Effects of dietary protein restriction on nephron number in the mouse

Hoppe et al. R-00442-2006.R1 29/01/2007

THE EFFECTS OF DIETARY PROTEIN RESTRICTION ON NEPHRON NUMBER IN

THE MOUSE

Chantal C Hoppe1, Roger G Evans2, John F Bertram1 and Karen M Moritz1.

Departments of 1Anatomy & Cell Biology and 2Physiology, Monash University,

Melbourne, Victoria, Australia.

Address for Correspondence:

Ms Chantal Hoppe,

Department of Anatomy & Cell Biology,

School of Biomedical Sciences,

Monash University,

Victoria, 3800,

Australia

Phone: 61-3-99052707

Fax: 61-3-99052766

E-mail: [email protected]

of 24Articles in PresS. Am J Physiol Regul Integr Comp Physiol (February 1, 2007). doi:10.1152/ajpregu.00442.2006

Copyright © 2007 by the American Physiological Society.

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Abstract

In rats, maternal protein restriction reduces nephron endowment and often leads to adult

hypertension. Sex differences in these responses have been identified. The molecular and genetic

bases of these phenomena can best be identified in a mouse model, but effects of maternal protein

restriction on kidney development have not been examined in mice. Therefore, we determined how

combined prenatal and postnatal protein restriction in mice affects organ weight, glomerular

number and dimensions and renal expression of angiotensin receptor mRNA, in both male and

female offspring. C57/Bl6/129sv mice received either a normal (20% w/w; NP) or low (9% w/w;

LP) protein diet during gestation and postnatal life. Offspring were examined at postnatal day 30.

Protein restriction retarded growth of the kidney, liver, spleen, heart and brain. All organs except

the brain weighed less in female than male offspring. Protein restriction increased normalized (to

body weight) brain weight, with females having relatively heavier brains than males. The effects of

protein restriction were not sex-dependent, except that normalized liver weight was reduced in

males but increased in females. Glomerular volume, but not number, was greater in female than

male mice. Maternal protein restriction reduced nephron endowment similarly in male and female

mice. Renal expression of AT1A receptor mRNA was ~6-fold greater in female than male NP mice,

but similar in male LP and female LP mice. We conclude that maternal protein restriction reduces

nephron endowment in mice. This effect provides a basis for future studies of developmental

programming in the mouse.

Abstract word count: 248

Key Words: stereology, nephron deficit, sex, mice, kidney

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Introduction

The hypothesis that adult disease can have developmental origins has been a major focus of

experimental and human studies in the last decade (36). There is now considerable evidence that the

in utero and early postnatal environment has a major impact on adult behaviour, physiology and

risk of adult disease (2, 7, 16, 29, 30, 36, 40, 41). There is also considerable evidence that the

effects of the perinatal environment on offspring development and adult renal and cardiovascular

function can be sex-specific (23, 30, 36, 41, 42). The most widely studied model of developmental

programming is maternal protein restriction in rats. Rat dams fed a low protein (8-9% w/w) diet

during pregnancy give rise to offspring with a 10-30% deficit in nephrons, a finding consistent

across a range of studies (14, 17, 39, 41, 43). This is often associated with hypertension in

adulthood (41). Nephron number data in female offspring following maternal protein restriction,

however, are somewhat conflicting. Some studies have shown females from protein restricted

mothers have a nephron deficit (17, 24, 39, 44). In contrast, Woods et al recently found that female

rats subjected to maternal protein restriction did not have a reduced nephron endowment (42).

Importantly, studies that have investigated both sexes following protein restriction have either used

a relatively severe form of protein restriction (39), maceration techniques (24) or pooled male and

female data, making it difficult to discern sex differences (17). Importantly, we are aware of no

previous studies that have specifically investigated whether the effects of modest protein restriction

on nephron endowment are sex-dependent in the mouse.

The increasing evidence that interactions between genotype, sex and the developmental

environment dominate adult cardiovascular health, calls for intense study of the molecular and

genetic bases of developmental programming. Currently, such studies can best be performed in

mice. A most powerful approach to assessing the pathophysiological role of potential genes

involved in developmental programming is to examine the consequences of gene disruption using

genetically modified animals. To date the mouse is the only mammalian species in which complex

investigations of molecular mechanisms can be easily performed. Before such studies can be

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performed we must define the effects of maternal protein restriction on nephron endowment in the

mouse. However, there are no previous published reports of the effects of maternal protein

restriction on renal development in mice. Therefore, in the current study we tested the primary

hypothesis that in mice, combined prenatal and postnatal protein restriction reduces nephron

endowment, alters glomerular morphology and retards the growth of major organs. There is strong

evidence from studies in rats that changes in the renin/angiotensin system contribute to reduced

nephron endowment associated with maternal protein restriction (41). Therefore, we also measured

renal expression of angiotensin receptors. Our secondary hypothesis was that the effects of

combined prenatal and postnatal protein restriction are sex-dependent, so we studied both male and

female offspring. Our findings suggest that maternal protein restriction reduces nephron endowment

in mice, and that the effects of protein restriction on the development of the kidney and other organs

are largely independent of sex. Thus, our current study characterizes a new model of maternal

protein restriction in mice.

Methods

Animals and their diets

Experiments were conducted in accordance with the National Health and Medical Research Council

of Australia ‘Australian Code of Practice for the Care and Use of Animals for Scientific Purposes’

(7th edition, 2004) and were approved by the Monash University, Biochemistry, Anatomy and

Microbiology Animal Ethics Committee. Previously unmated female C57/Bl6 mice were fed either

a normal-protein (20% w/w; Growth Diet AIN93G, Specialty Feeds, WA, Australia) (NP) or near-

isocaloric low-protein (9% w/w; 9% Protein Modified AIN93G, Specialty Feeds, WA, Australia)

(LP) diet from 2 weeks before mating with 129SV males. The LP diet was rendered near-isocaloric

by addition of sucrose and a small amount of starch, making it a high sucrose diet. After

compensation of low protein by sucrose-starch, the LP diet was nearly isocaloric with energy

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content being only 3.7% less than the NP diet. The compositions of the diets are given in Table 1.

Female breeders were maintained on their given diet throughout pregnancy and lactation. Only first

litters were studied. Protein restriction continued after birth to ensure that offspring were protein

restricted throughout the entire period of nephrogenesis, which in mice extends into the postnatal

period. Offspring were also maintained on their mother’s designated diet after weaning at 21 days,

to avoid the potentially confounding effects of altering dietary protein intake in the week before

animals were killed for post mortem measurements. We have previously used this approach in rats

(14), so our current observations allow a direct comparison across the two species. Male and female

offspring were used in all experiments. To minimize handling stress, animals were not weighed at

birth, but were weighed every 5 days of age, from postnatal day 5.

Morphometric measurements and stereological estimation of kidney volume, glomerular

number and glomerular volume

At postnatal day 30, mice were humanely killed via cervical dislocation and the kidneys, liver,

spleen, heart and brain were removed and weighed. Left kidneys were placed in 10% formalin for

stereological estimation of total nephron number and right kidneys were snap frozen and stored at -

70 ْC for analysis of gene expression. Total body weight and the weights of internal organs were

determined in a total of 12-16 animals from each diet and sex group, obtained from 4-6 different

litters.

Total glomerular number was estimated in 4-7 male and female animals per dietary group. Left

kidneys were decapsulated and sliced at 1 mm and every slice was processed for embedding in

glycolmethacrylate (Technovit 7100; Kulzer and Co, Germany). Tissue blocks were exhaustively

sectioned at 20 µm and every 10th and 11th section (section pair) collected and stained with

Periodic-Acid Schiff’s stain.

Total glomerular number (Nglom) and mean glomerular (Vglom) and mean renal corpuscle (Vcorp)

volumes were determined as previously described using unbiased stereological techniques (4). In

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brief, the areas of all sampled kidney sections were estimated using stereological point counting

with the aid of a microfiche reader using a magnification of 19.2x. Grid points overlying all

sections were designated Ps and those overlying complete sections were designated Pf. The total

number of glomeruli in a kidney was determined using physical disectors in a known fraction of the

kidney, the so-called physical disector/fractionator combination. Two light microscopes modified

for projection were employed, and each pair of sections was mounted on the microscopes.

Corresponding fields on the two sections were found, and glomeruli sampled by an unbiased

counting frame in one section that were not present in the section pair were counted (Q-). Nglom was

estimated using:

Nglom = 10 x (Ps/Pf) x [1/(2fa)] x Q-

where 10 is the inverse of the section sampling fraction, and Ps/Pf and 1/(2fa) give the fraction of the

total section area used to count glomeruli.

Kidney volume (Vkid) was estimated using the Cavalieri principle (4) through:

Vkid = 10 x t x a(p) x Ps

where t was section thickness and a(p) was the area of the stereological grid associated with each

grid point after adjustment for magnification.

Vglom was estimated using:

Vglom = VVglom,kid / NVglom,kid

where VVglom,kid was estimated by dividing the number of grid points overlying glomerular profiles

by the number of points overlying kidney sections. A similar point counting approach was used to

estimate Vcorp. NVglom,kid was obtained by dividing Nglom by Vkid. To calculate total glomerular and

total renal corpuscle volumes, mean volumes were multiplied by Nglom.

Gene expression studies

Total RNA was extracted from half of the right kidney using a commercially available kit (Qiagen).

Samples were DNase treated to eliminate any residual genomic DNA. Samples were reverse

transcribed to form cDNA and an ABI Prism 7700 Sequence Detector System (PE Biosystems,

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Foster City, CA) was used to perform real-time PCR (26). This assay was used to determine relative

levels of mRNA expression for the AT1A, AT1B and AT2 receptors. All primers and TaqMan®

probes were designed using Primer Express TM Version 1.0 (PE Biosystems) (see Table 2).

A comparative CT (cycle of threshold fluorescence) method was utilized with 18S used as an

endogenous reference. 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. The calibrator

used was the mean CT of the normal protein male group. This number was then inserted into the

formula 2- CT to give the expression level relative to the calibrator.

Statistics

Data are reported as mean ± SEM. Statistical analyses were performed using the software packages

SYSTAT (Version 11.0, SPSS Inc, Chicago, IL) and SPSS (Version 14.0, SPSS Inc). Mean and SEM of

data from individual mice were weighted by litter, via least squares regression. Data were analysed by

univariate analysis of variance, the factors comprising ‘protein intake’ and ‘sex’, but weighted for ‘litter’

as recommended by Festing (9). The main effect of ‘protein intake’ (PProtein) tested for effects of protein

restriction independent of sex. The main effect of ‘sex’ (PSex) tested for sex differences independent of

protein intake. The interaction term (PSex*Protein) tested whether the effects of protein restriction differed in

male and female mice. Analysis of changes in body weight over the first 30 days of postnatal life was

performed by analysis of covariance, and individual regression lines were determined by the least-

squares method (19). P ≤ 0.05 was considered statistically significant.

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Results

Body and organ weight, litter size

Effects of protein restriction: Litter size was similar for LP- (7.6 ± 1.8 pups) and NP- (6.6 ± 2.5

pups) dams (P = 0.25, unpaired t-test), and therefore no adjustment of litter size was performed.

There were no observed adverse effects of protein restriction on survival of pups to 30 days of age.

At postnatal day 30, body weight and the absolute weights of the kidneys, liver, spleen, heart and

brain were less in LP than NP offspring (Table 3A). The smaller weights of all organs in LP

offspring, except the brain, were proportional to the deficit in body weight, as evidenced by the fact

that protein restriction did not significantly alter the ratios of organ weight to body weight (Table

3B). In contrast, the ratio of brain weight to body weight was significantly increased by protein

restriction.

Sex differences: Body weight and the weight of all organs except the brain were less in females than

males (Table 3A). Sex differences in the weights of the liver, spleen and heart were in proportion to

differences in body weight, as evidenced by the fact that sex did not significantly affect the

normalized weights of these organs. In contrast, normalized kidney weight was significantly less in

female mice than male mice. Normalized brain weight, on the other hand, was significantly greater

in female than male mice.

The effects of protein restriction on body weight and the weights of internal organs were similar in

male and female mice, with the exception of the liver. Protein restriction reduced liver weight by

28% in male mice, but only by 11% in female mice. Consequently, protein restriction reduced

normalized liver weight in males (by 9%) but increased it in females (by 12%).

Covariant analysis of body weight up to postnatal day 30: There was a highly significant positive

relationship between postnatal age and body weight (r2 = 0.90-0.93 in the 4 groups of mice; PAge <

0.001). The rate of growth was greater in male than in female mice (PAge*Sex = 0.008). Protein

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restriction significantly reduced the slope of the growth curve (PAge*Protein < 0.001), but the

magnitude of this effect was similar in male and female mice (PAge*Protein*Sex = 0.511) (Figure 1).

Nephron Number

Total glomerular number, and therefore nephron number, was similar in male and female NP mice

(male = 10755 ± 937, female = 10695 ± 864). Glomerular number was significantly less in protein

restricted male offspring (by 22 %, 8364 ± 468) and female offspring (by 16%, 8977 ± 494) (Figure

2A). Similarly, kidney volume was significantly less in LP than NP mice (PProtein < 0.001). Mean

and total glomerular and renal corpuscle volumes were greater in female than in male mice. There

were no apparent interactions between the respective effects of sex and protein intake (Figure 2).

Renal gene expression

In male mice, relative expression of AT1A receptor mRNA was similar in LP and NP animals. AT1A

receptor mRNA was ~6-fold greater in NP female mice than in corresponding male mice, but

similar in LP female mice compared with the corresponding male mice (Figure 3). There were no

significant effects of protein or sex on AT1B receptor expression (Figure 3). Low levels of AT2

receptor mRNA were detected in all mice, but the level of expression was highly variable between

animals. There were no apparent effects of sex or protein intake on AT2 receptor expression (data

not shown). A positive control from E17.5 kidney tissue revealed a significantly greater expression

of AT2 receptor mRNA compared to mice from postnatal day 30.

Discussion

The most important finding of the present study was that combined pre- and post-natal protein

restriction led to a reduction in nephron endowment, of similar magnitude in male and female mice.

Furthermore, growth retardation in this model of protein restriction, both in terms of total body

weight and the weights of individual organs, was with the exception of the liver, of a similar

magnitude in male and female offspring. However, we did identify underlying sex differences in

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gross body and renal morphology. Interestingly, both mean glomerular volume and total glomerular

volume were greater in female mice than male mice. Furthermore, renal AT1A-receptor gene

expression was greater in female than male NP-mice, although similar in female and male LP mice.

The results of this study highlight the importance of analyzing offspring of both sexes in

experimental models of maternal protein restriction.

To our knowledge, this is the first report of reduced nephron endowment in a mouse model of

maternal protein restriction. We employed a relatively mild degree of protein restriction, similar to

that we have employed previously in rats (14). However, it is important to note that the LP diet

contains approximately 3.7% less energy than the NP diet, and contains a higher sucrose content,

and different starch: sucrose ratio. Nevertheless, in the present study in the mouse, the LP diet did

not reduce litter size or offspring survival. Our current observations do not address the impact of

combined pre- and post-natal protein restriction on adult cardiovascular health. However, our

characterization of this new mouse model of maternal protein restriction provides a basis for future

studies to examine both adult cardiovascular outcomes and the molecular mechanisms that mediate

renal developmental programming in the mouse. A nephron deficit, as seen in our study in both

male and female LP mice, has been implicated in the development of adult diseases, via Brenner’s

hypothesis (5). For example, maternal protein restriction in rats can reduce offspring nephron

endowment and lead to adult hypertension (17, 41). The nephron deficit seen in protein restricted

rats is characterized by a 10-30% reduction in glomerular number (14, 17, 41, 43). Thus, at least in

this respect, our findings in mice replicate previous observations in the rat. Interestingly, estimates

of female offspring nephron number following maternal protein restriction, have produced

somewhat conflicting data (17, 39, 42, 43). For example, Zimanyi et al, using unbiased techniques,

found that female protein restricted rats had a nephron deficit compared to controls (44). In contrast,

Woods et al. found that female offspring of moderately protein restricted rats dams had normal

nephron endowment (42). Strain and diet variation may account for these disparate findings in rats.

Of particular importance to the study of nephron number is the use of unbiased stereology.

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Alternatives to this ‘gold-standard’ technique are subject to considerable bias, leading to inaccurate

results. In our current study using unbiased stereology, we have shown that maternal protein

restriction can lead to a nephron deficit in both male and female mice. We were unable to detect a

sex-dependent difference in the magnitude of this effect.

However, we were able to detect sex-dependent differences in underlying renal morphology in

mice, in that female mice were found to have larger glomeruli and renal corpuscles than males, yet

have smaller kidneys. Total glomerular volume and total renal corpuscle volume was also found to

be greater in female than male mice. Two previous studies investigated sex differences in

glomerular morphology in mice at a similar age, yet failed to detect sex differences in renal

corpuscle diameter or glomerular area (28) (18), although neither study employed unbiased

stereological techniques. Okada et al found no sex differences in cortical corpuscle diameter in

DBA/2CrSlc mice at 30 days of age, although corpuscle diameter was greater in males from 90

days of age (28). Lovegrove et al found no sex differences in glomerular area in wild type mice at

70 days of age (18). Differences between our current observations, and those of these previous

studies, might be explained by strain differences, or by differences in the stereological techniques

employed. Our findings merit further investigation of adult mice of both sexes, to determine

whether the greater glomerular size in females confers a greater or decreased survival advantage,

under certain physiological conditions.

Total body weight, and the weight of all organs studied, was less in LP than NP mice. These

observations are consistent with the outcomes of previous studies of combined pre-natal and post-

natal protein restriction in rats (6, 14). In our study, retarded growth, of all organs except the brain,

was in proportion to the retardation in total body weight. This contrasts with the effects of

combined pre- and post-natal protein restriction in the rat, where the weights of the spleen and the

liver were found to be reduced proportionally more than was total body weight (14). ‘Brain-sparing’

following protein restriction occurred in both sexes in mice, consistent with previous findings in the

rat (3, 6). Similarly, brain sparing has been shown in rabbits and sheep subjected to intrauterine

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growth restriction (12, 29) and occurs in human growth restricted infants (10, 11, 20). Brain sparing

may confer an immediate survival advantage to the offspring, whilst growth of other organs is

compromised.

The effects of protein restriction on gross organ morphology were not sex-dependent, except in the

case of the liver. Protein restriction retarded growth of the liver far more in male mice than in

female mice. Sex-dependent effects of maternal protein restriction on hepatic glycogen stores have

been identified in rats (1). The significance of these sex-dependent effects of protein restriction on

liver development remains to be determined. They may reflect the potential for sex-dependent

developmental programming of metabolic disorders.

Female mice in our study had a significantly lower kidney and body weight than males, consistent

with observations from previous studies (18, 28). Brain weight, as a proportion of total body

weight, was greater in female than male mice. This has not previously been demonstrated in mice,

although our observations are consistent with the results of previous studies in rats (21, 31).

In the current study, we measured renal expression of AT1A, AT1B, and AT2 receptor mRNA in 30

day old mice. AT1A-receptor mRNA was detected in all kidneys, as was AT1B- and AT2-receptor

mRNA expression. Compared to our positive control of fetal kidney tissue, where the AT2 receptor

is constitutively highly expressed, the levels of AT2 receptor in postnatal day 30 tissues was

considerably lower and highly variable. This is perhaps explained by the fact that after birth, levels

of AT2 receptor expression reduce markedly (35), and the timing of this reduction to very low levels

may vary between individual mice. In the mouse kidney, the AT1 receptor is expressed in

developing glomeruli and S-shaped bodies, and in proximal and distal tubules, where it is thought to

mediate cellular differentiation and proliferation during nephrogenesis (15). Expression is greatest

at the end of gestation and decreases following birth. We found considerably greater renal AT1A

receptor mRNA levels in female NP mice compared with male NP mice at postnatal day 30. This

observation is consistent with the results of a previous study in rats (23), although other studies

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have failed to replicate this observation (24). The greater levels of renal AT1A receptor expression in

NP female than NP male mice might reflect sex differences in the time-course of renal

development, in that they are ‘leftover’ from the higher levels of AT1A receptor expression present

during nephrogenesis in these relatively young (adolescent) mice. This phenomenon could possibly

contribute to the sex-differences in glomerular morphology observed in the current study. We might

also therefore expect to find an eventual decrease in AT1A-receptor expression in female mice

beyond 30 days of age. Studies are currently underway to investigate these possibilities.

There is now considerable evidence, from a number of models of developmental programming, for

roles of abnormalities in the renal renin/angiotensin system (32). For example, maternal protein

restriction in rats reduced renal renin mRNA levels and renal tissue angiotensin II levels in newborn

male pups and resulted in adult hypertension (41). Moreover, AT1 receptor density was increased,

while AT2 receptor density was reduced, in protein restricted male rats at 4 weeks of age (33). We

found a clear effect of maternal protein restriction on renal AT1A receptor expression, but only in

female offspring. Thus, renal AT1A receptor expression was considerably less in LP compared to NP

female mice, but at a similar level to both LP and NP male mice. Previous studies of the effects of

maternal protein restriction on AT1A-receptor expression have produced conflicting results (22-24,

33, 34). Our current data raise the possibility that the sex-dependent effects of maternal protein

restriction on renal AT1A-receptor expression might lead to sex differences in the impact of

maternal protein restriction on adult disease. This hypothesis merits further investigation.

One important caveat must be applied to our current findings. The genetic background of the male

breeder mice (129sv) differed from that of the female breeders (C57/Bl6). We took this approach to

ensure hybrid vigor in the offspring, since we are currently undertaking intensive studies of

cardiovascular and renal physiology in these animals in adulthood. The advantages of a hybrid

strain include offspring vigor, uniformity, and litter sizes that are often larger than those of pure

isogenic strains (9). It does, however, mean that the single X-chromosomes of the male offspring

are exclusively of the C57/Bl6, whereas the females have both the 129sv and C57/Bl6 X-

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chromosome. However, we believe this is unlikely to have greatly confounded our current

observations since, in mice, X inactivation is imprinted with preferential silencing of the paternal X

chromosome (37). This means that the C57/Bl6 X chromosome should dominate in both male and

female mice. There is a small possibility that some X-linked genes are still expressed from the

inactive X chromosome. However, this phenomenon is less frequent in mice than humans (13).

Perspectives

Sex and gender have important influences on adult risk of disease (38). It might therefore be

expected that sex could greatly influence adult cardiovascular and renal function. Indeed, there is

now evidence that this is the case in the model of maternal protein restriction in rats (42), whilst

other studies have identified important differences between the sexes in basal renal haemodynamics

(25, 27) and renal function (8). Our current study is, to our knowledge, the first to examine the

effects of maternal protein restriction in mice on offspring renal morphology, and incorporates

measurements in both male and female offspring. Our results indicate that maternal protein

restriction reduces nephron endowment in the offspring of mice, as is the case in rats (14). This

effect, and many of the other effects of maternal protein restriction, was similar in male and female

offspring. However, there were sex-dependent effects of protein restriction on renal AT1A receptor

expression and liver weight, the significance of which requires further study. We also identified

sex-differences in underlying glomerular morphology that have not previously been described. The

mouse model we describe here should facilitate future study of the molecular bases of

developmental programming, and its sex-dependence.

Acknowledgements

The authors acknowledge the expert technical assistance of Mrs. Sue Connell and Ms Debbie

Arena.

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Grants:

This work was supported by grants from the National Health and Medical Research Council of

Australia (384207, 384101, 384279) and by an Australian Postgraduate Award (CCH) through the

Commonwealth Government of Australia. RGE is also supported by a NH&MRC Senior Research

Fellowship and KMM by a NH&MRC Career Development Award.

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16. Langley-Evans SC, Phillips GJ, Benediktsson R, Gardner DS, Edwards CR, Jackson AA, and Seckl JR. Protein intake in pregnancy, placental glucocorticoid metabolism and the programming of hypertension in the rat. Placenta 17: 169-172, 1996. 17. Langley-Evans SC, Welham SJ, and Jackson AA. Fetal exposure to a maternal low protein diet impairs nephrogenesis and promotes hypertension in the rat. Life Sci 64: 965-974, 1999. 18. Lovegrove AS, Sun J, Gould KA, Lubahn DB, Korach KS, and Lane PH. Estrogen receptor alpha-mediated events promote sex-specific diabetic glomerular hypertrophy. Am J Physiol Renal Physiol 287: F586-591, 2004. 19. Ludbrook J. Comparing methods of measurement. Clinical and Experimental Pharmacology and Physiology 24: 193-203, 1997. 20. Lumbers ER, Yu ZY, and Gibson KJ. The selfish brain and the barker hypothesis. Clin Exp Pharmacol Physiol 28: 942-947, 2001. 21. McArthur S, Siddique ZL, Christian HC, Capone G, Theogaraj E, John CD, Smith SF, Morris JF, Buckingham JC, and Gillies GE. Perinatal glucocorticoid treatment disrupts the hypothalamo-lactotroph axis in adult female, but not male, rats. Endocrinology 147: 1904-1915, 2006. 22. McMullen S, Gardner DS, and Langley-Evans SC. Prenatal programming of angiotensin II type 2 receptor expression in the rat. Br J Nutr 91: 133-140, 2004. 23. McMullen S, and Langley-Evans SC. Maternal low-protein diet in rat pregnancy programs blood pressure through sex-specific mechanisms. Am J Physiol Regul Integr Comp Physiol 288:R85-90, 2005. 24. McMullen S, and Langley-Evans SC. Sex-specific effects of prenatal low-protein and carbenoxolone exposure on renal angiotensin receptor expression in rats. Hypertension 46: 1374-1380, 2005. 25. Messow C, Gartner K, Hackbarth H, Kangaloo M, and Lunebrink L. Sex differences in kidney morphology and glomerular filtration rate in mice. Contrib Nephrol 19: 51-55, 1980. 26. Moritz K, Koukoulas I, Albiston A, and Wintour EM. Angiotensin II infusion to the midgestation ovine fetus: effects on the fetal kidney. Am J Physiol Regul Integr Comp Physiol 279:R1290-1297, 2000. 27. Munger K, and Baylis C. Sex differences in renal hemodynamics in rats. Am J Physiol 254: F223-231, 1988. 28. Okada A, Yabuki A, Matsumoto M, and Suzuki S. Development of gender differences in DBA/2Cr mouse kidney morphology during maturation. J Vet Med Sci 67: 877-882, 2005. 29. Oyama K, Padbury J, Martinez A, Chappell B, Stein H, and Humme J. Effects of fetal growth retardation on the development of central and peripheral catecholaminergic pathways in the sheep. J Dev Physiol 18: 217-222, 1992. 30. Ozaki T, Nishina H, Hanson MA, and Poston L. Dietary restriction in pregnant rats causes gender-related hypertension and vascular dysfunction in offspring. J Physiol 530: 141-152, 2001. 31. Rao S, Joshi S, Kale A, Hegde M, and Mahadik S. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring. Metabolism 55: 628-634, 2006. 32. Rasch R, Skriver E, and Woods LL. The role of the RAS in programming of adult hypertension. Acta Physiol Scand 181: 537-542, 2004. 33. Sahajpal V, and Ashton N. Increased glomerular angiotensin II binding in rats exposed to a maternal low protein diet in utero. J Physiol 563: 193-201, 2005. 34. Sahajpal V, and Ashton N. Renal function and angiotensin AT1 receptor expression in young rats following intrauterine exposure to a maternal low-protein diet. Clin Sci (Lond) 104: 607-614, 2003. 35. Shanmugam S, Llorens-Cortes C, Clauser E, Corvol P, and Gasc JM. Expression of angiotensin II AT2 receptor mRNA during development of rat kidney and adrenal gland. Am J Physiol 268: F922-930, 1995.

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36. Symonds ME, and Gardner DS. Experimental evidence for early nutritional programming of later health in animals. Curr Opin Clin Nutr Metab Care 9: 278-283, 2006. 37. Takagi N, and Sasaki M. Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 256: 640-642, 1975. 38. Torgrimson BN, and Minson CT. Sex and gender: what is the difference? J Appl Physiol 99: 785-787, 2005. 39. Vehaskari VM, Aviles DH, and Manning J. Prenatal programming of adult hypertension in the rat. Kidney Int 59: 238-245, 2001. 40. Vickers MH, Breier BH, McCarthy D, and Gluckman PD. Sedentary behavior during postnatal life is determined by the prenatal environment and exacerbated by postnatal hypercaloric nutrition. Am J Physiol Regul Integr Comp Physiol 285: R271-273, 2003. 41. Woods LL, Ingelfinger JR, Nyengaard JR, and Rasch R. Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats. Pediatr Res 49: 460-467, 2001. 42. Woods LL, Ingelfinger JR, and Rasch R. Modest maternal protein restriction fails to program adult hypertension in female rats. Am J Physiol Regul Integr Comp Physiol 289: R1131-1136, 2005. 43. Zimanyi MA, Bertram JF, and Black MJ. Does a nephron deficit in rats predispose to salt-sensitive hypertension? Kidney Blood Press Res 27: 239-247, 2004. 44. Zimanyi MA, Denton KM, Forbes JM, Thallas-Bonke V, Thomas MC, Poon F, and Black MJ. A developmental nephron deficit in rats is associated with increased susceptibility to a secondary renal injury due to advanced glycation end-products. Diabetologia 49: 801-810, 2006.

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Table 1: Composition of the diets

_______________________________________________

Constituent (g/kg

of diet)

Normal Protein

Diet (NP)

Low Protein

Diet (LP)

_______________________________________________

Casein 200 87

Canola Oil 70 70

Sucrose 100 200

Wheat Starch 404 417

Cellulose 50 50

D/L-Methionine 3 3

Sodium Chloride 2.6 2.6

Protein (%) 19.5 8.4

Total Fat (%) 7 7

Energy (MJ/kg) 16.1 15.5

_______________________________________________

Semi-pure diets were obtained from Specialty Feeds, Western Australia. Feed was manufactured as

a 12 mm diameter cube, vacuum packed, and stored at -20 ˚С until use. Low protein diets were

rendered isocaloric by addition of sucrose/starch.

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Table 2: Primer and probe sequences

Gene of Interest Concentrations Used

Primer and Probe Sequence

AT1A 50 Forward 5- GGG CTG TCT ATA CCG CTA

TGG AA -3

GenBank accession no. NM_177322

300 Reverse 5- GCC GAA GCG ATC TTA CAT AGG TG -3

75 Probe 5- ACC GCT GGC CCT TCG GCA A -3

AT1B 900 Forward 5- GGG CAG TTT ATA CCG CTA TGG A -3

GenBank accession no. NM 175086

900 Reverse 5- TGG CCG AAG CGA TCT TAC AT -3

200 Probe 5- TAC CAG TGG CCC TTC GGC AAT CA -3

AT2 300 Forward 5- TGT GTT GGC ATT CAT CAT TTG -3

GenBank accession no. NM_007429

900 Reverse 5- CCA TCC AAG CTA GAG CAT CCA -3

125 Probe 5- TGG CTT CCC CAT GTT CTG ACC TTC -3

AT1A = angiotensin receptor 1A; AT1B= angiotensin receptor 1B; AT2 = angiotensin receptor 2.

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http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=31982509



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Table 3: Effects of protein restriction and sex on body and organ weights at postnatal day 30

Values represent body and organ weights in 30 day old male and female mice exposed to normal (n =

4-6 litters) or low protein diet (n = 6 litters) both in utero and postnatally. Data are expressed both as

absolute values (A), and normalised to body weight (B). Data are presented as mean ± standard error

of the mean, determined by the least squares method, weighted by litter. P values are the outcomes of

univariate analysis of variance, testing for the main effects of ‘protein diet’ and ‘sex’ and for

interactions between these factors.

(A) Absolute Values (g)

Male Female PProtein PSex PSex*Protein

Normal

Protein Low Protein

Normal

Protein Low Protein

Body 19.6 ±0.4 15.6 ±0.5 17.1 ±0.3 13.7 ±0.3 <0.001 <0.001 0.628

Left Kidney 0.129 ±0.006 0.101 ±0.003 0.100 ±0.006 0.077 ±0.003 <0.001 <0.001 0.561

Right

Kidney 0.131 ±0.005 0.103 ±0.003 0.106 ±0.005 0.081 ±0.003 <0.001 <0.001 0.789

Total

Kidney 0.260±0.011 0.204 ±0.006 0.207 ±0.010 0.158 ±0.005 <0.001 <0.001 0.648

Liver 1.14 ±0.051 0.821 ±0.028 0.884 ±0.049 0.783 ±0.025 <0.001 <0.001 0.010

Spleen 0.109 ±0.006 0.081 ±0.003 0.082 ±0.006 0.071 ±0.003 <0.001 <0.001 0.099

Heart 0.110 ±0.004 0.093 ±0.002 0.093 ±0.004 0.081 ±0.002 <0.001 <0.001 0.480

Brain 0.436 ±0.008 0.411 ±0.005 0.433 ±0.008 0.420 ±0.004 0.004 0.685 0.349

(B) Corrected Values (mg/g)

Left Kidney 6.6 ±0.4 6.5 ±0.2 5.9 ±0.4 5.7 ±0.2 0.557 0.013 0.868

Right

Kidney 6.7 ±0.3 6.6 ±0.1 6.2 ±0.3 5.9 ±0.1 0.437 0.008 0.690

Total

Kidney 13.3 ±0.6 13.1 ±0.3 12.1 ±0.6 11.6 ±0.3 0.494 0.007 0.783

Liver 57.9 ±2.7 52.4 ±1.4 51.2 ±2.6 57.5 ±1.3 0.860 0.712 0.009

Spleen 5.6 ±0.5 5.3 ±0.2 4.8 ±0.5 5.2 ±0.2 0.818 0.223 0.350

Heart 5.6 ±0.3 6.0 ±0.2 5.5 ±0.3 6.0 ±0.1 0.051 0.683 0.814

Brain 22.3 ±1.1 26.2 ±0.6 25.4 ±1.0 30.7 ±0.5 <0.001 <0.001 0.378

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Figure 1:

Male

Postnatal Age (day)

0 5 10 15 20 25 30

Body

Wei

ght(

g)

0

5

10

15

20

25 Female

0 5 10 15 20 25 30

Normal Protein (NP)Low Protein (LP)

Fig. 1: Growth curves for male and female mice. Male and female mice were exposed to normal or

low protein diet both in utero and postnatally until postnatal day 30. Data are presented as within

litter averages in a scatter plot, and fitted to lines of best fit by the method of least squares using

individual animals as the experimental unit. Univariate analysis of covariance was performed on

these data, weighted by litter. See text for statistical analysis.

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Figure 2: G

lom

erul

arN

umbe

r

0

2000

4000

6000

8000

10000

12000

14000

PSex = 0.706 PProtein = 0.010

PSex*Protein = 0.647

PSex = 0.030

PProtein = 0.614


TCV

(mm

3 )

0

1

2

3

4

Vco

rp(1

0-4m

m3 )

0

1

2

3

4

PSex = 0.011

PProtein = 0.199


Vgl

om(1

0-4m

m3 )

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5



A

B

C

D

E

Low Protein MalesNormal Protein Males

Normal Protein FemalesLow Protein Females

TGV

(mm

3 )

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5



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Fig. 2: Nephron number and glomerular dimensions. Male and female mice were exposed to normal

or low protein diets both in utero and postnatally until postnatal day 30. Unbiased stereological

techniques were used to determine total glomerular (nephron) number (A); mean glomerular

volume (Vglom) (B); total glomerular volume (TGV) (C); mean renal corpuscle volume (Vcorp) (D);

and total renal corpuscle volume (TCV) (E). For mice exposed to a normal protein diet n=4-6

animals from n=3-4 litters. For mice exposed to a low protein diet n=6-7 animals from n=4 litters.

Values are mean ± standard error of the mean. P values are the outcomes of univariate analysis of

variance weighted for litter, testing the main effects of ‘protein diet’ and ‘sex’ and for interactions

between these factors.

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Figure 3:

A

B

Rel

ativ

eA

T 1a

rece

ptor

mR

NA

expr

essi

on

0

2

4

6

8

PSex = 0.013PProtein = 0.023PSex*Protein = 0.011

Rel

ativ

eA

T1b

rece

ptor

mR

NA

expr

essi

on

0

1

2

3

4

5

PSex = 0.388PProtein = 0.288PSex*Protein = 0.913

Normal Protein MalesLow Protein MalesNormal Protein FemalesLow Protein Females

Fig. 3: Relative expression of AT1A and AT1B receptor mRNA. Male and female mice were exposed

to a normal or low protein diet both in utero and postnatally until postnatal day 30. Real-time PCR

was performed using tissue from the left kidneys of these animals. Data are presented as mean ±

standard error of the mean, analyzed statistically as for Figure 2.

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Effects of dietary protein restriction on nephron number in the mouse

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