Placenta (2001), 22, 776–781 doi:10.1053 plac.2001.0717, available online at http://www.idealibrary.com on Water Channel Proteins AQP3 and AQP9 are Present in Syncytiotrophoblast of Human Term Placenta A. Damianoa,b, E. Zottab, J. Goldsteinb, I. Reisina and C. Ibarraa,b,c a Laboratorio de Canales Io´ nicos, Departamento de Fisicoquı´mica y Quı´mica Analı´tica, Facultad de Farmacia y Bioquı´mica, and b Laboratorio de Fisiopatogenia, Departamento de Fisiologı´a, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Paper accepted 7 June 2001 The syncytiotrophoblast of human term placenta (HST) is a continuous, multinucleated structure with minimal tight junctions, which results from the fusion of the underlying cytotrophoblast cells. Consequently, the transport of metabolites, ions and water from mother to fetus could take place primarily via transcellular routes. Transcellular water flux may be facilitated by aquaporins, membrane proteins functioning as water channels that are widely expressed in cells and tissues. Here, we report the presence of AQP3 and AQP9 in the apical membranes of HST using RT-PCR, immunoblotting and immunohistochemistry. Since AQP3 is not only a water channels, but also permits the rapid passage of both urea and glycerol, while AQP9 also mediates the passage of carbamides, polyols, purines, and pyrimidines, we have speculated that these proteins could be involved in the transport of water and solutes from mother to fetus. 2001 Harcourt Publishers Ltd Placenta (2001), 22, 776–781 INTRODUCTION pathway (Jansson and Illsley, 1993; Jansson et al., 1993). The syncytiotrophoblast of human term placenta (HST) is a However, the molecular mechanisms of these processes are continuous, multinucleated structure with minimal tight junc- little known. tions, which results from the fusion of the underlying cyto- Transcellular water flux may be facilitated by aquaporins trophoblast cells. Consequently, the transport of metabolites, (AQPs), membrane proteins functioning as water channels that ions and water from mother to fetus should take place are widely expressed in cells and tissues (Verkman et al., 1996; primarily via transcellular routes. Knepper et al., 1996). So far, ten isoforms of the AQP family Nevertheless, a significant gradient of osmotic or hydro- have been identified in mammals, each varying in its per- static pressure has not been demonstrated (Seed, 1965; meability to small molecules such as urea and glycerol, and Nicolini et al., 1989), and the possibility exists that wide, in its susceptibility to inhibition by mercurial compounds non-specific, paracellular channels, allowing the passage of (Yamamoto and Sasaki, 1998). AQP1, a protein exclusively large hydrophilic molecules, may also be present (Faber and water permeable, was the first water channel identified in Thornburg, 1983). It has been postulated that sodium is human red cells (Preston and Agre, 1991) and was also shown actively transported to the fetus in excess of the needs for fetal to be constitutively expressed in several secretory and resorp- growth, and that water follows transcellularly through channels tive epithelia and in continuous endothelia of capillaries with a high reflection coefficient. An increase of hydrostatic (Nielsen et al., 1993). AQP1 is also located in the human, rat, pressure on the fetal side drives both water and sodium, and ovine placentae (Hasegawa et al., 1994; Umenishi et al., together with other solutes through wide paracellular pathways 1996; Johnston et al., 2000). In addition, AQP3, an aquaporin back from the fetal to the maternal side (Stulc, 1997); a highly permeable to glycerol and urea in addition to water, has transcellular route requiring permeation of both microvillous also been found in trophoblast cells from ovine placenta and basal plasma membranes. Previous research suggested that (Johnston et al., 2000), while AQP8 strongly expressed in the the movement of water, urea and glycerol across human rat placenta, is permeable to water and urea but not glycerol syncytiotrophoblast membranes occurs by a lipid diffusion (Ma et al., 1997). c To whom correspondence should be addressed at: Departamento In the present study, we have examined the mRNA and de Fisiologı´a, Facultad de Medicina, Paraguay 2155, 7 piso, 1121, protein expression of the AQPs family in human syncytio- Buenos Aires, Argentina. E-mail: [email protected]. trophoblast membranes in order to determine whether the 0143–4004/01/080776+06 $35.00/0 2001 Harcourt Publishers Ltd Damiano et al.: Water Channel Proteins AQP3 and AQP9 777 Table 1. Primers for PCR designed from hAQP3 and hAQP9 Product Primer Product Primers Sequence lengh location name I Sense 5-CCTGAACCCTGCGGTGACC-3 398 bp 307-325 HST-AQP3 Antisense 5-GGCATAGCCGGAGTTGAAGC-3 703-684 II Sense 5-CATCAACCCAGCTGTGTCT-3 393 bp 462-481 HST-AQP9 Antisense 5-CAGCCACTGTTCAGTCCCA-3 855-836 movement of water and solutes across these membranes can be 11 000 g and the resulting supernatant centrifuged 70 min explained by the presence of aquaporins. at 16 000 g. The pellet was then resuspended in HES buffer containing 10 m MgCl2 to selectively precipitate non-apical membranes. MATERIALS AND METHODS The suspension was then incubated 10 min with constant RT-PCR assay stirring, after which it was centrifuged 30 min at 5000 g. The basal membrane-enriched pellet was redissolved in HES buffer Normal term placenta was obtained immediately after spon- containing protease inhibitors, and stored at 80C. Finally, taneous vaginal delivery. Several cotyledons were removed the supernatant was centrifuged 70 min at 16 000 g and the from underlying fibrous elements and rinsed thoroughly in 0.9 apical membrane-enriched pellet was resuspended in HES per cent NaCl at 4C. Soft villous material from the maternal buffer containing proteases inhibitors, and stored at 80C surface enriched in HST was cut away from connective tissue until assayed for biochemical markers. Alkaline phosphatase and vessels until approximately 1 g were collected. The tissue activity (an apical plasma membrane enzymatic marker) in the was rinsed again in order to remove most of the blood cells and final membrane suspension was >20 while ratios for those then homogenized 30 sec on ice using an Ultra-Turrax T25 enzymes that are markers of non-apical membranes were <1.5 blender at maximum speed. (Grosman et al., 1997). Total RNA from HST, rat kidney homogenate and leuko- For immunoblot studies, 10–20 g of membrane fraction cyte suspension was isolated using a SV Total RNA isolation proteins were dissolved in loading buffer (4 per cent sodium system (Promega). Reverse transcription was performed dodecil sulphate, 0.125 Tris-HCl pH 6.8, 0.2 dithio- 60 min on 5 g of total RNA from HST using moloney murine threitol, 0.02 per cent bromophenol blue, 20 per cent glycerol), heated to 90C for 2 min, resolved on 15 per cent polyacryla- leukemia virus reverse transcriptase, oligo (dT)15 primer and 400 of each deoxyribonucleotide triphosphate (dNTP) at mide gel and electrotransferred onto nitrocellulose mem- 42C. PCR (30 cycles at 94C 60 sec, 58C 60 sec and 72C branes (Hybond ECL, Amersham Pharmacia Biotech Ltd, 60 sec, followed by a final extension of 10 min at 72C) was UK). Membranes were blocked 30 min with 1 (w/v) bovine carried out using 5 of two specific primers (one for AQP3 serum albumin in PBS for 30 min at room temperature, and the other for AQP9) designed on the basis of a highly and incubated overnight with either polyclonal anti-AQP3 conserved region flanking by Asn-Pro-Ala (NPA) in the (Alpha Diagnostic International Inc, USA) or anti-AQP9 aquaporin family (Agre et al., 1998)(Table 1). The RT-PCR antibody diluted 1 : 400 (kindly providing by Dr van Hoek). product of HST using the primer pair II (HST-AQP9) was Membranes were washed with PBS-Tween 0.1 per cent, and cloned and sequenced. incubated 1 h at room temperature with a goat anti-rabbit IgG (Jackson ImmunoResearch) (1 : 1000) conjugated to peroxi- dase. Filters were washed and incubated 5 min in enhanced Immunoblotting chemiluminescence detection reagents (ECL plus, Amersham Pharmacia Biotech Ltd, UK) before exposure to X-ray film Full term normal human placenta was obtained within 20 min (Kodak Biomax MS) for 30 sec. In some experiments, apical- of vaginal delivery and immediately processed according to the enriched membranes were incubated with endoglycosidase H, method previously described (Grosman et al., 1997). Briefly, according to the manufacturer’s instructions (New England human chorionic villi were fragmented, and washed with Biolabs, Inc, Beverly, USA), to assess the extent of AQP9 unbuffered 150 m NaCl. The tissue was then shaken 1 h with glycosylation. 1.5 volumes of HES buffer (10 m HEPES-KOH, 0.1 m EGTA, 250 m sucrose) pH 7.4, with protease inhibitors Immunohistochemistry (0.2 m PMSF, 25 g/ml p-aminobenzamidine, 20 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin), follow- Human villous tissues were cut into small pieces, fixed over- ing by filtration and centrifugation for 10 min at 3100 g. night in 10 per cent formaldehyde in 0.1 sodium phosphate The supernatant was then further centrifuged 10 min at buffer, pH 7.4, dehydrated, and embedded in paraffin. 778 Placenta (2001), Vol. 22 showed 100 per cent identity to human AQP9 (Gene Bank Accession, BAA24864) and 83 per cent identity to rat AQP9 (Gene Bank Accession, BAA33680) in their respective NPA- NPA regions (Figure 2). Expression of AQP3 and AQP9 in HST To demonstrate that the mRNA was translated into protein, apical-enriched membrane preparation from HST was immu- noblotted using antisera for AQP3 and AQP9. AQP3 appeared as a major band of 28 kDa and a minor band of 35 kDa [Figure 3(A)], a pattern similar to that reported for rat kidney medulla, where the upper band represents the glycosylated form of AQP3, and the lower band represents the nonglycosylated form (Frigeri et al., 1995).