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Expression of Peptide Transporter Following Intestinal Transplantation in the Rat
Journal of Surgical Research 99, 294 –300 (2001) doi:10.1006/jsre.2001.6163, available online at http://www.idealibrary.com on
Expression of Peptide Transporter Following Intestinal Transplantation in the Rat 1 Hideyuki Motohashi, Ph.D.,* Satohiro Masuda, Ph.D.,* Toshiya Katsura, Ph.D.,* Hideyuki Saito, Ph.D.,* Seisuke Sakamoto, M.D.,† Shinji Uemoto, M.D.,† Koichi Tanaka, M.D.,† and Ken-ichi Inui, Ph.D.* ,2 *Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, and †Department of Transplantation and Immunology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan Submitted for publication November 30, 2000; published online June 28, 2001
Background. The absorptive function of the intestinal graft is one of the most important factors for successful intestinal transplantation. To clarify whether the intestinal H ⴙ/peptide cotransporter (PEPT1) was expressed in the transplanted intestine, we examined the expression of PEPT1 in an experimental model of rat small intestinal transplantation in comparison with expression of Na ⴙ/glucose cotransporter (SGLT1). Materials and methods. Heterotopic intestinal transplantation was performed in allogeneic and syngeneic rat strain combinations. An additional group of allogeneic recipients was treated with tacrolimus (1 mg/ kg) prior to transplantation, then daily for 7 days. Intestinal grafts were examined for histopathology and PEPT1 and SGLT1 expression. Results. In the isografts, the levels of messenger RNA (mRNA) encoding both transporters were not changed, while the amount of SGLT1 protein was decreased and that of PEPT1 protein was increased. In the allografts, mRNA level and protein amount of both transporters and the amount of villin protein were decreased, and microscopic examination revealed histopathological features of rejection on day 7. Tacrolimus treatment ameliorated the histopathological features and prevented the decrease in villin protein expression. However, the decreases in PEPT1 and SGLT1 expression (both mRNA and protein) were partially prevented by tacrolimus treatment. Conclusions. This study indicated that the expression of transporters should be determined to evaluate 1
This work was supported in part by a Grant-in-Aid from Japan Health Science Foundation and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. 2 To whom correspondence and reprint requests should be addressed. Fax: 81-75-751-4207. E-mail: [email protected].
0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
intestinal graft function in addition to histopathological examination of the mucosa and that the levels of mRNA encoding intestinal nutrient transporters in biopsy specimens may be useful for evaluating the intestinal graft function for intestinal transplant patients. © 2001 Academic Press Key Words: intestinal transplantation; tacrolimus; peptide transporter; rejection. INTRODUCTION
Small intestinal transplantation (SIT) is a potentialtreatment option for patients with irreversible intestinal failure, including those with short-bowel syndrome [1–3]. Absorptive function of nutrients following SIT is the most clinically important physiological determinant of graft function. However, intestinal function has been reported to be impaired following SIT. Nutrient absorption from isolated loops [4] and the electrophysiological characteristics [5] have been studied in the transplanted bowel. Intestinal function, as measured by these parameters, was uniformly reduced. Histopathological examination of mucosal biopsy specimens from intestinal allografts is the primary method used to assess mucosal integrity and monitor the onset of rejection. Then the pathological changes observed during the rejection have been studied. Although many nutrient transport systems exist and plays a important role for efficient absorption of nutrients in the small intestine, relatively little is known regarding the expression of intestinal nutrient transporters following SIT. Digestion of dietary proteins is carried out by gastric and pancreatic peptidase, and digested dietary peptides are absorbed via the intestine. Analysis of lumen contents showed that amino acids are present in the
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lumen primarily in peptide form rather than in free form [6]. The peptides in the lumen consist mostly of two to six amino acids, and the concentrations of peptide-bound amino acids are as high as 80% of total amino acids. Thus, it is suggested that intestinal peptide transporter, which mediates the cellular uptake of di- and tripeptide, plays an important role in the absorption of digested proteins, as compared to amino acid transporters. Molecular cloning studies have identified the intestinal peptide transporter (PEPT1) from various species, and many studies have been carried out to delineate the functional characteristics of PEPT1 [7, 8]. Immunological studies have demonstrated that PEPT1 is localized at the brush– border membrane of intestinal epithelial cells [9]. Na ⫹/glucose cotransporter (SGLT1) is also present in the brush– border membrane and plays a critical role in glucose absorption [10]. This transporter has been cloned, which permits the analysis of SGLT1 at the molecular level. In the present study, to clarify PEPT1 expression in the transplanted intestine, we investigated the expression of PEPT1 in grafts using the rat SIT model, in comparison with SGLT1 expression. MATERIALS AND METHODS
Animals Male Lewis rats (250 –300 g) and male Brown Norway (BN) Rats (250 –300 g) were purchased from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan). Lewis rats were used as both recipients and donors, and BN rats were used as allograft donors. All rats were allowed free access to water and food during the postoperative periods. All operations were carried out under ether anesthesia. The animal experiments were performed in accordance with the Guidelines for Animal Experiments of Kyoto University.
TABLE 1 Nucleotide Sequences of PCR Primers Used in Competitive PCR Analysis Primers
Positions a
For PEPT1 Sense 5⬘-GTGTGGGGCCCCAATCTATACCGT-3⬘ 1442–1465 Antisense 5⬘-GTTTGTCTGTGAGACAGGTTCCAA-3⬘ 2153–2176 For SGLT1 Sense 5⬘-ATGGACAGTAGCACCTTGAGCC-3⬘ 170–191 Antisense 5⬘-TAGCCCCAGAGAAGATGTCTGC-3⬘ 647–668 For GAPDH Sense 5⬘-CCTTCATTGACCTCAACTAC-3⬘ 131–150 Antisense 5⬘-GGAAGGCCATGCCAGTGAGC-3⬘ 705–724 a
From the GenBank database.
Histological Examination Tissue samples for histological examination, which were obtained from the transplanted intestine after the animals were sacrificed at day 7, were embedded in paraffin, sectioned at 4 m, and stained with hematoxylin and eosin. Rejection was graded histologically according to the phase of acute intestinal rejection established by Rosemurgy and Schraut [12]. Briefly, phase 0 showed no signs of rejection. In phase 1, lymphocytes and plasma cells were seen in the lamina propria, but there were no changes in villus architecture. Phase 2 was characterized by more diffuse infiltration extending into the submucosa and muscularis propria together with villus blunting and scattered epithelial sloughing. The end point of graft rejection was reached in phase 3 with complete mucosal destruction.
RNA Extraction Total cellular RNA was isolated from the intestine using TRIZOL (GIBCO, Life Technologies, Grand Island, NY). The isolated RNA was digested with RQ1 RNase free DNase I (Promega Corp., Madison, WI) and purified by phenol/chloroform extraction. The amount of total RNA was determined spectrophotometrically.
Transplantation
Competitive Polymerase Chain Reaction (PCR)
Heterotopic SIT was carried out as follows. The jejunal segment from the ligament of Treitz to the middle of the small intestine (length, approximately 30 cm) was removed from the donor on a vascular pedicle consisting of an aortic conduit including the superior mesenteric artery and portal vein. The aortic conduit and portal vein of the graft were anastomosed to the renal artery and vein, respectively, of the recipient using the cuff technique [11]. Both ends of the intestinal graft were exteriorized as stomata. Biopsy specimens were obtained before transplantation and on days 4 and 7, and the animals were sacrificed on day 7.
We performed competitive PCR according to the method of Siebert and Larrick [13] with some modifications. The competitor DNA for rat SGLT1 or rat PEPT1 was constructed according to the method described previously [14] with some modifications using a v-erbB retrovirus complementary DNA fragment as neutral DNA. The specific primer sets are summarized in Table 1. Aliquots of 1 g of total RNA, isolated from the biopsy specimens, were reverse-transcribed in 20-l reaction mixtures by SuperScript II reverse transcriptase (GIBCO) and after reverse transcription, the reaction mixture was diluted to 200 l (10-fold dilution). Aliquots of 5 l of diluted reaction mixtures, in combination with semilogarithmic serial dilutions of mimic competitor DNA from 1 to 250 zmol (10 ⫺21 mol), were amplified by PCR according to the following method: PCR was performed with sense and antisense primers at 0.5 M with an initial denaturation step of 95°C for 3 min, followed by 34 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 1 min, and a final incubation at 72°C for 10 min. PCR products were then size-fractionated by 1.5% agarose gel electrophoresis. The amount of competitor DNA yielding equal molar amounts of products gave that of target messenger RNA (mRNA). To verify the quality of total RNA, we quantified the glyceraldehyde-3phosphate dehydrogenase mRNA in total RNA by competitive PCR, and then PEPT1 and SGLT1 mRNA were normalized.
Experimental Groups In this study, three groups were compared: Group I (for allogeneic transplantation), BN rats were used as donors and Lewis rats were used as recipients; Group II (for allogeneic transplantation plus tacrolimus), BN rats were used as donors and Lewis rats were used as recipients, and recipients were administered orally by gavage 1 mg/kg body wt of tacrolimus (Fujisawa Pharmaceutical Co., Osaka, Japan) just prior to transplantation, then daily for 7 days; Group III (for syngeneic transplantation), Lewis rats were used as donors and recipients.
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FIG. 1. Sections of isografts (A) and untreated (B) and tacrolimus-treated (C) allografts on day 7 following SIT. No histopathological changes were detected in the isograft (A). Inflammatory infiltrates distributed throughout the intestine and extensive necrosis and ulceration were seen in the transplanted intestine (Group I) (B). Allografts obtained from tacrolimus-treated recipients (Group II) had intact villi, and the infiltrate was minimal (C).
Antibodies and Western Blotting Rabbit anti-PEPT1 antibody was raised against the 15 carboxyterminal amino acids of rat PEPT1 [15]. Rabbit anti-SGLT1 antibody (a gift of Prof. M. Kasahara) was raised against synthetic peptides corresponding to amino acids 564 –575 of rabbit intestinal SGLT1 [16]. Goat anti-villin polyclonal immunoglobulin G (IgG) and antiNa ⫹/K ⫹-ATPase monoclonal IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY), respectively. Crude plasma membrane fraction of the intestine was purified from biopsy specimens as described previously [9]. Briefly, each fresh intestinal segment was homogenized in a buffer composed of 0.23 M sucrose, 5 mM Tris–HCl, pH 7.5, 2 mM ethylenediamine tetraacetic acid, and 1% protease inhibitor cocktail III (Calbiochem, San Diego, CA). The homogenate was centrifuged at 3000g for 15 min, and the supernatant was further centrifuged at 100,000g for 30 min. The resultant pellet was referred to as the crude membrane fraction. Western blot analysis was performed as described previously [15]. The crude plasma membrane fraction was solubilized in loading buffer (2% sodium dodecyl sulfate, 125 mM Tris–HCl, 20% glycerol, and 5% 2-mercaptoethanol). The samples were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA) by semidry electroblotting for 30 min. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline containing 1% Tween 20 for 16 h at 4°C. The blots were then incubated with anti-PEPT1 serum (1:1000), anti-SGLT1 serum (1:1000), anti-villin polyclonal IgG (1:1000), or anti-Na ⫹/K ⫹-ATPase monoclonal IgG (1:10,000) for 1 h at 25°C. The blots were washed and the bound antibodies were detected on X-ray film by enhanced chemiluminescence with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech, Uppsala, Sweden), antigoat IgG antibody (Seikagaku Corp., Tokyo, Japan) or anti-mouse IgG antibody (Amersham Pharmacia Biotech) using ECL Western blotting detection reagent (Amersham Pharmacia Biotech).
Statistical Analysis Data were analyzed for statistical significance by nonpaired t test or one-way ANOVA followed by Sheffe´’s test.
RESULTS
Histology of Small Intestinal Grafts Microscopic examination of the isografts at day 7 showed nonspecific histopathological changes. Villi and crypts were intact and no inflammatory infiltrate was detected in the isografts (phase 0 of rejection, Fig. 1A). In the allograft without tacrolimus treatment, severe rejection changes characterized by complete villus flatting, epithelial apoptosis, and a transmural cellular infiltrate were apparent (phase 3 of rejection, Fig. 1B). Tacrolimus treatment ameliorated the histopathological features of rejection. Villi were intact but showed some blunting. Crypts had no necrosis and the lymphocyte infiltrate was minimal (phase 0 of rejection, Fig. 1C). PEPT1 and SGLT1 mRNA Expression in the Transplanted Small Intestine Then, we examined the expression of PEPT1 and SGLT1 mRNA by competitive PCR. In competitive PCR, a DNA fragment containing the same primer template sequences as the target (competitor) competed for primer binding and amplification with target. The PCR products amplified from target and competitor were distinguished by size, and the amount of products generated by the competitor and target was compared. Representative results of agarose-gel electrophoresis at day 0 from Groups I, II, and III are shown in Fig. 2. The relative band density in each reaction was determined densitometrically and the amount of mRNA was quantified. The amount of competitor yielding equal molar amounts of products gave the initial amount of target. Figure 3 shows the levels of PEPT1 and SGLT1 mRNA in allografts following transplantation. PEPT1 mRNA level in the donor (BN rat) small intestine was 5.1 ⫾ 1.0 amol/g total RNA
MOTOHASHI ET AL.: PEPT1 AFTER SIT IN THE RAT
FIG. 2. Competitive PCR assay of PEPT1 and SGLT1 in the rat small intestine on day 0 (Group I, (A); Group II, (B); Group III, (C)). Total RNA was extracted from allografts and reverse-transcribed. After reverse transcription, the reaction mixtures were diluted 10fold, and aliquots of 5 l of diluted reaction mixtures were amplified by PCR in combination with semilogarithmic serial dilutions of mimic competitor DNA. Representative results of agarose-gel electrophoresis are shown. Lanes 1–5; 1, 2.5, 10, 25, 100, and 250 zmol (10 ⫺21 mol) of PEPT1 and SGLT1 mimic competitor complementary DNA, respectively. The positions of the 735 (PEPT1 mRNA origin) and 607 bp (competitor origin) for PEPT1 and the position of the 626 (competitor origin) and 499 bp (SGLT1 mRNA origin) for SGLT1 fragments are indicated.
(mean ⫾ SE of six experiments) before transplantation (day 0). After transplantation, the level of PEPT1 mRNA in the allografts without tacrolimus (Group I)
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FIG. 4. The mRNA expression of PEPT1 (A) and SGLT1 (B) in the isografts. Total RNA was extracted from the isografts on day 0, day 4, and day 7. Extracted RNA was reverse-transcribed and PEPT1 and SGLT1 mRNA were quantified by competitive PCR. Each point represents the mean ⫾ SE of four experiments.
was decreased to 0.5 ⫾ 0.3 amol/g total RNA (mean ⫾ SE of six experiments) at day 7. SGLT1 mRNA level was also decreased after transplantation (day 0, 33.9 ⫾ 6.9 amol/g total RNA; day 7, 4.4 ⫾ 2.2 amol/g total RNA; mean ⫾ SE of six to seven experiments). While tacrolimus treatment (Group II) prevented the decrease of PEPT1 mRNA level at day 4, PEPT1 mRNA level in Group II was decreased to the same level as in Group I on day 7. The decrease of SGLT1 mRNA level was not prevented by tacrolimus treatment. Figure 4 shows PEPT1 and SGLT1 mRNA levels of the isograft (Group III). PEPT1 and SGLT1 mRNA levels in the donor (Lewis rat) small intestine were 21.1 ⫾ 6.9 amol/g total RNA and 28.3 ⫾ 8.8 amol/g total RNA (mean ⫾ SE of four experiments), respectively, before transplantation (at day 0). Levels of both transporters’ mRNA were not changed following transplantation up to day 7 (PEPT1 17.1 ⫾ 5.8 amol/g total RNA, and SGLT1 28.0 ⫾ 2.9 amol/g total RNA; mean ⫾ SE of four experiments). FIG. 3. The mRNA expression of PEPT1 (A) and SGLT1 (B) in the allografts. Total RNA was extracted from allografts with (E) or without (F) tacrolimus treatment on day 0, day 4, and day 7. Extracted RNA was reverse-transcribed and PEPT1 and SGLT1 mRNA were quantified by competitive PCR. Each point represents the mean ⫾ SE of three to six experiments. **P ⬍ 0.01, *P ⬍ 0.05, significantly different from the mRNA level on day 0. †P ⬍ 0.05, significantly different from the tacrolimus-untreated group.
Western Blot Analysis of SGLT1, PEPT1, and Na ⫹/ K ⫹-ATPase in the Transplanted Small Intestine Figure 5 shows the results of Western blot analysis of PEPT1, SGLT1, and villin proteins. Proteins at 75-, 81-, and 93-kDa, corresponding to PEPT1, SGLT1, and villin, respectively, were detected. In the allografts (Group I), levels of both PEPT1 and SGLT1 proteins
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DISCUSSION
FIG. 5. Western blot analysis of crude plasma membranes from transplanted intestine for PEPT1 (A), SGLT1 (B), and villin (C). Crude membranes (25 g) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8%) and blotted onto polyvinylidene difluoride membranes. The antisera for PEPT1 and SGLT1 and goat anti-villin polyclonal IgG (1:1,000 dilution) were used as primary antibodies. Horseradish peroxidase-conjugated anti-rabbit and anti-goat IgG antibodies were used for detection of bound antibodies. The arrowheads indicate the positions of each transporter.
were decreased, and these proteins were not detected on day 7. Tacrolimus treatment partially prevented the decrease in PEPT1 protein level, and it was detected weakly at day 7. On the other hand, tacrolimus treatment did not prevent the decrease in SGLT1 protein level. In the isograft, PEPT1 protein level was increased on days 4 and 7 compared to the amount at day 0. In contrast to PEPT1, SGLT1 protein was decreased on days 4 and 7. The level of villin, which was used as a marker of intestinal epithelial cells, was not changed following transplantation in Group II or Group III. In allografts without tacrolimus treatment (Group I), the signal for villin was decreased and it was not detected on day 7. Figure 6 shows the result of Western blot analysis of Na ⫹/K ⫹-ATPase, which is known to be expressed in the basal membrane of intestinal epithelial cells [17]. The band at 98-kDa corresponding to Na ⫹/K ⫹-ATPase protein was detected. In the isograft (Group III), the level of Na ⫹/K ⫹-ATPase protein was not decreased. In the allografts, the level of Na ⫹/K ⫹-ATPase was decreased on day 7, whereas tacrolimus treatment prevented the decrease in Na ⫹/K ⫹-ATPase expression on day 7.
The absorptive function of the intestinal graft is an important factor for recipients of intestinal transplantation. Morphological changes of biopsy specimens have been verified as a means of monitoring the SIT allografts [18]. Nutrient absorption via the small intestine is mainly mediated by the intestinal transport systems. However, the expression of the intestinal transporters, which mediate absorption of nutrients, has not been investigated in the intestinal graft. PEPT1 is involved in efficient absorption of small peptides contributing to the maintenance of protein nutrition [7, 8]. The present study represented an attempt to delineate the expression of PEPT1 as well as SGLT1 in the grafted intestine in the rat SIT experimental model. In the nonrejected rat small intestine (Group III), the expression of villin, used as a marker of intestinal epithelial cells, was not changed following transplantation, and microscopic examination revealed no features of rejection. These observations imply that the numbers of differentiated epithelial cells were not decreased following syngeneic transplantation. However, Sigalet et al. showed that the glucose-stimulated intestinal short circuit current was reduced by syngeneic SIT [19] and that intestinal transport of 3-O-methyl glucose from the mucosal to the serosal side was decreased following syngeneic SIT [20]. We also found that the SGLT1 protein level was decreased on days 4 and 7 after transplantation (Fig. 5B). These results suggest that anatomical factors such as ischemia or cold injury trigger the decrease in SGLT1 protein expression without affecting its mRNA expression. In contrast to SGLT1, the expression of PEPT1 protein was increased after syngeneic SIT with no changes in villin expression (Figs. 5A and 5C). These results sug-
FIG. 6. Western blot analysis of crude plasma membranes from transplanted intestine for Na ⫹/K ⫹-ATPase. Crude membranes (5 g) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8%) and blotted onto polyvinylidene difluoride membranes. The mouse monoclonal anti-Na ⫹/K ⫹-ATPase IgG (1:10,000 dilution) was used as a primary antibody. Horseradish peroxidaseconjugated anti-mouse IgG antibody was used for detection of bound antibody. The arrowheads indicate the positions of Na ⫹/K ⫹-ATPase.
MOTOHASHI ET AL.: PEPT1 AFTER SIT IN THE RAT
gest that the levels of PEPT1 protein in the intestinal epithelial cells may be increased following transplantation. PEPT1 protein is probably more resistant to anatomical damage than SGLT1 protein in the intestinal epithelial cells. Tanaka et al. showed that PEPT1 was resistant to 5-fluorouracil-induced injury and that the resistance may be attributable to increased mRNA synthesis [21]. In the present study, however, PEPT1 mRNA level was not increased following syngeneic SIT, suggesting that the resistance of PEPT1 protein to transplantation may be attributable to the prevention of protein degradation and not to increased synthesis of its mRNA. To investigate the effects of immunological factors on PEPT1 expression, we evaluated the expression of PEPT1 in the allografts. The levels of both PEPT1 and villin proteins were decreased in Group I (allograft, without tacrolimus treatment). Histopathological examination showed that severe rejection occurred in the allografts (Group I) on day 7, consistent with the observations reported previously [18]. Therefore, the dissolution of intestinal epithelial cells by rejection was considered to be responsible for the decrease in the level of PEPT1 protein. Tacrolimus treatment ameliorated the histopathological changes associated with rejection, and villin protein was preserved by this treatment (Group II). These results indicate that the intestinal epithelial cells were preserved by tacrolimus treatment. However, decreases in both PEPT1 mRNA and protein expression in the allografts were not prevented by tacrolimus treatment, and the levels of SGLT1 mRNA and protein expression were reduced from day 4. These results suggest that biosynthesis of PEPT1 and SGLT1 was decreased by mechanisms other than rejection itself, such as reduction of mRNA transcription or stability. In contrast to PEPT1 and SGLT1, Na ⫹/K ⫹-ATPase, which is known to be expressed in the basal membrane of the intestine, was preserved on day 7 in the tacrolimus-treated allografts (Group II). It is suggested that the decrease in the protein expression in tacrolimus-treated allografts may be specific for the transporters expressed in the brush– border membrane. On days 4 and 7, the molecular masses of PEPT1 and SGLT1 proteins were lower than those on day 0. We reported previously that the 75-kDa PEPT1 protein band shifted to a 63-kDa product after digestion with endoglycosidase H in the in vitro translation study [15]. It is likely that the glycosylation of these transporters was changed after allogeneic SIT. N-Glycosylation of protein has been demonstrated to play a variety of roles, including intracellular targeting, protein folding, and maintenance of protein stability. Martin et al. reported that missense mutations in SGLT1, which made the glycosylated SGLT1 undetectable, blocked the transfer of SGLT1 protein from the
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endoplasmic reticulum to the plasma membrane [22]. Although the relationship between N-glycosylation and protein expression of PEPT1 and SGLT1 is not yet clear, the decrease in levels of PEPT1 and SGLT1 protein in the allografts could be related to the changes in molecular mass of these proteins. In summary, we demonstrated novel observations in the present study. (1) Changes in the levels of expression of PEPT1 and SGLT1 were different in isografts. (2) The dissolution of epithelial cells by rejection occurred in the intestine of allograft recipients reducing the expression of PEPT1 and SGLT1 in the intestinal graft. (3) Decreases in the levels of PEPT1 and SGLT1 expression were not prevented by tacrolimus treatment, although it suppressed the rejection. These results suggest that histopathological examination of biopsy specimens for monitoring rejection of intestinal grafts is insufficient to evaluate graft function. The mRNA levels in biopsy specimens can be measured with a competitive PCR method, and therefore measurement of the mRNA levels of intestinal nutrient transporters in biopsy specimens may provide useful information for evaluating intestinal graft function. ACKNOWLEDGMENT We thank Professor M. Kasahara, Ph.D., Laboratory of Biophysics, School of Medicine, Teikyo University, for providing the rabbit antiSGLT1 antibody.
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Hauri, H. P. (Na ⫹ ⫹ K ⫹)-ATPase and plasma membrane polarity of intestinal epithelial cells: Presence of a brush border antigen in the distal large intestine that is immunologically related to  subunit. J. Cell Biol. 109: 1057, 1989. Hayashi, M., Martinez, O. M., Krams, S. M., Burns, W., and Esquivel, C. O. Characterization of allograft rejection in an experimental model of small intestinal transplantation. J. Gastrointest. Surg. 2: 325, 1998. Sigalet, D. L., Kneteman, N. M., Fedorak, R. N., Kizilisik, A. T., and Thomson, A. B. R. Intestinal function following allogeneic small intestinal transplantation in the rat. Transplantation 53: 264, 1992. Sigalet, D. L., Kneteman, N. N., Fedorak, R. N., Kizilisik, T., Madsen, K. E., and Thomson, A. B. R. Small intestinal function following syngeneic transplantation in the rat. J. Surg. Res. 61: 379, 1996. Tanaka, H., Miyamoto, K., Morita, K., Haga, H., Segawa, H., Shiraga, T., Fujioka, A., Kouda, T., Taketani, Y., Hisano, S., Fukui, Y., Kitagawa, K., and Takeda, E. Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury. Gastroenterology 114: 714, 1998. Martin, M. G., Lostao, M. P., Turk, E., Lam, J., Kreman, M., and Wright, E. M. Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects. Gastroenterology 112: 1206, 1997.
Expression of Peptide Transporter Following Intestinal Transplantation in the Rat 1 Hideyuki Motohashi, Ph.D.,* Satohiro Masuda, Ph.D.,* Toshiya Katsura, Ph.D.,* Hideyuki Saito, Ph.D.,* Seisuke Sakamoto, M.D.,† Shinji Uemoto, M.D.,† Koichi Tanaka, M.D.,† and Ken-ichi Inui, Ph.D.* ,2 *Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, and †Department of Transplantation and Immunology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan Submitted for publication November 30, 2000; published online June 28, 2001
Background. The absorptive function of the intestinal graft is one of the most important factors for successful intestinal transplantation. To clarify whether the intestinal H ⴙ/peptide cotransporter (PEPT1) was expressed in the transplanted intestine, we examined the expression of PEPT1 in an experimental model of rat small intestinal transplantation in comparison with expression of Na ⴙ/glucose cotransporter (SGLT1). Materials and methods. Heterotopic intestinal transplantation was performed in allogeneic and syngeneic rat strain combinations. An additional group of allogeneic recipients was treated with tacrolimus (1 mg/ kg) prior to transplantation, then daily for 7 days. Intestinal grafts were examined for histopathology and PEPT1 and SGLT1 expression. Results. In the isografts, the levels of messenger RNA (mRNA) encoding both transporters were not changed, while the amount of SGLT1 protein was decreased and that of PEPT1 protein was increased. In the allografts, mRNA level and protein amount of both transporters and the amount of villin protein were decreased, and microscopic examination revealed histopathological features of rejection on day 7. Tacrolimus treatment ameliorated the histopathological features and prevented the decrease in villin protein expression. However, the decreases in PEPT1 and SGLT1 expression (both mRNA and protein) were partially prevented by tacrolimus treatment. Conclusions. This study indicated that the expression of transporters should be determined to evaluate 1
This work was supported in part by a Grant-in-Aid from Japan Health Science Foundation and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. 2 To whom correspondence and reprint requests should be addressed. Fax: 81-75-751-4207. E-mail: [email protected].
0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
intestinal graft function in addition to histopathological examination of the mucosa and that the levels of mRNA encoding intestinal nutrient transporters in biopsy specimens may be useful for evaluating the intestinal graft function for intestinal transplant patients. © 2001 Academic Press Key Words: intestinal transplantation; tacrolimus; peptide transporter; rejection. INTRODUCTION
Small intestinal transplantation (SIT) is a potentialtreatment option for patients with irreversible intestinal failure, including those with short-bowel syndrome [1–3]. Absorptive function of nutrients following SIT is the most clinically important physiological determinant of graft function. However, intestinal function has been reported to be impaired following SIT. Nutrient absorption from isolated loops [4] and the electrophysiological characteristics [5] have been studied in the transplanted bowel. Intestinal function, as measured by these parameters, was uniformly reduced. Histopathological examination of mucosal biopsy specimens from intestinal allografts is the primary method used to assess mucosal integrity and monitor the onset of rejection. Then the pathological changes observed during the rejection have been studied. Although many nutrient transport systems exist and plays a important role for efficient absorption of nutrients in the small intestine, relatively little is known regarding the expression of intestinal nutrient transporters following SIT. Digestion of dietary proteins is carried out by gastric and pancreatic peptidase, and digested dietary peptides are absorbed via the intestine. Analysis of lumen contents showed that amino acids are present in the
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lumen primarily in peptide form rather than in free form [6]. The peptides in the lumen consist mostly of two to six amino acids, and the concentrations of peptide-bound amino acids are as high as 80% of total amino acids. Thus, it is suggested that intestinal peptide transporter, which mediates the cellular uptake of di- and tripeptide, plays an important role in the absorption of digested proteins, as compared to amino acid transporters. Molecular cloning studies have identified the intestinal peptide transporter (PEPT1) from various species, and many studies have been carried out to delineate the functional characteristics of PEPT1 [7, 8]. Immunological studies have demonstrated that PEPT1 is localized at the brush– border membrane of intestinal epithelial cells [9]. Na ⫹/glucose cotransporter (SGLT1) is also present in the brush– border membrane and plays a critical role in glucose absorption [10]. This transporter has been cloned, which permits the analysis of SGLT1 at the molecular level. In the present study, to clarify PEPT1 expression in the transplanted intestine, we investigated the expression of PEPT1 in grafts using the rat SIT model, in comparison with SGLT1 expression. MATERIALS AND METHODS
Animals Male Lewis rats (250 –300 g) and male Brown Norway (BN) Rats (250 –300 g) were purchased from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan). Lewis rats were used as both recipients and donors, and BN rats were used as allograft donors. All rats were allowed free access to water and food during the postoperative periods. All operations were carried out under ether anesthesia. The animal experiments were performed in accordance with the Guidelines for Animal Experiments of Kyoto University.
TABLE 1 Nucleotide Sequences of PCR Primers Used in Competitive PCR Analysis Primers
Positions a
For PEPT1 Sense 5⬘-GTGTGGGGCCCCAATCTATACCGT-3⬘ 1442–1465 Antisense 5⬘-GTTTGTCTGTGAGACAGGTTCCAA-3⬘ 2153–2176 For SGLT1 Sense 5⬘-ATGGACAGTAGCACCTTGAGCC-3⬘ 170–191 Antisense 5⬘-TAGCCCCAGAGAAGATGTCTGC-3⬘ 647–668 For GAPDH Sense 5⬘-CCTTCATTGACCTCAACTAC-3⬘ 131–150 Antisense 5⬘-GGAAGGCCATGCCAGTGAGC-3⬘ 705–724 a
From the GenBank database.
Histological Examination Tissue samples for histological examination, which were obtained from the transplanted intestine after the animals were sacrificed at day 7, were embedded in paraffin, sectioned at 4 m, and stained with hematoxylin and eosin. Rejection was graded histologically according to the phase of acute intestinal rejection established by Rosemurgy and Schraut [12]. Briefly, phase 0 showed no signs of rejection. In phase 1, lymphocytes and plasma cells were seen in the lamina propria, but there were no changes in villus architecture. Phase 2 was characterized by more diffuse infiltration extending into the submucosa and muscularis propria together with villus blunting and scattered epithelial sloughing. The end point of graft rejection was reached in phase 3 with complete mucosal destruction.
RNA Extraction Total cellular RNA was isolated from the intestine using TRIZOL (GIBCO, Life Technologies, Grand Island, NY). The isolated RNA was digested with RQ1 RNase free DNase I (Promega Corp., Madison, WI) and purified by phenol/chloroform extraction. The amount of total RNA was determined spectrophotometrically.
Transplantation
Competitive Polymerase Chain Reaction (PCR)
Heterotopic SIT was carried out as follows. The jejunal segment from the ligament of Treitz to the middle of the small intestine (length, approximately 30 cm) was removed from the donor on a vascular pedicle consisting of an aortic conduit including the superior mesenteric artery and portal vein. The aortic conduit and portal vein of the graft were anastomosed to the renal artery and vein, respectively, of the recipient using the cuff technique [11]. Both ends of the intestinal graft were exteriorized as stomata. Biopsy specimens were obtained before transplantation and on days 4 and 7, and the animals were sacrificed on day 7.
We performed competitive PCR according to the method of Siebert and Larrick [13] with some modifications. The competitor DNA for rat SGLT1 or rat PEPT1 was constructed according to the method described previously [14] with some modifications using a v-erbB retrovirus complementary DNA fragment as neutral DNA. The specific primer sets are summarized in Table 1. Aliquots of 1 g of total RNA, isolated from the biopsy specimens, were reverse-transcribed in 20-l reaction mixtures by SuperScript II reverse transcriptase (GIBCO) and after reverse transcription, the reaction mixture was diluted to 200 l (10-fold dilution). Aliquots of 5 l of diluted reaction mixtures, in combination with semilogarithmic serial dilutions of mimic competitor DNA from 1 to 250 zmol (10 ⫺21 mol), were amplified by PCR according to the following method: PCR was performed with sense and antisense primers at 0.5 M with an initial denaturation step of 95°C for 3 min, followed by 34 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 1 min, and a final incubation at 72°C for 10 min. PCR products were then size-fractionated by 1.5% agarose gel electrophoresis. The amount of competitor DNA yielding equal molar amounts of products gave that of target messenger RNA (mRNA). To verify the quality of total RNA, we quantified the glyceraldehyde-3phosphate dehydrogenase mRNA in total RNA by competitive PCR, and then PEPT1 and SGLT1 mRNA were normalized.
Experimental Groups In this study, three groups were compared: Group I (for allogeneic transplantation), BN rats were used as donors and Lewis rats were used as recipients; Group II (for allogeneic transplantation plus tacrolimus), BN rats were used as donors and Lewis rats were used as recipients, and recipients were administered orally by gavage 1 mg/kg body wt of tacrolimus (Fujisawa Pharmaceutical Co., Osaka, Japan) just prior to transplantation, then daily for 7 days; Group III (for syngeneic transplantation), Lewis rats were used as donors and recipients.
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FIG. 1. Sections of isografts (A) and untreated (B) and tacrolimus-treated (C) allografts on day 7 following SIT. No histopathological changes were detected in the isograft (A). Inflammatory infiltrates distributed throughout the intestine and extensive necrosis and ulceration were seen in the transplanted intestine (Group I) (B). Allografts obtained from tacrolimus-treated recipients (Group II) had intact villi, and the infiltrate was minimal (C).
Antibodies and Western Blotting Rabbit anti-PEPT1 antibody was raised against the 15 carboxyterminal amino acids of rat PEPT1 [15]. Rabbit anti-SGLT1 antibody (a gift of Prof. M. Kasahara) was raised against synthetic peptides corresponding to amino acids 564 –575 of rabbit intestinal SGLT1 [16]. Goat anti-villin polyclonal immunoglobulin G (IgG) and antiNa ⫹/K ⫹-ATPase monoclonal IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY), respectively. Crude plasma membrane fraction of the intestine was purified from biopsy specimens as described previously [9]. Briefly, each fresh intestinal segment was homogenized in a buffer composed of 0.23 M sucrose, 5 mM Tris–HCl, pH 7.5, 2 mM ethylenediamine tetraacetic acid, and 1% protease inhibitor cocktail III (Calbiochem, San Diego, CA). The homogenate was centrifuged at 3000g for 15 min, and the supernatant was further centrifuged at 100,000g for 30 min. The resultant pellet was referred to as the crude membrane fraction. Western blot analysis was performed as described previously [15]. The crude plasma membrane fraction was solubilized in loading buffer (2% sodium dodecyl sulfate, 125 mM Tris–HCl, 20% glycerol, and 5% 2-mercaptoethanol). The samples were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA) by semidry electroblotting for 30 min. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline containing 1% Tween 20 for 16 h at 4°C. The blots were then incubated with anti-PEPT1 serum (1:1000), anti-SGLT1 serum (1:1000), anti-villin polyclonal IgG (1:1000), or anti-Na ⫹/K ⫹-ATPase monoclonal IgG (1:10,000) for 1 h at 25°C. The blots were washed and the bound antibodies were detected on X-ray film by enhanced chemiluminescence with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech, Uppsala, Sweden), antigoat IgG antibody (Seikagaku Corp., Tokyo, Japan) or anti-mouse IgG antibody (Amersham Pharmacia Biotech) using ECL Western blotting detection reagent (Amersham Pharmacia Biotech).
Statistical Analysis Data were analyzed for statistical significance by nonpaired t test or one-way ANOVA followed by Sheffe´’s test.
RESULTS
Histology of Small Intestinal Grafts Microscopic examination of the isografts at day 7 showed nonspecific histopathological changes. Villi and crypts were intact and no inflammatory infiltrate was detected in the isografts (phase 0 of rejection, Fig. 1A). In the allograft without tacrolimus treatment, severe rejection changes characterized by complete villus flatting, epithelial apoptosis, and a transmural cellular infiltrate were apparent (phase 3 of rejection, Fig. 1B). Tacrolimus treatment ameliorated the histopathological features of rejection. Villi were intact but showed some blunting. Crypts had no necrosis and the lymphocyte infiltrate was minimal (phase 0 of rejection, Fig. 1C). PEPT1 and SGLT1 mRNA Expression in the Transplanted Small Intestine Then, we examined the expression of PEPT1 and SGLT1 mRNA by competitive PCR. In competitive PCR, a DNA fragment containing the same primer template sequences as the target (competitor) competed for primer binding and amplification with target. The PCR products amplified from target and competitor were distinguished by size, and the amount of products generated by the competitor and target was compared. Representative results of agarose-gel electrophoresis at day 0 from Groups I, II, and III are shown in Fig. 2. The relative band density in each reaction was determined densitometrically and the amount of mRNA was quantified. The amount of competitor yielding equal molar amounts of products gave the initial amount of target. Figure 3 shows the levels of PEPT1 and SGLT1 mRNA in allografts following transplantation. PEPT1 mRNA level in the donor (BN rat) small intestine was 5.1 ⫾ 1.0 amol/g total RNA
MOTOHASHI ET AL.: PEPT1 AFTER SIT IN THE RAT
FIG. 2. Competitive PCR assay of PEPT1 and SGLT1 in the rat small intestine on day 0 (Group I, (A); Group II, (B); Group III, (C)). Total RNA was extracted from allografts and reverse-transcribed. After reverse transcription, the reaction mixtures were diluted 10fold, and aliquots of 5 l of diluted reaction mixtures were amplified by PCR in combination with semilogarithmic serial dilutions of mimic competitor DNA. Representative results of agarose-gel electrophoresis are shown. Lanes 1–5; 1, 2.5, 10, 25, 100, and 250 zmol (10 ⫺21 mol) of PEPT1 and SGLT1 mimic competitor complementary DNA, respectively. The positions of the 735 (PEPT1 mRNA origin) and 607 bp (competitor origin) for PEPT1 and the position of the 626 (competitor origin) and 499 bp (SGLT1 mRNA origin) for SGLT1 fragments are indicated.
(mean ⫾ SE of six experiments) before transplantation (day 0). After transplantation, the level of PEPT1 mRNA in the allografts without tacrolimus (Group I)
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FIG. 4. The mRNA expression of PEPT1 (A) and SGLT1 (B) in the isografts. Total RNA was extracted from the isografts on day 0, day 4, and day 7. Extracted RNA was reverse-transcribed and PEPT1 and SGLT1 mRNA were quantified by competitive PCR. Each point represents the mean ⫾ SE of four experiments.
was decreased to 0.5 ⫾ 0.3 amol/g total RNA (mean ⫾ SE of six experiments) at day 7. SGLT1 mRNA level was also decreased after transplantation (day 0, 33.9 ⫾ 6.9 amol/g total RNA; day 7, 4.4 ⫾ 2.2 amol/g total RNA; mean ⫾ SE of six to seven experiments). While tacrolimus treatment (Group II) prevented the decrease of PEPT1 mRNA level at day 4, PEPT1 mRNA level in Group II was decreased to the same level as in Group I on day 7. The decrease of SGLT1 mRNA level was not prevented by tacrolimus treatment. Figure 4 shows PEPT1 and SGLT1 mRNA levels of the isograft (Group III). PEPT1 and SGLT1 mRNA levels in the donor (Lewis rat) small intestine were 21.1 ⫾ 6.9 amol/g total RNA and 28.3 ⫾ 8.8 amol/g total RNA (mean ⫾ SE of four experiments), respectively, before transplantation (at day 0). Levels of both transporters’ mRNA were not changed following transplantation up to day 7 (PEPT1 17.1 ⫾ 5.8 amol/g total RNA, and SGLT1 28.0 ⫾ 2.9 amol/g total RNA; mean ⫾ SE of four experiments). FIG. 3. The mRNA expression of PEPT1 (A) and SGLT1 (B) in the allografts. Total RNA was extracted from allografts with (E) or without (F) tacrolimus treatment on day 0, day 4, and day 7. Extracted RNA was reverse-transcribed and PEPT1 and SGLT1 mRNA were quantified by competitive PCR. Each point represents the mean ⫾ SE of three to six experiments. **P ⬍ 0.01, *P ⬍ 0.05, significantly different from the mRNA level on day 0. †P ⬍ 0.05, significantly different from the tacrolimus-untreated group.
Western Blot Analysis of SGLT1, PEPT1, and Na ⫹/ K ⫹-ATPase in the Transplanted Small Intestine Figure 5 shows the results of Western blot analysis of PEPT1, SGLT1, and villin proteins. Proteins at 75-, 81-, and 93-kDa, corresponding to PEPT1, SGLT1, and villin, respectively, were detected. In the allografts (Group I), levels of both PEPT1 and SGLT1 proteins
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DISCUSSION
FIG. 5. Western blot analysis of crude plasma membranes from transplanted intestine for PEPT1 (A), SGLT1 (B), and villin (C). Crude membranes (25 g) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8%) and blotted onto polyvinylidene difluoride membranes. The antisera for PEPT1 and SGLT1 and goat anti-villin polyclonal IgG (1:1,000 dilution) were used as primary antibodies. Horseradish peroxidase-conjugated anti-rabbit and anti-goat IgG antibodies were used for detection of bound antibodies. The arrowheads indicate the positions of each transporter.
were decreased, and these proteins were not detected on day 7. Tacrolimus treatment partially prevented the decrease in PEPT1 protein level, and it was detected weakly at day 7. On the other hand, tacrolimus treatment did not prevent the decrease in SGLT1 protein level. In the isograft, PEPT1 protein level was increased on days 4 and 7 compared to the amount at day 0. In contrast to PEPT1, SGLT1 protein was decreased on days 4 and 7. The level of villin, which was used as a marker of intestinal epithelial cells, was not changed following transplantation in Group II or Group III. In allografts without tacrolimus treatment (Group I), the signal for villin was decreased and it was not detected on day 7. Figure 6 shows the result of Western blot analysis of Na ⫹/K ⫹-ATPase, which is known to be expressed in the basal membrane of intestinal epithelial cells [17]. The band at 98-kDa corresponding to Na ⫹/K ⫹-ATPase protein was detected. In the isograft (Group III), the level of Na ⫹/K ⫹-ATPase protein was not decreased. In the allografts, the level of Na ⫹/K ⫹-ATPase was decreased on day 7, whereas tacrolimus treatment prevented the decrease in Na ⫹/K ⫹-ATPase expression on day 7.
The absorptive function of the intestinal graft is an important factor for recipients of intestinal transplantation. Morphological changes of biopsy specimens have been verified as a means of monitoring the SIT allografts [18]. Nutrient absorption via the small intestine is mainly mediated by the intestinal transport systems. However, the expression of the intestinal transporters, which mediate absorption of nutrients, has not been investigated in the intestinal graft. PEPT1 is involved in efficient absorption of small peptides contributing to the maintenance of protein nutrition [7, 8]. The present study represented an attempt to delineate the expression of PEPT1 as well as SGLT1 in the grafted intestine in the rat SIT experimental model. In the nonrejected rat small intestine (Group III), the expression of villin, used as a marker of intestinal epithelial cells, was not changed following transplantation, and microscopic examination revealed no features of rejection. These observations imply that the numbers of differentiated epithelial cells were not decreased following syngeneic transplantation. However, Sigalet et al. showed that the glucose-stimulated intestinal short circuit current was reduced by syngeneic SIT [19] and that intestinal transport of 3-O-methyl glucose from the mucosal to the serosal side was decreased following syngeneic SIT [20]. We also found that the SGLT1 protein level was decreased on days 4 and 7 after transplantation (Fig. 5B). These results suggest that anatomical factors such as ischemia or cold injury trigger the decrease in SGLT1 protein expression without affecting its mRNA expression. In contrast to SGLT1, the expression of PEPT1 protein was increased after syngeneic SIT with no changes in villin expression (Figs. 5A and 5C). These results sug-
FIG. 6. Western blot analysis of crude plasma membranes from transplanted intestine for Na ⫹/K ⫹-ATPase. Crude membranes (5 g) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (8%) and blotted onto polyvinylidene difluoride membranes. The mouse monoclonal anti-Na ⫹/K ⫹-ATPase IgG (1:10,000 dilution) was used as a primary antibody. Horseradish peroxidaseconjugated anti-mouse IgG antibody was used for detection of bound antibody. The arrowheads indicate the positions of Na ⫹/K ⫹-ATPase.
MOTOHASHI ET AL.: PEPT1 AFTER SIT IN THE RAT
gest that the levels of PEPT1 protein in the intestinal epithelial cells may be increased following transplantation. PEPT1 protein is probably more resistant to anatomical damage than SGLT1 protein in the intestinal epithelial cells. Tanaka et al. showed that PEPT1 was resistant to 5-fluorouracil-induced injury and that the resistance may be attributable to increased mRNA synthesis [21]. In the present study, however, PEPT1 mRNA level was not increased following syngeneic SIT, suggesting that the resistance of PEPT1 protein to transplantation may be attributable to the prevention of protein degradation and not to increased synthesis of its mRNA. To investigate the effects of immunological factors on PEPT1 expression, we evaluated the expression of PEPT1 in the allografts. The levels of both PEPT1 and villin proteins were decreased in Group I (allograft, without tacrolimus treatment). Histopathological examination showed that severe rejection occurred in the allografts (Group I) on day 7, consistent with the observations reported previously [18]. Therefore, the dissolution of intestinal epithelial cells by rejection was considered to be responsible for the decrease in the level of PEPT1 protein. Tacrolimus treatment ameliorated the histopathological changes associated with rejection, and villin protein was preserved by this treatment (Group II). These results indicate that the intestinal epithelial cells were preserved by tacrolimus treatment. However, decreases in both PEPT1 mRNA and protein expression in the allografts were not prevented by tacrolimus treatment, and the levels of SGLT1 mRNA and protein expression were reduced from day 4. These results suggest that biosynthesis of PEPT1 and SGLT1 was decreased by mechanisms other than rejection itself, such as reduction of mRNA transcription or stability. In contrast to PEPT1 and SGLT1, Na ⫹/K ⫹-ATPase, which is known to be expressed in the basal membrane of the intestine, was preserved on day 7 in the tacrolimus-treated allografts (Group II). It is suggested that the decrease in the protein expression in tacrolimus-treated allografts may be specific for the transporters expressed in the brush– border membrane. On days 4 and 7, the molecular masses of PEPT1 and SGLT1 proteins were lower than those on day 0. We reported previously that the 75-kDa PEPT1 protein band shifted to a 63-kDa product after digestion with endoglycosidase H in the in vitro translation study [15]. It is likely that the glycosylation of these transporters was changed after allogeneic SIT. N-Glycosylation of protein has been demonstrated to play a variety of roles, including intracellular targeting, protein folding, and maintenance of protein stability. Martin et al. reported that missense mutations in SGLT1, which made the glycosylated SGLT1 undetectable, blocked the transfer of SGLT1 protein from the
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endoplasmic reticulum to the plasma membrane [22]. Although the relationship between N-glycosylation and protein expression of PEPT1 and SGLT1 is not yet clear, the decrease in levels of PEPT1 and SGLT1 protein in the allografts could be related to the changes in molecular mass of these proteins. In summary, we demonstrated novel observations in the present study. (1) Changes in the levels of expression of PEPT1 and SGLT1 were different in isografts. (2) The dissolution of epithelial cells by rejection occurred in the intestine of allograft recipients reducing the expression of PEPT1 and SGLT1 in the intestinal graft. (3) Decreases in the levels of PEPT1 and SGLT1 expression were not prevented by tacrolimus treatment, although it suppressed the rejection. These results suggest that histopathological examination of biopsy specimens for monitoring rejection of intestinal grafts is insufficient to evaluate graft function. The mRNA levels in biopsy specimens can be measured with a competitive PCR method, and therefore measurement of the mRNA levels of intestinal nutrient transporters in biopsy specimens may provide useful information for evaluating intestinal graft function. ACKNOWLEDGMENT We thank Professor M. Kasahara, Ph.D., Laboratory of Biophysics, School of Medicine, Teikyo University, for providing the rabbit antiSGLT1 antibody.
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