intrauterine growth restriction and postnatal high-protein diet affect the kidneys in adult rats

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Nutrition 27 (2011) 364–371

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Nutrition

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Basic nutritional investigation

Intrauterine growth restriction and postnatal high-protein diet affectthe kidneys in adult rats

Qian Shen M.S. a, Hong Xu M.D. a,*, Li-Ming Wei M.S. b, Jing Chen M.D. a, Hai-Mei Liu M.S. a

a Department of Nephrology and Rheumatology, Children’s Hospital of Fudan University, Shanghai, People’s Republic of Chinab Institutes for Biomedical Sciences, Fudan University, Shanghai, People’s Republic of China

a r t i c l e i n f o

Article history:Received 5 July 2009Accepted 16 March 2010

Keywords:Intrauterine growth restrictionKidneyProteomicsNutritional interventionProteinuriaHypertension

This research was funded by grant 30672242 from thFoundation of China.

* Corresponding author. Tel: þ86-21-6493-1006; faE-mail address: [email protected] (H. Xu).

0899-9007/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.nut.2010.03.003

a b s t r a c t

Objective: Intrauterine growth restriction (IUGR) is associated with hypertension and chronickidney disease in adulthood. Postnatal overnutrition after IUGR may be of pathogenic importancefor the development of diabetes and cardiovascular disease. This study was to identify the effects ofIUGR and a postnatal high-protein diet on the kidneys in adult rats.Methods: Intrauterine growth restriction was induced in Sprague-Dawley rats by isocaloric proteinrestriction in pregnant dams. IUGR pups were divided into two groups that were a standard-protein diet (IUGR group) or a high-protein diet (HP group). A comparative proteomic methodwas used to study the differences of protein expression profiles between normal adult rats andadult rats with IUGR and the effects of a postnatal high-protein diet on the protein expressionprofiles of the kidneys.Results: The IUGR adults had higher urinary excretion of protein and blood pressure than controlsand the HP diet caused more severe hypertension and proteinuria than IUGR itself. The differentialproteomic expression analysis found 12 proteins that had significantly differential expressionbetween the IUGR and control groups, which were transcription regulators and structural mole-cules. The differential proteomic expression analysis between the HP and control groups found 13proteins that had significantly differential expression and were involved primarily in bodymetabolism, oxidation reduction, and apoptosis regulation.Conclusion: An HP diet intervention after IUGR worsens the severity of hypertension andproteinuria, and this study may provide valuable experimental evidence of proteins involved in thepathogenesis of kidney disease in IUGR and the effect of postnatal overnutrition.

� 2011 Elsevier Inc. All rights reserved.

Introduction

Intrauterine growth restriction (IUGR) has long-term effectson various organisms through fetal programming [1,2]. Humanstudies of the association between IUGR and renal diseases haveindicated that, in contrast to the normal fetal kidney, the IUGRfetal kidney has a smaller volume with a significant decrease inglomerular number [3,4]. In addition, long-term follow-up afterbirth has shown a significantly lower glomerular filtrationfunction and a higher incidence of proteinuria in the IUGR groupcompared with the control group [5,6]. Our previous animalstudies have also shown a decreased glomerular number in IUGR

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rats, with an increased incidence of proteinuria and hypertensionin the postnatal follow-up period [7]. However, the pathogenesisof IUGR-induced postnatal kidney diseases has not been fullyclarified.

Previous theories have suggested that nutritional supple-ments, especially a high-protein diet for infants in whom IUGR isdiagnosed, at an early postnatal stage may assist rapid postnatalgrowth, but the high-protein diet may further exacerbate kidneyinjury and lead to deterioration of renal function. Currently, thereis no definite conclusion on the proper postnatal nutritionalintervention for fetal IUGR.

This study was designed to observe the effects of a postnatalhigh-protein nutritional intervention on kidney developmentand function in IUGR rats. Moreover, a comparative proteomicmethod was used to study the differences of protein expressionprofiles between normal adult rats and adult rats with IUGR andthe effects of a postnatal high-protein diet on the protein

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Q. Shen et al. / Nutrition 27 (2011) 364–371 365

expression profiles of kidney to explore the possible pathogen-esis of IUGR-induced kidney injury and the effects of a high-protein diet intervention on the kidney.

Materials and methods

This work was performed with the approval of Children’s Hospital of FudanUniversity’s institutional animal use and care committee.

Establishment of the IUGR animal model

Twelve female Sprague-Dawley rats (clean level, body weight 250–300 g,provided by the Department of Experimental Animals, Fudan University,Shanghai, China) were randomly divided into two groups after mating with malerats. The normal control group was fed with a conventional pregnancy diet (22%protein) until natural delivery, and the newborn rats were fed with a conven-tional diet (22% protein, control group) until 12 wk after birth. The study groupwas fed with a low-protein isocaloric diet consisting of 6% protein throughout theentire pregnancy until natural labor [8]. Newborn rats with a birth weight 2standard deviations lower than the average birth weight of the normal newbornrats were defined as IUGR pups and fed with a conventional diet (containing 22%protein, IUGR group) or a high-protein diet (containing 30% protein, HP group)until 12 wk after birth. The detailed ingredients of different diets are listed inTable 1.

Measurement of urine protein and blood pressure

Eight male rats were selected from each group at 4, 8, and 12 wk, respec-tively. The rats were weighed and 24-h urine was collected and the volume wasrecorded. Urine protein content was determined using colorimetry. Systolicblood pressure was measured using a tail artery measuring instrument. Thekidney weight and body weight of each group of rats were measured at 12 wk ofage.

Determination of glomerular number and kidney morphology

The Sprague-Dawley rats were sacrificed at 12 wk of age by jugular punctureafter anesthesia. The kidneys of eight rats in each group were collected at 12 wkof age and were conventionally fixed, embedded, sliced, and stained withhematoxylin and eosin. The glomerular number was counted and the glomerularvolume was measured. The methods for measuring the glomerular number andglomerular volume have been previously reported [9,10].

Two-dimensional gel electrophoresis and image analysis

At 12 wk of age, six kidneys were selected from each group and were mixedfor total protein extraction. Tissues were homogenized in a lysis buffer, spundown to collect the supernatant, and subjected to the Bradford assay to deter-mine protein concentration. Two-dimensional gel electrophoresis (2-DE) wasperformed according to the manufacturer’s instruction (Amersham Biosciences,Buckinghamshire, UK). An Immobiline pH gradient DryStrip gel (18 cm, non-linearpH 3w10; Amersham Biosciences) was rehydrated with re-swelling buffer con-taining a 1-mg protein sample at 20�C for 12 h, focused by gradient increasingvoltage to 8000 V, and continued until total voltage-hour reached 52 000 Vh.After focusing, the strip was first equilibrated in the equilibration solution con-taining 1% dithiothreitol and 2.5% iodine acetamide for 15 min, transferred to thetop of a 12.5% polyacrylamide gel for sodium dodecyl sulfate polyacrylamide gelelectrophoresis. The electrophoresis was performed at 25�C until bromophenolblue reached the bottom of the gel. The gel was stained by Coomassie brilliantblue and then scanned for image analysis. Each 2-DE experiment was performedin triplicate to confirm the reproducibility. The scanned gel images were thenanalyzed by Pdquest 7.3.0 (Bio-Rad, Hercules, CA, USA).

Table 1Diet make-up

Ingredients Protein (%)

Conventionaldiet

corn 26%, wheat 34%, alfalfa meal 2%,soybean meal 27%, fish meal 5%,vegetable oil 1%, premix 5%

22

Low-proteindiet

corn 50%, sucrose 13%, amylum 26.5%,vegetable oil 2.5%, fish meal 3%, premix 5%

6

High-proteindiet

corn 15%, amylum 21%, soybean meal 51%,vegetable oil 1%, fish meal 2.5%,alfalfa meal 2%, casein 2.5%, premix 5%

30

In-gel digestion and mass spectrometry

Protein spots with significant differences were excised from the gels andplaced into a 96-well microtiter plate. Gel pieces were detained with a solution of15 mM of potassium ferricyanide and 50 mM of sodium thiosulfate (1:1) for20 min at room temperature. Then they were washed twice with deionized waterand shrunk by dehydration in acetone cyanohydrin. The samples were thenswollen in a digestion buffer containing 20 mM of ammonium bicarbonate and12.5 ng/mL of trypsin (Roche, Indianapolis, IN, USA) at 4�C. After a 30-min incu-bation, the gels were digested longer than 12 h at 37�C. Peptides were thenextracted twice using 0.1% trifluoroacetic acid in 50% acetone cyanohydrin. Theextracts were dried under the protection of N2.

For matrix-assisted laser desorption ionization/time-of-flight mass spec-trometry, the peptides were eluted onto the target with 0.7 mL of matrix solution(a-cyano-4-hydroxy-cinnamic acid in 0.1% trifluoroacetic acid and 50% acetonecyanohydrin). Then the solution was spotted on a stainless-steel target with 192wells (Applied Biosystems, Framingham, MA, USA). Samples were allowed to air-dry before inserting them into the mass spectrometer. The matrix-assisted laserdesorption ionization mass spectrometer was an ABI 4700 TOF-TOF ProteomicsAnalyzer (Applied Biosystems) instrument. The ultraviolet laser was operated ata 200-Hz repetition rate with wavelength of 355 nm. The accelerated voltage wasoperated at 20 kV. The data were searched by GPS Explorer (Applied Biosystems)using MASCOT (Matrix Science, London, UK) as a search engine. Data frommatrix-assisted laser desorption ionization/time-of-flight mass spectrometrywere analyzed using MASCOT search software. The following parameters wereused in the search: retrieval species for rat, enzyme for trypsin, missed cleavageby 1, peptide tolerance of 0.2, tandem mass spectrometry (MS/MS) toleranceof 0.6 Da, and possible oxidation of methionine.

Immunohistochemistry

Three kidney tissue sections from each group were used to detect theexpression level of prohibitin by immunohistochemical staining. Paraffin-embedded tissue sections were first dewaxed and rehydrated. Microwaveantigen retrieval was done in the presence of citric acid buffer (pH 6.0). Sampleswere blocked with horse serum for 20 min before incubation with monoclonalmouse anti-prohibitin antibody (1:100; Lab Vision Corporation, Fremont, CA,USA) at 37�C for 1 h followed by incubation at 4�C overnight. Tissue sections werethen incubated with biotin-labeled secondary antibody (Kangchen, Shanghai,China) or phosphate buffered saline for the negative control at 37�C for 1 h,treated with diaminobenzidine for chromogenesis, and the nuclei counterstainedby hematoxylin.

Western blot

Two samples of renal tissue protein were collected from each group forwestern blot analysis. Total protein of 30 mg was added according to conventionalmethods and the samples were separated using 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The proteins were transferred onto the pol-yvinylidene fluoride membrane using the semidry electrophoretic transfermethod. The samples were blocked with 5% skim milk powder at roomtemperature for 1 h and the monoclonal mouse anti-prohibitin antibody (1:500;Lab Vision Corporation) was added and the samples were incubated at 4�Covernight. The samples were incubated with horseradish peroxidase–labeledsecondary antibody (1:2000; Abcam, Cambridge, UK) at room temperature for1 h. Protein was visualized by enhanced chemiluminescence and b-actin(Kangchen) was detected as the internal reference.

Statistical analysis

Quantitative data were presented as mean � standard deviation. Compari-sons among multiple groups used one-way analysis of variance, and thecomparisons between groups were made with the least significant differencetest. P < 0.05 was considered statistically significant.

Results

Comparisons of examination indexes among rat groups

Comparison of kidney weight among rat groupsAt 12 wk of age, the kidney weight of rats in the IUGR group

was lower than that in the control group (P < 0.05), but the ratioof kidney weight to body weight was higher than that in thecontrol group (P < 0.05). Kidney enlargement in IUGR rats feda postnatal high-protein diet (HP group) was more obvious,

Table 3Comparison of blood pressure

Blood pressure (mm Hg) Control group IUGR group HP group

4 wk of age 99.6 � 2.5 104.1 � 3.0 104.6 � 1.78 wk of age 109.4 � 3.1 125.2 � 2.3* 127.3 � 1.2*

12 wk of age 115.2 � 2.3 132.1 � 2.9* 138.6 � 2.8y,z

HP, high-protein; IUGR, intrauterine growth restrictionData are presented as mean � SD; n ¼ 8 per group.

* P < 0.01.y P < 0.001 compared with the control group.z P < 0.01 compared with the IUGR group.

Q. Shen et al. / Nutrition 27 (2011) 364–371366

and the kidney weight reached the level of the control group(P > 0.05). The ratio of kidney weight to body weight in theHP group was significantly higher than that in the control group(P < 0.001) and IUGR group (P < 0.001) as presented in Table 2.

Comparison of blood pressureCompared with the control group, the blood pressure of

rats in the IUGR group was significantly increased at 8 wk of age(P < 0.01), and this increasing trend was more obvious at 12 wkof age (P< 0.01). Compared with the control group, rats in the HPgroup also exhibited an increase in blood pressure at 8 wk of age(P < 0.01), and the severity of the blood pressure increase waseven greater at 12 wk of age (P < 0.001). At 12 wk of age, theblood pressure increase in the HP group was significantly higherthan that in the IUGR group (P < 0.01; Table 3).

Comparison of 24-h urine proteinCompared with control group, rats in the IUGR group showed

significantly increased 24-h urine protein at 12 wk of age (P <

0.05), and the increase in proteinuria was greater in the HP groupthan in the IUGR group (P < 0.001; Table 4).

Comparison of glomerular numberThe numbers of glomeruli in the IUGR and HP groups

(number per unilateral kidney) were significantly lower thanthose in the control group (IUGR group 22 900 � 926 versuscontrol group 28 861 � 1044, P < 0.01; HP group 23 043 � 595versus control group, P < 0.01). There was no significant differ-ence between the IUGR and HP groups (P > 0.05).

Comparison of renal tissue morphologyHematoxylin and eosin staining showed that the glomerular

volumes of rats in the IUGR and HP groups were significantlyincreased at 12 wk of age compared with that in the controlgroup (IUGR group 1.092� 0.083� 105 mm3 versus control group0.830� 0.044� 105 mm3, P < 0.01; HP group 1.508� 0.097� 105

mm3 versus control group, P < 0.001). The glomerular volume ofrats in the HP group was significantly larger than that in the IUGRgroup (P < 0.001). Electron microscopy results showed observ-able mild foot process fusion in the IUGR group, whereas rats inthe HP group showed obvious mesangial cell proliferation andpartial fusion of the foot.

Differences in kidney protein expression profiles between IUGRand normal rats

Results of 2-DE and mass spectrometry identificationTotal proteins were extracted from adult rat kidneys from six

rats in the IUGR and control groups, respectively, and then sub-jected to 2-DE three times to show reproducibility. Three sets of

Table 2Comparison of kidney weight at 12 wk of age

Control group IUGR group HP group

Body weight (g) 394.8 � 11.3 300.0 � 26.9y 296.3 � 26.5y

Kidney weight (g) 1.320 � 0.061 1.134 � 0.106* 1.316 � 0.204z

Kidney weight/body weight (%)

0.334 � 0.007 0.378 � 0.019* 0.443 � 0.043y,x


* P < 0.05.y P < 0.001 compared with control group.z P < 0.05.x P < 0.001 compared with IUGR group.

2-DE profiles were obtained (Fig. 1). With the application ofPdquest 7.3.0 for image analysis, these results showed that 727�58 protein spots were obtained in the IUGR group, with anaverage match rate of 85%. In the control group, 758� 53 proteinspots were obtained, with an average match rate of 78%. Thedifferential expression analysis found that one protein spot wasexpressed only in the IUGR group (no. 1). Seven protein spotswere upregulated more than five-fold (nos. 2w8) and four spotsdownregulated more than five-fold (nos. 9w12) in the IUGRgroup compared with those in the control group. These 12protein spots were picked for mass spectrometric analysis and allhad successful protein identifications (Table 5).

Retrieval and classification of differentially expressed proteinsThe Gene Ontology classification method was used to carry

out functional retrieval for the 12 differentially expressedproteins in three areas, i.e., biological process, cellular compo-nent, and molecular function (Table 6). These proteins wereinvolved primarily in oxidation reduction, body metabolism, andtranscriptional regulation.

Effects of postnatal high-protein diet on renal protein expressionprofiles in IUGR rats

Results of 2-DE and mass spectrometric identificationTotal proteins were extracted from adult rat kidneys from six

rats in the HP and control groups, respectively, and then sub-jected to 2-DE three times to show reproducibility. Three sets of2-DE profiles were obtained (Fig. 1). With the application ofPdquest 7.3.0 for image analysis, these results showed that 740�43 protein spots were obtained in the HP group, with an averagematch rate of 84%. In the control group, 758 � 53 protein spotswere obtained, with an average match rate of 78%. The differ-ential expression analysis found that five protein spots wereexpressed only in the control group (nos. 1w5). Five proteinspots were upregulated more than five-fold (nos. 6w10) andthree spots downregulated more than five-fold (nos. 11w13) inthe HP group compared with those in the control group. These 13

Table 4Comparison of 24-h urine protein

Proteinuria(mg. kg�1. d�1)


4 wk of age 26.28 � 9.78 26.92 � 5.75 25.62 � 7.188 wk of age 109.12 � 19.7 89.55 � 13.23 130.9 � 36.0512 wk of age 69.72 � 10.35 118.46 � 21.85* 202.61 � 62.55y,z


* P < 0.05.y P < 0.001 compared with the control group.z P < 0.001 compared with the IUGR group.

pH3 pH10

pH3 pH10

pH3 pH10

A

B

C

Fig. 1. Images of two-dimensional gel electrophoretic profiles from the (A) control,(B) intrauterine growth restriction, and (C) high-protein groups.


protein spots were picked for mass spectrometric analysis and allhad successful protein identifications (Table 7).

Retrieval and classification of differentially expressed proteinsThe Gene Ontology classification method was used to carry

out the functional retrieval for the 13 differentially expressed

proteins (Table 8). These proteins were involved primarily inbody metabolism, oxidation reduction, and apoptosis regulation.

Effects of different postnatal protein diets on kidney proteinexpression profiles in IUGR rats

Compared with the control group, six proteins showed thesame trend of expression changes in the IUGR and HP groups.These six proteins included glutathione-S-transferase a1,fructose-bisphosphate aldolase A, long-chain specific acyl-coenzyme A dehydrogenase, hydroxyacid oxidase-2, retinaldehydrogenase-1, and transketolase. Two proteins, cappingprotein and prohibitin, had consecutive changes among threegroups (ratio of control group to IUGR group >5 and no obviousexpression in the HP group). Other proteins showed differentialexpression only after high-protein nutritional intervention.These included chloride intracellular channel-1, ganglioside-2(GM2) ganglioside activator protein, aspartoacylase-2, isocitratedehydrogenase, and disulfide-isomerase A3.

Confirmation of prohibitin expression by immunohistochemistryand western blot

Immunohistochemistry results indicated that prohibitin wasprimarily expressed in renal tubular epithelial cells. Staining inthe IUGR group was significantly weaker than in the controlgroup and almost no staining was observed in the HP group(Fig. 2). Western blot showed that the expression of prohibitin inthe IUGR group was weaker than in the control group and theexpression in the HP group was even weaker (Fig. 3).

Discussion

Intrauterine growth restriction can cause long-term or life-long effects on the functions of various organs in the body byfetal programming. Studies have found that IUGR can causemetabolic disorders and imbalances of the hormone levels thataffect the development and function of multiple organs in thebody. IUGR has been associated with many adulthood diseasesincluding hypertension, coronary heart disease, diabetes, andchronic kidney disease [11,12].

Previous theories have suggested that nutritional supple-ments, especially a high-protein diet for newborns affected withIUGR, may promote rapid postnatal physical growth. However,a theory of predictive adaptive response has been recently beensuggested [13]. This theory postulates that the environment offetal development predicts the postnatal environment and cau-ses a series of adaptive changes. If the predictive adaptiveresponse is correct, the phenotype will be normal. If the pre-dicted environment is different from the actual environment,diseases will easily occur. One study found that the richer thediet of young rats undergoing intrauterine malnutrition, theshorter their life span [14]. Studies on the reproductive endo-crine function of IUGR rats also found that a postnatal high-fatdiet aggravated insulin resistance and reproductive endocrinedisorders of IUGR rats [15]. At the same time, a high-protein dietitself can damage the permeability of the glomerular basementmembrane and aggravate glomerular hyperfiltration andhyperperfusion phenomenon. Glomerular basement membranedamage can further aggravate proteinuria and stimulatemesangial cell proliferation. All of these factors may furtherworsen kidney damage. Therefore, this study was designed toobserve the effects of a postnatal high-protein nutritionalintervention on kidney development and function in IUGR rats.The results showed that a postnatal high-protein nutritionalintervention not only could not correct the decrease the number

Table 5Differentially expressed proteins between intrauterine growth-restricted rats and normal rats

No. Name Molecularweight

Isoelectric point Score Sequencecoverage (%)

1 phosphoglycerate kinase-1 44 394 7.52 121 292 ribonuclease UK114 14 475 6.21 250 623 catalase 59 588 7.15 173 394 liver carboxylesterase-4 62 234 6.29 82 455 hydroxyacid oxidase-2 39 045 7.90 325 636 transketolase 71 113 7.54 383 607 retinal dehydrogenase-1 54 292 7.90 195 328 glutathione S-transferase a1 25 459 8.87 205 339 Prohibitin 29 801 5.57 169 3710 long-chain specific acyl-coenzyme A dehydrogenase 47 842 7.63 92 2711 fructose-bisphosphate aldolase A 39 196 8.39 85 3112 capping protein (actin filament) muscle Z-line, a2 32 946 5.57 164 21


of nephrons but also worsened the severity of hypertension andproteinuria. We believe that in the current society with abundantmaterials, postnatal nutrient intake is usually adequate andexcessive, thus the control of high-protein intake is of certainsignificance in patients with IUGR.

Recent animal studies have shown that the expression of theproapoptotic gene Bax is significantly increased in IUGR animalkidneys, whereas the antiapoptotic gene Bcl-2 is clearlydecreased. In addition, the expression of proteins associated withglomerular sclerosis and tubulointerstitial damage such asfibronectin, angiotensin receptor, and sodium channel are alsoincreased in the IUGR animal [16–20]. However, the mechanismsof IUGR-induced postnatal kidney disease and the aggravation of

Table 6Function of differentially expressed proteins between intrauterine growth-restricted

No. Name Biological process

1 phosphoglycerate kinase-1 glycolysis; phosphorylation

2 ribonuclease UK114 d

3 catalase cell proliferation; hydrogenperoxide catabolic process;oxidation reduction

4 liver carboxylesterase-4 d

5 hydroxyacid oxidase-2 oxidation reduction

6 transketolase ribose phosphate biosyntheticprocess; pentose-phosphateshunt

7 retinal dehydrogenase-1 oxidation reduction

8 glutathione S-transferase a1 metabolic process9 prohibitin DNA replication

10 long-chain specific acyl-coenzyme A dehydrogenase

fatty acid metabolic process;oxidation reduction

11 fructose-bisphosphate aldolaseA

glycolysis

12 capping protein (actin filament)muscle Z-line, a2

actin cytoskeleton organization

FAD, flavin adenine dinucleotide

kidney disease induced by a high-protein diet intervention havenot been fully clarified at the present time. Studies on the effectsof IUGR on kidney development and the mechanisms ofIUGR-induced postnatal kidney disease may lead to a betterunderstanding of the pathogenesis of kidney diseases of fetalorigin and the discovery of therapeutic targets.

In recent years, proteomics has been widely applied in basicmedical research. It has been used to study systemic and quan-titative proteomic changes in tissues and cells at different diseaseprogression stages. In the field of kidney research, proteomicapproaches have been used to compare the differences in proteinexpression profiles between the renal cortex and medulla [21]. Atthe same time, this technology has been applied to pathogenic

rats and control rats

Cellular component Molecular function

cytosol; soluble fraction adenosine triphosphatebinding; phosphoglyceratekinase activity

mitochondrion; nucleus endonuclease activityGolgi apparatus; cytosol;

endoplasmic reticulum;lysosome; mitochondrialintermembrane space;plasma membrane

catalase activity; growth factoractivity; heme binding; ironion binding

endoplasmic reticulum;endoplasmic reticulumlumen

carboxylesterase activity

peroxisome hydroxyacid oxidase activity;flavin mononucleotidebinding; electron carrieractivity

endoplasmic reticulummembrane; microsome;peroxisome; soluble fraction

calcium ion binding;magnesium binding;monosaccharide binding;thiamin pyrophosphatebinding; transketolaseactivity

cytoplasm retinal dehydrogenase activity;3-chloroallyl aldehydedehydrogenase activity

cytoplasm glutathione transferase activitymitochondrial inner membrane protein binding; negative

regulation of apoptosismitochondrial matrix FAD binding; electron carrier

activity; long-chain acyl-coenzyme A dehydrogenaseactivity

mitochondrion fructose-bisphosphate aldolaseactivity

F-actin capping proteincomplex

actin binding

Table 7Differentially expressed proteins between rats on a high-protein diet and control rats

No. Name Molecular weight Isoelectric point Score Sequence coverage (%)

1 prohibitin 29 801 5.57 169 372 disulfide-isomerase A3 57 043 5.88 381 633 retinal dehydrogenase-1 54 292 7.90 218 394 isocitrate dehydrogenase 60 934 8.88 65 455 capping protein (actin filament) muscle Z-line, a2 32 946 5.57 66 296 hydroxyacid oxidase-2 39 045 7.90 203 607 transketolase 71 113 7.54 210 628 aspartoacylase-2 35 419 5.42 149 489 glutathione S-transferase a1 25 459 8.87 205 3310 chloride intracellular channel-1 26 963 5.09 140 3211 GM2 ganglioside activator protein 21 479 6.13 169 3912 long-chain specific acyl-coenzyme A dehydrogenase 47 842 7.63 101 2113 fructose-bisphosphate aldolase A 39 196 8.39 85 31

GM2, ganglioside-2


studies in diabetic nephropathy, focal segmental glomerularsclerosis, lupus nephritis, tumor, and acute rejection [22–24].However, due to limitations in sample quantity in animal andclinical studies, ‘‘sample pooling’’ by mixing 3 to 10 samplestogether with the same background before performing the 2-DEanalysis has become a popular technique [25–27]. These resultssuggest that analysis on mixed samples not only helps to decreasethe required number of repeat runs in 2-DE but also decreases thestandard deviation obtained from individual samples.

We applied the sample-pooling comparative proteomics methodto study the differences in kidney protein expression profiles

Table 8Function of differentially expressed proteins between rats consuming a high-protein

No. Name Biological process

1 prohibitin DNA replication

2 disulfide-isomerase A3 cell redox homeostasis

3 retinal dehydrogenase-1 oxidation reduction

4 isocitrate dehydrogenase glyoxylate cycle; isocitratemetabolic process; oxidationreduction; tricarboxylic acidcycle

5 capping protein (actin filament)muscle Z-line, a2

actin cytoskeleton organization

6 hydroxyacid oxidase-2 oxidation reduction

7 transketolase ribose phosphate biosyntheticprocess; pentose-phosphateshunt

8 aspartoacylase-2 metabolic process9 glutathione S-transferase a1 metabolic process10 chloride intracellular channel-1 chloride transport; apoptosis

regulation

11 GM2 ganglioside activator protein ganglioside catabolic process;learning or memory; lipidstorage; neuromuscular processcontrolling balance;oligosaccharide catabolic process

12 long-chain specific acyl-coenzymeA dehydrogenase

fatty acid metabolic process;oxidation reduction

13 fructose-bisphosphate aldolase A glycolysis

GM2, ganglioside-2; FAD, flavin adenine dinucleotide

between adult IUGR rats and normal rats. Our results revealed thata total of 12 proteins showed significant differences in expressionprofile. Functional classification of these differential proteinsshowed that they were involved primarily in biological processessuch as oxidation reduction, body metabolism, and transcriptionalregulation. We suggest that the differentially expressed proteinsparticipate in postnatal kidney disease in IUGR rats.

Compared with the control group, two proteins, cappingprotein and prohibitin, showed consecutive changes among thethree groups. Some other proteins showed differential expres-sions only after the high-protein nutritional intervention,

diet and control rats

Cellular component Molecular function

mitochondrial inner membrane protein binding; negativeregulation of apoptosis

endoplasmic reticulum;endoplasmic reticulum lumen;melanosome

protein disulfide isomerase activity

cytoplasm retinal dehydrogenase activity;3-chloroallyl aldehydedehydrogenase activity

mitochondrion isocitrate dehydrogenase activity;magnesium ion binding;manganese ion binding

F-actin capping protein complex actin binding

peroxisome hydroxyacid oxidase activity; flavinmononucleotide binding;electron carrier activity

endoplasmic reticulum membrane;microsome; peroxisome; solublefraction

calcium ion binding; magnesiumbinding; monosaccharidebinding; thiamin pyrophosphatebinding; transketolase activity

d hydrolase activitycytoplasm glutathione transferase activitycytoplasm; membrane; membrane

fraction; nuclear envelope;soluble fraction

voltage-gated channel activity

mitochondrion b-N-acetylhexosaminidase activity;enzyme activator activity

mitochondrial matrix FAD binding; electron carrieractivity; long-chain acyl-coenzyme A dehydrogenaseactivity

mitochondrion fructose-bisphosphate aldolaseactivity

A

B

C

Fig. 2. Immunohistochemistry analysis of prohibitin expression in kidneys from the(A) control, (B) intrauterine growth restriction, and (C) high-protein groups (dia-minobenzidine chromogenesis, magnification 400�). Prohibitin was primarilyexpressed in renal tubular epithelial cells. Staining in the intrauterine growthrestriction group was significantly weaker than in the control group and almost nostaining was observed in the high-protein group.


Prohibitin

β-actin

30kDa

43kDa

Fig. 3. Western blot analysis of prohibitin expression in the kidney showed thatprohibitin in the IUGR group was weaker than the control group and the expressionin the HP group was even weaker. HP, high-protein; IUGR, intrauterine growthrestriction.


including chloride intracellular channel 1 that participates inapoptosis regulation, GM2 ganglioside activator protein andaspartoacylase-2 that participate in body metabolism, and iso-citrate dehydrogenase and disulfide-isomerase A3 that

participate in oxidation reduction. We suggest that the differ-ential expression proteins are related to the effects of a high-protein diet intervention on the kidneys of IUGR rats.

Among the identified proteins with obviously differentialexpressions, prohibitin, the antiproliferative protein, showedconsecutive changes among the three groups (ratio of controlgroup to IUGR group >5 and no obvious expression in the HPgroup). Previous studies have indicated that prohibitin hasantiproliferation and antitumor functions, and that it plays animportant role in cell metabolism, growth, aging, and apoptosis[28]. Our previous study on prohibitin suggested that theexpression level of prohibitin in renal tissue could reflect thedegree of tubulointerstitial injury, and exogenous prohibitincould significantly inhibit the transforming growth factor-b–induced fibroblast proliferation and phenotypic changes [29].We further validated the differential expression of prohibitin inrenal tissue using western blot and immunohistochemistry, andthe results showed that prohibitin was mainly expressed in renaltubules and its expression level was lower in the IUGR groupthan in the control group and was further decreased by thepostnatal high-protein diet. These results were consistent withthe results of proteomics research, which further confirms theaccuracy of the results of mass spectrometry. We suggest that in-depth studies on these differentially expressed proteinsincluding protein expression, action mechanism, and proteininteraction are needed to uncover the pathogenic mechanisms ofpostnatal proteinuria and hypertension in patients with IUGRand the effects of a high-protein diet intervention on the kidney.

Conclusion

A high-protein diet intervention after birth cannot correct thedecrease in the number of nephrons resulting from IUGR, andinstead worsens the severity of hypertension and proteinuria.The comparative proteomic approach has provided new avenuesfor future research to explore the pathogenesis of IUGR-inducedkidney injury and the effects of a high-protein diet interventionon the kidney.

Acknowledgments

The authors are grateful to Xiu-Rong Zhang and Zhong-HuaZhao for technical assistance.

References

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intrauterine growth restriction and postnatal high-protein diet affect the kidneys in adult rats

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