Expression of endothelial nitric oxide synthase in the postnatal developing porcine kidney

The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.     

神经型一氧化氮合酶在生后小鼠肾脏发育中的表达

目的观察生后小鼠肾脏发育不同阶段神经型一氧化氮合酶(nNOS)的表达,以及新生小鼠与成年小鼠肾脏nNOS表达差异,探讨nNOS在小鼠生后肾脏发育中的意义。方法分别取新生(出生小于2h)、生后3、5、7、14、40d昆明小鼠各8只,共6组。用免疫组织化学及免疫印迹方法对小鼠肾脏内nNOS表达进行定性、定量分析。结果新生小鼠生肾区nNOS呈强阳性表达,肾小管也有表达;成年小鼠肾远端小管,特别是致密斑,nNOS呈强阳性表达,集合管及肾小管均有阳性表达;新生小鼠肾脏nNOS含量最多,随后逐渐减少,成年小鼠nNOS含量最低。结论新生小鼠与成年小鼠肾脏nNOS表达部位不同,且表达含量由新生时最高到成年时降至最低。     

Maturation of growth hormone stimulation of kidney ornithine decarboxylase in the rat

The effect of ovine growth hormone (GH) on kidney ornithine decarboxylase (ODC) was studied in newborn, preweanling and young adult rats. Basal kidney ODC activity was very low from 4 to 22 days after birth but rose 20-fold by day 25; it remained elevated through day 45. GH failed to stimulate ODC in the first two weeks after birth. GH did however stimulate ODC markedly from 20 through 45 days. Kidney ODC was stimulated in the neonate by vasopressin and by isoproterenol, but not angiotensin II. Liver ODC remained relatively low and stable during development, and was responsive to GH at all ages studied. We conclude that a) the pattern of development of basal kidney ODC appears to be unique to this tissue and may be related to the postnatal maturation of renal morphology and/or function, b) neonatal kidney ODC is unresponsive to certain hormones but is not completely refractory to stimulation. These findings may have implications for the role of hormones in the maturation of the kidney and in the regulation of early renal function.     

Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung

Endothelial nitric oxide (NO) synthase (eNOS) produces NO, which contributes to vascular reactivity in the fetal lung. Pulmonary vasoreactivity develops during late gestation in the ovine fetal lung, during the period of rapid capillary and alveolar growth. Although eNOS expression peaks near birth in the fetal rat, lung capillary and distal air space development occur much later than in the fetal lamb. To determine whether lung eNOS expression in the lamb differs from the timing and pattern reported in the rat, we measured eNOS mRNA and protein by Northern and Western blot analyses and NOS activity by the arginine-to-citrulline conversion assay in lung tissue from fetal, newborn, and maternal sheep. Cellular localization of eNOS expression was determined by immunohistochemistry. eNOS mRNA, protein, and activity were detected in samples from all ages, and eNOS was expressed predominantly in the vascular endothelium. Lung eNOS mRNA expression increases from low levels at 70 days gestation to peak at 113 days and remains high for the rest of fetal life. Newborn eNOS mRNA expression does not change from fetal levels but is lower in the adult ewe. Lung eNOS protein expression in the fetus rises and peaks at 118 days gestation but decreases before birth. eNOS protein expression rises in the newborn period but is lower in the adult. Lung NOS activity also peaks at 118 days gestation in the fetus before falling in late gestation and remaining low in the newborn and adult. We conclude that the pattern of lung eNOS expression in the sheep differs from that in the rat and may reflect species-related differences in lung development. We speculate that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time.     

Differential effects of maternal nutrient restriction through pregnancy on kidney development and later blood pressure control in the resulting offspring

The mechanisms whereby maternal nutritional manipulation through pregnancy result in altered blood pressure in the offspring may include changes in fetal and newborn and adult renal prostaglandin (PG) synthesis, metabolism, and receptor expression. Since the postnatal effects of nutrient restriction on the renal PG synthesis and receptor system during nephrogenesis in conjunction with nephron numbers and blood pressure have not been evaluated in the rat, the present study examined the effect of reducing maternal food intake by 50% of ad libitum through pregnancy on young male rats. Six control-fed mothers and eight nutrient-restricted pregnant rats with single litter mates were used at each sampling time point, most of which occurred during nephrogenesis. Offspring of nutrient-restricted dams were lighter from birth to 3 days. This was accompanied by reduced PGE2, with smaller kidneys up to 14 days. Nutrient restriction also decreased mRNA expression of the PG synthesis enzyme, had little effect on the PG receptors, and increased mRNA expression of the degradation enzyme during nephrogenesis and the glucocorticoid receptor in the adult kidney. These mRNA changes were normally accompanied by similar changes in protein. Nephron number was also reduced from 7 days up to adulthood when blood pressure (measured by telemetry) did not increase as much as in control offspring during the dark, active period. In conclusion, maternal nutrient restriction suppressed renal PG concentrations in the offspring, and this was associated with suppressed kidney growth and development and decreased blood pressure.     

Concentration of phosphoribosyl pyrophosphate in the kidney during development and in experimental diabetic hypertrophy.

The effect of developmental growth on the kidney content of phosphoribosyl pyrophosphate PPRibP was studied in rats at ages between the foetal animal and up to 100 days of age. In addition, the effect of short-term diabetes (up to 14 days) on the renal content of PPRibP was studied in immature rats and in adults aged approx. 60 days. The developmental pattern of PPRibP is such that the PPRibP content is lowest in the young rat and increases as the rate of kidney growth slows. In the adult rat, the early kidney hypertrophy of diabetes is accompanied by a fall in PPRibP content and, again, the PPRibP content returns to normal as the rate of kidney hypertrophy diminishes. Induction of diabetes in the immature rat causes a lesser degree of kidney hypertrophy and also a smaller depression of renal PPRibP content. The activity of PPRibP synthetase (EC 2.7.6.1) is not significantly affected by age or diabetes. The changes in PPRibP content are discussed in relation to the generation of ribose 5-phosphate by the pentose phosphate pathway and the utilization of PPRibP for nucleotide synthesis via the 'de novo' and salvage pathways.     

Maturation of central autonomic control of the circulation

Postnatal maturation of central neural regulation of cardiovascular function is being examined in developing swine. Three major types of investigations have been undertaken: 1) alterations of inputs from visceral and somatic afferents, e.g., baroreceptor manipulation, stimulation of sciatic nerves; 2) stimulation of central vasoactive sites; 3) subjecting the animals to the stresses of hemorrhage, hypoxia, or hypercapnia. Our findings indicate that cardiovascular reflexes mature at different postnatal ages. For example, the J-receptor reflex had almost the adult pattern of response at birth, whereas the Bezold-Jarisch reflex had a markedly delayed postnatal maturation. Recordings of spontaneous discharge in a major efferent sympathetic supply, i.e., the greater splanchnic nerve, have indicated that neural innervation to the adrenal medulla and splanchnic vasculature is present at birth in piglets.     

Hormonal regulation of postnatal development of renal tubular transport processes

Renal tubular transport of p-aminohippurate (PAH) is immature at birth. Repeated saturation of transport sites by treatment with various organic anions is without any influence on the postnatal development of kidney transport capacity. Hormonal regulation of postnatal maturation of PAH transport must therefore be taken into consideration. It was tried to stimulate immature PAH transport by treating rats of different ages with thyroid hormones, corticosteroids or testosterone, respectively. In rats with immature kidney function, renal PAH excretion can be stimulated by daily treatment with thyroid hormones. Experiments on renal cortical slices have shown that PAH excretion is preferentially stimulated by an increase of transport capacity. Whereas thyroid hormones stimulate the renal excretion of PAH both in young and in adult rats, dexamethasone treatment is more effective in rats with immature kidney function. Dexamethasone treatment is without any influence on PAH accumulation in renal cortical slices. Kidney weight and the protein content of kidney tissue was increased after dexamethasone treatment. Repeated testosterone administration did not stimulate the PAH transport in rats of different ages. The data have demonstrated the influence of thyroid hormones or of dexamethasone on renal tubular transport processes in rats with immature kidney function. Treatment with such hormones could be useful in the management of renal insufficiency in full-term and pre-term neonates with immature kidney function.     

Activity of gluconeogenetic enzymes in the kidney of pigs during pre- and post-natal development

In pig fetuses (19 of 8 dams) developed by Caesarean section the dry matter and protein content of the kidneys and their PEPCK activity remain constant during the last third (from 80th to 112th day) of gestation. After birth the dry matter content of the kidneys rises slowly, but their protein content remarkably. In the kidneys of suckling piglets (17 animals of 3 offsprings) the FDPase activity remains at the same level from birth to the 9th day of life, while in the same time the G6Pase activity rises 1.5-2 times. In the kidneys of newborn piglets the total PEPCK activity increases 3-4 times and the activity of the cytosolic enzyme 2-3 times during the first 12 hours of life. At the end of the first week of life the total PEPCK activity decreases by one-third, while the activity of the cytosolic enzyme remains stable. In the kidneys of slaughter pigs (n = 7) the dry matter content and the FDPase activity are significantly higher, the protein content and the G6Pase activity are the same as in the kidneys of piglets. The total PEPCK activity is one-half, the activity of the cytosolic enzyme one-third lower than in the kidneys of piglets. In the kidneys of adult pigs the PEPCK activity is localized to equal parts in the cytosol and in the mitochondria, but in some development stages the mitochondrial part exceeds that of the cytosol. In adult pigs the PEPCK activity of the renal cortex is 2.5-3 times higher than that of the renal medulla.     

Renal Response to L-Arginine in Diabetic Rats. A Possible Link between Nitric Oxide System and Aquaporin-2

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [¹⁴C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.     

Ontogenic changes in metabolism and transport of glutathione in the rat

Changes in the level of glutathione (GSH), the turnover rate, and gamma-glutamyltransferase (GGT) activity were examined in newborn, weanling, and adult male Wistar rats, the objective being to elucidate the mechanisms which control the hepatic GSH level during maturation as well as under conditions of different degrees of protein ingestion. The hepatic GGT activity in the newborn rats was high at birth, decreased within a few days to 1 to 2% of the initial level, and remained unchanged thereafter, when these rats were fed a normal diet after 3 weeks of age. In contrast, the hepatic GSH level increased 3-4-fold while total GGT activity in the kidney increased 6-8-fold. When weanling rats were fed a low protein diet (containing 10% soy protein) for 3 weeks, the hepatic GSH level decreased markedly while the GGT activity increased 5-6-fold. The turnover rate of hepatic GSH also increased, as determined by the use of buthionine sulfoximine, a specific inhibitor of GSH synthesis; a value of 2.1 h was obtained in comparison with 3.5 h for that of rats fed the normal laboratory chow (CRF-1). On the other hand, feeding adult rats on the low protein diet resulted in a marked decrease in hepatic GSH level with no effect on either hepatic or renal GGT activity. These results together with other observations may suggest that GSH translocated out of liver cells in the newborn rats is degraded mainly by these cells, while the tripeptide secreted by hepatocytes of adult rats is metabolized predominantly in extrahepatic tissues, such as the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental regulation of peptidylglycine alpha-amidating monooxygenase (PAM) in rat heart atrium and ventricle. Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.     

Ontogeny of surfactant apoprotein D, SP-D, in the rat lung

Surfactant protein D (SP-D) is a collagenous surfactant-associated glycoprotein synthesized by alveolar type II cells. Antiserum against rat SP-D was raised in rabbits and an enzyme-linked immunosorbant assay (ELISA) has been developed using anti-rat SP-D IgG. In the present study we examined the developmental profile of SP-D in the rat lung compared with that of surfactant protein A (SP-A). SP-A content in the lungs increased during late gestation and reached its maximum on day 1 of neonate, and then gradually decreased until at least day 5. SP-D content during early gestation was less than 10 ng/mg protein until day 18, but on day 19 there was a 4-fold increase in SP-D (compared to that on day 18). It increased twice between day 21 and the day of birth, when it reached the adult level of 250 ng/mg protein, which is about one fourth that of the adult level of SP-A. Unlike SP-A there seemed to be no decrease in SP-D content after birth. These results demonstrate that SP-D is regulated developmentally as are the other components of surfactant, but the inconsistency in the developmental profiles of SP-A and SP-D suggests that these proteins may play different roles in lung maturation.     

Localization of retinol-binding protein messenger RNA in the rat kidney and in perinephric fat tissue

The cellular localization of retinol-binding protein (RBP) messenger RNA (mRNA) in the kidney, and the developmental pattern of the renal expression of the RBP gene, were studied in the Sprague-Dawley rat. In situ hybridization studies were conducted with single-stranded cRNA probes, using sections of adult and young rat kidneys. These studies revealed specific localization of RBP mRNA in the outer stripe of the medulla, specifically localized in the S3 segment of the proximal tubules. Northern blot analysis demonstrated that RBP mRNA was not detectable in the kidney before birth or during the first week postpartum, but was clearly detected by the end of the second week of age. No RBP mRNA was observed in the kidney by in situ hybridization at 12 days of age. At 26 days of age, however, RBP mRNA was clearly detected by the in situ hybridization technique, localized in the same anatomic region as that observed in the adult kidney. Transthyretin mRNA was not detected in the adult kidney. Previous studies have shown that immunoreactive RBP is localized in the convoluted segment of the proximal tubules of the rat kidney. The present results demonstrate that RBP mRNA in the kidney is localized in an anatomic region (the S3 segment of the proximal tubules) different from that of immunoreactive RBP. In addition, an intense RBP mRNA hybridization signal was detected in the perinephric fat tissue of 26- and 40-day-old and adult rats. Further analysis of RNA from epididymal fat showed a level of RBP mRNA approximately 20% of that of liver. The function of RBP synthesized in the kidney and adipose tissue remains to be determined. We have previously hypothesized that RBP synthesized in extrahepatic tissue may function in the recycling of retinol back to the liver or to other target tissues.     

Expression profile of mouse BWF1, a protein with a BEACH domain, WD40 domain and FYVE domain

We isolated a mouse cDNA encoding a protein that contains a BEACH domain, 5 WD40 repeats and a FYVE domain, which we designated as BWF1. The mRNA is approximately 10 kb in size and encodes a protein consisting of 3508 amino acids with a predicted molecular weight of 385 kDa. BWF1 has 45% homology with the Drosophila protein, blue cheese (BCHS). The BWF1 gene consists of 67 exons, which span 270 kb of genomic sequence, and has been mapped to mouse chromosome 5. Northern blot analysis revealed that it was strongly expressed in the liver, moderately in the kidney and testis, and weakly in the brain of adult mice. During the development of the mouse brain, BWF1 mRNA was abundant on embryonic day (E) 14-16; after birth, the level of BWF1 mRNA expression decreased markedly to reach the adult level at postnatal day 3. In situ hybridization analysis revealed that the expressed BWF1 mRNA was restricted to the marginal region both in E14 and E16 embryonic brain, but became diffuse after birth. Confocal microscopy studies of the epitope-tagged BWF1 protein showed that the protein was a cytoplasmic one.     

Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane.

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.