help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0765
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buffat, C.
Right arrow Articles by Simeoni, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buffat, C.
Right arrow Articles by Simeoni, U.
Endocrinology Vol. 148, No. 11 5549-5557
Copyright © 2007 by The Endocrine Society

Kidney Gene Expression Analysis in a Rat Model of Intrauterine Growth Restriction Reveals Massive Alterations of Coagulation Genes

Christophe Buffat, Farid Boubred, Françoise Mondon, Sonia T. Chelbi, Jean-Marc Feuerstein, Martine Lelièvre-Pégorier, Daniel Vaiman and Umberto Simeoni

Laboratoire de Biochimie et de Biologie Moléculaire (C.B.), Hôpitaux La Conception, Assistance Publique-Hôpital de Marseille (AP-HM), and Unité Propre de Recherche de l’Enseignement Supérieur, Equipe d’Accueil 2193 Faculté de Médecine, 13005 Marseille, France; Service de Médecine Néonatale (F.B., U.S.), Hôpital de La Conception, AP-HM, 13005 Marseille, France, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 608, Université de la Méditerranée, 13284 Marseille, France; Département de Génétique et Développement (F.M., S.T.C., D.V.), Institut Cochin, INSERM Unité (U) 567, Centre National de la Recherche Scientifique UMR 8104, and Faculté de Médecine René Descartes, Université Paris V, 75014 Paris, France; Centre de Formation et de Recherche Expérimental Medico-Chirurgicale (J.-M.F.), Faculté de Médecine, 13385 Marseille, France; INSERM U872 (M.L.-P.), Centre de recherche des Cordeliers, Université René Descartes (Paris 5), 75006 Paris, France; and Institut National de la Recherche Agronomique Department of Animal Genetics (D.V.), 78352 Jouy-en Josas, France

Address all correspondence and requests for reprints to: Daniel Vaiman, Département de Génétique et Développement, Institut Cochin, Institut National de la Santé et de la Recherche Médicale Unité 567, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, and Faculté de Médecine René Descartes, Université Paris V UM 3, 75014 Paris, France. E-mail: vaiman{at}cochin.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
In this study, low birth weight was induced in rats by feeding the dams with a low-protein diet during pregnancy. Kidneys from the fetuses at the end of gestation were collected and showed a reduction in overall and relative weight, in parallel with other tissues (heart and liver). This reduction was associated with a reduction in nephrons number. To better understand the molecular basis of this observation, a transcriptome analysis contrasting kidneys from control and protein-deprived rats was performed, using a platform based upon long isothermic oligonucleotides, strengthening the robustness of the results. We could identify over 1800 transcripts modified more than twice (772 induced and 1040 repressed). Genes of either category were automatically classified according to functional criteria, making it possible to bring to light a large cluster of genes involved in coagulation and complement cascades. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites, suggesting an overrepresentation of the AP1R binding site, together with the transcription induction of factors actually binding to this site in the set of induced genes. The induction of coagulation cascades in the kidney of low-birth-weight rats provides a putative rationale for explaining thrombo-endothelial disorders also observed in intrauterine growth-restricted human newborns. These alterations in the kidneys have been reported as a probable cause for cardiovascular diseases in the adult.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
INTRAUTERINE GROWTH restriction (IUGR), one of the known causes of low birth weight (LBW), can be defined as a failure to reach the genetic growth potential of a given fetus. Maternal diet or placental dysfunction during pregnancy may induce altered placental nutrient transfer (1, 2, 3) and slow down fetus development, thus constituting possible causes of IUGR. Nevertheless, molecular mechanisms are at work to best exploit the available resources, according to the thrifty phenotype hypothesis (4, 5). These adaptations, which take place during a long period of pregnancy, may induce short-term and long-term cellular and tissue alterations that will be memorized until adulthood, possibly by epigenetic mechanisms. These tissue alterations can materialize as the so-called metabolic syndrome (6, 7, 8), associating hypertension, impaired glucose tolerance, type 2 diabetes, and obesity in adult life (7, 9, 10, 11, 12, 13, 14, 15). Although mechanistic causes explaining the links between fetal and adult alterations are largely unknown, a programmed effect on renal function can be suspected as possible grounds for such disorders. Consistently, in animal models and humans, LBW has been associated with a reduced number of nephrons at birth (16, 17). Nephrons are highly differentiated structures, whose number is fixed from birth, suggesting that their reduction in number constitutes a possible way toward cardiovascular dysfunction in the adult. Indeed, nephron reduction is reported as an important mechanism in the developmental origin of cardiovascular diseases (16, 18, 19, 20, 21, 22, 23, 24).

Kidney development is a complex process involving tightly controlled expression of a number of genes and constant remodeling, implicated in the development of specific areas in the kidney, from the glomeruli in the cortex to the collecting tubes in the inner medulla (25). Numerous factors can adversely affect nephrogenesis, and molecular mechanisms altering nephron number and function are not yet completely elucidated.

In the present study, we submitted pregnant female rats to a low-protein (LP) diet during pregnancy, thereby inducing LBW. Kidney size and structure were significantly altered.

cDNAs from fetal kidneys were analyzed on a Rattus norvegicus 60-mer oligonucleotide microarray (Nimblegen). Firstly, genes known to play an important function in renal development were analyzed; then, a more systematic analysis was performed, showing that over 1800 transcripts were found significantly modified (at a 2-fold threshold). These genes were automatically classified into functional groups showing that one of the most prominent features of the expressional alterations in the IUGR kidney is the massive induction of genes involved in coagulation and complement function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals and diets
The local ethical committee for animal research at Marseille Medical University approved the experiments. Female virgin Sprague Dawley rats (Charles Rivers, l’Abresle, France) weighing 225–250 g were mated with the same male rat. Gestation d 1 of pregnancy was determined by the presence of spermatozoa in a vaginal smear. The animals were housed with free access to food and water under controlled light (12-h light, 12-h dark cycle) and temperature (22 ± 3 C). Pregnant rats were randomly divided into two groups. Dams of the control group (n = 5) were then housed individually and fed throughout gestation a 22% protein diet, or an isocaloric 9% protein diet, representing the LP diet (n = 6). Both diets were isocaloric, because the protein deficiency in the LP diet was compensated by the addition of carbohydrates. Feeding of the LP diet began on the day of conception and continued throughout pregnancy. The detailed composition is given in supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), showing that the ration was supplemented in vitamins in the LP diet (premix vitamin 200, 1 vs. 0.5% in the normal diet). A total of 77 and 67 fetuses were weighed for the LP diet and the control group, respectively. The average number of pups per female was not different in the two groups (13.7 ± 3.6 vs. 13.8 ± 4.5, Student’s t test = 0.96). The distribution of the pup weights is presented as supplemental Fig. 1Go.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 1. Comparison of induction ratios between qRT-PCR and microarray data. For five genes of the coagulation/complement cascade (Cpb2, Fgg, Bdkr, ProC, and Cfi), two positions corresponding to Nimblegen features were analyzed by qRT-PCR. The primer positions are given by reference with the accession no. given in supplemental Table 2. The graph presents the correlation between the two methods of analyzing gene expression, suggesting a very good fit between qRT-PCR and the array data.

 
Tissue collection
For collection of placental and fetal tissues, pregnant rats were anesthetized with halothane/nitrous oxide on d 20 of gestation, and each fetus and placenta were removed and weighed. Neonates were killed at birth and were weighed. Placentas, left kidneys, livers, lungs, and hearts of each live pup were rapidly excised, weighed, snap-frozen in liquid nitrogen, and then stored at –80 C. Right kidneys were used for estimating glomerular number and stored in sodium azide buffer (NaN3).

Nephron count
The total number of nephrons was determined in the entire kidney, as previously described (26). Briefly, and following a classical protocol for glomeruli separation and counting, whole kidneys were incubated in 50% hydrochloric acid (6 M) for 45 min at 37 C, rinsed with tap water, and stored overnight at 4 C in a gauged flask. After mechanical dissociation, tubules and glomeruli were suspended in water. Three 0.5-ml aliquots were taken and placed in a hemocytometric-like chamber, and the glomeruli were counted under microscope by three different investigators who were unaware of the specimen origin. The three results were averaged, and then this value was used to determine the total number of glomeruli in the sample and therefore in the kidney.

RNA preparation, cDNA synthesis, and microarray hybridizations
Total RNA was extracted in Trizol according to standard protocols, treated with RNase-free DNase and quantified by spectrophotometry. For microarrays, 20 µg total RNA from a pool of 40 kidneys obtained from IUGR pups and a pool of 40 kidneys obtained from control pups were analyzed on the Nimblegen platform in Reykjavik, Iceland. The RNAs were prepared using pools of pups equilibrated between both genders and equilibrated between the different dams. Labeling, hybridization, data collection, and normalization were carried out according to standardized protocols. A total of 390,000 oligonucleotides representing 23,456 transcripts from the rat genome were hybridized with fluorescently labeled cDNAs from pools of kidneys from IUGR rat pups vs. pools of kidneys from control rat pups. In this system, each transcript was represented by eight oligonucleotides spotted randomly in duplicates. Induction ratios were calculated by dividing the fluorescence value observed in the LP diet by the fluorescence value obtained in the control diet. This was done for each oligonucleotide corresponding to a given gene, and then the average was taken as the induction ratio. This way, possible effects on specific isoforms are masked, but the complete dataset is available as GEO profiles on the GEO (Gene Expression Omnibus) database. This database processing was accomplished according to the MIAME quality control and was validated under the requirements of MIAME (reviewer account login, www.ebi.ac.uk/aerep/login Username:Reviewer_E-MEXP-1015; password, 1172841983358).

Primers for RT-PCR analysis
Primers (supplemental Table 2) were chosen using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3), designed from the coding sequence of each gene of interest and generally overlapping two consecutive exons (http://www.ncbi.nlm.nih.gov). The different couples were aligned against the rat genome using BLAST to check their specificity. Primer sequences are provided as supplemental information.

Quantitative RT-PCR (qRT-PCR) analysis
Quantitative PCR was carried out starting from 2 µl of a 10x dilution of the cDNA in a volume of 17 µl using a LightCycler thermocycler (Roche, Indianapolis, IN), and the amplification kit Platinum SYBR Green (Invitrogen, Carlsbad, CA). The MasterMix was complemented with MgCl2 (4 mM final concentration), BSA (0.05 g/liter), and primers (10–3 M). The PCR program was as follows: 50 C for 120 sec; 95 C for 120 sec; 35 cycles of 94 C for 5 sec, 58 C for 10 sec, and 72 C for 30 sec; and 65 C for 15 sec, followed by a raise to 99 C at 0.1 C/sec. This last step was systematically used to obtain the fusion curve, making it possible to check the homogeneity of the PCR products. This was further confirmed by agarose electrophoresis. Cycle threshold (Ct) values were collected with the LightCycler software in the exponential phase of the PCR. The Ct values of the gene of interest were normalized by the Ct value obtained for the 18S gene taken as a reporter gene, using the 2–{Delta}{Delta}Ct approach as outlined by Livak and Schmittgen (27).

Functional clustering by DAVID (Database for Annotation and Visualization and Integrated Discovery)
Two lists of genes induced or repressed twice or more in the kidneys were submitted to the DAVID database (28). Briefly, DAVID clusterizes genes from a list according to a series of keywords common to several genes from the list. The proportion of each keyword from the gene list submitted is compared with the proportion in the whole genome, making it possible to compute a P value. Then enrichment values are calculated as the geometric mean of the inverse log of each P value. These enrichment scores depend on the number of genes present in the list. Therefore, we used a simulation to define minimal enrichment thresholds enabling us to consider gene clusters given by DAVID for further analysis (60). The groups were considered as significant if they were above an enrichment value of 2.55 for the repressed genes and 2.01 for the induced genes.

Statistics
Two-way ANOVA was used to show that no dam-specific effects could be observed on the birth size of the pups inside the two groups analyzed (LP vs. control diet). The Student’s t test was used to compare the organ and body weights in the two conditions. For the organs analyzed, the tests were performed either without correcting for weight or after division by the body weight to correct for proportional organ reduction. The results are presented in Table 1Go. As shown in the table, although all the values were significant when considered without correction, only liver, heart, and kidneys remained significantly reduced in weight when the body weight is taken into consideration.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Body weight and placenta, liver, lung, heart, and kidney weights and number of nephrons in LP group and in control pups group

 
For identifying promoters significantly enriched in putative transcription factor binding sites (TFBS), the promoters of the 10 most repressed and the 10 most induced genes were recovered using the Genomatix software (Genomatix.de), and putative TFBS were recovered using the Gene2Promoter procedure. Then, a table listing the possible TFBS in rows and the promoters in columns was constructed, where the number of putative TFBS present in each promoter was analyzed. Three independent statistical tests were performed. A correlation was calculated between the number of TFBS and the log2 of the induction ratio of each gene. The significance of these correlations was evaluated using the Vassarstat suite at http://faculty.vassar.edu/lowry/VassarStats.html (threshold: |r| > 0.36). Also, Student’s t tests were performed to compare the induced and repressed genes concerning their content in putative TFBS; finally, a permutation test (r test) was performed, and the P value was calculated from 10,000 iterations. In Table 3Go are presented the seven putative TFBS presenting a significant value for the three tests.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Putative TFBS enriched in genes induced or repressed in IUGR kidneys

 

   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Expression analysis of specific genes involved in kidney function and development
Body, lung, liver, kidney, heart, and placenta weights are presented in Table 1Go. IUGR effects were tested on the organ weight, with and without correction by the body weight. All absolute organ weights were significantly reduced in the LBW animals compared with normal fetuses. Relative organ weights remained significantly lower for liver, heart, and kidney, suggesting a specific allometric size/mass reduction. Lung and placenta relative weights were not significantly different between LBW and control groups, suggesting that the reduction in size of these organs is a direct consequence of the decreased body weight. The comparable weight reduction between the placenta and the fetus is consistent with the idea of a commensurate growth of the fetoplacental unit. It is known from the literature that imprinted genes regulate precisely the relative growth of the fetus and of the placenta. For instance, Igf2 (expressed from the male allele) is known to increase placental growth (29, 30), whereas h19 and the IGF-II receptor (Igf2r), expressed from the female allele, tend to counterbalance these genes by increasing fetal growth (31, 32). Concerning the other organs, the absolute and relative weight reduction suggests the existence of organ-specific mechanisms. The kidney was studied more extensively, and in particular, we were able to observe a significant diminution of the number of nephrons, consistent with a few reports of human cases of IUGR (23, 33).

In the microarray dataset, the fluorescence level was consistent between the two duplicates (r2 of 0.996 and 0.996 for the fluorescence values of the 23,456 transcripts in IUGR and controls, respectively). Considering a threshold of 2:1 as significant for induction or repression, 1040 transcripts were found transcriptionally repressed, whereas 772 were found transcriptionally induced in the IUGR kidney compared with the controls. To validate these findings, qRT-PCR analysis was performed on 10 genes (Fig. 1Go). For five of them, two regions of the gene corresponding to two oligonucleotides on the arrays were analyzed. Overall, a very good fit was found between the two approaches, suggesting a high robustness of microarrays based on long isothermic oligonucleotides. The calculated correlation r was estimated at 0.79 (r2 = 0.62) yielding a P value of 0.00027 as evaluated using the Vassarstat software.

As a first attempt to interpret these data in relation with kidney function, literature was used to identify relevant target genes putatively modified by diet-induced LBW. Fifty-four genes reported as crucial for kidney development and function were selected (Table 2Go). Odd1, Slit2, Wnt4, Igf1, and Cdh11 were found modified above the chosen threshold (2.2-, 2.2-, 2.0-, 2.2-, and 2.4-fold, respectively), whereas the forty-nine other genes were not significantly changed by diet-induced IUGR in the kidney. Statistical comparison of these 49 genes with the microarray data concerning the remaining genes showed a significant increase in the proportion of modified transcripts (P = 0.016). However, the observed transcription alterations were mild. This prompted us to switch to a more systematic approach to bring to light more relevant putative transcriptional alterations in the IUGR kidney.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Analysis of genes involved in kidney development and function from the literature

 
Systematic analysis of the microarray data
To interpret the composition of the groups of induced and repressed genes, the automatic functional clustering tool developed by the National Institutes of Health, DAVID, was used (28). A threshold for the enrichment index given by DAVID was calculated by a simulation as described in Materials and Methods. The clusters above the calculated enrichment threshold are represented on Fig. 2Go, A and B. In repressed genes, a major cluster was identified encompassing mainly G protein-coupled receptors, themselves highly enriched in olfactory receptors, the largest superfamily of transcriptionally active genes in rodents. Such genes that are probably not relevant in the kidney are strongly repressed when environmental conditions are not optimal.


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 2. Functional clusters of genes induced in kidneys from LBW rat neonates after protein restriction of the mother’s diet (A) and genes repressed in the same kidneys (B). The left ordinate represents the enrichment factor as defined, represented by histograms (see text), and the right ordinate (line) represents the number of genes present in each cluster.

 
Functional clustering of induced genes reveals the existence of an important group of factors involved in coagulation.
An important cluster of genes involved in coagulation and complement was clearly discriminated in the induced genes (enrichment score = 11.2). This cluster corresponds to a group of 28 genes positioned at strategic positions in the coagulation cascade. A second cluster of genes presenting a related function (proteolysis and coagulation) encompassed 29 genes with an enrichment score of 3.8. These microarray results were validated by qRT-PCR for a series of genes, Cpb2, Fg (fibrinogen {gamma}-chain precursor), Bdkr, ProC (protein C), Cfi (complement factor and mannan-binding protein), presenting induction ratios varying on the array from a 9.6-fold induction for Cpb2 to a 2.2-fold reduction for Bdkr.

Pro- and antithrombotic genes are induced in the IUGR kidney.
A summary of these coagulation factors is represented on Fig. 3Go, based upon the coagulation/complement pathway available in the KEGG database (34). A critical reading and analysis of this representation suggests that only some specific coagulation pathways are enhanced in IUGR, whereas some others are strictly controlled. Indeed, many pivotal genes involved in coagulation and clot formation were transcriptionally induced, such as prothrombin (induced 4.2-fold) and the {alpha}-peptide of fibrinogen (induced 6.3-fold). Thrombin formation occurs when factor III (F3 or tissue factor 3), the initiator of the coagulation cascade, complexes with factor VII (F7) to directly or indirectly activate factor X (F10). As shown in the figure, the protein restriction induces a mild transcript increase for F3 and F7 (1.6- and 1.5-fold, respectively) and a strong increase of F10, downstream in the cascade (3.6-fold). Tfpi (tissue factor pathway inhibitor, induced 2-fold) acts at this level as a factor Xa-dependent inhibitor for the factor VIIa-tissue factor complex. However, as previously mentioned, the induction of prothrombin transcript (F2) is manifest (4.2-fold). Despite the inhibitory action of serine protease inhibitor (SERPIN) d1 (heparin cofactor II) susceptible at this level to prevent activation of fibrinogen by thrombin, there is probably a bypass allowing increased fibrinogen transcription (3.6-fold).


Figure 3
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 3. An example of pathway abnormally regulated in kidneys from LBW rats (complement and coagulation KEGG pathway), with induction ratios derived from the rat microarray data. Induced genes are represented in blue, repressed genes in red. Compared with the all-data set, the genes in this pathway are induced in average 2.44-fold (P = 5 x 10–88), demonstrating the specificity of coagulation alterations in LBW rat neonates.

 
In parallel, anticoagulating factors were found induced at the transcription level. Among those, a strong induction of Serpind1 (induced 6.3-fold), Serpinc1 (induced 4.5-fold), and Proc (induced 4.2-fold) was observed. As shown in the KEGG diagram, these genes are located at strategic nodes in the coagulation pathway and presumably help to inhibit clot formation in the kidney. Several studies have demonstrated that genes belonging to the SERPIN family are key homeostatic regulators (35). It has recently been shown that their transcriptional alterations in placental diseases are associated with a modified epigenetic memory [specific CpG methylations in preeclampsia and IUGR, for SERPIN a3 ({alpha}1-antitrypsin) (36) and SERPIN b5 (maspin) (37)].

Cpb2, one of the most highly induced factors in the LBW kidney, may increase the severity of kidney long-term alterations by favoring clotting.
Cpb2 (carboxypeptidase B2, also called TAFI for thrombin-activatable fibrinolysis inhibitor) (38), was induced 9.6-fold in LBW kidneys and therefore is one of the most highly activated genes in this organ. The KEGG image represents Cpb2 as an anticoagulation factor preventing fibrin monomers to polymerize, thus probably contributing to the circumvention of clot formation. However, literature suggests that the function of Cpb2 is rather prothrombotic, because most studies show that activated Cpb2 (TAFIa) protects the fibrin clot against lysis (39). TAFI inhibition decreased the time of onset of fibrinolysis by 70% (40). Consistently, epidemiologic studies and work in animals, suggested that elevated levels of plasma Cpb2 are associated with a mildly elevated risk for venous thrombosis (41), whereas the protein has been found significantly elevated (50%) in patients with type 2 diabetes (42). More generally, it has been suggested that variations of plasma Cpb2 antigen levels and potential alterations in TAFI regulation, constitute risk factors for the development of vascular diseases (43, 44). These converging findings may be slightly alleviated by the fact that several reports on single-nucleotide polymorphism association with Cpb2 levels and clot formation failed to yield consistent associations (45, 46, 47) (for a recent review, see Ref. 48). In summary, most data indicate that a high level of Cpb2 transcripts observed in LBW could play a deleterious role in the kidney by favoring clot stabilization and ultimately thrombus formation.

Also, Cpb2 acts as a major regulator of inflammation via its action on the bradykinin and complement systems, both actions not being represented in the KEGG pathway; Cpb2 cleaves the C-terminal basic amino acids, arginine and lysine, from inflammatory peptides such as complement C3a and C5a fractions, and bradykinin (49, 50, 51). In our study of diet-induced LBW, the down-regulation of the bradykinin receptor (2.1-fold) suggests the involvement of an additional level of inflammation control.

Therefore, alterations of Cpb2 in the LBW kidney makes it a relevant novel target for the understanding of the developmental origins of the metabolic syndrome, after the birth of small newborns. However, given that our study material is limited to newborn animals, these findings need to be confirmed by additional studies involving adult animals from LBW and control newborns. von Willebrand factor (a plasma glycoprotein that mediates platelet adhesion to damaged blood vessels and stabilizes blood coagulation factor VIII) was not modified in our study, whereas thrombomodulin mRNA level was increased 2.4-fold. Thrombomodulin is a cell surface glycoprotein that plays an important role in the protein C anticoagulant pathway. This latter protein is an important marker of endothelial cell injury, and the thrombin-thrombomodulin complex is known as an activator of Cpb2 (52, 53).

The induction of complement factors may increase cell lysis in the IUGR kidney.
Several elements of the complement cascade were strongly up-regulated by IUGR, namely BF (complement factor B), C3, C2, C4, C6, and C9, although inhibitory pathways were also activated such as IF (complement factor I). Because final components of the cascade, such as C9 were transcriptionally activated at a high level (5.2-fold), this suggests that cell lysis is favored in the IUGR kidney. This observation could be connected with the decrease in kidney size and nephrons number observed in the IUGR rats. This is consistent with the observation of complement component activation leading to DNA damage in podocytes, visceral epithelial cells of the glomeruli (54).

As shown above, part of the energetic resources allotted to the kidney of LBW rats is turned toward the prevention of blood clotting, especially with the induction of several SERPINs (Serpin a1, a3, a6, a8, b6, c1, and d1), thus suggesting that these genes are induced to protect the kidney from partial destruction. This dysregulation may well result in unwanted epigenetic modifications, programming the abnormal transcriptional activity of genes that should be silenced in the normal adult kidney.

Search for common features in the promoter of IUGR-induced genes
To find logic in the network of gene inductions observed in IUGR, we compared the composition in putative TFBS in the proximal promoters of the 10 most induced genes vs. the promoters of the 10 most repressed genes. The promoters were recovered using the Genomatix Gene2Promoter procedure (http://www.genomatix.de/), as described in Materials and Methods. Three independent statistical approaches were used to identify putative binding sites that were enriched in the promoters of induced genes. These sites were V$COMP, V$AP1R, V$MYBL, V$HAML, V$TALE, V$P53F, and V$NOLF (Table 3Go). Only the last two sites were enriched in repressed genes, whereas the five others were prominent in induced genes. We then interrogated the array data to discover whether transcription factors binding to these sites were transcriptionally increased by protein restriction in the kidney. Among these putative binding sites, V$AP1R is reported to be bound by BTB/POZ ZNF (BR-C, T+k and Bab/Pox virus Zinc Finger Proteins) proteins BACH (BTB and CNC homology, basic leucine zipper transcription factor) 1, BACH2, MAF [v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)], TCF11 [also called NFE2L1; nuclear factor (erythroid-derived 2)-like 1], NRF2 [also called NFE2L2; nuclear factor (erythroid-derived 2)-like 2], and NFE2 [nuclear factor (erythroid-derived 2)]. We found a transcriptional activation for two members of BTB/POZ ZNF proteins (Kbtbd3 and Btbd3, induced 2.4- and 2.2-fold, respectively), for NFE2 (2.7-fold) and a similar trend for BACH1 and NRF2 (1.63- and 1.63-fold, respectively). We did not find other cases of consistent transcriptional increase in transcription factors for the other binding sites. These results are much less clear-cut than the ones we observed in the IUGR placentas in the same rat model (60). Nevertheless the AP1R pathway is an interesting trail to explore a putative hierarchical structure of transcription alterations in the IUGR kidney.

Conclusions
Consistent with our present findings in the rat model, different reports show that human newborns are at increased risk for thrombosis due to unique properties of their hemostatic system (55, 56, 57) and that these deleterious effects are enhanced in pregnancies complicated with IUGR (58, 59). These observations suggest that the coagulation deregulation in the kidney of LBW rats, brought to light in our study, may also be relevant for human disease in adulthood.


   Acknowledgments
 
Prof. Laurence Camoin-Jau and Prof. Marie-Christine Alessi are warmly thanked for a critical reading of the manuscript as well as Dr. Ilyes Kamoun for English corrections.


   Footnotes
 
This work was partially funded by a Programe Hospitalier de Recherche Clinique "Programation of cardiovascular diseases during perinatal development." C.B. was funded by grants obtained from Chiesi, Astra-Zeneca, and APRESAN. S.T.C. is a recipient of a Ph.D. fellowship from the French Ministry of Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 16, 2007

Abbreviations: Ct, Cycle threshold; IUGR, intrauterine growth restriction; LBW, low birth weight; LP, low-protein; qRT-PCR, quantitative RT-PCR; SERPIN, serine protease inhibitor; TAFI, thrombin-activatable fibrinolysis inhibitor; TFBS, transcription factor binding sites.

Received June 11, 2007.

Accepted for publication August 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Matthews JC, Beveridge MJ, Malandro MS, Kilberg MS, Novak DA 1998 Response of placental amino acid transport to gestational age and intrauterine growth retardation. Proc Nutr Soc 57:257–263[CrossRef][Medline]
  2. Myatt L 2006 Placental adaptive responses and fetal programming. J Physiol 572(Pt 1):25–30
  3. Cross JC, Nakano H, Natale DR, Simmons DG, Watson ED 2006 Branching morphogenesis during development of placental villi. Differentiation 74:393–401[CrossRef][Medline]
  4. Neel JV 1962 Diabetes mellitus: a "thrifty" genotype rendered detrimental by "progress"? Am J Hum Genet 14:353–362[Medline]
  5. Hales CN, Barker DJ 1992 Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 35:595–601[CrossRef][Medline]
  6. Barker DJ, Osmond C 1986 Diet and coronary heart disease in England and Wales during and after the second world war. J Epidemiol Commun Health 40:37–44[Abstract/Free Full Text]
  7. Barker DJ 1989 Rise and fall of Western diseases. Nature 338:371–372[CrossRef][Medline]
  8. Barker DJ 1992 The fetal origins of adult hypertension. J Hypertens Suppl 10:S39–S44
  9. Lumey LH 1992 Decreased birthweights in infants after maternal in utero exposure to the Dutch famine of 1944–1945. Paediatr Perinat Epidemiol 6:240–253[Medline]
  10. Law CM, Martyn CN, Fall CH, Osmond C 1996 Raised adult blood pressure linked to failure to achieve growth potential in utero. BMJ 312:1479[Free Full Text]
  11. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS 1993 Fetal nutrition and cardiovascular disease in adult life. Lancet 341:938–941[CrossRef][Medline]
  12. Phipps K, Barker DJ, Hales CN, Fall CH, Osmond C, Clark PM 1993 Fetal growth and impaired glucose tolerance in men and women. Diabetologia 36:225–228[CrossRef][Medline]
  13. Ravelli AC, van der Meulen JH, Michels RP, Osmond C, Barker DJ, Hales CN, Bleker OP 1998 Glucose tolerance in adults after prenatal exposure to famine. Lancet 351:173–177[CrossRef][Medline]
  14. Barker DJ 2002 Fetal programming of coronary heart disease. Trends Endocrinol Metab 13:364–368[CrossRef][Medline]
  15. Ravelli GP, Stein ZA, Susser MW 1976 Obesity in young men after famine exposure in utero and early infancy. N Engl J Med 295:349–353[Abstract]
  16. Langley-Evans SC, Sherman RC, Welham SJ, Nwagwu MO, Gardner DS, Jackson AA 1999 Intrauterine programming of hypertension: the role of the renin-angiotensin system. Biochem Soc Trans 27:88–93[Medline]
  17. Merlet-Benichou C, Gilbert T, Muffat-Joly M, Lelievre-Pegorier M, Leroy B 1994 Intrauterine growth retardation leads to a permanent nephron deficit in the rat. Pediatr Nephrol 8:175–180[CrossRef][Medline]
  18. Hoy WE, Hughson MD, Bertram JF, Douglas-Denton R, Amann K 2005 Nephron number, hypertension, renal disease, and renal failure. J Am Soc Nephrol 16:2557–2564[Free Full Text]
  19. Hoy WE, Kondalsamy-Chennakesavan S, McDonald S, Wang Z 2006 Renal disease, the metabolic syndrome, and cardiovascular disease. Ethn Dis 16(Suppl 2):46–51
  20. Keller G, Zimmer G, Mall G, Ritz E, Amann K 2003 Nephron number in patients with primary hypertension. N Engl J Med 348:101–108[Abstract/Free Full Text]
  21. Ortiz LA, Quan A, Zarzar F, Weinberg A, Baum M 2003 Prenatal dexamethasone programs hypertension and renal injury in the rat. Hypertension 41:328–334[Abstract/Free Full Text]
  22. Woods LL 1999 Neonatal uninephrectomy causes hypertension in adult rats. Am J Physiol 276:R974–R978
  23. Manalich R, Reyes L, Herrera M, Melendi C, Fundora I 2000 Relationship between weight at birth and the number and size of renal glomeruli in humans: a histomorphometric study. Kidney Int 58:770–773[CrossRef][Medline]
  24. Brenner BM, Chertow GM 1994 Congenital oligonephropathy and the etiology of adult hypertension and progressive renal injury. Am J Kidney Dis 23:171–175[Medline]
  25. Chabardes-Garonne D, Mejean A, Aude JC, Cheval L, Di Stefano A, Gaillard MC, Imbert-Teboul M, Wittner M, Balian C, Anthouard V, Robert C, Segurens B, Wincker P, Weissenbach J, Doucet A, Elalouf JM 2003 A panoramic view of gene expression in the human kidney. Proc Natl Acad Sci USA 100:13710–13715[Abstract/Free Full Text]
  26. Amri K, Freund N, Vilar J, Merlet-Benichou C, Lelievre-Pegorier M 1999 Adverse effects of hyperglycemia on kidney development in rats: in vivo and in vitro studies. Diabetes 48:2240–2245[Abstract]
  27. Livak K, Schmittgen T 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCt method. Methods 25:402–408[CrossRef][Medline]
  28. Dennis Jr G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA 2003 DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4:P3
  29. DeChiara TM, Robertson EJ, Efstratiadis A 1991 Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64:849–859[CrossRef][Medline]
  30. Constancia M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, Fundele R, Stewart F, Kelsey G, Fowden A, Sibley C, Reik W 2002 Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 417:945–948[CrossRef][Medline]
  31. Ripoche MA, Kress C, Poirier F, Dandolo L 1997 Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element. Genes Dev 11:1596–1604[Abstract/Free Full Text]
  32. Wang ZQ, Fung MR, Barlow DP, Wagner EF 1994 Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372:464–467[CrossRef][Medline]
  33. Hinchliffe SA, Lynch MR, Sargent PH, Howard CV, Van Velzen D 1992 The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obstet Gynaecol 99:296–301[Medline]
  34. Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M 1999 KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 27:29–34[Abstract/Free Full Text]
  35. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, Irving JA, Lomas DA, Luke CJ, Moyer RW, Pemberton PA, Remold-O’Donnell E, Salvesen GS, Travis J, Whisstock JC 2001 The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276:33293–33296[Free Full Text]
  36. Chelbi ST, Mondon F, Jammes H, Buffat C, Mignot TM, Tost J, Busato F, Gut I, Rebourcet R, Laissue P, Tsatsaris V, Goffinet F, Rigourd V, Carbonne B, Ferre F, Vaiman D 2007 Expressional and epigenetic alterations of placental serine protease inhibitors: SERPINA3 is a potential marker of preeclampsia. Hypertension 49:76–83[Abstract/Free Full Text]
  37. Chim SS, Tong YK, Chiu RW, Lau TK, Leung TN, Chan LY, Oudejans CB, Ding C, Lo YM 2005 Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci USA 102:14753–14758[Abstract/Free Full Text]
  38. Bajzar L, Manuel R, Nesheim ME 1995 Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. J Biol Chem 270:14477–14484[Abstract/Free Full Text]
  39. Mutch NJ, Thomas L, Moore NR, Lisiak KM, Booth NA 2007 TAFIa, PAI-1 and {alpha}-antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots. J Thromb Haemost 5:812–817[CrossRef][Medline]
  40. Nielsen VG, Ellis TC 2007 Quantification of the effects of thrombin activatable fibrinolysis inhibitor and {alpha}2-antiplasmin on fibrinolysis in normal human plasma. Blood Coagul Fibrinolysis 18:29–33[Medline]
  41. van Tilburg NH, Rosendaal FR, Bertina RM 2000 Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood 95:2855–2859[Abstract/Free Full Text]
  42. Yano Y, Kitagawa N, Gabazza EC, Morioka K, Urakawa H, Tanaka T, Katsuki A, Araki-Sasaki R, Hori Y, Nakatani K, Taguchi O, Sumida Y, Adachi Y 2003 Increased plasma thrombin-activatable fibrinolysis inhibitor levels in normotensive type 2 diabetic patients with microalbuminuria. J Clin Endocrinol Metab 88:736–741[Abstract/Free Full Text]
  43. Juhan-Vague I, Renucci JF, Grimaux M, Morange PE, Gouvernet J, Gourmelin Y, Alessi MC 2000 Thrombin-activatable fibrinolysis inhibitor antigen levels and cardiovascular risk factors. Arterioscler Thromb Vasc Biol 20:2156–2161[Abstract/Free Full Text]
  44. Leurs J, Hendriks D 2005 Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease. Thromb Haemost 94:471–487[Medline]
  45. Morange PE, Aillaud MF, Nicaud V, Henry M, Juhan-Vague I 2001 Ala147Thr and C+1542G polymorphisms in the TAFI gene are not associated with a higher risk of venous thrombosis in FV Leiden carriers. Thromb Haemost 86:1583–1584[Medline]
  46. Kostka H, Kuhlisch E, Schellong S, Siegert G 2003 Polymorphisms in the TAFI gene and the risk of venous thrombosis. Clin Lab 49:645–647[Medline]
  47. Martini CH, Brandts A, de Bruijne EL, van Hylckama Vlieg A, Leebeek FW, Lisman T, Rosendaal FR 2006 The effect of genetic variants in the thrombin activatable fibrinolysis inhibitor (TAFI) gene on TAFI-antigen levels, clot lysis time and the risk of venous thrombosis. Br J Haematol 134:92–94[CrossRef][Medline]
  48. Boffa MB, Koschinsky ML 2007 Curiouser and curiouser: recent advances in measurement of thrombin-activatable fibrinolysis inhibitor (TAFI) and in understanding its molecular genetics, gene regulation, and biological roles. Clin Biochem 40:431–442[CrossRef][Medline]
  49. Aso Y, Wakabayashi S, Yamamoto R, Matsutomo R, Takebayashi K, Inukai T 2005 Metabolic syndrome accompanied by hypercholesterolemia is strongly associated with proinflammatory state and impairment of fibrinolysis in patients with type 2 diabetes: synergistic effects of plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor. Diabetes Care 28:2211–2216[Abstract/Free Full Text]
  50. Saibeni S, Bottasso B, Spina L, Bajetta M, Danese S, Gasbarrini A, de Franchis R, Vecchi M 2004 Assessment of thrombin-activatable fibrinolysis inhibitor (TAFI) plasma levels in inflammatory bowel diseases. Am J Gastroenterol 99:1966–1970[CrossRef][Medline]
  51. Myles T, Nishimura T, Yun TH, Nagashima M, Morser J, Patterson AJ, Pearl RG, Leung LL 2003 Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem 278:51059–51067[Abstract/Free Full Text]
  52. Bajzar L, Jain N, Wang P, Walker JB 2004 Thrombin activatable fibrinolysis inhibitor: not just an inhibitor of fibrinolysis. Crit Care Med 32:S320–S324
  53. Nesheim M, Wang W, Boffa M, Nagashima M, Morser J, Bajzar L 1997 Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thromb Haemost 78:386–391[Medline]
  54. Pippin JW, Durvasula R, Petermann A, Hiromura K, Couser WG, Shankland SJ 2003 DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes. J Clin Invest 111:877–885[CrossRef][Medline]
  55. Nowak-Gottl U, Kosch A, Schlegel N, Salem M, Manco-Johnson M 2002 Thromboembolism in children. Curr Opin Hematol 9:448–453[CrossRef][Medline]
  56. Nowak-Gottl U, Junker R, Kreuz W, von Eckardstein A, Kosch A, Nohe N, Schobess R, Ehrenforth S 2001 Risk of recurrent venous thrombosis in children with combined prothrombotic risk factors. Blood 97:858–862[Abstract/Free Full Text]
  57. Nowak-Gottl U, von Kries R, Gobel U 1997 Neonatal symptomatic thromboembolism in Germany: two year survey. Arch Dis Child Fetal Neonatal Ed 76:F163–F167
  58. von Kries R, Junker R, Oberle D, Kosch A, Nowak-Gottl U 2001 Foetal growth restriction in children with prothrombotic risk factors. Thromb Haemost 86:1012–1016[Medline]
  59. Brenner B 2004 Haemostatic changes in pregnancy. Thromb Res 114:409–414[CrossRef][Medline]
  60. Buffat C, Mondon F, Rigourd V, Boubred F, Bessières B, Fayol L, Feuerstein JM, Gamerre M, Jammes H, Rebourcet R, Miralles F, Basire A, Dignat-Georges F, Carbonne B, Simeoni U, Vaiman D, A hierarchical analysis of transcriptome alterations in intrauterine growth restriction (IUGR) reveals common pathophysiological pathways in mammals. J Pathol, in press
  61. Dressler GR 2006 The cellular basis of kidney development. Annu Rev Cell Dev Biol 22:509–529[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
F. Boubred, L. Daniel, C. Buffat, J.-M. Feuerstein, M. Tsimaratos, C. Oliver, F. Dignat-George, M. Lelievre-Pegorier, and U. Simeoni
Early postnatal overfeeding induces early chronic renal dysfunction in adult male rats
Am J Physiol Renal Physiol, October 1, 2009; 297(4): F943 - F951.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buffat, C.
Right arrow Articles by Simeoni, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buffat, C.
Right arrow Articles by Simeoni, U.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals