2003 Harry M. Vars Research Award

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0148-6071/03/2703-0198$03.00/0 JOURNAL OF PARENTERAL AND ENTERAL NUTRITION Copyright © 2003 by the American Society for Parenteral and Enteral Nutrition

Vol. 27, No. 3 Printed in U.S.A.

2003 Harry M. Vars Research Award Keratinocyte Growth Factor Improves Epithelial Function After Massive Small Bowel Resection Hua Yang, MD, PhD; Barbara E. Wildhaber, MD; and Daniel H. Teitelbaum, MD From the Section of Pediatric Surgery, Department of Surgery, University of Michigan Medical School and C. S. Mott Children’s Hospital, Ann Arbor

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ABSTRACT. Background: Massive small bowel resection with subsequent short bowel syndrome (SBS) leads to the acute loss of epithelial cell (EC) absorptive function. Keratinocyte growth factor (KGF) has been shown to improve EC growth, although little is known about KGF activity on EC function after SBS. We hypothesized that KGF would improve epithelial function in a mouse SBS model. Methods: Adult C57BL/6J mice were randomized to a 55% mid-small bowel resection (SBS), SBS with KGF administration (SBSKGF), or sham-operated (Control) group, and were killed at 7 days. Ussing chambers were used to study epithelial function. Short circuit current (Isc) was monitored. EC absorption was studied by measuring (1) glucose [3-O-methyl-D-[1-3H]glucose (3-OMG)] absorption; (2) sodium coupled amino acid (alanine) absorption; and (3) changes in Isc by using the absorptive agent D-glucose (stimulated Na⫹ absorption). Epithelial barrier function was measured with transepithelial resistance (TER) and transmural passage of

H-mannitol (Papp). ECs were separated along the cryptvillus axis with laser capture microdissection. Epithelial KGF receptor (KGFR) mRNA expression was studied using real time reverse transcriptase-polymerase chain reaction (RT-PCR). Results: KGF administration increased the basic ion transport activity and net transepithelial absorption of 3-OMG and sodium-coupled alanine absorption. SBS significantly decreased epithelial ion transport, including the Na⫹ absorption stimulated by D-glucose and L-alanine. KGF administration partially improves Na⫹ absorption. KGF had no apparent effect on the TER and 3H-mannitol permeability in this study. KGF upregulated EC KGFR mRNA expression, predominately in the crypt and lower portion of the villus. Conclusions: KGF administration improves epithelial absorptive function and stimulates intestinal proliferation after SBS. This suggests that KGF improves intestinal adaptation after SBS and may have clinical applicability. ( Journal of Parenteral and Enteral Nutrition 27:198 –207, 2003)

Short bowel syndrome (SBS) produces significant morbidity because of malabsorption of nutrients, fluid, and electrolytes, leading to intractable diarrhea, weight loss, dehydration, and malnutrition.1 Successful intestinal adaptation after massive enterectomy is dependent on increased efficiency of nutrient transport.2– 4 The absorptive capacity of individual enterocytes decreases transiently after massive bowel resection in both humans and rodents only to increase several weeks later.5,6 It has been reported that epithelial transport activities of sodium, chloride, water, and galactose decreased in a rat SBS model 2 weeks after bowel resection.7,8 However, the mechanisms for this decreased mucosal transport specific activities is unclear. Several factors have been found to affect epithelial transporters. Avissar et al found that epithelial growth factor (EGF) and growth hormone (GH) treatment enhanced sodium-dependent amino acid transporter expression.2 A study from Lannoli et al found that EGF treatment concomitant with GH upregulated glucose, glutamine and leucine, and alanine and arginine

transport in a rabbit SBS model.9 These authors also found that glucocorticoid treatment increased glucose, leucine, and several amino acid transport, but there was no effect on serum insulin-like growth factor 1 (IGF-1).10 Recently, a study from our group found that keratinocyte growth factor (KGF) administration has significant effects on epithelial ion transport and increasing the epithelial barrier function in a total parenteral nutrition (TPN) mouse model.11 Our laboratory also found that intestinal intraepithelial lymphocyte (IEL)-derived KGF expression is significantly increased in a mouse SBS model.12 Several studies have shown that KGF administration upregulates alveolar epithelial fluid transport, Na⫹ pump expression, and transepithelial Na⫹ transport across the alveolar epithelium in an acute lung injury rat model and in a cell culture model.13,14 More recently, Johnson et al15 found that KGF supplementation significantly improved mucosal morphology and increased mucosal DNA and protein content in a rat SBS model. All these results suggest that KGF may play an important role in the epithelial proliferation and ion transport function. In this study, we hypothesized that KGF administration would not only stimulate intestinal growth, but also have an effect on enhancing nutrient absorption during the observed downregulation of ion transport that occurs in the early stages after intestinal resection. Therefore, we investigated the effect of KGF

Received for publication, December 19, 2002. Accepted for publication, January 31, 2003. Correspondence: Daniel H. Teitelbaum, MD, Section of Pediatric Surgery, University of Michigan Hospitals, Mott F3970, Box 0245, Ann Arbor, MI 48109. Electronic mail may be sent to [email protected].

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on epithelial function in a mouse model of SBS. By measuring epithelial glucose, amino acid, sodium absorption, and epithelial barrier function, we were able to determine the effects of KGF administration on epithelial function. We also measured intestinal growth using intestinal morphology and cell proliferation. Finally, epithelial cell KGFR gene expression was measured. MATERIALS AND METHODS

Animals The studies reported here conformed to the guidelines for the care and use of laboratory animals established by the University Committee on Use and Care of Animals at the University of Michigan, and protocols were approved by that committee. Male, 2-month-old, specific pathogen-free, C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were maintained in temperature, humidity, and light-controlled conditions. During the experiments, mice were housed in metabolic cages. Operative Procedures Mice were anesthetized with sodium pentobarbital (50 mg/kg body weight, IP). Mice were randomly divided into 3 groups. In the SBS group (n ⫽ 6), mice were submitted to a 55% mid-small bowel resection.16,17 This resection consisted of removing all bowels between 7 cm distal to the ligament of Treitz and 7 cm proximal to the ileocecal value. After resection, an end-to-end jejuno-ileal anastomosis was performed with 6 interrupted sutures of 6-0 Dexon. For the control group (n ⫽ 6), mice underwent a transection at the midportion of the small intestine, followed by a similar reanastomosis. After operation, all mice had free access to water for 12 hours. Mice were then given standard laboratory mouse chow, except that chow was in liquid form that improved survival and prevented obstructive problems (Microstabilized rodent diet; Purina Mills, Inc, Richmond, IN) and water ad libitum.17 No antibiotics were used. Recombinant human KGF (rHuKGF) administration to SBS mice (SBSKGF group, n ⫽ 6) was given daily by IV injection (5 mg/kg per day) after the first day of SBS and continued for 7 days. Recombinant human KGF (rHuKGF) was a gift from Amgen Inc (Thousand Oaks, CA). All animals were killed at 7 days using CO2. Histologic and Proliferative Analysis At the time of death, 0.5 cm of jejunum was excised, the luminal contents were removed, and this segmental of intestine was fixed with 10% formalin and used for histology and 5-bromo-2-deoxyuridine (BrdU) incorporation studies. A 0.5 cm of jejunum was fixed in 10% formaldehyde for histologic sectioning (5 ␮m thickness). Tissues were then dehydrated with ethanol and embedded in paraffin. Sections were cut and stained with hematoxylin-eosin. The villus height and depth of crypt were measured using a calibrated micrometer. Each measurement of villus height and crypt depth consisted of the mean of 7 different fields.

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EC Proliferation Assay Mice were injected intraperitoneally with BrdU (50 mg/kg; Roche Diagnostic Corp, Indianapolis, IN) 1 hour before the mice were killed. Paraffin-embedded sections of 5 ␮m thickness were deparaffinized with xylene. Immunohistochemistry was done by using a BrdU In-Site Detection kit according to the manufacturer’s guidelines (BD PharMingen, San Diego, CA). Briefly, endogenous peroxidase was quenched with 3% H2O2. Slides were then incubated with biotinylated anti-BrdU antibody in a 1:10 dilution and streptavidinHPR. Slides were exposed to diaminobenzine substrate. Finally, slides were counterstained with hematoxylin. An index of the crypt cell proliferation rate was calculated by the ratio of the number of crypt cells incorporating BrdU to the total number of crypt cells. The total number of proliferating cells per crypt was defined as a mean of proliferating cells in 10 crypts (counted at 45⫻ magnification). EC KGFR Gene Expression Mucosa RNA isolation. ECs were isolated by using a modified nonenzymatic technique.18,19 Briefly, a section of the small intestine was removed and placed in tissue culture media (RPMI 1640, with 10% fetal calf serum). The intestine was opened longitudinally to expose the entire mucosal surface. Mucus and debris were removed. The section was cut into 5 mm pieces and incubated in phosphate-buffered saline (PBS) containing 1 mmol/L ethylenediamine tetraacetic acid plus 1 mmol/L dithiothreitol (DTT) with continuous brisk stirring at 37°C for 25 minutes. After this incubation, the cells suspended in isolation buffer were immediately passed through filtering cylinders prepared by nylon wool to a tissue culture bottle and centrifuged at 4°C. Cells were then reconstituted in RPMI tissue culture media at 4°C. A guanidine isothiocyanate/chloroform RNA extraction method was used with Trizol (Gibco BRL, Gaithersburg, MD) following the manufacturer’s guidelines for total RNA isolation. Isolation of the ECs Along the Crypt-Villus Axis. Isolation of the ECs was also examined along the length of the villi to gain a better understanding of KGF receptor (KGFR) expression at various levels of the crypt-villus axis. For this, the crypt-villus axis was dissected using laser capture microdissection (LCM) techniques. Briefly, small pieces of jejunum were embedded in paraffin before histologic sectioning (7 ␮m) and were mounted onto glass slides. Staining with hematoxylin and eosin was performed before LCM. Microdissection was performed with a PixCell II Image Archiving Working station instrument (Arcturus Engineering, Mountain View, CA).20 The intestinal mucosa levels were divided into 3 portions: upper one-third of the villus; lower one-third of the villus; and the crypt portion (Fig. 1). After microdissection of each specimen, the thermoplastic film-coated cap containing the captured tissue was placed in a microtube. RNA was extracted by using a total RNA Microprep kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Total RNA (poly A positive) was then

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FIG. 1. Schematic diagram of an intestinal villus that shows how the intestinal mucosal levels were divided into 3 portions: top one-third of the villus; bottom one-third of the villus; and the crypt portion.

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the preparation of test cDNA. cDNA of KGFR and ␤-actin genes were amplified by real-time PCR. PCR product was then documented with gel electrophoresis to purify cDNA. Moreover, the single product gel was excised, and amplified cDNA was extracted using a centrifugal filter device (Milipore Corp, Bedford, MA) and quantified by spectrophotometry (OD260). The number of cDNA copies was calculated using the gene sequence size of each gene amplicon. Serial dilutions of the amplified gene at known concentrations were tested to make a standard curve. Normalization of values was performed by dividing the number of copies of the KGFR gene by the number of copies of the ␤-actin gene. Because of the high baseline expression of ␤-actin, ratio results are multiplied by 105. Epithelial Function Studies

reversed transcribed (RT) into cDNA as described below and real-time polymerase chain reaction (PCR) was performed for detection of KGFR mRNA expression. Real-Time RT-PCR to detect KGFR mRNA expression. RNA was reversed transcribed into cDNA by adding 50 ␮g/mL of total RNA to the following mixture: nucleotides (ATP, CTP, TTP, and GTP; Boehringer Mannheim, Mannheim, Germany; each at 1 mmol/L), MMLV (Gibco BRL; 8U/␮l), Oligo dT (New England Biolabs, Beverly, MA; 2.5 ␮mol/L); and RNAase inhibitor (Boehringer Mannheim; 2 U/␮l). Diethylpyrocarbonate-treated H2O was added to yield an appropriate final concentration. Samples were incubated at 39°C for 1 hour, and the reaction was stopped by incubating at 95°C for 5 minutes. A mastermix of the following reaction components was prepared to the indicated end concentration: 2.5 ␮L ddH2O, 6.5 ␮L MgCl2 (25 mmol/L), 2.5 ␮L 10⫻ PCR buffer, 1 ␮L forward primer (12.5 ␮mol/L). 1 ␮L reverse primer (12.5 ␮mol/L), 1.5 ␮L dNTP, 1 ␮L 1/600 SYBR Green I (Roche, Mannheim, Germany), 5 ␮L 5⫻ additive reagent, 0.5 ␮L Ampli Taq Polymerase, and 2.5 ␮L RT product. The sequences of the KGFR (GeneBank accession no. NM_010207) and ␤-actin (GeneBank accession no. M12481) oligonucleotide primers were as follows. KGFR: the forward primer was 5⬘-CCCTCACAGAGACCCACATT-3⬘ and the reverse primer was 5⬘-AAACACAGAATCGTCCCCTG-3⬘. ␤-actin primers were previously described. The forward primer was GAGGGAAATCGTGCGTGACAT and the reverse primer was AGAAGGAAGGCTGGAAAAGAG.21 The following Smart Cycler (Cepheid Corp, Sunnyvale, CA) experimental run protocol was used: denaturation program (94°C for 2 minutes), amplification and quantification program repeated 43 times (94°C for 15 s, 66°C for 15 s, 72°C for 25 s. Detection of the PCR products was made in real-time by measuring the fluorescent signal emitted by the intercalating of SYBR威 green into double-stranded DNA at the beginning of each annealing step. Gel bands were analyzed by DNA sequencing technology (University of Michigan’s Sequencing Core) to ensure the correct product. To create a standard curve, the cDNA was generated by RT using a technique identical with the one used for

Ion transport experiment. 1: Basal ion transport. Intestinal segments were taken approximately 1 cm proximal to the anastomosis and were mounted in modified Ussing chambers (Physiologic Instruments, San Diego, CA), with an exposed tissue surface area of 0.3 cm2. Each half cell (mucosal and serosal) was filled with 5 mL of preheated 37°C Krebs-buffer. The Krebs buffer contained NaCl, 110.0 mmol/L; CaCl2, 3.0 mmol/L; KCl, 5.5 mmol/L; KH2PO4, 1.4 mmol/L; NaHCO3, 29.0 mmol/L; MgCl2, and 1.2 mmol/L, and was adjusted to a pH of 7.4. Each chamber was continuously oxygenated with O2/CO2 (95/5%) and stirred by gas flow in the chambers. The serosal buffer included 10 mmol/L glucose as an energy source and was osmotically balanced with 10 mmol/L mannitol in the mucosal side. One pair of Ag/AgCl-electrodes (Physiologic Instruments) with 3 M KCl in 3% agar bridges was used for measurement of transepithelial potential difference, and another pair of Pt-electrodes was used for current passage. The spontaneous potential difference across the intestinal membrane was maintained at 5 mV by an automated voltage clamp, and the injected short circuit current (Isc) was continuously monitored as an indication of net active ion transport. The transmembrane resistance (TER), which is a measure of intestinal epithelial barrier function and tissue viability,22,23 was determined using Ohm’s law. Baseline Isc was determined as an indication of the ion transport state of the tissue and recorded after a 30-minute equilibration period. 2: Sodium coupled amino acid absorption detection. Alanine was used as an indicator of sodium-coupled amino acid absorption.24 –26 Alanine increases Isc and net sodium absorption by stimulating (mucosa to serosal) sodium flux through a sodium-amino acid cotransporter.25 Five milliliters L-alanine (10 mmol/L) was added to mucosal side, and 5 mL fresh KB buffer was added to the serosal side. Isc was measured by subtracting the basal current (before the addition of the agonist) from the peak current after the addition of the alanine. 3: Sodium absorption detection. Glucose uptake into enterocytes is linked to serosal sodium absorption, thus leading to changes in Isc with glucose absorption.27,28 To assess this, D-glucose (stimulated Na⫹

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TABLE I Effect of rHuKGF on mucosal histological parameters in the small intestine 7 days after surgery Group

Villus height (␮m)

Crypt depth (␮m)

Total height (␮m)

Control SBS SBSKGF

310 ⫾ 42* 512 ⫾ 77 582 ⫾ 42*

93 ⫾ 18* 156 ⫾ 17 185 ⫾ 12*

439 ⫾ 51* 680 ⫾ 84 773 ⫾ 56*

Data are expressed as mean ⫾ SD. *Compared with the SBS group, P ⬍ .05. KGF, keratinocyte growth factor; SBS, massive small bowel resection; SBSKGF, KGF administration to SBS mice.

absorption) was used. Basal electrical parameter measurements were taken after the equilibration period. The changes in Isc induced by the mucosal addition of 10 mmol/L D-glucose in Ussing chambers were registered on a chart recorder. The change in Isc was measured by subtracting the basal current (before the addition of D-glucose) from the peak current after the addition of D-glucose. 4: Transepithelial Glucose Absorption. Transepithelial glucose net fluxes from mucosal 3 serosal (M 3 S) was determined with the non-metabolizable glucose analog 3-O-methylglucose (3-OMG) to avoid metabolism of 25,26 D-glucose in enterocytes. Unidirectional 3-OMG fluxes were measured under short circuit conditions.24 After a 30-minute equilibration period to achieve isotopic steady state, 3-O-methyl-D-[1-3H] glucose (4 ␮Ci/mL; Sigma, St. Louis, MO) was added to the mucosal or serosal compartment. One-milliliter samples were then taken every 15 minutes from the serosal or mucosal compartment for analysis of 3-O-methyl-D-[13 H] glucose and replaced with fresh Krebs buffer. Incubations were carried out for 60 minutes. The radioactivity of 3-O-methyl-D-[1-3H] glucose was measured in a scintillation counter (Beckman LS-1801; Beckman Instrument Inc, Fullerton, CA). Net fluxes of 3-Omethyl-D-[1-3H] glucose (M 3 S) was calculated.22 Intestinal permeability experiments. The permeability of the small intestine was studied by TER (as stated before) and further assessed with 3H-mannitol. After a 30-minute equilibration period in Ussing chambers, 3 H-mannitol (3 ␮Ci/mL; Sigma) was added to the mucosal compartment. One-milliliter samples were taken every 10 minutes from the serosal compartment for analysis of 3H-mannitol and replaced with 1 mL fresh Krebs-buffer. Incubations were carried out for 90 minutes after equilibration. Radioactivity of 3H-mannitol was measured in a scintillation counter. Permeability of isotopes was assessed by measuring the appearance of the marker on the serosal side during the experiments. The apparent permeability coefficient (Papp) was calculated by standard equations.29,30 Statistics All data are expressed as mean ⫾ SD. Bonferonni 1-factor analysis of variance (ANOVA) was used for statistical analysis. Differences were considered significant at the p ⬍ .05 level.

RESULTS

Intestinal Morphology and Proliferation Intestinal histology. SBS led to significant intestinal hypertrophy. There was an increase in both villus height and crypt depth (total height) in the SBS group. Massive bowel resection led to a 161.9% increase in villus height compared with controls. Crypt depth was also greater in the SBS group than controls (Table I). rHuKGF treatment had a significant impact on both villus height and crypt depth. Villus height in the SBS mice supplemented with rHuKGF significantly increased by 15.9% compared with SBS mice, and increased 87.7% compared with control mice. Crypt depth also significantly increased (18.6% compared with nontreated SBS mice) in the SBS mice treated with rHuKGF (Table I). Intestinal epithelial proliferation rates. There was no difference in labeled cell position between groups of mice injected with BrdU. BrdU-positive cells were all distributed in the crypt of Lieberkuhn of the small intestine. The number of BrdU-positive cells was significantly (p ⬍ .05) greater in mice subjected to massive bowel resection than controls. Massive bowel resection caused a 37.5% increase of BrdU-positive ECs compared with controls. rHuKGF administration further increased the BrdU-positive ECs in SBS mice compared with the nontreated SBS group. EC proliferation with rHuKGF increased by 34.4% compared with SBS mice and increased by 84.8% compared with control mice (Fig. 2). ECs KGFR Gene Expression Mucosal expression of KGFR. Because of the observed increase in EC proliferation (BrdU-positive cells) and mucosal morphology with SBS, we hypothesized that intestinal mucosa KGFR expression would be increased with massive bowel resection, and rHuKGF administration would further increase KGFR expression. KGFR mRNA was measured by real time RTPCR. Results showed that SBS resulted in a signifi-

FIG. 2. 5-Bromo-2-deoxyuridine (BrdU)-positive cells in mouse jejunum with immunohistochemical staining. Mice were injected with BrdU, 50 mg/kg body weight (BW), through the intraperitoneal route 1 hour before death. An index of the crypt cell proliferation rate was calculated by the ratio of the number of crypt cells incorporating BrdU to the total number of crypt cells and expressed as the percent of positive cells. Epithelial cell proliferation significantly increased after 7 days in the SBS group compared with sham-operated controls. Keratinocyte growth factor (KGF) administration (SBSKGF) further (p ⬍ .05) increased the rate of epithelial proliferation.

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FIG. 3. Changes in keratinocyte growth factor (KGF) receptor (KGFR) mRNA in intestinal mucosa specimens measured by realtime reverse transcriptase-polymerase chain reaction (RT-PCR). Results are expressed as the ratio of the number of copies of the KGFR gene to the number of copies of the ␤-actin gene. Expression of KGFR was markedly increased in the SBS mice compared with control mice (*p ⬍ .05). KGF administration further (p ⬍ .05) increased mucosal KGFR expression.

cantly increased KGFR mRNA expression (14.9% over controls). rHuKGF administration further upregulated epithelial KGFR mRNA expression. KGFR mRNA levels in SBS mice supplemented with rHuKGF significantly increased compared with SBS mice (46.8% increase), and control mice (15.6% increase; Fig. 3). Mucosal expression of KGFR along the crypt-villus axis. LCM was used to dissect ECs at different levels of the crypt-villus axis (Fig. 1). This allowed a better understanding of EC KGFR expression at various levels of this axis and how KGF activity affected growth and maturation of ECs compared with EC proliferation, as measured by BrdU (see above). We found that baseline KGFR mRNA expression levels in the crypt and lower portion of the villus was significantly higher than for the upper portion of the villus (772 ⫾ 169 versus 499 ⫾ 166 versus 425 ⫾ 105 in the crypt and lower and upper villus, respectively; values are expressed as KGFR mRNA copies/105 ␤-actin copies). We found that SBS caused a 96% increase of crypt KGFR mRNA expression and a 96.1% increase of KGFR mRNA expression in the lower portion of villus compared with control mice (Fig. 4). No significant change in EC KGFR mRNA expression was found in the upper portion of villus. rHuKGF administration further increased the crypt and lower portion of KGFR mRNA expression. SBS mice receiving KGF had a 29% increase in crypt KGFR mRNA levels and a 26.1% increase in KGFR in the lower portion of the villi compared to the same location in SBS mice (Fig. 4). Baseline levels of KGFR were low in the villus tips, and little change in expression was noted in this area with rHuKGF administration.

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FIG. 4. Changes in epithelial cell (EC) keratinocyte growth factor (KGF) receptor (KGFR) mRNA expression at different levels of the crypt-villus axis. Results are expressed by dividing the number of KGFR gene copies into the number of copies of ␤-actin gene. Expression of KGFR mRNA in the crypt and lower portion of the villus was significantly increased in SBS compared with control mice; *p ⬍ .05. KGF administration further (p ⬍ .05) increased EC KGFR expression in these sites. There was no significant change in EC KGFR mRNA expression in the upper portion of villus after bowel resection or bowel resection with KGF administration.

uated the decrease in Isc caused by bowel resection. Basic Isc in the SBSKGF group was 28.9 ⫾ 5.8 ␮A/cm2 (Fig. 5). Epithelial nutrient absorption. Two methods were used to measure these parameters. First, changes in Isc after mucosal addition of either D-glucose or L-alanine. Second, measurement of transepithelial flux of 3-OMG. A: Stimulated ion transport. Glucose uptake into enterocytes is linked to sodium absorption, thus leading to changes in Isc with glucose absorption. Glucose evoked a significantly (p ⬍ .01) lower increase in Isc in intestinal segments from SBS mice compared with controls. rHuKGF administration statistically significantly increased the Isc response stimulated by L-alanine, although still lower than control levels (Fig. 6A). Alanine was used as an indicator of sodium-coupled amino acid absorption.24 –26 Alanine increases Isc and net sodium absorption by stimulating a mucosal to serosal (M 3 S) sodium flux through a sodium-amino acid co-transporter.25 Intestinal resection decreased the L-alanine-evoked increase in Isc. rHuKGF administration significantly increased the L-alanine– evoked Isc response, at the level of the control group (Fig. 6B).

Epithelial Function Epithelial basal ion transport. Isc is an indicator of active ion transport. Bowel resection led to significant Isc transport abnormalities. The baseline Isc significantly (p ⬍ .05) decreased in intestinal tissues from mice subjected to a massive bowel resection (21.3 ⫾ 4.9 ␮A/cm2) compared with controls (34.1 ⫾ 4.7 ␮A/cm2). rHuKGF administration significantly (p ⬍ .05) atten-

FIG. 5. Baseline short circuit current (Isc; ␮A/cm2) at 0 minutes after mounting mouse intestine from different study groups. Massive bowel resection significantly downregulated the intestinal baseline Isc. KGF administration attenuated the decreased in Isc induced by intestinal resection. *p ⬍ .05 compared with the massive bowel resection (SBS) group. Values are means ⫾ SD.

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TABLE II Changes in intestinal permeability and TER in mice after surgery

Papp of mannitol TER (⍀ 䡠 cm2)

Control

SBS

SBSKGF

7.5 ⫾ 1.3 86.7 ⫾ 10.0

6.7 ⫾ 1.0 95.9 ⫾ 10.1

6.8 ⫾ 1.4 94.3 ⫾ 12.0

Papp is expressed in 10⫺6 䡠 cm/s. All values are expressed as means ⫾ SD. P ⬎ .05 compared with the SBS group. TER, transepithelial resistance; SBS, massive small bowel resection; SBSKGF, rHuKGF administration to SBS mice.

FIG. 6. Changes in intestinal short circuit current (Isc) induced by mucosal addition of 10 mmol/L D-glucose (A), mucosal addition of 10 mmol/L L-alanine (B). Values are means ⫾ SD. ⌬Isc (␮mol/L/cm2) was measured by subtracting the basal current before the addition of the agonist from the peak current after the addition of the agonist. Values were then normalized to the serosal area exposed in flux chambers. *p ⬍ .05 compared with the massive bowel resection (SBS) group.

B: Epithelial 3-OMG transport. Net transepithelial absorption of the non-metabolizable glucose analog 3-OMG (M 3 S) was measured under short circuit conditions. The net 3-OMG flux significantly decreased in the SBS group compared with controls. The net 3-OMG flux was 0.73 ⫾ 0.08%䡠cm⫺2/h in the SBS group and 1.08 ⫾ 0.19% 䡠 cm⫺2/h in controls. rHuKGF administration significantly (p ⬍ .05) increased net 3-OMG net flux (M 3 S) in SBS mice (0.91 ⫾ 0.11% 䡠 cm⫺2/h; Fig. 7). Epithelial barrier function. Epithelial barrier function was detected by transepithelial resistance. It was fur-

FIG. 7. Transepithelial 3-O-methyl-D-[1-3H] glucose (3-OMG) flux across small intestine from different groups. Massive bowel resection significantly downregulated the transepithelial 3-OMG (mucosal to serosal) net flux. Keratinocyte growth factor (KGF) administration attenuated the decrease in transepithelial 3-OMG net flux induced seen with intestinal resection. *p ⬍ .05 compared with the SBS group.

ther assessed by determining the permeation of radiolabeled inert marker 3H-mannitol (183 Da). A: TER. TER is a measure of intestinal epithelial integrity and tissue viability.22 TER did not decrease in SBS mice compared with controls. Indeed, TER increased in the SBS group, although this increase was not significant compared with controls. Baseline TER (⍀ 䡠 cm2) at 0 minutes after mounting mouse intestine was 86.7 ⫾ 10.0 and 95.9 ⫾ 10.1 ⍀ 䡠 cm2 in control and SBS groups, respectively. rHuKGF administration did not significantly change the TER after massive bowel resection (Table II). B: Epithelial permeability. Mannitol was selected because it is reported to diffuse across the epithelium through intracellular and paracellular pathways. It has also been shown that mannitol passes through tight junctions at both the level of the villus and crypt.31 After the equilibration period, there was a constant permeation of 3H-mannitol in the SBS, control, and SBSKGF groups in the small intestine throughout the 90-minute incubation period. The permeability coefficients (Papp) for 3H-mannitol in the small intestine are shown in Table II. Interestingly, massive bowel resection did not cause an increase in permeability values of 3H-mannitol compared with controls. Similarly, rHuKGF administration did not significantly change the 3H-mannitol permeability in SBS mice. DISCUSSION

In this study, the effects of rHuKGF administration on gut epithelial function and growth in a mouse SBS model were investigated. The results of this study demonstrate that epithelial nutrient absorption function is downregulated at 7 days after massive bowel resection. rHuKGF administration demonstrated several beneficial effects in the modulation of epithelial function. In particular, rHuKGF enhanced nutrient absorption during this period of decreased epithelial absorption function. Additionally, rHuKGF administration increased mucosal crypt cell proliferation and intestinal morphology. Finally, rHuKGF led to an upregulation in small intestine EC KGFR expression. This latter effect may play an important role in epithelial proliferation and adaptation. The regulation of gastrointestinal cell growth and differentiation is complex and influenced by many factors.32 KGF has been shown to regulate the proliferative response of the intestinal tract.33 KGF is a known mitogenic growth factor, which is expressed in the mucosal layer by IELs12,34,35 and stromal cells in the

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underlying lamina propria.36 Because KGFRs have been detected in high numbers in the gastrointestinal tract,37 KGF seems to play a critical role in intestinal epithelial growth and maintenance.11,36,38 Recent studies have found that KGF administration can have a number of beneficial effects. Administration of exogenous KGF can prevent mucositis during the administration of chemotherapy and radiation to the intestine.39 KGF has also been shown to ameliorate mucosal injury in an experimental model of intestinal inflammation in rats.40,41 Fernadez Estivariz et al found recently that KGF administration can enhance trefoil factor 2 (TFF2) expression in the proximal small bowel and increases goblet cell number and TFF3 protein content throughout the intestine in a rat starvation model.42 Additionally, KGF given to rodents in a starvation model has been shown to prevent villus atrophy.11,43,44 In this study, we found that rHuKGF administration increased BrdU-positive ECs and increased mucosa morphology (both villus height and depth of crypt) in a mouse SBS model. Our results are supported by a study from Johnson et al.15 Their results showed that KGF administration significantly increased villus height, mucosal DNA, and protein contents in a rat SBS model. These results show that KGF is an important factor for adaptive intestinal growth and regeneration after bowel resection. However, there are no reports as to whether KGF administration has an effect on intestinal epithelial function, particularly with regard to epithelial nutrient absorption after bowel resection.5,15 Transport capacity is the essential functional measurement that determines the nutritional status of an animal. Baseline Isc was determined as an indication of the ion transport state of the tissue.45 We found that rHuKGF administration attenuated the marked decline in basal Isc after bowel resection. Similarly, Wolvekamp et al46 found that massive bowel resection caused a significantly decrease in ion transport in a rat SBS model 3 weeks after resection. This suggests that rHuKGF resulted in a higher level of active ion transport across the intestinal epithelium in the basal state, indicated by the higher Isc. rHuKGF administration also increased the absorptive capacity of SBS mice for 2 different Na⫹-coupled nutrients: D-glucose and L-alanine. Glucose uptake by the apical membrane of enterocytes is linked to sodium and increases Isc by stimulating mucosal to sodium flux (ie, stimulating transepithelial sodium absorption). However, the increase of Isc evoked by D-glucose in SBS mice still remained significantly lower than in the control animals. Alanine was used as an indicator of sodium-coupled amino acid absorption.24 –26 Alanine increases Isc and net sodium absorption by stimulating (mucosal to serosal) sodium flux through a sodium-amino acid cotransporter. The increase in Isc induced by the mucosal addition of either nutrient was higher in rHuKGFtreated SBS mice than in untreated SBS mice. Because both nutrients are co-transported with Na⫹, the D-glucose– and L-alanine–stimulated changes in Isc are indirect measures of each nutrient’s absorption rate.24 Epithelial nutrient absorption was also studied by the net transepithelial absorption of 3-OMG. The net

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3-OMG flux significantly decreased after massive bowel resection. rHuKGF administration significantly increased epithelial glucose absorption. The barrier function of the gut permits the absorption of nutrients while preventing systemic contamination by luminal toxins and microbial products.47 TER is considered to reflect tissue tight junction integrity. It was interesting to note that we did not find any decrease of epithelial TER or increase of 3H-mannitol permeability 7 days after massive bowel resection. These findings differ from another study that reported that epithelial TER decreased and polyethylene glycol 4000 fluxes increased in a rat SBS model 8 weeks after resection.48 Indeed, the epithelial permeability in our SBS group was higher than controls, although the change was not significant. Additionally, it is possible that the difference in animal models (mouse vs rat) may also explain some of the difference between our findings and these investigators. rHuKGF administration did not significantly affect epithelial permeability. Our results are supported by studies from O’Brien et al.49,50 These investigators performed a 50% proximal massive bowel resection in a rat model. They found that epithelial TER significantly increased at day 4 but decreased to sham levels by day 7. Epithelial permeability to FITC-dextran was significantly decreased in this same model at 3 days after bowel resection. Urban and Michel8 reported that they were unable to detect differences in permeability at 2 and 4 weeks after a 50% or 70% proximal massive bowel resection. The reason for this lack of change in epithelial TER and mannitol permeability is unclear. A study from Freeman et al51 found that upregulation of D-glucose transport in rats SBS model did not occur for 2 weeks after resection, suggesting that complete intestinal functional adaptation, including factors that affect intestinal permeability, may be delayed. Our study was performed at an early time point during postresection. We intentionally aimed for this early time period to investigate the effect of KGF on the epithelial nutrient absorption in the early stage of SBS. Such a time period is when the intestine would be predicted most dysfunctional and may be in the greatest need for growth factor support. Future studies using longer postoperative time periods would be necessary to observe the associated changes of epithelial barrier after massive bowel resection. KGFR has been found to be ubiquitously expressed in the mucosal epithelium of all gastrointestinal tract segments. Using immunofluorescent staining of KGFR, Chailler et al37 found that KGFR is located at the basolateral membrane and cytoplasm in differentiated enterocytes and in crypt cells. In this study, we found that rHuKGF administration significantly increased KGFR mRNA expression in a SBS mouse model. The increase in KGFR expression is coincident with an increase in epithelial cell proliferation and increase in villus height. Furthermore, we found that this increase in KGFR expression is located at the crypt and lower portion of villus. A study from Estivariz et al52 found that administration of recombinant KGF significantly increased small intestinal mucosa KGFR mRNA expression in rats subjected to 3 days of

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starvation or provision of a low level of luminal nutrition (25% of control group intake) for 3 days after 3 days of starvation. A previous study from our laboratory also found that rHuKGF administration significantly increased KGFR mRNA expression in a TPN mouse model.11 The increased expression of KGFR correlated with EC proliferation and supports the hypothesis that KGFR signaling is crucial for the mitogenic stimulus for gut adaptation. A study from Jonas et al44 found that KGF markedly enhanced glutathione (GSH) levels and GSH antioxidant capacity in the small intestinal mucosa in 25% refed rats compared with salinetreated controls. Their results demonstrated that KGF may upregulate tissue GSH by an endocrine mechanism, which may represent another mechanism for KGF-induced mucosal growth.43 The finding that the maximal expression of KGFR was located in the crypt and lower one-third of the villus was quite intriguing. A number of epithelial functions are differentially expressed from the crypt base to villus tip.53 Although these changes in EC maturation have been well-studied, an understanding of how growth factors affect these maturational changes is not well understood. Our observations suggest that the maximal effect of rHuKGF is in the crypt and lower portion of the villus. This suggests that KGF is more important for the proliferative effects of ECs as opposed to the maturational effects of the cells that are found to predominate in the upper portion of the villi.53 In conclusion, this study demonstrated that epithelial function was downregulated in a mouse SBS model. KGF administration enhanced epithelial Na⫹ and Na⫹-coupled nutrient absorptive function and stimulated EC growth. Additionally, rHuKGF administration upregulated the expression of epithelial KGFR. This effect may play an important role in epithelial function. Use of rHuKGF may have beneficial effects in patients with SBS and may eventually have clinical applicability. ACKNOWLEDGMENTS

This research was supported by National Institutes of Health Grant AI44076-01, UM-Comprehensive Cancer Center NIH CA46592, and the Laser Capture Microdissection Core. Amgen, Inc, Thousand Oaks, CA, supplied the rHuKGF. REFERENCES 1. Wang HT, Miller JH, Avissar N, et al: Small bowel adaptation is dependent on site of massive enterectomy. J Surg Res 84:94 – 100, 1999 2. Avissar NE, Ziegler TR, Wang HT, et al: Growth factors regulation of rabbit sodium-dependent neutral amino acid transporter ATB0 and oligopeptide transporter 1 mRNAs expression after enteretomy. JPEN 25:65–72, 2001 3. Iannoli P, Miller JH, Sax HC: Epidermal growth factor and human growth hormone induce two sodium-dependent arginine transport systems after massive enterectomy. JPEN 22:326 – 330, 1998 4. Iannoli P, Miller JH, Ryan CK, et al: Enterocyte nutrient transport is preserved in a rabbit model of acute intestinal ischemia. JPEN 22:387–392, 1998 5. Ray EC, Avissar NE, Sax HC: Growth factor regulation of enterocyte nutrient transport during intestinal adaptation. Am J Surg 183:361–371, 2002

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6. Sarac TP, Seydel AS, Ryan CK, et al: Sequential alterations in gut mucosal amino acid and glucose transport after 70% small bowel resection. Surgery 120:503–508, 1996 7. O’Connor TP, Lam MM, Diamond J: Magnitude of functional adaptation after intestinal resection. Am J Physiol 276:R1265– R1275, 1999 8. Urban E, Michel AM: Separation of adaptive mucosal growth and transport after small bowel resection. Am J Physiol 244: G295–G300, 1983 9. Iannoli P, Miller JH, Ryan CK, et al: Epidermal growth factor and human growth hormone accelerate adaptation after massive enterectomy in an additive, nutrient-dependent, and site-specific fashion. Surgery 122:721–728, 1997 10. Iannoli P, Miller JH, Ryan CK, et al: Glucocorticoids upregulate intestinal nutrient transport in a time-dependent and substratespecific fashion. J Gastrointest Surg 2:449 – 457, 1998 11. Yang H, Wildhaber B, Tazuke Y, et al: Keratinocyte growth factor stimulates the recovery of epithelial structure and function in a mouse model of total parenteral nutrition. JPEN 26:333–341, 2002 12. Antony PA, Yang H, Fan YY, et al: Altered expression of intraepithelial lymphocyte (IEL) keratinocyte growth factor (KGF) mRNA in the mouse. Gastroenterology 118:S658, 2000 13. Wang Y, Folkesson HG, Jayr C, et al: Alveolar epithelial fluid transport can be simultaneously upregulated by both KGF and ␤-agonist therapy. J Appl Physiol 87:1852–1860, 1999 14. Borok Z, Danto SI, Dimen LL, et al: Na⫹-K⫹-ATPase expression in alveolar epithelial cells: Upregulation of active ion transport by KGF. Am J Physiol 274:L149 –L158, 1998 15. Johnson W, DiPalma C, Ziegler T, et al: Keratinocyte growth factor enhances early gut adaptation in a rat model of short bowel syndrome. Vet Surg 29:17–27, 2000 16. Sonnino R, Teitelbaum D, Dunaway D, et al: Small bowel transplantation permits survival in rats with lethal short gut syndrome. J Pediatr Surg 24:959 –962, 1989 17. Helmrath MA, VanderKolk WE, Can G, et al: Intestinal adaptation following massive small bowel resection in the mouse. J Am Coll Surg 183:441– 449, 1996 18. Grossmann J, Maxson J, Whitacre C, et al: New isolation technique to study apoptosis in human intestinal epithelial cells. Am J Pathol 153:53– 62, 1998 19. Mosley RL, Klein JR: A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity. J Immunol Methods 156:19 –26, 1992 20. Curran S, McKay J, McLeod H, et al: Laser capture microscopy. J Clin Path Mol Pathol 53:64 – 68, 2000 21. Kiristioglu I, Teitelbaum DH: Alteration of the intestinal intraepithelial lymphocytes during total parenteral nutrition. J Surg Res 79:91–96, 1998 22. Smith PL: Methods for evaluating intestinal permeability and metabolism in vitro. Pharm Biotechnol 8:13–34, 1996 23. Tomita M, Menconi MJ, Delude RL, et al: Polarized transport of hydrophilic compounds across rat colonic mucosa from serosa to mucosa is temperature dependent. Gastroenterology 118:535– 543, 2000 24. Alexander AN, Carey HV: Oral IGF-I enhances nutrient and electrolyte absorption in neonatal piglet intestine. Am J Physiol 277:G619 –G625, 1999 25. Carey HV, Cooke HJ: Effect of hibernation and jejunal bypass on mucosal structure and function. Am J Physiol 261:G37–G44, 1991 26. Carey HV: Seasonal changes in mucosal structure and function in ground squirrel intestine. Am J Physiol 259:R385–R392, 1990 27. Peterson C, Ney D, Hinton P, et al: Beneficial effects of insulinlike growth factor i on epithelial structure and function in parenterally fed rat jejunum. Gastroenterology 111:1501–1508, 1996 28. Peterson CA, Carey HV, Hinton PL, et al: GH elevates serum IGF-I levels but does not alter mucosal atrophy in parenterally fed rats. Am J Physiol 272:G1100 –G1108, 1997 29. Madara JL, Trier JS: Structure and permeability of goblet cell tight junctions in rat small intestine. J Membr Biol 66:145–157, 1982 30. Grass G, Sweetana S: In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharmaceut Res 6:372–376, 1988 31. Bjarnason I, MacPherson A, Hollander D: Intestinal permeability: An overview. Gastroenterology 108:1566 –1581, 1995

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32. Klein RM, McKenzie JC: The role of cell renewal in the ontogeny of the intestine. II. Regulation of cell proliferation in adult, fetal, and neonatal intestine. J Pediatr Gastroenterol Nutr 2:204 –228, 1983 33. Dignass A, Lynch-Devaney K, Kindon H, et al: Trefoil peptides promote epithelial migration through a transforming growth factor ␤-independent pathway. J Clin Invest 94:376 –383, 1994 34. Boismenu R, Havran WL: Modulation of epithelial cell growth by intraepithelial ␥␦ T cells. Science 266:1253–1255, 1994 35. Chen Y, Chou K, Fuchs E, et al: Protection of the intestinal mucosa by intraepithelial ␥␦ T cells. Proc Natl Acad Sci USA 99:14338 –14343, 2002 36. Housley R, Morris C, Boyle W, et al: Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J Clin Invest 94:1764–1777, 1994 37. Chailler P, Basque JR, Corriveau L, et al: Functional characterization of the keratinocyte growth factor system in human fetal gastrointestinal tract. Pediatr Res 48:504 –510, 2000 38. Simmons J, Pucilowska J, Lund P: Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors. Am J Physiol 39:G817–G827, 1999 39. Farrell CL, Bready JV, Rex KL, et al: Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality. Cancer Res 58:933–939, 1998 40. Byrne FR, Farrell CL, Aranda R, et al: rHuKGF ameliorates symptoms in DSS and CD4⫹CD45RBHi T cell transfer mouse models of inflammatory bowel disease. Am J Physiol 282:G690 – G701, 2002 41. Zeeh JM, Procaccino F, Hoffmann P, et al: Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats. Gastroenterology 110:1077–1083, 1996 42. Fernandez-Estivariz C, Gu LH, Gu L, et al: Trefoil peptide expression and goblet cell number in rat intestine: Effects of KGF and fasting/refeeding. Am J Physiol Regul Integr Comp Physiol 284:R569 –R573, 2002

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43. Estivariz CF, Jonal CR, Gu LH, et al: Gut-trophic effects of keratinocyte growth factor in rat small intestine and colon during enteral refeeding. JPEN 22:259 –267, 1998 44. Jonas CR, Estivariz CF, Jones DP, et al: Keratinocyte growth factor enhances glutathione redox state in rat intestinal mucosa during nutritional repletion. J Nutr 129:1278 –1284, 1999 45. Saunders PR, Kosecka U, McKay DM, et al: Acute stressors stimulate ion secretion and increase epithelial permeability in rat intestine. Am J Physiol 267:G794 –G799, 1994 46. Wolvekamp MC, Durante NM, Meyssen MA, et al: The value of in vivo electrophysiological measurements for monitoring functional adaptation after massive small bowel resection in the rat. Gut 34:637– 642, 1993 47. Aranow JS, Fink MP: Determinants of intestinal barrier failure in critical illness. Br J Anaesth 77:71– 81, 1996 48. Schulzke JD, Fromm M, Bentzel CJ, et al: Ion transport in the experimental short bowel syndrome of the rat. Gastroenterology 102:497–504, 1992 49. O’Brien DP, Nelson LA, Kemp CJ, et al: Intestinal permeability and bacterial translocation are uncoupled after small bowel resection. J Pediatr Surg 37:390 –394, 2002 50. O’Brien DP, Nelson LA, Stern LE, et al: Epithelial permeability is not increased in rats following small bowel resection. J Surg Res 97:65–70, 2001 51. Freeman HJ, Ellis ST, Johnston GA, et al: Sodium-dependent D-glucose transport after proximal small intestinal resection in rat. Am J Physiol 255:G292–G297, 1988 52. Estivariz CF, Gu LH, Scully S, et al: Regulation of keratinocyte growth factor (KGF) and KGF receptor mRNAs by nutrient intake and KGF administration in rat intestine. Dig Dis Sci 45:736 –743, 2000 53. Chandrasekaran C, Coopersmith CM, Gordon JI: Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators. J Biol Chem 271:28414–28421, 1996

Discussant Thomas Ziegler: It’s a real pleasure to have the opportunity to discuss this paper from Drs Yang, Wildhaber, and Teitelbaum. It’s another in a series of well-designed and comprehensive studies in a very difficult model of short gut. As shown by Dr Yang, they use state-of-the-art methods for the study of the intestine, looking at gene expression in different portions of the crypt-villus axis. We will be seeing a number of studies using these techniques in the future. As pointed out by Dr Yang in their paper, the ultimate success of gut adaptation after enterectomy depends on the efficiency of nutrient transport across the shortened bowel over time. There’s lots of data in animal models showing a gut growth response, but ultimately the important endpoint is nutrient transport function. Dr Yang confirms the work of other investigators who used different models of bowel resection. Despite a robust growth response, there is downregulation in nutrient transport, at least in this early phase after resection. That’s a very important point with regard to clinical models of short gut, especially in terms of serial changes in gut absorption over time. The drop in nutrient absorption shortly after surgery represents a therapeutic opportunity. One of the therapies they’ve used here is the administration of KGF in an attempt to improve this downregulated absorptive function that occurs after resection. As Dr Yang pointed out, stromal cells in the gut and many other tissues synthesize KGF. It’s unique because it has specific activity in ECs, where it stimulates cell proliferation and differentiation and inhibits apopto-

sis. Others as well as ourselves have shown that KGF has effects in various models, including short bowel syndrome, inflammatory bowel disease, ischemia reperfusion, and chemotherapy. This is a very interesting growth factor, but until now, no one has investigated the effects of KGF on gut epithelial transport function. Dr Yang has also presented data on bacterial translocation and gut permeability. Previously published data in animal models are quite mixed. This paper adds to the data and confirms the work of others, suggesting that there is no major effect of small bowel resection on permeability and that KGF has no effects on permeability. The interesting data on mRNA expression, particularly along the crypt and lower villus, suggests that the KGF action pathway in these regions may be involved in the endogenous gut growth response to resection. One of the mechanisms by which KGF does have these effects may be indicated by upregulation of the KGF action pathway. After that long preamble, several questions come to mind. An identical study in KGF knockout mice or ␥␦ T-cell knockout mice would help establish the role of KGF action on the postresection gut growth and function. So first, do the authors have plans to use such models? Second, it would also be of interest to evaluate colonic growth in transport function, because we know clinically that the colon residual colonic function is very critical in terms of the adaptive absorptive response. Do the authors have any data on colonic growth or function in this small bowel resection model? Third, the authors measured KGF receptor messenger as one

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index of the KGF action pathway. Do they have any data on KGF receptor protein or KGF itself? Fourth, given the importance of food intake on both growth and transport function in this model, do the authors have any data on food intake in their mice? If not, then it may be of interest to perform subsequent studies using a pair-feeding regimen. Fifth and last, given the effects of KGF both on apoptosis and on regulation of goblet cells and trefoil factors, do they have any data on that in this short bowel model? Hua Yang: Thank you, Dr Ziegler. Regarding the larger

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issues of knockout mice, that’s a fantastic question. We haven’t done it. Second, we did give KGF to rule out an independent effect from the KGF administration. That’s a very good point. Third, to investigate the role of intraepithelial lymphocyte-derived KGF on epithelial proliferation, we have that data in the short bowel syndrome model. Fourth, regarding the colon, we do not have this data. But transport function would be a useful thing to evaluate in the colon. Last, KGF should have the effect of stimulating GSH and maybe epithelial proliferation. Thank you.