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Genetics and Development |
* Department of Physiology, University of Melbourne, Melbourne, Victoria, School of Paediatrics and Reproductive Health, Disciplines of Obstetrics and Gynaecology, University of Adelaide, South Australia, and Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia
Address correspondence to: Dr. Mary Wlodek, Department of Physiology, University of Melbourne, Parkville, 3010, Australia. Phone: +613-8344-8801; Fax: +613-8344-5818; E-mail: m.wlodek{at}unimelb.edu.au
Received for publication January 5, 2007. Accepted for publication March 5, 2007.
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Abstract |
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Introduction |
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Many experimental perturbations have been used to investigate the underlying mechanisms of the association between low birth weight and adult disease, the most common being exposure of the mother to a low-protein diet or global calorie restriction throughout all or part of pregnancy. However, in Western society, intrauterine growth restriction occurs in approximately 10% of pregnancies and is most commonly due to a poorly functioning placenta, rather than maternal undernutrition. Bilateral uterine vessel ligation in the rat that results in uteroplacental insufficiency causes a decrease in oxygen and nutrient supply to the fetus, restricting fetal growth and litter size (7–9). In addition, uteroplacental insufficiency in the rat increases basal and stressed BP while reducing the number of glomeruli (and therefore nephron number) (10,11). This is consistent with the consequences of other prenatal challenges in the rat, in which maternal undernutrition or glucocorticoid treatment at crucial stages of renal development reduces nephron number and induces adult hypertension (12,13). Growth-restricted human infants also have fewer nephrons (14), with a strong association found between nephron endowment and birth weight (15). Early postnatal growth, as indicated by weight at 1 yr, is also a predictor of adult BP in humans (16), suggesting that the postnatal environment also influences cardiovascular function in the longer term. Recently we found that placental insufficiency in the rat impairs mammary adaptation during pregnancy in preparation for lactation and causes reduced milk production and altered milk composition (17–19). This in turn alters postnatal development and growth and potentially contributes to the later adverse cardiovascular outcomes that others have described (10,11).
In this study, our aim was to assess growth, nephron endowment, and BP of male offspring that were born to placentally restricted mothers. Furthermore, we aimed to determine whether these parameters could be modulated by altering the postnatal (lactational) environment by cross-fostering. We hypothesized that placental restriction would reduce nephron endowment in adult offspring, which would be associated with an increase in BP. Because nephrogenesis in the rat continues for at least 1 wk postnatally, we further hypothesized that cross-fostering a pup from a placentally restricted mother onto a control mother at birth would overcome the prenatally induced nephron deficit by restoring early postnatal nutrition during lactation and thereby normalize adult BP.
In addition, the effect of placental restriction on the renal expression of angiotensin receptors was examined. A range of prenatal perturbations alter the expression of the angiotensin II type 1 and 2 receptors (AT1R, AT2R) in the fetus and adult, which are required for normal kidney development and regulation of BP and renal function in the adult (20,21). We hypothesized that alterations in the renal renin-angiotensin system (RAS) would occur after placental insufficiency because this is a common underlying mechanism through which an in utero perturbation can contribute to adult hypertension.
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Materials and Methods |
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A vaginal impedance reader (Model MK-10B; Muromachi Kikai Co. Ltd., Osaka, Japan) was used to determine whether female rats were in the appropriate stage of the estrus cycle for mating as described previously (22). The next morning, presence of sperm in vaginal smears was taken as day 1 of pregnancy. On day 18 of gestation, pregnant rats were randomly allocated into restricted or control (sham surgery) groups. The restricted group underwent bilateral uterine vessel (artery and vein) ligation to induce placental insufficiency as described previously (22). At birth, half of the litters from the control (sham surgery) group had their litter size randomly reduced to five to match the restricted group (reduced litter size of control from 10 to 14 pups to five pups).
Cross-Fostering Protocol
Pups from each of the three groups—control, reduced, and restricted (placental restriction)—were cross-fostered 1 d after birth onto a control (sham surgery) or restricted (placental restriction surgery) mother. This resulted in six experimental groups: Control-on-control, control-on-restricted, reduced-on-restricted, reduced-on-control, restricted-on-control, and restricted-on-restricted (n = 7 to 10 mothers per group). There were even gender ratios across all litters. After cross-fostering, mothers remained undisturbed in the same cage as they had delivered. Pups were toe-clipped on postnatal day 3 for identification purposes. Because the lactational environment is a key part of this study, pups were allowed to wean naturally rather than being forced to wean at 21 to 25 d as is performed in many studies. We have preliminary evidence to suggest that some groups (those on restricted mothers) suckle past day 25; therefore, pups were removed from the dams at day 35. The duration of lactation is not likely to affect nephron endowment because nephrogenesis is complete within 10 d of birth.
Body Weight and BP Measurements
One to two male pups per litter (n = 7 to 10 litters per group) resulted in 9 to 10 males being studied per group. Body weight was measured on postnatal days 1, 14, 21, and 28 and at post mortem (6 mo). Systolic BP was measured at 8, 12, and 20 wk by an indirect, tail-cuff method using a programmed electrosphygmomanometer with a pneumatic pulse transducer (PE-300; Narco Bio-System, Houston, TX) (23,24).
Tissue Collection
At 6 mo, rats were anesthetized with an intraperitoneal injection of a mixed solution that contained Ketamine (Parnell Laboratories, Pty. Ltd., Alexandria, NSW, Australia; 50 mg/kg body wt) and Ilium Xylazil-20 (Troy Laboratories, Pty. Ltd., Smithfield, NSW, Australia; 10 mg/kg body wt). The right kidney was weighed and fixed in 10% neutral-buffered formalin for subsequent analysis of nephron number. The left kidney was frozen in liquid nitrogen and stored at –80°C for subsequent extraction of RNA and protein.
Estimation of Total Nephron Number and Glomerular Tuft and Renal Corpuscle Volumes
The kidney was cut into 1-mm slices; every second slice was taken for processing, and the first slice chosen at random. The slices were dehydrated, then infiltrated with Technovit 7100 resin, Harderner I (Haraeus Kulzer, Hanau, Germany) for 2 to 3 d and embedded into glycolmethacrylate (Technovit 7100 resin, Harderner II, Haraeus Kulzer). Blocks were exhaustively sectioned at 20 µm. Every 10th and 11th section was sampled, the first of which was chosen randomly. Sections were stained with periodic acid-Schiff reagent.
Total kidney volume (Vkid) was estimated using the Cavalieri principle, and total nephron number per kidney was estimated using the Physical Dissector method as described previously (25): Nglom, kid = (Q–/2) x (1/fa) x (PS/PF) x 10 x 2, where Q– is the number of glomeruli counted, 1/fa is the inverse fraction of area used to count glomeruli, PS is the total number of points overlying all tissue, PF is the total number of points overlying tissue for complete sections, 10 is the inverse of section sampling fraction, and 2 is the inverse of slice sampling fraction.
Mean glomerular (Vglom) and renal corpuscle (Vcorp) volumes were estimated using the following equations:
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Total glomerular [Vglom(tot)] and renal corpuscle [Vcorp(tot)] volumes were estimated using the following equations:
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Gene and Protein Expression Analysis
Total RNA was extracted using the Micro-to-Midi Total RNA Purification System kit (Invitrogen, Life Technologies, Carlsbad, CA). Reverse transcription and real-time PCR were performed as described previously (22) for AT1AR, AT1BR, and AT2R. Primer and probe sequences along with optimal concentrations are shown in Table 1. Real-time PCR results were analyzed using the sequence detector software (Rotor-Gene v6; Corbett Research, Mortlake, Australia). Relative quantification of gene expression was performed by the comparative CT (
CT) method with ribosomal 18S as the endogenous control.
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Statistical Analyses
For group comparisons, data were analyzed by one-way ANOVA followed by Student-Newman-Keuls test for post hoc comparisons (SPSS-X; SPSS. Encinitas, CA). Data are presented as means ± SEM, and P < 0.05 was taken as statistically significant. No more than two littermates from one mother were used in each cross-foster group.
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Results |
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Discussion |
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One recent study used a similar uteroplacental insufficiency model in the rat and also showed a reduction in nephron endowment (11). The same group recently showed that uteroplacental insufficiency also results in elevated BP in male offspring at 6, 9, and 12 mo of age (10). However, these studies did not separate the effects of placental insufficiency from that of its effects on mammary function and thus postnatal environment; neither was the timing of hypertension onset examined. In this study, we used the tail-cuff method to perform sequential BP measurements in the same animals. Uteroplacental insufficiency combined with lactational restraint or lactational restraint alone caused onset of hypertension in offspring between 3 and 5 mo. The use of tail-cuff BP measurements means that BP was being examined under at least mildly stressed condition, indicating that the outcomes for basal BP remain unknown at this stage. Future studies that incorporate the use of telemetry for accurate measurement of basal BP are therefore warranted.
A low nephron endowment has been suggested as an underlying causative factor in the development of essential hypertension (26,27). Recent studies in humans have shown a strong association between glomerular (thus nephron) number and BP (15,28). There is also a strong correlation between birth weight and glomerular number in humans, with an estimated increase of approximately 250,000 nephrons for each kilogram increase in birth weight (15). Because no new nephrons can be formed after birth, this suggests that human infants that are born small are likely to be born with fewer nephrons and be more susceptible to cardiovascular complications, including hypertension.
Outcomes from studies that used a range of animal models of developmental programming of cardiovascular function have in general supported this aforementioned concept (for review, see reference [29]). Exposure of the mother to a low-protein diet throughout all or part of pregnancy often results in offspring that are born small, have a reduced nephron endowment, and develop hypertension (30). However, this is not universally true, with some studies demonstrating that a decrease in nephron endowment does not necessarily result in hypertension (27). The final outcome depends on gender of the offspring as well as the timing, severity, and quality of diet provided. We chose to use a placental insufficiency model to investigate the long-term consequences of being born small, because this is the major cause of low birth weight in Western society. Induction of placental insufficiency in rats on day 18 of pregnancy (term 22 d) coincides with the early stages of nephrogenesis in the rat, and at birth, <20% of nephrons are formed (31). Nephrogenesis continues into early lactation (up to postnatal day 10), when the remaining 80% of nephrons are formed.
In our model, placental restriction is likely to have an impact on early nephrogenesis, reducing nephron endowment at birth. Studies have highlighted the importance of the perinatal period in the programming of a low nephron number and subsequent hypertension in many models, including maternal glucocorticoid exposure (sheep and rat) and maternal undernutrition (sheep and rat) (27). The mechanisms through which early nephrogenesis may be affected are unknown but could involve increased apoptosis (32) or a decrease in the rate of branching morphogenesis. The 26% reduction in nephron number that was observed here in adult offspring at 6 mo of age was associated with an increase in glomerular volume. This is in agreement with other studies in the rat of maternal undernutrition models in which a reduced nephron endowment is associated with glomerular hypertrophy (30). This occurs presumably to maintain glomerular filtration surface area and thus GFR. In this study, nephron endowment was examined at 6 mo of age, allowing us to characterize nephron endowment, growth, and BP in the same individual animals. Therefore, although it is most likely that the nephron deficits that were observed after restriction in utero were established before birth, this requires direct confirmation. It is also possible that the lower nephron number in those that developed hypertension and had enlarged glomeruli may be due in part to glomerular loss (glomerulosclerosis). However, a study at 21 wk of age in male rat offspring after maternal exposure to low protein and a similar reduction in nephron number and elevations in BP, as seen in our study, demonstrated no glomerulosclerosis (33). This suggests that glomerular loss is unlikely to play a large role in the nephron deficit. To resolve these issues, future studies should be performed to determine the time of onset of nephron deficits, whether it occurs before the development of hypertension and glomerular hypertrophy, and the extent, if any, of glomerulosclerosis.
In contrast, the impaired lactational environment that was present in rats that were cross-fostered onto a restricted mother after birth is likely to affect the later stages of nephrogenesis and renal development. Our results suggest that both prenatal and postnatal periods are important in the programming of hypertension in the rat, acting through distinctly different mechanisms. The most severe phenotype results when all stages of nephrogenesis are challenged—that is, when both the prenatal and postnatal environments are suboptimal (restricted-on-restricted). This resulted in offspring that were born small, remained small throughout life, and in adulthood had a reduced nephron endowment (by 26%) and overt hypertension. Similar decreases in nephron number have been reported in studies that used maternal low-protein exposure in the rat (30,34). Furthermore, this study shows that impairment of the postnatal environment alone after nephrogenesis is complete (control or reduced-on-restricted) does not reduce nephron endowment, although it can lead to altered growth and elevations in BP (reduced-on-restricted group).
However, the most astounding finding of this study was that cross-fostering a growth-restricted pup onto a mother with normal lactation (restricted-on-control) was sufficient to restore nephron endowment and prevent the onset of adult hypertension. These rats received improved nutrition throughout the period of late nephrogenesis and underwent accelerated growth during early lactation, such that they were of similar size to control-on-control rats by 4 wk of age. This suggests that whereas a poor lactational environment may not impair nephron formation, an improved environment can enhance nephrogenesis. Although it is tempting to attribute the prevention of hypertension to the restoration of nephron endowment as a result of postnatal nutritional rescue, we cannot discount that this is coincidental rather than causal. Therefore, it is possible that the improved lactational environment has affected other aspects of development, including that of the cardiovascular system or factors that influence this and so prevented the onset of hypertension via other pathways. However, it would be of potential clinical interest to understand the mechanism that controls the compensatory increase in nephron number that we observed in our restricted-on-control male pups. One theory is that it may involve an extension in the nephron formation period, which has been demonstrated in a sheep model of increased nephrogenesis after fetal unilateral nephrectomy (35).
Most other studies of cardiovascular outcomes for offspring of prenatal perturbations in the rat have not used the cross-fostering techniques that we used. In nearly all cases, studies that used protein-restricted diets during pregnancy had a normal diet immediately after birth (30,33,36–39). Mammary development is nevertheless likely to be substantially altered in these mothers, adversely affecting lactation and hence postnatal nutrition in pups. Furthermore, the role of the early postnatal environment alone in programming later hypertension has not been previously examined. In the few studies in which cross-fostering was used, offspring that were born of normal birth weight and were nursed by mothers that had a low-protein diet during lactation remained smaller throughout life (40,41). This highlights that accurate determination of mammary function is required to understand postnatal growth consequences in many experimental models. Human studies have demonstrated that greater weight gain in infancy is associated with decreased risk for later disease (16,42,43). This and our results suggest that accelerated growth after being born small may be advantageous if it occurs during early postnatal life. In contrast, accelerated growth after a period of slow growth in late lactation may be detrimental.
An unexpected finding of this study was the increased BP in the reduced-on-restricted group at 5 mo of age, suggesting that hypertension may be a consequence of an altered postnatal environment, even if the offspring were exposed to a normal prenatal environment, were of normal birth weight, and had a normal nephron endowment. This group showed signs of slowed growth during lactation, as evidenced by decreased weight at 14 to 21 d (after nephrogenesis is complete), but underwent accelerated growth after weaning when this lactational restriction was removed. When a restricted mother with impaired lactation suckles fewer control pups (reduced-on-restricted), pup weight is reduced in late lactation as a result of poor milk quality and quantity (17). In contrast, the suckling stimulus of a normal litter size of control pups that are cross-fostered onto a mother with impaired lactation (control-on-restricted) stimulates increased lactational nutrition, resulting in normal postnatal growth and adult normotension. This suggests that even with normal intrauterine development and a normal nephron endowment, hypertension can result if postnatal growth is poor (after nephrogenesis is complete) but there is accelerated growth at a later time point (after weaning). This study demonstrates that a reduced nephron endowment is not necessarily causal in programming hypertension. This is of great relevance to the human, in whom nephron endowment is complete at birth. It could be speculated that children who are born of normal birth weight and have a full complement of nephrons could still develop hypertension if their growth is inhibited in the postnatal period but they undergo accelerated growth later. Rapid weight gain in early childhood is known to be associated with negative cardiovascular consequences, including hypertension (16,44).
The increase in BP in the reduced-on-restricted group was associated with increased expression of both subtypes of AT1R. Changes in expression of components of the renal RAS are also altered in protein-restricted and glucocorticoid exposure models of developmental programming (21,45). However, these changes have been demonstrated primarily in fetal or relatively young animals, in which they may contribute to alterations in renal development. Fewer studies have focused on older animals, in which alterations in expression of key elements of the renal RAS may contribute to the development of hypertension. There is now evidence to suggest that increased activation of this system may cause sodium retention and contribute to sustained elevations in BP (46).
It is of note that all groups that were cross-fostered onto restricted mothers showed an increase in expression of AT1R, suggesting that an impaired lactational environment can have an impact on the long-term regulation of the renal RAS. Postnatal growth of control-on-restricted pups was not affected by lactational restriction as a result of the ability of a larger number of normal pups to increase the total suckling stimulus on the mammary glands, promoting improved mammary function (M.E.W., unpublished observations, 2005). These pups had increased AT1AR mRNA expression but no signs of hypertension at 5 mo of age. However, these offspring may have other programmed deficits or diseases that may emerge at a later age or when exposed to an insult such as a high-fat or high-salt diet. The ability of the postnatal environment to alter AT1R mRNA expression is supported in a study in which renal AT1R mRNA expression was increased in rats that were fed a low-protein diet as adults (47). It cannot be discounted that the increase in adult renal AT1R expression at 6 mo of age is a consequence of hypertension rather than having resulted from the placental insufficiency that is induced during pregnancy. This question will be addressed by future studies in which renal AT1 expression will be quantified before the development of hypertension.
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Conclusion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresReferences |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresReferences |
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Acknowledgments |
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We also thank Debbie Bartal for performing Western blot analysis.
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Footnotes |
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See the related editorial, "Developmental Hypertension, Nephrogenesis, and Mother's Milk: Programming the Neonate," on pages 1626–1629.
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