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Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3010, Australia
Address all correspondence and requests for reprints to: Dr. Karen Moritz, Howard Florey Institute, University of Melbourne, Parkville, 3010, Australia. E-mail: k.moritz{at}hfi.unimelb.edu.au.
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Abstract |
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Introduction |
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This concept of a fetus being programmed for adult disease has led to an enormous amount of research into possible causes and mechanisms by which this may occur. Maternal undernutrition has been used to cause restriction in fetal growth and in some, but not all cases, this results in elevated blood pressure in the offspring (5). Other treatments that may result in programming include maternal anemia, and exposure to excess natural or synthetic glucocorticoids (6). Studies from our laboratory have shown that, in sheep, high blood pressure resulted in the adult after maternal infusion of the synthetic glucocorticoid, dexamethasone (D) for only 48 h between d 26 and 28 of gestation, even though lambs were of a normal birth weight (7). Recently, we have demonstrated that maternal infusions of the natural glucocorticoid, cortisol (F), at high physiological concentrations at the same gestational age resulted in hypertensive offspring (8).
The kidney, and in particular the renal renin-angiotensin system (RAS), may be critically affected in models of fetal programming and the subsequent development of disease. Growth-retarded infants have particularly small kidneys and may have elevated cord blood renin and angiotensin II concentrations (9, 10) as well as elevated renin gene expression in the kidney (11), suggesting the intrarenal RAS may be elevated. Perinatal treatment of rat pups with high doses of losartan, a specific AT1 receptor antagonist, resulted in increased blood pressure possibly due to fewer glomeruli and pathological changes in the adult kidney (12). Recently, it has been shown the AT1 receptor is up-regulated in other models of fetal programming. Offspring of rats maintained on a low protein diet during pregnancy had increased expression of the AT1 receptor at birth and at 16 wk of age. It was possible to prevent the development of hypertension in these rats by treating pups with losartan between wk 2 and 4 after birth (13). In an ovine maternal undernutrition model, where ewes were undernourished between d 28 and 77 of gestation, expression of the AT1 receptor was elevated in the kidney as well as the adrenal, liver, and lung in lambs at birth (14).
The RAS plays important roles in the development and functioning of the fetal kidney in utero (15, 16, 17). All components of the system (angiotensinogen, renin, angiotensin converting enzyme, AT1 and AT2 receptors) are expressed from very early in gestation in the human (18) and sheep meso- and metanephros (19, 20). Inhibition of the fetal RAS can lead to decreases in fetal urine production and may result in oligohydramnios. Elevated levels of angiotensin II in the fetus can cause a diuresis and increase in blood pressure from at least midgestation (21), which becomes more marked in later gestation (22, 23).
The major aim of this study was to examine the effect of early maternal D treatment for 48 h on gene expression and protein levels for key components of the RAS in the late gestation ovine kidney (130 d). The functional significance of altered late-gestation intrarenal RAS was then tested by angiotensin II infusions in chronically cannulated fetuses from ewes treated with S or D in early pregnancy. We were also interested in determining if treatment with the natural glucocorticoid, F, had similar effects on gene expression. Recently, we have reported that early maternal F and D treatment results in significant changes in fetal mesonephric function indicated by changes in allantoic fluid composition (24). This was found in fetuses killed immediately after treatment. This study examined if there were any long-term alterations in the metanephros, in particular, the renal RAS, some 100 d after the treatment regime had been completed. To determine whether any changes in the AT1 or AT2 receptor gene expression were specific for the kidney, we examined levels of expression of these receptors in the adrenal.
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Materials and Methods |
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3 yr) and weight (48 ± 2 kg) were serviced by a single ram from the Institute farm (Tooradin, Victoria, Australia). At 23–25 d of gestation, a cannula (inner diameter, 0.58 mm; outer diameter, 0.97 mm) was inserted into a jugular vein under local anesthetic. At d 26/27, ewes were infused iv with S (0.19 ml/h, n = 11), D (Decadron; Merck, Sharp and Dohme, Granville, New South Wales, Australia, 0.48 mg/h, n = 12), or F (5 mg/h, n = 5) for 48 h. During the infusion, ewes were housed in individual cages with free access to food and water. At completion of the infusion protocol, the cannula was removed and ewes returned to pasture. Twin pregnancies were identified by ultrasound at 50–60 d of gestation. At 130–134 d of gestation, ewes carrying twin fetuses (S, n = 5; D, n = 6; F, n = 5) were killed with an overdose of Lethobarb (Arnolds, Reading, UK). A sample of cord blood was obtained (10 ml) and a sample of urine (3 ml) obtained from the fetal bladder. Amniotic and allantoic fluid volumes were measured and samples taken for electrolyte analysis. Fetal body weight and kidney weights were obtained. One kidney was immediately frozen in liquid nitrogen for subsequent extraction of RNA or protein. The other kidney was taken into 4% paraformaldehyde and underwent standard processing into paraffin. The fetal adrenal glands were frozen for RNA extraction. Twin fetuses were counted as individual animals giving a total number of 10 S-exposed fetuses, 12 D-exposed fetuses, and 10 F-exposed fetuses.
Ewes carrying single fetuses (S, n = 6; D, n = 6) underwent general anesthesia at 115 d of gestation, at which time cannulae were placed in the fetal carotid artery, jugular vein, and bladder (23). A cannula was also placed in the amniotic fluid as the uterus was closed. Cannulae were flushed daily with heparinized S to maintain patency.
Angiotensin II infusion
At 127 ± 1 d, basal urine flow, glomerular filtration rate (GFR) and blood pressure were measured as described previously (21, 23) in all cannulated fetuses for a 3-h period. Fetuses were then infused iv with angiotensin II at the rate of 10 µg/h for 3 d. Fetal body weight at this age is approximately 3 kg, which equates to approximately 3 µg/kg·h. Blood pressure was measured every 10 min over the subsequent 3 d, and urine flow rate and solute excretion were measured after 24 h. After 72 h, the measurement of GFR was repeated, and ewes and fetuses were then killed as described above.
Sample analysis
Samples of amniotic and allantoic fluid, along with fetal and maternal plasma and urine, were assayed for sodium, chloride, potassium, total carbon dioxide, urea, creatinine, calcium, magnesium, phosphate, protein, glucose, fructose, and lactate using a Synchron CX5 clinical system (Beckman, Fullerton, CA). Osmolality of these fluids was measured by freezing point depression using an Advanced Osmometer (Advanced Instruments, Needham Heights, MA). Maternal plasma ACTH was measured using a DYNO kit (Brahms Diagnostica, Berlin, Germany).
Gene expression studies
In all cases, total RNA was extracted from a cross-section of fetal kidney containing both cortex and medulla or the entire adrenal using the method of Chomezynski and Saachi (25) and treated with deoxyribonuclease to remove any residual genomic DNA. Samples were reverse transcribed to form cDNA (20) and an ABI Prism 7700 Sequence Detector System (PE Biosystems, Foster City, CA) was used to perform real-time PCR. This assay was used to determine relative levels of mRNA expression for angiotensinogen, renin, and the AT1 and AT2 receptors (20, 21, 26). The primer and probe sequences, along with the optimal conditions for use, have been published (8, 21). The intraassay coefficients of variation for these genes are 14% (renin), 7% (AT1 receptor), 5% (AT2 receptor), and 10% (angiotensinogen).
A comparative CT (cycle of threshold fluorescence) method was used with 18S used as an endogenous reference. The calibrator to which all other samples were compared was a sample of kidney or adrenal from one of the S-treated fetuses. This sample was run five times in every assay and the mean value was used. To calculate the relative expression levels in each sample, the CT value for 18S was subtracted from the CT value of the gene of interest to give a 
CT value. The CT value of the calibrator was then subtracted from each individual sample to give a CT value. This number was then inserted into the formula 2-CT to give the expression level relative to the calibrator.
Hybridization histochemistry
The ovine cDNA for the AT1 and AT2 receptors were provided by Dr. J. Robillard and have been described previously (20). Probes were synthesized using a Promega Corp. riboprobe kit (Promega Corp., Madison, WI) and labeled with 35S-uridine triphosphate. Before use, probes were hydrolyzed, resuspended in 10 mmol dithiothreitol, and then diluted to a final concentration of 0.02 ng/µl in hybridization buffer as described previously (20).
Paraffin sections of kidney (5 µm) were cut and mounted on 2% silanized slides. These were dried overnight at 37 C, dewaxed, and rehydrated. Prehybridization with Pronase E (125 µg/ml, Sigma, St. Louis, MO) was performed at 37 C for 10 min, and then slides were postfixed in 4% paraformaldehyde. Hybridization was performed by treating sections with 80 µl of riboprobe and allowing them to incubate overnight at 55 C in a humidified chamber. Slides were washed and treated with ribonuclease A (150 µg/ml, Sigma) for 2 h at 37 C to remove any unhybridized probe. After dehydrating and air-drying, slides were placed on a Fuji Photo Film Co., Ltd. (Tokyo, Japan) phosphor imaging plate (BAS III) overnight and images scanned on a Fujix BAS 2000. Slides were then dipped in liquid emulsion (Ilford, Essex, UK) and exposed at room temperature for 10 d. Autoradiographs were developed in D19 (Kodak, Rochester, NY) and fixed in Hypam (Ilford) before undergoing staining with hematoxylin and eosin. All sections were run in duplicate.
Immunohistochemistry
Immunodetection of angiotensinogen protein was performed in a similar manner to that described previously (19). Sections of 5 µm were placed on silane-coated cells, dewaxed, and placed in 0.3% hydrogen peroxide in methanol for 30 min to inhibit endogenous peroxidase. After rinsing in 0.1 M phosphate buffer sections were incubated in 10% normal horse serum for 30 min at room temperature. Antibodies raised in rabbits against angiotensinogen were used at 1:1000 (27) diluted in normal horse serum and phosphate buffer. Slides were incubated overnight at 4 C. Immunodetection was then achieved using a DAKO Corp. (Carpinteria, CA) LSAB 2 System horseradish peroxidase kit. Diaminobenzidine at a concentration of 0.5 g/ml was used as the chromogen. Meyer’s hematoxylin was used as the counterstain.
Western blot
Homogenates of fetal kidney (from 6 S- and 6 D-exposed fetuses) were made and 15 µg of protein was loaded onto a 15% polyacrylamide gel as described previously (28). After transfer to a polyvinylidene difluoride-Sequi-Blot (BioRad, Hercules, CA) filter, the membrane was blocked with 10% nonfat dry milk powder in Tris-buffered S for 2 h and incubated overnight with the primary antibody to the AT1 receptor at a 1:1500 dilution (29). On the following day, filters were washed and incubated with affinity purified peroxidase conjugated antirabbit IgG diluted 1:3000 (Bio-Rad Laboratories, Inc.). After washing, bands were visualized using enhanced chemiluminescence detection reagents (Amersham, Buckinghamshire, UK) for 2 min, after which they were exposed to an x-ray film. Relative density of bands was assessed using a Bio-Rad Laboratories, Inc. GS-710 calibrated imaging densitometer.
Statistics
Data are reported as mean ± SEM. ANOVA was used to compare fetal fluids, organ weights, and values obtained in the real-time PCR analysis from the twins killed at 130 d. A t test was used to compare the body and organ weights after the angiotensin II infusion. Data obtained from cannulated fetuses over 3 d of angiotensin II infusion were assessed by repeated measures ANOVA. Where appropriate a post hoc test (Tukey test) was used to examine all pairwise comparisons. In some instances a nonparametric (Mann-Whitney rank sum) test was used if data were not normally distributed (gene expression studies). In this case, data are reported as median with the 25% and 75% confidence limits.
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Results |
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Effect of D and F on twin fetuses at 130 d
Body weights and crown rump lengths at postmortem were not different across the treatment groups (see Table 1
). There were seven females and three males in the S group, six females and six males in the D group, and six females and four males in the F group. One female fetus in the D-treated group weighed only 1.9 kg and had significantly lower volumes of fluids than all other fetuses and a high urine osmolality. This fetus was excluded from all further analysis. Kidney, adrenal, and placental weights are shown in Table 1 and were similar in all treatment groups
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Plasma concentrations of all ions was similar in all treatment groups (data not shown). The urinary concentration of chloride was significantly higher in fetuses of the D-treated group (25 ± 3 mmol/liter) compared with the S group (16 ± 2 mmol/liter, P < 0.05), although the urinary osmolalities were similar (126 ± 5 mOsmol/kg water, S; 138 ± 5 mOsmol/kg water, D). Other parameters were similar in all groups. All fetal urine osmolalities were less than 160 mosmol/kg water.
Gene expression using real-time PCR
Preliminary analysis showed that there was no difference between male and female fetuses in expression levels of any gene in any treatment group. Data from both sexes are thus pooled for each treatment. The mRNA expression levels of angiotensinogen, renin, and the AT1 and AT2 receptors in the fetal kidney are shown in Fig. 1. There was significantly higher expression of angiotensinogen in the D group compared with the S-treated group using the Mann-Whitney test (P < 0.01). In addition, expression of the AT1 and AT2 receptor was also higher (P < 0.05) in the D-exposed group, but there was no difference in mRNA expression levels of renin. In the F-treated group, there was a significant increase in the AT1 receptor mRNA compared with the S-treated group (P < 0.05) but no difference in the other genes examined. There was no difference in gene expression levels in any of the groups for the AT1 or AT2 receptor in the fetal adrenal gland (data not shown).
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, upper panels) compared with the S control animals. This was also apparent in the F-treated fetuses. Labeling for the AT1 receptor mRNA in the kidneys of fetuses exposed to D or F also appeared to be more restricted to the inner stripe of the outer medulla whereas it was more widespread throughout the medulla and cortex of those fetuses exposed to S.
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Immunohistochemistry
Immunoreactivity for angiotensinogen in the kidney of S- and D-exposed fetuses can be seen in Fig. 3. Staining was observed in proximal tubules. No attempt was made to quantify relative levels of protein, but there appeared to be more cells expressing high levels of angiotensinogen in the kidneys of fetuses from the D treatment group.
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Effect of angiotensin II infusion in cannulated fetuses at 130 d
There were three females and three males in the S group and five females and one male in the D group. Basal systolic, diastolic, mean arterial pressure, and heart rate were similar in both groups. Mean arterial pressure increased in the S fetuses from 46 ± 2 to 51 ± 2, 52 ± 2, and 56 ± 2 mm Hg over the 3 d of angiotensin II infusion. Corresponding values in the D-treated group were not different: 43 ± 1, 46 ± 2, 50 ± 2, and 52 ± 2 mm Hg.
Urine flow rate increased in both groups with angiotensin II infusion as shown in Fig. 4. However, ANOVA showed that there was a significant difference in the treatment groups (P < 0.05). Post hoc analysis showed the increase in urine flow was greater in the S group compared with the D-treated group on d 3 of infusion (P < 0.01). Due to cannula failure, it was not possible to obtain GFR measurements in two animals from the D group on d 3 of infusion. Thus, analysis comparing GFRs was performed on six animals from the S group and four from the D group. GFR increased in from 152 ± 8 to 230 ± 12 ml/h in the S group and from 170 ± 22 to 203 ± 29 ml/h in the D group. The increase was significantly larger in the S group than in the D group (P < 0.05). When calculated as a percentage change from basal, the S group increased GFR by 51 ± 3%, whereas the D group changed by only 19 ± 7% (P < 0.01). Sodium excretion tended to increase more in the S group, but this did not reach significance (data not shown). However, free water clearance increased to a greater extent in the S group (P < 0.05, data not shown) on d 3 of infusion. At postmortem, body, organ, and placental weights were similar between the groups (Table 2). However, when expressed as gram per kilogram of body weight, the kidneys of the fetuses from the D-exposed group were significantly heavier (P < 0.05) after the 3-d angiotensin II infusion.
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Discussion |
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In some models of fetal programming, particularly in the rat, only male offspring develop high blood pressure in adulthood (31). As yet there is little evidence as to whether there are any differences between sexes in utero. In this study, we did not find any differences in gene expression between males and females within the same treatment group in the fetal kidney at 130 d. Although limited numbers did not make it possible to assess if there were differences between sexes in the in vivo studies, we have recently reported that both adult male and female offspring of ewes treated with F (8) as well as D (32) develop hypertension.
The period of maternal glucocorticoid infusion in this study coincides with the first branching of the ureteric bud in the ovine fetus, so during the period of treatment the metanephros is simply a ureteric bud surrounded by metanephric mesenchyme (16). Many genes expressed within the ureteric bud and the undifferentiated metanephric mesenchyme have been identified to be critical for normal renal development (33), including components of the RAS (16). In many species including sheep, blockade of the RAS during the perinatal period with angiotensin converting enzyme inhibitors causes structural abnormalities as well as severely affecting renal function (15). It is unknown whether gene expression for angiotensinogen and the AT receptors was altered in the D group during the entire development of the metanephric kidney or whether the up-regulation only developed late in gestation. In either case, the alteration in gene expression is present some 100 d after the treatment was completed.
The expression pattern of the mRNA for the AT1 receptor in the D- and F-exposed groups is very similar to that seen in the 2-d lamb where expression has become restricted to glomeruli and the inner stripe of the outer medulla (20). This implies that the fetal kidney of the D- and F-treated groups has matured and more closely resembles that of a neonatal rather than fetal kidney. The similar results obtained with D and F treatment suggest that this effect is likely to be mediated via the GR. Increased levels of glucocorticoids have been suggested as a possible common mechanism through which a wide variety of maternal perturbations may cause programming effects (30).
No difference was observed in the protein levels for the AT1 receptor in this study between the S and D groups although levels tended to be higher in the D-exposed group. A discrepancy was also found between mRNA and protein levels for the GR and MR (34). In this study, it most likely reflects a higher degree of sensitivity in the real-time PCR methodology compared with the Western blot analysis. However, the altered functional response in the D group to infused angiotensin II, both in causing increased growth of the kidney and affecting renal function suggest that the increased AT1 mRNA is likely to have been translated to protein.
The AT2 receptor is expressed at high levels in interstitial cells of the cortex in the developing ovine kidney and in the macula densa during the period of active nephrogenesis (20). Increased expression in the D group, and in some fetuses of the F group, was found specifically in the macula densa (see Fig. 2
) with expression in cortical interstitial cells similar in all groups. These results suggest that within the kidney, the glomerulus and associated structures (macula densa) have been most affected by the maternal steroid treatment. The long term effects of this are unknown but it is interesting that up-regulation of the AT2 receptor has also been found in the kidney during the development of renal failure in the rat, although it is not known in which cells increased expression appeared (35). It could be speculated that high expression of the AT2 receptor in macula densa indicates that nephrogenesis is not yet complete in the D group. However, nephrogenesis is complete by 130 d in the sheep (16), and there was no evidence of a nephrogenic zone in any of the kidneys examined. It is unlikely there has been any delay in the completion of nephrogenesis as has been observed in ovine fetuses undergoing compensatory nephrogenesis after unilateral nephrectomy (36). In fact, if the kidney has matured prematurely, nephrogenesis may have been completed before a full complement of nephrons could be formed. Up-regulation of the angiotensin receptors may have occurred in the glomerulus and associated structures to compensate for a low number of glomeruli. Low nephron number may then be a significant factor in the development of hypertension later in life (37). Preliminary data suggest that animals exposed to prenatal D have a lower number of glomeruli (Moritz, K. M., and M. Dodic, unpublished observations).
Increased expression levels for angiotensinogen as seen in the D-exposed group may reflect an increase in the length of the proximal tubules, a further sign of increased renal maturity. It would appear that more cells were expressing the angiotensinogen protein rather than increased expression in any particular cell. Gene expression and protein levels for angiotensinogen increase significantly between midgestation and term in the sheep (38). This effect was specific for the D-treated fetuses however and was not observed in the F-exposed fetuses. The reasons for this are not known, but may reflect that D is more effective on the GR than F and/or the ability of D to bind to other receptors, such as the PXR (39). It may be also that the effective dose of glucocorticoid crossing the placenta and reaching the fetus is higher in the D treatment. Differences between the F and D treatments have been observed for other genes. The renal GR and MR were also up-regulated by maternal D treatment but not altered by F treatment (34). Conversely, gene expression in the late gestation fetal hippocampus was altered by maternal F (8) but not D treatment (39).
Chronic up-regulation of the angiotensinogen gene in the proximal tubules of a transgenic mouse model caused elevations in blood pressure without raising the circulating concentrations of angiotensin II (40). This suggests, as seen in our model, that hypertension may result from overexpression of the intrarenal RAS without changes in the peripheral angiotensin II concentration (41). The effects of D in this study appear to be quite specific to the kidney as no changes in expression of the receptors was seen in the adrenal gland or the heart (42). This is in contrast to the effects of undernutrition in the ovine fetus where increased AT1 expression was found in all organs examined including the adrenal gland, implying in that model, there may be changes in the peripheral renin-angiotensin system leading to a general up-regulation of receptors (13).
Examination of the chronically cannulated fetus in late gestation after early exposure to S or D showed there was no alteration in basal renal function or blood pressure. This demonstrates for the first time that the hypertension in this model most likely develops in the early postnatal period as it has been shown female offspring exposed to D have elevated blood pressure by 4 months of age (7). As seen in the adult offspring, the blood pressure responses to infused angiotensin II were not different between the groups. However, the renal response to infused angiotensin II demonstrated significant differences between the treatment groups. After 24–48 h, both groups had increased urine flow rate in response to angiotensin II to a similar degree. However, on d 3 of infusion, both the urine flow rate and GFR was significantly lower in the D-treated group. It has been well established in the ovine fetus that infusions of angiotensin produce a diuresis and natriureis and do not generally stimulate aldosterone production (20, 22, 23) unless close to term (43). The neonatal/adult response to infused angiotensin II is an increase in aldosterone production and a subsequent retention of fluid and sodium (43). Thus, in response to angiotensin II infusion, the fetuses that had been exposed to D early in gestation responded more like a neonatal kidney than a fetal kidney.
The infusion of angiotensin II caused a significant increase in renal growth in fetuses of the D group. Angiotensin II is known to act as a renal growth factor via the AT1 receptor (44) causing proliferation in mesangial cells (45) as well as renomedullary interstitial cells (46). The growth response seen in the D group is thus likely to be due to increased expression of the AT1 receptor in these kidneys. Although we cannot discount some differences may exist between single fetuses (as used in the in vivo study) and twins (used for gene expression studies) the altered functional responses seen in the single fetuses make it highly likely that there were also changes in gene and protein expression in this group. An increase in kidney weight was observed in lambs exposed to maternal undernutrition between d 28 and 77 of gestation (13), which as noted above also increased expression of the AT1 receptor. This provides further evidence that increased AT1 mRNA expression along with increased peripheral angiotensin II, can influence renal growth in two different models of fetal programming.
In conclusion, maternal D treatment around 27 d of gestation in the sheep resulted in significant alterations both in vitro and in vivo within the fetal kidney some 100 d after the treatment was concluded. Changes in gene expression for key components of the renin angiotensin system within the kidney were also present after maternal F treatment at a similar stage of gestation suggesting that modification of this system by glucocorticoids may play a critical role in the development of adult disease. Taken together, the gene expression and functional changes suggest that early prenatal treatment with D caused premature maturation of the fetal kidney. It is speculated this may possibly limit the process of nephrogenesis and cause permanent alterations in the functioning of the kidney. Examination of renal function and structure (including nephron number) in offspring subject to this treatment is necessary to further elucidate the importance of the kidney and/or intrarenal renin-angiotensin system in fetal programming.
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Acknowledgments |
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Footnotes |
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Abbreviations: CT, Cycle of threshold fluorescence; D, dexamethasone; F, cortisol; GFR, glomerular filtration rate; RAS, renin-angiotensin system; S, saline.
Received May 21, 2002.
Accepted for publication July 31, 2002.
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