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Intrauterine growth restriction modifies the normal gene expression in kidney from rabbit fetuses
Early Human Development 88 (2012) 899–904
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Intrauterine growth restriction modifies the normal gene expression in kidney from rabbit fetuses Horacio Figueroa a, b, Mauricio Lozano c, Cristian Suazo c, Elisenda Eixarch d, e, f, Sebastian E. Illanes a, b, Juan Eduardo Carreño c, Sandra Villanueva c, Edgar Hernández-Andrade d, e, f, g, Eduard Gratacós d, e, f, Carlos E. Irarrazabal c,⁎ a
Department of Obstetrics & Gynecology and Laboratory of Reproductive Biology, Faculty of Medicine, Universidad de los Andes, Santiago, Chile Department of Maternal-Fetal Medicine, Davila Clinic, Santiago, Chile c Laboratory of Molecular Physiology, Faculty of Medicine, Universidad de los Andes, Santiago, Chile d Department of Maternal-Fetal Medicine, Institut Clinic de Ginecologia, Obstetricia i Neonatologia (ICGON), Hospital Clinic, Barcelona, Spain e Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain f Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain g National Institute of Perinatal Medicine, Mexico b
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Article history: Received 15 March 2012 Received in revised form 30 June 2012 Accepted 8 July 2012 Keywords: IUGR Rabbit Inflammation Hypoxia Osmotic stress
a b s t r a c t The aim of this work was to study the effect of intrauterine growth restriction (IUGR) on fetal kidneys. The IUGR was induced by uteroplacental vessels ligature in a model of pregnant rabbit. We centralized the study in the gene expression of essential proteins for fetal kidney development and kidney protection against hypoxia, osmotic stress, and kidney injury. The gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM were studied by qRT-PCR and Western blot in kidneys from control and IUGR fetuses. Experimental IUGR fetuses were significantly smaller than the control animals (39 vs. 48 g, p b 0.05). The number of glomeruli was decreased in IUGR kidneys, without morphological alterations. IUGR increased the gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM (p b 0.05) in kidneys of fetuses undergoing IUGR, suggesting that fetal blood flow restriction produce alterations in gene expression in fetal kidneys. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The IUGR can be defined as a failure to reach the genetic growth potential of a given fetus [1,2]. Maternal health and placental dysfunction during pregnancy may induce altered fetal development. IUGR occur in about 6% of all pregnancies and is associated with increased risk of neonatal complications [3,4], stillbirth [4,5], birth hypoxia [3], and impaired neurodevelopment [5,6]. Numerous evidence in animals and also in humans suggest an association between IUGR and adult diseases, such as diabetes [7,8], hypercholesterolemia [9], cardiovascular diseases and hypertension [10]. During IUGR, normal fetal circulation is modified in a defined pattern. The final stage of this cascade is cardiac hypoxia and heart failure [11]. A considerable number of fetuses with IUGR are associated with altered renal development and present different degree of damage after birth, increasing the risk of hypertension and renal disease.
⁎ Corresponding author at: Molecular Physiology Laboratory, Universidad de los Andes, S. Carlos Apoquindo 2200-Las Condes, Santiago, Chile. Tel.: +56 2 4129607; fax: +56 2 2141756. E-mail address: [email protected] (C.E. Irarrazabal). 0378-3782/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2012.07.010
Using a rat model of IUGR, induced by low-protein diet during pregnancy, the kidney gene expression has been studied. Over 1800 transcripts modified at a 2-fold threshold (722 induced) were identified from IUGR rat kidneys. The up-regulated genes are involved in complement/coagulation, immune system, apoptosis, heme/globin and Calycin/lipocalin/retinol. The induction of coagulation cascade in kidney provides a putative explanation of thrombo-endothelial disorders observed in intrauterine growth-restricted human newborns and increased risk of cardiovascular diseases in adult [12], suggesting that impair fetal nutrition induces kidney injury. The mammalian kidney has a gradient of tonicity and partial pressure of oxygen (PO2). The kidney medulla is hypertonic (800 mosmol) and hypoxic (10–18 mm Hg) compared with cortex (300 mosmol and 40–50 mm Hg). The kidney cells have molecular mechanisms to adapt and survive to this extreme environment. A declination in PO2 (hypoxia) induces the transcription factor, HIF-1α (Hypoxia induced factor). On the other hand, an increase in tonicity produces the activation of the transcription factor NFAT5 (nuclear factor of activated T-cells 5), mediated in part by ATM (Ataxia Telangiectasia mutated gene) [13]. Recently, we have found that NFAT5 is also activated by hypoxia in cell culture (HEK293 cells) and in the kidney of rat undergoing to renal ischemia/reperfusion (I/R) [14]. These two transcription factors
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have been implicated in the normal kidney development: 1. NFAT5 knock-out mice results in embryonic lethality [15]. The few surviving homozygous NFAT5 null-mice have profound, progressive atrophy of the kidney medulla [15]. Other study, show that mouse expressing a dominant negative form of NFAT5 in kidney induces a progressive atrophy of the renal medulla, cortical thinning and impaired ability to concentrate urine [16]. 2. Mice genetically deficient in HIF-1α die at midgestation (embryonic day 9.5 [E9.5]) from vascular and neural tube defects [17]. VEGF, whose expression is regulated by HIF, seems to have a pivotal function in development and preservation of functional glomeruli in mice [17]. Another factor described to response to kidney injury and related with kidney development is NGAL. It was demonstrate that the local expression of NGAL can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures [18]. NGAL may play an important role in adult kidney, due it is highly up-regulated in transient renal ischemia [19]. Infusion of NGAL can protect against ischemia-mediated tubular injury in mouse [20]. Interestingly, the activation of NGAL and its receptor (NGALR) is under the control of IL-1β [21]. However, NGAL has been described as essential for chronic kidney disease progression in mice and human through of EGFR activation (Epidermal growth factor receptor) in a dependent way of HIF-1α [22]. The aim of this work was to study the effect of uteroplacental vessels ligature on fetal kidney in pregnant rabbit. We centralized the study in gene expression of essential proteins implicated in fetal kidney development and kidney protection against hypoxia (HIF-1α, NFAT5 and ATM) [14,23], osmotic stress (NFAT5 and ATM) [13,24], and kidney injury (IL-1β and NGAL) [25,26]. 2. Material and methods 2.1. Animals The Animal Experimental Ethics Committees of the University of Barcelona and University Los Andes approved animal experimentation of this study. Animal handling and all the procedures were performed following all applicable regulations and guidelines of the Animal Experimental Ethics Committee of the University of Barcelona. Dams were housed for 1 week before surgery in separate cages on a reversed 12/ 12 h light cycle, with free access to water and standard chow. IUGR model was performed as previously described [27]. In briefly, pregnant rabbits at twenty-five days of gestation (25 D) were used to create two experimental groups of fetal growth restriction: 20–30% ligature (mild restriction) and 40–50% ligature (severe restriction) of uteroplacental vessels. For each dam, fetuses from one uterine horn were treated and contralateral horn one was considered as controls. A total of 3 study groups resulted for comparisons: 13 controls, 10 mild, and 7 severe restriction fetuses. 2.2. Surgical procedure Prior to surgery tocolysis (progesterone 0.9 mg/Kg intramuscularly) and antibiotic prophylaxis (penicillin G 300.000 UI intravenous) were administered. Ketamine 35 mg/Kg and xylazine 5 mg/Kg were given intramuscularly for anesthetic induction. Inhaled anesthesia was maintained with a mixture of 1–5% isoflurane and 1–1.5 L/min oxygen. An abdominal midline laparotomy was performed for unilateral uteroplacental vessels ligation with silk suture (4/0) in a proportion of 20–30% or 40–50% to induce middle and severe blood flow restriction, respectively. After the ligature procedure the abdomen was closed in two layers with a single suture of silk (3/0). Animals were kept under a warming blanket until awake and active, and postoperative analgesia was administered (meloxicam 0.4 mg/Kg/24 h subcutaneous for 48 h). Animals were again housed and well-being was controlled each day.
2.3. Sample collection and processing At day 30 (120 h after surgery) newborns were obtained by cesarean section under the same anesthetic conditions. After delivery, living newborns were weighed and measured. Dams were sacrificed by pentobarbital 200 mg/Kg endovenous administration and fetuses were sacrificed by decapitation. Kidneys were obtained and frozen to −80 °C. 2.4. Histological analysis Kidneys of control and vascular-restricted fetuses were fixed in 0.1 M sodium phosphate-buffered formaldehyde (pH 7.4, 4% formaldehyde) for 48 h before being embedded whole in paraffin. Three parallel longitudinal sections were performed per kidney so as to include both poles and the renal pelvis. Sections of 3 μm were stained with hematoxylin and eosin, and the two best sections were chosen and examined. 2.5. Glomeruli count The total number of glomeruli was determined in the entire kidney, as previously described [28,29,29]. Briefly, and following a classical protocol for glomeruli separation and counting, each whole kidney was incubated in 2 mL of 50% hydrochloric acid (6 M) for 45 min at 37 °C. Kidneys were rinsed with distilled water, and stored in 5 mL of distilled water overnight at 4 °C. The kidneys were mechanically dissociated and three homogenized 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. 2.6. Western blot analysis Kidneys were homogenized with an Ultra-Turrax homogenizer in lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, protease inhibitor (Complete Mini, Roche Applied Science, Indianapolis, IN)], and phosphatase inhibitor cocktails (phosphatase inhibitor cocktails 1 and 2, Sigma, St. Louis, MO). Homogenized kidney sections were centrifuged (15,000 g × 10 min) and 100 micrograms of soluble proteins were separated on 7.5 or 10% Tris–Glycine gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Western blot analysis was performed according to standard conditions. In brief, nonspecific binding was blocked with 5% non-fat milk for 45 min at room temperature. The membranes were then incubated with rabbit anti-NFAT5 (Affinity BioReagents, Golden, CO), rabbit anti-IL1β (Cell signaling), mouse anti-HIF-1α (Abcam), mouse anti-NGAL ( R & D SYSTEM), mouse anti-ATM (Cell signalling, MA), and rabbit anti-GAPDH (Cell signalling, MA) over night at 4 °C. After washing with 0.1% Tween-20 in PBS, blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. Proteins were detected using enhanced chemiluminescence techniques (PerkinElmer, Life Sciences, Boston, MA). The blots were scanned and densitometric analysis was performed using ImageJ (US National Institutes of Health, http://rsbweb.nih.gov/ij/). 2.7. Real time PCR Around 30 mg of frozen kidney tissue was placed with Lysis Reagent (RNeasy, Qiagen) and homogenized with Ultra-Turrax homogenizer for 30s. The sample was purified with RNeasy Mini Kit (Qiagen). RNA was then quantitated using an UV–Vis spectrophotometer (Nanodrop 2000) and RNA purity was conform by 280/260 ratio. The cDNA was prepared from 1.0 μg of total RNA using reverse transcription system (random hexamers, Improm II Reverse Transcriptase System; Promega). Amplicons were detected for Real-Time Fluorescence Detection (Rotor-Gene Q, Qiagen). Primers to qRT-PCR were used to NFAT5
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5′-ACCTGACTGAGGGCAGCCGT-3′ and 5′-TTCACTCGACCGGAATCGTTG CC-3′; HIF-1α 5′-GCACCGCCACCACTGACGATT-3′ and 5′-GTTTGGTGAG GCTGTCCGACTCG-3′; and 18 S (housekeeping) 5′-GCCGCTAGAGGTG AAATTCTTGGA-3′ and 5′-ATCGCCAGTCGGCATCGTTTAT-3′. The detection system records the number of PCR cycles (Ct) required to produce an amount of product equal to a threshold value, which is a constant and it was determinate by standard curve. Ct values were used to calculate the mRNA abundance in each experimental condition, relative to the appropriate control. 2.8. Statistical analysis The variables were analyzed by Pearson χ2 test. For quantitative variables, normality was assessed by the Shapiro-Francia W′ test [17]. Normal-distributed quantitative variables were analyzed by one-way ANOVA. Additionally, a linear polynomial contrast was used to analyze linear trends across the experimental groups, where the weighted p value was considered. Non-normal distributed variables were analyzed with the non-parametric Kruskall–Wallis test. The SPSS 15.0 (SPSS Inc., Chicago, IL, USA) statistical software was used for the statistical analysis. 3. Results As expected, fetuses undergoing to IUGR, induced by uteroplacental vessels ligation, showed a reduction in body weight compared with controls animals (39 vs. 48 g, p b 0.05, Fig. 1A). Histological analysis of fetal kidneys showed no significant differences between size/development of cortex versus medulla. The IUGR did not produce apparently medullar atrophy or evidence of inflammatory cell infiltration in kidneys. However, the total number of glomerulus was significantly reduced in kidneys of IUGR fetuses (Fig. 1B). We studied the mRNA (qRT-PCR) and protein (Western blot) abundance in kidney of control and IUGR fetuses. We measured the levels of 18 S ribosomal RNA and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to normalize the mRNA and protein abundance, respectively. The abundance of 18 S or GAPDH did not change in kidneys of both groups of fetuses (control and IUGR kidneys). The mRNA and protein
Fig. 1. The body weight and total number of glomeruli was reduced in IUGR fetuses. A) Birth weight fetuses of controls (n=13) and undergoing to IUGR [20–30% (n=10) and 40–50% (n=7)]. B) Number of glomeruli. Mean±SD. *pb 0.05.
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abundance of studied gene were measured from 1.0 μg of total RNA or 100 μg of total protein. The data were normalized (18 S for RNA or GAPDH for protein) and expressed as arbitrary units (AU). The normalized protein abundance of HIF-1α was increased in kidney of fetuses undergoing to IUGR in a dose–response manner (Fig. 2A). However, the qRT-PCR analysis showed that mRNA abundance of HIF-1Α in kidneys did not show significant differences between control and IUGR fetuses (Fig. 2B), suggesting that HIF-1Α induction was associated to mRNA and/or protein stabilization. The transcription factor NFAT5 was increased in kidney of IUGR fetuses group (Fig. 3A). The mRNA abundance of NFAT5 was also increased in severe IUGR (Fig. 3B). These data indicate that the transcription factors normally activated by hypoxia and hypertonicity were up-regulated in kidneys of fetuses undergoing to intrauterine growth restriction. Inflammatory genes were also analyzed in the fetal kidney. The results showed a direct relationship between the severity of the blood flow restriction and NGAL protein abundance in fetal kidney (Fig. 4). The analysis of IL-1β protein abundance showed that the protein levels of this cytokine was increased in the kidney of fetuses undergoing to IUGR, but none differences was observed between middle and severe blood flow restriction (Fig. 4), suggesting a potential activation of inflammatory process in kidney of fetuses under a growth restriction inside of the uterus. We tested the ataxia telangiectasia mutated (ATM) gene due that this kinase is activated by DNA damage and it has been described to responds to a variety of insults including hypoxia [30] and osmotic stress [13]. The normalized protein abundance of ATM was increased in the kidneys of fetuses exposed to IUGR in a dose–response manner (Fig. 5), as HIF-1α and NGAL. Interestingly, it was observed that the electromobility of ATM in the electrophoresis was retarded suggesting a post translation modification in the protein induced by IUGR, such as phosphorylation (Fig. 5). Due to that abundance of 18 S or GAPDH did not change in the kidneys of control and IUGR fetuses, it possible to suggest that the
Fig. 2. Intrauterine growth restriction induces HIF-1α in fetal kidney. A) HIF-1α Western blot of controls (n = 8) and IUGR (20–30%, n = 10 and 40–50%, n = 7). B) HIF-1α qRT-PCR of controls (n = 8) and IUGR (40–50%, n = 7). Mean ± SD. *p b 0.05.
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Fig. 3. Intrauterine growth restriction induces NFAT5 in fetal kidney. A) NFAT5 Western blot of controls (n = 8) and IUGR (20–30%, n = 10 and 40–50%, n = 7). B) NFAT5 qRT-PCR of controls (n = 9) and IUGR (40–50%, n = 7). Mean ± SD. *p b 0.05.
changes observed in the gene expression of NFAT5, HIF-1α, IL-1β, NGAL and ATM are related with the pathophysiological condition and not associated to fetal kidney size or numbers of glomeruli.
4. Discussion 4.1. Kidney injury during IUGR A rat model of IUGR induced by low-protein diet during pregnancy showed that an important numbers of genes (1800 transcripts) were modified in its expression, suggesting that fetal kidneys response to IUGR at molecular levels [12]. On the other hand, using a rabbit model
Fig. 5. Intrauterine growth restriction induces NGAL and IL-1β in fetal kidney. Western blot of controls (n = 9) and IUGR (20–30%, n = 10 and 40–50%, n = 7) to NGAL (A) and IL-1β (B), Mean ± SD. *p b 0.05.
of IUGR induced by selective ligature of utero-placental vessels it was demonstrated a gradable model of intrauterine growth restriction [27,31]. Thus, we decide to study if the gene expression is modified in terms of the severity of ligature of utero-placental vessels. This model mimics the situation occurring in human pregnancy in terms of oxygen and nutrient deprivation [27,32]. Using this model we observed fetal weight reduction (Fig. 1A) as compare with controls. Histological analysis of fetal kidney did not show medullar atrophy or evidence of inflammatory cell infiltration in kidneys of fetuses undergoing to IUGR. We did not found significant differences between size/development of cortex versus medulla. However, the total number of glomeruli was significantly reduced in kidneys of IUGR fetuses (Fig. 1B), conforming the previous results described by Bassan et al. [32]. These data suggest that IUGR induces changes in the filtration capability (reduced number of glomeruli) without apparently inductions of the histological sign of inflammation or morphological alterations.
4.2. Effect of ischemic IUGR on fetal kidney
Fig. 4. Intrauterine growth restriction induces ATM in fetal kidney. ATM Western blot of controls (n = 9) and IUGR (20–30%, n = 10 and 40–50%, n= 7). Mean ± SD. *p b 0.05.
Despite of normal levels of hypoxia present in the intrauterine life of fetuses, physiopathological hypoxia can be produced during fetal intrauterine development. A persistence hypoxia, could induces a blood flow
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redistribution, increasing blood flow to brain, heart, and adrenals and decreased blood supply to the rest of fetal organs [33], such as kidney, inducing a renal ischemia condition. In the present study, we found that the protein abundance of HIF-1α, NFAT5, and ATM were induced in kidneys of fetuses undergoing to ischemic condition induced by 5 days of partial utero-placental vessels ligature, suggesting that these proteins participate in the complex mechanisms of kidney responses against to ischemic growth restriction. The master regulator of the hypoxia response is HIF-1α. However, several process activated by hypoxia are independently of HIF-1α [14]. Recently, we have published that NFAT5 is activated by hypoxia in kidney epithelial cells (cell culture) and protect the cells against cell death by hypoxia [14]. The NFAT5 activation by hypoxia was independent of HIF-1α activation [14]. Additionally, NFAT5 was found activated in kidneys undergoing to renal ischemia/reperfusion in rat [14]. On the other hand, using cell line, we and others have found that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage [14,34]. Additionally, the current hypothesis propose that NFAT5 is a transcription factor necessary for survival of renal cells in the challenging conditions of hypertonicity [35–37], in a dependent way of ATM activation [13]. Differences in the electromobility of ATM suggest a post transcriptional modification of ATM in kidney under IUGR that might be related with its activation. The ATM activation by hypoxia induces the pathways associated with cell cycle checkpoints and cause a replication arrest [34]. Thus, the ATM induction observed in kidney of fetal IUGR suggests a potential role of ATM in the observed reduction in the number of glomeruli. 4.3. Effect of ischemic IUGR on fetal kidney inflammation We found the protein induction of IL-1β and NGAL in kidneys undergoing to IUGR. These results, strongly suggests the participation of these two factors in the kidney response to fetal IUGR. NGAL was previously implicated in epithelial morphogenesis through organization of tubular structures [18] suggesting a protective role of NGAL in the ischemic kidney. However, NGAL expression was implicated in the progression chronic kidney disease mediated by HIF-1α. The right understood of NGAL function in fetal development is more complex considering that this factor was incremented in the serum of preeclampsia patient [38,39]. Moreover, the activation of NGAL and its receptor NGALR has been mediated thorough IL-1β [21,40], suggesting a potential mechanism of NGAL activation in kidney of fetuses subjected to IUGR. An interesting study demonstrated that HIF-1α, is not only activated by hypoxia but also by peptides such as IL-1β in normoxia [41], suggesting that our observation of HIF-1α induction in kidneys from IUGR fetuses might be mediated by IL-1β activation. Despite the induction of inflammatory cytokines, the histological analysis did not show sign of inflammation suggesting that the observed induction of IL-1β and NGAL can be related to others process, like morphogenesis. 4.4. Fetal programming Fetal programming occurs when the normal pattern of fetal development is disrupted by an abnormal stimulus or an insult is applied at a critical point of the uterus development. Malnutrition and/or hypoxia might program physiology and function of organs due to alteration of the proliferation and/or differentiation steps in tissue and organ development [42]. Since proliferative and differentiation timing is variable between tissues, the timing and intensity of placental dysfunction would have tissue-specific effects. It has been proposed that adverse conditions during the proliferative phase have an impact mainly on the total number of cells within a tissue. In contrast, a tissue undergoing to adverse conditions during differentiation phase would have altered profiles of cell types and potentially fewer functional units [43]. We
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have found that kidneys from IUGR fetuses modified important genes implicated in kidney development and function. In conclusion, we found that essential proteins involved in fetal kidney development and adult kidney maintenance were upregulated in kidneys of fetuses undergoing to growth restriction in the last period of intrauterine gestation. We do not know the consequences in kidney development or kidney function of HIF-1α, NFAT5, ATM, NGAL, and IL-1β protein induction, but this information encourages conduct further research to understand the role of these proteins in kidney function and its relation with others diseases. Conflict of interest statement There is no conflict of interest. Acknowledgments This research was supported by FAI-MED 003‐09 and FAI‐MED 001‐10 from the University of Los Andes—Chile. FONDECYT-1100885, FONDECYT-1110869 and FONDECYT-1080373. E.E. was supported by an Emili Letang fellowship by the Hospital Clinic and a Rio Hortega grant from the Carlos III Institute of Health (Spain) (CM08/00105). References [1] Soothill PW, Bobrow CS, Holmes R. Small for gestational age is not a diagnosis. Ultrasound Obstet Gynecol 1999;13(4):225–8. [2] Bernstein IM, Horbar JD, Badger GJ, Ohlsson A, Golan A. Morbidity and mortality among very-low-birth-weight neonates with intrauterine growth restriction. The Vermont Oxford Network. Am J Obstet Gynecol 2000;182(1 Pt 1):198–206. [3] Gagnon R. Placental insufficiency and its consequences. Eur J Obstet Gynecol Reprod Biol 2003;110(Suppl. 1):S99–S107. [4] Marsal K. Intrauterine growth restriction. Curr Opin Obstet Gynecol 2002;14(2): 127–35. [5] Pardi G, Marconi AM, Cetin I. Placental–fetal interrelationship in IUGR fetuses— a review. Placenta 2002;23(Suppl. A):S136–41. [6] Ferrazzi E, Bozzo M, Rigano S, Bellotti M, Morabito A, Pardi G, et al. Temporal sequence of abnormal Doppler changes in the peripheral and central circulatory systems of the severely growth-restricted fetus. Ultrasound Obstet Gynecol 2002;19(2):140–6. [7] De Prins FA, Van Assche FA. Intrauterine growth retardation and development of endocrine pancreas in the experimental rat. Biol Neonate 1982;41(1–2):16–21. [8] Fall CH, Stein CE, Kumaran K, Cox V, Osmond C, Barker DJ, et al. Size at birth, maternal weight, and type 2 diabetes in South India. Diabet Med 1998;15(3): 220–7. [9] Barker DJ, Martyn CN, Osmond C, Hales CN, Fall CH. Growth in utero and serum cholesterol concentrations in adult life. BMJ 1993;307(6918):1524–7. [10] Martyn CN, Lever AF, Morton JJ. Plasma concentrations of inactive renin in adult life are related to indicators of foetal growth. J Hypertens 1996;14(7):881–6. [11] Barker DJ, Eriksson JG, Forsen T, Osmond C. Fetal origins of adult disease: strength of effects and biological basis. Int J Epidemiol 2002;31(6):1235–9. [12] Buffat C, Boubred F, Mondon F, Chelbi ST, Feuerstein JM, Lelievre-Pegorier M, et al. Kidney gene expression analysis in a rat model of intrauterine growth restriction reveals massive alterations of coagulation genes. Endocrinology 2007;148(11): 5549–57. [13] Irarrazabal CE, Liu JC, Burg MB, Ferraris JD. ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci U S A 2004;101(23):8809–14. [14] Villanuevan S, Suazo C, Santapau D, Perez F. QuirozM, Careño JE, Illanes S, Lavandero S, Michea L, and Irarrazabal CE (2012) NFAT5 is activated by hypoxia: role in ischemia and reperfusion in the rat kidney. PLoS One 2012;7(7):e39665. [15] Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, et al. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci U S A 2004;101(8):2392–7. [16] Lam AK, Ko BC, Tam S, Morris R, Yang JY, Chung SK, et al. Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism. J Biol Chem 2004;279(46):48,048–54. [17] Gunaratnam L, Bonventre JV. HIF in kidney disease and development. J Am Soc Nephrol 2009;20(9):1877–87. [18] Gwira JA, Wei F, Ishibe S, Ueland JM, Barasch J, Cantley LG. Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro. J Biol Chem 2005;280(9):7875–82. [19] Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003;14(10):2534–43. [20] Mishra J, Mori K, Ma Q, Kelly C, Yang J, Mitsnefes M, et al. Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2004;15(12):3073–82.
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[32] Bassan H, Trejo LL, Kariv N, Bassan M, Berger E, Fattal A, et al. Experimental intrauterine growth retardation alters renal development. Pediatr Nephrol 2000;15(3–4): 192–5. [33] Martin Jr CB. Normal fetal physiology and behavior, and adaptive responses with hypoxemia. Semin Perinatol 2008;32(4):239–42. [34] Bencokova Z, Kaufmann MR, Pires IM, Lecane PS, Giaccia AJ, Hammond EM. ATM activation and signaling under hypoxic conditions. Mol Cell Biol 2009;29(2): 526–37. [35] Sykes E, Cosgrove JF. Acute renal failure and the critically ill surgical patient. Ann R Coll Surg Engl 2007;89(1):22–9. [36] Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM. Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci U S A 1999;96(5):2538–42. [37] Woo SK, Nahm O, Kwon HM. How salt regulates genes: function of a Rel-like transcription factor TonEBP. Biochem Biophys Res Commun 2000;278(2):269–71. [38] D'Anna R, Baviera G, Giordano D, Russo S, Dugo N, Santamaria A, et al. First trimester serum PAPP-A and NGAL in the prediction of late-onset pre-eclampsia. Prenat Diagn 2009;29(11):1066–8. [39] D'Anna R, Baviera G, Giordano D, Todarello G, Russo S, Recupero S, et al. Neutrophil gelatinase-associated lipocalin serum evaluation through normal pregnancy and in pregnancies complicated by preeclampsia. Acta Obstet Gynecol Scand 2010;89(2): 275–8. [40] Mao S, Jiang T, Shang G, Wu Z, Zhang N. Increased expression of neutrophil gelatinase-associated lipocalin receptor by interleukin-1beta in human mesangial cells via MAPK/ERK activation. Int J Mol Med 2011;27(4):555–60. [41] Qian D, Lin HY, Wang HM, Zhang X, Liu DL, Li QL, et al. Normoxic induction of the hypoxic-inducible factor-1 alpha by interleukin-1 beta involves the extracellular signal-regulated kinase 1/2 pathway in normal human cytotrophoblast cells. Biol Reprod 2004;70(6):1822–7. [42] Brameld JM, Buttery PJ, Dawson JM, Harper JM. Nutritional and hormonal control of skeletal-muscle cell growth and differentiation. Proc Nutr Soc 1998;57(2):207–17. [43] Langley-Evans SC. Nutritional programming of disease: unravelling the mechanism. J Anat 2009;215(1):36–51.
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Intrauterine growth restriction modifies the normal gene expression in kidney from rabbit fetuses Horacio Figueroa a, b, Mauricio Lozano c, Cristian Suazo c, Elisenda Eixarch d, e, f, Sebastian E. Illanes a, b, Juan Eduardo Carreño c, Sandra Villanueva c, Edgar Hernández-Andrade d, e, f, g, Eduard Gratacós d, e, f, Carlos E. Irarrazabal c,⁎ a
Department of Obstetrics & Gynecology and Laboratory of Reproductive Biology, Faculty of Medicine, Universidad de los Andes, Santiago, Chile Department of Maternal-Fetal Medicine, Davila Clinic, Santiago, Chile c Laboratory of Molecular Physiology, Faculty of Medicine, Universidad de los Andes, Santiago, Chile d Department of Maternal-Fetal Medicine, Institut Clinic de Ginecologia, Obstetricia i Neonatologia (ICGON), Hospital Clinic, Barcelona, Spain e Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain f Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain g National Institute of Perinatal Medicine, Mexico b
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Article history: Received 15 March 2012 Received in revised form 30 June 2012 Accepted 8 July 2012 Keywords: IUGR Rabbit Inflammation Hypoxia Osmotic stress
a b s t r a c t The aim of this work was to study the effect of intrauterine growth restriction (IUGR) on fetal kidneys. The IUGR was induced by uteroplacental vessels ligature in a model of pregnant rabbit. We centralized the study in the gene expression of essential proteins for fetal kidney development and kidney protection against hypoxia, osmotic stress, and kidney injury. The gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM were studied by qRT-PCR and Western blot in kidneys from control and IUGR fetuses. Experimental IUGR fetuses were significantly smaller than the control animals (39 vs. 48 g, p b 0.05). The number of glomeruli was decreased in IUGR kidneys, without morphological alterations. IUGR increased the gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM (p b 0.05) in kidneys of fetuses undergoing IUGR, suggesting that fetal blood flow restriction produce alterations in gene expression in fetal kidneys. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The IUGR can be defined as a failure to reach the genetic growth potential of a given fetus [1,2]. Maternal health and placental dysfunction during pregnancy may induce altered fetal development. IUGR occur in about 6% of all pregnancies and is associated with increased risk of neonatal complications [3,4], stillbirth [4,5], birth hypoxia [3], and impaired neurodevelopment [5,6]. Numerous evidence in animals and also in humans suggest an association between IUGR and adult diseases, such as diabetes [7,8], hypercholesterolemia [9], cardiovascular diseases and hypertension [10]. During IUGR, normal fetal circulation is modified in a defined pattern. The final stage of this cascade is cardiac hypoxia and heart failure [11]. A considerable number of fetuses with IUGR are associated with altered renal development and present different degree of damage after birth, increasing the risk of hypertension and renal disease.
⁎ Corresponding author at: Molecular Physiology Laboratory, Universidad de los Andes, S. Carlos Apoquindo 2200-Las Condes, Santiago, Chile. Tel.: +56 2 4129607; fax: +56 2 2141756. E-mail address: [email protected] (C.E. Irarrazabal). 0378-3782/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2012.07.010
Using a rat model of IUGR, induced by low-protein diet during pregnancy, the kidney gene expression has been studied. Over 1800 transcripts modified at a 2-fold threshold (722 induced) were identified from IUGR rat kidneys. The up-regulated genes are involved in complement/coagulation, immune system, apoptosis, heme/globin and Calycin/lipocalin/retinol. The induction of coagulation cascade in kidney provides a putative explanation of thrombo-endothelial disorders observed in intrauterine growth-restricted human newborns and increased risk of cardiovascular diseases in adult [12], suggesting that impair fetal nutrition induces kidney injury. The mammalian kidney has a gradient of tonicity and partial pressure of oxygen (PO2). The kidney medulla is hypertonic (800 mosmol) and hypoxic (10–18 mm Hg) compared with cortex (300 mosmol and 40–50 mm Hg). The kidney cells have molecular mechanisms to adapt and survive to this extreme environment. A declination in PO2 (hypoxia) induces the transcription factor, HIF-1α (Hypoxia induced factor). On the other hand, an increase in tonicity produces the activation of the transcription factor NFAT5 (nuclear factor of activated T-cells 5), mediated in part by ATM (Ataxia Telangiectasia mutated gene) [13]. Recently, we have found that NFAT5 is also activated by hypoxia in cell culture (HEK293 cells) and in the kidney of rat undergoing to renal ischemia/reperfusion (I/R) [14]. These two transcription factors
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have been implicated in the normal kidney development: 1. NFAT5 knock-out mice results in embryonic lethality [15]. The few surviving homozygous NFAT5 null-mice have profound, progressive atrophy of the kidney medulla [15]. Other study, show that mouse expressing a dominant negative form of NFAT5 in kidney induces a progressive atrophy of the renal medulla, cortical thinning and impaired ability to concentrate urine [16]. 2. Mice genetically deficient in HIF-1α die at midgestation (embryonic day 9.5 [E9.5]) from vascular and neural tube defects [17]. VEGF, whose expression is regulated by HIF, seems to have a pivotal function in development and preservation of functional glomeruli in mice [17]. Another factor described to response to kidney injury and related with kidney development is NGAL. It was demonstrate that the local expression of NGAL can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures [18]. NGAL may play an important role in adult kidney, due it is highly up-regulated in transient renal ischemia [19]. Infusion of NGAL can protect against ischemia-mediated tubular injury in mouse [20]. Interestingly, the activation of NGAL and its receptor (NGALR) is under the control of IL-1β [21]. However, NGAL has been described as essential for chronic kidney disease progression in mice and human through of EGFR activation (Epidermal growth factor receptor) in a dependent way of HIF-1α [22]. The aim of this work was to study the effect of uteroplacental vessels ligature on fetal kidney in pregnant rabbit. We centralized the study in gene expression of essential proteins implicated in fetal kidney development and kidney protection against hypoxia (HIF-1α, NFAT5 and ATM) [14,23], osmotic stress (NFAT5 and ATM) [13,24], and kidney injury (IL-1β and NGAL) [25,26]. 2. Material and methods 2.1. Animals The Animal Experimental Ethics Committees of the University of Barcelona and University Los Andes approved animal experimentation of this study. Animal handling and all the procedures were performed following all applicable regulations and guidelines of the Animal Experimental Ethics Committee of the University of Barcelona. Dams were housed for 1 week before surgery in separate cages on a reversed 12/ 12 h light cycle, with free access to water and standard chow. IUGR model was performed as previously described [27]. In briefly, pregnant rabbits at twenty-five days of gestation (25 D) were used to create two experimental groups of fetal growth restriction: 20–30% ligature (mild restriction) and 40–50% ligature (severe restriction) of uteroplacental vessels. For each dam, fetuses from one uterine horn were treated and contralateral horn one was considered as controls. A total of 3 study groups resulted for comparisons: 13 controls, 10 mild, and 7 severe restriction fetuses. 2.2. Surgical procedure Prior to surgery tocolysis (progesterone 0.9 mg/Kg intramuscularly) and antibiotic prophylaxis (penicillin G 300.000 UI intravenous) were administered. Ketamine 35 mg/Kg and xylazine 5 mg/Kg were given intramuscularly for anesthetic induction. Inhaled anesthesia was maintained with a mixture of 1–5% isoflurane and 1–1.5 L/min oxygen. An abdominal midline laparotomy was performed for unilateral uteroplacental vessels ligation with silk suture (4/0) in a proportion of 20–30% or 40–50% to induce middle and severe blood flow restriction, respectively. After the ligature procedure the abdomen was closed in two layers with a single suture of silk (3/0). Animals were kept under a warming blanket until awake and active, and postoperative analgesia was administered (meloxicam 0.4 mg/Kg/24 h subcutaneous for 48 h). Animals were again housed and well-being was controlled each day.
2.3. Sample collection and processing At day 30 (120 h after surgery) newborns were obtained by cesarean section under the same anesthetic conditions. After delivery, living newborns were weighed and measured. Dams were sacrificed by pentobarbital 200 mg/Kg endovenous administration and fetuses were sacrificed by decapitation. Kidneys were obtained and frozen to −80 °C. 2.4. Histological analysis Kidneys of control and vascular-restricted fetuses were fixed in 0.1 M sodium phosphate-buffered formaldehyde (pH 7.4, 4% formaldehyde) for 48 h before being embedded whole in paraffin. Three parallel longitudinal sections were performed per kidney so as to include both poles and the renal pelvis. Sections of 3 μm were stained with hematoxylin and eosin, and the two best sections were chosen and examined. 2.5. Glomeruli count The total number of glomeruli was determined in the entire kidney, as previously described [28,29,29]. Briefly, and following a classical protocol for glomeruli separation and counting, each whole kidney was incubated in 2 mL of 50% hydrochloric acid (6 M) for 45 min at 37 °C. Kidneys were rinsed with distilled water, and stored in 5 mL of distilled water overnight at 4 °C. The kidneys were mechanically dissociated and three homogenized 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. 2.6. Western blot analysis Kidneys were homogenized with an Ultra-Turrax homogenizer in lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, protease inhibitor (Complete Mini, Roche Applied Science, Indianapolis, IN)], and phosphatase inhibitor cocktails (phosphatase inhibitor cocktails 1 and 2, Sigma, St. Louis, MO). Homogenized kidney sections were centrifuged (15,000 g × 10 min) and 100 micrograms of soluble proteins were separated on 7.5 or 10% Tris–Glycine gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Western blot analysis was performed according to standard conditions. In brief, nonspecific binding was blocked with 5% non-fat milk for 45 min at room temperature. The membranes were then incubated with rabbit anti-NFAT5 (Affinity BioReagents, Golden, CO), rabbit anti-IL1β (Cell signaling), mouse anti-HIF-1α (Abcam), mouse anti-NGAL ( R & D SYSTEM), mouse anti-ATM (Cell signalling, MA), and rabbit anti-GAPDH (Cell signalling, MA) over night at 4 °C. After washing with 0.1% Tween-20 in PBS, blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. Proteins were detected using enhanced chemiluminescence techniques (PerkinElmer, Life Sciences, Boston, MA). The blots were scanned and densitometric analysis was performed using ImageJ (US National Institutes of Health, http://rsbweb.nih.gov/ij/). 2.7. Real time PCR Around 30 mg of frozen kidney tissue was placed with Lysis Reagent (RNeasy, Qiagen) and homogenized with Ultra-Turrax homogenizer for 30s. The sample was purified with RNeasy Mini Kit (Qiagen). RNA was then quantitated using an UV–Vis spectrophotometer (Nanodrop 2000) and RNA purity was conform by 280/260 ratio. The cDNA was prepared from 1.0 μg of total RNA using reverse transcription system (random hexamers, Improm II Reverse Transcriptase System; Promega). Amplicons were detected for Real-Time Fluorescence Detection (Rotor-Gene Q, Qiagen). Primers to qRT-PCR were used to NFAT5
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5′-ACCTGACTGAGGGCAGCCGT-3′ and 5′-TTCACTCGACCGGAATCGTTG CC-3′; HIF-1α 5′-GCACCGCCACCACTGACGATT-3′ and 5′-GTTTGGTGAG GCTGTCCGACTCG-3′; and 18 S (housekeeping) 5′-GCCGCTAGAGGTG AAATTCTTGGA-3′ and 5′-ATCGCCAGTCGGCATCGTTTAT-3′. The detection system records the number of PCR cycles (Ct) required to produce an amount of product equal to a threshold value, which is a constant and it was determinate by standard curve. Ct values were used to calculate the mRNA abundance in each experimental condition, relative to the appropriate control. 2.8. Statistical analysis The variables were analyzed by Pearson χ2 test. For quantitative variables, normality was assessed by the Shapiro-Francia W′ test [17]. Normal-distributed quantitative variables were analyzed by one-way ANOVA. Additionally, a linear polynomial contrast was used to analyze linear trends across the experimental groups, where the weighted p value was considered. Non-normal distributed variables were analyzed with the non-parametric Kruskall–Wallis test. The SPSS 15.0 (SPSS Inc., Chicago, IL, USA) statistical software was used for the statistical analysis. 3. Results As expected, fetuses undergoing to IUGR, induced by uteroplacental vessels ligation, showed a reduction in body weight compared with controls animals (39 vs. 48 g, p b 0.05, Fig. 1A). Histological analysis of fetal kidneys showed no significant differences between size/development of cortex versus medulla. The IUGR did not produce apparently medullar atrophy or evidence of inflammatory cell infiltration in kidneys. However, the total number of glomerulus was significantly reduced in kidneys of IUGR fetuses (Fig. 1B). We studied the mRNA (qRT-PCR) and protein (Western blot) abundance in kidney of control and IUGR fetuses. We measured the levels of 18 S ribosomal RNA and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to normalize the mRNA and protein abundance, respectively. The abundance of 18 S or GAPDH did not change in kidneys of both groups of fetuses (control and IUGR kidneys). The mRNA and protein
Fig. 1. The body weight and total number of glomeruli was reduced in IUGR fetuses. A) Birth weight fetuses of controls (n=13) and undergoing to IUGR [20–30% (n=10) and 40–50% (n=7)]. B) Number of glomeruli. Mean±SD. *pb 0.05.
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abundance of studied gene were measured from 1.0 μg of total RNA or 100 μg of total protein. The data were normalized (18 S for RNA or GAPDH for protein) and expressed as arbitrary units (AU). The normalized protein abundance of HIF-1α was increased in kidney of fetuses undergoing to IUGR in a dose–response manner (Fig. 2A). However, the qRT-PCR analysis showed that mRNA abundance of HIF-1Α in kidneys did not show significant differences between control and IUGR fetuses (Fig. 2B), suggesting that HIF-1Α induction was associated to mRNA and/or protein stabilization. The transcription factor NFAT5 was increased in kidney of IUGR fetuses group (Fig. 3A). The mRNA abundance of NFAT5 was also increased in severe IUGR (Fig. 3B). These data indicate that the transcription factors normally activated by hypoxia and hypertonicity were up-regulated in kidneys of fetuses undergoing to intrauterine growth restriction. Inflammatory genes were also analyzed in the fetal kidney. The results showed a direct relationship between the severity of the blood flow restriction and NGAL protein abundance in fetal kidney (Fig. 4). The analysis of IL-1β protein abundance showed that the protein levels of this cytokine was increased in the kidney of fetuses undergoing to IUGR, but none differences was observed between middle and severe blood flow restriction (Fig. 4), suggesting a potential activation of inflammatory process in kidney of fetuses under a growth restriction inside of the uterus. We tested the ataxia telangiectasia mutated (ATM) gene due that this kinase is activated by DNA damage and it has been described to responds to a variety of insults including hypoxia [30] and osmotic stress [13]. The normalized protein abundance of ATM was increased in the kidneys of fetuses exposed to IUGR in a dose–response manner (Fig. 5), as HIF-1α and NGAL. Interestingly, it was observed that the electromobility of ATM in the electrophoresis was retarded suggesting a post translation modification in the protein induced by IUGR, such as phosphorylation (Fig. 5). Due to that abundance of 18 S or GAPDH did not change in the kidneys of control and IUGR fetuses, it possible to suggest that the
Fig. 2. Intrauterine growth restriction induces HIF-1α in fetal kidney. A) HIF-1α Western blot of controls (n = 8) and IUGR (20–30%, n = 10 and 40–50%, n = 7). B) HIF-1α qRT-PCR of controls (n = 8) and IUGR (40–50%, n = 7). Mean ± SD. *p b 0.05.
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Fig. 3. Intrauterine growth restriction induces NFAT5 in fetal kidney. A) NFAT5 Western blot of controls (n = 8) and IUGR (20–30%, n = 10 and 40–50%, n = 7). B) NFAT5 qRT-PCR of controls (n = 9) and IUGR (40–50%, n = 7). Mean ± SD. *p b 0.05.
changes observed in the gene expression of NFAT5, HIF-1α, IL-1β, NGAL and ATM are related with the pathophysiological condition and not associated to fetal kidney size or numbers of glomeruli.
4. Discussion 4.1. Kidney injury during IUGR A rat model of IUGR induced by low-protein diet during pregnancy showed that an important numbers of genes (1800 transcripts) were modified in its expression, suggesting that fetal kidneys response to IUGR at molecular levels [12]. On the other hand, using a rabbit model
Fig. 5. Intrauterine growth restriction induces NGAL and IL-1β in fetal kidney. Western blot of controls (n = 9) and IUGR (20–30%, n = 10 and 40–50%, n = 7) to NGAL (A) and IL-1β (B), Mean ± SD. *p b 0.05.
of IUGR induced by selective ligature of utero-placental vessels it was demonstrated a gradable model of intrauterine growth restriction [27,31]. Thus, we decide to study if the gene expression is modified in terms of the severity of ligature of utero-placental vessels. This model mimics the situation occurring in human pregnancy in terms of oxygen and nutrient deprivation [27,32]. Using this model we observed fetal weight reduction (Fig. 1A) as compare with controls. Histological analysis of fetal kidney did not show medullar atrophy or evidence of inflammatory cell infiltration in kidneys of fetuses undergoing to IUGR. We did not found significant differences between size/development of cortex versus medulla. However, the total number of glomeruli was significantly reduced in kidneys of IUGR fetuses (Fig. 1B), conforming the previous results described by Bassan et al. [32]. These data suggest that IUGR induces changes in the filtration capability (reduced number of glomeruli) without apparently inductions of the histological sign of inflammation or morphological alterations.
4.2. Effect of ischemic IUGR on fetal kidney
Fig. 4. Intrauterine growth restriction induces ATM in fetal kidney. ATM Western blot of controls (n = 9) and IUGR (20–30%, n = 10 and 40–50%, n= 7). Mean ± SD. *p b 0.05.
Despite of normal levels of hypoxia present in the intrauterine life of fetuses, physiopathological hypoxia can be produced during fetal intrauterine development. A persistence hypoxia, could induces a blood flow
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redistribution, increasing blood flow to brain, heart, and adrenals and decreased blood supply to the rest of fetal organs [33], such as kidney, inducing a renal ischemia condition. In the present study, we found that the protein abundance of HIF-1α, NFAT5, and ATM were induced in kidneys of fetuses undergoing to ischemic condition induced by 5 days of partial utero-placental vessels ligature, suggesting that these proteins participate in the complex mechanisms of kidney responses against to ischemic growth restriction. The master regulator of the hypoxia response is HIF-1α. However, several process activated by hypoxia are independently of HIF-1α [14]. Recently, we have published that NFAT5 is activated by hypoxia in kidney epithelial cells (cell culture) and protect the cells against cell death by hypoxia [14]. The NFAT5 activation by hypoxia was independent of HIF-1α activation [14]. Additionally, NFAT5 was found activated in kidneys undergoing to renal ischemia/reperfusion in rat [14]. On the other hand, using cell line, we and others have found that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage [14,34]. Additionally, the current hypothesis propose that NFAT5 is a transcription factor necessary for survival of renal cells in the challenging conditions of hypertonicity [35–37], in a dependent way of ATM activation [13]. Differences in the electromobility of ATM suggest a post transcriptional modification of ATM in kidney under IUGR that might be related with its activation. The ATM activation by hypoxia induces the pathways associated with cell cycle checkpoints and cause a replication arrest [34]. Thus, the ATM induction observed in kidney of fetal IUGR suggests a potential role of ATM in the observed reduction in the number of glomeruli. 4.3. Effect of ischemic IUGR on fetal kidney inflammation We found the protein induction of IL-1β and NGAL in kidneys undergoing to IUGR. These results, strongly suggests the participation of these two factors in the kidney response to fetal IUGR. NGAL was previously implicated in epithelial morphogenesis through organization of tubular structures [18] suggesting a protective role of NGAL in the ischemic kidney. However, NGAL expression was implicated in the progression chronic kidney disease mediated by HIF-1α. The right understood of NGAL function in fetal development is more complex considering that this factor was incremented in the serum of preeclampsia patient [38,39]. Moreover, the activation of NGAL and its receptor NGALR has been mediated thorough IL-1β [21,40], suggesting a potential mechanism of NGAL activation in kidney of fetuses subjected to IUGR. An interesting study demonstrated that HIF-1α, is not only activated by hypoxia but also by peptides such as IL-1β in normoxia [41], suggesting that our observation of HIF-1α induction in kidneys from IUGR fetuses might be mediated by IL-1β activation. Despite the induction of inflammatory cytokines, the histological analysis did not show sign of inflammation suggesting that the observed induction of IL-1β and NGAL can be related to others process, like morphogenesis. 4.4. Fetal programming Fetal programming occurs when the normal pattern of fetal development is disrupted by an abnormal stimulus or an insult is applied at a critical point of the uterus development. Malnutrition and/or hypoxia might program physiology and function of organs due to alteration of the proliferation and/or differentiation steps in tissue and organ development [42]. Since proliferative and differentiation timing is variable between tissues, the timing and intensity of placental dysfunction would have tissue-specific effects. It has been proposed that adverse conditions during the proliferative phase have an impact mainly on the total number of cells within a tissue. In contrast, a tissue undergoing to adverse conditions during differentiation phase would have altered profiles of cell types and potentially fewer functional units [43]. We
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have found that kidneys from IUGR fetuses modified important genes implicated in kidney development and function. In conclusion, we found that essential proteins involved in fetal kidney development and adult kidney maintenance were upregulated in kidneys of fetuses undergoing to growth restriction in the last period of intrauterine gestation. We do not know the consequences in kidney development or kidney function of HIF-1α, NFAT5, ATM, NGAL, and IL-1β protein induction, but this information encourages conduct further research to understand the role of these proteins in kidney function and its relation with others diseases. Conflict of interest statement There is no conflict of interest. Acknowledgments This research was supported by FAI-MED 003‐09 and FAI‐MED 001‐10 from the University of Los Andes—Chile. FONDECYT-1100885, FONDECYT-1110869 and FONDECYT-1080373. E.E. was supported by an Emili Letang fellowship by the Hospital Clinic and a Rio Hortega grant from the Carlos III Institute of Health (Spain) (CM08/00105). References [1] Soothill PW, Bobrow CS, Holmes R. Small for gestational age is not a diagnosis. Ultrasound Obstet Gynecol 1999;13(4):225–8. [2] Bernstein IM, Horbar JD, Badger GJ, Ohlsson A, Golan A. Morbidity and mortality among very-low-birth-weight neonates with intrauterine growth restriction. The Vermont Oxford Network. Am J Obstet Gynecol 2000;182(1 Pt 1):198–206. [3] Gagnon R. Placental insufficiency and its consequences. Eur J Obstet Gynecol Reprod Biol 2003;110(Suppl. 1):S99–S107. [4] Marsal K. Intrauterine growth restriction. Curr Opin Obstet Gynecol 2002;14(2): 127–35. [5] Pardi G, Marconi AM, Cetin I. Placental–fetal interrelationship in IUGR fetuses— a review. Placenta 2002;23(Suppl. A):S136–41. [6] Ferrazzi E, Bozzo M, Rigano S, Bellotti M, Morabito A, Pardi G, et al. Temporal sequence of abnormal Doppler changes in the peripheral and central circulatory systems of the severely growth-restricted fetus. Ultrasound Obstet Gynecol 2002;19(2):140–6. [7] De Prins FA, Van Assche FA. Intrauterine growth retardation and development of endocrine pancreas in the experimental rat. Biol Neonate 1982;41(1–2):16–21. [8] Fall CH, Stein CE, Kumaran K, Cox V, Osmond C, Barker DJ, et al. Size at birth, maternal weight, and type 2 diabetes in South India. Diabet Med 1998;15(3): 220–7. [9] Barker DJ, Martyn CN, Osmond C, Hales CN, Fall CH. Growth in utero and serum cholesterol concentrations in adult life. BMJ 1993;307(6918):1524–7. [10] Martyn CN, Lever AF, Morton JJ. Plasma concentrations of inactive renin in adult life are related to indicators of foetal growth. J Hypertens 1996;14(7):881–6. [11] Barker DJ, Eriksson JG, Forsen T, Osmond C. Fetal origins of adult disease: strength of effects and biological basis. Int J Epidemiol 2002;31(6):1235–9. [12] Buffat C, Boubred F, Mondon F, Chelbi ST, Feuerstein JM, Lelievre-Pegorier M, et al. Kidney gene expression analysis in a rat model of intrauterine growth restriction reveals massive alterations of coagulation genes. Endocrinology 2007;148(11): 5549–57. [13] Irarrazabal CE, Liu JC, Burg MB, Ferraris JD. ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci U S A 2004;101(23):8809–14. [14] Villanuevan S, Suazo C, Santapau D, Perez F. QuirozM, Careño JE, Illanes S, Lavandero S, Michea L, and Irarrazabal CE (2012) NFAT5 is activated by hypoxia: role in ischemia and reperfusion in the rat kidney. PLoS One 2012;7(7):e39665. [15] Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, et al. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci U S A 2004;101(8):2392–7. [16] Lam AK, Ko BC, Tam S, Morris R, Yang JY, Chung SK, et al. Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism. J Biol Chem 2004;279(46):48,048–54. [17] Gunaratnam L, Bonventre JV. HIF in kidney disease and development. J Am Soc Nephrol 2009;20(9):1877–87. [18] Gwira JA, Wei F, Ishibe S, Ueland JM, Barasch J, Cantley LG. Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro. J Biol Chem 2005;280(9):7875–82. [19] Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003;14(10):2534–43. [20] Mishra J, Mori K, Ma Q, Kelly C, Yang J, Mitsnefes M, et al. Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2004;15(12):3073–82.
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