Association of intraoperative changes in brain-derived neurotrophic factor and postoperative delirium in older adults.

BACKGROUND: Delirium is common after surgery, although the aetiology is poorly defined. Brain-derived neurotrophic factor (BDNF) is a neurotrophin important in neurotransmission and neuroplasticity. Decreased levels of BDNF have been associated with poor cognitive outcomes, but few studies have characterized the role of BDNF perioperatively. We hypothesized that intraoperative decreases in BDNF levels are associated with postoperative delirium. METHODS: Patients undergoing spine surgery were enrolled in a prospective cohort study. Plasma BDNF was collected at baseline and at least hourly intraoperatively. Delirium was assessed using rigorous methods, including the Confusion Assessment Method (CAM) and CAM for the intensive care unit. Associations of changes in BDNF and delirium were examined using regression models. RESULTS: Postoperative delirium developed in 32 of 77 (42%) patients. The median baseline BDNF level was 7.6 ng ml -1 [interquartile range (IQR) 3.0-11.2] and generally declined intraoperatively [median decline 61% (IQR 31-80)]. There was no difference in baseline BDNF levels by delirium status. However, the percent decline in BDNF was greater in patients who developed delirium [median 74% (IQR 51-82)] vs in those who did not develop delirium [median 50% (IQR 14-79); P =0.03]. Each 1% decline in BDNF was associated with increased odds of delirium in unadjusted {odds ratio [OR] 1.02 [95% confidence interval (CI) 1.00-1.04]; P =0.01}, multivariable-adjusted [OR 1.02 (95% CI 1.00-1.03); P =0.03], and propensity score-adjusted models [OR 1.02 (95% CI 1.00-1.04); P =0.03]. CONCLUSIONS: We observed an association between intraoperative decline in plasma BDNF and delirium. These preliminary results need to be confirmed but suggest that plasma BDNF levels may be a biomarker for postoperative delirium.

Feasibility of pulmonary artery pressure measurements in infants through aorto-pulmonary shunts using a micromanometer pressure wire.

Assessment of pulmonary artery pressure is an essential element in the evaluation of children palliated with surgical aorto-pulmonary shunts prior to definitive surgical repair. We report the ease of use and accuracy of a 0.014 inch micromanometer pressure wire for the measurement of pulmonary artery pressures in children with aorto-pulmonary artery shunts. The study population consisted of 11 infants and children with either a 3.5 mm modified Blalock-Taussig shunt from the subclavian artery to the branch pulmonary artery after stage 1 Norwood repair for hypoplastic left heart syndrome or palliative staged repair for tetralogy of Fallot, or a central shunt for pulmonary atresia or double outlet right ventricle. The unique features of the micromanometer pressure wire allowed rapid access and accurate measurement of pulmonary pressures in all patients studied. We conclude that the micromanometer pressure wire is a unique and accurate alternative device for rapid and safe determinations of pulmonary artery pressures in children with aorto-pulmonary artery shunts.

Identification, cloning, and developmental expression of hepatoma-derived growth factor in the developing rat heart.

Hepatoma derived growth factor (HDGF) was identified as a developmentally regulated cardiac gene by mRNA differential display using 12-day rat fetal conotruncus vs. newborn aorta. The full-length rat HDGF cDNA was cloned from a rat fetal heart cDNA library and found to be 94 and 88% homologous to the mouse and human sequence, respectively. The rat sequence, like the human and mouse, contains a highly conserved amino portion and putative bipartite nuclear localization sequence. By Northern analysis, HDGF is highly expressed in the fetal conotruncus, heart, kidney, brain, and gut. By immunocytochemistry, HDGF was first detected only in atrial myocytes, hind gut epithelia, and notochord of the E10 rat with a nuclear expression pattern. By E12, expression had broadened to include the ventricular myocytes, endocardial cells, and cells of the ventricular outflow tract. HDGF is unique in that it is the first described nuclear targeted growth factor in the developing heart. The early expression of HDGF in embryonic heart and fetal gut suggests that HDGF may play a role in cardiovascular growth and differentiation.

Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells.

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous HDGF only in the nucleus and target a green fluorescent protein (GFP)-HDGF fusion to the nucleus. To define the features of the HDGF molecule that are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus demonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys --> Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34(cdc2) kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.

Angiotensin II regulates phosphorylation of translation elongation factor-2 in cardiac myocytes.

Increased protein synthesis is the cardinal feature of cardiac hypertrophy. We have studied angiotensin II (ANG II)-dependent regulation of eukaryotic elongation factor-2 (eEF-2), an essential component of protein translation required for polypeptide elongation, in rat neonatal cardiac myocytes. eEF2 is fully active in its dephosphorylated state and is inhibited following phosphorylation by eEF2 kinase. ANG II treatment (10(-10) - 10(-7) M) for 30 min produced an AT(1) receptor-specific and concentration- and time-dependent reduction in the phosphorylation of eEF-2. Protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin, but not the PP2B inhibitor FK506, attenuated ANG II-dependent dephosphorylation of eEF-2. ANG II activated mitogen-activated protein kinase, (MAPK) within 10 min of treatment, and blockade of MAPK activation with PD-98059 (1--20 nM) inhibited eEF-2 dephosphorylation. The effect of ANG II on eEF-2 dephosphorylation was also blocked by LY-29004 (1-20 nM), suggesting a role for phosphoinositide 3-kinase, but the mammalian target rapamycin inhibitor rapamycin (10--100 nM) had no effect. Together these results suggest that the ANG II-dependent increase in protein synthesis includes activation of eEF-2 via dephosphorylation by PP2A by a process that involves both PI3K and MAPK.

Protein phosphatase inhibitors arrest cell cycle and reduce branching morphogenesis in fetal rat lung cultures.

Protein phosphatase 2A (PP2A) is a key signal transduction intermediate in the regulation of cellular proliferation and differentiation in vitro. However, the role of PP2A in the context of a developing organ is unknown. To explore the role of PP2A in the regulation of lung development, we studied the effect of PP2A inhibition on new airway branching, induction of apoptosis, DNA synthesis, and expression of epithelial marker genes in whole organ explant cultures of embryonic (E14) rat lung. Microdissected lung primordia were cultured in medium containing one of either two PP2A inhibitors, okadaic acid (OA, 0-9 nM) or cantharidin (Can, 0-3,600 nM), or with the PP2B inhibitor deltamethrin (Del, 0-10 microM) as a control for a PP2A-specific effect for 48 h. PP2A inhibition with OA and Can significantly inhibited airway branching and overall lung growth. PP2B inhibition with Del did not affect lung growth or new airway development. Histologically, both PP2A- and PP2B-inhibited explants were similar to controls. Increased apoptosis was not the mechanism of decreased lung growth and new airway branching inasmuch as OA-treated explant sections subjected to the terminal deoxynucleotidyltransferase dUTP nick end labeling reaction demonstrated a decrease in apoptosis. However, PP2A inhibition with OA increased DNA content and 5-bromo-2'-deoxyuridine uptake that correlated with a G(2)/M cell cycle arrest. PP2A inhibition also resulted in altered differentiation of the respiratory epithelium as evidenced by decreased mRNA levels of the early epithelial marker surfactant protein C. These findings suggest that inhibition of protein phosphatases with OA and Can halted mesenchymal cell cycle progression and reduced branching morphogenesis in fetal rat lung explant culture.

Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development.

Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8-24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope-tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury.

Developmental expression of protein phosphatase 2A in the kidney.

Although a number of growth and transcription factors are known to regulate renal growth and development, the signal transduction molecules necessary to mediate these developmental signals are relatively unknown. Therefore, the activity and mRNA and protein expression of the signal transduction molecule protein phosphatase 2A (PP2A) were examined during rat kidney development. Northern analysis of total kidney RNA or Western analysis of kidney protein homogenates from embryonic day 15 to 90-d-old adults demonstrated developmental regulation of the catalytic, major 55-kD B regulatory subunit and A structural subunit with the highest levels of expression in late embryonic and newborn kidneys. Similarly, okadaic acid-inhibitable phosphatase enzyme activity was highest in the embryonic and newborn kidney. To map cell-specific expression of PP2A in the developing kidney, in situ hybridization with a catalytic subunit digoxigenin-labeled cRNA was performed on embryonic day 20 and newborn kidneys. PP2A was found predominately in the nephrogenic cortex and particularly in the developing glomeruli and non-brush border tubules in the embryonic day 20 and newborn kidneys. Similarly, immunocytochemistry with a specific PP2A catalytic subunit polyclonal anti-peptide antibody demonstrated catalytic subunit protein particularly concentrated in the podocytes of glomeruli in the newborn kidney. In the adult kidney, PP2A protein was no longer detectable except in the nuclei of distal tubular cells. Therefore, the developmental regulation of PP2A activity and protein during kidney development and its mapping to the nephrogenic cortex, developing glomeruli, and tubules suggests a role for PP2A in the regulation of nephron growth and differentiation.

eNOS expression is not altered in pulmonary vascular remodeling due to increased pulmonary blood flow.

Congenital heart lesions resulting in increased pulmonary blood flow are common and if unrepaired often lead to pulmonary hypertension and heart failure. Therefore, we hypothesized that increased pulmonary blood flow without changes in pressure would result in remodeling of the pulmonary arterial wall. Furthermore, because the vasodilator nitric oxide is produced by the lung, is regulated by flow in the systemic circulation, and has been associated with the regulation of smooth muscle cell proliferation, we hypothesized that increased pulmonary blood flow would result in altered expression of endothelial nitric oxide synthase (eNOS). To study this hypothesis, 42-day-old Sprague-Dawley rats had creation of an aortocaval shunt to increase pulmonary blood flow for 6 wk. The shunt resulted in a significant increase in the heart- and lung-to-body weight ratios (>2-fold; P < 0.05) without significant alteration of pulmonary or systemic blood pressures. Significant thickening of the pulmonary arterial medial wall developed, with increased muscularization of small (50-100 micron)- and medium (101-200 micron)-sized arteries as evidenced by alpha-actin smooth muscle staining. Proliferating cell nuclear antigen staining and bromodeoxyuridine labeling did not detect proliferating smooth muscle cells in the vascular wall. eNOS Western and Northern blot analyses and immunohistochemical staining demonstrated that eNOS protein and mRNA levels were not altered in the shunt lungs compared with sham controls. Therefore, increased pulmonary flow without increased pressure resulted in pulmonary artery medial thickening, without ongoing proliferation. Unlike chronic hypoxia-induced vascular remodeling, the pulmonary vascular remodeling resulting from increased pulmonary blood flow is not associated with changes in eNOS.

Expression and mapping of protein phosphatase 2A alpha in the developing rat heart.

Protein phosphatase 2A (PP2A) is a second messenger involved in cell cycle regulation, cell transformation, and cell fate determination. We previously identified a gene encoding the alpha catalytic subunit of PP2A in the embryonic rat heart, but its role in cardiac morphogenesis was unknown. In this study, we examined the developmental expression of PP2A alpha mRNA and protein in the heart using Northern and Western analysis, in situ hybridization, and immumohistochemical staining. We found two major PP2A alpha transcripts in the rat heart (1.8 and 2.4 kb), at all stages examined. By Western blotting, PP2A alpha protein levels were twice as high in the embryonic rat heart compared with the adult. In situ hybridization on embryonic d 12 showed that PP2A alpha mRNA was expressed in the heart, brain, tail, and limb buds. Cardiac PP2A alpha expression was regionally restricted to the atrium, ventricle, and truncus arteriosus. PP2A alpha expression did not extend into the more distal aortic sac or aortic arches. Cross-sectional hybridization revealed PP2A alpha mRNA in the epicardium, pericardium, and endothelium. Later in development, mRNA expression was also detected at high levels in mesenchymal cells populating the endocardial cushions and in myocardium. At term, PP2A alpha was highly expressed in endothelial cells, but not in the underlying myocardium. PP2A alpha protein had a similar distribution at all embryonic stages examined. These results show that there is transcriptional, translational, and cell-specific regulation of PP2A alpha during heart development. We speculate on the role of PP2A alpha-mediated dephosphorylation in cardiac morphogenesis and suggest a number of possible molecular targets.

Developmental expression and localization of the catalytic subunit of protein phosphatase 2A in rat lung.

Protein phosphatase type-2A (PP2A) is a highly conserved serine/threonine phosphatase known to play a key role in cell proliferation and differentiation in vitro, but the role of PP2A in mammalian embryogenesis remains unexplored. No particular information exists as to the tissue or cell specific expression of PP2A or the relevance of PP2A expression to mammalian development in vivo. To examine expression of PP2A during mammalian lung development, we studied fetal rats from day 14 of gestation (the lung bud is formed on day 12 of gestation) to parturition. Western analysis with a specific PP2A catalytic subunit antibody identified a single 36 kDa protein, with protein levels two-fold higher in the 17 and 19 day embryonic lung as compared to the adult. With in situ hybridization and immunohistochemistry, both mRNA and protein for PP2A were localized equally to the epithelial lining of the embryonic lung airway and the surrounding mesenchyme in the 14 day embryonic lung. With maturation of the lung, PP2A becomes highly expressed in respiratory epithelium. The highest level of expression was in the earliest developing airways with columnar epithelium (the pseudoglandular stage, 15-18 days of gestation). There was a decrease in expression with the transformation to cuboidal epithelium by day 20 of gestation. This was most noticeable in the developing bronchial epithelium of the 19 and 20 day gestation lungs where only an occasional cell continues to express PP2A. Mesenchymal hybridization was most obvious in early endothelial cells of forming vascular channels at 17-19 days of gestation. PP2A respiratory epithelial expression mimicked the centrifugal development of the respiratory tree where the highest expression was in the peripheral columnar epithelium (15-18 days gestation) with only an occasional central bronchiolar cell continuing to express PP2A at 19 and 20 days gestation. Endothelial hybridization decreased with muscularization of large pulmonary arteries with low levels of expression detected in bronchial or vascular smooth muscle. In the newborn lung PP2A expression was decreased, but detectable in alveolar epithelium and vascular endothelium. In summary; 1) PP2A mRNA and protein exhibit cell specific expression during rat lung development; 2) PP2A is highly expressed in the respiratory epithelium of the fetal rat lung and is temporally related to the maturation of the bronchial epithelium; 3) and the PP2A subunit is highly expressed in early vascular endothelium, but not smooth muscle of the rat lung.

Developmental regulation of angiotensin type 1 and 2 receptor gene expression and heart growth.

Little is known regarding the developmental regulation of the cardiac angiotensin type 1 (AT1) and type 2 (AT2) receptor genes or their role in normal cardiac growth. Regulation of AT1 and AT2 receptor genes were examined using total and poly A + RNA isolated from whole Sprague-Dawley rat hearts. AT1 mRNA levels were 3.5-fold higher in the 19-day-old fetal heart compared to the 90-day-old adult as detected with 2 or 5 microg of poly A + RNA. AT2 mRNA was only detectable with 20 microg of poly A + RNA. AT2 mRNA levels were highest in the 19-day-old fetal heart with no detectable message in the 90-day-old adult heart. Qualitative PCR for AT2 mRNA also could not detect AT2 mRNA in the adult heart. Treatment with the AT1 receptor antagonist losartan for 3 weeks in the 21-day-old rat or for 4 days in the 38-day-old rat resulted in a significant decrease in heart/body weight in both groups and body weight in the 3-week treatment group. AT2 blockade for 4 days with PD123319 or beta-receptor blockade with propranolol for 3 weeks did not alter heart/body weights. Losartan treatment also resulted in a three-fold increase in cardiac AT1 mRNA levels in both the 4-day and 3-week treatment groups compared to controls. We conclude that Ang II, acting primarily, if not exclusively via the AT1 receptor plays a significant role in the regulation of normal cardiac growth in the young rat.

AT1 receptor gene regulation in cardiac myocytes and fibroblasts.

The regulation of the AT1 receptor gene was studied in neonatal cardiomyocytes and fibroblasts in vitro. Incubation with angiotensin II (Ang II) resulted in a time-dependent and dose-dependent decrease in AT1 mRNA levels in both cardiomyocytes and fibroblasts. Coincubation with Ang II and the specific AT1 antagonist losartan prevented the decrease in AT1 mRNA whereas the AT2 antagonist PD123319 was ineffective in preventing the decrease in AT1 mRNA. Because Ang II is known to decrease cAMP levels in cardiomyocytes, the role of cAMP in the regulation of the AT1 gene was examined. Treatment with the adenylyl cyclase stimulant forskolin or the cAMP stereoisomer Sp-cAMPS increased AT1 mRNA levels or prevented the Ang II mediated decrease in AT1 mRNA levels. The role of calcium in the regulation of the AT1 gene was determined by incubation with the calcium ionophores A23187 and ionomycin (0.0625-1 microM) which resulted in a profound, dose-dependent decrease in AT1 mRNA levels. Treatment with BAPTA, an intracellular chelator of calcium, prevented the Ang II-mediated decrease in AT1 mRNA. Therefore Ang II is a potent negative regulator of the AT1 gene in cardiomyocytes and fibroblasts via the AT1 receptor. This Ang II mediated decrease in AT1 mRNA is mediated by two complementary mechanisms involving cAMP and intracellular calcium.

Angiotensin II type 1 receptor: role in renal growth and gene expression during normal development.

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)

Angiotensin receptor regulates cardiac hypertrophy and transforming growth factor-beta 1 expression.

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 heart weight-body weight ratios were not different from sham or sham plus DuP 753 animals (3.9 +/- 0.4 versus 3.6 +/- 0.4 or 3.3 +/- 0.08, respectively). Type 1 receptor mRNA levels were significantly increased in the coarctation group, with the highest levels in the coarctation plus DuP 753 and sham plus DuP 753 groups. To determine whether growth factors were involved in the hypertrophic process, we measured transforming growth factor-beta 1 mRNA levels. Northern analysis demonstrated a twofold increase in coarctation animals compared with sham or coarctation plus DuP 753-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of renin and AT1 receptor in the rat.

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.

Decreased perfusion pressure modulates renin and ANG II type 1 receptor gene expression in the rat kidney.

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)

Ontogeny of type 1 angiotensin II receptor gene expression in the rat.

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, AT1 mRNA levels increased fivefold from 15 d gestation to 5 d of age. Liver AT1 mRNA levels at 5 d of age were similar to those of adult rats. In the kidney, AT1 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.

Renin and angiotensinogen expression during the evolution of diabetes.

The expression of renin and angiotensinogen genes and their proteins were studied during the progression of diabetes using adult BioBreeding spontaneously diabetic rats at 1 day and 2-12 months of diabetes. The number of renin-stained cells per juxtaglomerular apparatus was determined by immunocytochemistry. Initially, at 2 months of diabetes the number of renin-stained cells per juxtaglomerular apparatus increased significantly (p less than 0.0001, 2 months versus resistant groups) and was followed by a decrease in the number and intensity of renin-stained cells after 12 months of diabetes (p = 0.007, 2 months versus 12 months). A significant negative correlation was observed between the number of renin-containing cells and the duration of diabetes (r = 0.99, p = 0.014). Immunoreactive angiotensinogen was restricted to the proximal tubule and appeared increased after 4 and 8 months of diabetes as compared with the 2- and 12-month diabetic groups. Renin messenger RNA (mRNA) levels increased with the onset of diabetes and decreased markedly during chronic diabetes. At 1 day of diabetes, renin mRNA levels were 700% higher than at 12 months of diabetes. Angiotensinogen mRNA levels were unchanged. We conclude that diabetes results in an initial increase in renin gene expression, and as the duration of diabetes lengthens, there is a progressive decrease in renin gene expression and in the number of cells containing renin. These findings suggest that as the duration of diabetes and the age of the animal lengthens, there is a decrease in the number of cells expressing the renin gene.

Hepatic angiotensinogen gene regulation in the fetal and pregnant rat.

To determine whether expression of the hepatic angiotensinogen (Ao) gene is modulated by 1) ontogeny, 2) pregnancy, 3) glucocorticoids, or 4) triiodothyroxine (T3), time-dated pregnant Wistar-Kyoto rats were studied at different gestational ages (15, 17, and 20 d) without the influence of hormonal treatment or were given a daily intraperitoneal injection of dexamethasone (Dex) or T3 for 5 d (chronic Dex or T3) or a single injection of Dex (acute Dex). Maternal and fetal hepatic Ao mRNA levels were detected by dot and Northern blot analysis by using a full-length rat Ao cDNA. Fetal Ao mRNA levels were lower than in their maternal counterparts and lower than in adult rats of either sex. Maternal hepatic Ao mRNA levels were markedly diminished. mRNA levels were their lowest at 15 d of gestation and increased progressively as gestation advanced, reaching a peak at 20 d of gestation. Chronic Dex treatment resulted in a 190% increase in maternal and a 370% increase in the fetal hepatic Ao mRNA levels. Acute Dex treatment resulted in a 260% increase in maternal hepatic Ao mRNA levels with no change in the fetus. Hepatic Ao mRNA levels increased to the nonpregnant level with acute and chronic Dex treatment. Chronic T3 treatment resulted in a 20% increase in maternal hepatic Ao mRNA levels, without alteration of fetal Ao gene expression. We conclude that 1) the fetal and pregnant state results in a profound decrease in maternal and fetal hepatic Ao gene expression, 2) Ao gene expression is regulated by glucocorticoids in the term fetal and maternal liver, and 3) chronic T3 treatment results in a modest increase in maternal hepatic Ao gene expression.