation (3-6). It is only later during fetal life that the RAS becomes an important modulator of blood pressure and distinct A11 receptor subtypes (AT, and AT,) (3, 9, 10). The AT, receptor subtype is expressed early during embryonic life in the rat (11,12), predominates in the fetal mesenchyme (3), and shows a marked decrease in expression during fetal and postnatal maturation (3). On the other hand, the AT, receptor subtype appears later during fetal development in the rat (3, 9, 10) and seems t o b e localized mainly in areas related t o blood pressure regulation and fluid homeostasis (3). Recent studies in rats have also shown that the AT, receptor gene is expressed in the fetal kidney, liver, adrenal, and heart and that the expression of this gene is developmentally regulated (9, lo), in agreement with previous A11 radioligand and in situ rece...

The role of renal nerves in regulating changes in plasma renin activity (PRA) and renal renin gene expression was studied in intact (n = 6) and denervated (n = 6) fetal sheep before birth and during the first 24 h after delivery. Renal denervation completely blunted the rise in PRA observed 24 h after delivery in newborn lambs; in lambs with intact kidneys, PRA increased significantly (P less than 0.05) from 3.26 +/- 0.60 (predelivery) to 6.34 +/- 1.85 ng angiotensin I (ANG I).ml-1.h-1 (24 h postdelivery), while in lambs with denervated kidneys, predelivery and post-delivery values were 2.84 +/- 0.19 and 2.49 +/- 0.45 ng ANG I.ml-1.h-1, respectively. Renin mRNA levels were significantly lower (P less than 0.001) in denervated than in intact kidneys 24 h after birth. A close analysis of these results also revealed that renin mRNA levels were significantly higher (P less than 0.001) in intact kidneys of newborn lambs delivered vaginally (n = 3) than in newborn lambs delivered by cesarean section (n = 3). These results suggest that renal nerves play an important role in regulating renin gene expression and PRA during the transition from fetal to newborn life.

We have studied the role of glucocorticoids in inducing the maturation in activity of the proximal tubule Na+/H+ exchanger that follows birth. Renal cortical microvillus membrane vesicles were prepared from 132-day gestation sheep fetuses (n = 8) that had received intraperitoneal cortisol (13 micrograms.kg-1.h-1) for the previous 48 h. Membrane vesicles were also obtained from sham-operated twin controls (n = 8). Amiloride-sensitive uptake of 22Na+ by these vesicles was measured, and Woolf-Augustinsson-Hofstee plots were used to determine the Michaelis constant (Km) and maximal velocity (Vmax). There was no significant difference in Km; however, the Vmax was 61% higher in cortisol-treated fetuses. Posttreatment circulating cortisol levels were significantly higher in the treated fetuses. Total RNA was collected from renal cortex of the eight pairs of twins when killed. Renal cortex Na+/H+ exchanger 3 (NHE3) mRNA levels were approximately fourfold higher in cortisol-treated than in control fetuses. Although proximal tubule Na+/H+ exchanger activity and renal cortex NHE3 mRNA levels increased significantly in cortisol-treated fetuses, cortisol infusion did not stimulate renal sodium reabsorption in the fetus but rather produced a natriuresis. These results demonstrate that glucocorticoids can induce an increase in both Na+/H+ exchanger activity and NHE3 mRNA levels during the last trimester of gestation in sheep. However, these changes are not associated with an increased ability of the fetal kidney to reabsorb sodium.