- Email: [email protected]
Intrauterine growth restriction and postnatal high-protein diet affect the kidneys in adult rats
Nutrition 27 (2011) 364–371
Contents lists available at ScienceDirect
Nutrition journal homepage: www.nutritionjrnl.com
Basic nutritional investigation
Intrauterine growth restriction and postnatal high-protein diet affect the 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 b
Department of Nephrology and Rheumatology, Children’s Hospital of Fudan University, Shanghai, People’s Republic of China Institutes for Biomedical Sciences, Fudan University, Shanghai, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2009 Accepted 16 March 2010
Objective: Intrauterine growth restriction (IUGR) is associated with hypertension and chronic kidney disease in adulthood. Postnatal overnutrition after IUGR may be of pathogenic importance for the development of diabetes and cardiovascular disease. This study was to identify the effects of IUGR and a postnatal high-protein diet on the kidneys in adult rats. Methods: Intrauterine growth restriction was induced in Sprague-Dawley rats by isocaloric protein restriction in pregnant dams. IUGR pups were divided into two groups that were a standardprotein diet (IUGR group) or a high-protein diet (HP group). A comparative proteomic method was used to study the differences of protein expression profiles between normal adult rats and adult rats with IUGR and the effects of a postnatal high-protein diet on the protein expression profiles of the kidneys. Results: The IUGR adults had higher urinary excretion of protein and blood pressure than controls and the HP diet caused more severe hypertension and proteinuria than IUGR itself. The differential proteomic expression analysis found 12 proteins that had significantly differential expression between the IUGR and control groups, which were transcription regulators and structural molecules. The differential proteomic expression analysis between the HP and control groups found 13 proteins that had significantly differential expression and were involved primarily in body metabolism, oxidation reduction, and apoptosis regulation. Conclusion: An HP diet intervention after IUGR worsens the severity of hypertension and proteinuria, and this study may provide valuable experimental evidence of proteins involved in the pathogenesis of kidney disease in IUGR and the effect of postnatal overnutrition. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: Intrauterine growth restriction Kidney Proteomics Nutritional intervention Proteinuria Hypertension
Introduction Intrauterine growth restriction (IUGR) has long-term effects on various organisms through fetal programming [1,2]. Human studies of the association between IUGR and renal diseases have indicated that, in contrast to the normal fetal kidney, the IUGR fetal kidney has a smaller volume with a significant decrease in glomerular number [3,4]. In addition, long-term follow-up after birth has shown a significantly lower glomerular filtration function and a higher incidence of proteinuria in the IUGR group compared with the control group [5,6]. Our previous animal studies have also shown a decreased glomerular number in IUGR
This research was funded by grant 30672242 from the National Natural Science Foundation of China. * Corresponding author. Tel: þ86-21-6493-1006; fax: þ86-21-6493-1901. E-mail address: [email protected] (H. Xu). 0899-9007/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.03.003
rats, with an increased incidence of proteinuria and hypertension in the postnatal follow-up period [7]. However, the pathogenesis of IUGR-induced postnatal kidney diseases has not been fully clarified. Previous theories have suggested that nutritional supplements, especially a high-protein diet for infants in whom IUGR is diagnosed, at an early postnatal stage may assist rapid postnatal growth, but the high-protein diet may further exacerbate kidney injury and lead to deterioration of renal function. Currently, there is no definite conclusion on the proper postnatal nutritional intervention for fetal IUGR. This study was designed to observe the effects of a postnatal high-protein nutritional intervention on kidney development and function in IUGR rats. Moreover, a comparative proteomic method was used to study the differences of protein expression profiles between normal adult rats and adult rats with IUGR and the effects of a postnatal high-protein diet on the protein
Q. Shen et al. / Nutrition 27 (2011) 364–371
expression profiles of kidney to explore the possible pathogenesis of IUGR-induced kidney injury and the effects of a highprotein diet intervention on the kidney. Materials and methods This work was performed with the approval of Children’s Hospital of Fudan University’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 male rats. The normal control group was fed with a conventional pregnancy diet (22% protein) until natural delivery, and the newborn rats were fed with a conventional diet (22% protein, control group) until 12 wk after birth. The study group was fed with a low-protein isocaloric diet consisting of 6% protein throughout the entire pregnancy until natural labor [8]. Newborn rats with a birth weight 2 standard deviations lower than the average birth weight of the normal newborn rats 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 in Table 1. Measurement of urine protein and blood pressure Eight male rats were selected from each group at 4, 8, and 12 wk, respectively. The rats were weighed and 24-h urine was collected and the volume was recorded. Urine protein content was determined using colorimetry. Systolic blood pressure was measured using a tail artery measuring instrument. The kidney weight and body weight of each group of rats were measured at 12 wk of age. Determination of glomerular number and kidney morphology The Sprague-Dawley rats were sacrificed at 12 wk of age by jugular puncture after anesthesia. The kidneys of eight rats in each group were collected at 12 wk of age and were conventionally fixed, embedded, sliced, and stained with hematoxylin and eosin. The glomerular number was counted and the glomerular volume was measured. The methods for measuring the glomerular number and glomerular volume have been previously reported [9,10].
365
In-gel digestion and mass spectrometry Protein spots with significant differences were excised from the gels and placed into a 96-well microtiter plate. Gel pieces were detained with a solution of 15 mM of potassium ferricyanide and 50 mM of sodium thiosulfate (1:1) for 20 min at room temperature. Then they were washed twice with deionized water and shrunk by dehydration in acetone cyanohydrin. The samples were then swollen in a digestion buffer containing 20 mM of ammonium bicarbonate and 12.5 ng/mL of trypsin (Roche, Indianapolis, IN, USA) at 4 C. After a 30-min incubation, the gels were digested longer than 12 h at 37 C. Peptides were then extracted twice using 0.1% trifluoroacetic acid in 50% acetone cyanohydrin. The extracts were dried under the protection of N2. For matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, 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% acetone cyanohydrin). Then the solution was spotted on a stainless-steel target with 192 wells (Applied Biosystems, Framingham, MA, USA). Samples were allowed to airdry before inserting them into the mass spectrometer. The matrix-assisted laser desorption ionization mass spectrometer was an ABI 4700 TOF-TOF Proteomics Analyzer (Applied Biosystems) instrument. The ultraviolet laser was operated at a 200-Hz repetition rate with wavelength of 355 nm. The accelerated voltage was operated at 20 kV. The data were searched by GPS Explorer (Applied Biosystems) using MASCOT (Matrix Science, London, UK) as a search engine. Data from matrix-assisted laser desorption ionization/time-of-flight mass spectrometry were analyzed using MASCOT search software. The following parameters were used in the search: retrieval species for rat, enzyme for trypsin, missed cleavage by 1, peptide tolerance of 0.2, tandem mass spectrometry (MS/MS) tolerance of 0.6 Da, and possible oxidation of methionine. Immunohistochemistry Three kidney tissue sections from each group were used to detect the expression level of prohibitin by immunohistochemical staining. Paraffinembedded tissue sections were first dewaxed and rehydrated. Microwave antigen retrieval was done in the presence of citric acid buffer (pH 6.0). Samples were blocked with horse serum for 20 min before incubation with monoclonal mouse 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 were then 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 counterstained by hematoxylin.
Two-dimensional gel electrophoresis and image analysis
Western blot
At 12 wk of age, six kidneys were selected from each group and were mixed for total protein extraction. Tissues were homogenized in a lysis buffer, spun down to collect the supernatant, and subjected to the Bradford assay to determine protein concentration. Two-dimensional gel electrophoresis (2-DE) was performed according to the manufacturer’s instruction (Amersham Biosciences, Buckinghamshire, UK). An Immobiline pH gradient DryStrip gel (18 cm, non-linear pH 3w10; Amersham Biosciences) was rehydrated with re-swelling buffer containing a 1-mg protein sample at 20 C for 12 h, focused by gradient increasing voltage to 8000 V, and continued until total voltage-hour reached 52 000 Vh. After focusing, the strip was first equilibrated in the equilibration solution containing 1% dithiothreitol and 2.5% iodine acetamide for 15 min, transferred to the top of a 12.5% polyacrylamide gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The electrophoresis was performed at 25 C until bromophenol blue reached the bottom of the gel. The gel was stained by Coomassie brilliant blue and then scanned for image analysis. Each 2-DE experiment was performed in triplicate to confirm the reproducibility. The scanned gel images were then analyzed by Pdquest 7.3.0 (Bio-Rad, Hercules, CA, USA).
Two samples of renal tissue protein were collected from each group for western blot analysis. Total protein of 30 mg was added according to conventional methods and the samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred onto the polyvinylidene fluoride membrane using the semidry electrophoretic transfer method. The samples were blocked with 5% skim milk powder at room temperature for 1 h and the monoclonal mouse anti-prohibitin antibody (1:500; Lab Vision Corporation) was added and the samples were incubated at 4 C overnight. The samples were incubated with horseradish peroxidase–labeled secondary antibody (1:2000; Abcam, Cambridge, UK) at room temperature for 1 h. Protein was visualized by enhanced chemiluminescence and b-actin (Kangchen) was detected as the internal reference.
Table 1 Diet make-up
Results
Conventional diet Low-protein diet High-protein diet
Ingredients
Protein (%)
corn 26%, wheat 34%, alfalfa meal 2%, soybean meal 27%, fish meal 5%, vegetable oil 1%, premix 5% corn 50%, sucrose 13%, amylum 26.5%, vegetable oil 2.5%, fish meal 3%, premix 5% corn 15%, amylum 21%, soybean meal 51%, vegetable oil 1%, fish meal 2.5%, alfalfa meal 2%, casein 2.5%, premix 5%
22
6 30
Statistical analysis Quantitative data were presented as mean standard deviation. Comparisons among multiple groups used one-way analysis of variance, and the comparisons between groups were made with the least significant difference test. P < 0.05 was considered statistically significant.
Comparisons of examination indexes among rat groups Comparison of kidney weight among rat groups At 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 ratio of kidney weight to body weight was higher than that in the control group (P < 0.05). Kidney enlargement in IUGR rats fed a postnatal high-protein diet (HP group) was more obvious,
366
Q. Shen et al. / Nutrition 27 (2011) 364–371
and the kidney weight reached the level of the control group (P > 0.05). The ratio of kidney weight to body weight in the HP 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 pressure Compared 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 wk of age (P < 0.01). Compared with the control group, rats in the HP group also exhibited an increase in blood pressure at 8 wk of age (P < 0.01), and the severity of the blood pressure increase was even greater at 12 wk of age (P < 0.001). At 12 wk of age, the blood pressure increase in the HP group was significantly higher than that in the IUGR group (P < 0.01; Table 3). Comparison of 24-h urine protein Compared 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 group than in the IUGR group (P < 0.001; Table 4). Comparison of glomerular number The numbers of glomeruli in the IUGR and HP groups (number per unilateral kidney) were significantly lower than those in the control group (IUGR group 22 900 926 versus control group 28 861 1044, P < 0.01; HP group 23 043 595 versus control group, P < 0.01). There was no significant difference between the IUGR and HP groups (P > 0.05). Comparison of renal tissue morphology Hematoxylin and eosin staining showed that the glomerular volumes of rats in the IUGR and HP groups were significantly increased at 12 wk of age compared with that in the control group (IUGR group 1.092 0.083 105 mm3 versus control group 0.830 0.044 105 mm3, P < 0.01; HP group 1.508 0.097 105 mm3 versus control group, P < 0.001). The glomerular volume of rats in the HP group was significantly larger than that in the IUGR group (P < 0.001). Electron microscopy results showed observable mild foot process fusion in the IUGR group, whereas rats in the HP group showed obvious mesangial cell proliferation and partial fusion of the foot. Differences in kidney protein expression profiles between IUGR and normal rats Results of 2-DE and mass spectrometry identification Total proteins were extracted from adult rat kidneys from six rats in the IUGR and control groups, respectively, and then subjected to 2-DE three times to show reproducibility. Three sets of
Table 2 Comparison of kidney weight at 12 wk of age
Body weight (g) Kidney weight (g) Kidney weight/ body weight (%)
Control group
IUGR group
HP group
394.8 11.3 1.320 0.061 0.334 0.007
300.0 26.9y 1.134 0.106* 0.378 0.019*
296.3 26.5y 1.316 0.204z 0.443 0.043y,x
HP, high-protein; IUGR, intrauterine growth restriction Data are presented as mean SD; n ¼ 8 per group. * P < 0.05. y P < 0.001 compared with control group. z P < 0.05. x P < 0.001 compared with IUGR group.
Table 3 Comparison of blood pressure Blood pressure (mm Hg)
Control group
IUGR group
HP group
4 wk of age 8 wk of age 12 wk of age
99.6 2.5 109.4 3.1 115.2 2.3
104.1 3.0 125.2 2.3* 132.1 2.9*
104.6 1.7 127.3 1.2* 138.6 2.8y,z
HP, high-protein; IUGR, intrauterine growth restriction Data 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.
2-DE profiles were obtained (Fig. 1). With the application of Pdquest 7.3.0 for image analysis, these results showed that 727 58 protein spots were obtained in the IUGR group, with an average match rate of 85%. In the control group, 758 53 protein spots were obtained, with an average match rate of 78%. The differential expression analysis found that one protein spot was expressed only in the IUGR group (no. 1). Seven protein spots were upregulated more than five-fold (nos. 2w8) and four spots downregulated more than five-fold (nos. 9w12) in the IUGR group compared with those in the control group. These 12 protein spots were picked for mass spectrometric analysis and all had successful protein identifications (Table 5). Retrieval and classification of differentially expressed proteins The Gene Ontology classification method was used to carry out functional retrieval for the 12 differentially expressed proteins in three areas, i.e., biological process, cellular component, and molecular function (Table 6). These proteins were involved primarily in oxidation reduction, body metabolism, and transcriptional regulation.
Effects of postnatal high-protein diet on renal protein expression profiles in IUGR rats Results of 2-DE and mass spectrometric identification Total proteins were extracted from adult rat kidneys from six rats in the HP and control groups, respectively, and then subjected to 2-DE three times to show reproducibility. Three sets of 2-DE profiles were obtained (Fig. 1). With the application of Pdquest 7.3.0 for image analysis, these results showed that 740 43 protein spots were obtained in the HP group, with an average match rate of 84%. In the control group, 758 53 protein spots were obtained, with an average match rate of 78%. The differential expression analysis found that five protein spots were expressed only in the control group (nos. 1w5). Five protein spots were upregulated more than five-fold (nos. 6w10) and three spots downregulated more than five-fold (nos. 11w13) in the HP group compared with those in the control group. These 13
Table 4 Comparison of 24-h urine protein Proteinuria (mg. kg1. d1)
Control group
IUGR group
HP group
4 wk of age 8 wk of age 12 wk of age
26.28 9.78 109.12 19.7 69.72 10.35
26.92 5.75 89.55 13.23 118.46 21.85*
25.62 7.18 130.9 36.05 202.61 62.55y,z
HP, high-protein; IUGR, intrauterine growth restriction Data are presented as mean SD; n ¼ 8 per group. * P < 0.05. y P < 0.001 compared with the control group. z P < 0.001 compared with the IUGR group.
Q. Shen et al. / Nutrition 27 (2011) 364–371
pH3
pH10
A
pH3
367
proteins (Table 8). These proteins were involved primarily in body metabolism, oxidation reduction, and apoptosis regulation. Effects of different postnatal protein diets on kidney protein expression profiles in IUGR rats Compared with the control group, six proteins showed the same 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 acylcoenzyme A dehydrogenase, hydroxyacid oxidase-2, retinal dehydrogenase-1, and transketolase. Two proteins, capping protein and prohibitin, had consecutive changes among three groups (ratio of control group to IUGR group >5 and no obvious expression in the HP group). Other proteins showed differential expression only after high-protein nutritional intervention. These included chloride intracellular channel-1, ganglioside-2 (GM2) ganglioside activator protein, aspartoacylase-2, isocitrate dehydrogenase, and disulfide-isomerase A3.
pH10
B
Confirmation of prohibitin expression by immunohistochemistry and western blot Immunohistochemistry results indicated that prohibitin was primarily expressed in renal tubular epithelial cells. Staining in the IUGR group was significantly weaker than in the control group and almost no staining was observed in the HP group (Fig. 2). Western blot showed that the expression of prohibitin in the IUGR group was weaker than in the control group and the expression in the HP group was even weaker (Fig. 3). Discussion
pH3
pH10
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 all had successful protein identifications (Table 7). Retrieval and classification of differentially expressed proteins The Gene Ontology classification method was used to carry out the functional retrieval for the 13 differentially expressed
Intrauterine growth restriction can cause long-term or lifelong effects on the functions of various organs in the body by fetal programming. Studies have found that IUGR can cause metabolic disorders and imbalances of the hormone levels that affect the development and function of multiple organs in the body. IUGR has been associated with many adulthood diseases including hypertension, coronary heart disease, diabetes, and chronic kidney disease [11,12]. Previous theories have suggested that nutritional supplements, especially a high-protein diet for newborns affected with IUGR, may promote rapid postnatal physical growth. However, a theory of predictive adaptive response has been recently been suggested [13]. This theory postulates that the environment of fetal development predicts the postnatal environment and causes a series of adaptive changes. If the predictive adaptive response is correct, the phenotype will be normal. If the predicted environment is different from the actual environment, diseases will easily occur. One study found that the richer the diet of young rats undergoing intrauterine malnutrition, the shorter their life span [14]. Studies on the reproductive endocrine function of IUGR rats also found that a postnatal high-fat diet aggravated insulin resistance and reproductive endocrine disorders of IUGR rats [15]. At the same time, a high-protein diet itself can damage the permeability of the glomerular basement membrane and aggravate glomerular hyperfiltration and hyperperfusion phenomenon. Glomerular basement membrane damage can further aggravate proteinuria and stimulate mesangial cell proliferation. All of these factors may further worsen kidney damage. Therefore, this study was designed to observe the effects of a postnatal high-protein nutritional intervention on kidney development and function in IUGR rats. The results showed that a postnatal high-protein nutritional intervention not only could not correct the decrease the number
368
Q. Shen et al. / Nutrition 27 (2011) 364–371
Table 5 Differentially expressed proteins between intrauterine growth-restricted rats and normal rats No.
Name
Molecular weight
Isoelectric point
Score
Sequence coverage (%)
1 2 3 4 5 6 7 8 9 10 11 12
phosphoglycerate kinase-1 ribonuclease UK114 catalase liver carboxylesterase-4 hydroxyacid oxidase-2 transketolase retinal dehydrogenase-1 glutathione S-transferase a1 Prohibitin long-chain specific acyl-coenzyme A dehydrogenase fructose-bisphosphate aldolase A capping protein (actin filament) muscle Z-line, a2
44 14 59 62 39 71 54 25 29 47 39 32
7.52 6.21 7.15 6.29 7.90 7.54 7.90 8.87 5.57 7.63 8.39 5.57
121 250 173 82 325 383 195 205 169 92 85 164
29 62 39 45 63 60 32 33 37 27 31 21
of nephrons but also worsened the severity of hypertension and proteinuria. We believe that in the current society with abundant materials, postnatal nutrient intake is usually adequate and excessive, thus the control of high-protein intake is of certain significance in patients with IUGR. Recent animal studies have shown that the expression of the proapoptotic gene Bax is significantly increased in IUGR animal kidneys, whereas the antiapoptotic gene Bcl-2 is clearly decreased. In addition, the expression of proteins associated with glomerular sclerosis and tubulointerstitial damage such as fibronectin, angiotensin receptor, and sodium channel are also increased in the IUGR animal [16–20]. However, the mechanisms of IUGR-induced postnatal kidney disease and the aggravation of
394 475 588 234 045 113 292 459 801 842 196 946
kidney disease induced by a high-protein diet intervention have not been fully clarified at the present time. Studies on the effects of IUGR on kidney development and the mechanisms of IUGR-induced postnatal kidney disease may lead to a better understanding of the pathogenesis of kidney diseases of fetal origin and the discovery of therapeutic targets. In recent years, proteomics has been widely applied in basic medical research. It has been used to study systemic and quantitative proteomic changes in tissues and cells at different disease progression stages. In the field of kidney research, proteomic approaches have been used to compare the differences in protein expression profiles between the renal cortex and medulla [21]. At the same time, this technology has been applied to pathogenic
Table 6 Function of differentially expressed proteins between intrauterine growth-restricted rats and control rats No.
Name
Biological process
Cellular component
Molecular function
1
phosphoglycerate kinase-1
glycolysis; phosphorylation
cytosol; soluble fraction
2 3
ribonuclease UK114 catalase
d cell proliferation; hydrogen peroxide catabolic process; oxidation reduction
4
liver carboxylesterase-4
d
5
hydroxyacid oxidase-2
oxidation reduction
mitochondrion; nucleus Golgi apparatus; cytosol; endoplasmic reticulum; lysosome; mitochondrial intermembrane space; plasma membrane endoplasmic reticulum; endoplasmic reticulum lumen peroxisome
adenosine triphosphate binding; phosphoglycerate kinase activity endonuclease activity catalase activity; growth factor activity; heme binding; iron ion binding
6
transketolase
ribose phosphate biosynthetic process; pentose-phosphate shunt
endoplasmic reticulum membrane; microsome; peroxisome; soluble fraction
7
retinal dehydrogenase-1
oxidation reduction
cytoplasm
8 9
glutathione S-transferase a1 prohibitin
metabolic process DNA replication
cytoplasm mitochondrial inner membrane
10
long-chain specific acylcoenzyme A dehydrogenase
fatty acid metabolic process; oxidation reduction
mitochondrial matrix
11
fructose-bisphosphate aldolase A capping protein (actin filament) muscle Z-line, a2
glycolysis
mitochondrion
actin cytoskeleton organization
F-actin capping protein complex
12
FAD, flavin adenine dinucleotide
carboxylesterase activity
hydroxyacid oxidase activity; flavin mononucleotide binding; electron carrier activity calcium ion binding; magnesium binding; monosaccharide binding; thiamin pyrophosphate binding; transketolase activity retinal dehydrogenase activity; 3-chloroallyl aldehyde dehydrogenase activity glutathione transferase activity protein binding; negative regulation of apoptosis FAD binding; electron carrier activity; long-chain acylcoenzyme A dehydrogenase activity fructose-bisphosphate aldolase activity actin binding
Q. Shen et al. / Nutrition 27 (2011) 364–371
369
Table 7 Differentially expressed proteins between rats on a high-protein diet and control rats No.
Name
Molecular weight
1 2 3 4 5 6 7 8 9 10 11 12 13
prohibitin disulfide-isomerase A3 retinal dehydrogenase-1 isocitrate dehydrogenase capping protein (actin filament) muscle Z-line, a2 hydroxyacid oxidase-2 transketolase aspartoacylase-2 glutathione S-transferase a1 chloride intracellular channel-1 GM2 ganglioside activator protein long-chain specific acyl-coenzyme A dehydrogenase fructose-bisphosphate aldolase A
29 57 54 60 32 39 71 35 25 26 21 47 39
Isoelectric point
801 043 292 934 946 045 113 419 459 963 479 842 196
5.57 5.88 7.90 8.88 5.57 7.90 7.54 5.42 8.87 5.09 6.13 7.63 8.39
Score
Sequence coverage (%)
169 381 218 65 66 203 210 149 205 140 169 101 85
37 63 39 45 29 60 62 48 33 32 39 21 31
GM2, ganglioside-2
studies in diabetic nephropathy, focal segmental glomerular sclerosis, lupus nephritis, tumor, and acute rejection [22–24]. However, due to limitations in sample quantity in animal and clinical studies, ‘‘sample pooling’’ by mixing 3 to 10 samples together with the same background before performing the 2-DE analysis has become a popular technique [25–27]. These results suggest that analysis on mixed samples not only helps to decrease the required number of repeat runs in 2-DE but also decreases the standard deviation obtained from individual samples. We applied the sample-pooling comparative proteomics method to study the differences in kidney protein expression profiles
between adult IUGR rats and normal rats. Our results revealed that a total of 12 proteins showed significant differences in expression profile. Functional classification of these differential proteins showed that they were involved primarily in biological processes such as oxidation reduction, body metabolism, and transcriptional regulation. We suggest that the differentially expressed proteins participate in postnatal kidney disease in IUGR rats. Compared with the control group, two proteins, capping protein and prohibitin, showed consecutive changes among the three groups. Some other proteins showed differential expressions only after the high-protein nutritional intervention,
Table 8 Function of differentially expressed proteins between rats consuming a high-protein diet and control rats No.
Name
Biological process
Cellular component
Molecular function
1
prohibitin
DNA replication
mitochondrial inner membrane
2
disulfide-isomerase A3
cell redox homeostasis
3
retinal dehydrogenase-1
oxidation reduction
endoplasmic reticulum; endoplasmic reticulum lumen; melanosome cytoplasm
protein binding; negative regulation of apoptosis protein disulfide isomerase activity
4
isocitrate dehydrogenase
mitochondrion
5
F-actin capping protein complex
actin binding
6
capping protein (actin filament) muscle Z-line, a2 hydroxyacid oxidase-2
glyoxylate cycle; isocitrate metabolic process; oxidation reduction; tricarboxylic acid cycle actin cytoskeleton organization oxidation reduction
peroxisome
7
transketolase
ribose phosphate biosynthetic process; pentose-phosphate shunt
endoplasmic reticulum membrane; microsome; peroxisome; soluble fraction
8 9 10
aspartoacylase-2 glutathione S-transferase a1 chloride intracellular channel-1
metabolic process metabolic process chloride transport; apoptosis regulation
11
GM2 ganglioside activator protein
d cytoplasm cytoplasm; membrane; membrane fraction; nuclear envelope; soluble fraction mitochondrion
hydroxyacid oxidase activity; flavin mononucleotide binding; electron carrier activity calcium ion binding; magnesium binding; monosaccharide binding; thiamin pyrophosphate binding; transketolase activity hydrolase activity glutathione transferase activity voltage-gated channel activity
12
long-chain specific acyl-coenzyme A dehydrogenase
ganglioside catabolic process; learning or memory; lipid storage; neuromuscular process controlling balance; oligosaccharide catabolic process fatty acid metabolic process; oxidation reduction
13
fructose-bisphosphate aldolase A
glycolysis
GM2, ganglioside-2; FAD, flavin adenine dinucleotide
retinal dehydrogenase activity; 3-chloroallyl aldehyde dehydrogenase activity isocitrate dehydrogenase activity; magnesium ion binding; manganese ion binding
b-N-acetylhexosaminidase activity; enzyme activator activity
mitochondrial matrix
mitochondrion
FAD binding; electron carrier activity; long-chain acylcoenzyme A dehydrogenase activity fructose-bisphosphate aldolase activity
370
Q. Shen et al. / Nutrition 27 (2011) 364–371
Control group
A Prohibitin
β-actin
IUGR group
HP group
30kDa
43kDa
Fig. 3. Western blot analysis of prohibitin expression in the kidney showed that prohibitin in the IUGR group was weaker than the control group and the expression in the HP group was even weaker. HP, high-protein; IUGR, intrauterine growth restriction.
B
C
participate in oxidation reduction. We suggest that the differential expression proteins are related to the effects of a highprotein diet intervention on the kidneys of IUGR rats. Among the identified proteins with obviously differential expressions, prohibitin, the antiproliferative protein, showed consecutive changes among the three groups (ratio of control group to IUGR group >5 and no obvious expression in the HP group). Previous studies have indicated that prohibitin has antiproliferation and antitumor functions, and that it plays an important role in cell metabolism, growth, aging, and apoptosis [28]. Our previous study on prohibitin suggested that the expression level of prohibitin in renal tissue could reflect the degree of tubulointerstitial injury, and exogenous prohibitin could significantly inhibit the transforming growth factorb–induced fibroblast proliferation and phenotypic changes [29]. We further validated the differential expression of prohibitin in renal tissue using western blot and immunohistochemistry, and the results showed that prohibitin was mainly expressed in renal tubules and its expression level was lower in the IUGR group than in the control group and was further decreased by the postnatal high-protein diet. These results were consistent with the results of proteomics research, which further confirms the accuracy of the results of mass spectrometry. We suggest that indepth studies on these differentially expressed proteins including protein expression, action mechanism, and protein interaction are needed to uncover the pathogenic mechanisms of postnatal proteinuria and hypertension in patients with IUGR and the effects of a high-protein diet intervention on the kidney.
Conclusion A high-protein diet intervention after birth cannot correct the decrease in the number of nephrons resulting from IUGR, and instead worsens the severity of hypertension and proteinuria. The comparative proteomic approach has provided new avenues for future research to explore the pathogenesis of IUGR-induced kidney injury and the effects of a high-protein diet intervention on the kidney. Fig. 2. Immunohistochemistry analysis of prohibitin expression in kidneys from the (A) control, (B) intrauterine growth restriction, and (C) high-protein groups (diaminobenzidine chromogenesis, magnification 400). Prohibitin was primarily expressed in renal tubular epithelial cells. Staining in the intrauterine growth restriction group was significantly weaker than in the control group and almost no staining was observed in the high-protein group.
Acknowledgments
including chloride intracellular channel 1 that participates in apoptosis regulation, GM2 ganglioside activator protein and aspartoacylase-2 that participate in body metabolism, and isocitrate dehydrogenase and disulfide-isomerase A3 that
References
The authors are grateful to Xiu-Rong Zhang and Zhong-Hua Zhao for technical assistance.
[1] Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993;341:938–41.
Q. Shen et al. / Nutrition 27 (2011) 364–371 [2] Armitage JA, Taylor PD, Poston L. Experimental models of developmental programming: consequences of exposure to an energy rich diet during development. J Physiol 2005;565:3–8. [3] Silver LE, Decamps PJ, Korst LM, Platt LD, Castro LC. Intrauterine growth restriction is accompanied by decreased renal volume in the human fetus. Am J Obstet Gynecol 2003;188:1320–5. [4] Latini G, De Mitri B, Del Vecchio A, Chitano G, De Felice Z, Zetterstro¨m R. Foetal growth of kidneys, liver and spleen in intrauterine growth restriction: ‘‘programming’’ causing ‘‘metabolic syndrome’’ in adult age. Acta Paediatr 2004;93:1635–9. [5] Rodrı´guez-Soriano J, Aguirre M, Oliveros R, Vallo A. Long-term renal followup of extremely low birth weight infants. Pediatr Nephrol 2005;20:579–84. [6] Hoy WE, Rees M, Kile E, Mathews JD, Wang Z. A new dimension to the Barker hypothesis: low birthweight and susceptibility to renal disease. Kidney Int 1999;56:1072–7. [7] Chen J, Xu H, Shen Q, Guo W, Sun L, Lin SY. Influence of intrauterine growth retardation on blood pressure and renal function in rats. Zhanghua Shen Zang Bing Za Zhi 2006;22:706–7. [8] Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71:1344S–52. [9] Martins JP, Monteiro JC, Paixa˜o AD. Renal function in adult rats subjected to prenatal dexamethasone. Clin Exp Pharmacol Physiol 2003;30:32–7. ˜ alich R, Reyes L, Herrera M, Melendi C, Fundora I. Relationship between [10] Man weight at birth and the number and size of renal glomeruli in humans: a histomorphometric study. Kidney Int 2000;58:770–3. [11] Thame M, Osmond C, Wilks RJ, Bennett FI, McFarlane-Anderson N, Forrester TE. Blood pressure is related to placental volume and birth weight. Hypertension 2000;35:662–7. [12] Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer TE. Maternal nutrition and fetal development. J Nutr 2004;134:2169–72. [13] Gluckman PD, Hanson MA. Living with the past: evolution, development and patterns of disease. Science 2004;305:1773–6. [14] Ozanne SE, Hales CN. Catch-up growth and obesity in male mice. Nature 2004;427:411–2. [15] Xu W, Zhu MW, Lin JF. Fetal growth retardation and postnatal high fat diet on the development of insulin resistance and fertility: experiment with rats. Zhonghua Yi Xue Za Zhi 2007;87:1633–6. [16] Gil FZ, Lucas SR, Gomes GN, Cavanal Mde F, Coimbra TM. Effects of intrauterine food restriction and long-term dietary supplementation with Larginine on age-related changes in renal function and structure of rats. Pediatr Res 2005;57:724–31.
371
[17] Sahajpal V, Ashton N. Renal function and angiotensin AT1 receptor expression in young rats following intrauterine exposure to a maternal low-protein diet. Clin Sci 2003;104:607–14. [18] do Carmo Pinho Franco M, Nigro D, Fortes ZB, Tostes RC, Carvalho MH, Lucas SR, et al. Intrauterine undernutritiondrenal and vascular origin of hypertension. Cardiovasc Res 2003;60:228–34. [19] Alexander BT. Intrauterine growth restriction and reduced glomerular number: role of apoptosis. Am J Physiol Regul Integr Comp Physiol 2003;285:R933–4. [20] Pham TD, MacLennan NK, Chiu CT, Laksana GS, Hsu JL, Lane RH. Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney. Am J Physiol Regul Integr Comp Physiol 2003;285:R962–70. [21] Arthur JM, Thongboonkerd V, Scherzer JA, Cai J, Pierce WM, Klein JB. Differential expression of proteins in renal cortex and medulla: a proteomic approach. Kidney Int 2002;62:1314–21. [22] Thongboonkerd V, Barati MT, McLeish KR, Benarafa C, Remold-O’Donnell E, Zheng S, et al. Alterations in the renal elastin-elastase system in type 1 diabetic nephropathy identified by proteomic analysis. J Am Soc Nephrol 2004;15:650–62. [23] Thongboonkerd V. Proteomics in nephrology: current status and future directions. Am J Nephrol 2004;24:360–78. [24] Schaub S, Rush D, Wilkins J, Gibson IW, Weiler T, Sangster K, et al. Proteomic-based detection of urine proteins associated with acute renal allograft rejection. J Am Soc Nephrol 2004;15:219–27. [25] Weinkauf M, Hiddemann W, Dreyling M. Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background. Electrophoresis 2006;27:4555–8. [26] Karp NA, Lilley KS. Investigating sample pooling strategies for DIGE experiments to address biological variability. Proteomics 2009;9:388–97. [27] Jia SQ, Niu ZJ, Zhang LH, Zhong XY, Shi T, Du H, et al. Identification of prognosis-related proteins in advanced gastric cancer by mass spectrometry-based comparative proteomics. J Cancer Res Clin Oncol 2009;135:403–11. [28] Merkwirth C, Langer T. Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis. Biochim Biophys Acta 2009;1793:27–32. [29] Guo W, Xu H, Chen J, Yang Y, Jin JW, Fu R, et al. Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1. Mol Cell Biochem 2007;295: 167–77.
Contents lists available at ScienceDirect
Nutrition journal homepage: www.nutritionjrnl.com
Basic nutritional investigation
Intrauterine growth restriction and postnatal high-protein diet affect the 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 b
Department of Nephrology and Rheumatology, Children’s Hospital of Fudan University, Shanghai, People’s Republic of China Institutes for Biomedical Sciences, Fudan University, Shanghai, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2009 Accepted 16 March 2010
Objective: Intrauterine growth restriction (IUGR) is associated with hypertension and chronic kidney disease in adulthood. Postnatal overnutrition after IUGR may be of pathogenic importance for the development of diabetes and cardiovascular disease. This study was to identify the effects of IUGR and a postnatal high-protein diet on the kidneys in adult rats. Methods: Intrauterine growth restriction was induced in Sprague-Dawley rats by isocaloric protein restriction in pregnant dams. IUGR pups were divided into two groups that were a standardprotein diet (IUGR group) or a high-protein diet (HP group). A comparative proteomic method was used to study the differences of protein expression profiles between normal adult rats and adult rats with IUGR and the effects of a postnatal high-protein diet on the protein expression profiles of the kidneys. Results: The IUGR adults had higher urinary excretion of protein and blood pressure than controls and the HP diet caused more severe hypertension and proteinuria than IUGR itself. The differential proteomic expression analysis found 12 proteins that had significantly differential expression between the IUGR and control groups, which were transcription regulators and structural molecules. The differential proteomic expression analysis between the HP and control groups found 13 proteins that had significantly differential expression and were involved primarily in body metabolism, oxidation reduction, and apoptosis regulation. Conclusion: An HP diet intervention after IUGR worsens the severity of hypertension and proteinuria, and this study may provide valuable experimental evidence of proteins involved in the pathogenesis of kidney disease in IUGR and the effect of postnatal overnutrition. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: Intrauterine growth restriction Kidney Proteomics Nutritional intervention Proteinuria Hypertension
Introduction Intrauterine growth restriction (IUGR) has long-term effects on various organisms through fetal programming [1,2]. Human studies of the association between IUGR and renal diseases have indicated that, in contrast to the normal fetal kidney, the IUGR fetal kidney has a smaller volume with a significant decrease in glomerular number [3,4]. In addition, long-term follow-up after birth has shown a significantly lower glomerular filtration function and a higher incidence of proteinuria in the IUGR group compared with the control group [5,6]. Our previous animal studies have also shown a decreased glomerular number in IUGR
This research was funded by grant 30672242 from the National Natural Science Foundation of China. * Corresponding author. Tel: þ86-21-6493-1006; fax: þ86-21-6493-1901. E-mail address: [email protected] (H. Xu). 0899-9007/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.03.003
rats, with an increased incidence of proteinuria and hypertension in the postnatal follow-up period [7]. However, the pathogenesis of IUGR-induced postnatal kidney diseases has not been fully clarified. Previous theories have suggested that nutritional supplements, especially a high-protein diet for infants in whom IUGR is diagnosed, at an early postnatal stage may assist rapid postnatal growth, but the high-protein diet may further exacerbate kidney injury and lead to deterioration of renal function. Currently, there is no definite conclusion on the proper postnatal nutritional intervention for fetal IUGR. This study was designed to observe the effects of a postnatal high-protein nutritional intervention on kidney development and function in IUGR rats. Moreover, a comparative proteomic method was used to study the differences of protein expression profiles between normal adult rats and adult rats with IUGR and the effects of a postnatal high-protein diet on the protein
Q. Shen et al. / Nutrition 27 (2011) 364–371
expression profiles of kidney to explore the possible pathogenesis of IUGR-induced kidney injury and the effects of a highprotein diet intervention on the kidney. Materials and methods This work was performed with the approval of Children’s Hospital of Fudan University’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 male rats. The normal control group was fed with a conventional pregnancy diet (22% protein) until natural delivery, and the newborn rats were fed with a conventional diet (22% protein, control group) until 12 wk after birth. The study group was fed with a low-protein isocaloric diet consisting of 6% protein throughout the entire pregnancy until natural labor [8]. Newborn rats with a birth weight 2 standard deviations lower than the average birth weight of the normal newborn rats 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 in Table 1. Measurement of urine protein and blood pressure Eight male rats were selected from each group at 4, 8, and 12 wk, respectively. The rats were weighed and 24-h urine was collected and the volume was recorded. Urine protein content was determined using colorimetry. Systolic blood pressure was measured using a tail artery measuring instrument. The kidney weight and body weight of each group of rats were measured at 12 wk of age. Determination of glomerular number and kidney morphology The Sprague-Dawley rats were sacrificed at 12 wk of age by jugular puncture after anesthesia. The kidneys of eight rats in each group were collected at 12 wk of age and were conventionally fixed, embedded, sliced, and stained with hematoxylin and eosin. The glomerular number was counted and the glomerular volume was measured. The methods for measuring the glomerular number and glomerular volume have been previously reported [9,10].
365
In-gel digestion and mass spectrometry Protein spots with significant differences were excised from the gels and placed into a 96-well microtiter plate. Gel pieces were detained with a solution of 15 mM of potassium ferricyanide and 50 mM of sodium thiosulfate (1:1) for 20 min at room temperature. Then they were washed twice with deionized water and shrunk by dehydration in acetone cyanohydrin. The samples were then swollen in a digestion buffer containing 20 mM of ammonium bicarbonate and 12.5 ng/mL of trypsin (Roche, Indianapolis, IN, USA) at 4 C. After a 30-min incubation, the gels were digested longer than 12 h at 37 C. Peptides were then extracted twice using 0.1% trifluoroacetic acid in 50% acetone cyanohydrin. The extracts were dried under the protection of N2. For matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, 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% acetone cyanohydrin). Then the solution was spotted on a stainless-steel target with 192 wells (Applied Biosystems, Framingham, MA, USA). Samples were allowed to airdry before inserting them into the mass spectrometer. The matrix-assisted laser desorption ionization mass spectrometer was an ABI 4700 TOF-TOF Proteomics Analyzer (Applied Biosystems) instrument. The ultraviolet laser was operated at a 200-Hz repetition rate with wavelength of 355 nm. The accelerated voltage was operated at 20 kV. The data were searched by GPS Explorer (Applied Biosystems) using MASCOT (Matrix Science, London, UK) as a search engine. Data from matrix-assisted laser desorption ionization/time-of-flight mass spectrometry were analyzed using MASCOT search software. The following parameters were used in the search: retrieval species for rat, enzyme for trypsin, missed cleavage by 1, peptide tolerance of 0.2, tandem mass spectrometry (MS/MS) tolerance of 0.6 Da, and possible oxidation of methionine. Immunohistochemistry Three kidney tissue sections from each group were used to detect the expression level of prohibitin by immunohistochemical staining. Paraffinembedded tissue sections were first dewaxed and rehydrated. Microwave antigen retrieval was done in the presence of citric acid buffer (pH 6.0). Samples were blocked with horse serum for 20 min before incubation with monoclonal mouse 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 were then 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 counterstained by hematoxylin.
Two-dimensional gel electrophoresis and image analysis
Western blot
At 12 wk of age, six kidneys were selected from each group and were mixed for total protein extraction. Tissues were homogenized in a lysis buffer, spun down to collect the supernatant, and subjected to the Bradford assay to determine protein concentration. Two-dimensional gel electrophoresis (2-DE) was performed according to the manufacturer’s instruction (Amersham Biosciences, Buckinghamshire, UK). An Immobiline pH gradient DryStrip gel (18 cm, non-linear pH 3w10; Amersham Biosciences) was rehydrated with re-swelling buffer containing a 1-mg protein sample at 20 C for 12 h, focused by gradient increasing voltage to 8000 V, and continued until total voltage-hour reached 52 000 Vh. After focusing, the strip was first equilibrated in the equilibration solution containing 1% dithiothreitol and 2.5% iodine acetamide for 15 min, transferred to the top of a 12.5% polyacrylamide gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The electrophoresis was performed at 25 C until bromophenol blue reached the bottom of the gel. The gel was stained by Coomassie brilliant blue and then scanned for image analysis. Each 2-DE experiment was performed in triplicate to confirm the reproducibility. The scanned gel images were then analyzed by Pdquest 7.3.0 (Bio-Rad, Hercules, CA, USA).
Two samples of renal tissue protein were collected from each group for western blot analysis. Total protein of 30 mg was added according to conventional methods and the samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred onto the polyvinylidene fluoride membrane using the semidry electrophoretic transfer method. The samples were blocked with 5% skim milk powder at room temperature for 1 h and the monoclonal mouse anti-prohibitin antibody (1:500; Lab Vision Corporation) was added and the samples were incubated at 4 C overnight. The samples were incubated with horseradish peroxidase–labeled secondary antibody (1:2000; Abcam, Cambridge, UK) at room temperature for 1 h. Protein was visualized by enhanced chemiluminescence and b-actin (Kangchen) was detected as the internal reference.
Table 1 Diet make-up
Results
Conventional diet Low-protein diet High-protein diet
Ingredients
Protein (%)
corn 26%, wheat 34%, alfalfa meal 2%, soybean meal 27%, fish meal 5%, vegetable oil 1%, premix 5% corn 50%, sucrose 13%, amylum 26.5%, vegetable oil 2.5%, fish meal 3%, premix 5% corn 15%, amylum 21%, soybean meal 51%, vegetable oil 1%, fish meal 2.5%, alfalfa meal 2%, casein 2.5%, premix 5%
22
6 30
Statistical analysis Quantitative data were presented as mean standard deviation. Comparisons among multiple groups used one-way analysis of variance, and the comparisons between groups were made with the least significant difference test. P < 0.05 was considered statistically significant.
Comparisons of examination indexes among rat groups Comparison of kidney weight among rat groups At 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 ratio of kidney weight to body weight was higher than that in the control group (P < 0.05). Kidney enlargement in IUGR rats fed a postnatal high-protein diet (HP group) was more obvious,
366
Q. Shen et al. / Nutrition 27 (2011) 364–371
and the kidney weight reached the level of the control group (P > 0.05). The ratio of kidney weight to body weight in the HP 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 pressure Compared 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 wk of age (P < 0.01). Compared with the control group, rats in the HP group also exhibited an increase in blood pressure at 8 wk of age (P < 0.01), and the severity of the blood pressure increase was even greater at 12 wk of age (P < 0.001). At 12 wk of age, the blood pressure increase in the HP group was significantly higher than that in the IUGR group (P < 0.01; Table 3). Comparison of 24-h urine protein Compared 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 group than in the IUGR group (P < 0.001; Table 4). Comparison of glomerular number The numbers of glomeruli in the IUGR and HP groups (number per unilateral kidney) were significantly lower than those in the control group (IUGR group 22 900 926 versus control group 28 861 1044, P < 0.01; HP group 23 043 595 versus control group, P < 0.01). There was no significant difference between the IUGR and HP groups (P > 0.05). Comparison of renal tissue morphology Hematoxylin and eosin staining showed that the glomerular volumes of rats in the IUGR and HP groups were significantly increased at 12 wk of age compared with that in the control group (IUGR group 1.092 0.083 105 mm3 versus control group 0.830 0.044 105 mm3, P < 0.01; HP group 1.508 0.097 105 mm3 versus control group, P < 0.001). The glomerular volume of rats in the HP group was significantly larger than that in the IUGR group (P < 0.001). Electron microscopy results showed observable mild foot process fusion in the IUGR group, whereas rats in the HP group showed obvious mesangial cell proliferation and partial fusion of the foot. Differences in kidney protein expression profiles between IUGR and normal rats Results of 2-DE and mass spectrometry identification Total proteins were extracted from adult rat kidneys from six rats in the IUGR and control groups, respectively, and then subjected to 2-DE three times to show reproducibility. Three sets of
Table 2 Comparison of kidney weight at 12 wk of age
Body weight (g) Kidney weight (g) Kidney weight/ body weight (%)
Control group
IUGR group
HP group
394.8 11.3 1.320 0.061 0.334 0.007
300.0 26.9y 1.134 0.106* 0.378 0.019*
296.3 26.5y 1.316 0.204z 0.443 0.043y,x
HP, high-protein; IUGR, intrauterine growth restriction Data are presented as mean SD; n ¼ 8 per group. * P < 0.05. y P < 0.001 compared with control group. z P < 0.05. x P < 0.001 compared with IUGR group.
Table 3 Comparison of blood pressure Blood pressure (mm Hg)
Control group
IUGR group
HP group
4 wk of age 8 wk of age 12 wk of age
99.6 2.5 109.4 3.1 115.2 2.3
104.1 3.0 125.2 2.3* 132.1 2.9*
104.6 1.7 127.3 1.2* 138.6 2.8y,z
HP, high-protein; IUGR, intrauterine growth restriction Data 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.
2-DE profiles were obtained (Fig. 1). With the application of Pdquest 7.3.0 for image analysis, these results showed that 727 58 protein spots were obtained in the IUGR group, with an average match rate of 85%. In the control group, 758 53 protein spots were obtained, with an average match rate of 78%. The differential expression analysis found that one protein spot was expressed only in the IUGR group (no. 1). Seven protein spots were upregulated more than five-fold (nos. 2w8) and four spots downregulated more than five-fold (nos. 9w12) in the IUGR group compared with those in the control group. These 12 protein spots were picked for mass spectrometric analysis and all had successful protein identifications (Table 5). Retrieval and classification of differentially expressed proteins The Gene Ontology classification method was used to carry out functional retrieval for the 12 differentially expressed proteins in three areas, i.e., biological process, cellular component, and molecular function (Table 6). These proteins were involved primarily in oxidation reduction, body metabolism, and transcriptional regulation.
Effects of postnatal high-protein diet on renal protein expression profiles in IUGR rats Results of 2-DE and mass spectrometric identification Total proteins were extracted from adult rat kidneys from six rats in the HP and control groups, respectively, and then subjected to 2-DE three times to show reproducibility. Three sets of 2-DE profiles were obtained (Fig. 1). With the application of Pdquest 7.3.0 for image analysis, these results showed that 740 43 protein spots were obtained in the HP group, with an average match rate of 84%. In the control group, 758 53 protein spots were obtained, with an average match rate of 78%. The differential expression analysis found that five protein spots were expressed only in the control group (nos. 1w5). Five protein spots were upregulated more than five-fold (nos. 6w10) and three spots downregulated more than five-fold (nos. 11w13) in the HP group compared with those in the control group. These 13
Table 4 Comparison of 24-h urine protein Proteinuria (mg. kg1. d1)
Control group
IUGR group
HP group
4 wk of age 8 wk of age 12 wk of age
26.28 9.78 109.12 19.7 69.72 10.35
26.92 5.75 89.55 13.23 118.46 21.85*
25.62 7.18 130.9 36.05 202.61 62.55y,z
HP, high-protein; IUGR, intrauterine growth restriction Data are presented as mean SD; n ¼ 8 per group. * P < 0.05. y P < 0.001 compared with the control group. z P < 0.001 compared with the IUGR group.
Q. Shen et al. / Nutrition 27 (2011) 364–371
pH3
pH10
A
pH3
367
proteins (Table 8). These proteins were involved primarily in body metabolism, oxidation reduction, and apoptosis regulation. Effects of different postnatal protein diets on kidney protein expression profiles in IUGR rats Compared with the control group, six proteins showed the same 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 acylcoenzyme A dehydrogenase, hydroxyacid oxidase-2, retinal dehydrogenase-1, and transketolase. Two proteins, capping protein and prohibitin, had consecutive changes among three groups (ratio of control group to IUGR group >5 and no obvious expression in the HP group). Other proteins showed differential expression only after high-protein nutritional intervention. These included chloride intracellular channel-1, ganglioside-2 (GM2) ganglioside activator protein, aspartoacylase-2, isocitrate dehydrogenase, and disulfide-isomerase A3.
pH10
B
Confirmation of prohibitin expression by immunohistochemistry and western blot Immunohistochemistry results indicated that prohibitin was primarily expressed in renal tubular epithelial cells. Staining in the IUGR group was significantly weaker than in the control group and almost no staining was observed in the HP group (Fig. 2). Western blot showed that the expression of prohibitin in the IUGR group was weaker than in the control group and the expression in the HP group was even weaker (Fig. 3). Discussion
pH3
pH10
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 all had successful protein identifications (Table 7). Retrieval and classification of differentially expressed proteins The Gene Ontology classification method was used to carry out the functional retrieval for the 13 differentially expressed
Intrauterine growth restriction can cause long-term or lifelong effects on the functions of various organs in the body by fetal programming. Studies have found that IUGR can cause metabolic disorders and imbalances of the hormone levels that affect the development and function of multiple organs in the body. IUGR has been associated with many adulthood diseases including hypertension, coronary heart disease, diabetes, and chronic kidney disease [11,12]. Previous theories have suggested that nutritional supplements, especially a high-protein diet for newborns affected with IUGR, may promote rapid postnatal physical growth. However, a theory of predictive adaptive response has been recently been suggested [13]. This theory postulates that the environment of fetal development predicts the postnatal environment and causes a series of adaptive changes. If the predictive adaptive response is correct, the phenotype will be normal. If the predicted environment is different from the actual environment, diseases will easily occur. One study found that the richer the diet of young rats undergoing intrauterine malnutrition, the shorter their life span [14]. Studies on the reproductive endocrine function of IUGR rats also found that a postnatal high-fat diet aggravated insulin resistance and reproductive endocrine disorders of IUGR rats [15]. At the same time, a high-protein diet itself can damage the permeability of the glomerular basement membrane and aggravate glomerular hyperfiltration and hyperperfusion phenomenon. Glomerular basement membrane damage can further aggravate proteinuria and stimulate mesangial cell proliferation. All of these factors may further worsen kidney damage. Therefore, this study was designed to observe the effects of a postnatal high-protein nutritional intervention on kidney development and function in IUGR rats. The results showed that a postnatal high-protein nutritional intervention not only could not correct the decrease the number
368
Q. Shen et al. / Nutrition 27 (2011) 364–371
Table 5 Differentially expressed proteins between intrauterine growth-restricted rats and normal rats No.
Name
Molecular weight
Isoelectric point
Score
Sequence coverage (%)
1 2 3 4 5 6 7 8 9 10 11 12
phosphoglycerate kinase-1 ribonuclease UK114 catalase liver carboxylesterase-4 hydroxyacid oxidase-2 transketolase retinal dehydrogenase-1 glutathione S-transferase a1 Prohibitin long-chain specific acyl-coenzyme A dehydrogenase fructose-bisphosphate aldolase A capping protein (actin filament) muscle Z-line, a2
44 14 59 62 39 71 54 25 29 47 39 32
7.52 6.21 7.15 6.29 7.90 7.54 7.90 8.87 5.57 7.63 8.39 5.57
121 250 173 82 325 383 195 205 169 92 85 164
29 62 39 45 63 60 32 33 37 27 31 21
of nephrons but also worsened the severity of hypertension and proteinuria. We believe that in the current society with abundant materials, postnatal nutrient intake is usually adequate and excessive, thus the control of high-protein intake is of certain significance in patients with IUGR. Recent animal studies have shown that the expression of the proapoptotic gene Bax is significantly increased in IUGR animal kidneys, whereas the antiapoptotic gene Bcl-2 is clearly decreased. In addition, the expression of proteins associated with glomerular sclerosis and tubulointerstitial damage such as fibronectin, angiotensin receptor, and sodium channel are also increased in the IUGR animal [16–20]. However, the mechanisms of IUGR-induced postnatal kidney disease and the aggravation of
394 475 588 234 045 113 292 459 801 842 196 946
kidney disease induced by a high-protein diet intervention have not been fully clarified at the present time. Studies on the effects of IUGR on kidney development and the mechanisms of IUGR-induced postnatal kidney disease may lead to a better understanding of the pathogenesis of kidney diseases of fetal origin and the discovery of therapeutic targets. In recent years, proteomics has been widely applied in basic medical research. It has been used to study systemic and quantitative proteomic changes in tissues and cells at different disease progression stages. In the field of kidney research, proteomic approaches have been used to compare the differences in protein expression profiles between the renal cortex and medulla [21]. At the same time, this technology has been applied to pathogenic
Table 6 Function of differentially expressed proteins between intrauterine growth-restricted rats and control rats No.
Name
Biological process
Cellular component
Molecular function
1
phosphoglycerate kinase-1
glycolysis; phosphorylation
cytosol; soluble fraction
2 3
ribonuclease UK114 catalase
d cell proliferation; hydrogen peroxide catabolic process; oxidation reduction
4
liver carboxylesterase-4
d
5
hydroxyacid oxidase-2
oxidation reduction
mitochondrion; nucleus Golgi apparatus; cytosol; endoplasmic reticulum; lysosome; mitochondrial intermembrane space; plasma membrane endoplasmic reticulum; endoplasmic reticulum lumen peroxisome
adenosine triphosphate binding; phosphoglycerate kinase activity endonuclease activity catalase activity; growth factor activity; heme binding; iron ion binding
6
transketolase
ribose phosphate biosynthetic process; pentose-phosphate shunt
endoplasmic reticulum membrane; microsome; peroxisome; soluble fraction
7
retinal dehydrogenase-1
oxidation reduction
cytoplasm
8 9
glutathione S-transferase a1 prohibitin
metabolic process DNA replication
cytoplasm mitochondrial inner membrane
10
long-chain specific acylcoenzyme A dehydrogenase
fatty acid metabolic process; oxidation reduction
mitochondrial matrix
11
fructose-bisphosphate aldolase A capping protein (actin filament) muscle Z-line, a2
glycolysis
mitochondrion
actin cytoskeleton organization
F-actin capping protein complex
12
FAD, flavin adenine dinucleotide
carboxylesterase activity
hydroxyacid oxidase activity; flavin mononucleotide binding; electron carrier activity calcium ion binding; magnesium binding; monosaccharide binding; thiamin pyrophosphate binding; transketolase activity retinal dehydrogenase activity; 3-chloroallyl aldehyde dehydrogenase activity glutathione transferase activity protein binding; negative regulation of apoptosis FAD binding; electron carrier activity; long-chain acylcoenzyme A dehydrogenase activity fructose-bisphosphate aldolase activity actin binding
Q. Shen et al. / Nutrition 27 (2011) 364–371
369
Table 7 Differentially expressed proteins between rats on a high-protein diet and control rats No.
Name
Molecular weight
1 2 3 4 5 6 7 8 9 10 11 12 13
prohibitin disulfide-isomerase A3 retinal dehydrogenase-1 isocitrate dehydrogenase capping protein (actin filament) muscle Z-line, a2 hydroxyacid oxidase-2 transketolase aspartoacylase-2 glutathione S-transferase a1 chloride intracellular channel-1 GM2 ganglioside activator protein long-chain specific acyl-coenzyme A dehydrogenase fructose-bisphosphate aldolase A
29 57 54 60 32 39 71 35 25 26 21 47 39
Isoelectric point
801 043 292 934 946 045 113 419 459 963 479 842 196
5.57 5.88 7.90 8.88 5.57 7.90 7.54 5.42 8.87 5.09 6.13 7.63 8.39
Score
Sequence coverage (%)
169 381 218 65 66 203 210 149 205 140 169 101 85
37 63 39 45 29 60 62 48 33 32 39 21 31
GM2, ganglioside-2
studies in diabetic nephropathy, focal segmental glomerular sclerosis, lupus nephritis, tumor, and acute rejection [22–24]. However, due to limitations in sample quantity in animal and clinical studies, ‘‘sample pooling’’ by mixing 3 to 10 samples together with the same background before performing the 2-DE analysis has become a popular technique [25–27]. These results suggest that analysis on mixed samples not only helps to decrease the required number of repeat runs in 2-DE but also decreases the standard deviation obtained from individual samples. We applied the sample-pooling comparative proteomics method to study the differences in kidney protein expression profiles
between adult IUGR rats and normal rats. Our results revealed that a total of 12 proteins showed significant differences in expression profile. Functional classification of these differential proteins showed that they were involved primarily in biological processes such as oxidation reduction, body metabolism, and transcriptional regulation. We suggest that the differentially expressed proteins participate in postnatal kidney disease in IUGR rats. Compared with the control group, two proteins, capping protein and prohibitin, showed consecutive changes among the three groups. Some other proteins showed differential expressions only after the high-protein nutritional intervention,
Table 8 Function of differentially expressed proteins between rats consuming a high-protein diet and control rats No.
Name
Biological process
Cellular component
Molecular function
1
prohibitin
DNA replication
mitochondrial inner membrane
2
disulfide-isomerase A3
cell redox homeostasis
3
retinal dehydrogenase-1
oxidation reduction
endoplasmic reticulum; endoplasmic reticulum lumen; melanosome cytoplasm
protein binding; negative regulation of apoptosis protein disulfide isomerase activity
4
isocitrate dehydrogenase
mitochondrion
5
F-actin capping protein complex
actin binding
6
capping protein (actin filament) muscle Z-line, a2 hydroxyacid oxidase-2
glyoxylate cycle; isocitrate metabolic process; oxidation reduction; tricarboxylic acid cycle actin cytoskeleton organization oxidation reduction
peroxisome
7
transketolase
ribose phosphate biosynthetic process; pentose-phosphate shunt
endoplasmic reticulum membrane; microsome; peroxisome; soluble fraction
8 9 10
aspartoacylase-2 glutathione S-transferase a1 chloride intracellular channel-1
metabolic process metabolic process chloride transport; apoptosis regulation
11
GM2 ganglioside activator protein
d cytoplasm cytoplasm; membrane; membrane fraction; nuclear envelope; soluble fraction mitochondrion
hydroxyacid oxidase activity; flavin mononucleotide binding; electron carrier activity calcium ion binding; magnesium binding; monosaccharide binding; thiamin pyrophosphate binding; transketolase activity hydrolase activity glutathione transferase activity voltage-gated channel activity
12
long-chain specific acyl-coenzyme A dehydrogenase
ganglioside catabolic process; learning or memory; lipid storage; neuromuscular process controlling balance; oligosaccharide catabolic process fatty acid metabolic process; oxidation reduction
13
fructose-bisphosphate aldolase A
glycolysis
GM2, ganglioside-2; FAD, flavin adenine dinucleotide
retinal dehydrogenase activity; 3-chloroallyl aldehyde dehydrogenase activity isocitrate dehydrogenase activity; magnesium ion binding; manganese ion binding
b-N-acetylhexosaminidase activity; enzyme activator activity
mitochondrial matrix
mitochondrion
FAD binding; electron carrier activity; long-chain acylcoenzyme A dehydrogenase activity fructose-bisphosphate aldolase activity
370
Q. Shen et al. / Nutrition 27 (2011) 364–371
Control group
A Prohibitin
β-actin
IUGR group
HP group
30kDa
43kDa
Fig. 3. Western blot analysis of prohibitin expression in the kidney showed that prohibitin in the IUGR group was weaker than the control group and the expression in the HP group was even weaker. HP, high-protein; IUGR, intrauterine growth restriction.
B
C
participate in oxidation reduction. We suggest that the differential expression proteins are related to the effects of a highprotein diet intervention on the kidneys of IUGR rats. Among the identified proteins with obviously differential expressions, prohibitin, the antiproliferative protein, showed consecutive changes among the three groups (ratio of control group to IUGR group >5 and no obvious expression in the HP group). Previous studies have indicated that prohibitin has antiproliferation and antitumor functions, and that it plays an important role in cell metabolism, growth, aging, and apoptosis [28]. Our previous study on prohibitin suggested that the expression level of prohibitin in renal tissue could reflect the degree of tubulointerstitial injury, and exogenous prohibitin could significantly inhibit the transforming growth factorb–induced fibroblast proliferation and phenotypic changes [29]. We further validated the differential expression of prohibitin in renal tissue using western blot and immunohistochemistry, and the results showed that prohibitin was mainly expressed in renal tubules and its expression level was lower in the IUGR group than in the control group and was further decreased by the postnatal high-protein diet. These results were consistent with the results of proteomics research, which further confirms the accuracy of the results of mass spectrometry. We suggest that indepth studies on these differentially expressed proteins including protein expression, action mechanism, and protein interaction are needed to uncover the pathogenic mechanisms of postnatal proteinuria and hypertension in patients with IUGR and the effects of a high-protein diet intervention on the kidney.
Conclusion A high-protein diet intervention after birth cannot correct the decrease in the number of nephrons resulting from IUGR, and instead worsens the severity of hypertension and proteinuria. The comparative proteomic approach has provided new avenues for future research to explore the pathogenesis of IUGR-induced kidney injury and the effects of a high-protein diet intervention on the kidney. Fig. 2. Immunohistochemistry analysis of prohibitin expression in kidneys from the (A) control, (B) intrauterine growth restriction, and (C) high-protein groups (diaminobenzidine chromogenesis, magnification 400). Prohibitin was primarily expressed in renal tubular epithelial cells. Staining in the intrauterine growth restriction group was significantly weaker than in the control group and almost no staining was observed in the high-protein group.
Acknowledgments
including chloride intracellular channel 1 that participates in apoptosis regulation, GM2 ganglioside activator protein and aspartoacylase-2 that participate in body metabolism, and isocitrate dehydrogenase and disulfide-isomerase A3 that
References
The authors are grateful to Xiu-Rong Zhang and Zhong-Hua Zhao for technical assistance.
[1] Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993;341:938–41.
Q. Shen et al. / Nutrition 27 (2011) 364–371 [2] Armitage JA, Taylor PD, Poston L. Experimental models of developmental programming: consequences of exposure to an energy rich diet during development. J Physiol 2005;565:3–8. [3] Silver LE, Decamps PJ, Korst LM, Platt LD, Castro LC. Intrauterine growth restriction is accompanied by decreased renal volume in the human fetus. Am J Obstet Gynecol 2003;188:1320–5. [4] Latini G, De Mitri B, Del Vecchio A, Chitano G, De Felice Z, Zetterstro¨m R. Foetal growth of kidneys, liver and spleen in intrauterine growth restriction: ‘‘programming’’ causing ‘‘metabolic syndrome’’ in adult age. Acta Paediatr 2004;93:1635–9. [5] Rodrı´guez-Soriano J, Aguirre M, Oliveros R, Vallo A. Long-term renal followup of extremely low birth weight infants. Pediatr Nephrol 2005;20:579–84. [6] Hoy WE, Rees M, Kile E, Mathews JD, Wang Z. A new dimension to the Barker hypothesis: low birthweight and susceptibility to renal disease. Kidney Int 1999;56:1072–7. [7] Chen J, Xu H, Shen Q, Guo W, Sun L, Lin SY. Influence of intrauterine growth retardation on blood pressure and renal function in rats. Zhanghua Shen Zang Bing Za Zhi 2006;22:706–7. [8] Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71:1344S–52. [9] Martins JP, Monteiro JC, Paixa˜o AD. Renal function in adult rats subjected to prenatal dexamethasone. Clin Exp Pharmacol Physiol 2003;30:32–7. ˜ alich R, Reyes L, Herrera M, Melendi C, Fundora I. Relationship between [10] Man weight at birth and the number and size of renal glomeruli in humans: a histomorphometric study. Kidney Int 2000;58:770–3. [11] Thame M, Osmond C, Wilks RJ, Bennett FI, McFarlane-Anderson N, Forrester TE. Blood pressure is related to placental volume and birth weight. Hypertension 2000;35:662–7. [12] Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer TE. Maternal nutrition and fetal development. J Nutr 2004;134:2169–72. [13] Gluckman PD, Hanson MA. Living with the past: evolution, development and patterns of disease. Science 2004;305:1773–6. [14] Ozanne SE, Hales CN. Catch-up growth and obesity in male mice. Nature 2004;427:411–2. [15] Xu W, Zhu MW, Lin JF. Fetal growth retardation and postnatal high fat diet on the development of insulin resistance and fertility: experiment with rats. Zhonghua Yi Xue Za Zhi 2007;87:1633–6. [16] Gil FZ, Lucas SR, Gomes GN, Cavanal Mde F, Coimbra TM. Effects of intrauterine food restriction and long-term dietary supplementation with Larginine on age-related changes in renal function and structure of rats. Pediatr Res 2005;57:724–31.
371
[17] Sahajpal V, Ashton N. Renal function and angiotensin AT1 receptor expression in young rats following intrauterine exposure to a maternal low-protein diet. Clin Sci 2003;104:607–14. [18] do Carmo Pinho Franco M, Nigro D, Fortes ZB, Tostes RC, Carvalho MH, Lucas SR, et al. Intrauterine undernutritiondrenal and vascular origin of hypertension. Cardiovasc Res 2003;60:228–34. [19] Alexander BT. Intrauterine growth restriction and reduced glomerular number: role of apoptosis. Am J Physiol Regul Integr Comp Physiol 2003;285:R933–4. [20] Pham TD, MacLennan NK, Chiu CT, Laksana GS, Hsu JL, Lane RH. Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney. Am J Physiol Regul Integr Comp Physiol 2003;285:R962–70. [21] Arthur JM, Thongboonkerd V, Scherzer JA, Cai J, Pierce WM, Klein JB. Differential expression of proteins in renal cortex and medulla: a proteomic approach. Kidney Int 2002;62:1314–21. [22] Thongboonkerd V, Barati MT, McLeish KR, Benarafa C, Remold-O’Donnell E, Zheng S, et al. Alterations in the renal elastin-elastase system in type 1 diabetic nephropathy identified by proteomic analysis. J Am Soc Nephrol 2004;15:650–62. [23] Thongboonkerd V. Proteomics in nephrology: current status and future directions. Am J Nephrol 2004;24:360–78. [24] Schaub S, Rush D, Wilkins J, Gibson IW, Weiler T, Sangster K, et al. Proteomic-based detection of urine proteins associated with acute renal allograft rejection. J Am Soc Nephrol 2004;15:219–27. [25] Weinkauf M, Hiddemann W, Dreyling M. Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background. Electrophoresis 2006;27:4555–8. [26] Karp NA, Lilley KS. Investigating sample pooling strategies for DIGE experiments to address biological variability. Proteomics 2009;9:388–97. [27] Jia SQ, Niu ZJ, Zhang LH, Zhong XY, Shi T, Du H, et al. Identification of prognosis-related proteins in advanced gastric cancer by mass spectrometry-based comparative proteomics. J Cancer Res Clin Oncol 2009;135:403–11. [28] Merkwirth C, Langer T. Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis. Biochim Biophys Acta 2009;1793:27–32. [29] Guo W, Xu H, Chen J, Yang Y, Jin JW, Fu R, et al. Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1. Mol Cell Biochem 2007;295: 167–77.