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H+ Exchanger Isoforms after Enterectomy
Journal of Surgical Research 86, 192–197 (1999) Article ID jsre.1999.5720, available online at http://www.idealibrary.com on
Differential Expression of Ileal Na 1/H 1 Exchanger Isoforms after Enterectomy 1 Richard A. Falcone, Jr., M.D., Cathy E. Shin, M.D., Lawrence E. Stern, M.D., Zhaohui Wang, Ph.D.,* Christopher R. Erwin, Ph.D., Manoocher Soleimani, M.D.,* and Brad W. Warner, M.D. Division of Pediatric Surgery, Department of Surgery, Children’s Hospital Medical Center, and *Division of Nephrology, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229 Submitted for publication January 26, 1999
Background. Na 1/H 1 exchangers (NHE) are transporters involved in the absorption of NaCl along the gastrointestinal tract. The aim of this study was to determine the expression pattern of the intestinal brush border NHE isoforms 2 and 3 following massive small bowel resection (SBR). Additionally, the effect of epidermal growth factor (EGF) and salivarectomy (removal of the primary source of EGF) on the expression pattern was studied. Materials and methods. ICR mice underwent a proximal SBR or sham surgery and then received either orogastric saline or EGF (50 mg/kg/day). In separate experiments mice underwent salivarectomy followed by SBR or sham. Postoperatively the remaining ileum was isolated and levels of NHE-2 and NHE-3 mRNA and protein were resolved. Results. Following SBR, the expression of both mRNA and protein for NHE-3 increased by ;2.5-fold. Treatment with EGF enhanced NHE-3 mRNA in sham animals with further elevation following SBR. The expression of NHE-2 mRNA demonstrated minimal change while protein marginally increased (40%) following SBR. EGF did not affect the expression of NHE-2 mRNA. Salivarectomy did not influence NHE-2 protein expression and inhibited the increased NHE-3 protein expression following SBR. Conclusions. Following SBR, the expression pattern for brush border NHE isoforms is distinctive. Increased expression of NHE-3 secondary to SBR and/or EGF treatment with loss of this increase following salivarectomy implies a common mechanism to enhance enterocyte proliferation and luminal absorp1
This work was supported by a Trustees Grant from the Children’s Hospital Research Foundation, Children’s Hospital Medical Center, Cincinnati, Ohio, and by National Institutes of Health RO-1 DK 53234-01 (Dr. Warner), RO-1 DK 46789 and RO-1 DK 52821 (Dr. Soleimani), and 1F32DK09882 (Dr. Stern).
0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
tion of NaCl and water. These results suggest that NHE-3 is an important ileal exchanger following SBR. © 1999 Academic Press Key Words: intestinal resection; adaptation; mouse; transport. INTRODUCTION
Intestinal adaptation is a crucial response to massive small bowel resection (SBR) that counteracts the loss of mucosal absorptive and digestive surface area. Adaptation consists of both hypertrophy and hyperplasia of all layers of the bowel wall, an increase in both caliber and length of the intestine, and augmented absorptive and digestive capacity per unit length [1–3]. Understanding the mechanism(s) of intestinal adaptation is fundamental toward the development of management strategies directed to augment this response. While multiple factors are involved during the adaptive response to SBR, there is compelling evidence to suggest a central role for epidermal growth factor (EGF). Following SBR, exogenous EGF has been shown to enhance ileal length, protein and DNA content, crypt depth and villus height, glucose transport, and EGF receptor expression [4 –7]. Adaptation is also accentuated in transgenic mice with intestinal overexpression of EGF [8]. Alternatively, adaptation is impaired in mice with perturbed EGF receptor signaling (waved-2) [9] or if the major endogenous source of EGF (salivary glands) is removed [6]. Since diarrhea is an important consequence of massive enterectomy, it is important to delineate the changes in Na 1 and water absorption that occur during adaptation. The majority of the sodium transport in the intestine occurs by passive Na 1 channels, active Na 1/substrate transporters, or Na 1/H 1 exchangers
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(NHE). Adaptation following intestinal resection results in increases in expression of mRNA for the Na 1glucose cotransporter [10] and NHE activity [11]; however, the effect of SBR on the expression of specific isoforms of NHE has not previously been described. Currently, there are four known NHE isoforms that are discriminated on the basis of tissue distribution, subcellular location, potential for glycosylation, amiloride sensitivity, and regulation by various protein kinases (reviewed in Ref. [12]). The mRNA for NHE-1 has been detected in all mammalian cells and is designated a housekeeping isoform and located in the basolateral location of polarized cells [12]. In contrast the NHE-2 and NHE-3 isoforms are primarily located at the luminal side of polarized epithelial cells and distributed throughout the intestine [13]. The NHE-2 isoform is considered to be sensitive to amiloride and positively regulated by protein kinase C [12]. Alternatively, NHE-3 is considered to be amiloride resistant, negatively regulated by protein kinase C, and affected by various growth factors and serum [14, 15]. Information regarding NHE-4 is limited and it appears to have restricted tissue and species expression [12]. This study therefore focused on the analysis of the brush border-localized NHE-2 and -3 isoforms to determine the effect of SBR on their ileal expression. Additionally, the effects of orally administered EGF and salivarectomy (removal of the major source of EGF) on this expression pattern were investigated. MATERIALS AND METHODS Animals. A protocol for this study was approved by the Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (Children’s Hospital Medical Center, Cincinnati, OH). Male ICR mice (weight range 25–29 g; The Harlan Laboratory, Indianapolis, IN) were housed in groups of four at 21°C on 12-h day–night cycles (6 AM to 6 PM). Before experimentation, the mice acclimated for 5 days. One day prior to operation, the diet was changed from regular chow to liquid rodent diet (Micro-Stabilized Rodent Liquid Diet LAD 101/101A; Purina Mills, St. Louis, MO). Experimental design. The mice were randomized to undergo either a 50% proximal SBR or a sham operation (n 5 5 for each group). The details of our murine model for SBR have already been presented [1]. Briefly, under inhaled isoflurane anesthesia and with the aid of an operating microscope, the small bowel was transected 12 cm proximal to the ileocecal valve. In sham-operated mice a reanastomosis was performed. In mice undergoing SBR, approximately 12.5 cm of proximal intestine (50% resection) was resected and an anastomosis performed. The anastomoses in both groups were accomplished using an end-to-end, single-layer technique with interrupted 9-O monofilament suture (a gift from Ethicon, Inc., Somerville, NJ). Postoperatively, the mice were resuscitated with a 3-ml intraperitoneal injection of 0.9% saline and allowed to recover in a warm incubator (30°C). Water was provided ad libitum for the first 24 h. Mice from each group were then pair fed with liquid diet. Mice were then randomized to receive either saline or EGF (50 mg/kg) by twice daily orogastric gavage. This dosage and route of exogenous EGF have previously been shown to maximally enhance adaptation in our murine SBR model [16]. After 1, 4, and 7 days, the remnant ileum was harvested and Northern blotting accomplished to measure expression of NHE-2 and NHE-3 mRNA. Protein expression of these
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isoforms was measured by Western blotting of ileal samples harvested on postoperative day 4. In separate experiments, a bilateral submandibular gland excision (salivarectomy) was done and the mice then underwent either sham or SBR. In prior studies from our laboratory, this has been an effective model for attenuation of intestinal adaptation following SBR [6]. Tissue harvest. Mice were sacrificed with an intramuscular injection of ketamine, xylazine, and acepromazine (4:1:1 proportion) followed by cervical dislocation. The abdomen was opened and the remnant ileum 2 cm from the anastomosis was collected for Northern or Western analysis after luminal contents were removed. RNA isolation. Total cellular RNA was extracted from the distal ileum and quantitated spectrophotometrically as we have previously described [17]. The total RNA was stored at 280°C until used for Northern analysis. Northern blotting. Total RNA samples (30 mg/lane) for each animal were fractionated on a 1.2% agarose–formaldehyde gel and transferred to a nylon membranes (MSI) using 103 SSPE as transfer buffer. Separate membranes for each postoperative day consisted of RNA from individual mice that underwent SBR or sham operation with or without EGF. Membranes were then cross-linked by UV light and baked as described previously [18]. Membranes were prehybridized for 1 h in 0.13 SSPE/1% SDS solution at 65°C and then for 3 h at 65°C with 0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA, and 100 mg/ml sonicated carrier DNA. The NHE-2 and NHE-3 probes were labeled with [ 32P]deoxynucleotides using the RadPrime DNA labeling kit (Gibco BRL, Grand Island, NY) as described previously [19]. After hybridization, the membranes were washed, blotted dry, and exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA). The expression for each isoform was normalized to 28S ribosomal mRNA and quantitated for each animal. The following rat NHE PCR product fragments were used as isoform-specific probes in the Northern blot analysis: (1) NHE-3, nucleotides 1883–2217, and (2) NHE-2, nucleotides 1899 – 2215. Western blotting. Crude membrane preparations were isolated from pooled samples of ileum taken from sham (n 5 5) and SBR (n 5 5) animals as previously described [20]. Fifty micrograms of protein from each sample was added to an equal volume of 23 protein sample buffer (250 mM Tris–HCl, pH 6.8, 4% SDS, 10% glycerol, 0.003% bromophenol blue, 2% BME). The samples were then run on a 10 –20% gradient polyacrylamide gel (Page-One; Owl Separation Systems, Portsmouth, RI) at 4°C with standard protein running buffer (0.192 M glycine, 0.025 M Tris base, 0.10% SDS). Protein was transferred to a PVDF-Plus membrane (Micron Separations, Inc., Westboro, MA) and after blocking in 5% nonfat milk, exposed for 2 h at room temperature to 10 mg of either rat anti-NHE-2 or antiNHE-3 antibody (Alpha Diagnostic Intl., Inc., San Antonio, TX). After five washings, antibody detection was accomplished by incubating the membrane for 1 h at room temperature in a 1:10,000 dilution of horseradish peroxidase–avidin goat anti-rabbit IgG (Calbiochem, La Jolla, CA) followed by use of a chemiluminescence system (Renaissance; NEN Life Science Products, Boston, MA) and exposure to X-ray film (Biomax ML; Eastman Kodak Co., Rochester, NY). Band intensity was quantified using ImageQuant 5.0 software. Statistical analysis. Results are presented as mean values (6SEM). When experiments included only two groups, unpaired Student’s t test was used for statistical comparisons. A P value of less than 0.05 was considered significant.
RESULTS
In the first series of experiments the effects of SBR and EGF on the expression of NHE-2 and NHE-3 mRNA in the distal ileum were examined. As shown in Fig. 1, SBR had no significant effect on the ileal ex-
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DISCUSSION
In this study, we have for the first time demonstrated that adaptation following massive SBR results in a significant increase in NHE-3 mRNA and protein expression. Additionally, the administration of EGF further enhances the mRNA expression of this particular isoform while the removal of the majority of the endogenous EGF (salivarectomy) inhibits this change. However, neither SBR nor the mitogenic effects of exogenous EGF or salivarectomy appear to significantly alter the expression of NHE-2. This differential pattern of NHE isoform expression is distinctive and, to our knowledge, the first such response demonstrated following SBR, EGF administration, or salivarectomy. Electrolyte and water absorption increases as an adaptive response to massive enterectomy [21]. Previous studies have shown an increase in the expression of the Na 1/glucose cotransporter mRNA [10] probably as a result of increased mucosal mass, rather than increased function of the individual enterocytes [21]. Since another major mechanism for the absorption of sodium and water includes the sodium/hydrogen ex-
FIG. 1. Mean 6 SEM expression of NHE-2 mRNA at 1, 4, and 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal small bowel resection (SBR). Mice from each group were further randomized to receive either EGF (50 mg/kg/day) or an equivalent volume of saline (control) by twice daily orogastric gavage. N 5 5 for each group. There were no significant differences between SBR and sham-operated mice, with or without EGF, at any time point.
pression of NHE-2 mRNA at any time point. EGF did not significantly affect the expression for NHE-2 mRNA in either sham or SBR mice. In contrast to the negative effect of SBR or EGF on NHE-2 transcript expression, SBR resulted in a significantly increased expression (;2.5-fold) of NHE-3 mRNA levels as early as the first postoperative day. This effect persisted for the duration of the experimental period (Fig. 2). EGF significantly augmented the already increased NHE-3 mRNA expression that occurred following SBR at each time point. Representative Northern blots for each isoform taken on postoperative day 4 are depicted in Fig. 3. Western analysis revealed a modest (1.4-fold) increase in NHE-2 protein expression on postoperative day 4 (Fig. 4). In comparison, a magnitude of increase similar to that noted with the transcript (;2.5-fold) for NHE-3 protein expression was recorded after SBR. Removal of the major source of endogenous EGF (submandibular glands) had little effect on the protein expression of NHE-2 (Fig. 5). On the other hand, salivarectomy blocked the increased protein expression of NHE-3 following SBR.
FIG. 2. Mean 6 SEM expression of NHE-3 mRNA at 1, 4, and 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal small bowel resection (SBR). Mice from each group were further randomized to receive either EGF (50 mg/kg/day) or an equivalent volume of saline (control) by twice daily orogastric gavage. N 5 5 for each group; #P , 0.05 control–SBR versus control–sham, *P , 0.05 SBR–EGF versus SBR– control.
FALCONE ET AL.: Na 1/H 1 EXCHANGES DURING ADAPTATION
FIG. 3. Representative Northern blots of ileal tissue for NHE-2 and NHE-3 isoforms at 4 days following either SBR or sham operation. Two individual mice from each group are demonstrated for each isoform. There were no significant differences in NHE-2 expression following SBR; however, there was a roughly 2.5-fold increase in the expression of NHE-3 following SBR.
changers, we sought to determine the effect of SBR on the expression of these isoforms. In contrast to the function of the Na 1/H 1 exchangers in the luminal absorption of Na 1 and water following intestinal resection, another significant role for the NHE is to generate an alkaline intracellular pH, an effect which appears to be permissive for DNA synthesis [15, 22, 23]. Sacks et al. have shown an increase in NHE activity in ileal brush border membrane vesicles
FIG. 4. Relative protein levels of NHE-2 and NHE-3 from pooled membrane preps following either sham operation (n 5 5) or 50% proximal small bowel resection (n 5 5; SBR) harvested on postoperative day 4. 90-kDa bands from Western blot demonstrated.
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FIG. 5. Relative protein levels of NHE-2 and NHE-3 from pooled membrane preps following salivarectomy and either sham operation (n 5 5) or 50% proximal small bowel resection (n 5 5; SBR) harvested on postoperative day 4. 90-kDa bands from Western blot demonstrated.
following a 70% SBR in rats [11]. In that study, the changes noted in NHE activity were reported to be amiloride sensitive, thus suggesting that NHE-2 is the primary isoform to change in response to SBR. However, the concentration of amiloride used in their study (1 mM) does not permit discrimination between the various isoforms [12]. Further, and perhaps more importantly, they studied NHE activity at 1 week following SBR, which corresponds with a plateau period of intestinal adaptation [24]. The interval after SBR in our experiments (1–7 days) evaluated time points prior to this plateau of proliferation [1]. It is possible that there is a differential expression of the NHE isoforms that is dependent on the timing after intestinal resection (either prior to 24 h or beyond 1 week). It will be important in future studies to detail the changes in expression of various NHE isoforms at these other time points following SBR. In a rodent model of absorptive diarrhea induced by lactulose ingestion, Amlal and Soleimani demonstrated a lack of change in either NHE-2 or NHE-3 mRNA expression [25]. The lack of change in these parameters using a hyperosmolar diarrhea model is distinctive compared with the findings of the present study. The unique pattern of NHE isoform expression observed following SBR suggests that a mechanism for the regulation of NHE isoforms is likely directed by factors other than a simple increase in solute load presented to the ileal lumen. It is intriguing that intestinal adaptation results in an increase in the expression of NHE-3 without affecting the expression of NHE-2. The observation that
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exogenous EGF further enhances the upregulation of NHE-3 while salivarectomy inhibits this upregulation implies an important role for EGF in the modulation of NHE-3 during adaptation. A comparable responsiveness of NHE-3 to EGF has been previously demonstrated [14]. Further, EGF has been shown to upregulate NHE activity in intestinal brush border membrane preparations [26]. This pattern of NHE isoform expression during intestinal adaptation is consonant with the hypothesis that endogenous EGF is a critical component of this response. Increased EGF receptor activation and expression in the remnant ileum [7] as well as increased salivary output of EGF following SBR has recently been identified [27]. The increased receptor expression during adaptation may partially explain the exaggerated NHE-3 response to exogenous EGF that was observed following intestinal resection. These findings, along with the observation of inhibited adaptation in mice with defective EGF receptor function [9] or salivary gland excision [6], endorse an important role for endogenous EGF during this essential response. It will be important in future studies to exploit our murine model of small bowel resection [1] to test the significance of the various NHE isoforms during intestinal adaptation. A mutant NHE-1-null mouse has recently been described [28]. Similarly, NHE-3 [29] knockout mice have been generated. The application of SBR procedures and studies of the adaptive response of these genetically perturbed strains may pave the way toward dissecting out the contributions of the various exchangers during adaptation. These studies will be necessary to elucidate a mechanism(s) for the adaptive response of the intestine to massive enterectomy.
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Hines, O. J., Bilchik, A. J., Zinner, M. J., Skotzko, M. J., Moser, A. J., McFadden, D. W., and Ashley, S. W. Adaptation of the Na 1/glucose cotransporter following intestinal resection. J. Surg. Res. 57: 22, 1994.
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Sacks, A. I., Acra, S. A., Dykes, W., Polk, D. B., Barnard, J. A., Pietsch, J., and Ghishan, F. K. Intestinal Na 1/H 1 exchanger activity is up-regulated by bowel resection in the weanling rat. Pediatr. Res. 33: 215, 1993.
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Soleimani, M., and Singh, G. Physiologic and molecular aspects of the Na 1/H 1 exchangers in health and disease processes. J. Invest. Med. 43: 419, 1995.
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Hoogerwerf, W. A., Tsao, S. C., Devuyst, O., Levine, S. A., Yun, C. H., Yip, J. W., Cohen, M. E., Wilson, P. D., Lazenby, A. J., Tse, C. M., and Donowitz, M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am. J. Physiol. 270: G29, 1996.
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Tse, M., Levine, S., Yun, C., Brant, S., Counillon, L. T., Pouyssegur, J., and Donowitz, M. Structure/function studies of the epithelial isoforms of the mammalian Na 1/H 1 exchanger gene family. J. Membr. Biol. 135: 93, 1993.
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Grinstein, S., Rotin, D., and Mason, M. J. Na 1/H 1 exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim. Biophys. Acta 988: 73, 1989.
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Shin, C. E., Helmrath, M. A., Falcone, R., Jr., Fox, J. W., Duane, K. R., Erwin, C. R., and Warner, B. W. Epidermal growth factor augments adaptation following small bowel resection: Optimal dosage, route, and timing of administration. J. Surg. Res. 77: 11, 1998.
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Helmrath, M. A., Shin, C. E., Erwin, C. R., and Warner, B. W. Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression. J. Pediatr. Surg. 33: 980, 1998.
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Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77: 5201, 1980.
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Wang, Z., Rabb, H., Craig, T., Burnham, C., Shull, G. E., and Soleimani, M. Ischemic–reperfusion injury in the kidney: Overexpression of colonic H 1-K 1-ATPase and suppression of NHE-3. Kidney Int. 51: 1106, 1997.
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Vaandrager, A. B., Schulz, S., De Jonge, H. R., and Garbers, D. L. Guanylyl cyclase C is an N-linked glycoprotein receptor that accounts for multiple heat-stable enterotoxin-binding proteins in the intestine. J. Biol. Chem. 268: 2174, 1993.
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Warner, B. W. The distribution of endogenous epidermal growth factor after small bowel resection suggests increased intestinal utilization during adaptation. J. Pediatr. Surg. 34: 22, 1999. 28. Cox, G. A., Lutz, C. M., Yang, C. L., Biemesderfer, D., Bronson, R. T., Fu, A., Aronson, P. S., Noebels, J. L., and Frankel, W. N. Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice. Cell 91: 139, 1997. 29. Schultheis, P. J., Clarke, L. L., Meneton, P., Miller, M. L., Soleimani, M., Gawenis, L. R., Riddle, T. M., Duffy, J. J., Doetschman, T., Wang, T., Giebisch, G., Aronson, P. S., Lorenz, J. N., and Shull, G. E. Renal and intestinal absorptive defects in mice lacking the NHE3 Na 1/H 1 exchanger. Nat. Genet. 19: 282, 1998.
Differential Expression of Ileal Na 1/H 1 Exchanger Isoforms after Enterectomy 1 Richard A. Falcone, Jr., M.D., Cathy E. Shin, M.D., Lawrence E. Stern, M.D., Zhaohui Wang, Ph.D.,* Christopher R. Erwin, Ph.D., Manoocher Soleimani, M.D.,* and Brad W. Warner, M.D. Division of Pediatric Surgery, Department of Surgery, Children’s Hospital Medical Center, and *Division of Nephrology, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229 Submitted for publication January 26, 1999
Background. Na 1/H 1 exchangers (NHE) are transporters involved in the absorption of NaCl along the gastrointestinal tract. The aim of this study was to determine the expression pattern of the intestinal brush border NHE isoforms 2 and 3 following massive small bowel resection (SBR). Additionally, the effect of epidermal growth factor (EGF) and salivarectomy (removal of the primary source of EGF) on the expression pattern was studied. Materials and methods. ICR mice underwent a proximal SBR or sham surgery and then received either orogastric saline or EGF (50 mg/kg/day). In separate experiments mice underwent salivarectomy followed by SBR or sham. Postoperatively the remaining ileum was isolated and levels of NHE-2 and NHE-3 mRNA and protein were resolved. Results. Following SBR, the expression of both mRNA and protein for NHE-3 increased by ;2.5-fold. Treatment with EGF enhanced NHE-3 mRNA in sham animals with further elevation following SBR. The expression of NHE-2 mRNA demonstrated minimal change while protein marginally increased (40%) following SBR. EGF did not affect the expression of NHE-2 mRNA. Salivarectomy did not influence NHE-2 protein expression and inhibited the increased NHE-3 protein expression following SBR. Conclusions. Following SBR, the expression pattern for brush border NHE isoforms is distinctive. Increased expression of NHE-3 secondary to SBR and/or EGF treatment with loss of this increase following salivarectomy implies a common mechanism to enhance enterocyte proliferation and luminal absorp1
This work was supported by a Trustees Grant from the Children’s Hospital Research Foundation, Children’s Hospital Medical Center, Cincinnati, Ohio, and by National Institutes of Health RO-1 DK 53234-01 (Dr. Warner), RO-1 DK 46789 and RO-1 DK 52821 (Dr. Soleimani), and 1F32DK09882 (Dr. Stern).
0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
tion of NaCl and water. These results suggest that NHE-3 is an important ileal exchanger following SBR. © 1999 Academic Press Key Words: intestinal resection; adaptation; mouse; transport. INTRODUCTION
Intestinal adaptation is a crucial response to massive small bowel resection (SBR) that counteracts the loss of mucosal absorptive and digestive surface area. Adaptation consists of both hypertrophy and hyperplasia of all layers of the bowel wall, an increase in both caliber and length of the intestine, and augmented absorptive and digestive capacity per unit length [1–3]. Understanding the mechanism(s) of intestinal adaptation is fundamental toward the development of management strategies directed to augment this response. While multiple factors are involved during the adaptive response to SBR, there is compelling evidence to suggest a central role for epidermal growth factor (EGF). Following SBR, exogenous EGF has been shown to enhance ileal length, protein and DNA content, crypt depth and villus height, glucose transport, and EGF receptor expression [4 –7]. Adaptation is also accentuated in transgenic mice with intestinal overexpression of EGF [8]. Alternatively, adaptation is impaired in mice with perturbed EGF receptor signaling (waved-2) [9] or if the major endogenous source of EGF (salivary glands) is removed [6]. Since diarrhea is an important consequence of massive enterectomy, it is important to delineate the changes in Na 1 and water absorption that occur during adaptation. The majority of the sodium transport in the intestine occurs by passive Na 1 channels, active Na 1/substrate transporters, or Na 1/H 1 exchangers
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(NHE). Adaptation following intestinal resection results in increases in expression of mRNA for the Na 1glucose cotransporter [10] and NHE activity [11]; however, the effect of SBR on the expression of specific isoforms of NHE has not previously been described. Currently, there are four known NHE isoforms that are discriminated on the basis of tissue distribution, subcellular location, potential for glycosylation, amiloride sensitivity, and regulation by various protein kinases (reviewed in Ref. [12]). The mRNA for NHE-1 has been detected in all mammalian cells and is designated a housekeeping isoform and located in the basolateral location of polarized cells [12]. In contrast the NHE-2 and NHE-3 isoforms are primarily located at the luminal side of polarized epithelial cells and distributed throughout the intestine [13]. The NHE-2 isoform is considered to be sensitive to amiloride and positively regulated by protein kinase C [12]. Alternatively, NHE-3 is considered to be amiloride resistant, negatively regulated by protein kinase C, and affected by various growth factors and serum [14, 15]. Information regarding NHE-4 is limited and it appears to have restricted tissue and species expression [12]. This study therefore focused on the analysis of the brush border-localized NHE-2 and -3 isoforms to determine the effect of SBR on their ileal expression. Additionally, the effects of orally administered EGF and salivarectomy (removal of the major source of EGF) on this expression pattern were investigated. MATERIALS AND METHODS Animals. A protocol for this study was approved by the Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (Children’s Hospital Medical Center, Cincinnati, OH). Male ICR mice (weight range 25–29 g; The Harlan Laboratory, Indianapolis, IN) were housed in groups of four at 21°C on 12-h day–night cycles (6 AM to 6 PM). Before experimentation, the mice acclimated for 5 days. One day prior to operation, the diet was changed from regular chow to liquid rodent diet (Micro-Stabilized Rodent Liquid Diet LAD 101/101A; Purina Mills, St. Louis, MO). Experimental design. The mice were randomized to undergo either a 50% proximal SBR or a sham operation (n 5 5 for each group). The details of our murine model for SBR have already been presented [1]. Briefly, under inhaled isoflurane anesthesia and with the aid of an operating microscope, the small bowel was transected 12 cm proximal to the ileocecal valve. In sham-operated mice a reanastomosis was performed. In mice undergoing SBR, approximately 12.5 cm of proximal intestine (50% resection) was resected and an anastomosis performed. The anastomoses in both groups were accomplished using an end-to-end, single-layer technique with interrupted 9-O monofilament suture (a gift from Ethicon, Inc., Somerville, NJ). Postoperatively, the mice were resuscitated with a 3-ml intraperitoneal injection of 0.9% saline and allowed to recover in a warm incubator (30°C). Water was provided ad libitum for the first 24 h. Mice from each group were then pair fed with liquid diet. Mice were then randomized to receive either saline or EGF (50 mg/kg) by twice daily orogastric gavage. This dosage and route of exogenous EGF have previously been shown to maximally enhance adaptation in our murine SBR model [16]. After 1, 4, and 7 days, the remnant ileum was harvested and Northern blotting accomplished to measure expression of NHE-2 and NHE-3 mRNA. Protein expression of these
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isoforms was measured by Western blotting of ileal samples harvested on postoperative day 4. In separate experiments, a bilateral submandibular gland excision (salivarectomy) was done and the mice then underwent either sham or SBR. In prior studies from our laboratory, this has been an effective model for attenuation of intestinal adaptation following SBR [6]. Tissue harvest. Mice were sacrificed with an intramuscular injection of ketamine, xylazine, and acepromazine (4:1:1 proportion) followed by cervical dislocation. The abdomen was opened and the remnant ileum 2 cm from the anastomosis was collected for Northern or Western analysis after luminal contents were removed. RNA isolation. Total cellular RNA was extracted from the distal ileum and quantitated spectrophotometrically as we have previously described [17]. The total RNA was stored at 280°C until used for Northern analysis. Northern blotting. Total RNA samples (30 mg/lane) for each animal were fractionated on a 1.2% agarose–formaldehyde gel and transferred to a nylon membranes (MSI) using 103 SSPE as transfer buffer. Separate membranes for each postoperative day consisted of RNA from individual mice that underwent SBR or sham operation with or without EGF. Membranes were then cross-linked by UV light and baked as described previously [18]. Membranes were prehybridized for 1 h in 0.13 SSPE/1% SDS solution at 65°C and then for 3 h at 65°C with 0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA, and 100 mg/ml sonicated carrier DNA. The NHE-2 and NHE-3 probes were labeled with [ 32P]deoxynucleotides using the RadPrime DNA labeling kit (Gibco BRL, Grand Island, NY) as described previously [19]. After hybridization, the membranes were washed, blotted dry, and exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA). The expression for each isoform was normalized to 28S ribosomal mRNA and quantitated for each animal. The following rat NHE PCR product fragments were used as isoform-specific probes in the Northern blot analysis: (1) NHE-3, nucleotides 1883–2217, and (2) NHE-2, nucleotides 1899 – 2215. Western blotting. Crude membrane preparations were isolated from pooled samples of ileum taken from sham (n 5 5) and SBR (n 5 5) animals as previously described [20]. Fifty micrograms of protein from each sample was added to an equal volume of 23 protein sample buffer (250 mM Tris–HCl, pH 6.8, 4% SDS, 10% glycerol, 0.003% bromophenol blue, 2% BME). The samples were then run on a 10 –20% gradient polyacrylamide gel (Page-One; Owl Separation Systems, Portsmouth, RI) at 4°C with standard protein running buffer (0.192 M glycine, 0.025 M Tris base, 0.10% SDS). Protein was transferred to a PVDF-Plus membrane (Micron Separations, Inc., Westboro, MA) and after blocking in 5% nonfat milk, exposed for 2 h at room temperature to 10 mg of either rat anti-NHE-2 or antiNHE-3 antibody (Alpha Diagnostic Intl., Inc., San Antonio, TX). After five washings, antibody detection was accomplished by incubating the membrane for 1 h at room temperature in a 1:10,000 dilution of horseradish peroxidase–avidin goat anti-rabbit IgG (Calbiochem, La Jolla, CA) followed by use of a chemiluminescence system (Renaissance; NEN Life Science Products, Boston, MA) and exposure to X-ray film (Biomax ML; Eastman Kodak Co., Rochester, NY). Band intensity was quantified using ImageQuant 5.0 software. Statistical analysis. Results are presented as mean values (6SEM). When experiments included only two groups, unpaired Student’s t test was used for statistical comparisons. A P value of less than 0.05 was considered significant.
RESULTS
In the first series of experiments the effects of SBR and EGF on the expression of NHE-2 and NHE-3 mRNA in the distal ileum were examined. As shown in Fig. 1, SBR had no significant effect on the ileal ex-
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DISCUSSION
In this study, we have for the first time demonstrated that adaptation following massive SBR results in a significant increase in NHE-3 mRNA and protein expression. Additionally, the administration of EGF further enhances the mRNA expression of this particular isoform while the removal of the majority of the endogenous EGF (salivarectomy) inhibits this change. However, neither SBR nor the mitogenic effects of exogenous EGF or salivarectomy appear to significantly alter the expression of NHE-2. This differential pattern of NHE isoform expression is distinctive and, to our knowledge, the first such response demonstrated following SBR, EGF administration, or salivarectomy. Electrolyte and water absorption increases as an adaptive response to massive enterectomy [21]. Previous studies have shown an increase in the expression of the Na 1/glucose cotransporter mRNA [10] probably as a result of increased mucosal mass, rather than increased function of the individual enterocytes [21]. Since another major mechanism for the absorption of sodium and water includes the sodium/hydrogen ex-
FIG. 1. Mean 6 SEM expression of NHE-2 mRNA at 1, 4, and 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal small bowel resection (SBR). Mice from each group were further randomized to receive either EGF (50 mg/kg/day) or an equivalent volume of saline (control) by twice daily orogastric gavage. N 5 5 for each group. There were no significant differences between SBR and sham-operated mice, with or without EGF, at any time point.
pression of NHE-2 mRNA at any time point. EGF did not significantly affect the expression for NHE-2 mRNA in either sham or SBR mice. In contrast to the negative effect of SBR or EGF on NHE-2 transcript expression, SBR resulted in a significantly increased expression (;2.5-fold) of NHE-3 mRNA levels as early as the first postoperative day. This effect persisted for the duration of the experimental period (Fig. 2). EGF significantly augmented the already increased NHE-3 mRNA expression that occurred following SBR at each time point. Representative Northern blots for each isoform taken on postoperative day 4 are depicted in Fig. 3. Western analysis revealed a modest (1.4-fold) increase in NHE-2 protein expression on postoperative day 4 (Fig. 4). In comparison, a magnitude of increase similar to that noted with the transcript (;2.5-fold) for NHE-3 protein expression was recorded after SBR. Removal of the major source of endogenous EGF (submandibular glands) had little effect on the protein expression of NHE-2 (Fig. 5). On the other hand, salivarectomy blocked the increased protein expression of NHE-3 following SBR.
FIG. 2. Mean 6 SEM expression of NHE-3 mRNA at 1, 4, and 7 days following either sham operation (bowel transection with reanastomosis) or 50% proximal small bowel resection (SBR). Mice from each group were further randomized to receive either EGF (50 mg/kg/day) or an equivalent volume of saline (control) by twice daily orogastric gavage. N 5 5 for each group; #P , 0.05 control–SBR versus control–sham, *P , 0.05 SBR–EGF versus SBR– control.
FALCONE ET AL.: Na 1/H 1 EXCHANGES DURING ADAPTATION
FIG. 3. Representative Northern blots of ileal tissue for NHE-2 and NHE-3 isoforms at 4 days following either SBR or sham operation. Two individual mice from each group are demonstrated for each isoform. There were no significant differences in NHE-2 expression following SBR; however, there was a roughly 2.5-fold increase in the expression of NHE-3 following SBR.
changers, we sought to determine the effect of SBR on the expression of these isoforms. In contrast to the function of the Na 1/H 1 exchangers in the luminal absorption of Na 1 and water following intestinal resection, another significant role for the NHE is to generate an alkaline intracellular pH, an effect which appears to be permissive for DNA synthesis [15, 22, 23]. Sacks et al. have shown an increase in NHE activity in ileal brush border membrane vesicles
FIG. 4. Relative protein levels of NHE-2 and NHE-3 from pooled membrane preps following either sham operation (n 5 5) or 50% proximal small bowel resection (n 5 5; SBR) harvested on postoperative day 4. 90-kDa bands from Western blot demonstrated.
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FIG. 5. Relative protein levels of NHE-2 and NHE-3 from pooled membrane preps following salivarectomy and either sham operation (n 5 5) or 50% proximal small bowel resection (n 5 5; SBR) harvested on postoperative day 4. 90-kDa bands from Western blot demonstrated.
following a 70% SBR in rats [11]. In that study, the changes noted in NHE activity were reported to be amiloride sensitive, thus suggesting that NHE-2 is the primary isoform to change in response to SBR. However, the concentration of amiloride used in their study (1 mM) does not permit discrimination between the various isoforms [12]. Further, and perhaps more importantly, they studied NHE activity at 1 week following SBR, which corresponds with a plateau period of intestinal adaptation [24]. The interval after SBR in our experiments (1–7 days) evaluated time points prior to this plateau of proliferation [1]. It is possible that there is a differential expression of the NHE isoforms that is dependent on the timing after intestinal resection (either prior to 24 h or beyond 1 week). It will be important in future studies to detail the changes in expression of various NHE isoforms at these other time points following SBR. In a rodent model of absorptive diarrhea induced by lactulose ingestion, Amlal and Soleimani demonstrated a lack of change in either NHE-2 or NHE-3 mRNA expression [25]. The lack of change in these parameters using a hyperosmolar diarrhea model is distinctive compared with the findings of the present study. The unique pattern of NHE isoform expression observed following SBR suggests that a mechanism for the regulation of NHE isoforms is likely directed by factors other than a simple increase in solute load presented to the ileal lumen. It is intriguing that intestinal adaptation results in an increase in the expression of NHE-3 without affecting the expression of NHE-2. The observation that
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exogenous EGF further enhances the upregulation of NHE-3 while salivarectomy inhibits this upregulation implies an important role for EGF in the modulation of NHE-3 during adaptation. A comparable responsiveness of NHE-3 to EGF has been previously demonstrated [14]. Further, EGF has been shown to upregulate NHE activity in intestinal brush border membrane preparations [26]. This pattern of NHE isoform expression during intestinal adaptation is consonant with the hypothesis that endogenous EGF is a critical component of this response. Increased EGF receptor activation and expression in the remnant ileum [7] as well as increased salivary output of EGF following SBR has recently been identified [27]. The increased receptor expression during adaptation may partially explain the exaggerated NHE-3 response to exogenous EGF that was observed following intestinal resection. These findings, along with the observation of inhibited adaptation in mice with defective EGF receptor function [9] or salivary gland excision [6], endorse an important role for endogenous EGF during this essential response. It will be important in future studies to exploit our murine model of small bowel resection [1] to test the significance of the various NHE isoforms during intestinal adaptation. A mutant NHE-1-null mouse has recently been described [28]. Similarly, NHE-3 [29] knockout mice have been generated. The application of SBR procedures and studies of the adaptive response of these genetically perturbed strains may pave the way toward dissecting out the contributions of the various exchangers during adaptation. These studies will be necessary to elucidate a mechanism(s) for the adaptive response of the intestine to massive enterectomy.
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Hines, O. J., Bilchik, A. J., Zinner, M. J., Skotzko, M. J., Moser, A. J., McFadden, D. W., and Ashley, S. W. Adaptation of the Na 1/glucose cotransporter following intestinal resection. J. Surg. Res. 57: 22, 1994.
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Sacks, A. I., Acra, S. A., Dykes, W., Polk, D. B., Barnard, J. A., Pietsch, J., and Ghishan, F. K. Intestinal Na 1/H 1 exchanger activity is up-regulated by bowel resection in the weanling rat. Pediatr. Res. 33: 215, 1993.
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Soleimani, M., and Singh, G. Physiologic and molecular aspects of the Na 1/H 1 exchangers in health and disease processes. J. Invest. Med. 43: 419, 1995.
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Shin, C. E., Helmrath, M. A., Falcone, R., Jr., Fox, J. W., Duane, K. R., Erwin, C. R., and Warner, B. W. Epidermal growth factor augments adaptation following small bowel resection: Optimal dosage, route, and timing of administration. J. Surg. Res. 77: 11, 1998.
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Helmrath, M. A., Shin, C. E., Erwin, C. R., and Warner, B. W. Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression. J. Pediatr. Surg. 33: 980, 1998.
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Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77: 5201, 1980.
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Wang, Z., Rabb, H., Craig, T., Burnham, C., Shull, G. E., and Soleimani, M. Ischemic–reperfusion injury in the kidney: Overexpression of colonic H 1-K 1-ATPase and suppression of NHE-3. Kidney Int. 51: 1106, 1997.
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