- Original Articles
- Open Access
Induction of Aquaporin-1 mRNA following Cardiopulmonary Bypass and Reperfusion
© Picower Institute Press 1997
- Accepted: 27 June 1997
- Published: 1 September 1997
Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are important components of congenital cardiac surgery. Ischemia/reperfusion injury and inflammatory cascade activation result in endothelial damage and vascular leak, which are clinically manifested as pulmonary edema and low cardiac output postoperatively. Newborns are particularly susceptible. Subtraction cloning is a useful method of isolating induced genes and can be applied to CPB/HCA.
Materials and Methods
We used a newborn lamb model replicating infant CPB with HCA to obtain tissues during various periods of reperfusion. We utilized subtraction cloning to identify mRNA induced in lung following CPB/HCA and reperfusion. Ribonuclease protection was used to quantify mRNA levels.
We isolated a cDNA encoding ovine aquaporin-1 in a subtracted cDNA screen comparing control lung with lung exposed to CPB/HCA and reperfusion. Aquaporin-1 mRNA levels increased 3-fold in lung (p =.006) exposed to CPB/HCA and 6 hr of reperfusion. No induction was observed immediately following bypass or after 3 hr of reperfusion. We found no significant induction of aquaporin-1 mRNA following bypass, arrest, and reperfusion in other tissues surveyed, including ventricle, atrium, skeletal muscle, kidney, brain, and liver.
Our finding that aquaporin-1 mRNA is reproducibly induced in lung following CPB/HCA with 6 hr of reperfusion suggests an important role for the water channel in the setting of pulmonary edema. Induction of Aquaporin-1 is late compared with other inflammatory mediators (ICAM-1, E-selectin, IL-8). Further studies are needed to determine if aquaporin-1 contributes to the disease process or if it is part of the recovery phase.
Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA), which are support techniques for pediatric cardiac surgery, invariably result in vascular injury and inflammatory activation, due in part to ischemia and reperfusion. Clinically, this translates to postoperative morbidity, including pulmonary edema, a transient low cardiac output state, and occasionally, profound capillary leakage. Identification of the inflammatory mediators induced by CPB/HCA is necessary to understand the mechanism of the associated vascular injury.
We have previously shown that genes for proinflammatory adhesion molecules ICAM-1 and E-selectin, as well as the potent neutrophil chemoattractant interleukin-8 (IL-8), are induced immediately following CPB in atrium and skeletal muscle in humans (1,2). Using a lamb model of bypass with circulatory arrest, we can obtain several tissues under varying conditions of bypass and varying times of reperfusion. We used subtraction cloning techniques to learn which genes might contribute to pulmonary vascular injury with bypass, circulatory arrest, and reperfusion. In a preliminary screening of genes induced in lung, we have identified the ovine homolog of a known water channel protein, aquaporin 1 (AQP1).
Other studies have employed subtraction techniques to demonstrate that major intrinsic protein (a member of the family of aquaporins) is a delayed early response gene following growth factor stimulation of mouse fibroblast cells (3) and that AQP1 expression is lost during repeated passage of aortic vascular smooth muscle cells in culture (4).
AQP1 (initially named CHIP 28) was first identified in erythrocytes and renal proximal tubules (5,6). It belongs to a family of at least five highly conserved membrane water channels, each with characteristic tissue distribution and physiology. AQP1 is a homotetramer of 30 kD subunits which act as functionally independent transmembrane water channels that are sensitive to inhibition by mercury salts (7–10). In rat tissue surveys, AQP1 is found in several epithelia: in ocular ciliary bodies and cornea, choroid plexus, hepatic bile ducts (11), and descending thin limbs of the loop of Henle in the kidney (12). AQP1 has also been localized by immunohistochemical techniques to endothelium of cardiac and skeletal muscle capillaries, and to peribronchiolar and perialveolar capillaries in the lung (11). There are no previous reports of the in vivo impact of vascular injury on the expression of AQP1 or other aquaporins. We have found significant elevation of AQP1 mRNA at 6 hr of reperfusion after cardiopulmonary bypass with circulatory arrest. We propose that this water channel gene may participate in pulmonary vascular leakage or response to injury following cardiopulmonary bypass.
Cardiopulmonary Bypass with Hypothermic Circulatory Arrest
All animals were treated in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” (13). The experimental protocol was approved by the Animal Care and Use Committee at Children’s Hospital, Boston, MA.
Neonatal lambs (age 2–7 days) were anesthetized with intramuscular ketamine (40 mg/ kg, followed by 0.5 mg/kg/hr continuous infusion) and pancuronium bromide (0.3 mg/kg), and mechanically ventilated with 100% oxygen. Femoral artery access was obtained for pressure measurements and arterial blood sampling. An electromagnetic flow probe (Nihon, Kodon, Tokyo, Japan) was placed around the main pulmonary artery for cardiac output measurements. A left atrial sheath (5 Fr) and pulmonary artery catheter (5 Fr) were placed for pressure measurements. Baseline cardiac output, pressure measurements, and arterial blood gases were obtained. After systemic heparinization, a pressure transducer (5 Fr) was placed in the left ventricular apex. A right femoral artery cannula (8 Fr) and a right atrial cannula (24 Fr) were used for cardiopulmonary bypass.
Animals who had undergone CPB/HCA were rewarmed and sacrificed after 0, 3, or 6 hr of reperfusion. Control animals underwent anesthesia and sternotomy only, at room temperature, and were sacrificed after 20 or 260 min. There were 1–3 animals per condition. Organs were harvested immediately and dissected tissue samples (approximately 5 × 5 × 10 mm) were placed directly in polypropylene tubes on dry ice. Long-term storage was at −80°C.
Tissue samples were made brittle in liquid nitrogen, broken into small pieces with a hammer, and transferred frozen to RNAzol-B (Cinna/Biotecx, Friendswood, TX) and immediately homogenized (Ultra-turrax T25, Janke & Kunkel, IKA, Germany). RNA was isolated by acid guanidinium-thiocyanate-phenol-chloroform extraction (14). Poly A+ RNA was isolated by hybridization with Oligo-dT-linked cellulose (Oligo-dT mRNA Kit, Qiagen, Inc., Chatsworth, CA). RNA was quantitated by spectrophotometry and stored at −80°C.
Sheep Lung Post-CPB cDNA Library
A cDNA library in λZAPII was constructed from poly A+ RNA isolated from neonatal lamb lung tissue harvested after CPB/HCA and 3 hr of reperfusion (Clontech Laboratories, Palo Alto, CA). Mixed oligo-dT and random primers were used for first-strand cDNA synthesis. The library contained 1.8 × 106 recombinants, with an average insert size of 1.5 kilobases.
First-strand cDNACPB was generated by reverse transcription of 1 µg of oligo-dT-selected RNA isolated from lamb lung after CPB/HCA and 6 hr of reperfusion, by incubating with oligo-dT primer and reverse transcriptase (Subtractor Kit, Invitrogen Corp., San Diego, CA). First-strand cDNACPB was isolated and used as the “tester” population. mRNACONTROL from lamb lung harvested after 20 min of anesthesia only was used as a “driver” population. mRNACONTROL was biotinylated by incubating with photobiotinacetate under a 300 watt light bulb. A large excess of 10 µg of “driver” mRNACONTROL was hybridized with the “tester” cDNACPB for 48 hr. After treatment with streptavidin, the biotin:mRNACONTROL:cDNACPB duplexes were removed by phenol-chloroform extraction, leaving the subtracted, first-strand cDNAcpb-control; this represented mRNAs induced in lung during CPB/HCA and reperfusion.
Isolation of Subtracted cDNA
A 32P-labeled subtraction probe was generated from the subtracted, first-strand cDNACPB-CONTROL using random primers and Klenow DNA polymerase (Boehringer-Mannheim, Indianapolis, IN) (15) in a reaction containing 50 µCi [α32P]dCTP (Amersham, Arlington Heights, IL). The sheep lung post-CPB cDNA library described above was plated sparsely (1000 phage per 150-mm plate) to allow isolation of single-phage plaques. Nitrocellulose filter lifts were performed in triplicate. Two lifts were screened with the 32P-labeled subtraction probe. One lift was screened with ovine actin cDNA to identify and discard this common cDNA. Remaining nonactin clones on duplicate filters were selected with a toothpick and amplified in host XL1-blue. Phagemids were excised with helper phage as recommended by the manufacturer (Strategene, La Jolla, CA). Inserts were sequenced by the Children’s Hospital sequencing facility using a PRISM instrument (ABI, Foster City, CA).
Synthesis of AQP1 Riboprobes
cDNA templates of ovine AQP1 and nonmuscle actin were chosen of different lengths to allow multiplex RNAse protection analysis in a single sample. A 125-bp fragment of AQP1 cDNA from within the coding region was amplified by polymerase chain reaction with artificial restriction sites placed in the primers to enable cloning into the BamH1 and EcoRI sites of the plasmid vector, pBluescript II KS (Stratagene). Transcription from 0.5 µg linearized AQP1 template produced a full-length, 168-nucleotide (nt) probe with a 125-nt protected sequence. Nonmuscle actin cDNA, which was transcribed from 0.5 µg plasmid vector pBluescript II KS and linearized with AccI, had a full-length, 136-nt probe with a 63-nt protected sequence. Transcription reactions contained 100 µCi [α32P]CTP (Amersham). T7 and T3 RNA polymerase (Promega, Madison, WI) were used for antisense and sense transcription, respectively.
Ribonuclease (RNAse) Protection Assay
The RNAse protection assay was modified from Kilbridge et al. as follows (2). Total RNA (30 µg) extracted and purified from lamb tissue was hybridized with 300,000 cpm AQP1 riboprobe and 50,000 cpm nonmuscle actin riboprobe. tRNA (30 µg) and sense riboprobes were run as negative controls. After overnight hybridization (37°C), single-stranded RNA was digested (37°C, 90 min) with 80 µg/ml RNase A (Boehringer) and 4 U/ml RNase T1 (Boehringer). Samples were analyzed on a 5% acrylamide-urea sequencing gel.
Protected bands were quantified by Phosphor-Imager analysis and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). After subtracting background levels from tRNA negative control lane, AQP1 signal was normalized to its internal nonmuscle actin control, [AQP1-tRNA]/[actin-tRNA]. Different tissue samples from the same experimental condition were combined (n = 2–5) and conditions compared using a Student’s t-test assuming unequal variances. All experiments were confirmed in duplicate.
Subtraction Cloning of Genes Induced during CPB/HCA and Reperfusion in Lung
Physiologic Parameters of Neonatal Lambs
Hemodynamic parameters of neonatal lambs at baseline and following CPB/HCA
Blood pressure (mm Hg)
PA pressure (mm Hg)
LA pressure (mm Hg)
CVP (mm Hg)
Cardiac output (1/min)
Heart rate (bpm)
Tissue Expression of AQP1 mRNA
Induction of AQP1 mRNA during CPB/HCA and Reperfusion
Induction of AQP1 mRNA during cardiopulmonary bypass (3 hr) with hypothermic circulatory arrest (2 hr at 15°C) followed by reperfusion (up to 6 hr at 37°C) was determined by RNAse protection assay of parallel samples from the same tissue type but harvested under different experimental conditions (Fig. 1). Samples included: 20 and 260 min, sternotomy only, controls; and CPB/HCA followed by 0, 3, or 6 hr of reperfusion (37°C). Two to three animals per condition were evaluated and 1 to 4 separate tissue samples per animal were analyzed. All AQP1 mRNA levels were normalized to an internal nonmuscle actin control and background was subtracted from tRNA negative control.
Ventricle AQP1 mRNA levels were not induced in neonatal lambs during CPB/HCA or during 6 hr of reperfusion. AQP1 mRNA levels normalized to internal actin control were not significantly different between the 20-min control (0.203; ± 0.037) and CPB/HCA with no reperfusion (0.198; ± 0.023, p value, .90), CPB/ HCA with 3 hr of reperfusion (0.157; ± 0.007, p value, .27), or CPB/HCA with 6 hr of reperfusion (0.285; ± 0.035, p value, .21).
There was considerable variance in kidney AQP1 mRNA levels without a specific trend during CPB/HCA and reperfusion. This variability most likely reflects the random harvesting of tissue from within the kidney with samples containing various amounts of proximal tubules and thin descending limbs where AQP1 is known to be localized. More specific harvesting in the future would be required to obtain comparable samples and to determine if AQP1 mRNA is induced.
Because AQP1 mRNA levels are known to increase in rat lung 12 hr following corticosteroid administration (17), it is possible that the induction we observed in lamb lung was a result of an endogenous stress response to anesthesia, surgery, bypass, and/or hypothermia. We compared AQP1 mRNA levels in lung from a lamb treated with 30 mg/kg methylprednisolone, followed by 260 min of anesthesia, to control animals with 260 min of anesthesia alone. No difference was observed (steroid lamb 70%, and 260 min control lamb 60%, of 20 min control AQP1 mRNA levels).
The recent discovery of a highly conserved family of water channels, aquaporins, has significantly increased understanding of rapid trans membrane water fluxes. A majority of research has focused on the role of aquaporins in the kidney, where mutations in AQP2 cause nephrogenic diabetes insipidus (18–20). High-resolution immunoelectron microscopy studies have demonstrated reversible migration of AQP2 from intracellular vesicles to the apical plasma membrane in response to vasopressin (21). AQP1 is expressed at the cell surface and is sensitive to inhibition by mercury salts (7,8,10). In studies of sheep lung, Folkesson et al. demonstrated that a mercury-sensitive water channel plays an important role in water permeability across capillary endothelium and into alveolar spaces (22). Using an isolated lung perfused continuously with an iso-osmotic dilute blood solution, they measured the movement of water (determined by dilution of radiolabeled albumin) from the capillaries to the alveolar spaces which contained hypertonic (900 mOsm/1) fluid instilled bronchoscopically. In the control lung, the osmotically induced water permeability had a t1/2 of .85 min. Water permeability in the contralateral lung was reversibly attenuated (t1/2 2.7 min) by mercury (0.5 mM HgCl2). In addition, in isolated rat alveolar type H epithelial cells, they measured an osmotic water permeability (Pf 0.015 ± .002 cm/sec, 10°C) which is comparable to the water permeability reported in AQP1-rich erythrocytes (Pf, 0.02 cm/sec) (23) and in isolated renal tubules (Pf, 0.007 – 0.06 cm/sec) (21). As a known mercury-sensitive water channel, AQP1 may play an important role in water permeability between alveolar spaces and perialveolar capillaries. Additional studies demonstrated a significant induction in AQP1 mRNA and protein expression (17,24) in perinatal rat lung, implying a potential role for AQP1 in lung water clearance at birth. This effect was augmented by corticosteroids, suggesting that under some circumstances, AQP1 induction may be part of an intrinsic stress response (17).
Using subtraction cloning techniques, we isolated full-length ovine AQP1 cDNA from neonatal lamb lung. This ovine cDNA was highly homologous to bovine and human AQP1. In the setting of cardiopulmonary bypass in neonatal lambs, we found that AQP1 mRNA is increased 3-fold in lung after cardiopulmonary bypass and circulatory arrest (15°C) followed by 6 hr of reperfusion (37°C). This increase was not seen after 3 hr of reperfusion, nor was it seen to a significant degree in the other tissues. Substantial increases in AQP1 mRNA levels in lung have been demonstrated during the perinatal period in rats as a function of age (17,24). In our experiments, mean lamb age among different conditions was similar (control, mean = 3 days; no reperfusion, mean = 5 days; 3 hr reperfusion, mean = 4.5 days; 6 hr reperfusion mean = 5 days). Therefore, age was not the sole determinant of increases in AQP1 mRNA levels, but it may have contributed to variation among samples. We demonstrated that high-dose corticosteroids do not result in AQP1 mRNA induction in lung over a 4-hr time course in an anesthetized lamb. Further study would be needed to determine if endogenous stress response hormones contribute to AQP1 induction observed at a later time following CPB/HCA.
Nieslen et al., studying tissue distribution of AQP1 in rats, demonstrated an abundance of AQP1 in the kidney and erythrocytes, moderate levels in lung, lesser amounts in the heart and liver, and none in the brain (11). In lambs, we found that AQP1 mRNA was essentially undetectable in brain and liver, minimally expressed in skeletal muscle and atrial muscle, and expressed significantly in ventricle, kidney, and lung.
Identification of AQP1 by differential gene expression after CPB/HCA is important and relevant for understanding bypass-related lung injury for two reasons. First, AQP1 mRNA levels are significantly induced by the subtraction conditions (CPB/HCA and 6 hr of reperfusion versus control). Second, increased AQP1 mRNA levels during a period of susceptibility to bypass-related, acute pulmonary edema is functionally consistent with the observation of mercury-sensitive water movement from capillaries to the alveolar space (22) and the observation of induction of AQP1 mRNA and protein expression in rat lung during the perinatal period (17,24). Further studies are needed to document increases in AQP1 protein expression and localization to perialveolar capillary endothelium associated with pulmonary vascular leak.
Induction of AQP1 mRNA in lung following CPB/HCA with reperfusion is late compared with induction of inflammatory mediators (ICAM-1, E-selectin and interleukin-8) (1,2), which participate in leukocyte recruitment, vascular injury, and associated capillary leak. Other studies imply a role for AQP1 in clearance of lung water (17,22,24). The late induction of AQP1 mRNA in lung may reflect a response to the pulmonary edema that occurs as a result of endothelial damage following CPB/HCA. Further investigations into the role of AQP1 in the lung could have significant clinical impact on understanding and managing other processes involving pulmonary vascular endothelial or alveolar epithelial damage, such as sepsis, neonatal respiratory distress syndrome, or adult respiratory distress syndrome.
We thank Eric Wang for rescreening AQP1 cDNA, Marc Schermerhorn for assistance with the lamb experiments. Amy Stagg for help with the RNAse protection of the steroid lamb, and Cristina Tufarelli and Janae Donady for their helpful suggestions. This work was supported by NIH program project grant HL48675 (P.R.H., J.E.M.), an American Heart Association Massachusetts Affiliate Grant-in-Aid (E.J.N.), and an NIDR Training Grant T35 DE07268 (N.T.). E.J.N, is a fellow of the Lucille P. Markey Charitable Trust.
- Burns SA, Newburger JW, Xiao M, Mayer JE, Walsh AZ, Neufeld EJ. (1995) Induction of Interleukin-8 messenger mRNA in heart and skeletal muscle during pediatric cardiopulmonary bypass. Circulation (Suppl. II) 92:II 315–317.View ArticleGoogle Scholar
- Kilbridge PM, Mayer JE, Newburger JW, Hickey PR, Walsh AZ, Neufeld EJ. (1994) Induction of ICAM-1 and E-selectin mRNA in heart and skeletal muscle of pediatric patients undergoing cardiopulmonary bypass. J. Thorac. Cardiovasc. Surg. 107: 1183–1192.PubMedGoogle Scholar
- Lanahan A, Williams JB, Sanders LK, Nathans D. (1991) Growth factor-induced delayed early response genes. Mol. Cell. Biol. 12: 3919–3929.View ArticleGoogle Scholar
- Shanahan CM, Weissberg PL, Metcalfe JC. (1993) Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ. Res. 73: 193–204.View ArticlePubMedGoogle Scholar
- Denker BM, Smith BL, Kuhajda FP, Agre P. (1988) Identification, purification and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J. Biol. Chem. 263: 15634–15642.PubMedGoogle Scholar
- Preston GM, Agre P. (1991) Isolation of the cDNA for erythrocyte integral membrane protein of 28 kD: Member of an ancient channel family. Proc. Natl. Acad. Sci. U.S.A. 88: 11110–11114.View ArticlePubMedPubMed CentralGoogle Scholar
- Nielsen S, Agre P. (1995) The aquaporin family of water channels in kidney. Kidney Int. 48: 1057–1068.View ArticlePubMedGoogle Scholar
- Knepper MA. (1994) The aquaporin family of molecular water channels. Proc. Natl. Acad. Sci. U.S.A. 91: 6255–6258.View ArticlePubMedPubMed CentralGoogle Scholar
- Moon C, Preston GM, Griffin CA, Jabs EW, Agre P. (1993) The human aquaporin-CHIP gene. J. Biol. Chem. 268: 15772–15778.PubMedGoogle Scholar
- Preston GM, Jung JS, Guggin WB, Agre P. (1993) The mercury-sensitive residue at cysteine 189 in CHIP 28 water channel. J. Biol. Chem. 268: 17–20.PubMedGoogle Scholar
- Nielsen S, Smith BL, Christensen EI, Agre P. (1993) Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia. Proc. Natl. Acad. Sci. U.S.A. 90: 7275–7279.View ArticlePubMedPubMed CentralGoogle Scholar
- Nielsen S, Pallone T, Smith BL, Christensen EI, Agre P, Maunsbach AB. (1995) Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney. Am. J. Physiol. 268: F1023–F1039.PubMedGoogle Scholar
- National Institutes of Health. (1985) Guide for the Care and Use of Laboratory Animals. NIH Publication No. 86-23, Bethesda, MD.Google Scholar
- Chomczynkski P, Sacci N. (1987) Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159.Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (eds). (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990) Basic local alignment search tool. J. Mol. Biol. 215: 403–410.View ArticlePubMedGoogle Scholar
- King LS, Nielsen S, Agre P. (1996) Aquaporin-1 water channel protein in lung. J. Clin. Invest. 97: 2183–2191.View ArticlePubMedPubMed CentralGoogle Scholar
- Merendino JJ, Spiegel AM, Crawford JD, O’Carroll AM, Brownstein MJ, Lolait SJ. (1993) Brief report: A mutation in the vasopressin V2 receptor gene in a kindred with X-linked nephrogenic diabetes insipidus. N Engl. J. Med. 328: 1538–1541.View ArticlePubMedGoogle Scholar
- Lolait SJ, O’Carroll AM, McBride OW, Konig M, Morel A, Brownstein MJ. (1992) Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature 357: 336–339.View ArticlePubMedGoogle Scholar
- Holtzman EJ, Harris HW, Kolakowski LF, Guay-Woodford LM, Botelho B, Ausiello DA. (1993) Brief report: A molecular defect in the vasopressin V2-receptor gene causing nephrogenic diabetes insipidus. N. Engl. J. Med. 328: 1534–1537.View ArticlePubMedGoogle Scholar
- Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA. (1995) Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc. Natl. Acad. Sci. U.S.A. 92: 1013–1017.View ArticlePubMedPubMed CentralGoogle Scholar
- Folkesson HG, Matthay MA, Hasegawa H, Kheradmand F, Verkman AS. (1994) Trans-cellular water transport in lung alveolar epithelium through mercury sensitive water channels. Proc. Natl. Acad. Sci. U.S.A. 91: 4970–4974.View ArticlePubMedPubMed CentralGoogle Scholar
- Macey RI, Farmer REL. (1970) Inhibition of water and solute permeability in human red cells. Biochem. Biophys. Acta 211: 104–106.View ArticlePubMedGoogle Scholar
- Umenishi F, Carter EP, Yang B, Oliver B, Matthay MA, Verkman AS. (1996) Sharp increase in rat lung water channel expression in the perinatal period. Am. J. Respir. Cell. Mol. Biol. 15: 673–679.View ArticlePubMedGoogle Scholar