Urea transport in kidney brush-border membrane vesicles from an elasmobranch, Raja erinacea
Department of Zoology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
* Author for correspondence (e-mail: patwrigh{at}uoguelph.ca)
Accepted 23 June 2003
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Summary |
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Key words: brush-border plasma membrane, phloretin, urea permeability, little skate, Raja erinacea
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Introduction |
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The gill, with its large surface area, is the dominant site of urea loss to
the environment, accounting for 9396% of urea excretion in
elasmobranchs (Payan et al.,
1973; Wood et al.,
1995
). The relatively low permeability of the gill to urea has
long been known (Boylan, 1967
),
and is estimated to be 82 times less permeable to urea relative to the rainbow
trout gill (Pärt et al.,
1998
). Recent studies have demonstrated the presence of a
secondary active gill basolateral Na+urea antiporter moving
urea from the gill back into the blood against the gradient
(Fines et al., 2001
). In
addition, the extraordinarily high cholesterol content in the basolateral
membrane would retard passive urea loss from the gill
(Fines et al., 2001
). The
other potential site of urea loss is the kidney, where approximately
9096% of the urea in the glomerular filtrate is reabsorbed
(Clark and Smith, 1932
;
Goldstein and Forster, 1971
;
Payan et al., 1973
),
ultimately resulting in only 47% of urea excretion to the environment
(Payan et al., 1973
;
Wood et al., 1995
).
The elasmobranch nephron consists of five loops, which appear to be
arranged in a countercurrent fashion, and can be separated into two distinct
anatomical regions: the dorsallateral bundle, enclosed by connective
tissue, and the ventral mass, where loops wind convolutedly in a blood sinus
(Lacy and Reale, 1985). A
possibly extensive capillary network and a `central vessel' are also present
in a countercurrent arrangement to many of the nephron segments
(Hentschel, 1988
). Based on
the countercurrent arrangement of tubules and micropuncture data, Boylan
(1972
) suggested a model of
passive reabsorption of urea, where the fluid in the terminal segment has a
higher urea concentration than the surrounding environment, allowing for
reabsorption passively down the concentration gradient.
Another aspect of urea reabsorption in the elasmobranch kidney that is not
clearly understood is the involvement of carrier-mediated urea transporters in
some or all tubule segments. Experiments on whole animals have indicated that
in the dogfish kidney, urea reabsorption is active and selective for amide or
amide-like compounds (Schmidt-Nielsen and
Rabinowitz, 1964), inhibitable by phloretin
(Hays et al., 1976
), and
appears to be linked to sodium reabsorption
(Schmidt-Nielsen et al.,
1972
). Although several models have been suggested for the
possible arrangement of urea transporters in the elasmobranch kidney
(Walsh and Smith, 2001
), these
models are based on very little experimental evidence. In a companion study,
we have isolated a partial cDNA for a skate kidney urea transporter (SkUT),
similar to other facilitated UTs, in both the dorsal and ventral regions of
the kidney (Morgan et al.,
2003
). Renal SkUT mRNA levels are downregulated with exposure to
environmental dilution, suggesting a possible role for SkUT in urea
reabsorption and retention.
To further characterize urea transport in the elasmobranch kidney, rates of urea uptake were measured using a rapid filtration method on resealed vesicles prepared from purified brush-border membranes from the two regions of the kidney. Characterization of urea transport measured the 14C-urea uptake in the presence of various urea concentrations, competitive and non-competitive urea transport inhibitors, and energy sources (ATP or ion gradients).
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Materials and methods |
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Kidney brush-border plasma membrane vesicles
Brush-border membrane vesicles (BBMV) were prepared using methods involving
calcium precipitation and differential centrifugation outlined by Bevan et al.
(1989) and Kipp et al.
(1997
), with some
modifications. All steps were carried out at 04°C. Skates were
killed by a blow to the head, followed by severance of the spinal cord, after
which both kidneys were rapidly excised and placed on ice. The kidneys were
then divided into dorsolateral and ventral sections, based on the appearance
of the sections as described by Hentschel et al.
(1986
). Approximately 0.75 g
tissue (wet mass) was homogenized in 15 ml of homogenizing buffer containing
10 mmol l1 D-mannitol, 2 mmol
l1 Tris-HCl, pH 7.1, using a Polytron (Brinkmann,
Mississauga, ON, Canada) homogenizer for 15 s, followed by a 30 s interval and
then an additional homogenization of 15 s. A sample (1 ml) was taken from this
homogenate and used immediately for enzymatic analysis. The rest of the
homogenate was filtered through cheesecloth and CaCl2 was added to
a final concentration of 30 mmol l1. After 15 min, the
preparation was centrifuged at 1400 g for 12 min, the
supernatant decanted into a clean centrifuge tube, and centrifuged for 20 min
at 15 800 g. The resulting pellet was resuspended in buffer
and homogenized in a glassTeflon homogenizer at low speed. The volume
was adjusted to a final volume of 15 ml with buffer and CaCl2 was
added to a final concentration of 30 mmol l1. After 15 min,
the preparation was centrifuged at 2200 g for 12 min, the
supernatant decanted and centrifuged for 20 min at 20 000 g.
The resulting pellet was suspended in 10 ml vesicle buffer containing 100 mmol
l1 D-mannitol, 1.5 mmol l1
CaCl2, 20 mmol l1 Hepes, 20 mmol
l1 Tris, pH 7.4, and centrifuged at 47 800 g
for 20 min. The final pellet of enriched brush-border membranes was
resuspended in 0.5 ml of vesicle buffer by passage through a 23-gauge needle
(10 times) to aid vesicle formation, and used immediately in further
studies.
Validation of membrane preparation
The relative purity of the final preparation and the relative contamination
of the membrane preparation by other cellular membranes were assessed by
enzymatic assays on the initial homogenate and the final pellet of enriched
BBMV. Alkaline phosphatase (Gasser and
Kirschner, 1987), Na+,K+-ATPase
(McCormick, 1993
), cytochrome
c oxidase (Blier and Guderley,
1988
), and glucose-6-phosphatase
(Stio et al., 1988
) were used
as marker enzymes for brush-border membrane, basolateral membrane, inner
mitochondrial membrane and endoplasmic reticulum, respectively. All
measurements were made in duplicate at 25°C using a spectrophotometer
(Hewlett Packard, Mississauga, ON, Canada).
Orientation of brush-border membrane vesicles
The orientation of the membrane vesicles was determined using the methods
of Giudicelli et al. (1985)
and Drai et al. (1990
). This
method is based on the inability of starch to cross the membrane and on the
orientation of maltaseglucoamylase in the brush-border membrane. In
brief, enriched BBMV were incubated for 10 min with or without detergent
(0.04% n-octyl ß-D-glucopyranoside). The activity of
maltase-glucoamylase (a brush-border membrane enzyme) was determined by
evaluating the hydrolysis of soluble starch (10 mg ml1) in
sodium citrate buffer (0.1 mol l1, pH 6.5). Glucose
production after 20 min was measured using a glucose oxidase reaction (Sigma
kit). The percentage of inside-out vesicles was calculated as the difference
in the enzyme activity with and without treatment in detergent. With detergent
treatment, all possible enzyme activity is revealed (i.e. right-side-out,
inside-out and leaky vesicles) while without detergent treatment only enzyme
activity in right-side-out and leaky vesicles will be present.
Urea transport assays
Transport of 14C-urea was performed at 10°C by a rapid
filtration method as previously described
(Fines et al., 2001). Freshly
prepared BBMV were suspended in 10 ml of buffer containing 300 mmol
l1 NaCl, 5.2 mmol l1 KCl, 2.7 mmol
l1 MgSO4, 5 mmol l1
CaCl2, 15 mmol l1 Tris-HCl, pH 7.4, 370 mmol
l1 D-mannitol and equilibrated on ice for 30 min.
The BBMV were then collected by centrifugation at 47800 g for
20 min and resuspended in the same buffer at a protein concentration of
approximately 0.3 mg ml1. Thorough mixing was achieved by
passage through a 23-gauge needle (10 times). Transport experiments were
initiated by the addition of 40 µl of radioactive elasmobranch isolation
medium (EIM) (containing 1.85 MBq 14C-urea) to 10 µl of BBMV
suspension. EIM contained 300 mmol l1 NaCl, 5.2 mmol
l1 KCl, 2.7 mmol l1 MgSO4, 5
mmol l1 CaCl2, 370 mmol l1
urea, 15 mmol l1 Tris-HCl, pH 7.4. Urea uptake was measured
over a range of urea concentrations (0.2370 mmol l1).
In EIM containing less than 370 mmol l1 urea, total
osmolarities of the EIM solutions were maintained using a balance of
D-mannitol, which functions as an osmotic replacement. Uptake was
terminated at 8 s (dorsal) and 5 s (ventral) intervals by rapid dilution of
the incubation solution with 1 ml of ice-cold stop solution (EIM containing
370 mmol l1 urea). These times were shown to provide valid
estimates of the initial rates of transport (data not shown). The diluted
sample was immediately filtered through pre-wetted filters (Millipore Isopore,
0.4 µm HTTP type). Filters were washed with 2x 3 ml of ice-cold stop
solution and placed in a scintillation vial with 15 ml of ScintiSafe Econo F
scintillation fluid (Fisher). Each preparation was measured in duplicate.
Inhibition assays
Inhibition of urea transport was examined to further define the properties
of the transporter. The urea analogues thiourea, acetamide,
N-methylurea and 1-(4-nitrophenyl)-2-thiourea (NPTU), were tested as
known competitive inhibitors in other urea transport systems. BBMVs were
prepared in the same manner as above, except vesicles were preincubated with
analogues, by adding thiourea, acetamide, or N-methylurea (370 mmol
l1) to EIM solution, instead of the mannitol suspension
buffer. These vesicles were then incubated with 0.5 mmol l1
urea (with 74 kBq of 14C-urea) and treated as described above. The
analogue NPTU was used at a final concentration of 0.08 mmol
l1 in EIM due to its low solubility. Prior to the addition
of the incubation mixture, 10 µl of the NPTU solution was added to the BBMV
and rapidly mixed. Urea uptake was then measured as described above.
The inhibitors phloretin (0.250.5 mmol l1) in ethanol (0.75%) and HgCl2 (0.3 mmol l1 in EIM solution) and a combination of both inhibitors (0.5 mmol l1 phloretin and 0.3 mmol l1 HgCl2 in 0.75% ethanol) were tested. Prior to the addition of 0.5 mmol l1 urea (containing 74 kBq of 14C-urea), 10 µl of the inhibitor or control (0.75% ethanol or EIM solution) was added to the BBMV and mixed. Urea uptake was measured as described above.
ATP dependence
The ATP dependence was determined to evaluate the requirement for ATP as an
energy source. In previous studies from our laboratories, elasmobranch gill
urea transport was found to be dependent on ATP and a Na+ gradient
(Fines et al., 2001). Urea
uptake was measured in EIM containing 4 mmol l1 urea
(containing 74 kBq of 14C-urea). This concentration of urea was
used to examine transport below the non-saturable range. Individual solutions
contained ATP (10 mmol l1), or ATP (10 mmol
l1) and ouabain (1 mmol l1), or ATP (10
mmol l1) and N-ethylmaleimide (NEM) (1 mmol
l1), and a control (no additions). If urea transport was
altered by NEM, this would indicate the presence of V-type and P-type ATPases
(i.e. proton pumps; Ehrenfeld,
1998
). Urea uptake was measured as described above.
Cation specificity
Cation specificity of urea transport was also examined in the BBMVs, using
modified resuspension buffer and radioactive mixture containing only one of
the following salts: KCl or NaCl. BBMV were prepared as described above and
then separated for use with individual cations.
Sodium
The final pellet was suspended in a medium containing 370 mmol
l1 D-mannitol, 2.7 mmol l1
MgSO4, 5 mmol l1 CaCl2, 15 mmol
l1 Tris-HCl, 25 mmol l1 NaCl and 225 mmol
l1 N-methyl-D-glucamine (NMDG, an
osmotic replacement). The BBMV were allowed to equilibrate on ice for 30 min
before being collected by centrifugation at 47800 g for 20 min
and resuspended in the same medium at a known protein concentration of
approximately 0.3 mg ml1. Control incubations used the same
medium in which the final pellet was resuspended. Gradient incubations
contained 250 mmol l1 NaCl and no NMDG, in order to produce
an inwardly-directed gradient across the BBM, similar to that found in the
skate kidney. The incubation medium contained 4 mmol l1 urea
and 366 mmol l1 mannitol in place of the 370 mmol
l1 mannitol and 1.85 MBq ml1 14C-urea.
Potassium
The final pellet was suspended in a medium containing 370 mmol
l1 D-mannitol, 2.7 mmol l1
MgSO4, 5 mmol l1 CaCl2, 15 mmol
l1 Tris-HCl, 250 mmol l1 KCl. The BBMV
were allowed to equilibrate on ice for 30 min before being collected by
centrifugation at 47800 g for 20 min and resuspended in the
same medium at a known protein concentration of approximately 0.3 mg
ml1. Control incubations used the same medium in which the
final pellet was resuspended. Gradient incubations contained 25 mmol
l1 KCl and 225 mmol l1 NMDG, in order to
produce an outwardly-directed gradient across the BBM, similar to that found
in the skate kidney. The incubation medium contained 4 mmol
l1 urea and 366 mmol l1 mannitol in place
of the 370 mmol l1 mannitol and 1.85 MBq ml1
14C-urea.
Protein determination
The protein concentration of the BBMV preparations was determined by the
method of Bradford (1976) using
a Bio-Rad kit (Richmond, CA, USA) with bovine serum albumin as the
standard.
Statistical analysis
Values are expressed as means ± standard error of the mean
(S.E.M.). An F-test for comparison of curves was used in
determination of best fit of regression lines. Data from functional vesicles
studies (analogs and inhibitors) were not normally distributed, and therefore
a log-transformation was performed to satisfy the assumption of normality
before further statistical analysis. Statistical comparisons were made by
one-way analysis of variance (ANOVA), and secondary tests were performed using
the TukeyKramer multiple comparison test or Student's t-test.
Values were considered statistically significant if P<0.05.
Source of chemicals
N-methylurea was obtained from Fluka through Sigma-Aldrich
Chemicals. 14C-urea was obtained either from Amersham Life Science
(Baie d'Urfé, Quebec) or Sigma Chemical (St Louis, MO, USA). All other
chemical were obtained from either Fisher Scientific (Whitby, ON, Canada) or
Sigma Chemicals (Oakville, ON, Canada) and were of reagent grade.
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Results |
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In tissue from the dorsal section of the kidney, the specific activities of glucose-6-phosphatase (endoplasmic reticulum) and cytochrome c oxidase (mitochondria) indicated a contamination of these marker enzymes of 0.44-fold and 0.35-fold, respectively (Table 1). The Na+,K+-ATPase (basolateral membrane) was slightly enriched in the BBMV preparation with an increase in specific activity of 1.12-fold (Table 1). However, based on the total activity of Na+,K+-ATPase, there was only 0.34% recovery of the initial amount of enzyme and 0.47% contamination (percentage of total alkaline phosphatase activity) (Table 2). Marker enzymes for the endoplasmic reticulum and inner mitochondrial membrane had recoveries of 0.15% and 0.11% of the initial amount of enzyme and contributed 0.03% and 0.49% contamination, respectively (Table 2). Analysis of maltoseglucoamylase activity (Table 3) indicates that only 5.0% of vesicles are sealed inside-out, the remaining percentage of vesicles were right-side-out or leaky.
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In the ventral section of the kidney, the specific activities of cytochrome c oxidase (mitochondria) indicated a contamination with inner mitochondrial membrane enzyme of 0.23-fold (Table 1). The marker enzymes for the basolateral membrane and endoplasmic reticulum were slightly enriched in the BBMV preparation with an increase in specific activity of 1.12- and 1.06-fold, respectively (Table 1). However, based on the total activity of Na+,K+-ATPase (basolateral membrane) and glucose-6-phosphatase (endoplasmic reticulum) there was only 0.42% and 0.44% recovery of the initial amount of enzyme and 0.38% and 0.09% contamination (percentage of total alkaline phosphatase activity), respectively (Table 2). Cytochrome c oxidase had a recovery of 0.09% of the initial amount of enzyme and contributed 0.37% contamination (Table 2). The activity of maltoseglucoamylase revealed that 4.6% of vesicles have an inside-out orientation (Table 3), so the majority of vesicles are right-side-out or leaky.
Final recovery of brush-border membrane was 20.52% and 23.94% for dorsal
and ventral sections, respectively (Table
2), which is similar to that reported by Bijvelds et al.
(1997).
Concentration dependence of urea uptake by BBMV
Urea uptake when measured over a range of urea concentrations in the
incubation medium revealed one component of urea uptake in the dorsal BBMV and
two components in the ventral BBMV (Figs
1,
2). At high concentrations of
urea (5370 mmol l1), the uptake was linearly
dependent upon the urea concentration in both regions (Figs
1A,
2A). At low concentrations of
urea (0.22 mmol l1), when the linear rate at high
concentrations of urea has been subtracted from the curve, urea uptake
exhibits saturation-like kinetics in the ventral BBMV
(Fig. 2B). In the dorsal BBMV,
however, there is a linear relationship at low urea concentrations, indicating
non-saturation (F-test, Fig.
1B); however the apparent linear relationship may be the result of
multiple saturation curves. When the ventral data are transformed using a
LineweaverBurk plot, urea uptake at urea concentrations of 0.22
mmol l1 had a Km of 0.70±0.20
mmol l1, and a Vmax of 1.18±0.39
µmol h1 mg1 protein for the ventral
BBMV (Fig. 2C).
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Inhibition of urea uptake
Urea uptake by both dorsal and ventral BBMV demonstrated sensitivity to the
non-competitive inhibitors phloretin and mercury chloride
(Fig. 3). There was a
dose-dependent phloretin inhibition of urea uptake by BBMV, with inhibition at
0.25 mmol l1 of 24% and 22%, for dorsal and ventral,
respectively (not shown), and at 0.50 mmol l1 of 37% and
55%, for dorsal and ventral, respectively (P<0.05;
Fig. 3). Mercury chloride
inhibited urea uptake in both dorsal and ventral BBMV (P<0.05;
Fig. 3). As well, the addition
of phloretin and mercury chloride together also significantly inhibited urea
uptake compared to control rates (P<0.05,
Fig. 3). In the dorsal section,
this inhibition was significantly greater than that of phloretin alone
(P<0.05, Fig. 3).
There was no change to the control rate of urea transport with the addition of
ethanol vehicle (data not shown). The use of urea analogues changed the rate
of urea uptake in the dorsal BBMV (Fig.
4). Here, nitrophenylthiourea (NPTU) significantly reduced urea
uptake, but this reduction was not seen in the ventral BBMV. There was no
significant effect on urea uptake in either section by the urea analogues,
acetamide and N-methylurea (Fig.
4). There was a significant difference between the dorsal and
ventral regions in response to acetamide (P<0.05,
Fig. 4).
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ATP independence of urea uptake by BBMV
The addition of ATP to the incubation medium had no significant effect on
the rate of urea uptake into dorsal (86.5±14.4%, N=5) and
ventral BBMV (92.7±19.4%, N=5). As well, the addition of
ouabain (dorsal 103.1±23.8%, N=5; ventral 80.1±14.3%,
N=5) and NEM (dorsal 79.4±15.1%, N=5; ventral
74.9±23.9%, N=5) had no effect on ATP-stimulated urea uptake
in BBMV (control dorsal 2.98±0.86 µmol h1
mg1 protein, N=5; control ventral 5.10±1.54
µmol h1 mg1 protein, N=5) (not
shown).
Cation dependence of urea uptake by BBMV
There was no significant difference between urea uptake in dorsal and
ventral BBMV in media containing only sodium or only potassium ions with no
concentration gradient present (data not shown). When urea uptake was measured
in the presence of an outwardly-directed potassium concentration gradient,
there was no significant change in the rate of urea uptake in either dorsal or
ventral BBMV (Fig. 5). As well,
in the dorsal BBMV there was no significant change in urea uptake in the
presence of an inwardly-directed sodium concentration gradient; however, in
the ventral BBMV the rate of urea uptake significantly increased when an
inward sodium concentration gradient was present (P<0.05;
Fig. 5).
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Discussion |
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Characteristics of urea transport
Our data on BBMV provide evidence for different urea transport
characteristics in the dorsal and ventral sections of the skate kidney. In the
dorsal section, urea transport was not apparently saturable at low or high
urea concentrations, but was inhibited by phloretin and HgCl2, and
significantly reduced in the presence of NPTU. These results suggest the
presence of one or more facilitated urea transporters, possibly coded for by
SkUT (skate urea transporter) or SkUT-homologs, which we have isolated in the
kidney of the little skate (Morgan et al.,
2003). In the ventral section, urea uptake by BBMV revealed
saturation kinetics at low urea concentration (Km=0.70
mmol l1, Vmax=1.18 µmol
h1 mg1 protein), but not at high urea
concentrations. Urea uptake was inhibited by phloretin and HgCl2,
but was stimulated by an inward Na+ gradient, suggesting the
presence of a Na+-linked urea transporter (UT). The fact that SkUT
was expressed in both the dorsal and ventral section
(Morgan et al., 2003
)
indicates that at least two distinct transporters may be active in the ventral
BBMV (i.e. a facilitative UT and a Na+-linked UT). Furthermore, the
high non-saturable rate of urea transport in the presence of relatively high
concentrations of urea (up to 370 mmol l1 urea), may
represent urea movement through nonspecialized aqueous channels, low
affinity/high capacity facilitated urea transporters similar to those
characterized in mammalian terminal inner medullar collecting ducts (IMCD),
the lipid-bilayer membrane, water channels (aquaporins), or possibly a
combination of two or more of these pathways.
Water channels facilitate the movement of lipophobic molecules by allowing
the molecule to remain in an aqueous phase as it diffuses through the channel,
and therefore represent a potential method of diffusion of urea across the
membrane. Inhibition of urea transport by mercurial compounds (e.g.
HgCl2, p-chloromercuribenezesulfonate, pCMBS), was shown
to occur in this study, and is generally considered diagnostic of aquaporins
(AQP) (Knepper, 1994;
Borgnia et al., 1999
). In
mammals, a subgroup of the aquaporins family (i.e. AQP3, AQP7 and AQP9) has
been shown to transport urea and/or glycerol as well as water
(Ishibashi et al., 1997
;
Tsukaguchi et al., 1998
). In
particular, aquaporin 3 (AQP3), which has been isolated in the basolateral
membrane of mammalian kidney tubule cells, has been shown to transport urea as
well as water, and this transport is inhibitable by phloretin and
HgCl2 (Tsukaguchi et al.,
1998
). In the present study, the addition of HgCl2 plus
phloretin did not inhibit transport relative to HgCl2 alone;
however, in the dorsal section, transport in the presence of HgCl2
plus phloretin was significantly lower than with phloretin alone. These
results suggest the possibility of AQP-linked urea transport and/or a
HgCl2-sensitive UT. Despite being well known for inhibiting
aquaporins, however, mercurial compounds are non-specific inhibitors and exert
their effects through cysteine residues. In the mammalian UT-B and the frog
facilitated urea transporter, pCMBS has been shown to inhibit urea transport
(Martial et al., 1996
;
Couriaud et al., 1999
). To our
knowledge, the inhibitory effect of mercurial compounds has not been
investigated on fish urea transporters, but a HgCl2-sensitive UT in
the marine elasmobranch kidney cannot be excluded.
The possibility of a low-affinity, high-capacity facilitated transporter is
supported by mammalian studies where the apparent Km
values of urea transporters are extremely high. When urea is present only in
the luminal perfusate of the terminal IMCD, urea reabsorption is linear with
urea concentrations as high as 800 mmol l1 (Chou et al.,
1989), a considerably higher urea concentration than that used in this study.
In Xenopus oocyte expression studies of the mammalian UT-A2, it was
shown that there was no saturation for a range of urea concentrations between
1 and 200 mmol l1 (You
et al., 1993). If SkUT is similar to the mammalian UT-A2 in terms
of transport kinetics, then it may be responsible for the non-saturable
component of urea uptake at high urea concentrations.
The effects of several known inhibitors of urea transport were examined to
try to further characterize the saturable component of urea uptake by BBMV.
This component of urea transport was examined by using a urea concentration
below the apparent Km. Inhibition by the non-competitive
inhibitor phloretin is considered diagnostic of both facilitated and secondary
active urea transport systems (Levine et
al., 1973; Knepper,
1994
; Kato and Sands,
1998
; Smith and Wright,
1999
; Fines et al.,
2001
). The inhibition of urea uptake in BBMVs by phloretin
observed in this study is consistent with a previous study on urine excretion
in free-swimming dogfish (Hays et al.,
1976
), which demonstrated that phloretin injected into the blood
system increased urinary urea excretion and decreased renal urea reabsorption.
In BBMVs from the dorsal section, the urea analogue NPTU (80 µmol
l1) significantly reduced urea uptake. This reduction is
comparable to that seen in the frog urinary bladder (IC50=79.4
µmol l1; Martial et
al., 1993
), indicating a high specificity for this analogue. In
the ventral section, acetamide elevated urea uptake above that of the ventral
control and the dorsal BBMV in the presence of acetamide. This result was
unexpected, but may further indicate that more than one transporter was under
study in this section of the kidney, and therefore the transport
characteristics are more complex. However, until the analogue concentrations
are optimized for this tissue, conclusions concerning analogue responses
cannot be drawn (Schmidt-Nielsen and
Rabinowitz, 1964
; Wood et al.,
1995
).
The results suggest that urea uptake is not energy dependent in skate renal
BBMV, since the addition of adenosine triphosphate (ATP) to the incubation
medium did not change the rate of urea uptake. In the presence of NEM, an
alkylating agent that binds selectively to sulfhydryl groups blocking V-type
and P-type ATPases (i.e. proton pumps;
Ehrenfeld, 1998), urea uptake
was unchanged. The addition of oubain, a specific inhibitor of
Na+,K+-ATPase, also had no effect. In contrast, gill
basolateral membranes in dogfish showed a significant stimulation of urea
uptake in the presence of ATP that was returned to control levels with the
addition of ouabain (Fines et al.,
2001
). In the present study, the lack of effect with ouabain and
ATP is not surprising since Na+,K+-ATPase is localized
to the basolateral, not the brush-border membrane. To examine the effect of
Na+,K+-ATPase on urea uptake in the BBM, an intact
tubule system is required in order to have both the brush-border and
basolateral membranes present in the same preparation.
In dorsal BBMV, urea uptake does not appear to be linked to the sodium or
potassium gradient. There is, altogether, little evidence to support the
presence of two different high-affinity facilitated transporters in the dorsal
section operating at different urea concentrations. This leads to the
hypothesis that urea uptake may be due to a single phloretin-sensitive
facilitated transporter, possibly that of the SkUT protein
(Morgan et al., 2003), and
similar to those isolated in the kidney of the dogfish shark and Atlantic
stingray (Smith and Wright,
1999
; Janech et al.,
2003
). In the ventral BBMV, the presence of an inwardly directed
sodium gradient significantly increased urea uptake relative to the control
and potassium gradient experiment. Despite the lack of information on the
energy requirements of urea uptake, it is possible that one component of urea
uptake in skate renal ventral BBMVs may be due to a Na+-coupled
secondary active urea transporter, similar to the active urea transport
described in the mammalian kidney (Kato
and Sands, 1998
) and in dogfish gills
(Fines et al., 2001
), while
another component of the urea uptake belongs to the same transporter found in
the dorsal section. Thus, the inward movement of urea and Na+
across the brush-border membrane would be linked to the active extrusion of
Na+ from the tubule cell across the basolateral membrane, back to
the blood.
In order to completely understand the mechanisms of urea reabsorption, further studies, possibly using tubule isolation techniques and basolateral membrane vesicles, are necessary to complete the hypothesized model. The physiological evidence suggests the presence of a facilitated urea transporter in the brush-border membrane of dorsal and ventral renal tubules, and the additional presence of a sodium-linked urea transporter in ventral tubules. Taken together, our data provide evidence that urea transporter(s) in the skate kidney play a role in urea retention.
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Acknowledgments |
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References |
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Bevan, C., Kinne, R. K. H., Shetlar, R. E. and Kinne-Saffran, E. (1989). Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish, Squalus acanthias. J. Comp. Physiol. B 159,339 -347.
Bijvelds, M. J. C., Kolar, Z., Wendelaar Bonga, S. E. and Flik,
G. (1997). Mg2+ transport in plasma membrane
vesicles of renal epithelium of the Mozambique tilapia (Oreochromis
mossambicus). J. Exp. Biol.
200,1931
-1939.
Blier, P. and Guderley, H. (1988). Metabolic responses to cold acclimation in the swimming musculature of lake whitefish, Coregonus clupeaformis. J. Exp. Zool. 246,244 -252.
Borgnia, M., Nielsen, S., Engel, A. and Agre, P. (1999). Cellular and molecular biology of the aquaporin water channels. Annu. Rev. Biochem. 68,425 -458.[CrossRef][Medline]
Boylan, J. W. (1967). Gill permeability in Squalus acanthias. In Sharks, Skates and Rays (ed. P. W. Gilbert, R. F. Mathewson and D. P. Rall), pp.197 -206. Baltimore: Johns Hopkins Press.
Boylan, J. W. (1972). A model for passive urea reabsorption in the elasmobranch kidney. Comp. Biochem. Physiol. 42A,27 -30.[CrossRef]
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Chou, C.-L. and Knepper, M. A. (1989). Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues. Am. J. Physiol. 257,F359 -F365.[Medline]
Clark, R. W. and Smith, H. W. (1932). Absorption and excretion of water and salts by the elasmobranch fishes. III The use of xylose as a measure of the glomerular filtrate in Squalus acanthias. J. Cell. Comp. Physiol. 1, 131-143.
Couriaud, C., Leroy, C., Simon, M., Silberstein, C., Bailly, P., Ripoche, P. and Rousselet, G. (1999). Molecular and functional characterization of an amphibian urea transporter. Biochim. Biophys. Acta 1421,347 -352.[Medline]
Drai, P., Albertini-Berhaut, J., Lafaurie, M., Sudaka, P. and Giudicelli, J. (1990). Simultaneous preparation of basolateral and brush-border membrane vesicles from sea bass intestinal epithelium. Biochim. Biophys. Acta 1022,251 -259.[Medline]
Ehrenfeld, J. (1998). Active proton and urea transport by amphibian skin. Comp. Biochem. Physiol. 119A,35 -45.
Fines, G. A., Ballantyne, J. S. and Wright, P. A. (2001). Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch. Am. J. Physiol. 280,R16 -R24.
Freire, C. A., Kinne-Saffran, E., Beyenbach, K. W. and Kinne, R. K. H. (1995). Na-D-glucose cotransport in renal brush-border membrane vesicles of an early teleost (Oncorhynchus mykiss). Am. J. Physiol. 269,R592 -R602.[Medline]
Gasser, K. W. and Kirschner, L. B. (1987). The response of alkaline phosphatase to osmoregulatory changes in the trout, Salmo gairdneri. J. Comp. Physiol. B. 157,469 -475.[Medline]
Giudicelli, J., Boudouard, M., Delqué, P., Vannier, C.
and Sudaka, P. (1985). Horse kidney neutral
-D-glucosidase: purification of the detergent-solubilized enzyme;
comparison with the proteinase-solubilized forms. Biochim. Biophys.
Acta 831,59
-66.[Medline]
Goldstein, L. and Forster, R. P. (1971).
Osmoregulation and urea metabolism in the little skate Raja erinacea.Am. J. Physiol. 220,742
-746.
Hays, R. M., Levine, S. D., Myers, J. D., Heinemann, H. O., Kaplan, M. A., Franki, N. and Berliner, H. (1976). Urea transport in the dogfish kidney. J. Exp. Zool. 199,309 -316.
Hentschel, H. (1988). Renal blood vascular system in the elasmobranch, Raja erinacea Mitchill, in relation to kidney zones. Am. J. Anat. 183,130 -147.[Medline]
Hentschel, H., Elger, M. and Schmidt-Nielsen, B. (1986). Chemical and morphological differences in the kidney zones of the elasmobranch, Raja erinacea, Mitch. Comp. Biochem. Physiol. 84A,553 -557.[CrossRef]
Ishibashi, K., Kuwahara, M., Gu, Y., Kageyama, Y., Tohsaka, A., Suzuki, F., Marumo, F. and Sasaki, S. (1997). Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol, and urea. J. Biol. Chem. 27,20782 -20786.[CrossRef]
Janech, M. G., Fitzgibbon, W. R., Chen, R., Nowak, M. W., Miller, D. H., Paul, R. V. and Ploth, D. W. (2003). Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray. Am. J. Physiol. 284,F996 -F1005.
Kato, A. and Sands, J. M. (1998). Active
sodium-urea counter-transport is inducible in the basolateral membrane of rat
renal initial inner medullary collecting duct. J. Clin.
Invest. 102,1008
-1015.
Kipp, H., Kinne-Saffran, E., Bevan, C. and Kinne, R. K. H. (1997). Characteristics of renal Na+-D-glucose cotransport in the skate (Raja erinacea) and shark (Squalus acanthias). Am. J. Physiol. 273,R134 -R142.[Medline]
Knepper, M. A. (1994). The aquaporin family of
molecular water channels. Proc. Natl. Acad. Sci. USA
91,6255
-6258.
Lacy, E. R. and Reale, E. (1985). The elasmobranch kidney. II. Sequence and structure of the nephrons. Anat. Embryol. 173,163 -186.[Medline]
Levine, S., Franki, N. and Hayes, R. M. (1973). Effect of phloretin on water and solute movement in the toad bladder. J. Clin. Invest. 52,1435 -1442.[Medline]
Martial, S., Neau, P., Degeilh, F., Lamotte, H., Rousseau, B. and Ripoche, P. (1993). Urea derivatives as tools for studying the urea-facilitated transport system. Pflügers Arch. 423,51 -58.[Medline]
Martial, S., Olivès, B., Abrami, L., Couriaud, C., Bailly, P., You, G., Hediger, M. A., Cartron, J.-P., Ripoche, P. and Rousselet, G. (1996). Functional differentiation of the human red blood cell and kidney urea transporters. Am. J. Physiol. 271,F1264 -F1268.[Medline]
McCormick, S. D. (1993). Methods for nonlethal gill biopsy and measurement of Na+, K+-ATPase activity. Can. J. Fish. Aquat. Sci. 50,656 -658.
Morgan, R. L., Ballantyne, J. S. and Wright, P. A.
(2003). Regulation of a renal urea transporter with reduced
salinity in a marine elasmobranch, Raja erinacea. J. Exp.
Biol. 206,3285
-3292.
Pärt, P., Wright, P. A. and Wood, C. M. (1998). Urea and water permeability in dogfish (Squalus acanthias) gills. Comp. Biochem. Physiol. 199A,117 -123.
Payan, P., Goldstein, L. and Forster, R. P.
(1973). Gills and kidneys in ureosmotic regulation in euryhaline
skates. Am. J. Physiol.
224,367
-372.
Schmidt-Nielsen, B. and Rabinowitz, L. (1964). Methylurea and acetamide: active reabsorption by elasmobranch renal tubules. Science 146,1587 -1588.[Medline]
Schmidt-Nielsen, B., Truniger, B. and Rabinowitz, L. (1972). Sodium-linked urea transport by the renal tubule of the spiny dogfish, Squalus acanthias. Comp. Biochem. Physiol. 42A,13 -25.[CrossRef]
Smith, C. P. and Wright, P. A. (1999). Molecular characterization of an elasmobranch urea transporter. Am. J. Physiol. 276,R622 -R626.[Medline]
Smith, H. W. (1929). The composition of the
body fluids of elasmobranchs. J. Biol. Chem.
81,407
-419.
Smith, H. W. (1936). The retention and physiological role of urea in the elasmobranchii. Biol. Rev. 11,49 -82.
Stio, M., Vanni, P. and Pinzauti, G. (1988). A continuous spectrophotometric assay for the enzymatic marker glucose-6-phosphatase. Anal. Biochem. 174, 32-37.[Medline]
Tsukaguchi, H., Shayakul, C., Berger, U. V., Mackenzie, B.,
Devidas, S., Guggino, W. B., van Hoek, A. N. and Hediger, M. A.
(1998). Molecular characterization of a broad selectivity neutral
solute channel. J. Biol. Chem.
273,24737
-24743.
Walsh, P. J. and Smith, C. P. (2001). Physiology of urea transport in fish. In Nitrogen Excretion, Vol. 20 (ed. P. A. Wright and P. M. Anderson), pp. 289-295. New York: Academic Press.
Wood, C. M., Pärt, P. and Wright, P. A.
(1995). Ammonia and urea metabolism in relation to gill function
and acidbase balance in a marine elasmobranch, the spiny dogfish
(Squalus acanthias). J. Exp. Biol.
198,1545
-1558.
You, G., Smith, C. P., Kanai, Y., Lee, W.-S., Steizner, M. and Hediger, M. A. (1993). Cloning and characterization of the vasopressin-regulated urea transporter. Nature 365,844 -847.[CrossRef][Medline]