Electrolyte and fluid secretion by cultured human inner
medullary collecting duct cells
Darren P.
Wallace1,2,3,
Marcy
Christensen1,
Gail
Reif1,
Franck
Belibi1,
Brantley
Thrasher4,
Duke
Herrell4, and
Jared J.
Grantham1,2,3
1 Kidney Institute and Departments of
2 Biochemistry and Molecular Biology,
3 Medicine, and 4 Urology, University
of Kansas Medical Center, Kansas City, Kansas 66160
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ABSTRACT |
Inner medullary collecting ducts (IMCD)
are the final nephron segments through which urine flows. To
investigate epithelial ion transport in human IMCD, we established
primary cell cultures from initial (hIMCDi) and terminal
(hIMCDt) inner medullary regions of human kidneys. AVP,
PGE2, and forskolin increased cAMP in both hIMCDi and hIMCDt cells. The effects of AVP and
PGE2 were greatest in hIMCDi; however,
forskolin increased cAMP to the same extent in hIMCDi and
hIMCDt. Basal short-circuit current
(ISC) of hIMCDi monolayers was
1.4 ± 0.5 µA/cm2 and was inhibited by benzamil, a
Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE2,
and forskolin increased ISC; the current was
reduced by blocking PKA, apical Cl
channels, basolateral
NKCC1 (a Na+-K+-2Cl
cotransporter), and basolateral Cl
/HCO
exchangers. In fluid transport studies, hIMCDi monolayers
absorbed fluid in the basal state and forskolin reversed net fluid
transport to secretion. In hIMCDt monolayers, basal current
was not different from zero and cAMP had no effect on
ISC. We conclude that AVP and PGE2
stimulate cAMP-dependent Cl
secretion by
hIMCDi cells, but not hIMCDt cells, in vitro.
We suggest that salt secretion at specialized sites along human
collecting ducts may be important in the formation of the final urine.
kidney; chloride transport; salt secretion
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INTRODUCTION |
INNER MEDULLARY COLLECTING
DUCTS (IMCD), the last intrarenal sites in contact with
the tubular fluid, are in a strategic location to have a significant
impact on the composition of the urine. Proteins and mRNA for diverse
electrolyte and fluid transport processes have been identified in
primary cultures of rat IMCD cells (16, 48) and mIMCD-K2,
a mouse IMCD cell line (52, 53). They include, but are not
limited to, the epithelial Na+ channel (ENaC) and the
cystic fibrosis transmembrane conductance regulator (CFTR)
Cl
channel, components that are critical to the
regulation of salt and fluid transport by epithelia including the
airway, intestine, and exocrine glands (2, 21). Whereas
most studies of collecting duct function have focused on the absorptive
pathways, recent studies demonstrated that collecting ducts have the
capacity to secrete as well as absorb solutes and fluid (39, 54,
55, 57). Net salt and fluid transport is the sum of absorptive
and secretory processes; consequently, changes in the rate of either salt absorption or secretion would determine the composition and the
volume of the final urine.
The regulation of transport mechanisms has been studied in rat IMCD and
cultured rat IMCD cells. Atrial natriuretic peptide (ANP) was shown to
inhibit renal Na+ absorption (63) involving
both ENaC phosphorylation and phosphorylation-independent mechanisms
(47). Vandorpe et al. (53) characterized a
cyclic nucleotide-gated (CNG) nonselective cation channel in IMCD cells that may also contribute to Na+ absorption. Although ENaC
and CNG channels were shown to have distinctive characteristics, both
were inhibited by benzamil (53). In many epithelia, cAMP
agonists modulate net NaCl and fluid transport by stimulating
Cl
secretion via CFTR, a cAMP-dependent Cl
channel, located in the apical membrane. Moreover, the activation of
CFTR may diminish Na+ absorption through inhibition of ENaC
(19, 44). In this regard, cAMP agonists may play an
important role in the regulation of net solute and fluid transport
within collecting ducts. To a large extent, our knowledge of
electrolyte transport by IMCD has come from studies of isolated rat
IMCD segments (39, 55, 57) and cultured rat and mouse IMCD
cells (16, 18, 22-24, 48, 52, 53). Although rodent
models are clearly important in defining electrolyte and fluid
transport mechanisms in renal collecting ducts, there are major
morphological and functional differences in collecting duct subsegments
among the different species (5, 8, 27, 31). Thus
mechanisms defined in rodent systems cannot be uniquely extrapolated to
human collecting ducts. In the current study, we examined the
functional contributions of classic modulators to electrolyte and fluid
transport by initial (IMCDi) and terminal (IMCDt) IMCD cultured from human kidneys.
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METHODS |
Human kidney tissue.
Discarded human kidney tissue was obtained with the assistance of two
surgeons in the urology section at the Kansas University Medical Center
(KUMC) and the Midwest Transplant Network (MTN; Kansas City, KS), an
organ retrieval agency. This protocol was approved by the KUMC Human
Subjects Committee. Eleven kidney specimens were obtained from normal
regions of kidneys removed for treatment of renal carcinomas. Renal
tissue judged to be normal by histological examination was placed in
sterile ice-cold PBS and brought to the lab for dissection. Four
kidneys obtained from MTN had been perfused with an electrolyte
preservation solution in preparation for transplantation. Kidneys that
were deemed unsuitable for transplantation because of anomalous
vasculature were maintained in ice-cold solution and delivered to the
laboratory within 24 h from the time the renal vessels were
clamped. We detected no difference in the quality or characteristics of
the cultures obtained from the two sources.
Kidneys were cut sagittally to expose papillae protruding into the
renal calyces. Papillae were isolated, and the adjoining cortical
tissue was removed ~2 mm below the cortico-medullary boundary,
leaving most of the medulla (Fig. 1) for
study. In most cases, the terminal 4 mm of the papilla was removed and
cultured separately, designated as hIMCDt. In kidneys
perfused for transplantation, the boundary between the outer medulla
(inner stripe) and the initial inner medulla was difficult to identify;
therefore, the absolute distance from the papillary tip was used to
define the initial inner medulla. A region of ~4 mm was removed from
the upper portion of the inner medulla and designated as
hIMCDi. Tissues used for morphological examination by
electron microscopy were fixed in cold 2% glutaraldehyde in HEPES
buffer. For lectin staining and immunolocalization of Na-K-ATPase,
tissues were fixed in 4% paraformaldehyde (PFA) in PBS overnight at
4°C.

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Fig. 1.
A: Schematic of a cross section through a
single renal papilla of a human kidney. The dimensions are estimated
based on tissue obtained from adult male kidneys. The human medulla is
much broader at the corticomedullary boundary than in the rat. The
transitional zones of the subsegments of human kidneys have variable
lengths, and the boundary between the outer medulla and the initial
inner medulla is often difficult to distinguish. Thus, in the present
study, ~11 mm of the inner medulla were removed from each papilla and
separated into initial (IMCDi, upper 4 mm) and terminal
(IMCDt, lower 4 mm) inner medulla collecting duct (IMCD).
The transitional region between IMCDi and IMCDt
was frequently discarded. B: schematic of a coronal section
of a rat kidney through the single papilla for comparison to that of
the human. The different regions of the medulla in the rat kidney are
readily identifiable. The drawings are not drawn to scale. OMCD, outer
medullary collecting duct. Anatomically, the rat IMCD can be subdivided
into the outermost one-third (IMCD1), the middle one-third
(IMCD2), and the terminal one-third
(IMCD3).
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Cell culture preparation.
The method for preparing primary cultures was published previously
(15, 56). Preparations of hIMCDt and
hIMCDi cells were handled identically. The tissue was
minced and digested in DME-F12 (1:1 vol/vol) mixture containing 220 IU/ml collagenase (type IV; Worthington Biochemical, Lakewood, NJ) and
100 IU/ml penicillin G-0.1 mg/ml streptomycin (P/S). The suspension of
tissue was incubated at 37°C for at least 6 h or until the
epithelial cells detached from the fibrous connective tissue.
Collagenase digestion was stopped by adding FBS to a final
concentration of 10%. The suspension was centrifuged, and the pellet
was rinsed twice and resuspended in DME-F12 supplemented with 5% FBS,
5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite
(ITS; Collaborative Biomedical Products, Bedford, MA) and P/S. The
cells were transferred to a T75 culture flask and allowed to attach to
the surface overnight. Unattached tissue and debris were removed the
next morning, and fresh medium was added to the flask. After the cells
had reached 70-80% confluence in the flask, they were lifted from
the plastic with a trypsin-EDTA solution (Sigma) and counted with a hemocytometer.
Lectin staining.
Normal human kidney tissue and cell monolayers grown either on
eight-well chamber slides or permeable supports (Snapwell-Clear, 12-mm
diameter; CoStar, Cambridge, MA) were examined with segment-specific lectins. Two lectins that bind selectively to collecting ducts, Dolichos biflorus agglutinin (DBA) and Arachis
hypogaea agglutinin (peanut agglutinin; PNA) were used for lectin
profiling of human IMCD cell cultures (14, 33). DBA and
PNA were conjugated to horseradish peroxidase (HRP) and visualized with
3,3'-diaminobenzidine (DAB) (see Figs. 3 and 4). To confirm lectin-HRP
staining, we used DBA and PNA conjugated to rhodamine (data not shown).
The concentration of the lectins used in the study was 50 µg/ml.
Specificity of lectin binding was determined by preincubating the
lectins with the appropriate inhibiting sugars (14).
Preparation of monolayers on permeable supports.
Primary cells were seeded at a density of 2.5 × 105
cells/1.13 cm2 on permeable supports (Snapwell-Tissue
Culture Treated, 12-mm diameter; CoStar) as described previously
(56). These supports have a lower inherent electrical
resistance than the Snapwell-Clear. After 3 days in culture, the
DME-F12, ITS, and P/S medium containing 5% FBS was changed to a medium
containing 1% FBS. The reduced serum concentration increased and
stabilized transepithelial electrical resistance (TER). Most
experiments were performed 7-10 days after the cells were seeded;
however, the monolayers responded to agonists after several weeks when
maintained in the 1% FBS medium.
Electrical measurements.
Snapwell supports containing confluent monolayers were inserted into
modified Ussing chambers (Harvard Apparatus, Hollison, MA), and both
surfaces of the cell monolayer were bathed in a HCO
-Ringer solution maintained at 37°C and
equilibrated in 5% CO2-95% O2
(56). Two dual-voltage-clamp devices (Warner Instruments,
Hamden, CT), each attached to four electrodes per chamber, were used to
measure transepithelial potential difference (PD), short-circuit
current (ISC), and TER in four monolayers
simultaneously. IMCDi cells developed TER ranging from 100 to 500
· cm2; TER values of the
IMCDt monolayers were lower (80-120
· cm2). Positive
ISC reflects the active transport of cations
(e.g., Na+) from the apical to basolateral media or the
active transport of anions (e.g., Cl
) from the
basolateral to apical media. ISC was
continuously monitored and recorded with a chart recorder.
Fluid transport measurements.
Dispersed hIMCDi cells were seeded (1 × 106 cells/4.52 cm2) on 24.5-mm-diameter cell
culture supports (Transwell-Col; Costar) and incubated for 3 days in
medium containing 5% FBS. After 3 days, the serum concentration was
reduced to 1%. hIMCD cell monolayers developed a uniform, cuboidal
appearance when grown on permeable supports (Fig.
2F). Rat IMCD cells grown in
this manner retain many of the characteristics expressed in IMCD in
situ (23).

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Fig. 2.
Morphology of human IMCD (hIMCD) in situ and cultured
hIMCD cell monolayers grown on permeable supports. Electron
microphotographs of initial hIMCD (hIMCDi; A),
terminal hIMCD (hIMCDt; B), cultured
hIMCDi cell monolayer (C), cultured
hIMCDt cell monolayer (D), and apical junctional
complex between two hIMCDi cells (E) are shown.
Light microphotographs of a hIMCDi cell monolayer viewed
with a phase microscope (F) and hematoxylin and eosin stain
of a paraffin section of a hIMCDi cell monolayer
(G) are also shown. In C, D, and
F, the cells were grown on a tissue culture-treated
Snapwell. The pores within the Snapwell are visible as parallel
vertical lines. In F, cells were grown on a more transparent
Transwell-Col.
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The procedure for measuring fluid transport across epithelial cell
monolayers was published previously (34, 59). Briefly, medium bathing the apical (upper) surface of the cell monolayer was
removed and replaced with 200 µl of DME-F12 medium containing 1%
FBS, ITS, and P/S. Sterile, water-saturated mineral oil was layered
above the fluid to prevent fluid evaporation. Transwell supports
containing the cell layer were placed in a six-well culture plate; each
well contained 2.5 ml of basolateral medium. The fluid-oil mixture was
collected after 24 h, and the volume of fluid was determined with
calibrated microcapillary tubes (Drummond, Broomall, PA). The volume of
fluid transported across the epithelium, expressed in microliters per
hour per square centimeter, was determined from the change in volume
over the 24-h period.
Immunostaining method.
A monoclonal antibody to the
1-subunit of Na-K-ATPase
(clone M7-PB-E9; Affinity BioReagents, Golden, CO) was used to examine membrane polarity of hIMCDi cell monolayers grown on
permeable supports. The immunostaining procedure was described
previously (13). Briefly, hIMCDi cell
monolayers were fixed in 4% PFA and permeabilized in 100% methanol at
20°C for 20 min. PBS containing 0.2% BSA, 5% goat serum, and 50 mM NH4Cl was used to block nonspecific binding sites.
Endogenous biotin was blocked with an avidin-biotin blocking kit
(Vector, Burlingame, CA). Monolayers were incubated in primary antibody
(diluted 1:50 in 0.1% goat serum in PBS) for 2 h at room
temperature. After several rinses, the secondary antibody (goat
anti-mouse conjugated to biotin) in PBS plus 0.1% goat serum was added
to the cell monolayers for 2 h at room temperature. Staining was
visualized by incubating the monolayers in ExtrAvidin conjugated
to peroxidase (Sigma) and developed with DAB (brown precipitate).
Similar results were obtained with a fluorescein isothiocyanate-conjugated secondary goat anti-mouse antibody. We
examined Na-K-ATPase localization in paraffin sections of fixed human
initial inner medulla with a method similar to that described above.
Immunoblot method.
Cell membrane extracts were prepared from fresh initial inner medullas
from two normal human kidneys and hIMCDi cell monolayers grown in plastic petri dishes. Fresh inner medullas were isolated, rapidly frozen in liquid N2, and kept frozen at
80°C
until cell membrane extracts were prepared. Crude cell lysates and
membrane extracts were prepared at 4°C. Tissues were homogenized with
a Polytron homogenizer (Brinkman, Westbury, NY) in lysate buffer containing 50 mM Tris (pH 7.4 with HCl), 10 mM imidazole, 0.3 M
sucrose, and protease inhibitors [104 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 0.08 mM aprotinin, 2.1 mM
leupeptin, 3.6 mM bestatin, 1.5 mM pepstatin A, 1 µg/ml antipain, 100 µM benzamidine, 1 µM dithiothreitol, and 1 mM phenylmethylsulfonyl
fluoride], and cell lysates were further homogenized with a glass
Dounce. The homogenates were centrifuged at 10,000 g for 15 min at 4°C to remove nuclei and debris. The supernatants were
collected and centrifuged at 100,000 g for 90 min at 4°C.
The cell membrane pellets were resuspended in 50 mM Tris (pH 7.4)
containing 1% (vol/vol) Nonidet P-40, 0.1% SDS, and 0.5% sodium
deoxycholate and protease inhibitors (same as above). Protein
concentrations in each of the membrane lysates were determined by
Pierce bicinchoninic acid protein assay (Rockford, IL).
Western blot analysis was used to determine the expression of NKCC1,
the secretory form of Na+-K+-Cl
cotransporter, and AE2 anion exchanger, a member of the
Cl
/HCO
exchanger family, in cell
membrane fractions of human initial inner medullas and cultured
hIMCDi cells. Immunoblotting for NKCC1 was performed with a
mouse monoclonal antibody (T4, supernatant; Developmental Studies
Hybridoma Bank, Iowa City, IA) directed against a 38-kDa fragment of
the COOH terminus of NKCC1 from human colonic crypt (T84 cells). This
antibody was described previously (10, 26). A T84 cell
lysate was used as a positive control. These cells were originally
purchased from American Type Culture Collection and maintained in
culture as previously described (58). For AE2
immunoblotting, we used an antibody raised against the COOH terminus of
mouse AE2 amino acids 1224-1237 (a generous gift from Dr. S. Alper, Harvard Medical School, Boston, MA). This antibody has been
shown to stain rat and mouse IMCD (1, 49)
T84 and primary cultures of IMCDi cells were grown to
confluence in petri dishes. Culture medium was removed, and the cells were washed three times in ice-cold PBS. Cells were scraped and collected and then homogenized with a glass Dounce in lysate buffer (same composition as used for tissue). Isolation of membrane proteins was carried out as described above.
A Mini-Protean III cell (Bio-Rad Laboratories) was used to resolve
membrane proteins by 7.5% SDS-PAGE with a method described previously
(61). Protein samples (10 µg for T84 and 20 µg for tissue and hIMCDi cells) were mixed with equal volume of
2× sample buffer (132 mM Tris · HCl pH 6.8, 4.2% SDS, 0.005% bromophenol blue, 21% glycerol, 0.72 M
-mercaptoethanol, 20 mM dithiothreitol) and denatured at 37°C for
30 min. Proteins were transferred to a nitrocellulose membrane and
blocked for 30 min at room temperature with blotting buffer containing
5% (wt/vol) nonfat powdered milk in TBS-T (20 mM
Tris · HCl, pH 8.0, 137 mM NaCl, and 0.05% Tween 20). The nitrocellulose membranes were incubated overnight at 4°C in
the blotting buffer containing antibody. The membranes were washed
several times in TBS-T and incubated in anti-mouse antibody conjugated
to HRP diluted in 5% nonfat milk in TBS-T for 60 min. The membranes
were washed several times, and NKCC1 and AE2 proteins were visualized
with an enhanced chemiluminescence system (ECL; Amersham Life Sciences,
Arlington Heights, IL).
cAMP measurements.
hIMCD cells were seeded (7.3 × 105 cells/0.33
cm2) on cell culture supports (Transwell-Col, 6.5-mm
diameter; Costar) and grown as confluent monolayers under growth
conditions similar to those used in the fluid transport experiments.
Medium was changed to Ringer solution containing 10 µM benzamil in
the apical reservoir for 15 min before the experiment (similar to
ISC experiments). Basolateral medium was changed
to a medium containing AVP, PGE2, or forskolin for 15 min.
The medium was removed, and a 80% methanol-20% water mixture was used
to extract cAMP from the cells. cAMP content was determined with an
enzyme immunoassay system (Amersham Pharmacia Biotech, Little Chalfont, UK).
Statistics.
Data are presented as means ± SE. Instat (GraphPad, San Diego,
CA), a statistical package, was used for data analysis. Where appropriate, Student's t-test or one-way ANOVA and the
Student-Newman-Keuls multiple-comparison posttest was used to determine
statistical significance. Data groups containing heterogeneous
variances indicated by Bartlett's test were analyzed with the
nonparametric tests Mann-Whitney U-test or the
Kruskal-Wallis nonparametric ANOVA and Dunn's posttest.
P < 0.05 was taken to indicate statistical significance.
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RESULTS |
Morphological properties of hIMCDi and
hIMCDt cell monolayers.
The initial and terminal regions of the inner medulla of human kidneys
were cultured separately to compare the functional response to cAMP
agonists in hIMCDi and hIMCDt cells. Primary cultures of IMCD cells grown as confluent monolayers on permeable supports for 10 days developed a "cobblestone" appearance (Fig. 2,
F and G). The cells appeared morphologically
similar to rat IMCD cell monolayers described by Light et al.
(23), and there were no measurable differences in the
appearance of the hIMCDi and hIMCDt monolayers
under low-magnitude light microscopy. Electron microscopy of the
ultrastructure of hIMCDi and hIMCDt monolayers grown on Snapwell revealed the cell monolayers to be polarized, with
basal surfaces of the cells attached to the filter supports (Fig. 2,
C and D) and adjacent cells joined by apical
junctional complexes (Fig. 2E). To a great extent, primary
cultures of hIMCDi and hIMCDt cells appeared to
have retained the basic cell appearance of collecting duct cells in
situ (Fig. 2, A and B).
hIMCDi cells grown on glass stained intensely for Pan
cytokeratin antibody (Sigma), indicating that the cells were of
epithelial origin (Fig. 3A).
Lectins bind to specific sugar residues that are uniquely distributed
in different parts of the nephron (14). Fluorochrome- and
HRP-labeled lectins were used as microscopic markers to characterize
the hIMCDi and hIMCDt cells in culture. DBA, a
lectin that binds preferentially to collecting duct cells in the inner
medulla (20, 33), showed strong, distinct staining in
hIMCDi cells (Fig. 3C). Nearly all cells were
stained with DBA conjugated with HRP (visualized with DAB); however,
the intensity of staining varied. Preincubation of DBA with 0.2 M
N-acetylgalactosamine, a competing sugar that has a high
affinity to DBA, inhibited staining (Fig. 3D). PNA, another
collecting duct lectin, bound to the apical surface of hIMCD cells
(Fig. 4A), but not proximal
tubules or glomerular cells, within the cortex (Fig. 4B).
PNA also showed strong, distinct staining of hIMCDi and
hIMCDt cell monolayers grown on glass (Fig. 4, D
and G) and a polarized hIMCDi monolayer grown on
a Snapwell (Fig. 4J). Preincubation of the lectin with 0.2 M
galactose, a competing sugar that has a high affinity to PNA, inhibited
staining (Fig. 4, E, H, and K). These
observations indicate that the cultures were highly enriched in IMCD
cells. Similar results were obtained with DBA and PNA conjugated to
rhodamine (not shown).

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Fig. 3.
Subconfluent culture of human IMCDi cells grown on
glass microscope chamber slides. A: cells were stained with
Pan anti-cytokeratin antibody (20 µg/ml) and visualized by a
secondary antibody conjugated to FITC. B: no
anti-cytokeratin antibody, but all other reagents were added.
C: cells stained with 50 µM Dolichos biflorus
agglutinin (DBA), a collecting duct-specific lectin. Most cells
stained positive for DBA; however, the intensity of the staining was
variable, suggesting that the cell culture was heterogeneous. Positive
staining was observed also in confluent hIMCDi cell
monolayers. D: mixture of 50 µM DBA and 200 µM
N-acetylgalactosamine, a competing sugar specific for
DBA.
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Fig. 4.
Arachis hypogaea agglutinin (peanut
agglutinin; PNA) staining of a collecting duct within the initial
region of a human inner medulla (A) and a distal tubule (red
arrow) situated juxtaposed to a glomerulus within the renal cortex
(B). In A, the red arrow denotes apical PNA
staining of the collecting duct (CD) cells consistent with a previous
report in rabbit CD (20). Confluent cultures of
hIMCDi (C, D, E) and
hIMCDt (F, G, H) cells
grown on glass microscope chamber slides are also shown.
hIMCDi and hIMCDt cells were incubated in the
absence of lectin and the presence of all other reagents (C
and F), 50 µM PNA (D and G), and a
mixture of 50 µM PNA and 200 µM galactose, a competing sugar
specific for PNA (E and H). Both cell types
stained specifically for PNA (brown). A hIMCDi cell
monolayer grown on a permeable support (Snapwell-Clear) also stained
for PNA. The monolayer was divided into 3 sections and incubated in
control medium (I), PNA (J), and PNA and
galactose (K). Cultured cells were counterstained with
Mayer's hematoxylin (blue of nuclei).
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Asymmetric distribution of Na-K-ATPase between the apical and
basolateral plasma membranes of polarized epithelial cells is necessary
for the establishment of vectorial transport of ions. As in many
transporting epithelia (2, 21, 31, 51, 52, 56, 57, 59),
the Na-K-ATPase was found to be restricted to the basolateral membrane
of human IMCDi in situ (Fig.
5A). Moreover, localization of
Na-K-ATPase in lateral membrane of hIMCDi monolayers (Fig.
5B) indicates that hIMCDi cells establish an orthodox polarity when cultured on permeable supports.

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Fig. 5.
Immunolocalization of Na-K-ATPase in a human initial IMCD
in situ (A) and a cultured hIMCDi cell monolayer
grown on a Snapwell-Clear support (B). A monoclonal antibody
directed against the 1-isoform of Na-K-ATPase was
visualized by 3,3'-diaminobenzidine (DAB; brown). Na-K-ATPase protein
was located on the basolateral cell membranes of hIMCDi.
Cellular localization of Na-K-ATPase to the lateral membranes of cells
within the polarized monolayer validates the establishment of an
orthodox polarity when hIMCDi cells are grown on permeable
filters.
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Comparison of effects of AVP and PGE2 on intracellular
cAMP in hIMCDi and hIMCDt cells.
We examined the effect of short-term incubation with media containing
AVP, PGE2, or forskolin on total intracellular cAMP levels
in confluent hIMCDi and hIMCDt cells grown on
permeable supports. Intracellular cAMP was measured in four groups
(n = 6 monolayers/group) treated for 15 min with
control medium, 100 mU/ml AVP, 25 ng/ml PGE2, or 10 µM
forskolin (Fig. 6). Benzamil (10 µM)
was added to the apical media of all groups for 10 min before the
experiment, to be consistent with the ISC
experiments. There were qualitatively similar increases in
intracellular cAMP levels in hIMCDi and hIMCDt
monolayers after incubation in the presence of AVP, PGE2,
and forskolin. These data indicate that cultured hIMCDi and
hIMCDt cells have AVP and PGE2 receptors
coupled to the cAMP pathway and that the capacity to generate cAMP by direct activation of adenylate cyclase with forskolin was not different
between hIMCDi and hIMCDt cells.

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Fig. 6.
Effect of 100 mU/ml AVP, 25 ng/ml PGE2, and
10 µM forskolin, an activator of adenylyl cyclase, on intracellular
cAMP levels in hIMCDi and hIMCDt cells grown on
collagen-coated permeable supports; n = 6/group, 2 kidney preparations. Total cAMP (pmol/monolayer) was determined with an
enzyme immunoassay system. Agonists were added 15 min before the cAMP
was extracted with 80% methanol. *P < 0.05 compared
with basal cAMP value.
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Bioelectrical properties of cultured hIMCDi and
hIMCDt cells.
We compared the electrical properties of hIMCDi and
hIMCDt cell monolayers (3 kidney preparations). Pairs of
hIMCDi and hIMCDt monolayers were mounted for
measurement of TER, transepithelial PD, and ISC
with two sets of chamber electrodes attached to a dual-voltage-clamp
device. hIMCDi cell monolayers (n = 10)
developed a TER of 258 ± 47
· cm2, a lumen-negative potential
(PD;
0.3 ± 0.1 mV), and a positive ISC
of 1.4 ± 0.5 µA/cm2. In contrast, the TER of
hIMCDt monolayers (n = 10) was 152 ± 16
· cm2 and PD and
ISC were not different from zero.
Evidence for Na+ absorption in human
IMCDi.
To investigate the contribution of Na+ absorption, through
ENaC and CNG channels, to the baseline current in the
hIMCDi cells, we examined the effect of adding ANP to the
basolateral medium and benzamil to the apical medium. In
hIMCDi monolayers (n = 13), ANP
(10-1,000 nM) caused a small but significant reduction in baseline
ISC (
ISC; 0.3 ± 0.1 µA/cm2, P < 0.05). The addition of
benzamil (10 µM), a high-affinity inhibitor of ENaC and CNG
(22, 53), to the apical fluid reduced baseline current
from 1.9 ± 0.2 to 1.5 ± 0.2 µA/cm2
(n = 28 monolayers, 4 kidney preparations;
P < 0.001). These data indicate that Na+
absorption via an ANP-sensitive apical Na+ conductance,
possibly ENaC and CNG channels, contributes to only a small fraction of
the basal current of cultured hIMCDi cell monolayers. In
experiments in which we tested the effect of cAMP agonists on
electrolyte secretion by IMCD monolayers, benzamil was included in the
apical medium to reduce the contribution of these Na+
absorptive pathways to ISC.
Effect of cAMP agonists on anion transport by human IMCD cells.
To investigate the effect of cAMP on anion secretion by human IMCD
cells, we incubated IMCDi monolayers in the presence of apical benzamil and monitored ISC after the
addition to apical and basolateral media of 200 µM 8-bromo-cAMP
(8-BrcAMP), a permeant analog of cAMP. A typical response of
IMCDi monolayers to benzamil and 8-BrcAMP is shown in Fig.
7. ISC increased
after the addition of 8-BrcAMP from 0.7 to 5.9 µA/cm2.
ISC reached a new steady state within 40 min. An
apically negative transepithelial PD hyperpolarized from
0.1 to
1.2
mV. In a group of four hIMCDi monolayers, 8-BrcAMP
increased benzamil-insensitive current from 2.2 ± 1.1 to 6.1 ± 0.7 µA/cm2 (P < 0.05; Fig.
8). In contrast, incubating human
hIMCDt monolayers (prepared from the same kidney and grown
under the same experimental conditions) in 8-BrcAMP did not
significantly change ISC
(
ISC = 0.0 ± 0.1 µA/cm2; n = 4).

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Fig. 7.
Effect of 8-bromo-cAMP (8-BrcAMP) and Cl
transport inhibitors on short-circuit current
(ISC) across hIMCDi cell monolayer.
Benzamil (10 µM) was added to the apical medium. 8-BrcAMP (200 µM)
was added to both apical and basolateral media. DIDS (50 µM) and
bumetanide (100 µM) were added to the basolateral medium.
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Fig. 8.
Effect of 8-BrcAMP on ISC in
collecting duct cells cultured from IMCDi (A)
and IMCDt (B); n = 4 monolayers/group. Benzamil (10 µM) was added to the apical media in
both groups. 8-BrcAMP (200 µM) was added to both apical and
basolateral media. *P < 0.01 compared with
control ISC; #P < 0.001 comparison between cAMP-stimulated ISC in
IMCDi and IMCDt.
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|
Forskolin, which rapidly penetrates plasma membranes and elevates cAMP
levels in hIMCDi cells (Fig. 6) by directly activating adenylyl cyclase, caused a peak increase in ISC
in hIMCDi monolayers within 5-10 min that declined to
a new steady-state current above baseline. Table
1 displays the changes in steady-state
current for 50 monolayers cultured from six nephrectomy specimens.
Forskolin stimulated an increase in benzamil-insensitive current in all hIMCDi cell monolayers (
ISC of
all monolayers was 3.3 ± 0.2 µA/cm2); however, the
magnitude of the response varied among the different preparations.
Forskolin did not significantly increase ISC in the IMCDt monolayers (data not shown).
Effect of AVP and PGE2 on anion secretion in
hIMCDi monolayers.
The data in Fig. 6 indicated that AVP and PGE2
significantly increased cAMP in hIMCDi and
hIMCDt cells; however, the response was much lower than
that of a maximally effective concentration of forskolin. In cultured
rat (16) and mouse (52) IMCD cells, the
cAMP-stimulated ISC in the presence of benzamil
was attributed to anion secretion involving apical CFTR
Cl
channels. To determine whether AVP and
PGE2 stimulated electrogenic anion secretion by the
hIMCDi monolayers, we tested the effect of these agonists
on ISC and PD (Table
2) after the addition of benzamil to the
apical medium. In most monolayers, benzamil caused a small decline in
baseline ISC. The addition of 100 mU/ml AVP to
the basolateral medium significantly increased the current (
ISC = 1.0 ± 0.1 µA/cm2) and hyperpolarized the cell monolayers.
PGE2, an important intrarenal autacoid involved in the
regulation of blood pressure, was examined to determine whether it also
affected electrolyte transport by hIMCD cells. PGE2
potently stimulated anion secretion by the hIMCDi cells.
ISC increased by 3.6 ± 0.5 µA/cm2, and the monolayer hyperpolarized by
1.1 ± 0.1 mV. These observations indicate that AVP and PGE2 are
important factors for promoting cAMP-dependent electrogenic
Cl
secretion by hIMCDi cells.
Effect of cAMP on fluid transport by hIMCDi cells.
To determine whether the cAMP-induced anion transport was coupled to
fluid secretion, we compared the direction and the rate of fluid
transport in three groups of hIMCDi monolayers
(n = 4 per group) incubated in 1) control
medium, 2) apical medium containing 10 µM benzamil, or
3) apical medium containing benzamil and basolateral medium
containing 10 µM forskolin. In Fig. 9,
negative values represent fluid absorption and positive values indicate
fluid secretion. In the control medium, human IMCDi
monolayers absorbed fluid at a rate of
0.13 ± 0.03 µl · h
1 · cm
2
(P < 0.02; 1-sample t-test). Benzamil added
to the apical medium had no effect on fluid absorption (
0.14 ± 0.02 µl · h
1 · cm
2),
whereas the addition of apical benzamil and basolateral forskolin reversed the net flux of fluid to secretion (0.40 ± 0.05 µl · h
1 · cm
2;
P < 0.001).

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Fig. 9.
Measurement of fluid transport rates across
hIMCDi cell monolayers. Values are means ± SE;
n = 4. Positive values indicate fluid secretion, and
negative values indicate fluid absorption. Significance was determined
by Kruskal-Wallis nonparametric analysis of variance and Dunn's test.
*P < 0.05 compared with previous period.
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|
Effect of Cl
channel blockers on
cAMP-dependent transport.
Mechanisms of cAMP-dependent Cl
secretion have been
investigated in cultured rat and mouse IMCD cell monolayers (16,
18); however, cAMP-dependent anion secretion has not been
previously characterized in hIMCD cells. Using pharmacological agents,
we determined whether cAMP activated CFTR Cl
channels. We
tested the effects of apical application of
diphenylamine-2-carboxylate (DPC) and glybenclamide, known
inhibitors of CFTR Cl
channels, on forskolin-stimulated
anion secretion in hIMCDi monolayers. DPC inhibited
91 ± 6% of the forskolin-stimulated current, whereas glybenclamide, a weak inhibitor of CFTR, reduced the stimulated ISC by 48 ± 5% (Fig.
10). Niflumic acid, a potent inhibitor
of epithelial Cl
channels including CFTR
(4), also attenuated cAMP-stimulated current in
hIMCDi monolayers (data not shown). DIDS is known to block Cl
/HCO
exchange and some types
of Cl
channels but is unable to block the
Cl
conductance of CFTR (42). Apical
application of DIDS did not significantly inhibit
ISC (data not shown). The pharmacological profile of cAMP-induced anion secretion by hIMCDi to
Cl
channel blockers is consistent with CFTR
Cl
channels in the apical plasma membrane
(42).

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Fig. 10.
Effect of Cl channel inhibitors on
forskolin-stimulated ISC in human IMCD cells.
Currents were measured in the presence of benzamil (10 µM) added to
the apical medium. Inhibitors were added after a steady state was
reached in the presence of basolateral forskolin (10 µM).
Diphenylamine-2-carboxylate (DPC) and glybenclamide were added to the
apical medium. *P < 0.05 compared with the control
value (benzamil alone); #P < 0.05 compared with the
forskolin-stimulated current.
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Mechanisms for Cl
uptake across
basolateral membrane during cAMP-dependent
Cl
secretion.
A mouse monoclonal antibody (T4) to NKCC1, the secretory isoform of
Na+-K+-Cl
cotransporter
(26), has been localized within the basolateral membranes
of a subfraction of rat collecting duct cells from the outer medulla
(10). In the current study, we detected by Western blot
abundant immunoreactivity of the T4 antibody to plasma membranes from
fresh human initial inner medulla (Fig.
11, lane 1). There was a
band at ~200 kDa and a broad band in the range of 150-170 kDa.
NKCC1 has been reported to migrate during electrophoresis in a broad
band of 145-205 kDa (10, 26). Multiple and broad bands for NKCC1 are often attributed to multiple states of
glycosylation. When the supernatant of cultured hybridoma cells not
expressing the relevant antibody was used as a control, no bands were
observed. In confluent monolayers enriched in hIMCDi cells,
we observed a major band at ~170 kDa in the membrane fraction (Fig.
11, lane 2). These results were confirmed with two
additional hIMCDi preparations from other kidneys. These
studies show that fresh human initial inner medulla and cultured
hIMCDi cells express NKCC1 protein.

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Fig. 11.
Immunoblots for NKCC1, a secretory form of the
Na+-K+-Cl cotransporter, and AE2,
an anion exchanger for HCO and Cl .
Membrane proteins were isolated from a freshly dissected human initial
inner medulla (lanes 1 and 3) and
hIMCDi cells grown as confluent monolayers in petri dishes
(lanes 2 and 4). A: human initial
inner medulla and hIMCDi cells expressed a protein of 170 kDa that was detected by NKCC1 antibody. A higher-molecular-mass
protein was also detected in the medullary tissue. B: an
antibody that detects both AE1 and AE2 revealed a 165-kDa band in both
medullary tissue and cultured cells, a protein consistent with AE2. A
second band was also detected at ~205 kDa in the medullary tissue.
Overexposure of the film revealed a weak band at ~ 100 kDa, the
expected molecular mass of AE1 protein.
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It is well established that AE anion exchangers, transporters that
exchange HCO
and Cl
, are present in
rodent IMCD (35). Measurements of mRNA and protein levels
for the three known AE anion exchangers have suggested that AE2 is the
most abundant form in renal cells (1). Recently, Alper and
co-workers used an epitope-unmasking technique to show that AE2
immunolocalized to basolateral plasma membranes but not apical
membranes of rat (1) and mouse (49) IMCD. To
investigate whether human initial inner medulla and cultured
hIMCDi cells express AE anion exchangers, we examined the
protein expression of AE1 (~100 kDa) and AE2 (~165-180 kDa) by
Western blot analysis with an antibody raised against the COOH terminus
of mouse AE2 (amino acids 1224-1237). The COOH termini of AE2 and
AE1 are sufficiently similar so that the antibody recognizes both
proteins (49). In membrane fractions of human initial
inner medulla (Fig. 11, lane 3) and cultured
hIMCDi cells (Fig. 11, lane 4), the antibody detected a major band at ~165 kDa. This is consistent with the expression of AE2 protein. Overexposure of the film also revealed a
band at ~100 kDa, indicating the presence of a less abundant AE1
anion exchanger.
To determine whether these Cl
transport mechanisms
participate in cAMP-dependent anion secretion by hIMCDi
cells, we tested the effect of basolateral application of bumetanide,
an inhibitor of NKCC1, and DIDS, an inhibitor of AE2, on
cAMP-stimulated ISC. In Fig. 7, we show a
typical response to these inhibitors after steady-state stimulation by
200 µM 8-BrcAMP. In other experiments, hIMCDi cell
monolayers (n = 6) incubated in medium containing benzamil exhibited a baseline ISC of 1.5 ± 0.6 µA/cm2 and forskolin increased this to a steady-state
current of 6.0 ± 0.5 uA/cm2 (P < 0.001; Fig. 12). Basolateral addition
of bumetanide (100 µM) reduced ISC to 3.8 ± 0.5 µA/cm2 (P < 0.01), and the
subsequent addition of DIDS caused a further reduction of current to
2.0 ± 0.4 µA/cm2 (P < 0.001 compared with forskolin alone). After bumetanide and DIDS were
combined, the current was not significantly different from the control
current in benzamil alone. These data indicate that NKCC1 and AE2 are
major pathways for Cl
entry across the basolateral
membrane during cAMP-dependent Cl
secretion.

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Fig. 12.
Effect of basolateral addition of Cl entry
inhibitors on forskolin-stimulated anion current. Benzamil (10 µM)
was added to the apical medium 10 min before control values were
recorded. Forskolin, added to the basolateral medium, increased
ISC in the 6 monolayers. All additions were made
after the ISC reached a steady state after the
prior addition. The combination of bumetanide, an inhibitor of
Na+-K+-Cl cotransport, and DIDS,
an inhibitor of Cl /HCO exchange,
reduced ISC to near the control level. See text
for mean ± SE values.
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|
 |
DISCUSSION |
In the present study we used an in vitro method to explore the
transport characteristics of primary cultures of cells enriched in
hIMCDi or hIMCDt. The key observations of the
study are as follows. 1) Benzamil caused a small decline in
ISC in hIMCDi cells; however, it did
not inhibit fluid absorption. 2) AVP, PGE2, and forskolin stimulated cAMP production in cells cultured from both initial and terminal regions of human IMCD. 3) cAMP
stimulated electrogenic Cl
and fluid secretion in
hIMCDi cell monolayers but not in hIMCDt monolayers. 4) NKCC1, a secretory form of
Na+-K+-Cl
cotransporter,
and AE2, a Cl
/HCO
anion exchanger, may
represent parallel pathways for Cl
entry into
hIMCDi cells across the basolateral membranes.
5) CFTR Cl
channels appear to mediate
cAMP-regulated Cl
efflux across the apical membranes of
hIMCDi cells.
Characteristics of hIMCDi and hIMCDt cell
monolayers.
The initial IMCD contain a heterogeneous population of mainly principal
or "light" cells and
-type intercalated or "dark" cells,
whereas the terminal IMCD contain a single cell type referred to as
"the IMCD cell" (5, 27, 32). To compare the
characteristics of hIMCDi and hIMCDt cells in
culture, we isolated regions of the human initial and terminal inner
medulla separately (Fig. 1) and prepared primary cultures with methods
similar to those published previously (15, 16, 23, 56).
Cells from both regions grew well in the presence of 5% FBS and formed
confluent monolayers on permeable supports within 5-7 days.
Adjacent cells in the monolayers formed apical junctional
complexes (Fig. 2E) that were electrically tight as gauged
by measurements of TER. The average resistance of
hIMCDi cell monolayers was greater than that of
hIMCDt monolayers. Moreover, the hIMCDi
monolayers generated an apically negative PD and positive
ISC, consistent with active ion transport in the
basal state (Fig. 8, Table 2). In contrast, PD and
ISC in IMCDt cells were not
different from zero. Because hIMCDt cells generated cAMP in
response to agonists as well as hIMCDi cells, the
differences in transport between them are probably due to a lower
abundance of solute transporters in hIMCDt. These results
are in accord with measurements of PD in isolated, perfused rat
collecting ducts (37, 40); however, we cannot exclude the
possibility that the hIMCDt cells would have different
basal transport characteristics in a medium with a composition
comparable to the interstitial fluid of terminal inner medulla.
Morphological examination of cultured hIMCDi and
hIMCDt monolayers showed a similar ultrastructure in the
two cell populations (Fig. 2, C and D). In
addition, assessment of the monolayers by electron microscopy showed a
relatively homogeneous population of cells. Unfortunately, principal
and intercalated cells could not be readily distinguished.
hIMCDi and hIMCDt monolayers expressed cytokeratin and stained for collecting duct lectins, indicating that
the cultures were derived from medullary collecting duct cells to a
major extent (Figs. 3 and 4).
Absorption of Na+ by
hIMCDi cells.
Na+ absorption occurs at many loci along the nephron;
however, the fine regulation of Na+ excretion is thought to
occur in the collecting duct. ENaC has been shown to mediate
Na+ absorption across the apical membrane of rat and mouse
IMCD cells (23, 48, 62); however, its role in
Na+ absorption by hIMCD cells has not been previously
explored. In the present study, benzamil, a specific inhibitor of the
ENaC and CNG channels (53), produced small but significant
decreases in baseline ISC, consistent with the
inhibition of Na+ absorption. On the other hand, benzamil
failed to inhibit solute-coupled fluid absorption over a 24-h period
(Fig. 9). Technical limitations in the method for measuring net fluid
transport across cell monolayers may have prevented detection of a
small inhibition of fluid absorption by benzamil. Moreover, our culture
medium, which may lack critical hormones (i.e., aldosterone) involved
in modulating Na+ absorption (62), may be
inappropriate for investigating Na+ absorptive pathways.
The results from this study, therefore, may underestimate the
contribution of NaCl absorption by hIMCDi cells.
Consequently, the molecular basis of fluid absorption by hIMCDi remains to be determined.
Salt secretion by IMCD cells.
The role of electrolyte and fluid secretion by IMCD and its
contribution to the composition and volume of urinary fluid have been
debated for several decades. Sonnenberg (46) was the first to demonstrate, with a microcatheterization approach, net salt secretion by renal collecting ducts in acutely volume-expanded rats.
Sonnenberg's data indicated that the IMCD secreted Na+ and
water in amounts equal to ~10% of the filtered load
(46). Recently, we confirmed (57) that,
indeed, rat IMCD have an intrinsic capacity for cAMP-dependent salt and
fluid secretion. Fluid secretion was inhibited by Cl
transport blockers, indicating that active Cl
secretion
was coupled to the osmotic flow of water in IMCD. Husted and associates
(16) showed that AVP and cAMP stimulated anion secretion
by cultured rat IMCD cells and that this was mediated through CFTR
Cl
channels present in the apical membrane. Thus the in
situ observations by Sonnenberg (46) may be attributed to
intrinsic salt and fluid secretion mechanisms in the initial region of
the IMCD. Although there is mounting evidence that supports a role for
cAMP-regulated salt secretion by the IMCD and, to a lesser extent,
other regions of the collecting duct of rat, mouse and rabbit (Table
3), few studies have examined electrolyte
transport by human collecting duct cells.
We found that the cAMP agonists AVP and PGE2, and
forskolin, increased the accumulation of intracellular cAMP in
hIMCDi and hIMCDt cell monolayers (Fig. 5).
Thus both cultured hIMCDi and hIMCDt cells have
receptors for AVP and PGE2 that are coupled to the
generation of cAMP. Furthermore, the capacity to maximally generate
cAMP through the direct activation of adenylyl cyclase by forskolin was
the same between the two cell lineages. On the other hand, the
electrolyte transport response to cAMP was distinctly different. AVP,
PGE2, forskolin, and 8-BrcAMP activated a
benzamil-insensitive current in IMCDi but not in
IMCDt cell monolayers. The current was inhibited by apical
application of DPC, glybenclamide, and niflumic acid, drugs that block
CFTR Cl
channels. Thus the evidence at hand indicates
that transepithelial Cl
transport has a clear role in the
secretion of fluid by hIMCDi but not by hIMCDt cells.
The Na+-K+-Cl
cotransporter has
been localized to the basolateral membranes of rat IMCD
(6), and it has been projected to mediate Cl
secretion in rat IMCD (39, 57) and outer medullary
collecting ducts (54). In the present study, NKCC1 protein
(175 kDa) was found to be expressed in the membrane fraction of tissue
isolated from human initial inner medulla and in cultured
hIMCDi cell monolayers (Fig. 11). Bumetanide applied to the
basolateral surface of the hIMCDi cells significantly
reduced cAMP-stimulated ISC, pointing to a
potential role for the NKCC1 transporter in Cl
entry
during cAMP-dependent secretion.
AE2 anion exchanger, a transporter that exchanges
HCO
and Cl
, has been immunolocalized
to the basolateral, but not apical, membranes of rat and mouse IMCD
cells (1, 49). Cl
/HCO
exchangers may contribute to the regulation of cell pH and cell volume.
Two observations in the present study indicate that AE2 transporters
may also contribute to transepithelial Cl
secretion in
human IMCD cells during cAMP stimulation: 1) Cell membranes
prepared from cell lysates of human initial inner medullary tissue and
cultured hIMCDi cells contained abundant AE2 protein. 2) DIDS, an inhibitor of the AE2 transporter, reduced
cAMP-stimulated anion secretion. Together, these findings indicate that
NKCC1 and AE2 may operate in concert to mediate Cl
uptake
across the basolateral membrane during cAMP-dependent Cl
secretion in hIMCDi.
Potential roles of collecting duct NaCl and fluid secretion.
In the evolution of excretory organs, NaCl-coupled fluid secretion
seems to be a conserved mechanism for regulating body fluid composition
and volume (12). Marine birds and reptiles possess specialized salt glands that secrete a highly concentrated NaCl fluid
(41), whereas marine teleosts, the bony fishes, secrete NaCl through "chloride cells" located in the gills (9)
and elasmobranchs secrete NaCl via rectal glands (45).
Beyenbach and Liu (3) demonstrated NaCl-coupled fluid
secretion in the proximal tubules of aglomerular and glomerular fish.
In the case of the aglomerular fish, urine formation was completely
dependent on tubular salt and fluid secretion. Apical Cl
channels mediate Cl
secretion in many of the excretory
organs of these nonmammalian species (3, 9, 12, 36, 41).
In fact, stellar cells of the insect malpighian tubules secrete
Cl
through CFTR-like channels (36).
Recent evidence of cAMP-dependent NaCl and fluid secretion in isolated
intact rat IMCD (39, 54, 57) and cultured rat (16), mouse (18), and human IMCD cells
demonstrates that this ancient secretory mechanism has been conserved
in the renal collecting system of mammals. The mechanism for salt and
fluid secretion is strategically located in the terminal portions of
the renal tubule system. Were the NaCl secretion process situated in
the proximal tubule exclusively, the contribution to urine formation of
the relatively low magnitudes of net solute and fluid secretion might
be nullified by reabsorption in more distal tubular segments. Previous
studies may be interpreted to demonstrate a role for tubular secretion
in the regulation of ECF. Luke (25) found in rats that
withholding water for 24 h led to natriuresis and chloruresis, an
effect on salt excretion that was mimicked by the administration of
AVP. It was suggested that the increase in NaCl excretion might reflect
a mechanism conserved in mammalian kidneys to maintain the tonicity of
ECF during dehydration. Martinez-Maldonado et al. (28)
showed similar natriuretic effects of AVP administered to dogs. It is
conceivable that dehydration, mediated by AVP, unmasks a cAMP-regulated
salt secretory mechanism in collecting ducts that blunts the elevation
in plasma hypertonicity during desiccation.
It is also conceivable that NaCl-coupled fluid secretion by the renal
tubule is important for maintaining patent lumens and propelling fluid
out of the kidney during conditions of reduced or complete cessation of
glomerular filtration, as may occur in acute renal failure,
dehydration, and sudden blood volume loss caused by hemorrhage. This
would enable the kidney to excrete to some extent potentially toxic
products during periods in which glomerular filtration is insufficient.
In this way, tubular secretion may serve as a "default" method of
urine formation that has been in place since the era of aglomerular
protovertebrates (12).
Role of NaCl and fluid secretion in renal cyst formation.
The contribution of salt secretion to urine formation is difficult to
assess clinically because massive quantities of salt and fluid are
filtered by glomeruli and reabsorbed by many nephron segments. On the
other hand, the intrinsic capacity to secrete NaCl is evident in renal
cyst formation. In autosomal dominant polycystic kidney disease
(ADPKD), cysts develop from the focal outgrowth of individual tubule
cells and fluid accumulates within isolated sacs. The mechanisms of
fluid secretion into ADPKD renal cysts were recently elucidated
(50, 51, 56). cAMP agonists regulate fluid secretion by
the mural epithelial cells through activation of apical CFTR and
basolateral NKCC1 cotransporters (56). In the present
study, cAMP-dependent NaCl and fluid secretion by renal cells from
individuals without ADPKD supports the view that secretion is a normal
physiological process within specific regions of the nephron. Thus the
accumulation of NaCl and fluid within renal cysts could simply be a
consequence of the fact that most cysts are isolated from their parent
tubules, an anatomic outcome of the polycystin gene mutation. The lack
of glomerular filtration into renal cysts may uncover a secretory
process normally masked by absorption. The new discovery that cAMP
agonists stimulate NaCl and fluid secretion in normal IMCD forces us to
reconsider the origin of urinary fluid that has been frequently and
most conveniently attributed to incomplete reabsorption of NaCl and water from the glomerular filtrate.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Tamio Yamaguchi for technical assistance and Dr. Larry
Sullivan for reading the manuscript and for helpful discussions. The T4
NKCC1 monoclonal antibody developed by Lytle and Forbush was obtained
from the Development Studies Hybridoma Bank developed under the
auspices of the National Institute of Child Health and Human
Development and maintained by the Department of Biological Sciences,
University of Iowa (Iowa City, IA). We thank Dr. S. Alper for the
generous gift of AE2 antibody.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants P01-DK-53763 and P50-DK-57301 (J. J. Grantham) and a National Research Service Award F32-DK-09929-01 (D. P. Wallace).
Portions of this study were published in abstract form (J Am
Soc Nephrol 11: 38A, 2000).
Address for reprint requests and other correspondence:
D. P. Wallace, Dept. of Medicine, Univ. of Kansas Medical
Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7382 (E-mail:
dwallace{at}kumc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 24, 2002;10.1152/ajprenal.00165.2002
Received 30 April 2002; accepted in final form 18 July 2002.
 |
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