Division of Renal Diseases and Hypertension, University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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In rat kidney the "secretory" isoform of the
Na+-K+-2Cl cotransporter (NKCC1)
localizes to the basolateral membrane of the
-intercalated cell. The
purpose of this study was to determine whether rat outer medullary
collecting duct (OMCD) secretes Cl
and whether
transepithelial Cl
transport occurs, in part, through
Cl
uptake across the basolateral membrane mediated by
NKCC1 in series with Cl
efflux across the apical
membrane. OMCD tubules from rats treated with deoxycorticosterone
pivalate were perfused in vitro in symmetrical HCO
secretion was observed in this segment, accompanied by
a lumen positive transepithelial potential. Bumetanide (100 µM), when added to the bath, reduced Cl
secretion by 78%, although
the lumen positive transepithelial potential and fluid flux were
unchanged. Bumetanide-sensitive Cl
secretion was
dependent on extracellular Na+ and either K+ or
NH
transport. In conclusion, OMCD tubules
from deoxycorticosterone pivalate-treated rats secrete Cl
into the luminal fluid through NKCC1-mediated Cl
uptake
across the basolateral membrane in series with Cl
efflux
across the apical membrane. The physiological role of NKCC1-mediated
Cl
uptake remains to be determined. However, the role of
NKCC1 in the process of fluid secretion could not be demonstrated.
ammonium; type 1 bumetanide-sensitive sodium-potassium-2 chloride cotransporter; type 2 bumetanide-sensitive sodium-potassium-2 chloride cotransporter; fluid flux; chloride-bicarbonate exchange; outer medullary collecting duct
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INTRODUCTION |
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TWO DISTINCT GENES
ENCODE the Na+-K+-2Cl
cotransporters, BSC-2 (NKCC1) and BSC-1 (NKCC2). NKCC2, or the
"absorptive" isoform, is kidney specific and localizes to the
apical membrane of the thick ascending limb (5, 25). In
contrast, NKCC1, or the "secretory" isoform, is widely distributed
(25). However, the distribution of NKCC1 in the kidney is
very species specific (13, 26). Recent cloning of NKCC1 in
the mouse has enabled development of antibodies that recognize this
cotransporter and has facilitated study of its distribution in various
tissues. In rat kidney, immunolocalization studies by Ginns and
colleagues (13) have detected the highest levels of
expression of the cotransporter along the basolateral membrane of
-intercalated cells, with low levels of protein expression in the
terminal inner medullary collecting duct (tIMCD). In contrast, in mouse
collecting duct (26) NKCC1 expression is highest in the
tIMCD, with no expression detected in either the cortical collecting
duct (CCD) or the outer medullary collecting duct (OMCD). Thus the
physiological role of the cotransporter in the collecting duct is
puzzling in view of the species-specific distribution of NKCC1 protein.
The cellular composition of rat OMCD is heterogeneous:
60-64% of cells are principal cells whereas 36-40% are
-intercalated cells (16). Although principal cells
mediate robust rates of Na+ transport (33),
they mediate little Cl
transport (34).
Therefore, it remains to be determined whether significant
transepithelial movement of Cl
occurs in rat OMCD. If
Cl
secretion is observed in rat OMCD, it might
occur through anion exchange- and/or NKCC1-mediated Cl
uptake across the basolateral membrane in series with Cl
movement across the apical membrane.
In rabbit OMCD, Cl is secreted in parallel with
H+ secretion (HCO
secretion and
HCO
and absorption of HCO
/HCO
across the apical membrane.
Like rabbit OMCD, rat OMCD absorbs HCO transport pathways in this segment and their possible
contribution to transepithelial Cl
transport are not
understood. The purpose of the present study was to determine whether
rat OMCD secretes Cl
and whether transepithelial
transport of Cl
is mediated, at least in part, by NKCC1.
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METHODS |
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Tubules from the inner stripe of the OMCD were dissected from pathogen-free male Sprague-Dawley rats weighing 65-120 g (Harlan, Indianapolis, IN). Animals were housed in microisolator cages and fed a low-Na+, 0.8% K+ diet (Zeigler Bros., Garners, PA) (41). Rats received 5 mg deoxycorticosterone pivalate (DOCP; CIBA-Geigy Animal Health, Greensboro, NC) by intramuscular injection 5-7 days before death. To induce a rapid diuresis, animals were injected with furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation. This furosemide-induced diuresis reduces the inner medullary axial solute concentration gradient (41) and attenuates changes in the extracellular osmolality of the tubule.
Coronal slices were cut from the kidneys and placed into a dissection
dish containing the chilled experimental solution (11°C). Solution
compositions are given in Table 1. The
dissection solution was either solution 1 or solution
2 as appropriate, to match the NH4Cl concentration of
the perfusate and bath solution used when measurements were performed.
To dissect OMCD tubules from the inner stripe of the outer medulla, a
cut was made between the inner and outer stripe of the outer medulla
using a razor blade, and OMCD tubules were dissected as reported
previously (9). Tubules were mounted on concentric glass
pipettes and perfused in vitro at 37°C.
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Experiments were performed with symmetrical solutions in the bath and
perfusate. Osmolality was measured in all solutions (41).
To maintain the desired CO2 concentration in
HCO
Because of time-dependent changes in transepithelial potential difference, VT (not shown), measurements in each tubule were made under a single experimental condition. All collections were begun 30 min, and terminated 75 min, after the tubule was warmed. Perfusate samples were collected continuously over this time period. VT was measured continuously; reported VT corresponds to that measured at the midpoint of this time period, or 50 min after the tubule was warmed.
Measurement of transepithelial Cl flux.
Cl
concentration in collected perfusate samples was
measured using a continuous-flow fluorometer with an assay developed by Garcia and colleagues (11) that utilizes 6 methoxy-N-(3-sulfopropyl) quinolinium (SPQ; Molecular
Probes, Eugene, OR), a Cl
-sensitive fluorophore. SPQ was
dissolved in water at a 0.20 mM concentration. The reagent was placed
in a pasteur pipette and drawn past the injection port and then through
stainless steel tubing into a cuvette with a constant-speed withdrawal
pump at 55 nl/s. Other details of the fluorometer design have been
reported previously (41). Samples were pipetted into the
flowing reagent. We have observed that the Cl
assay is
linear over a range of at least 0-5.3 nmol Cl
(not
shown). By using a 10-nl pipette, differences in Cl
concentration of 2 mM can be detected (2 × coefficient of
variation/slope) with this assay (11). Fluorescence of SPQ
is not affected by pH, Na+, or HCO
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Measurement of intracellular pH in
-intercalated cells.
Intracellualr pH (pHi) was measured using
2',7'-bis(carboxy-ethyl)-5(6)-carboxyfluorescein
acetoxymethyl ester (BCECF-AM). Tubules were perfused and bathed for 15 min at 37°C in solution 1. The perfusate was changed to
the same solution with the addition of 5 µM BCECF-AM. Tubules were
perfused with BCECF for 10 min. The perfusate was then changed to the
original solution but with BCECF removed. By using this technique,
-intercalated cells are preferentially loaded with BCECF
(35). pHi was determined by measuring the
ratio of emitted light at >530 nm when BCECF was excited alternatively
at 440 and 495 nm. Readings were calibrated by measuring fluorescence
when the tubule was perfused and bathed in a HEPES-buffered solution
containing 120 mM K+ and 14 µM nigericin. The pH of this
solution was varied between 7.0 and 7.8. The other details of
pHi measurement in tubules perfused in vitro were performed
as described previously by our laboratory (41).
Fluid flux.
To measure fluid flux (Jv), changes in raffinose
concentration of the luminal fluid were measured, using the assay
described by Garvin and Knepper (12). Raffinose
concentration in collected fluid samples was measured by using an
enzymatic assay in which raffinose is converted to galactonolactone and
NADH. This assay was purchased as a kit (Boehringer Mannheim, Mannheim,
Germany). Fluorescence of NADH was measured using a continuous-flow
ultramicrofluorometer. Samples were collected into the lower chamber
(17 nl) of a double-constriction pipette. An enzyme solution was
employed that contained 3.5 mg/ml NAD+ in citrate buffer
(pH 4.5) and 4.5 U/ml -galactosidase. This enzyme solution was drawn
into the pipette until the second chamber (237 nl) was filled. Because
the enzymatic conversion of raffinose to NADH and galactonolactone was
complete after 6 min at 37°C (data not shown), all samples were
incubated for 7-9 min at 37°C and then injected into a flowing
stream. The flowing stream was drawn by a constant-withdrawal pump at
166 nl/s and contained 0.3 U/ml
-galactose dehydrogenase in
potassium diphosphate buffer (pH 8.6). By using this assay, raffinose
concentration is linear from at least 0 to 203 pmol (not shown).
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VT. To measure VT, the solution in the perfusion pipette was connected to an electrometer (model KS-700, World Precision Instruments, New Haven, CT) through an agar bridge saturated with 0.16 M NaCl and a calomel cell as described previously (41). The reference was an agar bridge from the bath to a calomel cell.
Statistics.
In all experiments wherein either Cl or raffinose
concentration was assayed, two to three replicate measurements were
made in a single tubule. The mean of all measurements made in a single tubule was used in the statistical analysis, where n
represents the number of tubules studied. Statistical significance was
determined by using a paired or unpaired two-tailed Student's
t-test as appropriate. For multiple comparisons, ANOVA was
used with specific contrasts by the Bonferroni method. Statistical
significance was achieved with P < 0.05. Data are
displayed as means ± SE.
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RESULTS |
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Role of anion exchange in JCl.
Ion transport pathways along the collecting duct have been studied
extensively in DOCP-treated rats (9, 27, 41, 42). Therefore, to explore Cl transport pathways in rat OMCD,
DOCP-treated rats were employed both to facilitate comparison with
these previous studies and to stimulate Cl
transport
pathways such as anion exchange
(17).1
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Effect of bumetanide on JCl.
To determine the role of NKCC1-mediated Cl uptake in
the process of transepithelial transport of Cl
, the
effect of bumetanide on JCl was tested. Results
are shown in Fig. 2 (Table 2). Cl
secretion (
13.5 ± 0.8 pmol · mm
1 · min
1,
n = 5, solution 2) was attenuated in a
dose-dependent fashion with bumetanide, an inhibitor of NKCC1, when
added to the peritubular bath. JCl was inhibited
by 55% with 10 µM bumetanide (P < 0.05) and 78% by
100 µM bumetanide (P < 0.05).
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Effect of extracellular
Na+,
K+, and
NH was detected either in the presence
(
3.6 ± 2.2 pmol · mm
1 · min
1,
n = 5) or in the absence of bumetanide (
0.6 ± 2.7 pmol · mm
1 · min
1,
n = 5). Thus both total and bumetanide-sensitive
JCl are dependent on the presence of
Na+ in the bath and perfusate, which eliminates the
possibility that Cl
secretion sensitive to bumetanide is
mediated by KCl cotransport.
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Effect of bumetanide on Jv.
In other cell types, such as in salivary glands, NKCC1 mediates
Cl secretion, which gives rise to secretion of fluid
(28). We therefore tested whether fluid secretion is
observed in parallel with Cl
secretion in rat OMCD.
Tubules were perfused and bathed in the presence of 5 mM raffinose
(solution 7), which was employed as a volume marker. Results
are displayed in Table 3. In symmetrical solutions, low levels of fluid secretion were observed in this segment
(Jv =
0.042 ± 0.015 nl · mm
1 · min
1,
n = 5), which was unchanged with the application of
bumetanide to the bath. However, because of the variability in the
measurements, we cannot exclude a small effect of bumetanide on
Jv.
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DISCUSSION |
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This study demonstrates that in OMCD tubules perfused in vitro
from DOCP-treated rats, Cl is secreted into the luminal
fluid through Cl
uptake across the basolateral membrane,
mediated by both NKCC1 and anion exchange, in series with
Cl
efflux across the apical membrane. This contribution
of NKCC1 to Cl
secretion represents a novel mechanism of
transepithelial Cl
transport in rat OMCD.
Significant Cl secretion was observed in the OMCD in the
present in vitro study. In vivo studies suggest that Cl
secretion might occur in rat OMCD. After NaCl stress, Cl
delivery to the base of the collecting duct is greater than
Cl
delivery at the level of the superficial distal tubule
(18). One explanation for these results is that
Cl
secretion occurs along the collecting duct in vivo in
one or more nephron segments, which lie between the superficial distal tubule and the base of the collecting duct. The contribution of each
segment that falls between these two micropuncture sites to the
increment in Cl
delivered to the base of the collecting
duct is not known. The present study demonstrates that the OMCD is one
segment in this region of the nephron that secretes Cl
in
vitro. The pathway(s) that mediates Cl
uptake across the
basolateral membrane of the
-intercalated cell in rat OMCD were
therefore explored in more detail.
Anion exchange mediates Cl uptake and
HCO
/HCO
/HCO
-intercalated cell in rat OMCD (7). Because
Cl
secretion was not eliminated with 0.5 mM
H2DIDS, other Cl
uptake pathways along the
basolateral membrane of rat OMCD might also be important in the process
of transepithelial Cl
transport.
We demonstrated that JCl is reduced in a
dose-dependent fashion with bumetanide. These data are consistent with
previous reports of the dose response of bumetanide to rat NKCC1
(14, 28). At a concentration of <10 µM, bumetanide is a
relatively specific inhibitor of NKCC1 (15). Therefore,
significant Cl uptake across the basolateral membrane is
mediated by NKCC1 because >50% of JCl is
inhibited by low concentrations of bumetanide (10 µM) when added to
the bath.
In rat, NKCC1 is fully inhibited at a bumetanide concentration of 100 µM (14, 28). However, at this bumetanide concentration, at least partial inhibition of other Cl transporters,
such as Cl
/HCO
channels (19), and KCl
cotransport (19), has been observed. Cl
secretion sensitive to 100 µM bumetanide may therefore overestimate the contribution of NKCC1 to total transepithelial Cl
transport in rat OMCD. However, ion substitution experiments demonstrated that the bumetanide-sensitive component of
Cl
secretion is dependent on extracellular
Na+ and either K+ or NH
uptake mediated by
NKCC1 instead of through these other transporters. Although
Cl
uptake across the basolateral membrane can be
attributed to both anion exchange and NKCC1, the mechanism of
Cl
transport across the apical membrane is unknown.
Whether Cl uptake across the basolateral membrane of the
-intercalated cell in rat OMCD is mediated by NKCC1 or NKCC2 cannot be determined directly. In the rat outer medulla, the absorptive isoform of the Na-K-2Cl cotransporter, NKCC2, has been localized to the
thick ascending limb by in situ hybridization and immunolocalization studies (5, 25). Although NKCC2 message has also been
detected in rat OMCD (45), it is unlikely that the isoform
of the cotransporter detected in the present study is NKCC2. First,
NKCC2 protein expression has not been detected in rat OMCD
(5), although NKCC1 protein has been clearly demonstrated
in this segment (13). Therefore, NKCC2 is most probably
not as abundant in this segment as NKCC1. Second, bumetanide inhibits
Cl
secretion when applied to the bath, consistent with
localization of NKCC1 to the basolateral membrane (13).
In the present study it was observed that total and
bumetanide-sensitive JCl vary greatly over an
extracellular K+ concentration range of 2-20 mM.
Because the interstitium of the rat outer medulla is not accessible to
micropuncture, interstitial K+ concentration in vivo is not
known. However with medullary recycling of K+, interstitial
K+ concentration in the outer medulla is expected to be
greater than serum values (23), which vary in
rat3 between 2 and 7 mM
(3, 4), but less than interstitial values in the inner
medulla, which range from 6 to 54 mM (2). Thus interstitial K+ concentration in the interstitium of the
outer medulla is probably >2 but <50 mM. For NKCC1 the
Michaelis-Menten constant
(Km)4
for Rb+ (a K+ congener) is 2-15 mM (22,
30, 43). Thus NKCC1-mediated Cl uptake should vary
greatly over a K+ concentration range of 2-50
mM5. If so, changes in
K+ concentration in the interstitium of the rat outer
medulla in vivo should markedly alter Cl
secretion in the
OMCD through changes in NKCC1-mediated Cl
transport.
Although total and bumetanide-sensitive JCl in
rat OMCD vary greatly with changes in extracellular Na+
concentration, it is less likely that changes in interstitial Na+ concentration in vivo significantly regulate
NKCC1-mediated Cl uptake. In rat, serum Na+
concentration ranges from 95 to 200 mM (20). Through
countercurrent multiplication, interstitial Na+
concentration in rat outer medulla is therefore probably >95 mM,
although it has not been measured directly. Because the
Km for Na+ reported for mammalian
NKCC1 is generally less than 50 mM (22, 30), the
Na+ concentration of the rat outer medullary interstitium
probably always approaches maximal transport rate conditions for NKCC1. Thus changes in interstitial Na+ concentration over the
physiological range expected in vivo probably do not significantly
alter NKCC1-mediated Cl
uptake.
Although Cl transporters such as AE1 clearly participate
in transepithelial secretion of net H+ equivalents, the
role of NKCC1 in renal physiology is not known. The Na-K-2Cl
cotransporter has been implicated in a number of cell functions,
including the secretion of HCl (38) and KCl (32). However, its primary physiological function is in
volume regulation and the vectorial transport of water and NaCl
(25). Therefore, rat
-intercalated cells, which express
high levels of NKCC1 relative to other cells in rat kidney, may serve
physiological functions other than secretion of H+ equivalents.
In vivo studies have demonstrated secretion of NaCl and fluid along rat
IMCD (36, 40). However, more recent studies of Wallace and
co-workers (44) have reported secretion of fluid in vitro
in rat initial IMCD (44), a segment that contains
-intercalated cells and expresses NKCC1 (13). Fluid
secretion, determined by measuring luminal diameter over a 5- to 12-h
period in tubules with sealed ends, was attenuated with the addition of
bumetanide to the bath (44). In the present study, very
low levels of fluid secretion were observed in rat OMCD, with a
Jv similar to that reported previously in rat
CCD (1) and rat tIMCD (42). Low levels of
fluid secretion observed in rat OMCD in the present study are
compatible with observations of Wallace et al. (44) in
initial IMCD. However, in rat OMCD no change in
Jv was detected with inhibition of NKCC1.
Therefore, under the conditions of the present study a role of NKCC1 in
fluid secretion or absorption in OMCD tubules from DOCP-treated rats
could not be demonstrated.
Previous studies have suggested a role of the Na-K-2Cl cotransporter in
NaCl secretion in the rat IMCD (31). The natriuresis and
chloruresis observed in rats given a NaCl load occurs in part through
atrial natriuretic factor (ANF) (10, 37, 40). In rat
initial IMCD, Rocha and Kudo (31) observed that with the application of ANF to the bath fluid, bath-to-lumen flux of
Na+ and Cl is increased, whereas the
lumen-to-bath flux of these ions is reduced. This ANF-induced change in
Na+ and Cl
secretion was fully inhibited with
low concentrations of furosemide when added to the bath solution. The
authors concluded that ANF reduced Na+ and Cl
absorption through inhibition of apical Na+ channels while
stimulating Na+ and Cl
secretion mediated by
the Na-K-2Cl cotransporter. The possible role of NKCC1 in mediating
NaCl excretion after NaCl stress will require further study.
In conclusion, rat OMCD secretes Cl. Cl
secretion in this segment occurs in part through Cl
uptake across the basolateral membrane, mediated by NKCC1, in series
with Cl
efflux across the apical membrane. The
physiological role of this transport process remains to be determined.
However, under the conditions of the present in vitro study, a role of
NKCC1 in fluid secretion or absorption was not demonstrated in this segment. The cosecreted cation or counterion, which accompanies bumetanide-sensitive Cl
secretion, remains to be established.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46493 (to S. M. Wall).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M. Wall, Div. of Renal Diseases and Hypertension, University of Texas Medical School at Houston, 6431 Fannin, M.S.B. 4.148, Houston, TX 77030 (E-mail: Susan.M.Wall{at}uth.tmc.edu).
1
An effect of DOCP on JCl
could not be detected in rat OMCD. JCl was
2.9 ± 1.1 pmol · mm
1 · min
1
(n = 5) in untreated controls and
7.1 ± 1.6 pmol · mm
1 · min
1 in
tubules from DOCP-treated rats (solution 2,
n = 7, P = 0.075, unpaired Student's
t-test).
2 At an H2DIDS concentration of 0.5 mM, 66% of Na+-independent anion exchange is inhibited along the basolateral membrane of intercalated cells in rat CCD (6).
4 The Km, or apparent affinity, of a transport protein reflects the substrate concentration needed to achieve half the maximal transport rate (Vmax).
5
For mouse NKCC1, our laboratory has reported a
Km for K+ of 4.6 mM
(43). Assuming that NKCC1 follows Michaelis-Menton
kinetics, at an extracellular K+ concentration of 2 mM,
NKCC1-mediated Cl uptake should operate at ~28% of
Vmax. If a perfusate flow rate of 2 nl · mm
1 · min
1 is
employed, the predicted change in perfusate Cl
concentration with the application of bumetanide should be 0.8 mM,
which is beyond the limit of detection of this assay
(11).
3 Interstitial ion concentration in cortex is taken to be equivalent to serum levels. In inner medulla, interstitial ion concentrations are assumed to reflect values measured in vasa recta plasma at the same level along the corticomedullary axis (24).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 October 2000; accepted in final form 20 December 2000.
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