To test the hypothesis that angiotensin II (ANG II) is necessary for normal embryonic and postnatal kidney development, the effect of angiotensin receptor blockade or angiotensin converting enzyme inhibition on nephrovascular development was studied in newborn Sprague-Dawley rats and in Rana catesbeiana tadpoles undergoing prometamorphosis. Blockade of ANG II type 1 receptor (AT1) in newborn rats induced an arrest in nephrovascular maturation and renal growth, resulting in altered kidney architecture, characterized by fewer, thicker, and shorter afferent arterioles, reduced glomerular size and number, and tubular dilatation. Inhibition of ANG II generation in tadpoles induced even more marked developmental renal abnormalities. Blockade of ANG II type 2 receptor (AT2) in newborn rats did not alter renal growth or morphology. Results indicate that ANG II regulates nephrovascular development, a role that is conserved across species.

The role of angiotensin II via the angiotensin type 1 or type 2 receptor in the development of cardiac hypertrophy was determined in adult male Sprague-Dawley rats subjected to coarctation of the abdominal aorta. Five groups of animals were studied: coarctation, coarctation plus DuP 753, coarctation plus PD 123319, sham plus DuP 753, or sham operation. Type 1 receptor blockade was accomplished with DuP 753 given in the drinking water and type 2 blockade with PD 123319 delivered by osmotic minipumps beginning with the day of surgery until 72 hours after aortic coarctation. Mean carotid blood pressures and the carotid-femoral artery blood pressure gradients were not different among coarctation, coarctation plus DuP 753, and coarctation plus PD 123319 animals. However, ratios of heart weight to body weight were higher in coarctation (4.95 ±0.8) or coarctation plus PD 123319 (4.52±0.5) than in sham animals (3.6±0.4; P<.005 and .05, respectively). In coarctation plus DuP 753-treated animals

To determine whether low oxygen is a stimulus for endothelial cell differentiation and vascular development in the kidney, we examined the effect of low oxygen on rat metanephric organ culture, a model known to recapitulate nephrogenesis in the absence of vessels. After 6 days in culture in standard (20% O2) or low oxygen (1-3% O2) conditions, metanephric kidney growth and morphology were assessed by DNA measurement, and light and electron microscopy. DNA content was higher in 3% O2-treated explants (2.5 +/- 0.17 microgram/kidney, n = 9) than in 20% O2 explants (1.5 +/- 0.09 microgram/kidney, n = 9), P < 0.05. Low oxygen induced proliferation of tubular epithelial cells, resulting in enhanced number of tubules of similar size. Endothelial cells forming capillaries were localized in 3% O2 explants by light and electron microscopy and by immunocytochemistry using endothelial cell markers. Flt-1, Flk-1, and ACE-containing cells were detected in 3% O2-treated explants, whereas 20% O2 explants were virtually negative. VEGF mRNA levels were 10-fold higher in 3% O2-treated explants than in 20% O2-treated explants. Addition of anti-VEGF antibodies to 3% O2-treated explants prevented low oxygen-induced growth and endothelial cell differentiation and proliferation. Our data indicate that low oxygen stimulates growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture. Upregulation of VEGF expression by low oxygen and prevention of low oxygen-induced tubulogenesis and vasculogenesis by anti-VEGF antibodies indicate that these changes were mediated by VEGF. These data suggest that low oxygen is the stimulus to initiate renal vascularization.

To determine whether the expression of the type 1 angiotensin II receptor (AT1) gene is developmentally regulated and whether the regulation is tissue specific, AT1 mRNA levels were determined by Northern blot analysis in livers and kidneys from fetal, newborn, and adult rats, using a 1133-bp rat AT1 cDNA. In the liver, AT, mRNA levels increased fivefold from 15 d gestation to 5 d of age. Liver AT, mRNA levels at 5 d of age were similar to those of adult rats. In the kidney, AT, mRNA levels were higher in immature than in adult animals. The intrarenal distribution of AT1 mRNA was assessed by in situ hybridization to a 35S-labeled 24 residues oligonucleotide complementary to rat AT1 mRNA. In the adult, AT1 mRNA was present in glomeruli, arteries, and vasa recta, whereas in the newborn AT1 mRNA was observed also over the nephrogenic area of the cortex.We conclude that: (a) fetal kidney and liver express the AT1 gene; (b) the AT1 gene expression is developmentally regulated in a tissue-specific manner; (c) during maturation, localization of AT1 mRNA in the kidney shifts from a widespread distribution in the nephrogenic cortex to specific sites in glomeruli, arteries, and vasa recta, suggesting a role for the angiotensin receptor in nephron growth and development. (J. Clin. Invest. 1993. 91:530-537.)

To determine whether angiotensin II (ANG II) modulates renal growth and renin and angiotensin type 1 (AT1) gene expression via AT1 during development, weanling rats were given ANG II antagonist losartan (DuP 753) for 3 wk. Body weight (g), kidney weight (g), and kidney weight-to-body weight ratio were lower in losartan-treated rats (162 +/- 7, 1.6 +/- 0.06, and 9.5 +/- 0.1 x 10(-3)) than in control rats (184 +/- 5, 1.8 +/- 0.07, and 10.1 +/- 0.1 x 10(-3); P < 0.05). Renal DNA content (mg/kidney) was lower in losartan-treated (2.4 +/- 0.17) than in control rats (3.3 +/- 0.31; P < 0.05), whereas protein-to-DNA and RNA-to-DNA ratios were similar in losartan-treated and control rats. Renin mRNA levels were sevenfold higher in losartan-treated than in control rats, as determined by quantitative standardized dot blot analysis. In addition, blockade of AT1 with losartan induced recruitment of renin-synthesizing and renin-containing cells in the renal vasculature, as determined by immunocytochemistry and in situ hybridization. To establish whether AT1 blockade has a direct effect on renin gene expression, freshly isolated renin-producing cells were exposed in vitro to losartan (10(-6) M) or culture media (control). Losartan induced a twofold increase in steady-state renin mRNA levels above control (P < 0.05). Intrarenal AT1 mRNA levels were not altered by losartan given either in vivo or in vitro to freshly dispersed cells. To define whether immature renin-secreting cells are responsive to ANG II, renin release was determined by reverse hemolytic plaque assay.(ABSTRACT TRUNCATED AT 250 WORDS)

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The enzyme renin and the angiotensin II (Ang II), subtype I receptor (ATI) are developmentally regulated in a tissue-specific manner. In early life, renin is expressed widely along the renal vasculature. As maturation progresses, there is a decrease in renin mRNA levels and a shift in the localization of renin close to the glomerulus. In addition, in the newborn rat, the number of renin-secreting cells is higher than in the adult rat. Exposure of neonatal and adult cells to Ang II results in a decrease of similar magnitude in the number of renin-secreting cells. These findings suggest that the high levels of renin observed in immature animals are due to increased renin synthesis and release rather than to a blunted response to Ang II. Expression of the ATI gene is also developmentally regulated in a tissue-specific manner. With maturation, ATI mRNA levels decrease in the kidney while they increase in the liver. The localization of ATI transcripts in precursor cells of the nephrogenic cortex suggests a role for this receptor in nephron growth and development. Inhibition of ATI with DUP753 results in delayed kidney and somatic growth and in increased renin mRNA levels and recruitment of renin-containing cells. These observations suggest that Ang II exerts a tonic negative feedback on renin gene expression via the ATI receptor subtype. Further studies are necessary to delineate the molecular and cellular signals mediating these developmental changes.

To determine whether decreased perfusion pressure affects the abundance and distribution of renin and its mRNA and the expression of the angiotensin II type 1 (AT1) receptor gene within the kidney, adult male Sprague-Dawley rats were subjected to aortic coarctation proximal to the renal arteries (Coarc, n = 8) and compared with sham-operated rats (Sham, n = 6). Renal renin distribution was determined by immunocytochemistry using a specific polyclonal antibody against rat renin. Renin mRNA was assessed by in situ hybridization to a 35S-labeled oligonucleotide complementary to rat renin mRNA. Kidney AT1 mRNA levels were determined by Northern analysis using a 1,133-base pair rat AT1 cDNA. Femoral arterial blood pressure, measured 24 h after surgery, was lower in Coarc than in Sham rats (75 +/- 5.4 vs. 122 +/- 2.3 mmHg, P < 0.05). Aortic coarctation increased the percent of juxtaglomerular apparatuses (%JGA) containing renin and its mRNA (85 +/- 2.5 and 66 +/- 2.8 vs. 49 +/- 5.3 and 36 +/- 1.7%, Coarc vs. Sham, P < 0.05) and the intensity of hybridization signals (497 +/- 89 vs. 71 +/- 12 grains/JGA, Coarc vs. Sham, P < 0.05). In addition, recruitment of renin gene expressing cells was observed along afferent arterioles in Coarc rats, whereas renin and its mRNA were limited to the JGAs in Sham rats. Renal AT1 receptor gene expression was threefold lower in Coarc than in Sham rats. We conclude that reduction of perfusion pressure after abdominal aortic coarctation acutely enhances renin gene expression and downregulates AT1 receptor gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)