1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and 2 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
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With the aim of identifying
possible gene targets for direct or indirect regulation by vasopressin
in the renal medulla, we have carried out cDNA array experiments in
inner medullas of Brattleboro rats infused with the V2
receptor-selective vasopressin analog desamino-Cys1,D-Arg8
vasopressin (dDAVP) for 72 h. Of the 1,176 genes on the array, 137 transcripts were increased by 2-fold or more, and 10 transcripts were
decreased to 0.5-fold or less. Quantitative, real-time RT-PCR
measurements confirmed increases seen for six selected transcripts
(Wilms' tumor protein, -arrestin 2, neurofibromin, casein kinase
II
, aquaporin-3, and aquaporin-4). To correlate changes in mRNA
expression with changes in protein expression, we carried out
quantitative immunoblotting for 28 of the proteins whose cDNAs were
on the array. For several targets including aquaporin-2, transcript
abundance and protein abundance changes did not correlate. However, for
most genes examined, changes in mRNA abundances were associated with
concomitant protein abundance changes. Targets with demonstrated
increases in both protein and mRNA abundances included neurofibromin,
casein kinase II
, the
-subunit of the epithelial Na channel
(
-ENaC), 11
-hydroxysteroid dehydrogenase type 2, and c-Fos.
Additional cDNA arrays revealed that several transcripts that were
increased in abundance after 72 h of dDAVP were also increased
after 4 h, including casein kinase II
,
-ENaC, aquaporin-3,
UT-A, and syntaxin 2. These studies have identified several transcripts
whose abundances are regulated in the inner medulla in response to
infusion of dDAVP and that could play roles in the regulation of salt
and water excretion.
aldosterone; kidney; sodium; epithelia
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INTRODUCTION |
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VASOPRESSIN IS A
PEPTIDE HORMONE that controls systemic osmolality through
regulation of renal water excretion. Its main site of action in the
kidney is the collecting duct, where it regulates the transport of
water, urea, and Na+ (25). In collecting duct
principal cells, vasopressin binds to a Gs-coupled receptor
(the V2 receptor), which stimulates an increase in
intracellular cAMP content via adenylyl cyclase. Binding of vasopressin
to the V2 receptor is also associated with intracellular calcium mobilization mediated by calcium release from
ryanodine-sensitive intracellular stores via the type I ryanodine
receptor, which triggers calmodulin-dependent regulatory processes
within the cell (6). Many of the actions of vasopressin in
the collecting duct are short-term responses that do not involve
activation of gene transcription, such as stimulation of aquaporin-2
trafficking to the apical plasma membrane (35) and
activation of the urea transporter UT-A1 through phosphorylation
(58). However, vasopressin has clear-cut long-term actions
to alter the abundance of aquaporin-2 (8), aquaporin-3
(51), and the epithelial Na channel (ENaC) - and
-subunits (11). These long-term actions are thought to
be associated with regulatory processes at a transcriptional level,
involving either the transporter genes themselves or regulatory molecules that indirectly alter transporter protein abundance. In
addition, in renal medulla, vasopressin may have indirect effects, owing to altered interstitial osmolality, urea concentration, or ionic strength.
Here, we have carried out cDNA array experiments with the aim of identifying possible new direct or indirect gene targets for vasopressin action in renal inner medulla. For this, we have examined levels of 1,176 transcripts after infusion of the V2 receptor-selective vasopressin analog desamino-Cys1,D-Arg8 vasopressin (dDAVP) into Brattleboro rats, which lack endogenously circulating vasopressin.
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METHODS |
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Brattleboro rat model. Male Brattleboro rats (180-230 g) were obtained from Harlan-Sprague Dawley (Indianapolis, IN) and maintained in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle (approved ACUC protocol 9-KE-5). All animals were given free access to tap water and regular pelleted rat chow during the experiments. Under light anesthesia (isofluorane), osmotic minipumps (model 2001, Alza, Palo Alto, CA) were implanted subcutaneously in the rats to deliver 5 ng/h of the V2-selective vasopressin analog dDAVP (Rhone-Poulenc Rorer, Collegeville, PA). Control rats received osmotic minipumps loaded with isotonic saline. After dDAVP administration for time periods designated below, rats were killed and the inner medullas were isolated for RNA extraction, or cortices and inner medullas were isolated for protein analysis. In some experiments, serum was collected for determination of the aldosterone concentration by radioimmunoassay (Coat-A-Count, Diagnostic Products, Los Angeles, CA).
RNA isolation. Total RNA from rat inner medullas was isolated using Qiagen RNAeasy columns (74104, Qiagen, Valencia, CA) according to the manufacturer's directions. Inner medullary tissue was initially homogenized in the manufacturer's buffer solution, using an RNAase-free sawtooth tissue homogenizer (Omni 2000). Homogenates were then passed through the QiaShredder column (79654, Qiagen). RNA was treated with DNAse while bound to the RNAeasy column. Total RNA concentration was measured by spectrophotometry and run on agarose gels to assess RNA quality.
cDNA arrays. Full documentation of cDNA array procedures and results are presented according to minimum information about a microarray experiment (MIAME) guidelines (4) in the Supplemental Materials.1 Briefly, Clontech rat 1.2 nylon filter arrays (7854-1, Clontech Laboratories, Palo Alto, CA) were used for cDNA array analysis according to the manufacturer's instructions. For each experiment, two filters are used, one for control RNA samples and one for experimental RNA. Twenty-five micrograms of total RNA were used for each array. For the two Brattleboro rat experiments (72- and 4-h dDAVP infusion), RNA samples were pooled from the inner medullas from 3 rats, with identical amounts added from each sample (specifically, 8 µg from each inner medulla). 33P was used for labeling in the reverse-transcription reactions, and filters were hybridized overnight at 50°C. Filters were washed at a final stringency of 0.5% SDS, 0.1× SSC at 68°C. Images were captured as TIFF images using a PhosphorImager and analyzed using the National Institutes of Health software program pSCAN (http://mscl.cit.nih.gov). Results were normalized to the overall intensity of the individual filters. To do this, the normalizing variable was total hybridization signal for the whole filter (for all 1,176 spots), allowing the relative dot density to be calculated for each individual gene.
Northern blotting. Northern blots were run to assess relative aquaporin-2, UT-A1, and UT-A2 mRNA abundances in total RNA samples from Brattleboro rat kidney inner medullas and cortices. Aquaporin-2 Northern blots were labeled with a digoxigenin-labeled aquaporin-2 cDNA probe as previously described (12). UT-A Northern blots were probed with 32P-labeled cDNA probes corresponding to the entire length of the UT-A1 transcript (14).
Real-time RT-PCR. Quantitative, real-time RT-PCR was used to validate selected array results as previously described (5, 40). DNase-treated (Ambion) total RNA (1 µg) from rat kidney inner medulla samples from control or dDAVP-infused rats (5 vs. 5) was reverse transcribed using oligo-dT and Superscript II reverse transcriptase (Invitrogen) following the manufacturer's recommended protocol. RT-negative controls were performed to assess the presence of possible genomic contamination of RNA samples. PCR primers were designed to amplify targets between 80 and 150 bp in length, with minimal secondary structure. Sequences of specific primer pairs are listed in the Supplemental Materials. Real-time PCR was performed on an ABI Prism 7900HT system, using 1 µl of a 1:100 dilution of the original RT reaction product, 18 pmol (each) of gene-specific primers, and the Quantitect SYBR green PCR kit (Qiagen) according to the manufacturer's protocol. Specificity of the amplified product was determined using melting curve analysis (5). Relative quantitation of gene expression was determined using the comparative CT method, with validation experiments performed to determine that amplification efficiencies were equal between control and experimental groups (5) as outlined at http://docs.appliedbiosystems.com/pebiodocs/04303859. pdf. All experiments were repeated at least twice, on separate days, to validate results.
Antibodies.
The study utilized affinity-purified rabbit polyclonal antibodies
produced in our laboratory recognizing UT-A1, UT-A2, -ENaC,
-ENaC,
-ENaC, synaptotagmin 5, syntaxin-2, syntaxin-3,
syntaxin-4, vesicle-associated membrane protein (VAMP2), renin,
aquaporin-1, aquaporin-2, aquaporin-3, and the Na-K-Cl cotransporter
type 2 (NKCC2) (8, 13, 28-30, 36, 37, 51). Additional
antibodies were commercially obtained: mouse monoclonal antibodies to
Na-K-ATPase
1-subunit (05-369, Upstate
Biotechnology, Lake Placid, NY), casein kinase II
(sc-12739, Santa
Cruz Biotechnology, Santa Cruz, CA), calbindin D (C8666, Sigma, St.
Louis, MO) and
-arrestin 2 (sc-13140, Santa Cruz Biotechnology); a
sheep polyclonal antibody to 11
-hydroxysteroid dehydrogenase type 2 (11
-HSD2; AB1296, Chemicon, Temecula, CA); goat polyclonal
antibodies to the Wilms' tumor protein (WT1; sc-15422, Santa Cruz
Biotechnology) and CD5 (sc-6984, Santa Cruz Biotechnology) and rabbit
polyclonal antibodies to neurofibromin (sc-68, Santa Cruz
Biotechnology), the endothelin B receptor (AER-002, Alomone Labs, Jerusalem, Israel), endothelial nitric oxide synthase (eNOS; 160880, Cayman Chemical, Ann Arbor, MI), neuronal (n)NOS (160870, Cayman),
-actin (A2066, Sigma), c-Fos (06-341, Upstate) and
c-Jun (KAP-TF102E, StressGen Biotechnologies, Victoria, BC, Canada); and a phospho-specific rabbit antibody to c-Jun phosphorylated at Ser73
(06-659, Upstate).
Protein sample preparation, SDS-PAGE electrophoresis, and immunoblotting. Kidneys were dissected into regions and homogenized in ice-cold isolation solution (250 mM sucrose, 10 mM triethanolamine, pH 7.6, containing 1 mg/ml leupeptin, 0.1 mg/ml phenylmethylsulfonyl fluoride) using a tissue homogenizer (Omni 1000 fitted with a microsawtooth generator) at maximum speed for three 15-s intervals. Total protein concentrations were measured (BCA kit, Pierce, Rockford, IL), and the samples were solubilized in Laemmli sample buffer at 60°C for 15 min.
Semiquantitative immunoblotting was carried out as previously described (22, 51) to assess the relative abundances of individual proteins in the dDAVP-treated Brattleboro rats compared with control Brattleboro rats. To confirm that protein loading of the gels was equal, preliminary 12% polyacrylamide gels were stained with Coomassie blue, as previously described (51). Proteins were separated on 10 or 12% polyacrylamide gels by SDS-PAGE and transferred to nitrocellulose membranes electrophoretically (Bio-Rad Mini Trans-Blot Cell). Membranes were blocked for 1 h at room temperature with 5% nonfat dry milk and probed overnight at 4°C with the appropriate affinity-purified polyclonal antibody. Membranes were washed and exposed to one of the following horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature: donkey anti-sheep IgG (1713-035-147, diluted to 1:5,000, Jackson Laboratories), goat anti-rabbit IgG (31463, diluted to 1:5,000, Pierce), mouse anti-goat IgG (31400, diluted 1:5,000, Pierce), or rabbit anti-mouse IgG (31450, diluted to 1:5,000, Pierce). After a washing, bands were visualized using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and Perry Laboratories, Gaithersburg, MD). Band densities were determined by laser densitometry (Personal Densitometer SI, Molecular Dynamics, San Jose, CA).Immunocytochemistry.
Control and dDAVP-treated Brattleboro rats were prepared as
described above. The kidneys were fixed by perfusion with cold PBS (pH
7.4) for 15 s via the abdominal aorta, followed by cold 4%
paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 min. The
kidneys were removed and postfixed for 1 h, followed by 3 × 10-min washes with 0.1 M cacodylate buffer (pH 7.4). The tissue was
dehydrated in graded ethanol and left overnight in xylene. The tissue
was embedded in paraffin and 2-µm sections were cut on a rotary
microtome (Micron). Localization of 11-HSD2 was carried out using
indirect immunoperoxidase labeling as described (19). The
primary and secondary antibodies were the same as described above for
immunoblotting. For immunoperoxidase labeling, counterstaining was done
using Mayer's hematoxylin. Microscopy was carried out with a Leica
DMRE light microscope.
Comparative genomic analysis of 5'-flanking region of 11-HSD2
gene.
The human 11
-HSD2 mRNA sequence was downloaded from GenBank via
Entrez (http://www.ncbi.nlm.nih.gov/Entrez/index.html). The mRNA
sequence was used in a homology search of the human genome using the
genome browser available at the University of California, Santa Cruz
(Jim Kent curator,
http://genome.ucsc.edu/cgi-bin/hgBlat? command=start) to
identify the human gene. The browser was used to locate the 5'-flanking
region and a 7,000-bp length immediately upstream of the transcription
start site was chosen for further analysis. Regions that were conserved
between human and mouse were identified by the browser. The conserved
human and mouse sequences were further analyzed by a string search
using TESS (http://www.cbil.upenn.edu/tess/index.html) to compare
the conserved sequences with elements of a transcription factor binding
motif database (TRANSFAC version). The conserved sequences between
human and mouse were entered into TESS, which returned a list of
potential transcription factor binding sites. Only perfect matches with elements of the database were included, and sites not found in common
between the two species were eliminated.
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RESULTS |
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To generate hypotheses concerning the direct or indirect long-term
action of vasopressin in the renal collecting duct, we carried out cDNA
array experiments using mRNA isolated from Brattleboro rat inner
medullary tissue samples after 72 h of dDAVP or vehicle treatment.
Figure 1 summarizes the distribution of
dot-density ratio responses for an experiment that compared RNA pooled
from three dDAVP-infused Brattleboro rats with RNA pooled from three vehicle-infused rats. Of the 1,176 genes on the array, 137 transcripts were increased by 2-fold or more in response to dDAVP infusion, and 10 transcripts were decreased to 0.5-fold or less (dashed lines, Fig. 1).
Full results including TIFF images of the arrays are presented in
accordance with MIAME guidelines (4) in the Supplemental
Materials.
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Validation of array results.
Among the 1,176 genes on the arrays, several of them were selected for
further analysis because of the potential physiological significance of
changes (or lack of changes) that were detected. These targets were
studied further using both real-time RT-PCR to confirm the responses at
an mRNA level and semiquantitative immunoblotting to test whether the
mRNA changes are associated with corresponding changes in protein
abundance. The results of real-time RT-PCR determinations of mRNA
responses to dDAVP are shown in Table 1.
Real-time RT-PCR determinations were carried out in different total RNA
samples than used for the arrays. These samples were isolated from
inner medullas of separate, identically treated Brattleboro rats (5 dDAVP-treated vs. 5 vehicle-infused Brattleboro rats). As can be seen
in Table 1, the genes with the largest increases in mRNA in response to
dDAVP on the cDNA arrays (namely, WT1, -arrestin 2, neurofibromin,
and casein kinase II
) were found to be associated with significant
increases in mRNA in response to dDAVP infusion by real-time RT-PCR.
These are novel responses, which have potential significance regarding the mechanism of the cellular response to vasopressin in collecting duct cells (see DISCUSSION). In addition, among the three
aquaporins expressed in the renal collecting duct, aquaporin-2, -3, and
-4, there was reasonable agreement between cDNA array results and real-time RT-PCR results. Both aquaporin-3 and aquaporin-4 manifested substantial increases in mRNA in response to dDAVP, while, somewhat surprisingly, aquaporin-2 either did not change (cDNA array) or increased only modestly (real-time RT-PCR). Interestingly, GAPDH and
-actin, both of which are considered housekeeping genes, manifested
increases in mRNA in response to dDAVP when measured by real-time
RT-PCR.
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Correlation between mRNA changes and protein changes for selected
genes.
To correlate changes in mRNA expression detected on the cDNA arrays
with changes in protein expression, we carried out quantitative immunoblotting for 28 of the proteins corresponding to genes on the
array. For this, we utilized kidneys from a different set of
dDAVP-infused Brattleboro rats than those used for the arrays (Fig.
4, Table
2). As shown in Fig. 4, several
transcripts exhibiting very large increases in mRNA levels in response
to dDAVP infusion (based on cDNA array analysis) were also
associated with significant increases in the corresponding protein
abundances. This group included neurofibromin, casein kinase II, and
11
-HSD2. WT1 (24-fold increase in mRNA) was not detectable in the
inner medulla by immunoblotting despite positive controls showing
strong labeling of heterologously expressed WT1 protein (Sean Lee,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD, personal communication).
Two proteins previously recognized to be upregulated by vasopressin,
-ENaC (11) and aquaporin-3 (13, 51), also
showed substantial increases at the protein and mRNA levels in the
present study (Fig. 4). In addition, c-Fos showed a relatively modest
increase in mRNA abundance by cDNA array analysis (1.7-fold) but
exhibited a large increase in protein abundance (4.9-fold). Conversely,
two proteins (the
1-subunit of the Na-K-ATPase and the
type 2 Na-K-2Cl cotransporter) showed corresponding decreases in mRNA
and protein (Fig. 4). [Analysis of serum samples from these rats
revealed a significant increase in serum concentrations of aldosterone
in dDAVP-infused (1.9 ± 0.5 nM, n = 6) compared
with vehicle-infused Brattleboro rats (0.5 ± 0.1 nM,
n = 6), ruling out a decrease in circulating
aldosterone level as a cause of the decrease in Na-K-ATPase
1-subunit protein expression.]
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11-HSD2.
One of the upregulated transcripts was 11
-HSD2, whose dot density on
cDNA arrays was increased by 3.1-fold (Table 1) and whose protein
abundance was increased by 4.0-fold (Table 2) in response to long-term
dDAVP infusion. Because of its potential physiological importance in
the regulation of glucocorticoid action in the collecting duct
(16), we chose to study 11
-HSD2 regulation by
vasopressin in greater detail. Additional Brattleboro rats were studied
to localize the increase in 11
-HSD2 protein abundance in response to
dDAVP (Fig. 5). As shown in Fig.
5A, immunoblot analysis of inner medullary protein samples
from Brattleboro rats infused with dDAVP for 3 days again showed an
increase in 11
-HSD2 protein abundance compared with vehicle-infused
rats. Densitometry analysis of the immunoblots revealed that the
normalized band density was significantly increased to 169 ± 20 compared with 100 ± 25 in control inner medulla,
P < 0.05. Similar increases were seen in renal
cortical samples (not shown; dDAVP infused, 242 ± 9 vs.
vehicle-infused, 100 ± 12, P < 0.05). Figure
5B shows immunoperoxidase labeling of 11
-HSD2 in renal
inner medullas of vehicle- and dDAVP-infused Brattleboro rats. Labeling
conditions and exposure settings on the microscope were identical for
both images. The 11
-HSD2 labeling was present in the principal cells of the collecting ducts (arrows, Fig. 5B), whereas
intercalated cells were not labeled (arrowheads). dDAVP treatment
markedly increased 11
-HSD2 immunostaining in principal cells
(arrows, Fig. 5B-B) compared with vehicle-infused
Brattleboro rats (arrow, Fig. 5B-A). Similar observations
were made in two additional pairs of rats. Thus we conclude that
long-term dDAVP infusion increases the abundance of 11
-HSD2 protein
in the inner medullary collecting ducts of Brattleboro rats.
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Response to 4-h dDAVP infusion.
Figure 8 summarizes the overall pattern
of mRNA abundance changes in response to 4-h treatment with dDAVP
compared with 72-h treatment, as determined by cDNA microarray
analysis. Predictably, the range of response ratios is not as great
after 4-h compared with 72-h dDAVP infusion. Furthermore, in a large
number of cases, responding genes at 72 h were different from
responding genes at 4 h. Among the 18 transcripts that were
increased in Tables 1 or 2, only 5 of them were also increased
(dDAVP:vehicle > 1.5) after 4 h of dDAVP infusion
(casein kinase II, syntaxin-2,
-ENaC, UT-A, and aquaporin-3),
while 9 were unchanged after 4-h dDAVP treatment (neurofibromin, CD5,
11-
-HSD2, renin, synaptotagmin 5, c-Jun, calbindin 28,
-ENaC, and
c-Fos) and 4 were decreased (dDAVP:vehicle < 1.5) after 4-h
treatment (WT1, ET-B receptor,
-arrestin 2, and aquaporin-4). Full
results of the 4-h dDAVP/Brattleboro rat array experiments, including
TIFF images of the arrays, are presented in accordance with MIAME
guidelines (4) in the Supplemental Materials.
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DISCUSSION |
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Previous studies have established that vasopressin regulates gene expression in the kidney, at least with regard to the genes that code for aquaporin-2 and -3 (12, 51), the three ENaC subunits (11, 34), the urea transporter UT-A2 (55), and the Na-K-2Cl cotransporter NKCC2 (22). In this paper, we used cDNA arrays to broaden these observations, identifying several additional genes for which expression is altered in the renal inner medulla in response to administration of the V2 receptor-selective vasopressin analog dDAVP. The studies were done in the Brattleboro rat, which lacks circulating vasopressin due to a mutation in the vasopressin-neurophysin gene (46) and thus provides a vasopressin-free host in which to test responses to dDAVP infusion. Because these studies were done in vivo, responses to dDAVP infusion in this study could be either direct, i.e., responses to increased phosphorylation of transcription factors by vasopressin-activated kinases (protein kinase A or calmodulin-dependent kinases) in collecting duct cells, or indirect, i.e., due to a more complex response that is triggered by increased V2-receptor occupation but is not an immediate consequence of vasopressin receptor-mediated signaling. The latter would include several categories of responses including 1) activation of additional signaling cascades in collecting duct cells such as the MAP kinase pathway; 2) induction of hierarchical transcription factors downstream of transcription factors immediately activated by vasopressin-induced signaling; 3) secondary changes in circulating hormone levels; or 4) responses to an altered inner medullary interstitial environment. This paper does not attempt to discriminate these different types of responses. In the following, we discuss, first, the regulatory targets identified and then address some general issues raised by the results.
-Arrestin 2.
-Arrestin 2 is a member of a family of proteins involved in G
protein-coupled receptor desensitization (39). These
proteins bind to the phosphorylated COOH termini of G protein-coupled
receptors and mediate receptor desensitization in part by inducing
receptor endocytosis. In the present studies, identification of
-arrestin 2 upregulation in response to dDAVP infusion points to a
potential role for the induction of this protein in the
vasopressin-escape phenomenon (12), which has been found
to be due to V2 receptor downregulation (10,
53) and is crucial in limiting the degree of extracellular fluid
dilution in the syndrome of inappropriate antidiuresis. Recently, the
-arrestins have also been found to play a role as ligand-activated
scaffold proteins for two MAP kinase pathways, the ERK cascade
(7, 27) and the c-Jun NH2-terminal kinase 3 (JNK3) cascade (31). It is therefore conceivable that induction of
-arrestin 2 by vasopressin is involved in the observed activation of MAP kinase cascades in the renal inner medulla during antidiuresis (56) and may provide part of the explanation
for the dDAVP-induced increase in c-Jun phosphorylation demonstrated in
the present study (Fig. 7).
Neurofibromin. The renal inner medullary expression of neurofibromin was markedly increased at both the mRNA (Table 1) and protein (Table 2) levels in response to long-term dDAVP infusion. Neurofibromin was originally identified as the protein product of the disease gene responsible for the autosomal dominant genetic disease neurofibomatosis type 1 (18). It is a member of the GTPase-activating protein (GAP) family and has been implicated as a key factor in limiting of the growth-promoting action of the small GTP-binding protein Ras. Recently, however, an additional function has been identified for the protein, direct activation of CNS-specific adenylyl cyclase isoforms (17, 54). It is unknown whether neurofibromin interacts in a similar manner with renal isoforms of adenylyl cyclase. If it does, upregulation of neurofibromin expression in the kidney could contribute to long-term regulation of water, urea, and sodium ion transport in the renal tubule.
Casein kinase II.
Casein kinase II
expression was strongly increased in the renal
inner medulla in response to dDAVP infusion at both mRNA and protein
levels (Tables 1 and 2). A detectable increase in casein kinase II
mRNA after a 4-h dDAVP infusion suggests rapid, direct induction.
Casein kinase II
is a regulatory subunit of the protein kinase CK2,
a ubiquitous serine/threonine kinase, which is composed of two
regulatory
-subunits and two catalytic
-subunits. Many protein
substrates for CK2 have been identified, including growth factor
receptors, transcription factors, cytoskeletal proteins, cell cycle
regulatory proteins, and vesicle trafficking proteins. The latter
includes syntaxin-4, which is believed to play a role in
vasopressin-dependent aquaporin-2 trafficking to the plasma membrane
(41). Recent findings point to a role for CK2 in the
cellular response to various forms of cellular stress (1),
raising the possibility that its induction by dDAVP may play a role in
protecting inner medullary cells against osmotic stress.
11-HSD2.
11
-HSD2 was upregulated in response to long-term dDAVP
infusion at both mRNA and protein levels (Figs. 4 and 5). This enzyme is believed to play a central role in the regulation of ion transport in the renal collecting duct through its ability to break down glucocorticoids (cortisol in humans; corticosterone in rodents) to
inactive forms (16). In the absence of 11
-HSD2 in
collecting duct cells, glucocorticoids at circulating concentrations
would be expected to bind to and fully activate the mineralocorticoid receptor (MR), impairing the ability of regulated changes in
circulating aldosterone levels to alter gene expression. The
conventional view of the role of 11
-HSD2 is that it is
constitutively expressed at such high levels that only the
mineralocorticoid aldosterone can reach the MR. However, previous
studies identified 11
-HSD2 as a regulatory target for the short-term
actions of vasopressin by a nontranscriptional mechanism
(2), suggesting that regulation of 11
-HSD2 activity may
play a physiological role, perhaps by controlling glucocorticoid access
to the MR and glucocorticoid receptor in the collecting duct. The
present finding of upregulation of 11
-HSD2 gene expression in
response to long-term vasopressin treatment extends this view and could
have implications for the regulation of transporter proteins in the
collecting duct that are recognized targets for glucocorticoid or
mineralocorticoid regulation, including the UT-A1 urea transporter
(38), the Na-K-ATPase (15), and ENaC
(48).
WT1. Among all cDNAs on the array, WT1 manifested the largest increase in dot density in response to long-term infusion of dDAVP (Table 1). A large increase in mRNA abundance was confirmed by real-time quantitative RT-PCR. WT1 is a zinc-finger transcription factor expressed chiefly in kidney, gonads, uterus, and spleen, which functions as a tumor suppressor (26). In addition, WT1 appears to be involved in posttranscriptional processing of mRNA (45). Mutations in WT1 are associated with a high incidence of Wilms' tumor, a renal neoplasm arising from renal tissue of metanephric origin. In the mature kidney, WT1 is generally believed to be expressed only in the glomerular podocyte (45), although the possibility of its expression in the renal inner medulla of the adult kidney has not been investigated in detail. Available antibodies to WT1 could not convincingly demonstrate WT1 protein in the rat inner medulla (Table 2), although absolute expression levels of a functional transcription factor could conceivably be quite low. Consequently, further studies will be needed to localize WT1 expression in the renal inner medulla and to determine its role in vasopressin-mediated transcriptional regulation.
c-Fos and c-Jun.
c-Fos and c-Jun are immediate early genes,
whose products together constitute the transcription factor AP1. On
cDNA arrays, dot densities for both c-Jun and
c-Fos mRNA were observed to increase with long-term dDAVP
infusion (Fig. 4). Immunoblotting demonstrated that c-Fos protein
abundance was markedly increased in response to dDAVP administration,
consistent with the findings of Yasui et al. (57) in
LLC-PK1 cells. However, there was no demonstrable increase
in the abundance of c-Jun protein. Nevertheless, there was a marked
increase in the abundance of phosphorylated c-Jun, possibly resulting
from activation of MAP kinases in response to dDAVP. Thus, although
direct studies of transcriptional regulation are beyond the scope
of this study, we postulate a critical role of the AP1 binding motif in
mediating the long-term responses to vasopressin in the renal inner
medulla. One gene that may be transcriptionally upregulated by AP1 may
be 11-HSD2, which has a conserved AP1 binding site in its
5'-flanking region and is markedly upregulated by vasopressin (see above).
Synaptotagmin 5.
The synaptotagmins are postulated to play calcium-sensing roles in the
regulation of exocytosis (49). In the present study, we
found that synaptotagmin 5 (20) (also termed synaptotagmin IX) was upregulated in the inner medulla in response to long-term dDAVP
infusion at both mRNA and protein levels (Fig. 4). Synaptotagmin 5 has
recently been demonstrated to be a binding partner for the -subunit
of the protein serine/threonine kinase CK2 (9), another dDAVP-responsive protein (see above).
Aquaporin-2 and -3. Aquaporin-3, a basolateral water channel in collecting duct principal cells, was found to be upregulated at both the mRNA (Table 1) and protein (Table 2) levels, consistent with prior results (12, 13, 51). The inner medullary protein abundance of aquaporin-2, the apical water channel in collecting duct principal cells, was also increased (Table 2), consistent with previous findings (8, 51). In the context of these previous findings, we were surprised to find that there was little or no increase in aquaporin-2 mRNA abundance in the inner medulla in response to dDAVP infusion as demonstrated on cDNA arrays (Fig. 4), by Northern blotting (Fig. 2), and by real-time RT-PCR (Table 1). This result suggests that posttranscriptional mechanisms may be involved in the regulation of aquaporin-2 protein abundance in the renal inner medulla. In contrast, aquaporin-2 mRNA abundance was strongly increased in the cortex in response to dDAVP infusion (Fig. 2), consistent with previous findings (10).
Urea transporters. The UT-A gene codes for several urea transporter proteins expressed in inner medulla that arise from alternative splicing (33). UT-A1, the predominant form in the inner medullary collecting duct (37, 47), has been shown to be downregulated in the inner medulla in response to dDAVP infusion (52). Hence, we were somewhat surprised to find that the mRNA dot density for UT-A was increased 2.8-fold on the cDNA array (Table 1), whereas immunoblotting confirmed a lack of increase in UT-A1 protein in the inner medulla in response to dDAVP infusion (Fig. 4). Further analysis by real-time quantitative RT-PCR revealed a 56-fold increase in the abundance of a second splicing variant, UT-A2, which is expressed chiefly in the thin descending limbs of Henle in the outer medulla (37, 47) but is relatively nonabundant in the inner medulla (55). Interrogation of the commercial supplier of the array revealed that the sequence of the UT-A cDNA on the array overlaps both UT-A1 and UT-A2. Hence, it appears that a very large change in a relatively nonabundant splicing variant gave a result on cDNA array analysis that was not representative of changes in the most abundant splice variant.
General observations and conclusions. cDNA array analysis has revealed several genes that are upregulated in the inner medulla of the Brattleboro rat in response to dDAVP infusion. Further studies will be needed to investigate further the role of these genes and their protein products in the overall response to vasopressin. Clearly, the data presented here provide only an initial glimpse of the response to vasopressin, with a detailed view only of the long-term response, which may consist of both direct and indirect effects of vasopressin on gene expression. A general comparison of the response to vasopressin at a 4-h vs. a 72-h time point (Fig. 8) reveals a much different pattern of mRNA abundance changes at the pre-steady-state time point. Detailed time course studies will be required to work out the sequence of events involved in the vasopressin response and to determine which genes are upregulated in direct response to vasopressin-mediated signaling vs. secondary responses, which might be related to vasopressin-mediated changes in local osmolality, local calcium ion concentrations, luminal pH, luminal flow rate, and other factors altered by vasopressin.
An important component of the present study was a detailed comparison of mRNA abundance and protein abundance responses to dDAVP infusion, with an assessment of 28 different protein products by quantitative immunoblotting (Table 2, Fig. 4). For this element of the study, we used a large number of polyclonal antibodies developed in this laboratory for targeted proteomic studies (24) as well as a selection of antibodies from commercial sources for which the specificities were clearly documented. Although for many of the genes examined in this manner there was a clear correlation between changes in mRNA and those in protein, it is important to emphasize that several genes exhibited mRNA responses that were qualitatively different from protein responses. Some genes showed increases in protein with no change in mRNA levels, whereas some showed changes in mRNA abundance without coordinate changes in protein abundance. Similar observations have been made in studies in yeast using large-scale proteomics and gene expression arrays (21). Clearly, there are physiologically important mechanisms by which levels of specific proteins can change without changes in mRNA levels, including translational regulation and regulation of protein half-life (23). Hence, although cDNA arrays provide an important means of generating new hypotheses about physiological regulation at a molecular level, a complete evaluation of such mechanisms requires protein measurements. A previous study reported genes whose transcript abundances were upregulated or downregulated in response to exposure of cultured mpkCCDc14 to 10 ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Dr. Sean Lee (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) for advice regarding WT1 antibodies.
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FOOTNOTES |
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This study was funded by the Intramural Budget of the National Heart, Lung, and Blood Institute (National Institutes of Health, project no. Z01-HL-01282-KE, to M. A. Knepper). Studies at Aarhus University were supported by the Danish Medical Research Council, the Karen Elise Jensen Foundation, the Commission of the European Union (EU-TMR Program and K.A. 3.1.2 Program) and Dongguk University. The Water and Salt Research Center, Aarhus University, is supported by The Danish National Research Foundation (Danmarks Grundforskningsfond). A. Seth was supported by the Biomedical Engineering Student Internship Program of the Whitaker Foundation.
Present address of H. L. Brooks: Dept. of Physiology, College of Medicine, 1501 N. Campbell Ave., Univ. of Arizona, Tucson, AZ 85724.
Address for reprint requests and other correspondence: M. A. Knepper, National Institutes of Health, Bldg. 10, Rm. 6N260, 10 Center Dr., MSC 1603, Bethesda, MD 20892-1603 (E-mail: knep{at}helix.nih.gov).
1 Supplemental Materials for this study can be found at http://ajprenal.physiology.org/cgi/content/full/284/1/F218/DC1.
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.
September 11, 2002;10.1152/ajprenal.00054.2002
Received 7 February 2002; accepted in final form 3 September 2002.
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