Region-specific adaptation of apical Na/H exchangers after
extensive proximal small bowel resection
Mark W.
Musch1,
Cres
Bookstein1,
Flavio
Rocha1,
Alvaro
Lucioni1,
Hongyu
Ren1,
Janet
Daniel1,
Yue
Xie1,
Rebecca L.
McSwine1,
Mrinalini C.
Rao2,
John
Alverdy3, and
Eugene B.
Chang1
1 The Martin Boyer Laboratories,
3 Department of Surgery, University of Chicago,
Chicago 60637; and 2 Department of Physiology and
Biophysics, University of Illinois at Chicago, Chicago, Illinois
60612
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ABSTRACT |
After massive small bowel resection
(MSBR), the remnant small intestine adapts to restore Na absorptive
function. The possibility that this occurs through increases in
cellular Na absorptive capacity was examined by assessing the regional
effects of 50% proximal MSBR on the function and expression of the
apical membrane Na/H exchangers (NHEs) NHE2 and NHE3. Morphometric
analysis confirmed adaptive changes consistent with villus hypertrophy,
particularly distal to the anastomosis. Villus epithelium prepared by
light mucosal scrapings from 2-wk-postresected and -posttransected
control rats exhibited comparable brush-border hydrolase activities,
total cell protein per DNA, and villin expression but increased
basolateral Na-K-ATPase activity. Parallel increases of two- to
threefold in protein and mRNA abundance of NHE2 and NHE3 were observed
only in ileal regions distal to the anastomosis of resected rats.
Basolateral NHE1 expression was unchanged. After 80% resection,
increases in NHE2 and NHE3 became evident in proximal colon. We
conclude that increased enterocyte expression and function of apical
membrane NHEs in regions distal to the anastomosis play a role in the
adaptive process after MSBR. The increased luminal Na load to distal
bowel regions after proximal resection may stimulate increases in
apical membrane NHE gene transcription and protein expression.
sodium transport; sodium/hydrogen exchange; intestinal surgery; intestinal adaptation; diarrhea; malabsorption; epithelial cell; intestinal physiology
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INTRODUCTION |
SODIUM/HYDROGEN
EXCHANGERS (NHEs) comprise a family of highly related proteins
that mediate the electroneutral 1:1 exchange for extracellular Na with
intracellular H (1, 5-7, 11, 24, 46). However, the
various NHE isoforms of the family exhibit considerable cell- and
tissue-specific differences in their expression and regulation of
function. The mammalian small and large intestine, for instance,
express three NHE isoforms. NHE1 is present in virtually all cells but,
in epithelial cells, is selectively expressed in the basolateral
membrane where it is believed to have a role in maintaining pH and
volume homeostasis. NHE2 and NHE3, in contrast, exhibit
epithelial-specific expression and are predominantly found in the
apical membrane where they are believed to have an important role in
non-nutrient-dependent, vectorial absorption of Na. These two apical
isoforms differ in a number of ways. Although both are
amiloride-inhibitable, NHE2 is significantly more sensitive to
pharmacological inhibition by HOE-642 than NHE3, a functional property
used to distinguish them (24). NHE3 expression, in contrast to NHE2, is also sensitive to hormonal and metabolic signals.
Glucocorticoids, for instance, increase ileal NHE3 expression (6,
41, 46), whereas mineralocorticoids or hypokalemia increase NHE3
in rat proximal colon (5, 41). Although NHE2 expression is
ontologically regulated (7), few humoral or luminal signals have not been identified that regulate NHE2 expression in the
adult intestine except for epidermal growth factor (EGF; see Ref.
47).
After massive small bowel resection (MSBR), the remnant small
intestinal mucosa rapidly adapts to restore normal nutrient, water, and
electrolyte absorptive capacity (11, 29, 32, 34, 43-45).
Alterations in the smooth muscle have also been noted after bowel
resection (39). In addition, a number of studies have
demonstrated alterations in monosaccharide transporters after bowel
resection (1, 19, 36, 42). Increased overall electrolyte absorption has also been observed, which has been attributed to increases in mucosal mass and surface area (9, 25).
However, these results were based on extrapolated multipliers from
morphometric data, which are imprecise. Although a major component of
this adaptive process does involve villus hypertrophy (increase in villus length) and increased absorptive surface area, the possibility that increased cellular Na absorptive capacity contributed to this
process remains controversial (2, 27, 43-45).
Because a large portion of intestinal Na absorption is mediated by the
apical NHEs NHE2 and NHE3 (24, 46), we examined this
possibility by assessing the regional effects of 50% proximal small
bowel resection on their function and expression. It should be noted
that Sacks et al. (32) demonstrated increased brush-border membrane NHE activity in weanling rats after bowel resection but did
not determine at that time whether it was because of NHE2 or NHE3. More
recently, Falcone et al. (11) have demonstrated increased
NHE3 mRNA and protein levels in mice that had MSBR. No differences in
NHE2 were observed, and changes in NHE1 were not investigated. However,
this study did not determine whether these changes were a result of
mucosal hypertrophy (increase in villus length), cellular adaptation
(increased NHE activity/cell), or increased apical NHE expression along
the villus-crypt axis (recruitment). In this study, we report increased
expression and function of these apical membrane NHEs per villus cell
in regions downstream of the anastomosis as one mechanism of intestinal
adaptation after MSBR. The extent of downstream cellular adaptation was
proportional to the extent of MSBR. These conclusions were based on
data normalized to functional and biochemical parameters found not to
be changed in villus cell preparations of either resected or transected
animals. Moreover, we found no evidence of apical NHE recruitment along the villus-crypt axis.
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MATERIALS AND METHODS |
Surgical resection of the proximal 50% of rat small intestine.
All procedures were approved by the Institutional Animal Care Use
Committee of the University of Chicago. Rats (200-255 g) were
anesthetized, and a 40-cm segment of the small bowel was removed
beginning 15 cm distal to the ligament of Treitz. Great care was taken
to maintain blood supply to the remnant bowel. To control for the
effects of surgical manipulation of the bowel, paired rats underwent
intestinal transection and anastomosis at 15 cm distal to the ligament
of Treitz. The weight of the rats was monitored every day for the first
4 days and subsequently every 2 days. For the first 2 days, most rats
lost 8-12 g of their body weight, probably from postoperative
fluid losses and diminished oral intake. Thereafter, all rats began to
consume chow at comparable levels (20-25 g/day) and gained weight
in a similar fashion (5-7 g/day). With the use of the weight data
from a number of groups (since only certain experimental data could be
analyzed in consideration of the length of the region of the small
intestine proximal to the anastomosis), the presurigical weights were
244 ± 5 and 246 ± 7 in the transected and resected groups,
and 14 days after surgery the weights were 308 ± 14 and 301 ± 17 in the respective groups (n = 12). After 14 days,
the rats were killed, and the entire length of the small intestine was
measured, generally 85-95 cm in transected rats and 37-42 cm
in resected rats. The ligament of Treitz was noted, and 8-cm sections
of the small intestine were taken beginning either 5 cm proximal or 15 cm distal to the anastomosis (i.e., away from the anastomosis). A
section of the proximal large intestine was taken just after the cecum.
The sections of the intestine were processed as appropriate for the analysis.
For analysis of sucrase, villin, NHE2, and NHE3, brush-border membranes
were isolated from villus epithelial samples prepared from light
scrapings of the intestinal mucosa using glass slides (6).
Shearing at the villus-crypt junction by this technique was confirmed
by histological staining of the remaining gut segment from transected
and resected animals (see Fig. 1). In
using this approach, direct comparisons of the villus cell compartment
between resected and transected animals could be compared directly
without confounding variables such as admixture of crypt, muscle, or
mesenchymal cells. It is important to note that, when the epithelial
cell-specific protein villin was analyzed in homogenates from the
scrapings, the amount of villin expressed per microgram protein was not
different in the corresponding section of the transected vs. the
resected rats, suggesting a similar proportion of villus cells. Because DNA content per cell can be used to estimate cell number (assuming that
the amount of DNA/cell does not change between conditions), these data
suggest that the protein content per cell was not significantly different between the resected and transected animals, i.e., there was
no evidence for cellular hypertrophy (increased cell protein expression
while the cell remains the same size). Cellular hypertrophy can also be
interpreted to mean increased cell size. In this case, one may not
predict the amount of protein expressed per cell, and this information
must still be determined by measuring cell protein and then preferably
cell number (which is not possible in cells such as intestinal
enterocytes, which tend to clump) or by an indirect measure of cell
number (such as DNA). This does not discount the development of villus
hypertrophy (lengthening of villi), where there is an increased number
of enterocytes per villi, but argues against a generalized increase in
protein content of individual enterocytes. As will be discussed later,
no differences in villin content, brush-border hydrolase-specific
activities, and basolateral NHE1 expression were observed between
villus preparations of resected and transected animals. These data
strongly suggested the comparability of villus cell preparations of the
two groups, making any observed differences in NHE2 and NHE3 meaningful
and reflective of specific cellular adaptive changes. Additionally, any
increases observed in Na transport function would then be considered
somewhat specific, since we know that other transporters (e.g.,
Na-K-ATPase) are also increased by proximal bowel resection.

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Fig. 1.
Histological confirmation of villus cell population removed by
light scraping in rats that underwent transection and resection. The
small intestine was removed, and a section was removed for fixation in
formalin and hematoxylin and eosin staining. A section immediately
adjacent was lightly scraped, and the remaining mucosa and muscle were
fixed and also processed for staining. The images were obtained on a
Leitz microscope using Pixar software. The images shown are
representative of those on 3 occasions.
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Tissues for morphometric analysis were fixed in neutral buffered
formalin and stained with hematoxylin and eosin by the surgical pathology laboratories of the University of Chicago Hospitals. Morphometric analyses were collected from fixed sections of three separate rats, and the averages from each animal (n = 3 from each animal were first averaged) were used to determine the means
and SE. Protein was measured in the light mucosal scrapings by the bicinchonininc acid procedure (35) and DNA using
Hoechst 33258 reagent (4).
For immunohistochemical analysis of NHE2, paraffin sections were heated
to 65°C for 30 min, paraffin was removed by xylene, and slides were
rehydrated through graded ethanol washes. After a saline wash, antigen
recapture was performed by microwaving in 0.1 M citrate (pH 6.0) four
times. Slides were then treated using the Vector Elite staining
procedure (Vector Laboratories, Burlingame, CA). Nonspecific binding
was blocked using goat serum and both avidin and biotin blocking
solutions. Slides were incubated overnight with a 1:50 dilution of
affinity-purified anti-NHE2 polyclonal antiserum that was developed and
characterized by our laboratory (24). Slides were washed
and developed with the ABC Elite Vector kit.
Brush-border membranes were prepared as previously described
(6). A sample of both the crude homogenate and the
purified brush-border membranes was saved for the analysis of sucrase, alkaline phosphatase, and villin to determine enrichment factors. Sucrase and alkaline phosphatase were measured as previously described (5, 6), and villin was determined by Western blotting
using a specific monoclonal antibody (Transduction Laboratories,
Lexington, KY). An aliquot of the basolateral membranes was saved for
analysis of K-stimulated phosphatase activity. K-stimulated phosphatase was measured as previously described (15) in crude
homogenates and the basolateral membrane fraction, which also contained
endoplasmic reticulum and Golgi.
In a limited number of experiments, 80% of the small intestine was
resected where 60 cm of intestine was removed compared with 40 cm for
the 50% resections. These rats lost weight for the first 7 days
(generally 40-50 g of 225 g body wt) but by day 10 were gaining 5-7 g/day and eating chow at a comparable rate to
50% resected rats. For these studies, only NHE activities and protein
levels were measured.
Measurement of intestinal NHE2 and NHE3 activities.
The activity of NHE2 and NHE3 in the brush-border membranes was
measured as previously described (6). For the present
experiments, the 22Na uptakes were always performed with
both HOE-694 (30 µM) and dimethylamiloride (DMA, 500 µM) so that
NHE2 and NHE3 activities could be distinguished. Both exchangers are
sensitive to DMA. At 1 mM Na, NHE2 is completely inhibited by the HOE
amiloride analog at 30 µM, whereas NHE3 is inhibited <5%. Thus the
former was defined as the HOE-694-sensitive and the latter as the
HOE-694-insensitive component of the DMA-inhibitable unidirectional
22Na influx. Fluxes were measured under acid-loaded
conditions as previously described (6).
Measurement of NHE2 and NHE3 protein expression.
An aliquot of the brush-border membranes was analyzed using Western
blots. The brush-border proteins were solubilized in Laemmli stop
solution and resolved on a 7.5% SDS-PAGE. Proteins were immediately transferred to a polyvinylidene difluoride membrane and blocked with
5% wt/vol nonfat milk in Blotto [composition in mmol/l: 150 NaCl, 5 KCl, and 10 Tris (pH 7.4) with 0.05% Tween 20]. Blots were incubated
with specific polyclonal antisera developed and characterized in our
laboratory to NHE2 and NHE3 (1, 24). Blots were visualized
using an enhanced chemiluminescence system. Because NHE1 is a
basolateral and not a brush-border membrane protein (1), a
different membrane fraction was used. Scraped mucosa was homogenized
and spun at 500 g (5 min at 4°C) to remove nuclei and
unbroken cells. The supernatant was spun at 10,000 g (10 min
at 4°C) to pellet mitochondria, and the resulting supernatant was
spun at 100,000 g (20 min at 4°C) to obtain a membrane
fraction that contained the plasma membranes (both apical and
basolateral) and the endoplasmic reticulum and Golgi. Because of the
less purified nature of these membranes, 50 µg were generally
analyzed for Western blots.
Quantitation of NHE mRNA expression.
In some experiments, the intestinal scrapings were immediately placed
into Trizol and homogenized using an Ultra-Turrax at maximum speed for
20 s. RNA was isolated from Trizol according to the
manufacturer's instructions and extracted one additional time using
acid phenol-chloroform to remove any remaining DNA and protein.
Immediately before analysis, the RNA was repelleted and quantitated by
the absorbance at 260 nm. RNA (20 µg) was size-separated on a
formaldehyde-denaturing agarose gel using a MOPS buffer system described previously (1). The RNA was transferred to a
positively charged nylon membrane overnight, and the RNA was
cross-linked to the membrane by ultraviolet irradiation. The blots were
analyzed for NHE2, NHE3, and the constitutive probe
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using an XOTCH
hybridization solution (1). For NHE2, the probe consisted
of a section of coding region (bases 1564-2540), and, for NHE3, a
full-length probe of the coding region was used. The cDNA probes used
for NHE2 and NHE3 have been validated previously by our laboratory to
be specific for their respective isoform (1). GAPDH mRNA
abundance was used to normalize data, using a full-length murine GAPDH
cDNA probe obtained from American Tissue Culture Collection. Blots were
always hybridized overnight and washed up to a stringency of 0.5×
saline-sodium citrate and 0.5% SDS at 55°C.
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RESULTS |
Regional brush-border hydrolase and villin expression after
resection or transection.
Because MSBR is reported to cause hypertrophy of the intestine at and
around the anastomotic area (29, 30), samples were always
taken well away from this area and from the same position in all rats.
Morphometric analysis revealed adaptive changes similar to those
reported previously by other groups (43, 45),
characterized by villus hypertrophy distal to the anastomosis (see
Table 1). To further characterize the
adaptive changes, length and weight of the intact intestine and protein
and DNA of the mucosal scrapings (a predominantly villus cell
preparation) were analyzed. As shown in Table
2, there were no changes in length per
weight or protein per DNA in the proximal small intestine or in the
colon from rats that underwent 50% MSBR. In the small intestine distal
to the anastomosis, there was a small increase in the weight/length
that was not statistically significant. Accurate cell counts could not
be determined, since intestinal enterocytes scraped off as sheets tend
to clump. Therefore, DNA was measured in the homogenates to assess cell
number. As shown in Table 2, both protein and DNA per length increased
in the portion of the remnant small intestine corresponding to the
ileum (Table 2). No difference was observed in the protein-to-DNA ratio
from villus specimens harvested from corresponding segments (jejunum,
ileum, and proximal colon). Because cellular hypertrophy would be
associated with an increased protein-to-DNA ratio, these data argue
against this possibility. On the other hand, the results presented in
Table 1 (increased villus length) and Table 2 (increased protein and
DNA/length of distal small intestine remnant) are consistent with the
mucosal hypertrophy, particularly in villus regions.
Contributions caused by mucosal hypertrophy, such as altered numbers of
crypt cells, smooth muscle, and other stromal elements, increased
mucosal surface area, and changes in bowel length can be eliminated by
performing subsequent analyses only on villus cell preparations.
Observed changes in apical NHE function or activity will therefore
reflect specific changes in intestinal epithelial (villus cell)
adaptation and not increases in total function resulting from
generalized mucosal hypertrophy. Activities of the brush-border enzymes
sucrase and alkaline phosphatase and the protein expression of the
microvillus protein villin were then determined in crude homogenates
and purified brush borders prepared from ileum of both transected and
resected rats. When these results are normalized to milligram protein,
no differences in the specific activities of sucrase or alkaline
phosphatase activities of villus scrapings were observed in any regions
of the small bowel of either group. Of note, the degrees of enrichment of brush-border membranes prepared from both groups were similar (presented below each brush-border membrane value for alkaline phosphatase, sucrase, or villin in Fig.
2). The only enzyme marker that changed
significantly was Na-K-ATPase, a basolateral protein, which occurred
only in sections distal to the anastomosis of resected animals. These
data demonstrate the comparability of brush-border membrane
preparations between the two experimental groups. Thus the observed
changes corroborate many of those previously reported and indicate that
intestinal adaptation did indeed take place after MSBR. However, when
villus specimens prepared from light mucosal scrapings from the two
groups were compared, no differences in the specific activities of
epithelial cell markers (brush-border hydrolase, villin expression)
could be observed (Fig. 2C).

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Fig. 2.
Analysis of alkaline phosphatase, sucrase, villin, and Na-K-ATPase
from mucosal scrapings of 50% resected (R) vs. transected (T) rats.
Alkaline phosphatase, sucrase, and villin were analyzed in brush-border
membranes and Na-K-ATPase in basolateral membranes and crude homogenate
analysis for all as described in MATERIALS AND METHODS.
Values are means ± SE for 3 separate experiments. Enrichment
values for each group are presented underneath and represent means ± SE of increased enzyme activity (or protein level for villin) in
brush-border membranes [BBM; basolateral membranes (BLM) for
Na-K-ATPase] divided by activities in crude homogenates. JEJ, jejunum;
IL, ileum. *P < 0.05 compared with comparable segment
by paired Student's t-test.
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MSBR selectively increases brush-border membrane NHE2 and NHE3
activities and expression distal to the anastomosis.
To determine the effects of bowel resection on regional intestinal
mucosal NHE2 or NHE3 activity, measurements of brush-border NHE
activity were performed in brush-border membrane vesicles under
conditions of a transmembrane acid gradient. Extravesicular and
intravesicular pH values were maintained at 7.4 and 6.1, respectively. Fluxes were measured as previously described, where NHE2 and NHE3 activities were defined as the HOE-694-sensitive and -insensitive components of DMA-inhibitable 22Na influx. Resection of the
proximal 50% of bowel caused a large increase in the activities of
both NHE2 (Fig. 3A) and NHE3 (Fig. 3B) in the distal portion of
the remnant small intestine. NHE2 activity increased 92 ± 11%,
whereas NHE3 activity increased 89 ± 14%. These changes occurred
in distal ileal segments, since great care was taken to avoid inclusion
of segments immediately distal to the anastomosis (i.e., samples were
always taken 5 cm away from the anastomosis). Similarly, the proximal
small intestinal mucosal samples excluded regions immediately adjacent
to the anastomosis. No changes in NHE2 or NHE3 activities were observed
in the most proximal portion of the small intestine or in the colon.

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Fig. 3.
Na/H exchange (NHE) activities in brush borders from 50% resected
and transected rats. 22Na uptakes were measured as
described in MATERIALS AND METHODS under maximally
stimulating conditions. 22Na influx for NHE2 was defined as
that flux inhibitable by 30 µM HOE-694, whereas NHE3 was
defined as the flux further inhibited by 500 µM
dimethylamiloride. Data shown are means ± SE for 4 rats. For each
rat, each value was determined in triplicate. ++P < 0.001 compared with comparable segment by paired Student's
t-test.
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As shown in Fig. 4, the region-specific
increases in brush-border NHE2 and NHE3 activity were paralleled by
increases in brush-border membrane NHE2 and NHE3 protein expression.
Again, significant changes of greater than twofold were observed only
in the ileal regions distal to the anastomosis (P < 0.05, n = 4, densitometric analysis is shown in Fig. 4,
right). These changes were specific, since the villin
content of loaded samples was essentially no different in corresponding
regional segments of resected and transected groups (Fig. 2).
Furthermore, as mentioned previously, brush-border hydrolase activities
of corresponding segments from resected and transected rats were not
significantly different. In contrast to NHE2 and NHE3, no significant
changes in mucosal NHE1 protein expression were noted, as shown in the
representative blot shown in Fig. 4.

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Fig. 4.
Increase in NHE2 and NHE3 proteins in 50% resected rats.
Brush-border membranes from resected and transected rats were
solubilized and run on SDS-PAGE, and Western blots were generated.
Blots were probed with antibodies to NHE2, NHE3, and villin to
determine equivalence of loading of brush borders. For NHE1,
basolateral membranes were used for Western blots. Images shown for
NHE2 and NHE3 are representative of those of 4 separate experiments,
whereas for NHE1 the image is representative of 2 separate experiments.
+P < 0.01 compared with comparable segment by paired
Student's t-test.
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To determine if proximal resection altered the expression of
apical NHEs along the villus-to-crypt axis (recruitment phenomenon), immunohistochemical analyses using an affinity-purified anti-NHE2 polyclonal antiserum were performed. As can be observed in Fig. 5, NHE2 is specifically expressed at the
luminal and subapical membranes of villus cells but not by crypt cells
of transected and resected rat ileum, i.e., expression increasing
abruptly at the villus-crypt junctions. Because affinity-purified
anti-NHE3 was not available at the time of these studies, NHE3
immunohistochemistry was not performed.

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Fig. 5.
Immunohistochemical analysis of NHE2 in villus and crypt
regions of ileum of transected and resected rats. Paraffin sections
were prepared as described in MATERIALS AND METHODS, and
NHE2 was detected using affinity-purified polyclonal antiserum against
NHE2. Slides were developed using the Vector ABC Elite protocol
followed by copper enhancement. Images shown are representative of
those obtained in 3 resected and 3 transected rats.
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To determine if larger resection of the small intestine would
upregulate NHE2 and NHE3 to a greater extent or more distally, an 80%
bowel resection was performed. Brush-border NHE2 and NHE3 activities
and protein levels increased in the small intestine distal to the
anastomosis. The increases observed in the distal small intestine after
80% resection were slightly but not significantly greater compared
with the increases after 50% resection (increased ileal NHE2 105 ± 32% and NHE3 142 ± 28%, n = 3). However,
NHE2 and NHE3 activities were increased in the proximal colon after 80% resection (Fig. 6), whereas no
increases were observed in this region after 50% resection (increase
in the colon for NHE2 112 ± 52% and NHE3 161 ± 42%,
n = 3). Western blots of brush-border membranes from
rats that had 80% resection are shown in Fig.
7. Increases in both NHE2 and NHE3 were
noted in both ileal (117 ± 33%, n = 3) and
colonic (134 ± 39, n = 3) values in corresponding portions of the resected rats.

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Fig. 6.
NHE activities in brush borders from 80% resected and transected
rats. 22Na uptakes were measured as described in
MATERIALS AND METHODS under maximally stimulating
conditions. 22Na influx for NHE2 was defined as that flux
which was inhibitable by 30 µM HOE-694, whereas NHE3 was defined as
the flux further inhibited by 500 µM dimethylamiloride. Data shown
are means ± SE for 4 rats. For each rat, each value was
determined in triplicate. BBMV, brush-border membrane vesicles.
*P < 0.05 and +P < 0.01 compared with
comparable segment by paired Student's t-test.
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Fig. 7.
Increase in NHE2 and NHE3 proteins in 80% resected rats.
Brush-border membranes from resected and transected rats were
solubilized and run on SDS-PAGE, and Western blots were generated.
Blots were probed with antibodies to NHE2, NHE3, and villin to
determine equivalence of loading of brush borders. Images shown are
representative of those of 4 separate experiments. *P < 0.05 and +P < 0.01 compared with comparable segment
by paired Student's t-test.
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Fifty percent small bowel resection upregulates NHE2 and NHE3 mRNA
abundance.
Northern blots for NHE2 and NHE3 mRNA were performed to determine if
changes corresponding to altered protein and activity of the respective
isoforms occurred. As shown in Fig. 8,
the mRNA for both NHE2 and NHE3 increased only in the distal portion of the remnant small intestine, corresponding to the region where activity
and protein expression of these isoforms were observed. However,
densitometric quantitation of these changes revealed a slightly greater
degree of increase in NHE2 and NHE3 mRNA abundance relative to NHE3
protein or activity (NHE2 mRNA increased 190 ± 37% and NHE3 mRNA
increased 182 ± 40%). These data thus suggest that increased
mRNA, possibly through transcription activation of the NHE2 and NHE3
genes, plays a role in the NHE adaptive process after MSBR.

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Fig. 8.
Increase in NHE2 and NHE3 mRNA in 50% resected rats. Total RNA was
isolated from the three segments of resected or transected rats, and 20 µg were used for Northern blot analysis. Blots were probed for NHE2,
NHE3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a
constitutive marker. Image shown is representative of those of 3 separate occasions. +P < 0.01 and ++P < 0.001 compared with comparable segment by paired Student's
t-test.
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DISCUSSION |
The gastrointestinal tract has a remarkable adaptive capacity to
restore digestive and absorptive functions after injury or MSBR. This
process is in part mediated by increased mucosal hypertrophy, typified
by greater villus height and size and enlargement of crypt depth
(2, 27, 43, 44). In addition, studies have reported
increases in luminal circumference, size of intestinal folds, and cells
per unit length of bowel. All these findings would contribute to an
overall increase in absorptive surface area and capacity (2, 27,
44). However, controversy continues as to whether specific
cellular functions for nutrient and electrolyte transport are
upregulated as an additional mechanism of mucosal adaptation after
intestinal resection. Several studies have suggested that certain
cellular function may in fact be decreased despite an overall increase
in intestinal absorptive capacity. For instance, one study reported
decreased glucose and amino acid transport when data were normalized to
intestinal dry weight (25). Brush-border disaccharidase
abundance and activity have also been noted to be variably changed
after resection because of the normalization protocol used (3,
21). It has been speculated that the decrease in overall villus
cell function is a consequence of a hyperproliferative response of the
mucosa after resection, resulting in a greater proportion of relatively
less well-differentiated villus enterocytes, and this may relate to the
expression of proteins considered to be markers of the mature enterocyte.
Our studies, as well as those of others, would suggest that this
hypothesis that resection leads to enterocytes with diminished function
is incorrect. The present studies demonstrate upregulation of the two
apical exchangers required for small intestinal Na absorption. Several
investigators have reported increased cellular uptake of glucose in the
remnant small bowel, believed to be because of a specific upregulation
of glucose transporters, namely the apical Na-dependent transporter
SGLT1 (14) and the basolateral glucose transporter GLUT2
(36). In fact, the upregulation of GLUT2 has been
hypothesized to serve a dual function, facilitating glucose absorption
and enhancing the ability of the enterocyte to obtain glucose from the
blood. Furthermore, the specific activity of the Na-K-ATPase pump in
the remnant ileum after small bowel resection is significantly
increased compared with Mg-dependent ATPase, which was essentially
unchanged (40). This observation was later confirmed by
Hines et al. (18) and the present studies.
We believe that the apparent discrepancies in defining a role of the
enterocyte in intestinal adaptation may be attributed to differences in
methodology and experimental conditions. For instance, considerable
differences exist among studies attributable in part to the way data
were normalized. In some cases, conclusions were based on rough
extrapolations or estimates of highly variable reference data,
including measurements of crypt depth, villus length, and
circumference. Data outcome can also be influenced by timing of
observations after resection, choice of controls, and differences in
the types or length of intestinal resection. For these reasons, we took
great care to define experimental conditions to provide a definitive
answer regarding the effects of proximal intestinal resection on
enterocyte expression and function of apical Na/H exchange. Because of
the known effects of surgical manipulation of bowel on intestinal
mucosal function, rats undergoing intestinal transection were felt to
be the most appropriate controls for assessing changes after intestinal
resection. We also confined our studies to villus preparations made
from mucosal scrapings, a technique that is commonly employed to yield
a predominant villus cell harvest. Using this approach, we documented
that the enrichment of brush-border membrane preparations was identical
between resected and transected intestinal preparations and that the
villin content and brush-border hydrolase activities of brush-border
membranes from both groups were equivalent. Thus these findings provide justification for dependable comparative analysis of mucosal NHE function and expression between these two groups.
Our studies demonstrate several important findings. First, they show
that part of the intestinal adaptation after MSBR does involve
selective and region-specific upregulation of enterocyte NHE3 and NHE2,
but not NHE1, function and expression. No concomitant changes in
brush-border hydrolase, villin expression, or DNA or protein content
were noted. The increases in activity and protein levels for NHE2 and
NHE3 agreed well; however, larger changes in mRNA levels were observed
for both NHE isoforms. The changes in mRNA suggest that increased
enterocyte NHE2 and NHE3 result from increased gene activation, but the
possibility that additional posttranscriptional mechanisms are involved
in regulating NHE3 expression cannot be ruled out.
The immunohistochemical analysis of NHE2 expression along the
crypt-villus axis also argues against recruitment of NHE2-expressing enterocytes in crypt regions. Such a mechanism has been suggested for
the increased SGLT1 expression observed in chronically diabetic rats
that demonstrate villus hypertrophy (12). Our studies fail to show a similar phenomenon for apical NHEs in MSBR. Moreover, these
changes occurred in the absence of changes in specific activities of
brush-border sucrase and alkaline phosphatase, NHE1 expression, and
villin content. We therefore feel that the specific upregulation of
cellular apical NHE expression represents one form of intestinal adaptation after MSBR.
Previous studies in weanling rats have noted increased NHE activity in
the small intestine after bowel resection (32), and more
recent studies using a mouse model noted increased NHE3 protein and
mRNA without a change in NHE2 (11). Neither of these
studies investigated changes in the colon, which our studies would
suggest do not occur until >50% resection. After 50% resection, it
is unknown why the mouse did not upregulate NHE2 as we observed in the
rat. The differences could be species dependent and may relate to the
length of the bowel, the precise nature and placement of the resection,
or differences in hormonal regulation of NHE2 between the species. EGF
has been demonstrated to be a potent modulator of the response to
resection and in the rat potently upregulates NHE2 (47).
The studies in the mouse model did not report a detailed investigation
of morphological or biochemical changes; thus, our studies may be
useful, since NHE upregulation may be viewed as specific and since
other brush-border enzymes like sucrase and alkaline phosphatase and
the structural protein villin did not change. Our studies suggest that
there is a specific upregulation of apical NHE activity per cell (since
brush-border protein was used as a denominator) and also that
intestinal Na absorptive capacity is increased because of an increase
in the numbers of villus enterocytes in the distal remnant of the small
intestine (because of the increase in villus length).
The region-specific increases in apical membrane NHE expression and
function after resection are particularly noteworthy and deserve
comment. Increases were only observed in ileal segments distal to and
distant from the site of anastomosis. The latter is an important
distinction to make, since it is well known that persistent functional
changes occur in mucosal regions at the site of anastomosis of
transected or resected bowel. The increases we observed are therefore
not part of this phenomenon. The increases in ileal NHE2 and NHE3
function and expression are also not characteristic of
hyperaldosteronism, which can occur in response to prolonged volume
depletion. Aldosterone stimulates increased NHE3 expression only in the
proximal colon (5, 41) and has no effects on intestinal
NHE2. Furthermore, serum aldosterone levels were measured in both
transected and resected animals and were not found to be different
(data not shown). Likewise, chronic increases in serum glucocorticoids
can stimulate increases in ileal NHE3 expression but have no effects on
intestinal NHE2. Thus these changes are not likely to be secondary to
increased corticosteroid stimulation. In fact, intestinal NHE2
expression and function have been remarkably insensitive to most
physiological perturbations and have only been reported to change
during ontogeny (7). Therefore, the stimuli causing these
postresection changes in NHE2 appear to be unique, albeit not
understood presently.
One possibility for causing this phenomenon is the increased luminal Na
load to the distal segment resulting from massive proximal bowel
resection serving as a luminal cue. More distal, i.e., colonic NHE,
expression did not occur because the ileal adaptation adequately
compensated for the additional luminal Na load. Consistent with this
notion is that, in a few animals that underwent 80% resection,
increases in apical NHE2 and NHE3 were observed more distally, i.e., in
colonic segments distal to the small bowel anastomosis (Fig. 6). These
studies were not pursued because of the increased morbidity and
mortality attended with more extensive bowel resection.
Substrate-induced enhancement of membrane transporters has been
observed previously, most notably for sugar transporters (13, 16,
26, 28, 29, 33, 42); there may be such an effect for electrolyte
transporters. It should be noted that Dowling and Booth
(9) proposed in 1967 that the adaptive hyperfunction
response of the remnant ileum after proximal resection of the jejunum
might be in large part the result of the presence of a nutrient-rich
chyme never before seen by the ileum. The importance of humoral factors
in the upregulation of apical NHEs cannot be dismissed and may also
play an important role. A number of growth factors, including EGF
(10, 17), insulin-like growth factors (22, 48,
49), and glucagon-like peptides (36), have all been
demonstrated to be potent modulators of intestinal enterocyte growth
and gene expression. Additionally, many genes are turned on in the
enterocyte shortly after bowel resection (8, 31). These
genes may be required in the generation of humoral factors involved in
the adaptive response, or these genes may result in changes in
transcription factors involved in the adaptive response. The
nutritional status of the animal may also play a role in the adaptive
response, since modulation of glutamine or short-chain fatty acids in
the diet has also been demonstrated to modulate the adaptive response
(29, 30, 33-34, 36-38, 48). It cannot be
determined from these studies whether these effects are direct or
indirect (e.g., through a stimulated increase of growth factors).
Growth factors could contribute to upregulation of the apical NHEs and
increased basolateral Na-K-ATPase. Our studies, and previous studies
(18), have noted increased Na-K-ATPase after bowel
resection. Because the Na-K-ATPase is required to maintain low cell Na
and therefore is essential for secondary active transporters that use
the Na gradient, we speculate whether increases in total Na pump
activity can induce apical NHE activity and protein expression.
In summary, we believe these data provide strong support for a role of
selective functional adaptation of the enterocyte after MSBR. The
region-specific changes in apical membrane NHE2 and NHE3 are unique and
implicate a role for increased luminal Na or nutrient-rich chyme in
promoting this response in regions distal to the anastomosis. These
changes are likely to be important in allowing the intestinal mucosa to
compensate for decreased absorptive surface area resulting from
proximal resection or disease.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-38510 and DK-47722 (E. B. Chang) and DK-42086, the Gastrointestinal Research Foundation of
Chicago, and a grant from the Crohn's and Colitis Foundation of America.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
E. B. Chang, The Martin Boyer Laboratories, Univ. of
Chicago, MC 6084, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail:
echang{at}medicine.bsd.uchicago.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 3, 2002;10.1152/ajpgi.00528.2001
Received 18 December 2001; accepted in final form 21 June 2002.
 |
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