- Email: [email protected]
Expression of aquaporins early in human pregnancy
Early Human Development 88 (2012) 589–594
Contents lists available at SciVerse ScienceDirect
Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Expression of aquaporins early in human pregnancy Javier Escobar a, María Gormaz b, Alessandro Arduini c, Karien Gosens d, Anabel Martinez e, Alfredo Perales e, Raquel Escrig b, Enrique Tormos e, Mónica Roselló f, Carmen Orellana f, Máximo Vento a, b,⁎ a
Neonatal Research Unit, Research Institute Hospital La Fe, Valencia, Spain Division of Neonatology, University and Polytechnic Hospital La Fe, Valencia, Spain Department of Physiology, University of Valencia, Spain d Department of Obstetrics & Neonatology, UMC St Radboud, Holland, the Netherlands e Division of Obstetrics, University and Polytechnic Hospital La Fe, Valencia, Spain f Division of Genetics, University and Polytechnic Hospital La Fe, Valencia, Spain b c
a r t i c l e
i n f o
Article history: Received 30 June 2011 Received in revised form 6 January 2012 Accepted 7 January 2012 Keywords: Aquaporins Pregnancy Chorionic villi Amniotic fluid
a b s t r a c t Background: Aquaporins (AQPs) constitute a family of channel proteins implicated in transmembrane water transport. Thirteen different AQPs (AQP0–12) have been described but their precise biologic function still remains unclear. AQPs 1, 3, 4, 8, and 9 expression has been described in human chorion, amnion and placenta; however, AQP4 is the only that has been identified in the first trimester of human pregnancy. Objective: To assess multiplicity of AQPs expression from 10th to 14th week gestation. Population and methods: Chorionic villi samples (CVS) collected in pregnant women for prenatal diagnosis were analysed by real time-PCR to assess cDNA expression of AQPs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 11, and compared with AQPs expression in placentas from normal term pregnancies. Results: 26 CVS corresponding to 26 pregnant women (age: 32.7 ± 4.5 years; gestational age: 12.4± 0.9 weeks) and 10 placental samples corresponding to normal term pregnancies were analysed. In CVS karyotype was normal in 16 cases, trisomy in 6 cases, mosaicism in 1 and unknown in 1. We found high mRNA expression for AQPs 1, 3, 9 and 11, low for AQPs 4, 5, and 8, and non-detectable for AQPs 2, 6, and 7 in chorionic villi. Conclusions: This is the first study systematically assessing the expression of a multiplicity of AQPs in chorionic villi samples between 10th and 14th weeks of gestation. High expression of AQP11 has been identified for the first time in early stages of human pregnancy. Chromosomal abnormalities did not alter AQPs' expression. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Water is an indispensable constituent of biological systems. Aquaporins, first discovered in 1991, constitute a family of small hydrophobic intra-membranous proteins (26–30 kDa) functioning as cell membrane water channels [1,2]. Of note, compared to the traditionally described transmembrane transfer across the lipid by-layer water permeability across AQP water channels may be increased up to 50 fold [3]. In addition, a specific subset of AQPs namely AQPs 3, 7, 9 and 10 referred to as “aquaglyceroporins” is also permeable to water, urea and glycerol; moreover, AQP9 also facilitates the flux of neutral solutes such as monocarboxylates, purines and pyrimidines [4]. To date, 13 members of the AQP family have been described in humans [5]. The Abbreviations: AF, amniotic fluid; AQPs, aquaporins; CV, chorionic villi; CVS, chorionic villi sampling; RT-PCR, real time polymerase chain reaction; RNA, ribonucleic acid; mRNA, messenger ribonucleic acid; cDNA, complementary deoxy-ribonucleic acid; Ct, threshold cycle; NPA, asparagine–proline–alanine motif. ⁎ Corresponding author at: Division of Neonatology, University & Polytechnic Hospital La Fe, Bulevar Sur s/n, E46026 Valencia, Spain. Tel.: + 34 96 1245688; fax: + 34 96 3619999. E-mail address: [email protected] (M. Vento). 0378-3782/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2012.01.009
role of AQPs in the regulation of placental water transfer to the foetus and intramembranous resorption is still only poorly understood; however, experimental and clinical data support the hypothesis that AQPs play an important role in foetal water flow (for review [4]). Foetal weight increases exponentially during gestation and foetal water requirements do accordingly [6]. Hence, at the end of gestation up to 400 mL/day are transferred from the amniotic cavity across the foetal membranes into the foetal circulation [7]. Interestingly, mechanisms regulating AF volume are yet not fully understood it has been suggested that aquaporins (AQPs) could play an important role in the regulatory process [5]. Remarkably, abnormal placental transfer of fluid may result in excessive (polyhydramnios) or reduced (oligohydramnios) amniotic fluid volume putting the foetus at risk of significant morbidity [8,9]. Moreover, increased AQP9 expression, protein synthesis and functionality have been associated with preeclamptic placentas modulated by human chorionic gonadotropin via cAMP pathways [10]. AQPs 1, 3, 4, 8 and 9 have been located in human placenta and foetal membranes late in gestation and consistently associated with the regulation of placental, chorion and amnion water transfer [4]. In this regard, several studies have correlated AQP9 and AQP8 over-expression with polyhydramnios and decreased AQP1 and AQP3 expression with
590
J. Escobar et al. / Early Human Development 88 (2012) 589–594
oligohydramnios [11,12]. Notwithstanding, expression and role of AQPs early in human pregnancy when water transport to the foetus is still quantitatively not so relevant are lacking. Considering this, we hypothesized that AQPs expression towards the end of embryogenesis and initiation of organogenesis might be different than in later stages of pregnancy and could contribute to explain AQPs physiologic and pathophysiologic implications. The aim of this study was to analyse the expression of AQPs in chorionic villi sampled between 10th and 14th week gestation using real time polymerase chain reaction (RT-PCR) and compare it to placental expression in term pregnancies. We omitted the determination of AQPs 0, 10 and 12 because they are specifically and uniquely expressed in the eye, gastrointestinal tract and pancreas respectively [5,13,14]. 2. Material and methods 2.1. Study design This is a prospective clinical study performed in both the Division of Obstetrics and the Division of Neonatology of the University Hospital La Fe (UHLF; Valencia; Spain) between January 2006 and September 2008 approved by the Internal Board Review (Comité de Ética e Investigación Clínica) of the UHLF. All participating women received, understood, and signed an informed consent prior to sample collection. It should be underscored that chorionic villi sampling (CVS) was not performed exclusively to determine expression of AQPs but following medical indications. All participants accepted that a minimal quantity of the tissue sampled was used for research purposes. The inclusion criteria for the selection of the patients were: (i) spontaneous pregnancy; (ii) single pregnancy; (iii) pregnancy strictly controlled following routines of the Division of Obstetrics (UHLF). Exclusion criteria were: (i) co-morbidity or medication susceptible of influencing foetal fluid balance or AQP expression (e.g.: drugs interfering with renal function); (ii) multiple gestation; (iii) employment of assisted reproduction techniques. Patients' data and any other important co-morbidity were anonymously reported in a database. CVS was performed trans-vaginally. Indications for CVS are shown in Table 1. The outcomes of the prenatal diagnostic and the pregnancy were processed in the database as well. Placental tissue samples (trophoblast) from 5 normal term pregnancies were used as controls. 2.2. Tissue collection and processing Chorionic villi (CV) samples were soaked in RNAlater® solution (Ambion, Carlsbad, California, USA) and kept at 4 °C overnight. Thereafter, supernatant was removed and samples were frozen at − 80 °C. Placental (trophoblast) samples were collected from non-complicated term pregnancies (n = 5) and also soaked in RNAlater® for a few hours, and then stored as for CV. Human samples of liver, uterus, and cerebral artery were obtained from the Division of Pathology of Hospital La Fe (Valencia, Spain). PANC-1 (cells of human carcinoma of the exocrine pancreas) and HEK293 (human embryonic kidney) cells were grown in DMEM 4.5 g/L glucose supplemented with 10% FBS, penicillin and streptomycin. After 24 h culture medium was removed and cells were re-suspended in TRIzol (Invitrogen Corporation, Carlsbad, CA, USA). 2.3. RNA extraction and cDNA synthesis Total RNA was isolated using TRIzol (Invitrogen Corporation Carlsbad, CA, USA) reagent following the manufacturer's protocol. RNA concentration was quantified by spectrophotometry at 260 nm. Then, RNA was reverse transcribed to cDNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Canada) following manufacturer's instructions. All
Table 1 Summary of patients' characteristics and indications for chorionic villi sampling (CVS). Case #
Maternal age CVS performed at Indication for CVS (years) g. age (weeks+ days)
1
36
10+ 6
2 3 4
26 35 30
13+ 3 12+ 5 14+ 1
5
38
13+ 0
6
28
12+ 0
7
31
12+ 4
8
36
12+ 6
9 10
26 37
12+ 3 11+ 3
11
30
13+ 0
12
33
13+ 6
13 14
38 37
12+ 5 13+ 3
15
38
12+ 4
16
32
13+ 5
17
31
11+ 4
18
41
12
+4
19
37
11+ 6
20
36
12+ 4
29
12
+0
+1
21 22
32
13
23
39
12+ 4
24
27
13+ 5
25
41
12
+1
26
30
12+ 6
Nuchal translucency 6.8 mm Cystic hygroma Cystic hygroma Chromosomal abnormality in mother Previous son with mutation in NF1 Previous son with X-linked recessive inheritance Nuchal translucency 6 mm Nuchal translucency 6.3 mm Cystic hygroma Father carrier of IT-15mutation Chromosomal abnormality in mother Nuchal translucency 6.3 mm Cystic hygroma Nuchal translucency 3.8 mm Foetus suspected hydrops Foetus suspected hydrops Nuchal translucency 5.5 mm Increased risk for Down's syndrome 1st trimester Nuchal translucency 6.8 mm Nuchal translucency 4.8 mm Nuchal translucency 6.7 mm Foetus suspected hydrops Nuchal translucency 7.3 mm Nuchal translucency 5.6 mm Increased risk for Down's syndrome 1st trimester Nuchal translucency 4.3 mm
Karyotyping Trisomy 21 Normal Normal Normal Normal Normal
Trisomy 21 Normal Normal Normal Mosaicism Normal Normal Mosaicism Normal Normal Normal Trisomy 21
Trisomy 13 Normal Normal Normal Trisomy 18 Unknown Trisomy 21
Normal
samples were treated (reverse transcribed and PCR amplified) simultaneously to avoid batch-to-batch bias.
2.4. Real-time PCR The expression of ten AQPs (AQP1, 2, 3, 4, 5, 6, 7, 8, 9, 11) was evaluated by real-time PCR (iQ5, Bio-Rad Laboratories Inc., California, USA) using TaqMan® gene expression assays and TaqMan® 2X PCR Master Mix (Applied Biosystems, Life Technologies Corporation, Carlsbad, California, USA). A list of analysed genes and TaqMan probes is presented in Table 2. Ribosomal 18S gene was used as housekeeping gene. Real time PCR has been performed with 1 cycle of denaturation of 5 min at 95 °C, followed by 40 cycles of 15 s denaturation at 95 °C and 60 s annealing at 60 °C. The last cycle was followed by 10 min extension at 72 °C.
J. Escobar et al. / Early Human Development 88 (2012) 589–594 Table 2 TaqMan® gene expression assays (Applied Biosystems; Life Technologies Corporation; Carlsbad; California; USA) for aquaporins studied in chorionic villi samples of 26 pregnant women between 10th and 14th week of gestation. Gene name Human Human Human Human Human Human Human Human Human Human Human
Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin 18S
1 2 3 4 5 6 7 8 9 11
Gene symbol
Applied Biosystems TaqMan® gene expression assays (ID)
AQP1 AQP2 AQP3 AQP4 AQP5 AQP6 AQP7 AQP8 AQP9 AQP11 18S
Assay ID Hs00166067_m1 Assay ID Hs00166640_m1 Assay ID Hs00185020_m1 Assay ID Hs00242342_m1 Assay ID Hs00387048_m1 Assay ID Hs00364989_m1 Assay ID Hs00357359_m1 Assay ID Hs01086280_g1 Assay ID Hs00175573_m1 Assay ID Hs00542682_m1 Hs99999901_s1
2.5. cDNA sequencing Verification of specificity of TaqMan assay for AQP11 was performed by cDNA sequencing (ABI 3730; Applied Biosystems; Foster City; CA; USA). cDNA from CV was amplified by PCR using the TaqMan assay for AQP11 (Applied Biosystems; Foster City; CA; USA). PCR product was purified and sequenced from both strands using specific primers: FW (5′-3′)-TTG ATG AAG CAT TCC CTC AG, RW (5′3′) – TTC CTT CCA TGG CTG CAT A. Sequencing results were used to run a BLAST using NCBI software (http://blast.ncbi.nih.gov/Blast.cgi). 2.6. Data analysis mRNA expression was calculated by the delta Ct (ΔCt) method (ΔCt = Ct target gene — Ct 18S) were Ct is the threshold cycle of each sample. To assess relative abundance of different AQPs in CV data were also expressed as ΔΔCt; in this case AQP4, which was found to be the least expressed aquaporin in CV, was used as reference to calculate fold expression level of other mRNAs. ΔΔCt analysis was used to assess expression level of different AQPs in all tissue included in this study, and term gestation placenta was used as a reference tissue (Table 3). 2.7. Statistical analysis Patients' characteristics are reported as mean, standard deviation (mean ± SD) and confidence intervals (CI). Since AQPs do not have a normal distribution and the number of samples was limited nonparametric statistics were employed. Statistical parameters, mRNA expression level (ΔCt, ΔΔCt and standard deviation of ΔΔCt) and Mann-Whitney's U test were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences between groups were considered statistically significant for p b 0.05. Table 3 mRNA expression of aquaporins in chorionic villi. Aquaporins
Fold expression
AQP3 AQP1 AQP11 AQP9 AQP5 AQP8 AQP4 AQP2 AQP6 AQP7
1453 1199 465 102 10 8 1 0 0 0
mRNA expression has been normalized to 18S. To calculate relative expression of aquaporins studied, AQP4 has been arbitrarily assigned a mRNA expression of 1. mRNA expression of different aquaporins has been ordered from most to least abundant transcript level.
591
3. Results From 35 eligible CV samples 26 were finally processed (Table 1). Nine samples were rejected because they were not preserved in good conditions or insufficient for being adequately analysed. Duration of gestation at the time of sampling ranged from 10 weeks and 4 days to 14 weeks and 1 day with a mean gestational age of 12.4 ± 0.9 weeks (CI: 12.3–13.2). Maternal age was between 24 and 41 years, with a mean age of 32.7 ± 4.5 years (CI: 30.4–34.4) (Table 1). Routine karyotyping was performed at the Division of Genetics (UHLF). Final genetic reports informed normal karyotype in 16 cases (61.5%); trisomy was found in 6 cases (23.1%) corresponding to Down syndrome [4], trisomy 13 [1] and trisomy 18 [1]; mosaicism was described in 3 cases (11.6%), and unknown in 1 case (3.8%) (Table 1). Hence, we studied mRNA expression of 10 different aquaporins in 26 CV samples. Among expressed aquaporins higher mRNA levels were found for AQPs 1, 3, 9, and 11, and a lower level for AQPs 4, 5 and 8. No expression for AQPs 2, 6 and 7 mRNA in CV was detected (Table 3). In addition, AQP expression was analysed in other tissue considered as positive controls. For example, AQP2 was not detected in CV but was found expressed in uterus (Fig. 1). Similarly, AQP7 expression was not detected in CV; however, it expression was positive in all other analysed tissues especially in cerebral artery. Identically, although AQP6 expression was negative in placental tissue and CV it was significantly expressed in renal cells (HEK293). AQP11 had a 4 to 10 fold higher mRNA expression in CV than in the rest of the analysed tissues (Fig. 1). In addition, relative expression of AQPs was studied in CV samples (10–14 weeks gestation) and placentas (38–40 weeks gestation). CV and placenta had a similar mRNA expression level for AQPs 1, 3, 4 and 5; however, AQP8 mRNA expression was lower in CV than in placenta although differences did not reach statistical significance (p = 0.08). Furthermore, mRNA expression of AQP7 was absent in CV and of AQP9 significantly lower in CV than in placenta (p b 0.05), and mRNA expression of AQP1 was 4.2 fold higher in CV than in placenta (p b 0.01) (Fig. 2). AQP11 was for the first time described in samples of human CV and placenta. In order to confirm this finding we amplified placental and CV cDNA with the specific assay for human AQP11 (Taqman Assay_ID Hs00542682_m1, Applied Biosystems, California, USA). After purification of the PCR product, we performed the sequencing (ABI 3730, Applied Biosystems, California, USA) (see description in the subsection cDNA sequencing of the Material and methods section). Results allowed us to conclude that AQP11 was indeed amplified in placenta and CV. Finally, no differences regarding AQPs expression in samples with normal versus abnormal karyotyping were found (Table 4). 4. Discussion Water comprises more than 50% of adult human body weight and is necessary for most of the biochemical reactions which take place in living organisms [1]. Interestingly, in the embryonic stage of human development water can account for almost 90% of the total body composition. Moreover, at the end of gestation a significant volume of water is transferred daily from the placenta to the foetus [6,7]. Of note, accumulation or deficit of AF (polyhydramnios or oligohydramnios respectively) may be responsible for significant morbidity in the foetus and newborn infant. Hence, AQPs which significantly contribute to organize water distribution within different biological compartments constitute essential elements during human pregnancy [15,16,17]. In this regard, AQPs 1, 3, 4, 8 and 9 located in placental tissue and foetal membranes have been identified as especially relevant not only during normal pregnancy but also under pathologic situations such as idiopathic polyhydramnios or oligohydramnios late in gestation [4].
AQP6 2,0 1,5 1,0 0,5 0,0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
0,4 0,2 0,0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP7 12 10 8 6 4 2 0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
1,0 0,5 0,0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
12 10 8 2 1 0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP9 12 10 2
1
0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
mRNA fold expression (vs 18S)
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
0,6
1,5
AQP4 14
mRNA fold expression (vs 18S)
0
0,8
2,0
mRNA fold expression (vs 18S)
1
1,0
AQP3 2,5
mRNA fold expression (vs 18S)
2
1,2
mRNA fold expression (vs 18S)
3
AQP2 1,4
mRNA fold expression (vs 18S)
AQP1 20 18 16 14 4
mRNA fold expression (vs 18S)
J. Escobar et al. / Early Human Development 88 (2012) 589–594
mRNA fold expression (vs 18S)
mRNA fold expression (vs 18S)
mRNA fold expression (vs 18S)
592
AQP5 7 6 5 4 3 2 1 0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP11 6 5 4 3 2 1 0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
Fig. 1. Relative gene expression of aquaporins in different tissues. mRNA expression of 10 aquaporins was studied by real-time PCR using TaqMan probes in placenta, chorionic villi, liver, uterus, PANC-1 cells (pancreatic cancer), cerebral artery, and HEK293 cells (human embryonic kidney). mRNA fold expression was determined by the DDct method.
Our objective was to assess if AQPs' expression profile varied from the early stages (10th to 14th weeks) towards the end of gestation. Experimental studies in mouse models at a very early stage of pregnancy have shown a consistent expression of AQPs 3, 8 and 9 [18,19]. Furthermore, studies in mice models have also shown membrane and placental expression of AQPs 1, 3, 8 and 9 in the embryonic stage of gestation [19]. Interestingly, as gestation progresses changes in amniotic fluid (AF) volume are paralleled by concomitant changes in AQPs' expression. Thus, AF volume negatively correlated with foetal membrane AQP1 as well as with placental AQP1 and AQP9 expression, and positively correlated with placental AQP3 expression. Altogether these experimental findings suggest a role of AQPs 1, 3 and 9 as mediators in the regulation of placental and membrane water flow, and ultimately AF volume [9–12]. Interestingly, both AQPs 3 and 9 which were highly expressed in our CV samples pertain to the subgroup “aquaglyceroporins” and play a fundamental role in osmoregulation and energy metabolism through the transport of
Fig. 2. Relative gene expression of aquaporins in chorionic villi and placenta. mRNA expression was studied by real-time PCR using TaqMan probes. mRNA fold expression was determined by the ΔΔCt method. mRNAs of AQP2 and AQP6 were not amplified neither in chorionic villi nor in placenta. **p b 0.01 and *p b 0.05 versus placenta. ■ Placenta; □ chorionic villi.
glycerol [20]. Because glycerol is produced by fat degradation and used for gluconeogenesis, its transport will also modulate fat and glucose metabolism and contribute to the energy metabolism. Thus, the regulation of glycerol transport by aquaglyceroporins contributes to the control of fat accumulation, glucose homeostasis, cardiac energy production and pancreatic insulin secretion, among other functions [21]. Surprisingly, AQP9 also transports other solutes such as lactate and monocarboxylates related with energy provision and purines and pyrimidines related with DNA metabolism [4]. Seemingly, high expression of AQPs 3 and 9 would translate their relevance to foetal energy metabolism and DNA activity throughout gestation. However, interpretation of these results should be cautiously taken into consideration since the role of aquaglyceroporins in human gestation has not been yet studied in depth. In our study equilibrium in fluid exchange between mother and foetus was assessed. This could be related with an adequate expression of AQPs directly related with water transfer to the foetus specifically AQPs 1, 3 and 8. Hence, it has been shown that alterations both in AQP1 and AQP3 expression in foetal membranes and placenta may be important in the pathophysiology of isolated oligohydramnios while increased expression of Aquaporin 8 has been associated with idiopathic polyhydramnios [11,12]. De Falco et al. assessed the expression of AQP4 in 15 human CV samples and placentas from pregnancies between 5 and 40 weeks gestation by immuno-histochemical analysis [22]. They found decreased AQP4 expression in the syncytiotrophoblast from the first to the third semester. Contrarily, samples of endothelial cells and stroma of placental villi showed an increased expression of AQP4 from the first to the third semester [22]. Conversely, we found very low AQP4 expression in CV samples of 10 to 14 weeks of gestation which would be coincidental with the results in stromal and placenta villi cells, but in concurrence with the results in syncytiotrophoblast. Undoubtedly, the different methodological approaches employed in both studies make comparison of results difficult. In ovine foetal lung AQP5 is highly expressed well before birth even exceeding in foetuses the levels of adult animals [23]; however, expression of AQP5 during gestation is extremely variable depending
J. Escobar et al. / Early Human Development 88 (2012) 589–594
593
Table 4 Association between karyotype and aquaporin expression in chorionic villi obtained at 10–14 weeks of gestation. Karyotype
n
AQP1
Normal
16
19.3 (18.8–19.5) 18.7 (18–3–19.0) 19.2 (N/A) 19.5 (N/A) 19.2 (18.9–19.6) 18.9 (N/A) 19.5 (19.1–19.6)
Trisomy 21
4
Trisomy 13
1
Trisomy 18
1
Mosaicism
3
Unknown abnormality
1
All subjects
26
AQP2
N/C
AQP3
AQP4
AQP5
19.1 (18.7–19.4) 18.7 (18.3–19.1) 19.1 (N/A) 18.8 (N/A) 19.1 (18.7–19.4) 18.8 (N/A) 18.9 (18.6–19.6)
29.3 (28.4–31.0) 29.1 (28.6–29.4) 28.3 (N/A) 29.7 (N/A) 30.0 (28.7–31.4) 29.2 (N/A) 29.6 (29.1–30.0)
25.9 (25.1–26.7) 26.2 (25.8–26.5) 25.1 (N/A) 26.3 (N/A) 26.9 (26.4–27.3) 25.8 (N/A) 26.4 (25.9–26.7)
on the mammal species. No special attributes in relation to water homeostasis during gestation have been described [13]. In our samples, expression of AQP5 was similar in early (chorionic villi) as in late gestation (mature placenta) [13]. AQP11 together with AQP12 have very special characteristics. In fact, both have been proposed to form a new AQPs subfamily named “sub-cellular AQPs” due to differences in their structure to the rest of AQPS [24]. Both are intracellular but while AQP12 is specific of pancreatic zymogen membranes [14,25]. AQP11 gene expression has been found in the endoplasmic reticulum (ER) of rat kidney, liver, testis and brain, and structurally has a cysteine substituted for an alanine at a highly conserved asparagine-proline-alanine (NPA) motif [26]. Interestingly, AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA1, and cerebral cortical neurons [27]. Moreover, it has also been associated to intrauterine growth restriction, and very recently with salivary submandibular gland in developing mouse [28,29]. Importantly, when AQP11 is knocked out, mice die before weaning due to polycystic kidney disease [30]. In humans, AQP11 transcript has so far been detected in heart, ureter and bladder [29]. Moreover, although the water permeability of AQP11 has been widely discussed very recent studies in Sf9 cell membranes have shown that AQP11 could be involved in slow but constant water movements across membranes [26]. Remarkably, expression of AQP11 in human placenta, foetal membranes or amniotic fluid has not been previously described. Thus, to our understanding this study reveals for the first time AQP11 gene expression in CV obtained in human pregnancies between 10th and 14th weeks gestation (see Fig. 1). BLAST analysis confirmed the homology between our findings in placenta and CV, and human AQP11 (Material and methods section). A proposed physiological function for AQP11 has been to maintain suitable environment in the ER allowing translation and proper protein folding [29]. Enhanced expression of AQP11 in the early stages of gestation (embryogenesis) as shown in our study could be related to the development of certain organs such as salivary glands or kidney and excretory system (ureter, bladder) and slow water transfer to the embryo. However, during foetal development AQP11 expression becomes progressively lower and towards end of gestation when organ functionality has achieved maturity and other AQPs take over water regulation (AQP1, 3, 8, 9). The presence of cysteine in the NPA motif could render AQP11 vulnerable to oxidative stress which could be of relevance in case of preterm delivery [26]. Since biological functions of AQP11 are functionally distinct from the rest of aquaporins and aqua-glyceroporins its role during human gestation deserves further study. The small number of samples is a limiting factor for the generalization of our results; however it should be kept in mind that CV sampling for research studies is difficult and rejection to participate is understandable. In spite of this, our results show a different AQPs'
AQP6
N/C
AQP7
N/C
AQP8
AQP9
AQP11
26.1 (25.6–27.2) 27.1 (26.6–27.5) 26.4 (N/A) 26.9 (N/A) 25.9 (25.4–26.3) 27.1 (N/A) 26.6 (26.3–26.8)
22.3 (21.9–22.8) 23.4 (22.6–24.1) 23.3 (N/A) 22.1 (N/A) 22.5 (21.9–23.1) 22.0 (N/A) 22.7 (22.6–23.2)
19.7 (18.3–21.4) 21.2 (19.6–21.8) 21.5 (N/A) 20.3 (N/A) 19.6 (19.0–19.9) 20.5 (N/A) 20.9 (20.3–21.1)
profile and rate of expression in initial stages as compared to the end of pregnancy in placenta and foetal membranes. We conclude that AQPs 1, 3, 4, 5, 8, 9 and 11 are expressed in chorionic villi samples collected between 10th and 14th weeks of gestation. Of note, AQP11 expression has been described for the first time in the medical literature in this specific period of pregnancy, and it could play a role in slow pace water transport in the first weeks of pregnancy and development of excretory systems. Regulation of AF volume seems to be undertaken later on in gestation by AQPs 1, 3, 8 and 9 as has been shown in animal models and in the clinical setting. However, there are still many unanswered questions especially in their role under pathologic conditions and possible use as pharmacologic agents. Further experimental and clinical studies are needed to understand the physiologic role of AQP11 early in gestation. Finally, chromosomal abnormalities detected did not alter AQPs expression or amniotic fluid volume regulation. Conflict of interest statement None of the authors of this manuscript declares having any actual or potential conflict of interest including financial, personal or other relationships with other people or organizations within that could inappropriately bias the present manuscript. Acknowledgements We would like to thank our obstetric colleagues and nurses from the Prenatal Diagnostic Unit for their co-operation in chorionic villi sample collection and the members of the Genetic Division of our hospital for karyotyping the samples. This study received financial funding from the Institute Carlos III/ Spanish Ministry of Science and Technology grant RD08/072/022 to M Vento and A Perales, FIS PI08/0027 to M Vento, Consolider Grant CSD-2007-00020 to J Escobar and A Arduini, and CD11/00154 to J Escobar. References [1] Agre P. Nobel lecture. Aquaporin water channels. Biosci Rep 2004;24:127–63. [2] Carbrey JM, Agre P. Discovery of the aquaporins and the development of the field. Handb Exp Pharmacol 2009;190:3–28. [3] Gonen T, Walz T. The structure of aquaporins. Q Rev Biophys 2006;39:361–96. [4] Ishibashi K, Hara S, Kondo S. Aquaporin water channels in mammals. Clin Exp Nephrol 2009;13:107–17. [5] Damiano AE. Review: water channel proteins in the human placenta and foetal membranes. Placenta 2011;32:S207–11 Supplement B, Trophoblast Research, 25. [6] Stulc J. Placental transfer of inorganic ions and water. Physiol Rev 1997;77: 805–36. [7] Mann SE, Nijland MJ, Ross MG. Mathematic modelling of human amniotic fluid dynamics. Am J Obstet Gynecol 1996;175:937–44. [8] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Amniotic fluid water dynamics. Placenta 2007;28:816–23.
594
J. Escobar et al. / Early Human Development 88 (2012) 589–594
[9] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Regulation of amniotic fluid volume. Placenta 2007;28:824–32. [10] Marino GI, Castro-Parodi M, Dietrich V, Damiano AE. High levels of human chorionic gonadotropin (hCG) correlate with increased aquaporin-9 (AQP9) expression in explants from human preeclamptic placenta. Reprod Sci 2010;17:444–53. [11] Zhu X, Jiang S, Hu Y, Zheng X, Zou S, Wang Y, et al. The expression of aquaporin 8 and aquaporin 9 in foetal membranes and placenta in term pregnancies complicated by idiopathic polyhydramnios. Early Hum Dev 2010;86:657–63. [12] Zhu XQ, Jiang SS, Zhu XJ, Zou SW, Wang YH, Hu YC. Expression of aquaporin 1 and aquaporin 3 in foetal membranes and placenta in human term pregnancies with oligohydramnios. Placenta 2009;30:670–6. [13] Liu H, Wintour EM. Aquaporins in development — a review. Reprod Biol Endocrinol 2005;3:18–28. [14] Itoh T, Rai T, Kuwahara M, Ko SB, Uchida S, Sasaki S, et al. Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells. Biochem Biophys Res Commun 2005;330:832–8. [15] Zaccai G. The effect of water on protein dynamics. Philos Trans R Soc Lond B Biol Sci 2004;359:1269–75. [16] Zelenina M, Zelenin S, Aperia A. Water channels (aquaporins) and their role for postnatal adaptation. Pediatr Res 2005;57:47–53. [17] Huppertz B. The anatomy of the normal placenta. J Clin Pathol 2008;61:1296–302. [18] Barcroft LC, Offenberg H, Thomsen P, Watson AJ. Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev Biol 2003;256:342–54. [19] Beall MH, Wang S, Yang B, Chaudhri N, Amidi F, Ross MG. Placental and membrane aquaporin water channels: correlation with amniotic fluid volume and composition. Placenta 2007;28:421–8.
[20] Ishibashi K, Kondo S, Hara S, Morishita Y. The evolutionary aspects of aquaporin family. Am J Physiol Regul Integr Comp Physiol 2011;300:R566–76. [21] Rodriguez A, Catalán V, Gómez-Ambrosi J, Frühbeck G. Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control. Cell Cycle 2011;10:1548–56. [22] De Falco M, Cobellis L, Torella M, Acone G, Varano L, Sellitti A, et al. Down-regulation of aquaporin 4 in human placenta throughout pregnancy. In Vivo 2007;21:813–7. [23] Liu H, Hooper SB, Armugam A, Dawson N, Ferraro T, Jeyaseelan K, et al. Aquaporin gene expression and regulation in the ovine foetal lung. J Physiol 2003;551: 503–14. [24] Ishibashi K. Aquaporin subfamily with unusual NPA boxes. Biochim Biophys Acta 2006;1758:989–93. [25] Yakata K, Hiroaki Y, Ishibashi K, Sohara E, Sasaki S, Mitsuoka K, et al. Aquaporin-11 containing a divergent NPA motif has normal water channel activity. Biochim Biophys Acta 2007;1768:688–93. [26] Yakata K, Tani K, Fujiyoshi Y. Water permeability and characterization of aquaporin-11. J Struct Biol 2011;174:315–20. [27] Gorelick DA, Praetorius J, Tsunenari T, Nielsen S, Agre P. Aquaporin-11: a channel protein lacking apparent transport function expressed in brain. BMC Biochem 2006;7:14–28. [28] Scifres CM, Nelson DM. Intrauterine growth restriction, human placental development and trophoblast cell death. J Physiol 2009;587:3453–8. [29] Larsen HS, Ruus AK, Schreurs O, Galtung HK. Aquaporin 11 in the developing mouse submandibular gland. Eur J Oral Sci 2010;118:9–13. [30] Morishita Y, Matsuzaki T, Hara-chikuma M, Andoo A, Shimono M, Matsuki A, et al. Disruption of aquaporin 11 produces polycystic kidneys following vacuolization of the proximal tubule. Mol Cell Biol 2005;25:7770–9.
Contents lists available at SciVerse ScienceDirect
Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Expression of aquaporins early in human pregnancy Javier Escobar a, María Gormaz b, Alessandro Arduini c, Karien Gosens d, Anabel Martinez e, Alfredo Perales e, Raquel Escrig b, Enrique Tormos e, Mónica Roselló f, Carmen Orellana f, Máximo Vento a, b,⁎ a
Neonatal Research Unit, Research Institute Hospital La Fe, Valencia, Spain Division of Neonatology, University and Polytechnic Hospital La Fe, Valencia, Spain Department of Physiology, University of Valencia, Spain d Department of Obstetrics & Neonatology, UMC St Radboud, Holland, the Netherlands e Division of Obstetrics, University and Polytechnic Hospital La Fe, Valencia, Spain f Division of Genetics, University and Polytechnic Hospital La Fe, Valencia, Spain b c
a r t i c l e
i n f o
Article history: Received 30 June 2011 Received in revised form 6 January 2012 Accepted 7 January 2012 Keywords: Aquaporins Pregnancy Chorionic villi Amniotic fluid
a b s t r a c t Background: Aquaporins (AQPs) constitute a family of channel proteins implicated in transmembrane water transport. Thirteen different AQPs (AQP0–12) have been described but their precise biologic function still remains unclear. AQPs 1, 3, 4, 8, and 9 expression has been described in human chorion, amnion and placenta; however, AQP4 is the only that has been identified in the first trimester of human pregnancy. Objective: To assess multiplicity of AQPs expression from 10th to 14th week gestation. Population and methods: Chorionic villi samples (CVS) collected in pregnant women for prenatal diagnosis were analysed by real time-PCR to assess cDNA expression of AQPs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 11, and compared with AQPs expression in placentas from normal term pregnancies. Results: 26 CVS corresponding to 26 pregnant women (age: 32.7 ± 4.5 years; gestational age: 12.4± 0.9 weeks) and 10 placental samples corresponding to normal term pregnancies were analysed. In CVS karyotype was normal in 16 cases, trisomy in 6 cases, mosaicism in 1 and unknown in 1. We found high mRNA expression for AQPs 1, 3, 9 and 11, low for AQPs 4, 5, and 8, and non-detectable for AQPs 2, 6, and 7 in chorionic villi. Conclusions: This is the first study systematically assessing the expression of a multiplicity of AQPs in chorionic villi samples between 10th and 14th weeks of gestation. High expression of AQP11 has been identified for the first time in early stages of human pregnancy. Chromosomal abnormalities did not alter AQPs' expression. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Water is an indispensable constituent of biological systems. Aquaporins, first discovered in 1991, constitute a family of small hydrophobic intra-membranous proteins (26–30 kDa) functioning as cell membrane water channels [1,2]. Of note, compared to the traditionally described transmembrane transfer across the lipid by-layer water permeability across AQP water channels may be increased up to 50 fold [3]. In addition, a specific subset of AQPs namely AQPs 3, 7, 9 and 10 referred to as “aquaglyceroporins” is also permeable to water, urea and glycerol; moreover, AQP9 also facilitates the flux of neutral solutes such as monocarboxylates, purines and pyrimidines [4]. To date, 13 members of the AQP family have been described in humans [5]. The Abbreviations: AF, amniotic fluid; AQPs, aquaporins; CV, chorionic villi; CVS, chorionic villi sampling; RT-PCR, real time polymerase chain reaction; RNA, ribonucleic acid; mRNA, messenger ribonucleic acid; cDNA, complementary deoxy-ribonucleic acid; Ct, threshold cycle; NPA, asparagine–proline–alanine motif. ⁎ Corresponding author at: Division of Neonatology, University & Polytechnic Hospital La Fe, Bulevar Sur s/n, E46026 Valencia, Spain. Tel.: + 34 96 1245688; fax: + 34 96 3619999. E-mail address: [email protected] (M. Vento). 0378-3782/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2012.01.009
role of AQPs in the regulation of placental water transfer to the foetus and intramembranous resorption is still only poorly understood; however, experimental and clinical data support the hypothesis that AQPs play an important role in foetal water flow (for review [4]). Foetal weight increases exponentially during gestation and foetal water requirements do accordingly [6]. Hence, at the end of gestation up to 400 mL/day are transferred from the amniotic cavity across the foetal membranes into the foetal circulation [7]. Interestingly, mechanisms regulating AF volume are yet not fully understood it has been suggested that aquaporins (AQPs) could play an important role in the regulatory process [5]. Remarkably, abnormal placental transfer of fluid may result in excessive (polyhydramnios) or reduced (oligohydramnios) amniotic fluid volume putting the foetus at risk of significant morbidity [8,9]. Moreover, increased AQP9 expression, protein synthesis and functionality have been associated with preeclamptic placentas modulated by human chorionic gonadotropin via cAMP pathways [10]. AQPs 1, 3, 4, 8 and 9 have been located in human placenta and foetal membranes late in gestation and consistently associated with the regulation of placental, chorion and amnion water transfer [4]. In this regard, several studies have correlated AQP9 and AQP8 over-expression with polyhydramnios and decreased AQP1 and AQP3 expression with
590
J. Escobar et al. / Early Human Development 88 (2012) 589–594
oligohydramnios [11,12]. Notwithstanding, expression and role of AQPs early in human pregnancy when water transport to the foetus is still quantitatively not so relevant are lacking. Considering this, we hypothesized that AQPs expression towards the end of embryogenesis and initiation of organogenesis might be different than in later stages of pregnancy and could contribute to explain AQPs physiologic and pathophysiologic implications. The aim of this study was to analyse the expression of AQPs in chorionic villi sampled between 10th and 14th week gestation using real time polymerase chain reaction (RT-PCR) and compare it to placental expression in term pregnancies. We omitted the determination of AQPs 0, 10 and 12 because they are specifically and uniquely expressed in the eye, gastrointestinal tract and pancreas respectively [5,13,14]. 2. Material and methods 2.1. Study design This is a prospective clinical study performed in both the Division of Obstetrics and the Division of Neonatology of the University Hospital La Fe (UHLF; Valencia; Spain) between January 2006 and September 2008 approved by the Internal Board Review (Comité de Ética e Investigación Clínica) of the UHLF. All participating women received, understood, and signed an informed consent prior to sample collection. It should be underscored that chorionic villi sampling (CVS) was not performed exclusively to determine expression of AQPs but following medical indications. All participants accepted that a minimal quantity of the tissue sampled was used for research purposes. The inclusion criteria for the selection of the patients were: (i) spontaneous pregnancy; (ii) single pregnancy; (iii) pregnancy strictly controlled following routines of the Division of Obstetrics (UHLF). Exclusion criteria were: (i) co-morbidity or medication susceptible of influencing foetal fluid balance or AQP expression (e.g.: drugs interfering with renal function); (ii) multiple gestation; (iii) employment of assisted reproduction techniques. Patients' data and any other important co-morbidity were anonymously reported in a database. CVS was performed trans-vaginally. Indications for CVS are shown in Table 1. The outcomes of the prenatal diagnostic and the pregnancy were processed in the database as well. Placental tissue samples (trophoblast) from 5 normal term pregnancies were used as controls. 2.2. Tissue collection and processing Chorionic villi (CV) samples were soaked in RNAlater® solution (Ambion, Carlsbad, California, USA) and kept at 4 °C overnight. Thereafter, supernatant was removed and samples were frozen at − 80 °C. Placental (trophoblast) samples were collected from non-complicated term pregnancies (n = 5) and also soaked in RNAlater® for a few hours, and then stored as for CV. Human samples of liver, uterus, and cerebral artery were obtained from the Division of Pathology of Hospital La Fe (Valencia, Spain). PANC-1 (cells of human carcinoma of the exocrine pancreas) and HEK293 (human embryonic kidney) cells were grown in DMEM 4.5 g/L glucose supplemented with 10% FBS, penicillin and streptomycin. After 24 h culture medium was removed and cells were re-suspended in TRIzol (Invitrogen Corporation, Carlsbad, CA, USA). 2.3. RNA extraction and cDNA synthesis Total RNA was isolated using TRIzol (Invitrogen Corporation Carlsbad, CA, USA) reagent following the manufacturer's protocol. RNA concentration was quantified by spectrophotometry at 260 nm. Then, RNA was reverse transcribed to cDNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Canada) following manufacturer's instructions. All
Table 1 Summary of patients' characteristics and indications for chorionic villi sampling (CVS). Case #
Maternal age CVS performed at Indication for CVS (years) g. age (weeks+ days)
1
36
10+ 6
2 3 4
26 35 30
13+ 3 12+ 5 14+ 1
5
38
13+ 0
6
28
12+ 0
7
31
12+ 4
8
36
12+ 6
9 10
26 37
12+ 3 11+ 3
11
30
13+ 0
12
33
13+ 6
13 14
38 37
12+ 5 13+ 3
15
38
12+ 4
16
32
13+ 5
17
31
11+ 4
18
41
12
+4
19
37
11+ 6
20
36
12+ 4
29
12
+0
+1
21 22
32
13
23
39
12+ 4
24
27
13+ 5
25
41
12
+1
26
30
12+ 6
Nuchal translucency 6.8 mm Cystic hygroma Cystic hygroma Chromosomal abnormality in mother Previous son with mutation in NF1 Previous son with X-linked recessive inheritance Nuchal translucency 6 mm Nuchal translucency 6.3 mm Cystic hygroma Father carrier of IT-15mutation Chromosomal abnormality in mother Nuchal translucency 6.3 mm Cystic hygroma Nuchal translucency 3.8 mm Foetus suspected hydrops Foetus suspected hydrops Nuchal translucency 5.5 mm Increased risk for Down's syndrome 1st trimester Nuchal translucency 6.8 mm Nuchal translucency 4.8 mm Nuchal translucency 6.7 mm Foetus suspected hydrops Nuchal translucency 7.3 mm Nuchal translucency 5.6 mm Increased risk for Down's syndrome 1st trimester Nuchal translucency 4.3 mm
Karyotyping Trisomy 21 Normal Normal Normal Normal Normal
Trisomy 21 Normal Normal Normal Mosaicism Normal Normal Mosaicism Normal Normal Normal Trisomy 21
Trisomy 13 Normal Normal Normal Trisomy 18 Unknown Trisomy 21
Normal
samples were treated (reverse transcribed and PCR amplified) simultaneously to avoid batch-to-batch bias.
2.4. Real-time PCR The expression of ten AQPs (AQP1, 2, 3, 4, 5, 6, 7, 8, 9, 11) was evaluated by real-time PCR (iQ5, Bio-Rad Laboratories Inc., California, USA) using TaqMan® gene expression assays and TaqMan® 2X PCR Master Mix (Applied Biosystems, Life Technologies Corporation, Carlsbad, California, USA). A list of analysed genes and TaqMan probes is presented in Table 2. Ribosomal 18S gene was used as housekeeping gene. Real time PCR has been performed with 1 cycle of denaturation of 5 min at 95 °C, followed by 40 cycles of 15 s denaturation at 95 °C and 60 s annealing at 60 °C. The last cycle was followed by 10 min extension at 72 °C.
J. Escobar et al. / Early Human Development 88 (2012) 589–594 Table 2 TaqMan® gene expression assays (Applied Biosystems; Life Technologies Corporation; Carlsbad; California; USA) for aquaporins studied in chorionic villi samples of 26 pregnant women between 10th and 14th week of gestation. Gene name Human Human Human Human Human Human Human Human Human Human Human
Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin Aquaporin 18S
1 2 3 4 5 6 7 8 9 11
Gene symbol
Applied Biosystems TaqMan® gene expression assays (ID)
AQP1 AQP2 AQP3 AQP4 AQP5 AQP6 AQP7 AQP8 AQP9 AQP11 18S
Assay ID Hs00166067_m1 Assay ID Hs00166640_m1 Assay ID Hs00185020_m1 Assay ID Hs00242342_m1 Assay ID Hs00387048_m1 Assay ID Hs00364989_m1 Assay ID Hs00357359_m1 Assay ID Hs01086280_g1 Assay ID Hs00175573_m1 Assay ID Hs00542682_m1 Hs99999901_s1
2.5. cDNA sequencing Verification of specificity of TaqMan assay for AQP11 was performed by cDNA sequencing (ABI 3730; Applied Biosystems; Foster City; CA; USA). cDNA from CV was amplified by PCR using the TaqMan assay for AQP11 (Applied Biosystems; Foster City; CA; USA). PCR product was purified and sequenced from both strands using specific primers: FW (5′-3′)-TTG ATG AAG CAT TCC CTC AG, RW (5′3′) – TTC CTT CCA TGG CTG CAT A. Sequencing results were used to run a BLAST using NCBI software (http://blast.ncbi.nih.gov/Blast.cgi). 2.6. Data analysis mRNA expression was calculated by the delta Ct (ΔCt) method (ΔCt = Ct target gene — Ct 18S) were Ct is the threshold cycle of each sample. To assess relative abundance of different AQPs in CV data were also expressed as ΔΔCt; in this case AQP4, which was found to be the least expressed aquaporin in CV, was used as reference to calculate fold expression level of other mRNAs. ΔΔCt analysis was used to assess expression level of different AQPs in all tissue included in this study, and term gestation placenta was used as a reference tissue (Table 3). 2.7. Statistical analysis Patients' characteristics are reported as mean, standard deviation (mean ± SD) and confidence intervals (CI). Since AQPs do not have a normal distribution and the number of samples was limited nonparametric statistics were employed. Statistical parameters, mRNA expression level (ΔCt, ΔΔCt and standard deviation of ΔΔCt) and Mann-Whitney's U test were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences between groups were considered statistically significant for p b 0.05. Table 3 mRNA expression of aquaporins in chorionic villi. Aquaporins
Fold expression
AQP3 AQP1 AQP11 AQP9 AQP5 AQP8 AQP4 AQP2 AQP6 AQP7
1453 1199 465 102 10 8 1 0 0 0
mRNA expression has been normalized to 18S. To calculate relative expression of aquaporins studied, AQP4 has been arbitrarily assigned a mRNA expression of 1. mRNA expression of different aquaporins has been ordered from most to least abundant transcript level.
591
3. Results From 35 eligible CV samples 26 were finally processed (Table 1). Nine samples were rejected because they were not preserved in good conditions or insufficient for being adequately analysed. Duration of gestation at the time of sampling ranged from 10 weeks and 4 days to 14 weeks and 1 day with a mean gestational age of 12.4 ± 0.9 weeks (CI: 12.3–13.2). Maternal age was between 24 and 41 years, with a mean age of 32.7 ± 4.5 years (CI: 30.4–34.4) (Table 1). Routine karyotyping was performed at the Division of Genetics (UHLF). Final genetic reports informed normal karyotype in 16 cases (61.5%); trisomy was found in 6 cases (23.1%) corresponding to Down syndrome [4], trisomy 13 [1] and trisomy 18 [1]; mosaicism was described in 3 cases (11.6%), and unknown in 1 case (3.8%) (Table 1). Hence, we studied mRNA expression of 10 different aquaporins in 26 CV samples. Among expressed aquaporins higher mRNA levels were found for AQPs 1, 3, 9, and 11, and a lower level for AQPs 4, 5 and 8. No expression for AQPs 2, 6 and 7 mRNA in CV was detected (Table 3). In addition, AQP expression was analysed in other tissue considered as positive controls. For example, AQP2 was not detected in CV but was found expressed in uterus (Fig. 1). Similarly, AQP7 expression was not detected in CV; however, it expression was positive in all other analysed tissues especially in cerebral artery. Identically, although AQP6 expression was negative in placental tissue and CV it was significantly expressed in renal cells (HEK293). AQP11 had a 4 to 10 fold higher mRNA expression in CV than in the rest of the analysed tissues (Fig. 1). In addition, relative expression of AQPs was studied in CV samples (10–14 weeks gestation) and placentas (38–40 weeks gestation). CV and placenta had a similar mRNA expression level for AQPs 1, 3, 4 and 5; however, AQP8 mRNA expression was lower in CV than in placenta although differences did not reach statistical significance (p = 0.08). Furthermore, mRNA expression of AQP7 was absent in CV and of AQP9 significantly lower in CV than in placenta (p b 0.05), and mRNA expression of AQP1 was 4.2 fold higher in CV than in placenta (p b 0.01) (Fig. 2). AQP11 was for the first time described in samples of human CV and placenta. In order to confirm this finding we amplified placental and CV cDNA with the specific assay for human AQP11 (Taqman Assay_ID Hs00542682_m1, Applied Biosystems, California, USA). After purification of the PCR product, we performed the sequencing (ABI 3730, Applied Biosystems, California, USA) (see description in the subsection cDNA sequencing of the Material and methods section). Results allowed us to conclude that AQP11 was indeed amplified in placenta and CV. Finally, no differences regarding AQPs expression in samples with normal versus abnormal karyotyping were found (Table 4). 4. Discussion Water comprises more than 50% of adult human body weight and is necessary for most of the biochemical reactions which take place in living organisms [1]. Interestingly, in the embryonic stage of human development water can account for almost 90% of the total body composition. Moreover, at the end of gestation a significant volume of water is transferred daily from the placenta to the foetus [6,7]. Of note, accumulation or deficit of AF (polyhydramnios or oligohydramnios respectively) may be responsible for significant morbidity in the foetus and newborn infant. Hence, AQPs which significantly contribute to organize water distribution within different biological compartments constitute essential elements during human pregnancy [15,16,17]. In this regard, AQPs 1, 3, 4, 8 and 9 located in placental tissue and foetal membranes have been identified as especially relevant not only during normal pregnancy but also under pathologic situations such as idiopathic polyhydramnios or oligohydramnios late in gestation [4].
AQP6 2,0 1,5 1,0 0,5 0,0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
0,4 0,2 0,0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP7 12 10 8 6 4 2 0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
1,0 0,5 0,0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
12 10 8 2 1 0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP9 12 10 2
1
0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
mRNA fold expression (vs 18S)
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
0,6
1,5
AQP4 14
mRNA fold expression (vs 18S)
0
0,8
2,0
mRNA fold expression (vs 18S)
1
1,0
AQP3 2,5
mRNA fold expression (vs 18S)
2
1,2
mRNA fold expression (vs 18S)
3
AQP2 1,4
mRNA fold expression (vs 18S)
AQP1 20 18 16 14 4
mRNA fold expression (vs 18S)
J. Escobar et al. / Early Human Development 88 (2012) 589–594
mRNA fold expression (vs 18S)
mRNA fold expression (vs 18S)
mRNA fold expression (vs 18S)
592
AQP5 7 6 5 4 3 2 1 0
ta illi er us er A. en l V Liv ter anc ral U . c eb ac ria l P ho r er nc C C Pa
AQP11 6 5 4 3 2 1 0
. r a li r s nt Vil ive ru ce l A ce ial L Ute can bra a Pl hor r. ere nc C C Pa
Fig. 1. Relative gene expression of aquaporins in different tissues. mRNA expression of 10 aquaporins was studied by real-time PCR using TaqMan probes in placenta, chorionic villi, liver, uterus, PANC-1 cells (pancreatic cancer), cerebral artery, and HEK293 cells (human embryonic kidney). mRNA fold expression was determined by the DDct method.
Our objective was to assess if AQPs' expression profile varied from the early stages (10th to 14th weeks) towards the end of gestation. Experimental studies in mouse models at a very early stage of pregnancy have shown a consistent expression of AQPs 3, 8 and 9 [18,19]. Furthermore, studies in mice models have also shown membrane and placental expression of AQPs 1, 3, 8 and 9 in the embryonic stage of gestation [19]. Interestingly, as gestation progresses changes in amniotic fluid (AF) volume are paralleled by concomitant changes in AQPs' expression. Thus, AF volume negatively correlated with foetal membrane AQP1 as well as with placental AQP1 and AQP9 expression, and positively correlated with placental AQP3 expression. Altogether these experimental findings suggest a role of AQPs 1, 3 and 9 as mediators in the regulation of placental and membrane water flow, and ultimately AF volume [9–12]. Interestingly, both AQPs 3 and 9 which were highly expressed in our CV samples pertain to the subgroup “aquaglyceroporins” and play a fundamental role in osmoregulation and energy metabolism through the transport of
Fig. 2. Relative gene expression of aquaporins in chorionic villi and placenta. mRNA expression was studied by real-time PCR using TaqMan probes. mRNA fold expression was determined by the ΔΔCt method. mRNAs of AQP2 and AQP6 were not amplified neither in chorionic villi nor in placenta. **p b 0.01 and *p b 0.05 versus placenta. ■ Placenta; □ chorionic villi.
glycerol [20]. Because glycerol is produced by fat degradation and used for gluconeogenesis, its transport will also modulate fat and glucose metabolism and contribute to the energy metabolism. Thus, the regulation of glycerol transport by aquaglyceroporins contributes to the control of fat accumulation, glucose homeostasis, cardiac energy production and pancreatic insulin secretion, among other functions [21]. Surprisingly, AQP9 also transports other solutes such as lactate and monocarboxylates related with energy provision and purines and pyrimidines related with DNA metabolism [4]. Seemingly, high expression of AQPs 3 and 9 would translate their relevance to foetal energy metabolism and DNA activity throughout gestation. However, interpretation of these results should be cautiously taken into consideration since the role of aquaglyceroporins in human gestation has not been yet studied in depth. In our study equilibrium in fluid exchange between mother and foetus was assessed. This could be related with an adequate expression of AQPs directly related with water transfer to the foetus specifically AQPs 1, 3 and 8. Hence, it has been shown that alterations both in AQP1 and AQP3 expression in foetal membranes and placenta may be important in the pathophysiology of isolated oligohydramnios while increased expression of Aquaporin 8 has been associated with idiopathic polyhydramnios [11,12]. De Falco et al. assessed the expression of AQP4 in 15 human CV samples and placentas from pregnancies between 5 and 40 weeks gestation by immuno-histochemical analysis [22]. They found decreased AQP4 expression in the syncytiotrophoblast from the first to the third semester. Contrarily, samples of endothelial cells and stroma of placental villi showed an increased expression of AQP4 from the first to the third semester [22]. Conversely, we found very low AQP4 expression in CV samples of 10 to 14 weeks of gestation which would be coincidental with the results in stromal and placenta villi cells, but in concurrence with the results in syncytiotrophoblast. Undoubtedly, the different methodological approaches employed in both studies make comparison of results difficult. In ovine foetal lung AQP5 is highly expressed well before birth even exceeding in foetuses the levels of adult animals [23]; however, expression of AQP5 during gestation is extremely variable depending
J. Escobar et al. / Early Human Development 88 (2012) 589–594
593
Table 4 Association between karyotype and aquaporin expression in chorionic villi obtained at 10–14 weeks of gestation. Karyotype
n
AQP1
Normal
16
19.3 (18.8–19.5) 18.7 (18–3–19.0) 19.2 (N/A) 19.5 (N/A) 19.2 (18.9–19.6) 18.9 (N/A) 19.5 (19.1–19.6)
Trisomy 21
4
Trisomy 13
1
Trisomy 18
1
Mosaicism
3
Unknown abnormality
1
All subjects
26
AQP2
N/C
AQP3
AQP4
AQP5
19.1 (18.7–19.4) 18.7 (18.3–19.1) 19.1 (N/A) 18.8 (N/A) 19.1 (18.7–19.4) 18.8 (N/A) 18.9 (18.6–19.6)
29.3 (28.4–31.0) 29.1 (28.6–29.4) 28.3 (N/A) 29.7 (N/A) 30.0 (28.7–31.4) 29.2 (N/A) 29.6 (29.1–30.0)
25.9 (25.1–26.7) 26.2 (25.8–26.5) 25.1 (N/A) 26.3 (N/A) 26.9 (26.4–27.3) 25.8 (N/A) 26.4 (25.9–26.7)
on the mammal species. No special attributes in relation to water homeostasis during gestation have been described [13]. In our samples, expression of AQP5 was similar in early (chorionic villi) as in late gestation (mature placenta) [13]. AQP11 together with AQP12 have very special characteristics. In fact, both have been proposed to form a new AQPs subfamily named “sub-cellular AQPs” due to differences in their structure to the rest of AQPS [24]. Both are intracellular but while AQP12 is specific of pancreatic zymogen membranes [14,25]. AQP11 gene expression has been found in the endoplasmic reticulum (ER) of rat kidney, liver, testis and brain, and structurally has a cysteine substituted for an alanine at a highly conserved asparagine-proline-alanine (NPA) motif [26]. Interestingly, AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA1, and cerebral cortical neurons [27]. Moreover, it has also been associated to intrauterine growth restriction, and very recently with salivary submandibular gland in developing mouse [28,29]. Importantly, when AQP11 is knocked out, mice die before weaning due to polycystic kidney disease [30]. In humans, AQP11 transcript has so far been detected in heart, ureter and bladder [29]. Moreover, although the water permeability of AQP11 has been widely discussed very recent studies in Sf9 cell membranes have shown that AQP11 could be involved in slow but constant water movements across membranes [26]. Remarkably, expression of AQP11 in human placenta, foetal membranes or amniotic fluid has not been previously described. Thus, to our understanding this study reveals for the first time AQP11 gene expression in CV obtained in human pregnancies between 10th and 14th weeks gestation (see Fig. 1). BLAST analysis confirmed the homology between our findings in placenta and CV, and human AQP11 (Material and methods section). A proposed physiological function for AQP11 has been to maintain suitable environment in the ER allowing translation and proper protein folding [29]. Enhanced expression of AQP11 in the early stages of gestation (embryogenesis) as shown in our study could be related to the development of certain organs such as salivary glands or kidney and excretory system (ureter, bladder) and slow water transfer to the embryo. However, during foetal development AQP11 expression becomes progressively lower and towards end of gestation when organ functionality has achieved maturity and other AQPs take over water regulation (AQP1, 3, 8, 9). The presence of cysteine in the NPA motif could render AQP11 vulnerable to oxidative stress which could be of relevance in case of preterm delivery [26]. Since biological functions of AQP11 are functionally distinct from the rest of aquaporins and aqua-glyceroporins its role during human gestation deserves further study. The small number of samples is a limiting factor for the generalization of our results; however it should be kept in mind that CV sampling for research studies is difficult and rejection to participate is understandable. In spite of this, our results show a different AQPs'
AQP6
N/C
AQP7
N/C
AQP8
AQP9
AQP11
26.1 (25.6–27.2) 27.1 (26.6–27.5) 26.4 (N/A) 26.9 (N/A) 25.9 (25.4–26.3) 27.1 (N/A) 26.6 (26.3–26.8)
22.3 (21.9–22.8) 23.4 (22.6–24.1) 23.3 (N/A) 22.1 (N/A) 22.5 (21.9–23.1) 22.0 (N/A) 22.7 (22.6–23.2)
19.7 (18.3–21.4) 21.2 (19.6–21.8) 21.5 (N/A) 20.3 (N/A) 19.6 (19.0–19.9) 20.5 (N/A) 20.9 (20.3–21.1)
profile and rate of expression in initial stages as compared to the end of pregnancy in placenta and foetal membranes. We conclude that AQPs 1, 3, 4, 5, 8, 9 and 11 are expressed in chorionic villi samples collected between 10th and 14th weeks of gestation. Of note, AQP11 expression has been described for the first time in the medical literature in this specific period of pregnancy, and it could play a role in slow pace water transport in the first weeks of pregnancy and development of excretory systems. Regulation of AF volume seems to be undertaken later on in gestation by AQPs 1, 3, 8 and 9 as has been shown in animal models and in the clinical setting. However, there are still many unanswered questions especially in their role under pathologic conditions and possible use as pharmacologic agents. Further experimental and clinical studies are needed to understand the physiologic role of AQP11 early in gestation. Finally, chromosomal abnormalities detected did not alter AQPs expression or amniotic fluid volume regulation. Conflict of interest statement None of the authors of this manuscript declares having any actual or potential conflict of interest including financial, personal or other relationships with other people or organizations within that could inappropriately bias the present manuscript. Acknowledgements We would like to thank our obstetric colleagues and nurses from the Prenatal Diagnostic Unit for their co-operation in chorionic villi sample collection and the members of the Genetic Division of our hospital for karyotyping the samples. This study received financial funding from the Institute Carlos III/ Spanish Ministry of Science and Technology grant RD08/072/022 to M Vento and A Perales, FIS PI08/0027 to M Vento, Consolider Grant CSD-2007-00020 to J Escobar and A Arduini, and CD11/00154 to J Escobar. References [1] Agre P. Nobel lecture. Aquaporin water channels. Biosci Rep 2004;24:127–63. [2] Carbrey JM, Agre P. Discovery of the aquaporins and the development of the field. Handb Exp Pharmacol 2009;190:3–28. [3] Gonen T, Walz T. The structure of aquaporins. Q Rev Biophys 2006;39:361–96. [4] Ishibashi K, Hara S, Kondo S. Aquaporin water channels in mammals. Clin Exp Nephrol 2009;13:107–17. [5] Damiano AE. Review: water channel proteins in the human placenta and foetal membranes. Placenta 2011;32:S207–11 Supplement B, Trophoblast Research, 25. [6] Stulc J. Placental transfer of inorganic ions and water. Physiol Rev 1997;77: 805–36. [7] Mann SE, Nijland MJ, Ross MG. Mathematic modelling of human amniotic fluid dynamics. Am J Obstet Gynecol 1996;175:937–44. [8] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Amniotic fluid water dynamics. Placenta 2007;28:816–23.
594
J. Escobar et al. / Early Human Development 88 (2012) 589–594
[9] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Regulation of amniotic fluid volume. Placenta 2007;28:824–32. [10] Marino GI, Castro-Parodi M, Dietrich V, Damiano AE. High levels of human chorionic gonadotropin (hCG) correlate with increased aquaporin-9 (AQP9) expression in explants from human preeclamptic placenta. Reprod Sci 2010;17:444–53. [11] Zhu X, Jiang S, Hu Y, Zheng X, Zou S, Wang Y, et al. The expression of aquaporin 8 and aquaporin 9 in foetal membranes and placenta in term pregnancies complicated by idiopathic polyhydramnios. Early Hum Dev 2010;86:657–63. [12] Zhu XQ, Jiang SS, Zhu XJ, Zou SW, Wang YH, Hu YC. Expression of aquaporin 1 and aquaporin 3 in foetal membranes and placenta in human term pregnancies with oligohydramnios. Placenta 2009;30:670–6. [13] Liu H, Wintour EM. Aquaporins in development — a review. Reprod Biol Endocrinol 2005;3:18–28. [14] Itoh T, Rai T, Kuwahara M, Ko SB, Uchida S, Sasaki S, et al. Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells. Biochem Biophys Res Commun 2005;330:832–8. [15] Zaccai G. The effect of water on protein dynamics. Philos Trans R Soc Lond B Biol Sci 2004;359:1269–75. [16] Zelenina M, Zelenin S, Aperia A. Water channels (aquaporins) and their role for postnatal adaptation. Pediatr Res 2005;57:47–53. [17] Huppertz B. The anatomy of the normal placenta. J Clin Pathol 2008;61:1296–302. [18] Barcroft LC, Offenberg H, Thomsen P, Watson AJ. Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev Biol 2003;256:342–54. [19] Beall MH, Wang S, Yang B, Chaudhri N, Amidi F, Ross MG. Placental and membrane aquaporin water channels: correlation with amniotic fluid volume and composition. Placenta 2007;28:421–8.
[20] Ishibashi K, Kondo S, Hara S, Morishita Y. The evolutionary aspects of aquaporin family. Am J Physiol Regul Integr Comp Physiol 2011;300:R566–76. [21] Rodriguez A, Catalán V, Gómez-Ambrosi J, Frühbeck G. Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control. Cell Cycle 2011;10:1548–56. [22] De Falco M, Cobellis L, Torella M, Acone G, Varano L, Sellitti A, et al. Down-regulation of aquaporin 4 in human placenta throughout pregnancy. In Vivo 2007;21:813–7. [23] Liu H, Hooper SB, Armugam A, Dawson N, Ferraro T, Jeyaseelan K, et al. Aquaporin gene expression and regulation in the ovine foetal lung. J Physiol 2003;551: 503–14. [24] Ishibashi K. Aquaporin subfamily with unusual NPA boxes. Biochim Biophys Acta 2006;1758:989–93. [25] Yakata K, Hiroaki Y, Ishibashi K, Sohara E, Sasaki S, Mitsuoka K, et al. Aquaporin-11 containing a divergent NPA motif has normal water channel activity. Biochim Biophys Acta 2007;1768:688–93. [26] Yakata K, Tani K, Fujiyoshi Y. Water permeability and characterization of aquaporin-11. J Struct Biol 2011;174:315–20. [27] Gorelick DA, Praetorius J, Tsunenari T, Nielsen S, Agre P. Aquaporin-11: a channel protein lacking apparent transport function expressed in brain. BMC Biochem 2006;7:14–28. [28] Scifres CM, Nelson DM. Intrauterine growth restriction, human placental development and trophoblast cell death. J Physiol 2009;587:3453–8. [29] Larsen HS, Ruus AK, Schreurs O, Galtung HK. Aquaporin 11 in the developing mouse submandibular gland. Eur J Oral Sci 2010;118:9–13. [30] Morishita Y, Matsuzaki T, Hara-chikuma M, Andoo A, Shimono M, Matsuki A, et al. Disruption of aquaporin 11 produces polycystic kidneys following vacuolization of the proximal tubule. Mol Cell Biol 2005;25:7770–9.