Am J Physiol Gastrointest Liver Physiol 300: G647–G653, 2011.
First published January 20, 2011; doi:10.1152/ajpgi.00546.2010.
NHE2X3 DKO mice exhibit gender-specific NHE8 compensation
Hua Xu,1 Jing Li,1 Rongji Chen,1 Bo Zhang,1 Chunhui Wang,1,2 Nolan King,1 Huacong Chen,1
and Fayez K. Ghishan1
1
Department of Pediatrics, University of Arizona Health Sciences Center, Tucson, Arizona; and 2Department of
Gastroenterology, West China Hospital, Sichuan University, Chengdu, China
Submitted 15 December 2010; accepted in final form 18 January 2011
intestine; sodium/hydrogen exchanger 8; sex hormone; gender
the small intestine, and this
process is mediated by several transporter families, including
the sodium/hydrogen exchangers (NHEs). The NHEs are
plasma membrane-bound antiporters that mediate the movement of extracellular sodium ions in cells in exchange for
intracellular hydrogen ions. To date, nine mammalian NHEs
have been discovered. These proteins have broad physiological
functions, including intracellular pH homeostasis, cell volume
regulation, acid-base regulation, and electroneutral NaCl transport. Three out of the nine NHEs (NHE2, -3, and -8) are
expressed at the apical membrane of enterocytes (29, 31).
These apically expressed NHEs have distinct transporter kinetic characteristics and expression patterns during development. NHE2 is highly sensitive to the inhibitor HOE-694,
whereas NHE3 is highly sensitive to the inhibitor S-3226, but
NHE8 is sensitive to both inhibitors (8, 28). The expression of
NHE8 in the small intestine is higher at a young age and
declines in adults; in contrast, the expression of NHE2 and
NHE3 is higher after weanling but is lower at the young age (6,
7, 28, 29).
SODIUM ABSORPTION OCCURS IN
Address for reprint requests and other correspondence: F. K. Ghishan, Dept.
of Pediatrics, Steele Children’s Research Center, 1501 N. Campbell Ave.,
Tucson, AZ 85724 (e-mail: [email protected]).
http://www.ajpgi.org
NHE2 is expressed at the apical membrane of the intestinal
epithelia, and, at first, it was thought to play a role in intestinal
sodium absorption. However, interruption of NHE2 function in
mice displays no obvious small intestinal phenotype such as
diarrhea and sodium malabsorption (14, 24). Therefore, NHE2
does not participate in intestinal Na⫹ absorption. In contrast,
NHE3 knockout (KO) mice display diarrhea and impaired
acid-base balance, suggesting an important role for NHE3 in
intestinal and renal Na⫹ absorption (25). Furthermore, NHE3
does not play a compensatory role in the loss of NHE2 KO
mice and vice versa NHE2 cannot compensate for the loss of
NHE3 (14). Because NHE3 KO mice survive to adulthood
regardless of diarrhea and impaired acid-base balance, another
mechanism must compensate for the loss of NHE3. In fact, an
amiloride-sensitive Na⫹ absorption was detected in NHE3KO
mice that contributed to ⬃30% of Na⫹ absorption (11). Because this observation was reported before the discovery of
NHE8, we hypothesized that NHE8 might be the unidentified
amiloride-sensitive NHE activity observed in NHE3 KO mice,
and NHE8 could partially compensate for the loss of NHE3.
In the present study, we created NHE2X3 double-knockout
(DKO) mice to study the possible compensatory role of NHE8
in the small intestine. Our results indicated that NHE8 expression was decreased by ⬃54% in male mice and by ⬃30% in
female mice from 4 to 6 wk of age. In NHE2X3 DKO mice,
NHE8 expression was increased in both male and female mice
at 4 wk of age, but this increase was abolished in 6-wk-old
male mice. Furthermore, testosterone treatment inhibited
NHE8 expression at RNA and protein levels while estradiol
treatment had no effect on NHE8 expression in Caco-2 cells.
These observations suggested that NHE8 would partially compensate for the loss of NHE2 and NHE3, but this compensatory
mechanism was inhibited by testosterone.
MATERIALS AND METHODS
Animals. NHE2X3 DKO were produced by breeding NHE2⫹/⫺/
NHE3⫹/⫺ mice together. To increase the number of NHE2X3 DKO
mice, NHE2⫺/⫺/NHE3⫹/⫺ breeding pairs were also used. Body
weight was recorded at 3, 4, and 6 wk of age. Survival rate was
calculated by recoding the percentage of surviving mice at each age
group. Serum was collected for electrolyte analysis. Jejunal mucosa
scrapings were collected and used for mRNA purification and brushborder membrane vesicle (BBMV) isolation. Electrolyte measurement
was performed by the university animal care pathology services
laboratory (the University of Arizona, Tucson, AZ). All animal work
was approved by the University of Arizona Institutional Animal Care
and Use Committee.
Cell culture. Human intestinal epithelial cells (Caco-2) were purchased from American Type Culture Collection (ATCC, Manassas,
VA) and cultured according to ATCC guidelines. Cells were cultured
at 37°C in a 95% air-5% CO2 atmosphere and passaged every 48 –72
h. For hormone treatment experiments, cells were incubated with 100
0193-1857/11 Copyright © 2011 the American Physiological Society
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Xu H, Li J, Chen R, Zhang B, Wang C, King N, Chen
H, Ghishan FK. NHE2X3 DKO mice exhibit gender-specific
NHE8 compensation. Am J Physiol Gastrointest Liver Physiol
300: G647–G653, 2011. First published January 20, 2011;
doi:10.1152/ajpgi.00546.2010.—NHE8, the newest member of the
sodium/hydrogen exchanger family, is expressed in the epithelial cells
of the intestine and the kidney. Intestinal expression of NHE8 is
significantly higher than that of NHE2 and NHE3 at a young age,
suggesting that NHE8 is an important player for intestinal sodium
absorption during early development. The current study was designed
to explore if NHE8 plays a compensatory role for the loss of NHE2
and NHE3 function in NHE2X3 double-knockout (NHE2X3 DKO)
mice. We further explored the regulatory mechanism(s) responsible
for the change in NHE8 expression in NHE2X3 DKO mice. We found
that ⬎95% of NHE2X3 DKO mice survived through weanling.
However, only 60% of male NHE2X3 DKO mice and 88% of female
NHE2X3 DKO mice survived to 6 wk of life. We also found that the
expression of NHE8 in wild-type female mice was higher compared
with wild-type male mice after puberty. In NHE2X3 KDO mice,
NHE8 expression was increased in females but not in males. Using
Caco-2 cells as a model of the small intestine, we showed that
testosterone inhibited endogenous NHE8 expression by reducing
NHE8 mRNA synthesis, whereas estrogen had no effect on NHE8
expression. Thus our data show for the first time that intestinal NHE8
has a compensatory role in NHE2X3 DKO mice and this regulation is
gender-dependent.
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TESTOSTERONE INHIBITS INTESTINAL NHE8 EXPRESSION
tomaster FB 12; Berthold Detection System, Pforzheim, Germany).
Renilla luciferase activity driven by pRL-CMV (Promega) was used
as an internal control to calculate the relative luciferase activity. To
test the effect of testosterone on human NHE8 promoter activity,
transfected cells were treated with 100 nM testosterone for 18 h before
promoter reporter assay.
Statistical analysis. Student’s t-test was used to compare values of
the experimental data. P values ⬍0.05 were considered significant.
RESULTS
Growth and survival rate in NHE2X3 DKO mice. NHE2X3
DKO mice were produced by breeding NHE2⫹/⫺ ⫻ NHE3⫹/⫺
and NHE2⫺/⫺ ⫻ NHE3⫹/⫺ mice. About two hundred
NHE2X3 DKO mice were collected in this study. Body weight
and survival rate were recorded at the ages of 3, 4, and 6 wk.
Serum sodium level was also measured at the age of 4 and 6
wk. As shown in Fig. 1A, NHE2X3 DKO mice have lower
body weight compared with their wild-type littermates. The
body weight of male NHE2X3 DKO mice was reduced by
⬃22%, ⬃47%, and ⬃30% at 3, 4, and 6 wk of age, respectively, compared with male wild-type littermates. The body
weight of female NHE2X3 DKO mice was reduced by ⬃32%,
⬃45%, and ⬃16% at 3, 4, and 6 wk of age, respectively,
compared with female wild-type littermates. The greatest reduction on gaining body weight occurred between the age of 3
and 4 wk in NHE2X3 DKO mice. At the same time, the
survival rate is higher in the female compared with the male
double-knockout mice. In male NHE2X3 DKO mice, the
survival rate was reduced to 70% at the age of 4 wk and 60%
at the age of 6 wk. In female NHE2X3 DKO mice, the survival
rate was reduced to 92% at the age of 4 wk and 88% at the age
of 6 wk (Fig. 1B). Furthermore, the serum sodium level in
NHE2X3 DKO mice was lower than their wild-type littermates
at 4 and 6 wk of age (Table 1).
Fig. 1. Body weight and survival curve of
NHE2X3 DKO mice. Body weight and survival rate were recorded at the age of 3, 4, and
6 wk. A total of 83 NHE2X3 DKO mice and
128 wild-type mice were used. A: body weight
curve of male and female NHE2X3 DKO
mice. Data are presented as means ⫾ SE for
each age group. WT, wild-type mice; DKO,
NHE2X3 DKO mice. B: survival curve of
male and female NHE2X3 DKO mice. Data
are presented as percentage for each age
group. *P ⬍ 0.05 for NHE2X3 DKO mice vs.
wild type.
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nM estradiol (Sigma-Aldrich, St. Louis, MO) or testosterone (SigmaAldrich) for 18 h. For gene transcription study, cells were pretreated
with actinomycin D (100 nM) for 2 h before the addition of hormones.
Protein purification and western blot analysis. BBMVs were prepared from mouse jejunal mucosa as previously described (29). Total
protein was prepared from cultured Caco-2 cells with RIPA buffer (7).
BBMV protein (30 ␮g) and total protein (40 ␮g) were used for
Western blot. NHE8 antibody was used as a 1:3,000 dilution in these
experiments (29). A 1:5,000 dilution of the ␤-actin antiserum (SigmaAldrich) was used to determine ␤-actin protein abundance. Western
detection was performed with the BM Chemiluminescence Western
Blotting Kit (Roche Diagnostics, Indianapolis, IN). A ratio of NHE8
protein intensity over ␤-actin protein intensity was used for protein
expression quantitation.
RNA purification and PCR analysis. RNA was purified from mouse
jejunal mucosa and Caco-2 cells using Trizol reagent (Invitrogen,
Carlsbad, CA). Total RNA (500 ng) was reverse-transcribed using the
qScript kit (Quanta Biosciences, Gaithersburg, MD), and 10% of the
reverse transcription reaction was used for real-time PCR. TaqMan
technology was used to determine the expression levels of NHE8
using mouse and human NHE8 and TATA-binding protein (TBP)
primers from Applied Biosystems (Foster City, CA). Resulting data
were analyzed using the comparative cycle threshold (Ct) method. The
target gene Ct values are adjusted relative to a calibrator (normalized
Ct value obtained from control groups) and expressed as 2⫺⌬⌬Ct
(Applied Biosystems User Bulletin no. 2: Rev B “Relative Quantitation of Gene Expression”). TBP data were used as an endogenous
reference to normalize expression levels.
Transient transfection and functional promoter analysis. Caco-2
cells were cultured in 24-well plates. When cell density reached
60 –70%, Caco-2 cells were transfected with human NHE8 gene
promoter constructs (pGL3b/⫺89 and pGL3b/⫺671) (27) using Effectene (Qiagen, Valencia, CA) according to the manufacturer’s
instruction. Cells were harvested for promoter reporter assays 40 h
after transfection. Promoter reporter assay was performed using a dual
luciferase assay kit according to the manufacturer’s instruction (Promega). Luciferase activities were measured with a luminometer (Fem-
TESTOSTERONE INHIBITS INTESTINAL NHE8 EXPRESSION
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Table 1. Serum sodium ion concentration*
Age
Mouse
4wk
6wk
Wild type
NHE2X3 DKO
P Value
153 ⫾ 2 (n ⫽ 11)
133 ⫾ 5 (n ⫽ 12)
0.003
161 ⫾ 1 (n ⫽ 6)
138 ⫾ 6 (n ⫽ 6)
0.009
Values are means ⫾ SE; n, no. of mice. Units are mM. NHE2X3 DKO,
sodium/hydrogen exchanger double knockout. *The normal range of serum
sodium ion concentration is 140 –160 mM.
Fig. 3. NHE8 protein expression in wild-type male and female mice. Brushborder membrane vesicles (BBMVs) were isolated from the intestinal mucosa
of male and female mice at the age of 4, 6, and 12 wk. BBMV protein (30 ␮g)
was loaded on SDS-PAGE, and immunoblots were performed. The expression
of NHE8 protein is calculated by the optical density of NHE8 band over that
of the ␤-actin band. Bar chart shows the NHE8 protein expression indicated as
means ⫾ SE in the sum of four independent experiments. *P ⱕ 0.01 for female
mice vs. male mice. Inset: corresponding Western blot images.
(1.00 ⫾ 0.06 in wild-type male mice, 1.10 ⫾ 0.03 in NHE2x3
DKO male mice; n ⫽ 29 mice). In female mice, NHE8 mRNA
abundance was higher in NHE2X3 DKO mice compared with
wild-type mice (1.01 ⫾ 0.04 in wild-type female mice, 1.90 ⫾
0.33 in NHE2X3 DKO female mice; P ⬍ 0.01; n ⫽ 30 mice)
(Fig. 4A). The expression of BBM NHE8 protein was also
significantly increased in both male and female NHE2X3 DKO
mice compared with their wild-type littermates (1.0 ⫾ 0.05 and
1.0 ⫾ 0.10 in wild-type males and females, 1.5 ⫾ 0.10 and
1.75 ⫾ 0.45 in NHE2X3 DKO males and females; P ⬍ 0.01;
n ⫽ 4 groups, 5 mice in each group) (Fig. 4B).
Intestinal NHE8 expression in 6-wk-old NHE2X3 DKO
mice. Jejunal mucosa was collected from 6-wk-old wild-type
and NHE2X3 DKO mice. RNA was prepared and used for
real-time PCR. BBMV was isolated for Western blot. As
shown in Fig. 5A, NHE8 expression in male mice was similar
between wild-type mice and NHE2X3 DKO mice (1.015 ⫾
0.12 in wild-type male mice, 1.19 ⫾ 0.09 in NHE2x3 DKO
male mice; n ⫽ 18). The expression of NHE8 mRNA in
NHE2X3 DKO female mice was significantly higher compared
with their wild-type female mice (1.10 ⫾ 0.06 in wild-type
mice, 1.67 ⫾ 0.303 in NHE2X3 DKO; P ⬍ 0.01; n ⫽ 12).
Fig. 2. NHE8 mRNA expression in wild-type male and female mice. RNAs were isolated from the intestinal mucosa of male and female mice at the age of 4, 6, and
12 wk. Real-time PCR was performed using mouse-specific NHE8 and TATA-binding protein (TBP) primers. Data are means ⫾ SE for each age group. *P ⱕ 0.01
for female mice vs. male mice. Twenty-eight 4-wk-old mice (12 males, 16 females), 26 6-wk-old mice (13 for each gender), and 18 12-wk-old mice (9 for each gender)
were used. M, male mice; F, female mice.
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Intestinal NHE8 mRNA expression in wild-type mice. Four-,
6-, and 12-wk-old male and female mice were killed. RNA was
purified from mouse jejunal mucosa, and real-time PCR was
performed to determine the abundance of NHE8 mRNA. As
indicated in Fig. 2, NHE8 mRNA expression was similar
between 4-wk-old males and females (1.02 ⫾ 0.07 in male
mice and 1.10 ⫾ 0.07 in female mice; n ⫽ 28 mice). By 6 wk
of age, NHE8 expression was higher in female mice compared
with male mice (1.01 ⫾ 0.04 in male mice and 1.30 ⫾ 0.08 in
female mice; P ⬍ 0.01; n ⫽ 26 mice). At 12 wk of age, NHE8
expression was higher in female mice compared with male
mice (1.05 ⫾ 0.07 in male mice and 1.28 ⫾ 0.02 in female
mice; P ⬍ 0.01; n ⫽ 18 mice).
Intestinal NHE8 protein expression in wild-type mice. Male
and female mice at the age of 4, 6, and 12 wk were killed, and
BBMV protein was isolated from mouse jejunal mucosa.
BBMV protein was then used for Western blot to determine
NHE8 protein abundance. As shown in Fig. 3, NHE8 protein
level was the same between male and female mice at 4 wk of
age. The abundance of NHE8 at the age of 6 wk was reduced
by ⬃40% in male mice and by ⬃20% in female mice compared with values at 4 wk of age. At 12 wk of age, the
abundance of NHE8 was further reduced by ⬃60% in male
mice and by ⬃25% in female mice compared with values at 4
wk of age. The overall protein expression level of NHE8 is
reduced more dramatically in male mice than that in female
mice at 6 wk of age (P ⬍ 0.01, n ⫽ 4 groups, 4 –5 mice in each
group).
Intestinal NHE8 expression in 4-wk-old NHE2X3 DKO
mice. Jejunal mucosa was collected from 4-wk-old wild-type
and NHE2X3 DKO mice. RNA was prepared and used for
real-time PCR. BBMV was isolated for Western blot. As
shown in Fig. 4, the expression of NHE8 mRNA in male mice
was similar between wild-type and the NHE2X3 DKO mice
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TESTOSTERONE INHIBITS INTESTINAL NHE8 EXPRESSION
Fig. 4. NHE8 expression in 4-wk-old NHE2X3 DKO mice. A: NHE8 mRNA
expression in 4-wk-old male and female mice. RNA was isolated from the
intestinal mucosa of 4-wk-old male and female wild-type mice and NHE2X3
DKO mice. Real-time PCR was performed using mouse-specific NHE8 and
TBP primers. The changes in NHE8 gene expression are analyzed by the
comparative cycle threshold (Ct) method. Data are means ⫾ SE for each age
group. *P ⱕ 0.01 for NHE2X3 DKO mice vs. wild-type mice. Thirty wild-type
mice (15 males, 15 females) and 29 NHE2X3 DKO mice (14 males, 15
females) were used. B: NHE8 protein expression in 4-wk-old male and female
mice. BBMV was isolated from the intestinal mucosa of 4-wk-old male and
female wild-type mice and NHE2X3 DKO mice. Western blot was used to
detect NHE8 and ␤-actin protein abundances in BBMV preparations. The
expression of NHE8 protein is calculated by the optical density of the NHE8
band over that of the ␤-actin band. Data are means ⫾ SE for each gender
group. *P ⱕ 0.01 for NHE2X3 DKO mice vs. wild-type mice. Three independent experiments were performed. Inset: corresponding Western blot images.
NHE8 protein expression in female NHE2X3 DKO mice was
also increased compared with their female wild-type mice
(1.00 ⫾ 0.10 in wild-type mice, 1.50 ⫾ 0.25 in NHE2X3 DKO
mice; P ⬍ 0.01; n ⫽ 4 groups, 4 mice in each group). The
expression of NHE8 protein in male NHE2X3 DKO mice
remained unchanged compared with their wild-type mice
(1.0 ⫾ 0.10 in wild-type mice, 0.97 ⫾ 0.15 in NHE2X3 DKO
mice; n ⫽ 3 groups, 4 mice in each group) (Fig. 5B).
Effect of sex hormones on NHE8 expression in Caco-2 cells.
Real-time PCR and Western blotting were used to assess the
expression of endogenous NHE8 in Caco-2 cells. Testosterone
Fig. 5. NHE8 expression in 6-wk-old NHE2X3 DKO mice. A: NHE8 mRNA
expression in 6-wk-old NHE2X3 DKO mice. RNA was isolated from the
intestinal mucosa of 6-wk-old mice. Real-time PCR was performed using
mouse-specific NHE8 and TBP primers. The changes in NHE8 gene expression are analyzed by the comparative Ct method. Data are means ⫾ SE for each
age group. *P ⱕ 0.01 for NHE2X3 DKO mice vs. wild-type mice. Eighteen
wild-type mice (9 males, 9 females) and 12 NHE2X3 DKO mice (6 males, 6
females) were used. B: NHE8 protein expression in 6-wk-old NHE2X3 DKO
mice. BBMV was isolated from the intestinal mucosa of 6-wk-old mice.
Western blot was used to detect NHE8 and ␤-actin protein abundances in
BBMV preparations. The expression of NHE8 protein is calculated by the
optical density of NHE8 band over that of the ␤-actin band. Data are means ⫾
SE for each gender group. *P ⱕ 0.01 for NHE2X3 DKO mice vs. wild-type
mice. Three independent experiments were performed. Inset: corresponding
Western blot images.
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treatment (100 nM, 18 h) reduced NHE8 gene expression by
⬃40% in Caco-2 cells compared with untreated cells (n ⫽ 4;
p ⬍ 0.01) while estradiol treatment (100 nM, 18 h) had no
effect on NHE8 expression (Fig. 6A). At NHE8 protein levels,
testosterone treatment also reduced NHE8 protein expression
by ⬃50% in Caco-2 cells (1.0 ⫾ 0.05 in control cells; 0.35 ⫾
0.10 in testosterone cells; n ⫽ 3; P ⬍ 0.05) (Fig. 6B).
Effect of testosterone on human NHE8 gene transcription in
Caco-2 cells. To explore if testosterone-mediated NHE8 expression downregulation is due to reduced gene transcription,
Caco-2 cells were treated with 100 nM testosterone for 18 h in
the presence or absence of actinomycin D (100 nM). In the
absence of actinomycin D treatment, testosterone inhibited
NHE8 gene expression by ⬃40%. In the presence of actinomycin D, the inhibitory effect of testosterone on NHE8 mRNA
expression was completely abolished (Fig. 7A). Furthermore,
TESTOSTERONE INHIBITS INTESTINAL NHE8 EXPRESSION
transfection with NHE8 gene promoter constructs (pGL3b/
⫺89 and pGL3b/⫺671) showed that testosterone inhibited
NHE8 gene promoter activity by ⬃20% in transfected Caco-2
cells, and this inhibition occurred only in promoter construct
pGL3/⫺671 but not pGL3/⫺89 (Fig. 7B).
DISCUSSION
The nine identified mammalian NHE isoforms have different
functions, tissue distribution, and membrane localization (31).
Of these nine NHEs, only four of them are expressed in the
small intestine. NHE1 is expressed at the basolateral membrane of the intestinal epithelial cells, and NHE2, NHE3, and
NHE8 are expressed at the apical membrane of the intestinal
epithelial cells (30, 31). Knockout animal models revealed that
NHE2 plays little or no role in intestinal sodium absorption
while NHE3 is the main contributor of the intestinal and renal
sodium absorption (11, 14, 24, 25). NHE8 is highly expressed
at early life, and it has been suggested to have a role in
intestinal sodium absorption during early development when
NHE2 and NHE3 are expressed at very low levels (28, 29).
Disruption of NHE3 function results in diarrhea and impaired acid-base balance in mice (25). Studies in NHE3 KO
mice suggested that an unidentified NHE is expressed in the
gut, and this NHE contributes to ⬃30% of sodium absorption
(11, 14). In our initial work, we found that NHE8 expression
was increased in young NHE3 KO mice. To further study the
role of NHE8 in the intestine, we created a NHE2X3 DKO
mouse model. Body weight and serum sodium level of
NHE2X3 DKO mice were lower than their wild-type littermates. Almost no body weight was gained in NHE2X3 DKO
mice between 3 and 4 wk of age. This period in development
coincides with a dramatic increase in NHE3 expression (7).
These observations suggested that NHE3 is important for the
development after weanling. After 4 wk of age, the NHE2X3
DKO mice started to gain body weight again with the rate
similar to their wild-type littermates. By 6 wk of age, the body
weights of male and female NHE2X3 DKO mice were ⬃70%
and ⬃84% of the wild-type mice, respectively. Interestingly,
only 60% of male NHE2X3 DKO mice survived after 6 wk of
age while 88% of female NHE2X3 DKO mice survived after 6
wk of age, which indicated that NHE8 expression may be
regulated differently in male and female mice. A previous
study by another group reported ⬃79% survival in NHE2X3
DKO mice (14). However, this study used a very small number
of animals (24 NHE2X3 DKO mice), and there was no gender
description for these mice. If the male and the female mice in
our study were combined, the total survival rate would be 74%,
which is very close to the survival rate reported by the earlier
group.
To understand if the low survival rate in male NHE2X3
DKO mice was due to low NHE8 expression, we first compared the expression pattern of NHE8 in wild-type male and
female mice. Our data indicated that NHE8 expression indeed
differs between males and females. At 4 wk of age, NHE8
expression was the same between male and female mice.
However, at 6 wk of age, NHE8 expression in male mice was
30% lower than in female mice. By 12 wk of age, NHE8
expression in male mice was 50% lower than in female mice.
These observations suggested that NHE8 expression is regulated by a gender-specific mechanism and female mice have
higher NHE8 expression levels. We therefore compared NHE8
regulation in NHE2X3 DKO male and female mice. Our
results showed that NHE8 was upregulated in female NHE2X3
DKO mice, but this compensatory mechanism was absent in
male NHE2X3 DKO mice. In female NHE2X3 DKO mice,
NHE8 mRNA and protein expression increased ⬎50% compared with wild-type female mice. In male NHE2X3 DKO
mice, NHE8 mRNA expression level remained unchanged, but
the protein level increased by ⬃50% only in 4-wk-old mice.
The levels of NHE8 expression correlated with the survival
rate in NHE2X3 DKO mice. These observations indicated that
NHE8 was upregulated in NHE2X3 DKO mice, and this
compensatory mechanism was gender-specific. Interestingly,
NHE8 expression was also increased in young NHE3 KO mice
but not in adult NHE3 KO male mice (data not shown). It is
possible that other sodium transporters instead of NHE8 compensate for the loss of NHE3 in adult NHE3 KO mice, such as
the epithelial sodium channel protein (25).
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Fig. 6. Effect of sex hormone on NHE8 expression in human intestinal
epithelial cells. Caco-2 cells were cultured in standard medium or sex hormone-containing medium (100 nM) for 18 h before harvest. RNAs were
isolated from cells and were used for RT-PCR. Real-time PCR was performed
with human NHE8 or TBP primers in separate reactions. Total protein was
prepared from cells and used for Western blot. Results are means ⫾ SE from
3 separate experiments. *P ⬍ 0.01 for control (CT) vs. testosterone treatment.
A: effect of sex hormones on NHE8 mRNA expression in Caco-2 cells.
B: effect of testosterone on NHE8 protein expression in Caco-2 cells.
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Gender-dependent gene expression has been observed in
mammals, and this difference is linked to sex hormones.
Testosterone is the principal male sex hormone and is an
anabolic steroid. It is found in mammals, reptiles, birds, and
other vertebrates (9, 22). In mammals, testosterone is primarily secreted in the testes of males and the ovaries of
females. In men, testosterone plays a key role in the development of male reproductive tissues such as the testis and
prostate as well as promoting secondary sexual characteristics such as increased muscle, bone mass, and hair growth
(17). Although testosterone is a male-specific steroid hormone, it has been known to regulate certain transporter
expression via a gender-specific manner. In male rodents,
testosterone inhibits sodium-glucose cotransporter expression by reducing SGLT-1 mRNA abundance (23). Testosterone also stimulates epithelial sodium channel protein (10,
13, 18 –21), organic anion transporters (2–5, 12, 15, 16), and
organic cation transporters (1, 26). All of this testosteronemediated transporter gene regulation occurred in the kidney
but not in the small intestine.
In the current study, we observed the gender-specific
expression of NHE8 in the small intestine in mice. To
understand the mechanism of gender-specific NHE8 expression, we tested the effect of sex hormones on NHE8 expression in Caco-2 cells. Estradiol had no effect on NHE8
expression in Caco-2 cells, but testosterone significantly
reduced NHE8 expression at both mRNA and protein ex-
pression levels. These observations indicated that testosterone was indeed an important regulator of NHE8 expression
in the intestine. To further understand if this inhibition is
mediated by transcriptional regulation, we treated cells with
actinomycin D. Actinomycin D treatment completely
blocked the inhibitory effect of testosterone on NHE8 expression in Caco-2 cells, indicating that the inhibitory effect
of testosterone on NHE8 occurred at the gene transcription
level. Transfection with human NHE8 gene promoter constructs in Caco-2 cells showed a similar response of NHE8
promoter activity in the promoter construct pGL3/⫺671 but
not pGL3/⫺89, suggesting that the testosterone-responsive
region is located between ⫺671 and ⫺89 bp of the human
NHE8 gene. Interestingly, no androgen receptor (AR) binding motif was found in this human NHE8 gene promoter
region, which suggested an AR-independent mechanism for
testosterone’s regulation of NHE8 gene expression. Future
study is forthcoming to identify the mechanism involved.
In summary, we have shown that the intestinal NHE8
expression exhibits a gender difference after puberty, and
this difference was mediated by the inhibitory effect of
testosterone. Furthermore, NHE8 could partially compensate for the loss of NHE3 in NHE2X3 DKO female mice.
These studies, for the first time, directly demonstrated that
the intestinal NHE8 expression regulation was genderspecific, and testosterone is an important regulator of NHE8
gene in the intestine.
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Fig. 7. The mechanism of testosterone effect on human
NHE8 gene expression. A: actinomycin D’s effect on testosterone-mediated NHE8 gene expression inhibition.
Caco-2 cells were cultured in standard medium or testosterone-containing medium (100 nM) in the presence or
absence of actinomycin D for 18 h before harvest. RNAs
were isolated from these cells. Real-time PCR was performed with human NHE8 or TBP primers in separate
reactions. Results are means ⫾ SE from 3 separate experiments. *P ⬍ 0.01 for control vs. testosterone treatment. B:
cells were cotransfected with pRL-CMV and pGL3 basic
(pGL3b) or human NHE8 promoter constructs (pGL3b/
⫺89 and pGL3b/⫺671). Testosterone was applied 18 h
before measuring promoter activities. Promoter activity is
shown as a relative activity, which is a ratio of firefly
luciferase activity driven by NHE8 promoter over Renilla
luciferase activity driven by CMV promoter. The degree of
inhibition is shown as the ratio of luciferase activity in
testosterone-treated cells over luciferase activity in vehicletreated cells. Results are means ⫾ SE from 4 separate
experiments.
TESTOSTERONE INHIBITS INTESTINAL NHE8 EXPRESSION
GRANTS
This investigation was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-073638 and R01-DK-041274.
DISCLOSURES
No conflicts of interest are declared by the authors.
REFERENCES
15. Ljubojevic M, Herak-Kramberger CM, Hagos Y, Bahn A, Endou H,
Burckhardt G, Sabolic I. Rat renal cortical OAT1 and OAT3 exhibit
gender differences determined by both androgen stimulation and estrogen
inhibition. Am J Physiol Renal Physiol 287: F124 –F138, 2004.
16. Lu R, Kanai N, Bao Y, Wolkoff AW, Schuster VL. Regulation of renal
oatp mRNA expression by testosterone. Am J Physiol Renal Fluid Electrolyte Physiol 270: F332–F337, 1996.
17. Mooradian AD, Morley JE, Korenman SG. Biological actions of
androgens. Endocr Rev 8: 1–28, 1987.
18. Reckelhoff JF. Gender differences in the regulation of blood pressure.
Hypertension 37: 1199 –1208, 2001.
19. Reckelhoff JF, Granger JP. Role of androgens in mediating hypertension
and renal injury. Clin Exp Pharmacol Physiol 26: 127–131, 1999.
20. Reckelhoff JF, Zhang H, Srivastava K. Gender differences in development of hypertension in spontaneously hypertensive rats: role of the
renin-angiotensin system. Hypertension 35: 480 –483, 2000.
21. Reckelhoff JF, Zhang H, Srivastava K, Granger JP. Gender differences
in hypertension in spontaneously hypertensive rats: role of androgens and
androgen receptor. Hypertension 34: 920 –923, 1999.
22. Reed WL, Clark ME, Parker PG, Raouf SA, Arguedas N, Monk DS,
Snajdr E, Nolan V Jr, Ketterson ED. Physiological effects on demography: a long-term experimental study of testosterone’s effects on fitness.
Am Nat 167: 667–683, 2006.
23. Sabolic I, Skarica M, Gorboulev V, Ljubojevic M, Balen D, HerakKramberger CM, Koepsell H. Rat renal glucose transporter SGLT1
exhibits zonal distribution and androgen-dependent gender differences.
Am J Physiol Renal Physiol 290: F913–F926, 2006.
24. Schultheis PJ, Clarke LL, Meneton P, Harline M, Boivin GP, Stemmermann G, Duffy JJ, Doetschman T, Miller ML, Shull GE. Targeted
disruption of the murine Na⫹/H⫹ exchanger isoform 2 gene causes
reduced viability of gastric parietal cells and loss of net acid secretion. J
Clin Invest 101: 1243–1253, 1998.
25. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M,
Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch
G, Aronson PS, Lorenz JN, Shull GE. Renal and intestinal absorptive
defects in mice lacking the NHE3 Na⫹/H⫹ exchanger. Nat Genet 19:
282–285, 1998.
26. Urakami Y, Nakamura N, Takahashi K, Okuda M, Saito H,
Hashimoto Y, Inui K. Gender differences in expression of organic cation
transporter OCT2 in rat kidney. FEBS Lett 461: 339 –342, 1999.
27. Xu H, Chen H, Dong J, Li J, Chen R, Uno JK, Ghishan FK. Tumor
necrosis factor-␣ downregulates intestinal NHE8 expression by reducing
basal promoter activity. Am J Physiol Cell Physiol 296: C489 –C497,
2009.
28. Xu H, Chen H, Dong J, Lynch R, Ghishan FK. Gastrointestinal
distribution and kinetic characterization of the sodium-hydrogen exchanger isoform 8 (NHE8). Cell Physiol Biochem 21: 109 –116, 2008.
29. Xu H, Chen R, Ghishan FK. Subcloning, localization, and expression of
the rat intestinal sodium-hydrogen exchanger isoform 8. Am J Physiol
Gastrointest Liver Physiol 289: G36 –G41, 2005.
30. Yun CH, Tse CM, Nath SK, Levine SA, Brant SR, Donowitz M.
Mammalian Na⫹/H⫹ exchanger gene family: structure and function studies. Am J Physiol Gastrointest Liver Physiol 269: G1–G11, 1995.
31. Zachos NC, Tse M, Donowitz M. Molecular physiology of intestinal
Na⫹/H⫹ exchange. Annu Rev Physiol 67: 411–443, 2005.
AJP-Gastrointest Liver Physiol • VOL
300 • APRIL 2011 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on June 15, 2017
1. Alnouti Y, Petrick JS, Klaassen CD. Tissue distribution and ontogeny of
organic cation transporters in mice. Drug Metab Dispos 34: 477–482,
2006.
2. Buist SC, Cherrington NJ, Choudhuri S, Hartley DP, Klaassen CD.
Gender-specific and developmental influences on the expression of rat
organic anion transporters. J Pharmacol Exp Ther 301: 145–151, 2002.
3. Buist SC, Cherrington NJ, Klaassen CD. Endocrine regulation of rat
organic anion transporters. Drug Metab Dispos 31: 559 –564, 2003.
4. Buist SC, Klaassen CD. Rat and mouse differences in gender-predominant expression of organic anion transporter (Oat1-3; Slc22a6-8) mRNA
levels. Drug Metab Dispos 32: 620 –625, 2004.
5. Cheng X, Klaassen CD. Tissue distribution, ontogeny, and hormonal
regulation of xenobiotic transporters in mouse kidneys. Drug Metab
Dispos 37: 2178 –2185, 2009.
6. Collins JF, Kiela PR, Xu H, Zeng J, Ghishan FK. Increased NHE2
expression in rat intestinal epithelium during ontogeny is transcriptionally
mediated. Am J Physiol Cell Physiol 275: C1143–C1150, 1998.
7. Collins JF, Xu H, Kiela PR, Zeng J, Ghishan FK. Functional and
molecular characterization of NHE3 expression during ontogeny in rat
jejunal epithelium. Am J Physiol Cell Physiol 273: C1937–C1946, 1997.
8. Counillon L, Scholz W, Lang HJ, Pouyssegur J. Pharmacological
characterization of stably transfected Na⫹/H⫹ antiporter isoforms using
amiloride analogs and a new inhibitor exhibiting anti-ischemic properties.
Mol Pharmacol 44: 1041–1045, 1993.
9. Cox RM, John-Alder HB. Testosterone has opposite effects on male
growth in lizards (Sceloporus spp) with opposite patterns of sexual size
dimorphism. J Exp Biol 208: 4679 –4687, 2005.
10. Gambling L, Dunford S, Wilson CA, McArdle HJ, Baines DL. Estrogen and progesterone regulate alpha, beta, and gammaENaC subunit
mRNA levels in female rat kidney. Kidney Int 65: 1774 –1781, 2004.
11. Gawenis LR, Stien X, Shull GE, Schultheis PJ, Woo AL, Walker NM,
Clarke LL. Intestinal NaCl transport in NHE2 and NHE3 knockout mice.
Am J Physiol Gastrointest Liver Physiol 282: G776 –G784, 2002.
12. Isern J, Hagenbuch B, Stieger B, Meier PJ, Meseguer A. Functional
analysis and androgen-regulated expression of mouse organic anion transporting polypeptide 1 (Oatp1) in the kidney. Biochim Biophys Acta 1518:
73–78, 2001.
13. Kienitz T, Allolio B, Strasburger CJ, Quinkler M. Sex-specific regulation of ENaC and androgen receptor in female rat kidney. Horm Metab
Res 41: 356 –362, 2009.
14. Ledoussal C, Woo AL, Miller ML, Shull GE. Loss of the NHE2
Na⫹/H⫹ exchanger has no apparent effect on diarrheal state of NHE3deficient mice. Am J Physiol Gastrointest Liver Physiol 281: G1385–
G1396, 2001.
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