the rat nephron Sex-dependent expression of

Transcription

Am J Physiol Renal Physiol 308: F809–F821, 2015.
First published February 5, 2015; doi:10.1152/ajprenal.00368.2014.
CALL FOR PAPERS
Sex and Gender Differences in Renal Physiology
Sex-dependent expression of water channel AQP1 along the rat nephron
Carol M. Herak-Kramberger,1 Davorka Breljak,1 Marija Ljubojević,1 Mirela Matokanović,1 Mila Lovrić,2
Dunja Rogić,2 Hrvoje Brzica,1 Ivana Vrhovac,1 Dean Karaica,1 Vedran Micek,1 Jana Ivković Dupor,3
Dennis Brown,4 and Ivan Sabolić1
1
Molecular Toxicology, Institute for Medical Research and Occupational Health, Zagreb, Croatia; 2Clinical Institute of
Laboratory Diagnosis, University Hospital Center, Zagreb, Croatia; 3Department of Biotechnology, University of Rijeka,
Rijeka, Croatia; 4Program in Membrane Biology and Division of Nephrology, Center for Systems Biology, Massachusetts
General Hospital and Harvard Medical School, Boston, Massachusetts
Herak-Kramberger CM, Breljak D, Ljubojević M,
Matokanović M, Lovrić M, Rogić D, Brzica H, Vrhovac I,
Karaica D, Micek V, Dupor JI, Brown D, Sabolić I. Sex-dependent
expression of water channel AQP1 along the rat nephron. Am J
Physiol Renal Physiol 308: F809 –F821, 2015. First published February 5, 2015; doi:10.1152/ajprenal.00368.2014.—In the mammalian
kidney, nonglycosylated and glycosylated forms of aquaporin protein
1 (AQP1) coexist in the luminal and basolateral plasma membranes of
proximal tubule and descending thin limb. Factors that influence
AQP1 expression in (patho)physiological conditions are poorly
known. Thus far, only angiotensin II and hypertonicity were found to
upregulate AQP1 expression in rat proximal tubule in vivo and in
vitro (Bouley R, Palomino Z, Tang SS, Nunes P, Kobori H, Lu HA,
Shum WW, Sabolic I, Brown D, Ingelfinger JR, Jung FF. Am J
Physiol Renal Physiol 297: F1575–F1586, 2009), a phenomenon that
may be relevant for higher blood pressure observed in men and male
experimental animals. Here we investigated the sex-dependent AQP1
protein and mRNA expression in the rat kidney by immunochemical
methods and qRT-PCR in tissue samples from prepubertal and intact
gonadectomized animals and sex hormone-treated gonadectomized
adult male and female animals. In adult rats, the overall renal AQP1
protein and mRNA expression was ⬃80% and ⬃40% higher, respectively, in males than in females, downregulated by gonadectomy in
both sexes and upregulated strongly by testosterone and moderately
by progesterone treatment; estradiol treatment had no effect. In
prepubertal rats, the AQP1 protein expression was low compared with
adults and slightly higher in females, whereas the AQP1 mRNA
expression was low and similar in both sexes. The observed differences in AQP1 protein expression in various experiments mainly
reflect changes in the glycosylated form. The male-dominant expression of renal AQP1 in rats, which develops after puberty largely in the
glycosylated form of the protein, may contribute to enhanced fluid
reabsorption following the androgen- or progesterone-stimulated activities of sodium-reabsorptive mechanisms in proximal tubules.
aquaporin 1; castration; sex differences; immunocytochemistry; ovariectomy; sex steroids; Western blotting
AQP1 mediates bulk (⬃80%) water reabsorption in the mammalian kidney. The protein has been
localized to the luminal and basolateral plasma membranes of
epithelial cells in proximal tubules and thin descending limbs
of Henle (TDLH), where it exists in water-permeable nongly-
THE WATER CHANNEL
Address for reprint requests and other correspondence: I. Sabolić, Molecular
Toxicology, Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 10000 Zagreb, Croatia (e-mail: sabolic@imi.hr).
http://www.ajprenal.org
cosylated (NG; molecular weight, Mr, of ⬃28 kDa) and glycosylated (G; Mr 35–50 kDa) forms (11, 42, 59, 65).
AQP1 has long been considered a constitutive water channel, insensitive to vasopressin and other physiological and
pathophysiological stimuli that affect the “vasopressin-regulated” water channel AQP2 (40, 41). However, an increasing
number of studies shows that the activity and/or expression of
renal AQP1 can also be regulated to some extent, but the
relevant effectors and mechanisms of their actions are poorly
known. Thus, in experimental animals (mainly rats), the expression of renal AQP1 protein and/or mRNA and the associated water reabsorption were downregulated in ischemia/reperfusion injury (16, 20), gentamycin- and cisplatin-induced
nephrotoxicity (2, 25, 30), unilateral and bilateral ureteral
obstruction (24, 34, 35), denervated kidney (31), late gestation
(22), and high-sodium diet (13). Upregulation was demonstrated in rats in vivo in spontaneously hypertensive rats (SHR)
(29), in hypothyroid (70) animals, in contralateral kidney
following denervation of one kidney (31), in early gestation
(22), and in proximal tubule cells following 10 days of treatment with angiotensin II (ANG II) (5). In experiments in vitro,
AQP1 was upregulated in transferrin-treated human (63) and
ANG II- or hypertonic solution-treated rat proximal tubule cell
lines (5), whereas, in studies with AQP1-transfected Xenopus
oocytes, water permeability was stimulated by vasopressin via
a cAMP/PKA-dependent mechanism (17, 44) and by PKC
activation (72).
It is well established that mammalian kidneys are affected by
sex hormones, including their morphology, metabolism, secretory and reabsorptive functions, as well as pharmacology and
toxicology of various substances via affecting the expression
and/or activity of specific enzymes, channels, and membrane
transporters of inorganic and organic material (18, 39, 55–57,
61, 68). Previous data showed that sex hormones, particularly
androgens, can affect urine flow and osmolarity (Uosm) and
may play an important role in generating sex differences in
blood pressure, which is higher in men and male experimental
animals (14, 23, 49, 69). Some related mechanisms may
include the vasopressin-dependent processes that regulate the
expression of AQP2 in collecting duct principal cells (8, 40),
whereas the vasopressin-independent, androgen-driven transport of ions and water takes place in the proximal parts of the
nephron. The male-dominant sex differences and a role of
testosterone in elevating blood pressure via enhanced Na⫹ and
1931-857X/15 Copyright © 2015 the American Physiological Society
F809
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Submitted 30 June 2014; accepted in final form 3 February 2015
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SEX DIFFERENCES IN RENAL AQP1 EXPRESSION
MATERIALS AND METHODS
Animals and treatment. Age-matched prepubertal (3 wk old) and
adult (12–14 wk old) male and female Wistar rats were from the
breeding colony at the Institute in Zagreb. Animals were housed (4 per
cage) in air-conditioned rooms with controlled temperature (23 ⫾
1°C) and light (12-h:12-h light/dark cycle). Animal care and treatment
were conducted in conformity with institutional guidance that is in
compliance with international laws, and The Institutional Ethics
Committee approved the studies. Before and during experiments,
animals had free access to standard pelleted food (Mucedola) and tap
water. Prepubertal rats were used untreated, whereas the adult rats
were used either untreated, sham operated, or gonadectomized. Untreated adult rats were killed at an age of 12–13 wk. Gonadectomy
was performed at an age of 8 wk under general anesthesia (Narketan,
80 mg/kg body mass ip ⫹ Xylapan, 12 mg/kg body mass ip); males
were castrated by the scrotal route, whereas females were ovariectomized by a lumbal approach. The sham-operated animals underwent
the same procedure, except the gonads were not removed. The
gonadectomized or sham-operated rats were left to recover for 4 wk
before euthanasia. Some gonadectomized animals (both sexes; 4
animals/group) underwent subcutaneous treatment with either testosterone enanthate, estradiol dipropionate, or progesterone (each: 2.5
mg/kg body mass per day for 14 days; hormones dissolved in
sunflower oil) and were killed at an age of 14 wk. In these experiments, the gonadectomized controls (4 animals/group) were treated
with an equivalent amount of sunflower oil (0.5 ml/kg body mass sc
per day for 14 days).
Urine was collected in metabolic cages; 1– 8 days before euthanasia, rats were placed in individual metabolic cages with free access to
water but without (fasted) or with (nonfasted) food, and urine was
collected for 24 h. The fasted rats were placed in metabolic cages
without preceding accommodation, and 24-h urine collection was
started immediately, whereas the nonfasted animals were placed in
metabolic cages for 24 h before urine collection was started over the
next 24 h. Collection of urine under fasting conditions was performed
to avoid contamination of urine with food particles. As shown previously by Amlal et al. (1), fasting in rats causes downregulation of
AQP2 and results in polyuria, but it does not change the expression of
renal AQP1. Following removal from the metabolic cages, the mass of
each animal and the volume of each urine sample was measured.
Urine was centrifuged at 2,500 g for 15 min to remove debris;
osmolality (freezing point osmometer SLAMED 800 CL) and protein
(6) were determined in the clear supernatant. In experiments with
nonfasted animals, consumption of water and food was also measured
during their stay in metabolic cages.
Glomerular filtration rate (GFR) was estimated from creatinine
clearance using the following formula: (urine concentration ⫻ urine
volume)/plasma concentration, and was expressed per kilogram of
body mass. Blood was collected in heparinized tubes from the anesthetized animals by cutting the jugular blood vessels and then centrifuged at 1,500 g for 15 min to obtain plasma. Creatinine in blood
plasma and urine was determined by ion chromatography with a
conductivity detector (Dionex).
Antibodies and other reagents. The rabbit-raised polyclonal antibody (immune serum or affinity-purified antibody) against the human
AQP1 holoprotein (AQP1-Ab) and its use in immunochemical studies
with rat tissues and isolated membranes were previously described
(59). Monoclonal antibody against the Na/K-ATPase ␣1-subunit (Na/
K-ATPase-Ab) was purchased commercially from Santa Cruz Biotechnology (no. sc-48345). Secondary antibodies, including CY3labeled (GAR-CY3) and alkaline phosphatase-labeled goat anti-rabbit
(GAR-AP) or anti mouse (GAM-AP) IgG antibodies, were purchased
from Jackson ImmunoResearch Laboratories or Kirkegaard and Perry.
Anesthetics (Narketan and Xylapan) were purchased from Chassot.
Oil solutions of testosterone enanthate, estradiol dipropionate, and
progesterone were from RotexMedica and Galenika. All other chemicals and reagents were analytical grade and obtained commercially
from Sigma or Fisher Scientific. The sources of specific reagents and
equipment for RNA isolation, real-time RT-PCR, and Western blotting assays are indicated in the text related to these methods.
RNA isolation, cDNA synthesis, and quantitative RT-PCR. The
RT-PCR analysis was previously described in detail (7, 9). In short,
the animals were euthanized by decapitation, and a 1-mm-thick
middle sagittal slice of the decapsulated kidney was immediately
submerged in RNAlater solution (Sigma). In adult rats, the slice was
used either in toto, or the renal cortex and outer stripe tissues were
dissected manually and used separately for RNA isolation. In prepubertal rats, the whole kidneys were used. Total cellular RNA from
corresponding tissues was extracted using Trizol (Invitrogen); the
RNA concentration and its purity were determined spectrophotometrically (BioSpec Nano), and the quality of RNAs was estimated by
agarose gel electrophoresis and ethidium bromide staining. Firststrand cDNA synthesis was performed using High-Capacity cDNA
Reverse Transcription Kits (Applied Biosystems) following the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using TaqMan primers and probes designed by Applied
Biosystems (assay IDs were Rn00562834_m1 and Rn00667869_m1
for rAQP1 and r␤-actin, respectively; http://www.appliedbiosystems.com). Amplification and detection were performed using the 7500
Real-Time PCR System (Applied Biosystems). To standardize the
input of cDNA amount, the housekeeping gene ␤-actin was run and
quantified, and the results were normalized to these values. Quantification of rAQP1 mRNA amount (mean fold changes ⫾ SE of
duplicate measurements) was accomplished by comparative Ct
method using the Relative Quantification Study Software (Applied
Biosystems).
Tissue fixation, immunocytochemistry, and fluorescence quantification.
As described in detail previously (3, 58), adult animals were anesthetized, and the organs were fixed in vivo by vascular perfusion with 4%
paraformaldehyde. The kidneys were removed, sliced, postfixed overnight in the same fixative, and extensively washed in PBS. Four-
AJP-Renal Physiol • doi:10.1152/ajprenal.00368.2014 • www.ajprenal.org
fluid reabsorption in the proximal tubule have been established
in several animal models of hypertension (10, 12, 19, 50, 69)
and in humans (14, 23, 49). Studies in SHRs showed that
testosterone promotes the development of hypertension via an
androgen receptor-mediated mechanism that stimulates the
systemic renin-angiotensin system (RAS). The final effector in
the RAS, ANG II, promotes retention of Na⫹ and water in the
proximal tubule and thus contributes to higher blood pressure
in males (reviewed in Ref. 23). Recent reports have described
in rodents an autonomous, androgen-stimulated RAS system in
the proximal tubule, which produces and secretes ANG II that
acts as an autocrine and paracrine stimulator of Na⫹ and fluid
reabsorption (15, 48) by increasing the activity and/or expression of the Na⫹/H⫹ exchanger isoform 3 (NHE3),
sodium-phosphate cotransporter 2 (NaPi2) in the brushborder membrane (BBM), and Na/K-ATPase and the sodium bicarbonate cotransporter 1 (NBC1) in the basolateral
membrane (BLM) (33, 46, 54, 71). The increased Na⫹
reabsorption is accompanied by increased water transport
(reabsorption), which may be at least partly mediated by the
ANG II-driven upregulation of AQP1 expression in proximal tubules (5).
The goal of the present study was to determine whether sex
differences exist in the expression of renal AQP1 and whether
sex hormones, notably androgens, influence the expression of
proximal tubule AQP1 and thus support the observed maledominant sex differences in fluid reabsorption and blood pressure.
buffer containing the AQP1-Ab (1:1,000). In some experiments, to
check for proper loading and transfer, the upper part of the stained
transfer membrane was cut off and incubated overnight in the buffer
containing the Na/K-ATPase-Ab (1:1,000). The membranes were
finally washed with blotting buffer, incubated for 60 min at room
temperature in the same buffer that contained GAR-AP (0.1 ␮g/ml) or
GAM-AP (0.5 ␮g/ml), respectively, and stained for alkaline phosphatase activity using the color-developing BCIP/NBT assay (7). The
labeled AQP1-related protein bands of 28 kDa (NG form) and 35–50
kDa (G form) were evaluated by densitometry using ImageJ software.
Both AQP1-specific protein bands (NG ⫹ G) were encircled and
quantified as a single sample, or NG and G bands were encircled and
quantified separately. The density of the individual NG or G protein
bands or combined bands (NG ⫹ G) was expressed relative to the
respective strongest band density (1 arbitrary unit) in the membranes
from control animals.
Statistical evaluation. The immunocytochemical and immunoblotting data represent findings in one or two independent experiments
with different tissue or membrane samples and with similar results.
The numeric data, expressed as means ⫾ SE, were statistically
evaluated by use of Student’s t-test or ANOVA/Duncan test at the 5%
level of significance.
RESULTS
Sex differences in renal AQP1 protein and mRNA expression. As
found by immunoblotting of TCM isolated from the whole
kidneys of adult male and female rats (Fig. 1A) and by
densitometric evaluation of the combined AQP1-related protein bands (NG ⫹ G) (Fig. 1B), the density of these bands was,
overall, ⬃80% higher in males than in females. Interestingly,
the G band of 35–50 kDa showed a much bigger difference in
expression levels between males and females (⬃140%) than
the NG band of 28 kDa (⬃11%.) (Table 1, Fig. 1A). In
contrast, the ⬃100-kDa protein band of Na/K-ATPase ␣-subunit, which was used as a loading control, showed a femaledominant abundance. A similar pattern of Na/K-ATPase protein expression (males ⬍ females) was observed in all subsequent experiments in which TCM from the whole kidneys were
used (data not shown). Sex differences in the expression of
AQP1 were also observed at the mRNA level; the expression
was ⬃40% higher in males than in females (Fig. 1C). The
Western blot and mRNA data are further supported by immunostaining of AQP1 in cryosections of various kidney zones in
adult animals (Fig. 1D). In accordance with previous findings
(42, 59), the AQP1-Ab stained the luminal and contraluminal
membranes of proximal tubules in the cortex and outer stripe
and of TDLH, as well as capillaries in the inner stripe and
papilla (weakly; not shown). As shown in Fig. 1D, the intensity
of AQP1 staining in the respective nephron segments was
stronger in males than in females. This was confirmed by
measuring the pixel intensity of fluorescence staining (males ⬎
females; Fig. 1E) and density of AQP1 (NG ⫹ G) protein
bands (males ⬎ females; Fig. 1, F and G) in TCM from the
same tissue zones from male and female kidneys. When
compared at the level of individual protein bands (Table 1, Fig.
1F), in various tissue zones the difference between males and
females in the expression of NG form was on average 23
arbitrary units, whereas the difference in the expression of G
form was on average 43 arbitrary units. Therefore, in all tissue
zones, both NG and G bands exhibited a higher expression in
male rats, but the difference was much larger for the G form.
micron-thick cryosections were cut (Leica CM 1850 cryostat, Leica
Instruments) and collected on Superfrost/Plus Microscope slides (Fischer Scientific). Before applying the primary antibody, cryosections
underwent an antigen retrieval procedure by microwave heating in 10
mM citrate buffer, pH 3, and treatment with Triton X-100-containing
buffers (3). They were then incubated as follows: 1) bovine serum
albumin to block nonspecific antibody binding, at room temperature
for 15 min, 2) optimal dilutions of AQP1-Ab determined in preliminary experiments (to see sex differences in the staining and to quantify
the fluorescence intensity, the AQP1-Ab concentration had to be
significantly reduced and adapted to a nonsaturating range), in a
refrigerator overnight, and 3) in GAR-CY3, at room temperature for
60 min. The samples were then washed in PBS, mounted in
Vectashield (Vector Laboratories), and examined with an Opton III
RS fluorescence microscope (Opton Feintechnik). Images were taken
with a software-driven Spot RT Slider camera (Diagnostic Instruments). The software parameters were adjusted so that the strongest
fluorescence intensity in a respective set of cryosections was not
saturating, and all other pictures were then taken using the same
parameters. The photos were imported into Adobe Photoshop 6.0 for
processing and labeling. In the representative images shown here, the
red fluorescence of CY3 was converted into black and white mode
using Adobe Photoshop to obtain better contrast.
The pixel intensity of CY3 fluorescence was measured in the
original images using Image J software, version 1.4 (NIH). One
cryosection per kidney from each animal was stained, and four
representative images in each tissue zone [cortex, outer stripe, inner
stripe, inner medulla (papilla)] were taken with a ⫻25 objective. For
each image in the respective zone, the background fluorescence in the
empty tubular/peritubular space was subtracted; 10 –15 randomly
chosen AQP1-positive tubule profiles (excluding the lumen) were
then encircled (regions of interest; ROI); the average fluorescence
pixel intensity in each ROI was measured; and the medium value of
all measurements was calculated and used as one datum per tissue
zone. Thereafter, the average pixel intensity from five ROI in the
AQP1-negative surrounding tubule profiles (excluding the lumen) in
the same tissue zone was also measured, and this value was subtracted
from the average pixel intensity in the AQP1-positive tubule profiles.
These results, obtained from measurements in all four images per
kidney tissue zone from an animal, were then averaged and used as
one datum per zone per animal. The final tissue zone-related data from
three to four animals were expressed relative to the strongest fluorescence pixel intensity (1 fluorescence unit) measured in cryosections
from the respective control animal.
Preparation of cell membranes. The anesthetized animals were
killed by exsanguination, and the kidneys were removed and sagittally
sliced. The slices were used in toto, or their cortex, outer stripe, and
inner stripe plus papilla tissues were dissected manually and used as
separate tissue pools. The kidneys from prepubertal rats were used
intact. The respective tissue pools were homogenized in a protease
inhibitor-containing chilled buffer, and total cell membranes (TCM)
were isolated by differential centrifugation as a pellet between 6,000
g and 150,000 g, as described in detail previously (7). BBM and BLM
from the cortex or outer stripe tissue homogenates were isolated by
the Mg-EGTA-aggregation (4) and Percoll density gradient centrifugation method (60), respectively. Protein in isolated membranes was
measured by the dye-binding assay (6) using bovine serum albumin as
the standard.
SDS-PAGE and Western blotting. The membrane samples were
prepared in reducing Laemmli buffer (final: 1% SDS, 12% vol/vol
glycerol, 30 mM Tris·HCl, pH 6.8, with 5% ␤-mercaptoethanol),
denatured at 37°C for 30 min, and further processed by SDS-PAGE
and wet transfered to Immobilon membrane (Millipore), as described
in detail previously (7). For SDS-PAGE, the amount of protein per
lane was 10 ␮g for BBM and BLM and 20 ␮g for TCM. Following
transfer, the Immobilon membrane was blocked in milk-containing
blotting buffer and incubated in the refrigerator overnight in the same
F811
F812
Fig. 1. Sex differences in the expression of renal aquaporin 1 (AQP1) protein and mRNA in 3-mo-old rats. A: representative blots of AQP1 and Na/K-ATPase
in the whole kidney total cell membranes (TCM) from male (M) and female (F) animals. Both nonglycosylated (NG, molecular weight, Mr, of 28 kDa) and
glycosylated (G, Mr 35–50 kDa) AQP1 protein bands in M were stronger than in F, whereas the ⬃100-kDa protein band of Na/K-ATPase ␣-subunit showed an
opposite abundance (F ⬎ M). B: densitometric evaluation of the cumulative AQP1 protein bands (NG ⫹ G), collected from 2 independent experiments with
membrane preparations from separate animals (n ⫽ 6 in each group); density of the bands was ⬃80% stronger in M than in F (*P ⬍ 0.05). C: relative expression
of AQP1 mRNA in RNA preparations from the whole kidneys from M and F rats (n ⫽ 4 in each group); relative expression in M was ⬃40% higher than in
F (*P ⬍ 0.05). D: immunostaining of AQP1 in various tissue zones in M and F rats. Clearly stained were proximal convoluted tubules (S1/S2) in the cortex,
proximal straight tubules in the outer stripe, and descending thin limbs of Henle in the inner stripe and papilla. Capillaries in the inner stripe and papilla were
also weakly stained (not shown). In all zones, the staining intensity of the indicated nephron segments was visibly stronger in M than in F. Scale ⫽ 25 ␮m. E:
pixel intensity of the AQP1 immunostaining in various tissue zones of M and F kidneys. The highest pixel intensity recorded in the M cortex was taken as a
reference, and the pixel intensities in all other tissue zones of M and F kidneys were expressed relative to the highest value. In all tissue zones, the pixel intensity
(relative units) was weaker in F (*P ⬍ 0.05). F: Western blots of AQP1 in TCM from various tissue zones of M and F kidneys (CO, cortex; OS, outer stripe;
IS ⫹ P, inner stripe ⫹ papilla); both NG and G protein bands were weaker in all zones in F. G: densitometric evaluation of the cumulative (NG ⫹ G) AQP1
protein bands shown in F; density of the bands in various zones was 30 – 80% higher in M than in F (*P ⬍ 0.05); n ⫽ 7 in each zone.
F813
Table 1. Separate densitometric evaluation of nonglycosylated and glycosylated forms of the renal AQP1 protein in male
and female rats, as shown by protein bands in the respective figures
Relative Band Density, AU
Fig. No
Tissue/Zone (tissue sample)
Fig. 1A
Whole kidneys (TCM)
Fig. 1F
Cortex (TCM)
Outer stripe (TCM)
Inner stripe ⫹ papilla (TCM)
Fig. 2A
Cortex (BBM)
Cortex (BLM)
Outer stripe (BLM)
Fig. 3A
Whole kidneys (TCM)
n
Nonglycosylated
Glycosylated
6
6
7
7
7
7
7
7
8
8
8
8
8
8
8
8
4
4
0.84 ⫾ 0.06
0.76 ⫾ 0.09*
0.81 ⫾ 0.05
0.69 ⫾ 0.02*
0.95 ⫾ 0.02
0.58 ⫾ 0.01*
0.95 ⫾ 0.03
0.75 ⫾ 0.02*
0.91 ⫾ 0.03
0.80 ⫾ 0.01*
0.79 ⫾ 0.07
0.80 ⫾ 0.06
0.96 ⫾ 0.03
0.78 ⫾ 0.04*
0.91 ⫾ 0.03
0.83 ⫾ 0.03*
0.92 ⫾ 0.05
1.02 ⫾ 0.14
0.71 ⫾ 0.1
0.29 ⫾ 0.04*
0.69 ⫾ 0.07
0.38 ⫾ 0.04*
0.95 ⫾ 0.02
0.34 ⫾ 0.03*
0.92 ⫾ 0.06
0.54 ⫾ 0.01*
0.87 ⫾ 0.04
0.59 ⫾ 0.01*
0.91 ⫾ 0.05
0.52 ⫾ 0.04*
0.92 ⫾ 0.04
0.59 ⫾ 0.05*
0.85 ⫾ 0.06
0.45 ⫾ 0.03*
0.66 ⫾ 0.10
1.19 ⫾ 0.06*
Values are means ⫾ SE of the data measured in the number of animals indicated by n. AQP1, aquaporin 1; TCM, total cell membranes; BBM, brush-border
membranes; BLM, basolateral membranes. *P ⬍ 0.05 (t-test) vs. the same tissue sample in males. Other relations, NS.
The male-dominant sex differences in renal AQP1 protein
expression were further confirmed by Western blotting of
BBM and BLM isolated from the kidney cortex and outer
stripe of adult rats (Fig. 2, A–C); in both kinds of membranes
from both tissue zones, the cumulative band density (NG ⫹ G)
was 20 –30% higher in males than in females (P ⬍ 0.05). The data
in Table 1 (Fig. 2A) show that in isolated BBM and BLM the
male-dominant abundance of AQP1 protein was, overall, ex-
pressed more for the G then for the NG form. Except in BBM
from the outer stripe, where the relative density of NG form was
similar in males and females, the relative density of both forms
was significantly higher in males in other membrane preparations,
and this difference was larger for the G form. Therefore, the
preceding experiments demonstrated in rat kidneys the overall
presence of male-dominant sex differences in the expression of
both AQP1 protein (higher for the G form) and mRNA.
Fig. 2. Sex differences in the expression of AQP1
protein in brush-border membrane (BBM) and basolateral membrane (BLM) isolated from the kidney
CO and OS of 3-mo-old rats. A: representative
Western blots with independent membrane preparations from 4 M and 4 F animals. Both NG and G
bands in the membranes from both tissue zones
were stronger in M than in F. B and C: densitometric
evaluation of the bands shown in A in BBM and
BLM, respectively. The data were collected from 2
independent experiments with membrane preparations from separate animals (n ⫽ 8 in each group).
In the membranes from both tissue zones, the cumulative (NG ⫹ G) band density in both BBM and
BLM was stronger in M than in F (*P ⬍ 0.05).
Outer stripe (BBM)
Sex
Males
Females
Males
Females
Males
Females
Males
Females
Males
Females
Males
Females
Males
Females
Males
Females
Males
Females
F814
Fig. 3. Expression of AQP1 protein in TCM (A and B) and mRNA (C) from the
whole kidneys of prepubertal rats (3 wk old); comparison with the expression
in adult animals. A: representative blot of AQP1 protein in TCM preparations
from separate animals. The density of NG and G bands in prepubertal animals
was similar to that in adult F but much weaker than that in adult M. B: density
of cumulative (NG ⫹ G) bands, shown for prepubertal animals in A was ⬃30%
higher in F (P ⬍ 0.05; n ⫽ 4 in each bar). C: relative expression of AQP1
mRNA; the expression in prepubertal rats was similar in M and F and similar
to that in adult F but significantly (⬃60%; P ⬍ 0.05) weaker than that in M.
Each bar reflects RNA preparation from 4 separate animals. Statistics
(ANOVA/Duncan test): a vs. b, c, or d, P ⬍ 0.05; b vs. c or d, and c vs. d, NS.
Effect of gonadectomy in adult rats. AQP1 protein and
mRNA expression data in sham-operated and gonadectomized
adult rats are shown in Fig. 4. In male rats, castration visibly
downregulated the staining intensity in cortical and outer stripe
tubules (Fig. 4A), and in Western blots of TCM from the same
kidney zones; the relative density (arbitrary units) of protein
bands (NG ⫹ G) in castrated males decreased ⬃40% (Fig. 4,
B and C). As listed in Table 2 (Fig. 4B), a separate quantification of NG and G bands showed a significantly lower density
of both forms in TCM from both cortex and outer stripe of
castrated males, but the differences in the G form were larger.
A comparable pattern was observed in ovariectomized females.
As shown in Fig. 4D, ovariectomy diminished the staining
intensity in both tissue zones, and this phenomenon was
confirmed by weaker protein bands in Western blots of TCM
(Fig. 4E) and by densitometric evaluation of these bands (Fig.
4F); the cumulative band (NG ⫹ G) density (arbitrary units) in
ovariectomized females was ⬃40% and ⬃80% diminished in
the cortex and outer stripe, respectively. A separate quantification of the NG and G protein bands (Table 2, Fig. 4E)
revealed a significant downregulation of both protein forms in
TCM from both tissue zones in ovariectomized rats. These
protein data were only partially supported by mRNA data
shown in Fig. 4G. Whereas AQP1 mRNA expression was
hardly affected by gonadectomy in the cortex of both sexes,
unchanged in males and slightly increased in females, gonadectomy significantly downregulated (⬃40% in males and
⬃30% in females) the expression of AQP1 mRNA in the outer
stripe of both sexes.
Effects of in vivo treatment with sex hormones. The preceding experiments revealed testosterone as one of the sex hormones that potentially affects (increases) the expression of
AQP1 in rat kidneys. However, downregulation of AQP1
protein and mRNA expression in ovariectomized females indicated that the female sex hormones (estrogens and/or progesterone) can also be stimulatory to some extent. This issue
was addressed in castrated male and ovariectomized female
rats treated for 2 wk with oil (controls) or individual sex
hormones (Fig. 5). As shown by Western blots of isolated
TCM from the cortex and outer stripe (Fig. 5A) and by
densitometric evaluation of the protein bands (Fig. 5B), in both
tissue zones of castrated males, the cumulative (NG ⫹ G)
AQP1 protein bands were clearly upregulated by testosterone
treatment (cortex ⬍ outer stripe) but also by progesterone
treatment in the outer stripe, whereas treatment with estradiol
had no effect. The data on separate quantification of NG and G
protein bands (Table 2, Fig. 5A) indicate that 1) testosterone
treatment upregulated both forms in the cortex and outer stripe,
2) estradiol and progesterone treatment downregulated only the
G form in the cortex with the NG form unaffected, and 3)
progesterone, but not estradiol, treatment upregulated both
forms in the outer stripe. A similar pattern of AQP1 expression
(stimulation by testosterone and progesterone, absence of significant estrogen effect) was also visible by immunocytochemical staining in tissue cryosections of the cortex and outer stripe
(Fig. 5C). A comparable pattern of AQP1 protein expression in
isolated TCM was observed in ovariectomized females treated
with various sex hormones (Fig. 5, D and E); testosterone and
progesterone treatment upregulated the expression of AQP1
protein in both tissue zones, whereas estrogen had no effect. As
Renal AQP1 expression in prepubertal rats. The Western
blot in Fig. 3A shows that the NG and G bands of AQP1 in
TCM from the whole kidneys of prepubertal (3 wk old) rats
were weaker than those in adult (3 mo old) animals. However, the relative density (arbitrary unit) of (NG ⫹ G) bands
in prepubertal animals was ⬃30% higher in females than in
males (Fig. 3B). As shown in Table 1 (Fig. 3A), quantification of the specific protein bands revealed similar density of
the NG form in both sexes and female-dominant density of
the G form in females. However, the AQP1 mRNA expression in the whole kidney tissues in prepubertal male and
female rats was similar in both sexes, much lower (⬃50%)
than that in adult males, but similar to that in adult females
(Fig. 3C). This experiment indicates that the male-dominant
expression of renal AQP1 protein and mRNA in rats develops after puberty. The next experiments were performed to
determine the hormone(s) responsible for these differences
in adult rats.
F815
indicated in the blots (Fig. 5D), a dominant effect was again
observed in the expression of the G form.
Urine parameters of fluid excretion, sex differences, and
effect of gonadectomy and treatment with sex hormones. To
determine whether sex differences and related changes in the
renal expression of AQP1 were reflected in urine flow, osmolality, and a few other parameters, we performed several
experiments in age-matched adult rats of both sexes from
which 24-h urine was collected in fasting and nonfasting
conditions. In two independent preliminary experiments in
fasted animals, the data for urine osmolality (mosmol/kg H2O)
showed no significant sex difference [Exp. 1 (16 animals in
each group in the age of 14 wk): 825 ⫾ 45 in males and 839 ⫾
79 in females; Exp. 2 (10 animals in each group in the age of
12 wk): 601 ⫾ 88 in males and 707 ⫾ 87 in females].
However, the urine flow (ml/kg body mass per day) in Exp. 1
Fig. 4. Effect of gonadectomy in adult M and
F rats (3 mo old) on AQP1 expression in the
CO and OS. A: immunostaining in cryosections of kidney tissue from sham-operated
and castrated M. Castration downregulated
the staining intensity in both tissue zones.
Scale ⫽ 50 ␮m for all images. B: Western
blots of isolated TCM. Castration diminished the abundance of AQP1 NG and G
bands in TCM from both tissue zones. C:
densitometric evaluation of the bands shown
in B. In TCM from both tissue zones, the
cumulative band (NG ⫹ G) density was
⬃40% lower in castrated than in sham-operated M (*P ⬍ 0.05; n ⫽ 4 in each group).
D: Immunostaining in cryosections of the
kidney tissues from sham-operated and
ovariectomized F. Ovariectomy downregulated the staining intensity in both tissue
zones. E: Western blots of isolated TCM.
Ovariectomy diminished the abundance of
AQP1 NG and G bands in TCM from both
tissue zones (OS ⬎ CO). F: densitometric
evaluation of the bands shown in E. Compared with that in sham-operated F, the cumulative band (NG ⫹ G) density in the CO
and OS of ovariectomized F was ⬃40% and
⬃80%, respectively (n ⫽ 3 in each group;
*P ⬍ 0.05). G: relative expression of AQP1
mRNA in the CO and OS tissues from shamoperated M and F, castrated M (castr.), and
ovariectomized F (ovariect.). All the data
were calculated relative to the highest expression level in the OS of sham-operated
M. In the CO of both sexes, the AQP1
mRNA expression was not strongly affected
by gonadectomy, whereas, in the OS, gonadectomy in both sexes clearly downregulated
(⬃40% in M and ⬃30% in F) the expression
of this mRNA. n ⫽ 3 in each group. Statistics (ANOVA/Duncan test): a:b and a:e, NS;
c:d, c:g, e:f, g:h, P ⬍ 0.05.
F816
Table 2. Separate densitometric evaluation of nonglycosylated and glycosylated forms of the renal AQP1 protein in male
and female sham-operated and gonadectomized rats, and in sex hormone-treated castrated males, as shown by protein bands
in the respective figures
Relative Band Density, AU
Sex, Treatment, Tissue Zone (sample)
Fig. 4B
Males (TCM)
Sham operated, cortex
Castrated, cortex
Sham operated, outer stripe
Castrated, outer stripe
Females (TCM)
Sham operated, cortex
Ovariectomized, cortex
Sham operated, outer stripe
Ovariectomized, outer stripe
Cortex (TCM)
Oil
Testosterone
Estrogen
Progesterone
Outer stripe (TCM)
Oil
Testosterone
Estrogen
Progesterone
Fig. 4E
Fig. 5A
n
Nonglycosylated
Glycosylated
4
4
4
4
0.95 ⫾ 0.01
0.64 ⫾ 0.03*
0.86 ⫾ 0.04
0.56 ⫾ 0.03*
0.87 ⫾ 0.04
0.38 ⫾ 0.04*
0.83 ⫾ 0.05
0.41 ⫾ 0.04*
3
3
3
3
0.88 ⫾ 0.04
0.58 ⫾ 0.07*
0.86 ⫾ 0.04
0.19 ⫾ 0.01*
0.83 ⫾ 0.05
0.60 ⫾ 0.04*
0.71 ⫾ 0.09
0.10 ⫾ 0.01*
4
4
4
4
0.96 ⫾ 0.02
1.15 ⫾ 0.04†
0.88 ⫾ 0.04
0.99 ⫾ 0.05
0.84 ⫾ 0.06
1.11 ⫾ 0.04†
0.63 ⫾ 0.04†
0.68 ⫾ 0.07†
4
4
4
4
0.79 ⫾ 0.06
1.35 ⫾ 0.03†
0.91 ⫾ 0.05
1.20 ⫾ 0.06†
0.75 ⫾ 0.08
1.99 ⫾ 0.10†
0.86 ⫾ 0.04
1.49 ⫾ 0.19†
Values are means ⫾ SE of the data measured in the number of animals indicated by n. *P ⬍ 0.05 (t-test) vs. the same tissue sample in gonadectomized animals.
†P ⬍ 0.05 (ANOVA/Duncan test) vs. the same tissue sample in oil-treated castrates.
in females (61 ⫾ 6.4) was ⬃50% higher than in males (44 ⫾
4.0; P ⬍ 0.05), whereas, in Exp. 2, for unknown reasons the
data in both sexes were much higher and heterogeneous and
only slightly lower in males (94 ⫾ 13.5) than in females (103 ⫾
14.8; NS). We then compared various urinary and other parameters in the third experiment, using groups of age-matched
adult male and female rats, from which the 24-h urine was
collected first in fasting conditions 8 days before death. After
7 days of a recovery period with free access to food and water,
the urine was collected again from these rats in nonfasting
conditions 1 day before euthanasia. In addition to urine parameters, we measured water and food consumption during urine
collection, as well as urine protein, plasma, and urine creatinine to calculate the creatinine clearance. Body and kidney
mass was measured to calculate the kidney mass index. As
listed in Table 3, in fasted animals, 1) water consumption was
⬃90% higher in females; 2) proteinuria was 10-fold higher in
males, which is a well-known phenomenon in Wistar rats (53);
3) urine creatinine and osmolality showed a slight tendency to
be higher in males (vs. females, NS); 4) GFR was similar in
both sexes; 5) urine flow was ⬃85% higher in females; and 6)
in both sexes the urine flow was equal to water consumption.
In nonfasted animals, 1) the kidney mass was ⬃60% higher in
males, but the kidney mass index exhibited no sex difference;
2) water and food consumption was higher in females (35%
and 29%, respectively), with water consumption similar to that
in fasted animals of the same sex; 3) plasma creatinine was
similar in both sexes but with urine creatinine ⬃50% higher in
males, and in both sexes it was higher than in the respective
fasted animals; 4) GFR (creatinine clearance) was similar in
both sexes and comparable to that in fasted animals; 5) urine
flow was ⬃37% higher in females, ⬃40% lower than water
consumption in both sexes, and significantly (⬃30% in males
and ⬃50% in females) lower than in the respective fasted
animals; and 6) urine osmolality was slightly higher in males
(vs. females, NS), but in both sexes it was two- to threefold
higher than in the respective fasted animals.
Urine flow and osmolality were further tested in shamoperated and gonadectomized male and female rats and in
castrated males treated for 2 wk with various sex hormones
(Table 4). Proteinuria was a useful parameter for checking the
efficiency of gonadectomy and hormonal treatment. As shown
in Table 4, castration strongly decreased proteinuria; this
decrease was largely reversed by testosterone treatment but not
by estradiol and progesterone treatment. In sham-operated,
castrated, and hormone-treated castrated animals, the urine
flow was similar, as was urine osmolality, which was high and
exhibited considerable variability in all groups. On the other
hand, in females, 1) urine protein was low and unaffected by
ovariectomy, 2) urine flow was high in sham-operated and
⬃40% lower in ovariectomized animals, and 3) urine osmolality was low and similar in sham-operated and ovariectomized
animals.
DISCUSSION
By immunochemical studies in tissue cryosections and isolated membranes and by mRNA studies with real-time RTPCR, in rat kidneys we demonstrated a significantly higher
level of expression of AQP1 in male compared with female
rats, which occurs after puberty because of a strong androgen
and weaker progesterone stimulation. These differences were
localized in both the proximal tubule and TDLH and were most
evident in the proximal tubule S3 segment. The overall
changes in AQP1 protein expression in intact adult, prepubertal, and gonadectomized adult rats match the mRNA expression patterns, indicating that androgens are the major stimulators of transcription. These data confirm previous findings in
Fig. No.
F817
rats, where gonadectomy decreased blood pressure and RAS
activity and increased Na⫹ and fluid excretion, whereas the
treatment of gonadectomized animals with androgens had an
opposite effect, but the treatment with estradiol was ineffective
(48 –51). However, a diminished AQP1 expression in ovariectomized females and its limited upregulation by progesterone
treatment of castrated males, as shown here, suggest that
progesterone may be responsible for physiological and pathophysiological AQP1-mediated renal handling of water in various stages of gestation (22).
The presence of sex differences in renal AQP1 expression
was predicted on the basis of numerous previous studies, which
showed that androgens in several animal models and in humans
contribute to high blood pressure by promoting Na⫹ and fluid
Fig. 5. Effect of treatment with sex hormones in castrated M (A–C) and ovariectomized F (D and E) rats on renal AQP1
protein expression in CO and OS. A: Western blots of TCM isolated from CO and OS
of castrated M treated subcutaneously with
oil (O), testosterone (T), estradiol (E), or
progesterone (P) in oil for 14 days. B: densitometric evaluation of the cumulative (G ⫹
NG) bands shown in A. The data were collected from 2 independent experiments with
2 animals/group in each experiment (n ⫽ 4
in all groups). T treatment stimulated the
AQP1 protein expression in TCM from both
CO and OS (CO ⬍ OS), whereas P treatment
stimulated the protein abundance in the OS
membranes. Statistics: a:b, c:d, d:c, d:e, and
c:e, P ⬍ 0.05; a:a, b:b, and c:c, NS. C:
immunostaining of AQP1 in cryosections of
the kidney CO and OS tissues. The staining
intensity in both tissue zones largely confirmed the Western blot data, e.g., the T and
P treatment upregulated (T ⬎ P), whereas
the E treatment had no effect. Scale ⫽ 50
␮m. D: Western blots of TCM isolated from
CO and OS of ovariectomized F rats treated
subcutaneously with O, E, P, or T for 14
days. E: densitometric evaluation of the cumulative (G ⫹ NG) bands shown in D. The
data were collected from 2 independent experiments with 2 animals/group in each experiment (n ⫽ 4 in all groups). P and T
treatment stimulated the AQP1 protein expression in TCM from both CO and OS,
whereas E treatment had no effect in either
zone. Statistics: a:a, d:d, and e:f, NS; a:b,
a:c, b:c, d:e, and d:f, P ⬍ 0.05.
F818
Table 3. Various parameters in intact adult male and female rats
Fasted
Nonfasted
Parameters
Females
Males
Females
Males
Body mass, g
Kidney mass, g
Kidney mass index, mg/g body mass
Water consumption, ml/kg body mass per day
Food consumption, g/kg body mass per day
Plasma creatinine, ␮mol/l
Urine protein, mg/kg body mass per day
Urine creatinine, mmol/l
GFR, ml·min⫺1·kg⫺1 body mass
Urine flow, ml/kg body mass per day
Urine osmolality, mosmol/kgH2O
314 ⫾ 3
N.D.
N.D.
69 ⫾ 12.9
N.A.
N.D.
32 ⫾ 3.1
5.57 ⫾ 1.04
8.38 ⫾ 0.27§
73 ⫾ 10.3
691 ⫾ 112
192 ⫾ 2*
N.D.
N.D.
131 ⫾ 25.1*
N.A.
N.D.
3.1. ⫾ 0.4*
3.18 ⫾ 0.61
8.54 ⫾ 0.36§
135 ⫾ 21.1*
500 ⫾ 88
345 ⫾ 3
2.28 ⫾ 0.05
6.59 ⫾ 0.16
80 ⫾ 4.4
59 ⫾ 3.1
26.4 ⫾ 0.62
N.D.
7.90 ⫾ 0.69
9.78 ⫾ 0.66
49 ⫾ 3.8‡
1529 ⫾ 104‡
211 ⫾ 3†
1.40 ⫾ 0.04†
6.63 ⫾ 0.12
108 ⫾ 10.4†
76 ⫾ 5.3†
26.8 ⫾ 1.42
N.D.
5.26 ⫾ 0.71†‡
8.32 ⫾ 0.53
67 ⫾ 7.2†‡
1352 ⫾ 131‡
reabsorption in the renal proximal tubule (14, 23, 49, 69).
Although the mechanism of androgen action in increasing
blood pressure is not known in detail, studies in experimental
animals and humans suggest that androgens may work via
stimulation of the activity of systemic and/or autonomous RAS
in the proximal tubule, where the chain of reactions starts with
increase in plasma and renal renin and ends with the elevated
production of ANG II (14, 15, 23, 26, 49, 62, and references in
these reviews). In the proximal tubule, ANG II, via its AT1
receptors in the BBM and BLM, stimulates the activity and/or
expression of several Na⫹ transporters, thus enhancing transepithelial reabsorption of Na⫹, which is followed by fluid
movement (33, 46, 48, 54, 71). The increased fluid reabsorption in proximal tubules of androgen-treated rats was diminished by inhibitors of ANG II production and AT1 receptor
activity (48), whereas our recent studies in rats and in immortalized rat proximal tubule cells (IRPTC) in culture showed
that ANG II stimulates the expression of AQP1 protein in
proximal tubules and TDLH and both AQP1 protein and
mRNA in IRPTC. The ANG II-dependent elevated expression
of AQP1 was abolished by AT1 receptor blockers (5), thus
indicating ANG II as a possible direct mediator of androgen
action in the proximal tubule. However, ANG II may also act
indirectly, by stimulating production and release of aldosterone
in the human and rat adrenal cortical cells, which can upregulate NHE3 in the proximal tubule BBM and increase Na⫹ and
volume reabsorption and thus can contribute to elevation of
blood pressure (26, 27). However, treatment of adrenalectomized rats with aldosterone had no effect on the renal expression of AQP1 (28).
Although most data indicate that androgen-stimulated fluid
reabsorption in the proximal tubule may be mediated by ANG
II-dependent activities, we cannot exclude the possibility of a
genomic regulation of AQP1 via androgen receptor-mediated
transcription. This is possible for progesterone action also.
Androgen receptors are present in both rodent and human
proximal tubules (reviewed in Ref. 56), and the nonsteroidal
anti-androgen drug, flutamide, which blocks the activity of
androgen receptors, decreases blood pressure in male SHR (49,
52). However, although some other aquaporins, such as human
AQP5 (37), and possibly rat AQP9 (21, 43), do express
androgen-responsive elements (ARE) in their promoters, potential ARE sites in the 5=- and 3=-flanking DNA region of the
rat AQP1 gene could not be detected. Another possibility is
that androgens work indirectly, via the glucocorticoid-responsive element (GRE); the 4.4-kb promoter of mouse AQP1
contains several GREs that may bind androgens and be involved in the transcriptional regulation of AQP1 gene (38).
Table 4. Urine parameters in male and female rats and effect of gonadectomy and treatment with sex hormones in
gonadectomized males
Males
Sham operated ⫹ Oil
Castrated ⫹ Oil
Castrated ⫹ Testosterone
Castrated ⫹ Estradiol
Castrated ⫹ Progesterone
Females
Sham-operated
Ovariectomized
n
Urine Protein, mg/kg body mass per day
Urine Flow, ml/kg body mass per day
Urine Osmolality, mosmol/kgH2O
7
8
8
9
9
23.9 ⫾ 2.1
4.7 ⫾ 0.3*
18.0 ⫾ 1.9†
3.6 ⫾ 0.5
3.8 ⫾ 0.3
43.0 ⫾ 7.6
40.7 ⫾ 8.9
40.5 ⫾ 6.4
42.5 ⫾ 11.7
30.9 ⫾ 8.7
998 ⫾ 211
1,083 ⫾ 149
958 ⫾ 154
1,434 ⫾ 362
1,347 ⫾ 150
8
8
3.0 ⫾ 0.3
3.1 ⫾ 0.3
113 ⫾ 12.6
68 ⫾ 9.2‡
445 ⫾ 58
583 ⫾ 89
Values are means ⫾ SE of the data collected from the number of animals indicated by n. The indicated parameters were calculated from 24-h urine from fasted
animals. Statistically significant difference (P ⬍ 0.05): *compared with sham-operated ⫹ oil, †compared with castrated ⫹ oil (ANOVA/Duncan test), and
‡compared with sham-operated females (t-test). Other relations, not significant.
Values are means ⫾ SE. The same animals (n ⫽ 10 in each group) underwent 24-h urine collection in metabolic cages in the absence (fasted; age ⫽ 12 wk)
and presence (nonfasted; age ⫽ 13 wk) of pelleted food. Statistically significant difference (t-test; P ⬍ 0.05) was observed in comparison with fasted *or
nonfasted †females, or in comparison with the same sex in fasted animals ‡. Other relations are not significant. §Because blood creatinine is a fairly stable
parameter in healthy animals, and fasted and nonfasted rats exhibit similar blood creatinine (1), glomerular filtration rate (GFR, creatinine clearance) in fasted
animals was calculated using the data for urine creatinine from fasted animals and plasma creatinine from nonfasted animals. Urine protein in nonfasted animals
was not measured because the collected urine was variously contaminated with the food particles. The ionic content in these particles may have also contributed
with unknown extent to the level and variability of urine osmolality in nonfasted animals. N.D., not determined; N.A., not applied.
treatment for 2 wk with various sex hormones had any significant effect on urine flow and osmolality. However, the data in
Fig. 5 showed a significant testosterone- and progesteroneinduced upregulation of the AQP1 protein expression in TCM
from the renal cortex and outer stripe in castrated males and
ovariectomized females. Rather, the data may be related to the
previously described sexually dimorphic renal responsiveness
to endogenous vasopressin. Although both sexes have similar
levels of plasma vasopressin, the kidneys in male rats have a
higher density of vasopressin receptors and react with a significantly greater antidiuretic response to vasopressin, leading
to lower urine flow (higher fluid retention) and lower daily
water consumption in males, compared with higher urine flow
(higher fluid excretion) and greater water consumption in
females (66, 67). We also confirmed these prior findings that
castration in males had no effect on urine flow, whereas
ovariectomy in females decreased urine flow because the
estrogen-controlled inhibition of the vasopressin response in
the kidney was removed. Therefore, the male-dominant expression of AQP1 may be important locally, in the proximal parts
of the nephron, possibly serving ANG II-mediated, androgenstimulated Na⫹ reabsorption via the activity/expression of
several Na⫹-reabsorptive mechanisms (33, 46, 48, 54, 71), but
the sex-dependent, AQP1-mediated effects on fluid handling
along the proximal nephron may be masked by the also
sex-dependent vasopressin/AQP2-mediated activity in the collecting duct.
ACKNOWLEDGMENTS
The authors thank Mrs. Eva Heršak for technical assistance.
Present address M. Matokanović: Department of Medical Biochemistry and
Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia. Present address for H. Brzica: Department of Anatomy,
Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia.
GRANTS
This work was supported partially by grant no. 022-0222148-2146 (I.
Sabolić) from the Ministry for Science, Education, and Sports, Republic of
Croatia, and partially by grant no. 1481 (I. Sabolić) from the Croatian Science
Foundation. D. Brown was supported by NIH grant DK096586.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: C.M.H.-K., D. Breljak, M. Ljubojević;, M.M., M.
Lovrić, D.R., H.B., I.V., D.K., V.M., and J.I.D. performed experiments;
C.M.H.-K., D. Breljak, M. Ljubojević;, M.M., M. Lovrić, D.R., H.B., I.V.,
D.K., V.M., J.I.D., and I.S. analyzed data; C.M.H.-K., D. Breljak, M. Ljubojević;, M.M., M. Lovrić, D.R., V.M., J.I.D., D. Brown, and I.S. interpreted
results of experiments; C.M.H.-K., D. Breljak, M. Ljubojević;, M.M., I.V.,
D.K., and I.S. prepared figures; C.M.H.-K. and D. Breljak drafted manuscript;
C.M.H.-K., D. Brown, and I.S. edited and revised manuscript; D. Brown and
I.S. designed conception of research; D. Brown and I.S. approved final version
of manuscript.
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12: 331–336, 2008.
Regarding the upregulating effects of progesterone, intracellular progesterone receptors were demonstrated in mouse proximal tubules (32), but the presence of progesterone-responsive
elements in the AQP1 promoter could not be indicated by
searching the available literature.
Although most of our data showed that testosterone stimulates the expression of both NG and G forms of renal AQP1
protein, the effects on G form were always stronger, thus
indicating that androgens, not only enhanced the AQP1 gene
transcription, as shown by the increased expression of AQP1
mRNA in our RT-PCR studies, but also stimulated the glycosylation of AQP1 protein. Information on whether testosterone
stimulates glycosylation or inhibits deglycosylation of proteins
could not be found in the literature. However, previous studies
showed that glycosylation is not required for expression or
function of AQP1; both NG and G forms colocalized in the cell
membrane, forming homotetramers, and exhibited similar permeability for water (11, 46, 65). As reviewed elsewhere (45,
64), glycosylation is generally important for the folding, quality control, sorting, stability (resistance to proteolysis), interaction with other proteins, and intracellular trafficking. In most
cases, the glycosylation-defective proteins could not be directed to the cell membrane and accumulated in an intracellular
compartment and/or underwent faster degradation, indicating
that glycosylation affects intracellular targeting and protein
stability. Some proteins also exhibited impaired or lost affinity
for substrate and/or activity. Because NG and G forms of
AQP1 have similar water permeability, the stimulatory role of
testosterone (and, to a lesser extent, progesterone) in glycosylation of renal AQP1 is unclear.
Several functional parameters in the urine of fasted and
nonfasted rats were examined here to determine their possible
link with the sex-dependent expression of AQP1 protein. Some
related studies had been performed previously only in males
(1); fasting (starvation) in male rats impaired urinary concentration ability, resulting in polyuria and low urine osmolality.
The findings were associated with decreased abundance of the
AQP2 protein in the cortical collecting duct and unchanged
abundance of AQP1 protein in proximal tubules. In our study,
although the absolute values of urine flow exhibited some
heterogeneity, the GFR in fasted and nonfasted males and
females was similar, and, overall, fasted rats exhibited higher
urine flow and much lower urine osmolality. In our fasted rats,
water consumption and urine flow fully matched, both being
sex related (males ⬍ females), with a tendency toward a higher
urine osmolality in males. In nonfasted rats, a basically similar
pattern was observed for GFR (males ⫽ females), with water
consumption and urine flow both being higher in females.
However, the overall urine flow in both sexes was 40 –50%
lower than in fasted animals and significantly lower than water
consumption, whereas urine osmolality was two- to threefold
higher than in fasted animals. The preceding data on water
consumption and urine flow in nonfasted animals may be at
least partially related to food consumption, which was 29%
higher in females.
Although in the preceding studies nearly all functional
parameters except for urine osmolality were lower in males, it
is unclear whether these phenomena in either fasted or nonfasted rats have any connection with the male-dominant renal
expression of AQP1. This argument is particularly valid seeing
the data in Table 4, which showed that neither castration nor
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the rat nephron Sex-dependent expression of

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