Kidney International, Vol. 61 (2002), pp. 1231–1242

Protein restriction in pregnancy is associated withincreased apoptosis of mesenchymal cells at thestart of rat metanephrogenesis

SIMON J.M. WELHAM, ANGELA WADE, and ADRIAN S. WOOLF

Nephro-Urology and Paediatric Epidemiology and Biostatistics Units, Institute of Child Health,University College London, London, England, United Kingdom

Conclusions. Maternal low protein diets reduce final num-Protein restriction in pregnancy is associated with increasedbers of glomeruli in association with enhanced deletion ofapoptosis of mesenchymal cells at the start of rat metanephro-mesenchymal cells at the start of kidney development. Whethergenesis.aberrant nephrogenesis is a direct effect from deletion of neph-Background. In rats, offspring born to mothers supplied lowron precursors, or an indirect effect from loss of supportiveprotein diets during pregnancy have fewer glomeruli than nor-interstitial precursors, requires further investigation.mal. We hypothesized that such nephron deficits are associated

with altered cell turnover in the metanephros, the embryonicprecursor of the adult kidney.

Methods. Wistar rats were supplied with one of three iso-Certain adult diseases may originate by an, as yet, poorlycaloric diets from day 0 of pregnancy: control (18% protein) or

defined embryonic processes called ‘programming.’ Forlow protein (9% or 6%) diets. All had a normal chow afterbirth. Groups were compared by multilevel statistical modeling. example, a large body of epidemiological data is consis-

Results. At two weeks postnatally, when nephrogenesis has tent with the hypothesis that individuals born to mothersfinished, controls had 16.8 � 103 � 0.7 � 103 (mean � SEM) with poor diets are prone to develop hypertension andglomeruli/kidney, whereas offspring exposed to 9% diet had

cardiovascular disease in adulthood [1]. A human autopsy5.1 � 103 � 1.2 � 103 fewer and those exposed to 6% diet hadstudy reported fewer glomeruli in infants with intrauter-6.9 � 103 � 1.7 � 103 fewer glomeruli/kidney (P � 0.001, both

diets). At embryonic day 13 (E13), when the metanephros has ine growth retardation [2], and it has been suggestedjust formed, control metanephroi contained 2.35 � 104 � 0.15 � that congenital ‘nephron deficits’ predispose individuals104 cells, with no significant differences in low protein groups. to hypertension later in life [3]. Using animal experiments,At E15, when mesenchyme begins forming primitive nephrons

maternal nutrition has been established to be an impor-but glomeruli are still absent, controls had 2.00 � 106 � 0.13 �tant determinant of fetal growth. The imposition of a106 cells. E15 embryos exposed to 9% protein had 1.09 � 106 �

0.36 � 106 fewer cells/metanephros than controls, while those severe dietary protein restriction (5 to 6% diets) duringexposed to 6% diet had 1.45 � 106 � 0.37 � 106 fewer (P � pregnancy impairs somatic growth and reduces numbers0.01, both diets). Apoptotic cells were detected by molecular

of glomeruli/kidney measured postnatally when nephro-(in-situ end-labeling) and morphological (propidium iodidegenesis has finished [4–6]. Milder maternal protein re-staining) techniques. In all diets, apoptosis was noted in con-

densing mesenchyme (nephron precursors) and loose mesen- striction (9% diet) is also detrimental, leading to fewerchyme (interstitial precursors). Control E13 metanephroi had glomeruli in offspring and higher adult blood pressure63 � 7 apoptotic cells/mm2, whereas those exposed to 9% diet versus control rats [7, 8]. Congenital nephron deficits mayhad an increase of 77 � 26 cells/mm2 (P � 0.01) and those

interact with environmental (such as hyperglycemia) andexposed to 6% diet had an increase of 55 � 26 cells/mm2

genetic factors to generate glomerulosclerosis [9].(P � 0.05). By E15, apoptosis was similar in all groups butmetanephric mitosis was significantly increased in the 6% pro- The metanephros is the embryonic precursor of adulttein diet group. No change was found in the level of apoptosis mammalian kidney. Metanephric mesenchyme arises fromin E13 mesonephroi. the intermediate mesoderm, an embryonic compartment

that also forms the pronephros, and mesonephros, tran-sient embryonic organs. Metanephric mesenchyme un-Key words: cell death, diet, embryo, metanephros, nephron, proliferat-

ing cell nuclear antigen, protein. dergoes apoptotic death unless rescued by poorly definedfactors derived from the ureteric bud [10, 11]. In vivo,Received for publication May 29, 2001a subset of renal mesenchyme cells becomes induced toand in revised form November 9, 2001

Accepted for publication November 13, 201 differentiate into nephrons. A first step in this process in-volves aggregation into ‘mesenchymal condenses’ that 2002 by the International Society of Nephrology

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Welham et al: Maternal diet and metanephrogenesis1232

subsequently polarize to form epithelia, forming vesicles consumption during pregnancy, determined gravimetri-and S-shaped bodies; the head of the S-shaped body forms cally, does not vary as a result of maternal dietary proteinthe glomerulus. Cells in mesenchymal condensates are content [7]. Therefore, we supplied diets ad libitum.proliferative, have a low index of apoptosis and express Diets were supplied until delivery, when all mothers werePax-2 and Bcl-2 [12–14], cell survival molecules [15, 16]. fed standard laboratory chow. Rats were sacrificed atIn normal murine and human metanephrogenesis, apo- either day 13 or 15 of pregnancy, or were allowed toptosis occurs around mesenchymal condensates, where deliver offspring that were sacrificed two weeks after birth.the process may limit the numbers of cells being incorpo- Embryonic day 13 (E13) and E15 embryos and placentasrated into nascent nephrons [12, 13]. The hilum of the were removed and weighed to the nearest mg. E13 andmetanephros is another ‘hot-spot’ for apoptosis, where E15 metanephroi were used for histological analysis ofinterstitial cell detection is spatially related to blood ves- apoptosis and mitosis, for metanephric total cell countssel growth [17]. Camp and Martin [18] and Cale et al and for proliferating cell nuclear antigen (PCNA) West-[19] have produced histological evidence that apoptotic ern blot; embryos for analysis were randomly selectedmetanephric cells are cleared by macrophages. Apo- from each litter. E13 mesonephroi also were analyzedptotic deletion of specific tubule cells occurs during dif- histologically. Two-week postnatal kidneys of offspringferentiation of the loop of Henle and the collecting duct

were processed for histology and glomerular counting. In[20, 21]. With renal maturation, both mitosis and apopto-

the strain of rat used in the current study, our preliminarysis decrease [12–14, 18].observations (not shown) demonstrated that the meta-Kwong et al recently demonstrated that maternal lownephros forms between E12 and E13 in all three dietaryprotein diets reduced cell numbers in the inner cell massgroups, when the organ consists of the ureteric bud enve-of pre-implantation embryos, providing an insight intoloped by renal mesenchyme, and that the first metaneph-the ‘programming’ phenomenon [22]. We hypothesizedric glomeruli form at E16. In rats, nephron formation isthat maternal undernutrition might affect apoptosis orcomplete by ten days after birth [25].proliferation in the early stages of development of the

metanephros. The data presented in this study suggestMetanephric cell countsthat maternal protein restriction during pregnancy is as-

At E13 and E15, individual metanephroi were care-sociated with apoptotic deletion of metanephric progeni-fully dissected free from adherent connective tissues andtor cells at stages before glomeruli have formed.ureters. E13 and E15 organs were respectively incubatedin 28 �L or 60 �L of 0.05% trypsin/0.02% ethylenedi-

METHODS aminetetraacetic acid (EDTA) for 30 minutes at 37�C; inReagents were obtained from Sigma Chemical Com- this manner, tissues were dissociated to single cells. Tis-

pany (Poole, Dorset, UK), unless otherwise specified. sues were homogenized using a 25 gauge needle (Merck,Poole, Dorset, UK). The number of cells in the suspen-Animals and dietssion was then determined using a hemocytometer. Our

Experimental protocols were approved by the UK preliminary data (not shown) demonstrated intra- andHome Office. Female Wistar rats (Charles River, Mar- inter-assay variations of 9% and 8%.gate, Kent, UK) were time-mated by exposure to malesbetween 14:00 and 17:00 hours. At 09:00 hours the next Glomerular countsday, rats with vaginal plugs were selected for further study

Kidneys were harvested at two weeks postnatally, andand this time was designated embryonic day 0 (E0). Fe-analyzed as described [26]. Organs were incubated inmales were immediately randomly supplied one of three1 mol/L hydrochloric acid for 30 minutes at 37�C. Acidisocaloric diets (Dyets, Bethlehem, PA, USA) containingwas removed and replaced with 0.5 mL phosphate buf-either 18%, 9% or 6% protein by weight [22, 23]. Thefered saline (PBS; pH 7.4). The tissue was homogenizedcompositions of these respective diets were as follows:using a bench top homogenizer (Ultra-Turrax T25; BDH,casein (180 g/kg, 90 g/kg, 60 g/kg), maize starch (425 g/kg,Poole, Dorset, UK). A 20 �L sample was taken and placed485 g/kg, 505 g/kg) and sucrose (213 g/kg, 243 g/kg,on a slide and overlain with a cover slip. Using a �10253 g/kg). All diets contained: methionine (5 g/kg), cornobjective lens, the number of glomeruli in the aliquotoil (100 g/kg), vitamin mix AIN-76 (5 g/kg) [24], mineralwas counted. This was carried out in triplicate for eachmix AIN-76 (20 g/kg), choline chloride (2 g/kg) and cellu-sample. The three results were averaged and then thislose (50 g/kg). The diets were chosen because they repre-value was used to determine the total number of glomer-sent protein levels similar to standard laboratory chowuli in the sample, and therefore the kidney. Our prelimi-(18% control diet), a mild dietary protein restriction (9%nary data (not shown) demonstrated intra- and inter-protein diet) and a more severe dietary protein restriction

(6% protein diet). Previous work demonstrated that food assay variations of 10% and 11%.

Welham et al: Maternal diet and metanephrogenesis 1233

Histology However, it was possible to use individual E15 organs.Tissues were homogenized in lysis buffer [50 mmol/LEmbryonic day 13 and E15 metanephroi, and E13 meso-Tris pH 8.0, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5%nephroi, were examined in tissue blocks of caudal portionssodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)]of whole embryos. Two-week postnatal organs were re-containing protease inhibitors [1 �g/mL sodium ortho-moved from animals and processed further. Tissues werevanadate, 100 �g/mL phenylmethylsulfonyl fluoridefixed in 4% paraformaldehyde and paraffin embedded.(PMSF) and 30 �L/mL aprotinin]. Samples (20 �L) wereTissues were sectioned at 4 �m, dewaxed, rehydratedelectrophoresed through a 5% stacking and 12% resolv-and some were stained with hematoxylin and eosin. Usinging Tris-glycine SDS-polyacrylamide gel (Protogel, Na-these stains, nucleated erythrocytes have red cytoplasm,tional Diagnostics, Atlanta, GA, USA) and proteins wereproviding a simple marker for metanephric vessels, andelectrophoretically transferred to membranes (Hybond-P;dense, small pyknotic (that is, apoptotic) nuclei can beAmersham Pharmacia Biotech, Little Chalfont, Bucking-detected. Other sections from E13 and E15 metanephroihamshire, UK) by semi-dry blotting. Filters were placedwere examined for molecular evidence of apoptosis usingin block solution (10% marvel, 0.05% Tween in PBS)in situ end-labeling (In-situ Cell Death detection kit;overnight and incubated for one hour with mouse anti-Boerhinger Mannheim, Mannheim, Germany) based onhuman PCNA antibody (1:5000; BD-Pharmingen, Santhe technique of terminal deoxynucleotidyltransferase-Diego, CA, USA). Membranes were washed three timesmediated dUTP nick end-labeling (TUNEL), as describedfor ten minutes in blocking solution and incubated for[27]. In these same sections, all nuclei were stained withtwo hours with a sheep anti-mouse peroxidase-conjugated4 mg/L propidium iodide containing 100 mg/L RNase Aantibody (Amersham Pharmacia Biotech). After wash-[13]. Sections were mounted in Citifluor� (Chemical Labs,ing in PBS, signals were detected with enhanced chemilu-University of Kent, Canterbury, UK) and viewed withminescence reagent (Amersham Pharmacia Biotech).a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen,Chemiluminescence was quantified using Phoretix analy-Germany). Using appropriate wavelengths, TUNEL la-sis software. Blots were re-probed with mouse anti-humanbeled nuclei were detected as a green color (515 to 565 nm)�-actin (Sigma; A5441), as �-actin is a ‘housekeeping’

and also could be identified morphologically by propid-protein, and its quantification allows for correction of any

ium iodide staining (617 nm) as small and fragmented minor variation in protein loading and transfer between(pyknotic), bright nuclei; nucleated red blood cells also each lane. In control experiments the primary antibodieshave bright nuclei at the latter wavelength, but these are were omitted and no signal was obtained (not shown).larger and regular in outline. Cells undergoing mitosisare also easily identified using propidium iodide staining Statisticsby visualizing the mitotic spindle. To quantify apoptosis For descriptive purposes data were expressed as mean �and mitosis histologically, three sections spaced at least SEM. Litter size was compared between dietary groups20 �m through the whole of each E13 or E15 metaneph- using one-way analysis of variance. All other measure-ros were examined. The number of apoptotic and mitotic ments—apart from Western blots of E13 metanephroicells were counted in each section, and the three values (where small amounts of tissues necessitated pooling ofwere summed. The surface area of each of the sections organs)—were made on each embryo, placenta or meta-was determined using KS300 image analysis software nephros, and more complex statistical approaches were(Carl Zeiss): only the body of the metanephros was used, necessary to account for within litter grouping. It is un-and we excluded loosely adherent connective tissue and clear whether exposure to a maternal low protein dietthe area of embryonic ureter from analysis. The three affects all embryos from a litter equally (for example, asvalues for surface area were also summed. The total num- a consequence of a change in maternal physiology) andber of apoptotic nuclei in each sample was divided by the in this case, ignoring the grouping of values within litterstotal surface area and converted to give a value of apo- could be considered to represent ‘pseudoreplication.’ Al-ptotic nuclei per mm2. In addition, sections through E13 ternatively, analysis of only one animal per litter (thatmesonephric kidneys were analyzed in the same manner. is, representing around 10% of the litter population), or

taking averages within litters, would result in relativelyWestern blot small numbers for analysis and may be wasteful of the

Proliferating cell nuclear antigen (PCNA) is a DNA available data. Intralitter correlation is a measure of thepolymerase-�-associated protein expressed in S phase [28] extent to which fetuses within the same litter are moreand can be used as a surrogate marker of proliferation. alike than those from different litters and takes valuesPCNA expression is prominent in kidney development between 0 and 1. As the intralitter correlation approachesand is down-regulated with maturation [14]. For exami- 1, the value of inclusion of each additional littermate isnation of expression in E13 organs, we pooled metanephroi decreased and a value of 1 indicates that the outcome is

the same for all animals from a given litter. Our ongoingfrom each litter to obtain enough experimental material.

Welham et al: Maternal diet and metanephrogenesis1234

Fig. 1. Embryonic and placental weights. (A and B) Embryonic day 13 (E13) embryonic and placental weights. (C and D) E15 embryonic andplacental weights. For each diet, columns contain values for individual litters. Symbols are: (�) 18% protein; (�) 9% protein; (�) 6% protein.Significant differences between control and low protein diets are indicated.

experimental work on metanephric apoptosis and dietary nificantly affected by maternal low protein diets (Fig. 1A, B). At E15, control embryos weighed 0.50 � 0.01 gmanipulation indicated that the intralitter correlationand their placentas weighed 0.31 � 0.01 g (Fig. 1 C, D).was at most 0.4 but could be as low as 0.1. Hence, multi-Embryos exposed to a 9% protein diet were 0.08 � 0.02 glevel modelling was used to compare outcomes betweenlighter than controls (P � 0.05) and had placentas 0.02 �dietary groups while taking account of the intralitter0.03 g lighter than controls (NS). Embryos exposed to acorrelation [29]. Using this methodology, it is possiblematernal 6% protein diet were 0.09 � 0.02 g lighter thanto obtain valuable information from littermates while pre-controls (P � 0.001) and had placentas 0.06 � 0.03 gventing any pseudoreplicative bias. Three to seven litterslighter than controls (P � 0.001). These data demon-were examined in each experimental group. For simplic-strate that the low protein diets had biologically signifi-ity, significant P values were stratified into �0.05, �0.01cant effects on growth of the conceptus as a whole by E15.and �0.001.

Kidney developmentThe stages of kidney development examined are shownRESULTS

in Figure 2 for control animals, with no gross differencesLitter sizenoted in the low protein groups (not shown). At E13,

Females supplied a control diet had on average 10.6 � the ureteric bud had branched approximately twice and0.4 pups/litter and exposure to either a 9% protein diet the renal mesenchyme had begun to aggregate into con-(10.5 � 0.4 pups/litter) or a 6% protein diet (10.1 � 0.4 densates, the first step in nephron formation (Fig. 2 A,pups/litter) did not significantly affect litter size at any B). At E15, further ureteric bud branching had occurredtime examined (P � 0.66). and the most mature nephrons had reached the S-shaped

stage, but glomeruli had yet to form (Fig. 2 C, D). AtEmbryonic and placental growth two weeks after birth, a nephrogenic zone was no longer

Neither embryonic weight (control 0.17 � 0.01 g) nor evident and all glomeruli appeared mature, with capillaryloops (Fig. 2 E, F).placental weight (control 0.20 � 0.01 g) at E13 was sig-

Welham et al: Maternal diet and metanephrogenesis 1235

Fig. 2. Bright field images of kidney maturation stages examined. Sections from control animals were stained with hematoxylin and eosin. (A andB) E13 metanephros with ureteric bud branch tips (u) and condensing mesenchyme (m). Nearby vessels are indicated (v). (C and D) E15metanephros shows considerable growth and differentiation but the most mature nephron stages are S-shaped bodies (s) and an outer nephrogeniczone is still prominent. (E and F ) Two-week postnatal kidney. Note that there is no nephrogenic zone and that all glomeruli (g) appear maturewith capillary loops. Bars are: 100 �m in A, C and E, and 10 �m in B, D and F.

Glomerular counts at maturity

The number of glomeruli per kidney two weeks afterbirth was significantly affected by maternal diet (Fig. 3).Control animals possessed 16.8 � 103 � 0.7 � 103 glomer-uli/kidney. Kidneys of animals exposed to a maternal9% protein diet had 5.1 � 103 � 1.2 � 103 fewer glomerulithan controls, and those exposed to a maternal 6% pro-tein diet had 6.9 � 103 � 1.7 � 103 fewer glomeruli thancontrols (P � 0.001, both diets).

Metanephric cell numbers

At E13, the total numbers of cells in metanephroifrom control diets was 2.35 � 104 � 0.15 � 104, with nosignificant differences between control and low proteingroups (Fig. 4A). At E15 (Fig. 4B), control animals had2.00 � 106 � 0.13 � 106 cells/metanephros, representingan approximately hundred-fold increase versus E13.Fetuses exposed to a maternal 9% protein diet had1.09 � 106 � 0.36 � 106 fewer cells/E15 metanephros

Fig. 3. Glomerular number two weeks after birth. For each diet, col-than controls, while those exposed to a maternal 6%umns contain values for individual litters. Symbols are: (�) 18% protein;

protein diet had 1.45 � 106 � 0.37 � 106 less cells/kidney (�) 9% protein; (�) 6% protein. Significant differences between controland low protein diets are indicated.than those of control animals (P � 0.01, both low protein

Welham et al: Maternal diet and metanephrogenesis1236

to a maternal 6% protein diet had 55 � 26/mm2 morethan controls (P � 0.05). At E15 (Fig. 8B), the level ofapoptosis in control metanephroi was 30 � 2 cells/mm2,and there was no significant difference between the con-trol and the low protein groups at this stage. At E13,control metanephroi had 119 � 11 mitotic cells/mm2

(Fig. 8C). There was a trend for a lower level of mitosisin the 6% diet, but this did not reach significance (P �0.26). At E15, metanephroi of control animals had 47 � 3mitotic cells/mm2 (Fig. 8D). Embryonic kidneys exposedto a maternal 9% protein diet had 14 � 19 more mitoticcells/mm2 (NS), while those of animals exposed to amaternal 6% protein diet had 54 � 16 more mitoticcells/mm2 compared with controls (P � 0.001). We nextassessed the distribution of mitosis at E15, counting theproportion of mitotic cells in three tissue compartmentsat this stage: ‘condensed mesenchyme,’ representing cellsforming primitive nephrons, ‘loose mesenchyme’ repre-senting deeper cells between tubules which will form theinterstitial compartment, and ‘epithelia,’ which representbranches of the ureteric bud, vesicles and S-shaped bod-ies. In the 18% protein diet, the mean proportion of mi-

Fig. 4. Metanephric total cell number. (A) E13 metanephros. (B) E15 totic cells in these respective compartments was 35%,metanephros. For each diet, columns contain values for individual litters.

9% and 56%. There was no significant difference in theSymbols are: (�) 18% protein; (�) 9% protein; (�) 6% protein. Signifi-cant differences between control and low protein diets are indicated. distribution in either of the low protein diets versus the

controls (respective means of 38%, 9% and 53% for the9% protein diet, and 39%, 10% and 51% for the 6%protein diet). Hence, the increase of mitosis measureddiets). Histology of E15 metanephroi showed that organsin at E15 in the 6% protein diet was distributed evenlyfrom each of the three dietary groups had a similar gen-across these cell populations.eral development, including the attainment of the first

sets of S-shaped bodies (arrows; Fig. 5).Metanephric PCNA levels

Visualization of metanephric apoptosis and mitosis Western blots demonstrated single bands for PCNA(35 kD) and �-actin (42 kD) proteins at E13 and E15 inIn E13 and E15 metanephroi, apoptotic nuclei wereall diets (Fig. 9A). As assessed by the ratio of metaneph-detected in all dietary groups. An almost perfect correla-ric PCNA/�-actin, there was no significant differences be-tion between the TUNEL (Fig. 6A) and propidium iodidetween the dietary groups at E13 or at E15 (Fig. 9B).(Fig. 6B) methods was noted, and for simplicity we choseThis ratio fell in control diet metanephroi between E13to quantify apoptosis using the former method. The loca-and E15, but the significance was borderline (P � 0.06).tion of apoptotic nuclei was almost always in renal mesen-

chymal compartment, in all groups, with the cell deathAnalysis of the E13 mesonephrosrarely seen in the ureteric bud branches (Fig. 6). In all

The mesonephros is present, albeit actively involuting,diets, apoptosis often occurred in ‘clusters’ of cells lo-at E13. The distribution of apoptotic cells in this transientcated in loose mesenchyme and was also noted, lesskidney is shown in Figure 10. Note the marked apoptosisprominently, in condensing mesenchyme (Fig. 6C). Mi-in the epithelial tubules. E13 control mesonephroi (N �totic cells (Fig. 6B) were detected in both renal mesen-23) had 111 � 14 apoptotic cells/mm2; E13 metanephroichymal and ureteric bud lineages. Figure 7 shows brightin embryos (N � 8) of mothers supplied a 9% proteinfield histology of a low protein diet E13 metanephros,diet had 12 � 28 fewer than controls, and those exposedwith two pyknotic (that is, highly likely to be apoptotic)to a maternal 6% protein diet (N � 19) had 10 � 21nuclei located in the condensed mesenchyme, the com-fewer than controls (neither was significantly differentpartment that will form nephrons.vs. the control diet). E13 control mesonephroi had 96 �

Quantification of apoptosis and mitosis 10 mitotic cells/mm2; E13 metanephroi in embryos ofmothers supplied a 9% protein diet had 22 � 21 fewerEmbryonic day 13 control metanephroi had 63 � 7

apoptotic cells/mm2 (Fig. 8A). E13 metanephroi in em- than controls, and those exposed to a maternal 6% pro-tein diet had 5 � 14 fewer than controls (neither signifi-bryos of mothers supplied a 9% protein diet had 77 �

26/mm2 more than controls (P � 0.01), and those exposed cantly different vs. control diet).

Welham et al: Maternal diet and metanephrogenesis 1237

Fig. 5. Histology of metanephroi at E15. ‘Equa-torial’ sections (that is, through the central ure-ter) of an organ from each of the three dietarygroups at E15: (A) 18% protein, (B) 9% pro-tein, and (C ) 6% protein. The general develop-ment, including the attainment of the first setsof S-shaped bodies (arrows), is similar for allgroups. Abbreviations are: cm, condensed mes-enchyme; lm, loose mesenchyme; u, uretericbud. Bars are 100 �m.

Fig. 11. Programming in the metanephros. Summary of results of thecurrent study relating how maternal low protein diets might ‘program’the embryo to generate too few nephrons by altering metanephric cellturnover.

DISCUSSION

In these experiments, apoptosis was detected in nor-mal metanephroi, almost exclusively in the mesenchyme.In addition, we noted that low protein diets were associ-ated with the generation of significantly fewer nephronsthan controls, as assessed by glomerular counts at twoweeks after birth. The observations on apoptosis in thenormal metanephros and the effect of maternal low pro-tein diet on final nephron complement are consistentwith previous observations cited in the beginning of thisarticle. Figure 11 summarizes the current results in rela-

Fig. 6. Confocal laser scanning images of the E13 metanephros. Sections tion to how maternal low protein diet might ‘program’from control diets. (A and B) The same area under appropriate wave- the embryo to generate too few glomeruli. A novel obser-lengths for detection of TUNEL and propidium iodide labeled nuclei,

vation of our study is that low protein diets reduce finalrespectively. Two TUNEL labeled nuclei (bright green) associated withnumbers of glomeruli in association with increased dele-condensing mesenchyme are indicated in panel A (arrows) and appear

as pyknotic nuclei stained red with propidium iodide (arrows in panel B). tion of precursors at the start of metanephric develop-A ureteric bud branch (u) and mesenchyme (m) are indicated. The ment. In humans, the equivalent developmental timearrowhead indicates a mitotic cell. (C ) Whole E13 metanephros, out-

frame would be five to seven weeks’ gestation [30], andlined for clarity by a dotted line, and surrounding tissues. The photo-we postulate that this represents a window when kidneygraph was generated by merging the signals from both wavelengths, with

apoptotic TUNEL/propidium iodide-stained nuclei appearing bright morphogenesis could be affected by dietary influences.yellow. Note the apoptotic cells, often in clusters in loose mesenchyme An assessment of the final number of glomeruli was(*). Apoptosis was also seen, but less prominently, in condensed mesen-

performed only to demonstrate that the low protein dietchyme (cm) and was not detected in ureteric bud branches (u). Barsare 10 �m in A and B, and 30 �m in C. diets used had an important biological effect on kidney

Welham et al: Maternal diet and metanephrogenesis1238

Fig. 7. Bright field histology at E13. Hema-toxylin and eosin stained section from regionof E13 metanephros. (A) Note two pyknoticnuclei (arrows) in condensing mesenchyme.A capillary is apparent with nucleated eryth-rocytes (arrowheads). These respective areasare shown in higher power in B and C. Abbre-viations are: a, aorta; cm, condensed mesen-chyme; u, ureteric bud. Bars are 10 �m in Aand 5 �m in B and C.

Fig. 10. Confocal laser scanning images of theE13 mesonephros. (A and B) The same areaunder appropriate wavelengths for detectionof TUNEL and propidium iodide labeled nu-clei, respectively. In each frame, the meso-nephros is outlined by a yellow line and theadjacent gonad is indicated (g). Note promi-nent apoptosis in mesonephric tubules (t). Barsare 100 �m.

Welham et al: Maternal diet and metanephrogenesis 1239

Fig. 8. Quantification of metanephric apoptosis and mitosis. (A) Apoptosis in E13 metanephros. (B) Apoptosis E15 metanephros. (C ) Mitosisin E13 metanephros. (D) Mitosis in E15 metanephros. For each diet, columns contain values for individual litters. Symbols are: (�) 18% protein;(�) 9% protein; (�) 6% protein. Significant differences between control and low protein diets are indicated.

kidney) is at the lower end of a range of previouslypublished measurements. Examples include the follow-ing: Langley-Evans, Welham and Jackson, who used his-tology sections of Wistar rat kidneys and reported amean of 15 � 103 glomeruli/kidney [8]; Jones et al, usingstereology of Sprague Dawley rat kidneys, reported amean of 24 � 103 glomeruli/kidney [31]; Fassi et al, usingdissociation of Wistar rat kidneys, reported a mean of27 � 103 glomeruli/kidney [32]; and Lelievre-Pegorier etFig. 9. Proliferating cell nuclear antigen (PCNA) protein levels. (A)

A representative blot at E13 with lanes annotated according to maternal al, using a dissociation technique in Sprague-Dawley ratdiets. (B) Western blot signal intensities expressed as PCNA/�-actin. kidneys, reported a mean of 36 � 103 glomeruli/kidneyFor each diet, each column contains values for individual litters: note

[26]. Although some experts favor stereology over disso-that for E13 data, each point represents a pooled litter. Symbols are:(�) 18% protein; (�) 9% protein; (�) 6% protein. ciation, we chose to use the latter technique as it is simple

and rapid, and preliminary data demonstrated intra- andinter-assay variations of 10% and 11%, respectively.maturation. The results confirm the general relationship

We noted that an early effect of low protein diets onreported by several other studies, that is, that maternalmetanephrogenesis was a significant increase in the num-low protein diets significantly reduce the final proportionbers of apoptotic nuclei per unit area at E13, as assessedof glomeruli by up to 1/3. The numbers measured in our

control Wistar rats (approximately 17 � 103 glomeruli/ by both the molecular TUNEL and morphological pro-

Welham et al: Maternal diet and metanephrogenesis1240

pidium iodide techniques. Since the rate of clearance of susceptible to deletion from low protein diets, and thatthey could be deleted earlier or later, depending on whenapoptotic nuclei is unknown, a skeptic might argue that

low protein diet merely slows the clearance of dead cells, the low protein diet is administered. It is, however, diffi-cult to compare the main conclusions of current studyleaving more to be detected. However, the increased

number of apoptotic cells at E13 most likely is biologi- (that is, that maternal low protein diets are associatedwith apoptotic deletion of metanephric cells) with eithercally important since total metanephric cells numbers,

which were similar in all dietary groups at E13, were sig- of the above reports, since neither Vehaskari et al [34],nor Langley-Evans et al [8] measured markers of cellnificantly lower in the protein restriction groups two days

later, at E15. The observed cell death in all experimental death in prenatal kidney development.The mesonephric data presented in the current studygroups was almost totally restricted to renal mesenchyme,

a compartment that contains at least three types of cells: further suggest that: (1) the normal pattern of apoptosisis different in mesonephroi versus metanephroi (that is,(1) a population of uninduced ‘stem cells’ located in the

periphery of the organ; (2) condensing mesenchymal cells more in epithelia vs. mesenchyme); (2) the overall levelof apoptosis is higher in control mesonephroi than meta-that have been induced to differentiate into nephron epi-

thelia; and (3) loose mesenchymal cells that will form nephroi, as is appropriate for an involuting organ; (3)there was no difference in the level of apoptosis of mito-interstitial cells. Our study did not specifically measure

the amount of death in each mesenchymal subcompart- sis in the different diets. Therefore, although both meta-nephric mesenchyme and the mesonephros arise fromment, although our observations clearly showed that apo-

ptosis occurred in, or immediately adjacent to, condens- intermediate mesenchyme, the apoptotic effects of ma-ternal low protein diets were confined to the precursoring mesenchyme (Figs. 6A and 7B) and also prominently

in loose mesenchyme (Fig. 6C) in all three experimental of the adult kidney. With regard to other organs systems,it is notable that Petrik et al reported that a maternalgroups. We postulate that increased loss of renal mesen-

chymal cells caused by low protein diets could affect low protein diet increased apoptosis in the late gestationrat pancreas [35].nephron formation, and hence final glomerular numbers,

by at least two mechanisms. The first mechanism could Other experiments suggest that deregulated apoptosisperturbs kidney differentiation. Araki et al reported thatbe through a direct, or classical, pathway mediated by

deletion of nephron precursors (such as in the condens- caspase inhibitors prevented renal mesenchymal celldeath and reduced ureteric bud branching and nephro-ing mesenchyme). A second, more speculative, mecha-

nism could be through an indirect pathway involving genesis in mouse metanephric organ culture [36]. Simi-larly, a genetic strategy to diminish apoptosis in the de-deletion of loose interstitial mesenchyme, cells that sup-

port normal nephron growth, although they do not di- veloping urinary tract, by null mutation of AT2, resultedin a variety of kidney and ureter malformations [37]. Inrectly contribute cells to the epithelial lineage. Of note,

mutation of BF-2, a transcription factor expressed in meta- these experiments, it is possible that prevention of nor-mal mesenchymal cell death disrupts critical interactionsnephric interstitial stroma, results in a perturbation of

nephron formation, presumably by interfering with the between mesenchymal and epithelial cell populationsthat are necessary for normal organogenesis. In normalphysical or paracrine support that this compartment pro-

vides to nascent nephrons [33]. However, further study development, apoptosis can be associated with regres-sion of relatively large structures, such as the mammalianis necessary to definitively establish which of these two

pathways is operative in low protein diets. At E15, how- mesonephros [18]. A pathological excess of metanephriccell death can be triggered genetically, for example inever, the wave of enhanced death had passed, with no sig-

nificant differences between the dietary groups. Vehaskari, Pax2/ and Bcl2/ mutant mice [15, 38], or by expo-sure to specific cytokines such as tumor necrosis factor-�Aviles and Manning recently reported that offspring of

mothers subjected to 6% protein restriction exhibited [19]. Furthermore, a physical insult, in the form of experi-mental urinary flow impairment, also causes increasedincreased renal apoptosis when examined at eight weeks

postnatally [34]; this may represent a later wave of cell apoptosis in the maturing kidney [27, 39]. All these ma-nipulations result in hypoplastic organs, with fewer neph-death secondary to a reduction of nephron numbers.

Our study used maternal protein restriction through rons than normal. Based on data in the current study,we can add maternal dietary protein restriction to the listthe whole of gestation, whereas others have reported that

final numbers of nephrons are reduced with, for example, of causes of enhanced metanephric apoptosis associatedwith renal hypoplasia.maternal restriction from E12 to term [34], or low protein

diets administered between 8–14 and 15–22 days of ges- We assessed the effects of maternal dietary protein re-striction on proliferation by counting propidium iodidetation [8]. In rats, undifferentiated renal mesenchyme is

present from the inception of the organ until the end of stained mitotic nuclei and measuring PCNA protein. Inpreliminary experiments, we performed PCNA immuno-nephrogenesis in the first week after birth. Thus, it is spec-

ulated that a population of precursor cells exists that are histochemistry and found that most cells were positive

Welham et al: Maternal diet and metanephrogenesis 1241

at E13 and E15, reflecting the high rate of proliferation in early study by Avner and colleagues reported that gluco-these early stages. We elected, therefore, to semiquantify corticoids perturbed metanephric growth in organ cul-the PCNA data by Western blots. Using mitotic counts, ture [45]. In the future, it would be interesting to assessthere was no difference between the groups at E13, but the effects of glucocorticoids on apoptosis in this experi-a statistically significant increase was detected in the 6% mental setting.low protein group at E15 versus time-matched controls;the distribution of mitoses between the mesenchyme and ACKNOWLEDGMENTSepithelia did not differ between the diets. However, at We thank the Kidney Research Aid Fund and the National Kidneythis time point, PCNA protein levels were not different. Research Fund (R16/1/2001) for grant support, and Professor Mark

Hanson (University of Southampton, UK) for support and helpfulOne reason for this discrepancy could be that the immu-discussion at the start of the project.noblots are less sensitive than cell counts. Another rea-

son is suggested by Petrik et al, who reported that ma- Reprint requests to Professor Adrian S. Woolf, Nephro-Urology Unit,Institute of Child Health, University College London, 30 Guilford Street,ternal low protein diet decreased proliferation in theLondon WC1N 1EH, England, United Kingdom.maturing pancreas, as assessed by bromodeoxyuridineE-mail: A.Woolf@ich.ucl.ac.uk

incorporation, although PCNA expression was not re-duced [35]. The authors speculated that cells exposed to

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