Expression and localization of aquaporin 1 and 3 in human fetal membranes

Expression and localization of aquaporin 1 and 3 in human fetal membranes Stephanie E. Mann, MD, Emily A. Ricke, MS, Baxoue A Yang, PhD, Alan S. Verkman, MD, PhD, and Robert N. Taylor, MD, PhD San Francisco, Calif OBJECTIVES: Aquaporins are a family of water-selective channels that facilitate fluid movement across cell membranes. Specifically, aquaporin 1 (AQP1) and aquaporin 3 (AQP3) have been found to be important in osmotic water movement across membranes. Our goal in this study was (1) to determine whether AQP1 or AQP3 messenger RNA are expressed in the chorioamniotic membrane and, if present, (2) to determine the precise membrane location of these aquaporins. STUDY DESIGN: Placentas were collected from women with intact membranes not in labor who underwent elective cesarean sections at term (37-40 weeks). The membranes (amnion and chorion) directly overlying the placenta were sampled as well as the free-floating reflected membranes. RNA and protein were isolated from the amnion and chorion. Reverse transcriptase–polymerase chain reaction, Western analysis, and immunohistochemistry were used to determine expression and localization of AQP1 and AQP3. RESULTS: AQP1 messenger RNA was found in amnion and chorion from both membrane locations. Western analysis also yielded positive results for amnion and chorion from both locations. Immunohistochemical localization of AQP1 showed it to be present on the apical aspect of the chorionic plate amnion. AQP3 protein was not found in the fetal membranes. CONCLUSIONS: AQP1 is present in the fetal membranes. AQP1 may play a role in water movement from the amniotic cavity across the placenta into the fetal circulation. Further studies are needed to clarify our understanding of the role of fetal membrane aquaporins in amniotic fluid homeostasis. (Am J Obstet Gynecol 2002;187:902-7.)

Key words: Amnion, chorion, intramembranous pathway, aquaporin 1, aquaporin 3

The amniotic cavity is lined by a bilayer membrane that provides a large surface for fluid movement between the amniotic cavity and the fetal circulation. This membrane is composed of the inner amnion lining the amniotic cavity and the outer chorion, which directly abuts the fetal surface of the placenta as well as the underlying decidua. Two distinct regions of the fetal membranes have been described: the portion directly covering the fetal surface of the placenta is referred to as the chorionic plate and the region of the membranes directly juxtaposed to the underlying decidua is referred to as the reflected portion (Fig 1). The chorionic plate membranes are the main

From the Departments of Obstetrics, Gynecology, and Reproductive Sciences and Medicine, University of California San Francisco School of Medicine. Supported in part by grants from the National Institutes of Health (No. HD30367) and the University of California San Francisco Academic Senate. Presented at the Twenty-second Annual Meeting of the Society for Maternal-Fetal Medicine, New Orleans, La, January 14-19, 2002. Reprint requests: Stephanie Mann, MD, San Francisco General Hospital, 1001 Potrero Ave 6D13, San Francisco, CA 94110. © 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/6/127168 doi:10.1067/mob.2002.127168

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component of the intramembranous pathway, the primary route of water movement from the amniotic cavity across the fetal surface of the placenta into the fetal circulation (via intraplacental vessels).1,2 Under normal conditions, water flow progressively increases throughout gestation; by term, up to 400 mL per day are transferred from the amniotic cavity across the fetal membranes into the fetal circulation.3 Because of the osmotic pressure difference between amniotic fluid (AF) (255 mOsm/kg) and fetal blood (280 mOsm/kg), an osmotic gradient drives transport of fluid and solutes from the amniotic compartment into the fetal blood under normal conditions.4 Understanding the mechanisms of water transport between the amniotic cavity and the fetal circulation has become increasingly important because AF provides an essential fluid-filled compartment for normal fetal growth, movement, and development. Disorders of AF volume, either in excessive or deficient amounts, are associated with significant perinatal morbidity and mortality. Water can move from the AF to the fetal circulation by either paracellular or transcellular routes and under hydrostatic or osmotic gradients. In the sheep, one of the best studied species in terms of AF regulation, the transcellular route appears to most important.5 Transcellular

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water movement is facilitated by aquaporins (AQPs), which are a family of water-selective channels that increase plasma membrane permeability and provide a route for rapid fluid movement.6,7 At present, 10 distinct mammalian AQPs have been identified within numerous tissues throughout the body. Specifically, AQP1 and AQP3 are widely expressed in epithelia and capillary endothelia involved in fluid transport.8 Hence, these AQPs would be likely candidates for water channels in the human fetal amnion and or chorion. This study was undertaken to determine whether AQP1 or AQP3 messenger RNA (mRNA) is expressed in human fetal membranes and to determine the precise membrane location of these proteins. Material and methods Tissues. Human placenta and fetal membranes were obtained from five term (37-40 weeks) elective cesarean sections within 30 minutes after delivery. Membranes were not taken from placentas with fetal anomalies. The amnion was separated from the chorion by gentle traction. Tissue samples of amnion and chorion were taken from the chorionic plate and the reflected membranes. All tissues were rinsed thoroughly in phosphate-buffered saline solution (PBS) and immediately placed into RNA extraction buffer. Subjects provided written informed consent under a protocol approved by the University of California San Francisco (UCSF) Committee on Human Research. Molecular biology reagents were purchased from Life Technologies (Grand Island, NY). Reagents for sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and immunoblotting were purchased from Fisher Scientific (Fairlawn, NJ). Exceptions are noted. Reverse transcriptase–polymerase chain reaction analysis. Total RNA was extracted with Trizol reagent. One microgram of total RNA was reverse transcribed in a 10 µL of reaction containing 1 polymerase chain reaction (PCR) buffer, 6 mmol/L magnesium chloride, 1 mmol/L each deoxyadenosine triphosphatase, deoxycytosine triphosphatase, deoxyguanosine triphosphatase, and deoxythymidine triphosphatase, 2.5 mmol/L random hexamers, 0.3 U/mL ribonuclase inhibitor (Eppendorf Scientific, Westbury, NY), and 2.5 U/mL Moloney murine leukemia virus reverse transcriptase (RT). RT-PCR was carried out in a GeneAmp PCR system 2400 (Perkin Elmer) at 23°C for 10 minutes, 37°C for 20 minutes, 48°C for 60 minutes, and 95°C for 5 minutes and stored at 4°C until used. PCR was carried out in a final volume of 50 µL containing 1 µL of complementary DNA (cDNA), 1 PCR buffer, 1.5 mmol/L magnesium chloride, 0.2 mmol/L each deoxyadenosine triphosphatase, deoxycytosine triphosphatase, deoxyguanosine triphosphatase, and deoxythymidine triphosphatase, and 0.05 U/mL Taq plus Taqstart antibody (Clontech, Palo Alto, Calif). Amplifica-

Fig 1. The intramembranous route. Solid line, Chorionic plate; dotted line, reflected region of the fetal membranes. (Adapted from Seeds, 1968.)

tion of the AQPs was done in the following manner: A 300 bp AQP1 fragment was amplified with the forward primer, 5´-GCCATCGGCCTCTCTGTAGCC-3´, and the reverse primer, 5´-CTATTTGGGCTTCATCTCCAC-3´ (Operon, Alameda, Calif). A 117-bp AQP3 fragment was amplified with the forward primer, 5´-CCGCCTTTTTACAGCCCTTG-3´, and the reverse primer, 5´-GGCGGAAAAATGTCGGGAAC-3´ (Operon). cDNA was amplified with the following conditions: 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds followed by 72°C for 7 minutes. Five microliters of PCR product was mixed with DNA sample buffer and loaded onto 4% NuSieve (FMC, Rockland, Me) agarose gels containing 0.5 µg/mL ethidium bromide. Gels were photographed after electrophoresis. The identities of the resulting PCR products were confirmed by sequencing in the UCSF Biomolecular Resource Center. Western blotting. Samples were homogenized in 50 mmol/L TRIS (tris-[hydroxymethyl]-aminomethane), 120 mmol/L sodium chloride, and 1 Complete (Roche Molecular Biochemicals, Indianapolis, Ind) buffer. Tissue homogenates were centrifuged at 1000g for 10 minutes at 4°C to remove insoluble material, and supernatants were further centrifuged at 16,000g for 1 hour at 4°C. Protein pellets were resuspended in the original homogenization buffer and stored at –20°C. Total protein was measured spectrophotometrically with the BCA Protein Assay (Pierce Endogen, Rockford, Ill), using bovine serum albumin as the standard. Protein extracts were solubilized by heating for 10 minutes at 70°C in loading buffer (0.1 mol/L TRIS, 24% [wt/vol] glycerol, 8% [wt/vol] SDS, 0.2 mol/L dithiolthreitol, and 0.02% [wt/vol] Coomassie blue G-250). Membrane proteins were subjected to 12% SDS-PAGE (20 µg/lane) and transferred to nitrocellulose. Membranes were blocked in 10% nonfat dry milk and then incubated overnight at 4°C with anti-rabbit AQP1

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Fig 2. RT-PCR of RNA from fetal membranes. Total RNA from amnion and chorion directly overlying the placenta (chorionic plate) and from the reflected membranes was reverse transcribed and used for PCR analysis. The observed bands correspond to a 300-bp fragment of the AQP1 gene. The positive control is from human kidney (K). A, Amnion; C, chorion.

Fig 3. AQP1 and AQP3 expression in fetal membranes. Amnion and chorion were obtained from the chorionic plate and reflected membranes. Protein extract from a human monocytic cell line U937 was used as negative control. A, A major band at 28 kd corresponding to AQP1 can be seen in amnion and chorion from both membrane locations (arrow). B, A faint band at 28 kd is seen in the chorion from the chorionic plate and the reflected membranes (arrow); no signal is seen in the amnion from the chorionic plate or reflected membranes. Several nonspecific bands are seen for amnion and chorion from both membrane regions at 40 kd. No signal was seen in U937 cells.

and AQP3 antibodies (1:2000) (provided by Dr Alan Verkman). Bound antibodies were detected with use of LumiGlo chemiluminescent substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Md). Immunofluorescence histochemistry. The tissues were cut into small pieces and fixed in 4% paraformaldehyde for 4 hours. Samples were cryoprotected overnight with PBS containing 30% sucrose embedded in ornithine carbamoyltransferase compound and frozen in liquid nitrogen. Cryostat sections (4-6 µm) were incubated for 10 minutes with PBS containing 1% bovine serum albumin. Slides were rinsed with 2.7% sodium chloride and then with PBS and then incubated for 30 minutes with Cy3conjugated sheep anti-rabbit F(ab)2 fragment (1:200, Sigma) with a 1:500 dilution of immune or preimmune serum overnight at 4°C. Slides were then incubated with

a secondary fluorescein-conjugated sheep anti-rabbit antibody. Results RT-PCR. RT-PCR confirmed which AQP transcripts were detectable in amnion and chorion. AQP1 mRNA was expressed in both the reflected and chorionic plate amnion and chorion (Fig 2). AQP3 mRNA (not shown) was faintly detected in the chorion from both membrane regions but not the amnion. cDNA fragment identity was confirmed in each case by subcloning and sequence analysis. A positive control (cDNA from human kidney) is shown in the last lane (see Fig 2 legend). Western blot. Immunoblotting with anti-AQP1, and anti-AQP3 protein lysates of amnion and chorion from the chorion plate and reflected membrane region is

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Fig 4. Immunofluorescence localization of AQP1 (A) and AQP3 (B) in fetal membranes. Bright orange staining represents a positive signal. The strongest signal for AQP1 is in the amnion epithelium (ae). No staining is observed for AQP3. cy, Cytotrophoblast.

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shown in Fig 3. In Fig 3, A, a major band of 28 kd, which represents the unglycosylated form of AQP1, is observed in all four tissues. Faint bands between 35 and 45 kd also can be seen, which represent the various glycosylated forms of AQP1. Fig 3, B, shows the results after an identical blot was incubated with anti-AQP3 antibody. A faint band at 28 kd is observed in the chorion from both regions; no bands are observed in the amnion. We interpret the bands to represent nonspecific proteins because the highly sensitive and specific RT-PCR results were negative. Immunofluorescence histochemistry. Immunofluorescence histochemistry was carried out to determine the location(s) of AQP1 and AQP3 protein expression (Fig 4). Staining for AQP1 was seen in both the reflected and chorionic plate amnion (Fig 4, A); the AQP1 signal was strongest in the epithelium of the chorionic plate amnion (ae). AQP1 staining in the chorion and decidua was limited to endothelial cells (arrows). No AQP1 was seen in the trophoblast cells of the chorion. AQP3 was not detected in any region of the fetal membranes (Fig 4, B). Comment In this study, we have characterized the expression of AQP1 and AQP3 in human fetal membranes and found that only AQP1 is consistently present. Moreover, we have localized AQP1 in the amnion epithelium of both the chorionic plate and reflected region of the fetal membranes. Although faint AQP3 banding was noted on the immunoblot, AQP3 did not appear in the membrane by immunofluorescence. A possible explanation is contamination of the chorion from the underlying syncytiotrophoblast at the time of membrane dissection and separation as AQP3 protein has been found in human syncytiotrophoblast.9 The amnion and chorion comprise a complex bilayered membrane surrounding the amniotic cavity.10 The innermost part of the membrane directly interfacing with the amniotic fluid is the amnion. This layer is composed of a single cuboidal epithelium, a basement membrane that interdigitates with the overlying epithelium, a compact acellular zone, a fibroblast layer, and a spongy layer. The underlying chorion is composed of a cellular fibroblast layer, a reticular layer, a pseudobasement membrane, and trophoblast cells. The localization of AQP1 to the epithelial layer of the amnion suggests that the epithelium rapidly facilitates water transport between the amniotic cavity and the fetal circulation and that the chorion layer may play a more passive role. Although AQP1 has been found in many tissues involved in water transport, it has yet to be identified in human amnion and chorion. AQP1 has been found in microcapillary endothelial cells of several tissues and in the epithelia of tissues that maintain water permeability as a result of osmotically induced water movement. Such

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examples include peritoneum, alveolar epithelium, and renal tubules.11-13 Approximately 400 mL of water is transported across the fetal placental membranes on a daily basis. The importance of the fetal membranes to facilitate water transport has been repeatedly demonstrated in ovine and primate studies.1,14 Under normal conditions in pregnancy, water moves down its gradient from the hypotonic amniotic fluid into the fetal circulation. Measurement of the permeability coefficient (Pf) in cultured amnion epithelial cells yielded a transepithelial Pf value that is too high to be accounted for by simple diffusion of water across amnion and chorion overlying the placenta.15 This calculation is more consistent with the presence of water channels in the cuboidal layer of epithelium facing the amniotic cavity. One other study identified AQP3 but not AQP1 water channels in fetal membranes from the ovine placenta.16 Our contrasting findings for AQP1 may be accounted for by the difference in the vascularity in the membranes of these two species; in contrast to the avascular amnion and chorion of humans, sheep amnion and chorion are highly vascularized. Because AQP3 has been found in syncytiotrophoblast cells, it is possible that it may facilitate water transport across the trophoblast barrier, whereas AQP1 may be more important for the water transport across the amnion. The presence of water channels in human fetal membrane research is a new finding. Although the functional importance of AQP1 in AF transport remains to be determined, its presence in human fetal membranes suggests potential therapeutic strategies for the treatment of AF volume disorders. Inhibitors of AQP1 water channels could be useful for the treatment of oligohydramnios, whereas AQP1 water channel inducing agents could potentially benefit fetuses with polyhydramnios. Additional studies are required to determine the precise roles that AQP1 plays in water transport to and from the human amniotic cavity. REFERENCES

1. Gilbert WM, Brace RA. The missing link in amniotic fluid volume regulation: intramembranous absorption. Obstet Gynecol 1989;74:748-54. 2. Brace RA, Gilbert WM, Thornburg KL. Vascularization of the ovine amnion and chorion: a morphometric characterization of the surface area of the intramembranous pathway. Am J Obstet Gynecol 1992;167:1747-55. 3. Mann SE, Nijland MJ, Ross MG. Mathematic modeling of human amniotic fluid dynamics. Am J Obstet Gynecol 1996; 175:937-44. 4. Gillibrand PN. Changes in amniotic fluid volume with advancing pregnancy. J Obstet Gynaecol Br Commonw 1969;76:527-9. 5. Hedriana HL, Brace RA, Gilbert WM. Changes in blood flow to the ovine chorion and amnion across gestation. J Soc Gynecol Investig 1995;2:727-34. 6. Verkman AS, Mitra AK. Structure and function of aquaporin water channels. Am J Physiol Renal Physiol 2000;278:F13-28. 7. Verkman AS, Van Hoek AN, Ma T, et al. Water transport across mammalian cell membranes. Am J Physiol 1996;270:C12-30.

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8. Verkman AS, Yang B, Song Y, Manley GT, Ma T. Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice. Exp Physiol 2000; 85:233-41S. 9. Damiano A, Zotta E, Goldstein J, Reisin I, Ibarra C. Water channel proteins AQP3 and AQP9 are present in syncytiotrophoblast of human term placenta. Placenta 2001;22:776-81. 10. Bourne GL. The anatomy of the human amnion and chorion. Proc R Soc Med 1966;59:1127-8. 11. Verkman AS, Matthay MA, Song Y. Aquaporin water channels and lung physiology. Am J Physiol Lung Cell Mol Physiol 2000;278:L867-79. 12. Verkman AS, Shi LB, Frigeri A, et al. Structure and function of kidney water channels. Kidney Int 1995;48:1069-81.

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13. Yang B, Folkesson HG, Yang J, Matthay MA, Ma T, Verkman AS. Reduced osmotic water permeability of the peritoneal barrier in aquaporin-1 knockout mice. Am J Physiol 1999;276:C76-81. 14. Gilbert WM, Eby-Wilkens E, Tarantal AF. The missing link in rhesus monkey amniotic fluid volume regulation: intramembranous absorption. Obstet Gynecol 1997;89:462-5. 15. Lloyd SJ, Garlid KD, Reba RC, Seeds AE. Permeability of different layers of the human placenta to isotopic water. J Appl Physiol 1969;26:274-6. 16. Johnston H, Koukoulas I, Jeyaseelan K, et al. Ontogeny of aquaporins 1 and 3 in ovine placenta and fetal membranes. Placenta 2000;21:88-99.