Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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We employed two guanine nucleotide binding protein (G
protein)-coupled receptors known to be targeted to opposite domains in
renal epithelial cells to test the hypothesis that the polarized receptor expression of receptors regulates the activity of the receptor's effector molecule, adenylyl cyclase. We used
LLC-PK1 cells stably transfected
with cDNA encoding the
2B-adrenergic receptor
(
2B-AR) or
A1-adenosine receptor
(A1-AdR). Immunohistochemistry and
Western blot analysis confirmed the basolateral and apical expression
of
2B-ARs and
A1-AdRs, respectively. Adenylyl
cyclase activity was assessed by measuring cAMP accumulation following the addition of forskolin (10 µM) in the presence of
3-isobutyl-1-methylxanthine to apical or basolateral chambers of
confluent monolayers. A five- to sixfold increase in cAMP accumulation
occurred following apical (or basolateral) stimulation of
LLC-PK1 cells expressing apical (or basolateral) receptors in comparison to forskolin stimulation of
corresponding domains of untransfected cells. We conclude
1) adenylyl cyclase activity is
present at or near the apical and basolateral domains of
LLC-PK1 cells, and
2) factors that regulate the
polarized expression of inhibitory G protein-coupled receptors may also
regulate local adenylyl cyclase activity.
LLC-PK1 cells; adenosine receptors; adrenoceptors; adenosine 3',5'-cyclic monophosphate; polarity
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INTRODUCTION |
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DISTINCT CELL SURFACE proteins at apical and
basolateral domains of renal epithelial cells are vital to the
transport of ion and water across renal epithelium (4). With cloning of
renal epithelial cell transporters, much work has focused on the
polarized distribution of these transport proteins. Recently, however,
a growing body of literature suggests that receptors are also
asymmetrically expressed in renal epithelial cells. Although cell
surface receptors in renal epithelial cells are generally regarded as
being expressed at the basolateral domain, functional studies have
demonstrated their expression at the apical membrane as well.
Angiotensin II (23),
2-adrenergic (8), and adenosine
receptors (6, 15) are thought to mediate the effects of agonist
exposure at the apical domain. Filtered catecholamines or endogenous
ligands secreted into the tubular fluid may have distant or local
effects by activation of apically expressed receptors.
Although guanine nucleotide binding protein (G protein)-coupled
receptors are known to be expressed at both apical and basolateral domains, much less is known regarding the effector molecules to which
they couple. For example, despite the demonstration of inhibitory G
protein-coupled receptors at the apical membrane of epithelial cells
(16, 22), the mechanism by which these receptors couple to adenylyl
cyclase is not known. Adenylyl cyclase is thought to be localized to
the basolateral domain (24). If this indeed were the case, then it
would appear that adenylyl cyclase is spatially segregated from apical
receptors to which they couple. The purpose of our study were to
examine the influence of polarization of receptor expression on the
activity of their effector molecule, adenylyl cyclase. Toward this end
we employed A1-adenosine receptors (A1-AdRs), receptors known to be
localized to the apical membrane of renal epithelial cells (22), and
2B-adrenergic receptors (
2B-ARs), receptors known to be
expressed at the basolateral membrane of proximal tubule cells (10).
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METHODS |
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Cell culture. LLC-PK1 cells (American Type Culture Collection, Rockville, MD) were maintained in medium 199 (GIBCO-BRL Laboratories, Grand Island, NY) supplemented with 10% (vol/vol) dialyzed fetal bovine serum (FBS; GIBCO-BRL Laboratories) and penicillin (100 U/ml)/streptomycin (100 µg/ml) (GIBCO-BRL) under a 5% CO2 atmosphere at 37°C. Subculturing was performed every 4-5 days.
For experiments aimed at examining the distribution of receptors and their interaction with adenylyl cyclase, cells were seeded onto polycarbonate membrane permeable filter supports (24.5 mm, 0.45 µm, Transwell chambers; Costar, Cambridge, MA) and grown to confluence. The degree of monolayer confluence was assessed by two independent methods prior to each experiment. Membrane leakage rate of inulin was assessed by adding [3H]inulin (395 mCi/g; Dupont-NEN, Boston, MA) to the apical medium for 1 h at 37°C. Samples of medium from basolateral and apical chambers were removed, and radioactivity was determined by scintillation counting. Chambers were used only when apical to basolateral leakage rates were less than 2%/h. A second method used to assess the degree of confluence was established by the measurement of resistance across cells, using modified electrodes and an epithelial volt-ohmmeter (EVOM; World Precision Instruments, Sarasota, FL).
Expression of RNG
2B-AR and FLAG
A1-AdR in
LLC-PK1 cells.
RNG
2B-AR cDNA previously
subcloned into the plasmid RLDN (9) (provided by Dr. Kevin
R. Lynch, Department of Pharmacology, University of Virginia) and human
FLAG epitope-tagged
A1-AdR cDNA previously subcloned
into pDT (19) (provided by Dr. Joel Linden, Department of Medicine,
University of Virginia) were used to transfect LLC-PK1 cells as previously
described (7). The FLAG epitope consists of a 24-nucleotide sequence
5' to the coding region of the cDNA encoding the human
A1-AdR. cDNAs encoding
2B-ARs or A1-AdRs (25 µg) were suspended
in 1 ml of solution consisting of (in mM) 42 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 276 NaCl, 10 KCl, 3 Na2HPO4 · 7H2O,
and 11 dextrose (pH 7.1) and precipitated by the addition of 1 ml of
0.25 M CaCl2 and 40 µM
chloroquine for 15 min. Trypsinized
LLC-PK1 cells (1 × 106) were pelleted and
resuspended in 2 ml of precipitated cDNA suspension and allowed to
stand at room temperature for 15 min. Medium was supplemented with 12.5 mM CaCl2 and 2 µM chloroquine
(final concentration), and the suspended cells were seeded onto 100-mm
tissue culture dishes and incubated at 37°C, 5%
CO2 for 6 h. Following the
incubation period, the medium was aspirated, and cells were subjected
to osmotic shock with 5 ml of 20% dimethyl sulfoxide in complete medium 199 for 5 min at 37°C. Cells were washed and grown for 24 h
in complete medium 199 before selection in G-418 (0.5 g/l) (GIBCO-BRL).
Resistant colonies were isolated 2-3 wk after transfection and
screened for receptor expression. Receptor densities were determined by
using 3H-labeled MK-912 (81.3 Ci/mmol; Dupont-NEN), an
2-AR-selective antagonist, or
[3H]cyclopentyl-1,3-dipropylxanthine
(DPCPX; 120 Ci/mmol, Dupont-NEN), an
A1-AdR-selective antagonist, for
radioligand binding assays.
Immunohistochemical localization of
2B-ARs and
A1-AdRs in
LLC-PK1 cells.
LLC-PK1 cells were grown on
eight-well Lab-Tek chamber slides (Nunc, Naperville, IL) until
confluent and fixed in periodate-lysine-paraformaldehyde (final
concentration, 0.01 M NaIO4, 0.75 M lysine, 0.0375 M sodium phosphate buffer, pH 7.4, and 2%
formaldehyde) for 10 min at 4°C. Cells were rinsed in
phosphate-buffered saline (PBS) and blocked in avidin-biotin blocking
reagent (Vector Laboratories, Burlingame, CA). Cells were then washed
in PBS, blocked with PBS/6% FBS for 2 h followed by overnight
incubation with PBS/6% FBS, 0.1% Triton X-100, and a
well-characterized affinity-purified polyclonal antibody (2.5 µg/ml)
raised to the third intracellular loop of the
2B-AR (10) or an
anti-FLAG monoclonal antibody (27 µg/ml; International Biotechnologies, New Haven, CT). The following day the chambers were
rinsed with PBS/FBS and incubated with a biotinylated secondary antibody (1:1,000 dilution) followed by avidin-fluorescein
isothiocyanate (1:1,000 dilution). Cells were rinsed ×3 in
PBS/goat serum followed by 10 mM sodium phosphate (pH 7.4) and mounted
with an aqueous-based mounting solution consisting of
p-phenylenediamine (1 mg/ml) and 70%
glycerol. Cells were viewed under a Zeiss AxioSkop photofluorescence microscope.
Steady-state surface expression of
2B-AR and
A1-AR in
LLC-PK1 cells.
Apical and basolateral surface membrane proteins were isolated by a
procedure utilizing biotin long chain (LC) hydrazide or biotin 3-sulfo-N-hydroxysuccinimide
ester (sulfo-NHS-SS-Biotin; Pierce, Rockford, IL) (21).
Because RNG
2B-ARs are
nonglycosylated receptors (30), we used sulfo-NHS-SS-Biotin, a compound
that binds to surface lysine
-residues, to isolate
apical and basolateral surface membrane proteins. In contrast, because
A1-AdRs are glycosylated receptors, we employed biotin LC hydrazide, a compound that binds to
surface glycoproteins.
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RESULTS |
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2B-ARs
are expressed at the basolateral domain of
LLC-PK1 cells.
To test the hypothesis that polarization of receptor expression
influenced the activity of the receptor's effector molecule, LLC-PK1 cells were stably
transfected with cDNA encoding the rat
2B-AR. Saturation binding
studies utilizing
[3H]MK-912
demonstrated a receptor density of ~2 pmol/mg protein. No detectable
binding was found in untransfected
LLC-PK1 cells.
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Steady-state expression of
2B-ARs at the
basolateral membrane increases basolateral adenylyl cyclase
activity.
To determine whether the increase in expression of
2B-AR at the basolateral domain
of LLC-PK1 cells regulated its
effector, adenylyl cyclase, we stimulated apical and basolateral
membrane adenylyl cyclase separately with forskolin. In separate wells, apical surfaces and basolateral surfaces of untransfected
LLC-PK1 cells or
LLC-PK1 cells expressing
basolateral
2B-ARs were
incubated with forskolin. cAMP accumulation was measured as an index of adenylyl cyclase activity. Basal levels of cAMP accumulation were 11.1 ± 2.83 and 6.6 ± 0.56 pmol/mg protein for untransfected
LLC-PK1 cells and
LLC-PK1 cells transfected with
2B-ARs, respectively (n = 3;
P = NS). Stimulated levels of cAMP
accumulation following the application of forskolin separately to
apical and basolateral sides of untransfected cells were 113.8 ± 14.63 (n = 5) and 67.6 ± 13.6 (n = 6) pmol/mg protein
(P < 0.05), respectively. Unlike untransfected cells, basolateral addition of forskolin in
LLC-PK1 cells expressing
basolateral
2B-ARs produced a
dramatic increase in cAMP accumulation. When forskolin was applied to
the apical surface of
2B-AR
transfected cells, cAMP accumulation was similar to that observed in
untransfected cells. In
2B-AR
transfected LLC-PK1 cells, cAMP
accumulation in response to separate apical and basolateral incubation
with forskolin was 134.2 ± 8.41 (n = 5) and 471.6 ± 97.89 (n = 9)
pmol/mg protein (P < 0.05),
respectively. Table 1 summarizes the
effects of forskolin on cAMP accumulation in
LLC-PK1 cells transfected with
2B-ARs. These observations suggested that the steady-state expression of
2B-ARs at the basolateral domain of LLC-PK1 cells influenced
the activity of adenylyl cyclase at or near the basolateral domain.
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A1-AdRs are
expressed at the apical domain of
LLC-PK1 cells.
Our preceding results prompted us to determine whether apically
expressed receptors produce similar but opposite responses as those
with basolaterally expressed receptors. Previously, Saunders et al.
(22) demonstrated that the canine
A1-AdRs were expressed primarily
at the apical domain of renal epithelial cells. Using this information,
we expressed human FLAG epitope-tagged
A1-AdRs in
LLC-PK1 cells that we could
localize immunohistochemically with a commercially available anti-FLAG
antibody. Saturation binding studies using
[3H]DPCPX demonstrated
a receptor density of ~8 pmol/mg protein. LLC-PK1 cells transfected with the
epitope-tagged A1-AdRs showed a
diffusely punctate labeling sparing the lateral membranes (Fig. 5). This pattern of labeling is strikingly
different from the lateral labeling observed in
LLC-PK1 cells expressing
2B-ARs (see Fig. 3).
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Steady-state expression of
A1-AdRs at the apical domain increases
apical adenylyl cyclase activity.
The dramatic basolateral response of adenylyl cyclase in cells
transfected with the 2B-ARs led
us to examine whether the polarized response to forskolin could be
reversed when another inhibitory G protein-coupled receptor,
A1-AdR, was expressed at the
opposite domain. LLC-PK1 cells
demonstrating stable expression of apical membrane
A1-AdRs were grown on permeable
filter supports. In separate wells, apical and basolateral membranes
were incubated with forskolin and IBMX. In contrast to the results with
LLC-PK1 cells transfected with the
2B-AR, the expression of
A1-AdR at the apical membrane led
to a large increase in cAMP following apical membrane but not
basolateral membrane incubation with forskolin. cAMP accumulation
following apical vs. basolateral incubation with forskolin was
1,140.7 ± 144.25 (n = 9)
vs. 98.2 ± 10.64 pmol/mg protein
(n = 9)
(P < 0.001), respectively. In
contrast, the response of the apical vs. basolateral membranes to
forskolin in control untransfected cells was 176.5 ± 29.41 (n = 9) vs. 111.1 ± 10.94 pmol/mg
protein (n = 9)
(P = NS), respectively. Table
2 summarizes the effects of forskolin on
cAMP accumulation in LLC-PK1
cells trans- fected with
A1-AdRs. In an analogous but in an
opposite manner to
2B-ARs, the
steady-state expression of apical
A1-AdRs in
LLC-PK1 cells influenced the
activity of adenylyl cyclase following forskolin stimulation.
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DISCUSSION |
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The plasma membrane of polarized renal epithelial cells is characterized by domains that differ functionally and biochemically (for a review, see Refs. 5, 20). In the kidney, apical plasma membrane is exposed to plasma ultrafiltrate and thus has a large surface area composed of microvilli that contain specialized proteins that function mainly to reabsorb various ions, nutrients and water. For example, the Na/H exchanger in the apical membrane of proximal tubule cells is the major mechanism for sodium entry across this membrane. The plasma membrane on the basolateral cell surface is exposed to blood and contains proteins for basic cellular processes such as Na-K-adenosinetriphosphatase (Na-K-ATPase), transferrin receptors, and low-density lipoprotein receptors. The asymmetric distribution of these proteins suggests that unique functions are subserved by both apical and basolateral domains. The importance of maintenance of these distinct domains is emphasized by pathological conditions that result in the loss of polarity. For example, the abnormal targeting of Na-K-ATPase in polycystic kidney disease could contribute to the abnormal tubular fluid accumulation and cyst formation (28).
Accumulating evidence indicates that receptors are asymmetrically
expressed in renal epithelial cells and suggests that
compartmentalization of receptors to apical and basolateral domains
could provide an important mechanism for segregating receptor function
to their respective domains. Filtered catecholamines, adenosine, or
angiotensin II could regulate ion transport by stimulating apical
membrane receptors. Functional studies indicate that apical membranes
of renal epithelial cells are responsive to catecholamines (8, 17) and
adenosine agonists (6, 15), and other studies have demonstrated
directly the expression of A1-AdRs
(22) and 2A-ARs at the apical domain (16, 17). How apical receptors
couple to adenylyl cyclase is not well known, since it is thought that this enzyme is expressed exclusively at the basolateral domain (24).
One explanation to account for the coupling of apical G protein-coupled
receptors to basolateral adenylyl cyclase is that apical receptors
undergo endocytosis to couple to basolateral adenylyl cyclase (8). We
thought that another possible explanation for the apparent lack of
apical adenylyl cyclase in experiments in vivo could be the low
abundance of receptors that couple to this enzyme at the apical
membrane. We wondered whether receptor expression regulates the degree
of enzyme activity to which receptors are coupled.
To determine whether receptors that are expressed at opposite domains
have the capability to regulate the activity of their effectors, we
employed two receptors that have been shown to be targeted to opposite
domains in renal epithelial cells (22). 2B-ARs (10) have been shown to
be expressed at basolateral domains of renal epithelial cells. In
contrast, canine A1-AdRs, when
expressed in MDCK and LLC-PK1
cells, are localized primarily to the apical domain (22). We used
this information and transfected LLC-PK1 cells with cDNAs encoding
the rat
2B-AR and human
A1-AdR to determine whether
adenylyl cyclase activity is regulated by G protein-coupled receptors
expressed at opposite domains.
Morphological and biochemical studies performed with
LLC-PK1 cells transfected with the
2B-AR and
A1-AdR confirmed the anticipated localization of these two receptors in our cell culture system. Immunofluorescence demonstrated
2B-AR immunoreactivity at
lateral borders, consistent with findings of the localization of the
2B-ARs in rat kidneys (10) and
in transfected renal epithelial cells (29). Furthermore, our
surface biotinylation studies demonstrated that the majority of surface
2B-ARs were expressed at the
basolateral domain.
In a similar manner, both our immunofluorescence and biotinylation studies demonstrated that the A1-AdRs were expressed primarily at the apical plasma membrane of LLC-PK1 cells. It is interesting to note that these results are similar to those of Saunders et al. (22), despite the fact that we used the human ortholog of A1-AdR. Moreover, functional evidence in A6 cells indicates apical localization of amphibian A1-AdRs (6).
Our functional studies further substantiated our
biochemical and morphological studies. UK-14034, an
2-selective agonist, when
applied to the basolateral membranes, produced a decrease in
forskolin-stimulated cAMP accumulation. Thus, taken together, our
immunohistochemistry and Western blot analysis not surprisingly provide
evidence for the presence of
2B-ARs at the cell surface, thus confirming findings described previously (29). The results presented in the current study showed that
2B-ARs expressed at or near the
basolateral membrane surface were coupled to adenylyl cyclase through
inhibitory G proteins. In a similar manner but at the opposite domain,
we showed that apical A1-AdRs were
coupled to adenylyl cyclase at or near the apical membrane surface.
The results of our experiments assessing adenylyl cyclase activity at
apical and basolateral domains are noteworthy for two reasons. First,
our results provide indirect evidence for the local interaction of
receptors and the effector, adenylyl cyclase. The expression of apical
A1-AdRs led to a dramatic increase
in apical adenylyl cyclase activity in response to forskolin.
Furthermore, the rise in cAMP accumulation in response to apically
applied forskolin was inhibited to a large degree by the coapplication of an A1-agonist, CPA. Similar but
opposite results were obtained with
LLC-PK1 cells transfected with
2B-ARs. We believe that our results are consistent with the possibility that both
A1-AdRs and adenylyl cyclase are
coexpressed at the apical domain and
2B-ARs and adenylyl cyclase are
coexpressed at the basolateral domain. The spatially restricted
arrangement of receptor and effector could result in local
receptor/effector coupling and obviate the need to invoke more complex
mechanisms (8).
Although forskolin is a lipid-soluble molecule and could interact with
adenylyl cyclase expressed at distant sites, we believe that the
effects were limited to adenylyl cyclase at or near the domain to which
it was applied. In other cells, forskolin has been demonstrated to act
locally on adenylyl cyclase and not on adenylyl cyclase expressed in
other regions. Jurevicius and Fischmeister (11) recently examined the
effects of 30 µM forskolin on adenylyl cyclase-induced cAMP
accumulation. They found that the effects of forskolin were limited to
the region to which forskolin was applied. Diffusion of forskolin to
distant sites was not observed. Furthermore, in our experiments, if
diffusion of forskolin had occurred, then we would have seen similar
effects on adenylyl cyclase whether applied to apical or basolateral
domains. Instead, we observed a polarized response that was most
pronounced when forskolin was applied to the side to which recombinant
receptors were expressed. To determine whether the effect of forskolin
on cAMP accumulation was due to a distant effect of forskolin on adenylyl cyclase expressed at the opposite domain, we directly incubated the opposite domain with forskolin. As shown in Tables 1 and
2, the effect of forskolin when applied to plasma membrane domains that
did not express recombinant receptors was similar to the corresponding
domain in untransfected cells. Figure 7
illustrates the asymmetric response of forskolin in
LLC-PK1 cells transfected with
A1-AdRs or
2B-ARs. The apical/basolateral
ratio of cAMP accumulation following application of forskolin was 1.57 ± 0.26 for untransfected
LLC-PK1 cells. When recombinant
A1-AdRs were expressed in the
apical membrane, the apical-to-basolateral ratio of cAMP was 11.61 ± 1.47, an approximately sixfold increase in apical/basolateral
ratio of cAMP accumulation compared with untransfected cells. In a
similar manner but opposite in orientation, the basolateral/apical ratio of cAMP accumulation was greater in transfected than
untransfected LLC-PK1 cells. After
application of forskolin to untransfected LLC-PK1 cells, the
basolateral/apical ratio was 0.4 ± 0.13. Transfection of
recombinant
2B-ARs led to an
increase of basolateral/apical ratio to 3.52 ± 0.73, an
approximately eightfold increase above the ratio in untransfected
LLC-PK1 cells. These results
suggested that the asymmetric response to forskolin is consistent with
an effect at or near the domain to which receptors were expressed.
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For these reasons we believe that our results suggest that adenylyl cyclase is expressed and regulated at or near the apical or basolateral membranes. Definite proof will require separation of apical and basolateral membranes with intact adenylyl cyclase to permit detection of activity or immunoreactivity. The low abundance of adenylyl cyclase compared with other components of the adenylyl cyclase signaling cascade (1) makes these experiments very difficult. Indeed, this has been our experience in our attempts to demonstrate directly adenylyl cyclase activity or immunoreactivity from isolated apical and basolateral membranes.
Our results raise interesting issues that will need to be addressed in additional studies. First, it is interesting to speculate on the mechanism that might be responsible for this observation. It is possible that there is coordination of receptor and effector expression. Furthermore, because G proteins are heterogeneously localized on renal epithelia (25), polarized expression of receptors might influence the intracellular distribution of G proteins, leading to altered activity of adenylyl cyclase. Such coordinated expression of effects at specific cellular domains could lead to efficient cellular processing of external information. Alternatively, expression of inhibitory G protein-coupled receptors could lead to sensitization of adenylyl cyclase (26). Sensitization refers to a process whereby exposure of tissues or cells to agents that inhibit adenylyl cyclase results in an increase in activity of adenylyl cyclase following removal of the agent. In our system, we used dialyzed FBS and therefore the presence of endogenous agonist is unlikely; however, inhibitory G protein-coupled receptors can be activated even in the absence of agonist. Previous studies have demonstrated that certain inhibitory G protein-coupled receptors are constitutively active (13) and could render adenylyl cyclase sensitized to the effects of forskolin.
Second, our results raise the possibility that multiple adenylyl cyclase isoforms could be expressed and compartmentalized in renal epithelial cells and participate in specific signaling events spatially restricted to separate domains.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs. Joel Linden (Department of Medicine, University of Virginia) and Diane L. Rosin and Kevin R. Lynch (Department of Pharmacology, University of Virginia) for providing valuable reagents and advice throughout the study.
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FOOTNOTES |
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This work was supported in part from funds provided by the American Heart Association National Grant-in-Aid and Virginia Affiliate of the American Heart Association Grant-in-Aid. M. D. Okusa was a recipient of a National Kidney Foundation Clinical Scientist Award.
Portions of this work have been previously published in abstract form (J. Am. Soc. Nephrol., vol. 7, p. 1312, 1996).
Address for reprint requests: M. D. Okusa, Division of Nephrology, Box 133, Univ. of Virginia Health Sciences Center, Charlottesville, VA 22908.
Received 29 May 1997; accepted in final form 31 July 1997.
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