Cyclooxygenase-2 expression and function in the medullary
thick ascending limb
Nicholas R.
Ferreri,
Shao-Jian
An, and
John C.
McGiff
Department of Pharmacology, New York Medical College, Valhalla, New
York 10595
 |
ABSTRACT |
The medullary thick ascending limb (MTAL)
metabolizes arachidonic acid (AA) via cytochrome
P-450 (CyP450)- and cyclooxygenase (COX)-dependent pathways. In the present study, we demonstrated that
the COX-2-selective inhibitor, NS-398, prevented tumor necrosis factor-
(TNF)- and phorbol myristate acetate (PMA)-mediated
increases in PGE2 production by
cultured MTAL cells. Accumulation of COX-2, but not COX-1, mRNA
increased when cells were challenged with TNF (1 nM) or PMA (1 µM).
Pretreatment of cells for 30 min with actinomycin D (AcD, 1 µM) had
little effect on COX-2 mRNA accumulation in unstimulated cells or in
cells challenged with either TNF or PMA. Moreover, a
posttranscriptional mechanism(s) appears to contribute significantly to
COX-2 mRNA accumulation as pretreatment for 15 min with cycloheximide
(CHX, 1 µM) caused a superinduction of COX-2 mRNA accumulation in
unstimulated cells as well as in cells challenged with either TNF or
PMA. Expression of COX-2 protein in unstimulated MTAL cells was
attenuated by preincubation for 2 h with dexamethasone (Dex, 2 µM);
however, Dex had little or no effect on COX-2 expression in cells
challenged with either PMA or TNF. The time-dependent inhibition of
86Rb uptake by MTAL cells
challenged with TNF was diminished by pretreating cells with NS-398.
These data suggest that TNF-mediated induction of COX-2 protein
expression accounted for the lag-time required for this cytokine to
inhibit 86Rb uptake in MTAL cells.
tumor necrosis factor-
; kidney; cyclooxygenase-2; prostaglandin
H synthase-2; medullary thick ascending limb; prostaglandins; cytokines
 |
INTRODUCTION |
THE MEDULLARY THICK ascending limb (MTAL) is central to
the regulation of extracellular fluid volume and is responsible for establishing the osmotic gradient in the medulla, the critical event
for concentrating urine (15, 16). This nephron segment metabolizes
arachidonic acid (AA) via a cytochrome
P-450-dependent pathway (CyP450-AA) to
several products, including 20-hydroxyeicosatetraenoic acid (20-HETE),
which contribute importantly to MTAL function by regulating ion
transport mechanisms (5). Despite immunohistochemical evidence that
cyclooxygenase (COX) levels in the MTAL are low (38), several reports
have demonstrated substantial levels
(10
8-10
6
M) of PGE2 by MTAL preparations in
vitro (11, 17, 24). Indeed, PGE2
also may be an important regulator of ion transport in the MTAL, as
this prostanoid has been shown to inhibit basolateral Na+-K+-ATPase
(Na+ pump) and the apical
Na+-K+-2Cl
cotransporter, two important transepithelial ion transport mechanisms present in MTAL epithelial cells (18, 40, 44).
The presence of two distinct COX isozymes, COX-1 and COX-2, has been
well-documented. COX-1 is constitutively expressed in many cell types,
whereas COX-2 gene transcription is induced by mitogens, growth
factors, cytokines, and tumor promoters (4, 7, 8, 12, 33). Expression
of COX-2 in the kidney has been reported (14), and recent
immunohistochemical localization studies have shown that COX-2 is
present in a subset of tubular epithelial cells located in the cortex
and outer medulla (43). These cells contained both
Na+-K+-ATPase
and Tamm-Horsfall protein, indicating that they are TAL cells.
We previously demonstrated that the MTAL produces tumor necrosis
factor-
(TNF) when stimulated with lipopolysaccharide (LPS) or
angiotensin II (ANG II) (9, 26). This cytokine may be an important
mediator/modulator of ion transport in the MTAL (6), as exogenous TNF,
or TNF produced by the MTAL in response to LPS or ANG II, inhibited
ouabain-sensitive 86Rb uptake by
this nephron segment via a prostanoid-dependent mechanism, an effect
consistent with the reported natriuretic action of TNF (6, 42). The
prostanoid-dependent component of this mechanism required a latency
period of more than 4 h. As TNF increased the expression of COX-2 in
several cell types, the present study was designed to determine whether
MTAL cells express COX-2 after challenge with this cytokine and to test
the hypothesis that the latent period for inhibition of
86Rb uptake by TNF was related to
increases of COX-2 gene transcription and protein expression.
 |
METHODS |
Animals. Male Sprague-Dawley rats
(Charles River Lab, Wilmington, MA) weighing 100-115 g were
maintained on standard rat chow (Ralston-Purina, Chicago, IL) and given
tap water ad libitum.
Reagents. Tissue culture media and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were obtained
from Life Technologies (Grand Island, NY). Reagent-grade chemicals and
collagenase (type 1A) were from Sigma (St. Louis, MO). COX-1 and COX-2
antisera and primers were obtained from Cayman (Ann Arbor, MI). In
addition, another COX-2 primer set, obtained from Life Technologies,
was used in some experiments. TNF was purchased from Genzyme (Boston, MA), and NS-398 was from Biomol (Ann Arbor, MI). Polyvinylidene difluoride (PVDF) membranes were obtained from Amersham (Arlington Heights, IL).
Isolation of MTAL cells. MTAL cells
were isolated and characterized as previously described (6, 26).
Briefly, male Sprague-Dawley rats were anesthetized with an
intraperitoneal injection of pentobarbital (0.65 mg/100 g body wt). The
kidneys were perfused with sterile 0.9% saline, via retrograde
perfusion of the aorta, and cut along the corticopapillary axis. The
inner stripe of the outer medulla was excised, minced with a sterile
blade, and incubated for 10 min at 37°C in a 0.75% collagenase
solution gassed with 95% oxygen. The suspension was sedimented on ice,
mixed with Hanks' balanced salt solution (HBSS) containing 2% BSA,
and the supernatant containing the crude suspension of tubules was
collected. The collagenase digestion was repeated four times with the
remaining undigested tissue. The combined supernatants were spun,
resuspended in HBSS, and filtered through a 52-µm nylon mesh (Fisher
Scientific, Springfield, NJ). The filtrate was discarded, and the
tubules retained on the mesh were resuspended in DMEM (Life
Technologies). Combination of the perfusion and size
exclusion steps was done to eliminate blood elements and yielded a
tubule suspension that was ~95% MTAL, as previously described by our
laboratory (6, 26) as well as by other investigators (19, 41). The
cells were cultured in DMEM-Ham's F-12 medium (1:1) (GIBCO), 5% fetal
calf serum (FCS), epidermal growth factor (20 ng/ml; Life
Technologies), streptomycin-penicillin (100 U/ml), and Fungizone (1 µg/ml; Life Technologies). After ~5-7 days, monolayers of
cells were 80-90% confluent, and dome formation, indicative of
vectorial transport, was exhibited. The cells were quiesced
in DMEM containing 0.5% FCS for 18-24 h prior to
their use.
Isolation of total RNA/RT-PCR
analysis. Total RNA was isolated by lysing the cells in
Trizol reagent (Life Technologies) and precipitation with isopropyl
alcohol. A 3-µg aliquot of total RNA isolated from unstimulated or
stimulated MTAL cells was used for cDNA synthesis using the Superscript
Preamplification system (Life Technologies) in a 20-µl reaction
mixture containing Superscript II reverse transcriptase (200 U/µl)
and random hexamers (50 ng/µl). The reaction was incubated at room
temperature for 10 min to allow extension of the primers by reverse
transcriptase, then at 42°C for 50 min, 70°C for 15 min, and
4°C for 5 min. An aliquot of the cDNA was then amplified using
Taq DNA polymerase (2.5 U) in the
presence of sense and antisense primers (1 µM) for murine COX-1,
COX-2, or GAPDH. In control experiments, total RNA was amplified prior
to cDNA synthesis to exclude the possibility of contamination with
genomic DNA. The PCR primer sets for COX-1, COX-2, and GAPDH amplified
specific genes of transcript sizes 756 bp, 724 bp, and 983 bp,
respectively. A second primer set for COX-2 (Life Technologies) yielded
a PCR product that was 304 bp. The amplification (35 cycles) was
initiated by 1 min of denaturation at 94°C, 1 min of
annealing at 53°C, and polymerization for 2 min at 72°C
followed by autoextension at 72°C for 8 min. PCR products were
quantitated by normalizing mRNA accumulation for either COX-1 or COX-2
with GAPDH.
Southern blot analysis. The amplified
PCR product was analyzed by Southern blot analysis following
electrophoresis through a 1.5% agarose gel, transfer to
nitrocellulose, and hybridization with a
32P-labeled cDNA probe for COX-2.
Blots were analyzed using a Molecular Dynamics Storm Phosphorimager.
Western blot analysis of COX proteins.
After treatment with TNF (1 nM) or phorbol myristate acetate (PMA, 1 µM), the media were removed and cells washed twice with PBS. Cells
were harvested, centrifuged at 600 g
for 4 min in the cold room and lysed using 10 mM
Tris · HCl, pH 7.5, 1 mM EDTA, and 1% SDS for 5 min
on ice. The lysate was centrifuged at 10,000 g for 20 min at 4°C. Protein concentrations were determined using a detergent-compatible Bio-Rad protein assay kit. Thirty micrograms of cell lysate were mixed with an
equal volume of 2× SDS-PAGE sample buffer (100 mM Tris-Cl, pH
6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20%
glycerol) and boiled for 3 min. The proteins in the cell lysate were
separated on a 10% SDS-PAGE gel and transferred to nitrocellulose or
PVDF membranes. Nonspecific sites on the membrane were blocked by
incubating in blocking solution containing 5% nonfat dry milk in Tris
buffer saline + Tween (TBST) at room temperature for 30 min. Membranes
were immunoblotted with a rabbit anti-mouse COX-2 polyclonal antibody
or mouse anti-sheep COX-1 monoclonal antibody for 1 h at room
temperature. Membranes were washed with TBST and incubated with
horseradish peroxidase (HRP)-conjugated antisera (Santa Cruz, CA) for
30 min at room temperature. Membranes were washed, and COX proteins
were detected by the enhanced chemiluminescence system (ECL; Amersham,
Arlington Heights, IL). Alternatively, enhanced chemifluorescence
and phosphorimaging was used for the analysis of COX-2
protein expression.
PGE2
ELISA.
Quiescent MTAL cells were incubated with TNF (1 nM) or PMA (1 µM) in
media containing 0.5% serum for varying times, after which the
cell-free supernatants were assayed for
PGE2 by ELISA (Oxford Biomedical
Research, Oxford, MI). Briefly, 50 µl of diluted medium and 50 µl
of HRP-conjugated PGE2 were added
for 1 h to wells of a 96-well plate that had previously been coated
with anti-PGE2 antibody. Following
incubation, substrate for HRP was added to each well for 30 min, and
the reaction was stopped by addition of 1 N HCl. Quantitation was
achieved by measuring absorbance at 450 nm.
Rubidium (86Rb)
uptake.
MTAL cells grown in 24-well tissue culture plates were incubated in
buffer containing (in mM) 140 NaCl, 1.0 CaCl2, 1.0 MgCl2, 4 KCl, 20 HEPES, and 5.0 glucose. Uptake was initiated by adding 86Rb (0.5-1.0 µg, specific
activity 500 mg/mCi; Amersham) for 10 min at 37°C. Isotope uptake
was terminated by addition of a stop solution (10 mM HEPES and 100 mM
MgCl2), and cells were then
washed twice with stop solution. The cells were lysed with 1% SDS and 4 mM EDTA, and the radioactivity associated with the cell pellet was
determined in a gamma counter. The ouabain-sensitive component of total
86Rb uptake was calculated by
subtracting 86Rb uptake in the
presence of ouabain (1 mM) from uptake in the absence of ouabain, as
previously described (6).
Statistical analysis. The responses of
control and treated MTAL cells were compared by unpaired Student's
t-test. Multiple comparisons were made
using one-way analysis of variance (ANOVA) and Bonferroni
t-test. Data are presented as means ± SD; P
0.05 was considered
statistically significant.
 |
RESULTS |
Effects of COX-2-selective inhibition on
PGE2 production.
MTAL cells were preincubated for 15 min in the absence or presence of
the COX-2-selective inhibitor, NS-398 (13), and then challenged for
various times with TNF (1 nM) or PMA (1 µM, positive control).
Production of PGE2 increased
slightly in unstimulated (control) cells over the 20-h incubation
period (Fig. 1,
A and B). PMA significantly increased
PGE2 production at each of the time points tested; a twofold increase was observed after 2 h, whereas
a four- to fivefold increase was seen at 6-20 h (Fig. 1A). Incubation of MTAL cells with
TNF for 20 h increased PGE2 production approximately threefold (Fig.
1B). However,
PGE2 production did not increase
significantly when cells were incubated with TNF for either 2 or 6 h.
NS-398 abolished PMA- and TNF-mediated increases in
PGE2 production at 20 h and
PMA-induced increases at 6 h (Fig. 1,
A and
B). NS-398 did not inhibit basal
PGE2 production or PMA-mediated
increases in PGE2 production at 2 h, possibly reflecting production of this prostanoid by a
COX-2-independent mechanism. Indeed, pretreatment of cells with
indomethacin (1 µM) attenuated basal
PGE2 production by ~50% at 3 and 20 h (Fig. 2). Indomethacin also
completely inhibited PMA-mediated
PGE2 production and reduced
PGE2 to below basal levels (Fig.
2). These data suggest that induction of COX-2 protein by TNF and PMA
may be linked to PGE2 production
via this COX isoform and that both COX isoforms are active in the MTAL.


View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of NS-398 on phorbol myristate acetate (PMA)- and tumor
necrosis factor- (TNF)-mediated
PGE2 production. Medullary thick
ascending limb (MTAL) cells were quiesced in DMEM containing 0.5% FCS
for 18 h then preincubated for 15 min in absence or presence of NS-398
(0.1 µM). Cells were challenged with either PMA (1 µM,
A) or TNF (1 nM,
B) for the indicated times, and the
concentration of PGE2 was
determined by ELISA. Data are means ± SD of triplicate
determinations (n = 3, P 0.01).
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Differential effects of indomethacin and NS-398 on MTAL
PGE2 production. MTAL cells were
quiesced in DMEM containing 0.5% FCS for 18 h, then preincubated for
15 min in absence or presence of NS-398 (0.1 µM) or indomethacin
(Indo, 1 µM). Cells were challenged with PMA for the indicated times,
and the concentration of PGE2 was
determined by ELISA. Data are means ± SD of triplicate
determinations (n = 3).
* P 0.05. ** P 0.01.
|
|
RT-PCR identification of COX-1 and COX-2 mRNA in MTAL
cells. Accumulation of mRNA for COX isoforms was
assessed using specific PCR primers and RT-PCR analysis of total RNA
isolated from MTAL cells that were challenged with TNF or PMA. PMA was
used as a positive control for COX-2 gene transcription. COX-2 mRNA
accumulation was detected in untreated MTAL cells and increased
significantly after treatment with either PMA (1 µM) or TNF (1 nM)
(Figs. 3A and 4A,
respectively). The specificity of the COX-2 PCR primers was confirmed
by Southern blot analysis, which demonstrated that the 756-bp PCR
fragment hybridized with a COX-2-specific cDNA probe (Fig.
5). Accumulation of COX-2 mRNA exhibited
distinct kinetics after stimulation with either PMA or TNF. PMA
increased COX-2 mRNA accumulation approximately twofold at each of the
time points tested (Fig. 3, A and
B). COX-2 mRNA accumulation
increased approximately twofold after challenge with TNF for 2 h (Fig.
4, A and
B). Moreover, the increase in COX-2
mRNA level was maximal after a 2-h exposure to the cytokine and was not
observed after incubation for either 4 or 8 h (Fig. 4,
A and
B). Significant levels of COX-1 mRNA
were present in untreated MTAL cells; however, neither PMA (Fig.
3A) nor TNF (Fig.
4A) affected COX-1 mRNA
accumulation. GAPDH mRNA accumulation (used as a control) also was not
changed after challenge with either PMA or TNF (Figs.
3A and
4A). Thus, neither COX-1 nor GAPDH
mRNA accumulation was affected by TNF or PMA, suggesting that the
increased COX-2 mRNA accumulation was not due to differences in the RNA
concentrations in each sample. It should be noted, however, that COX-1
mRNA accumulation did increase with time, possibly reflecting an effect
of the low levels of FCS (0.5%) in the media.


View larger version (71K):
[in this window]
[in a new window]
|
Fig. 3.
RT-PCR of cyclooxygenase-1 (COX-1) and COX-2 mRNA accumulation in MTAL
cells challenged with PMA. A: MTAL
cells were quiesced for 18 h and then challenged with PMA (1 µM) for
the indicated times. Total RNA was extracted and mRNA accumulation was
assessed by RT-PCR using primer sets specific for COX-2, COX-1, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
B: relative intensities of bands was
determined by scanning densitometry, and data were normalized based on
the amounts of GAPDH mRNA detected in each sample.
* P 0.05 and
** P 0.01;
n = 3.
|
|


View larger version (58K):
[in this window]
[in a new window]
|
Fig. 4.
RT-PCR of COX-1 and COX-2 mRNA accumulation in MTAL cells challenged
with TNF. A: MTAL cells were quiesced
for 18 h and then challenged with TNF (1 nM) for the indicated times.
Total RNA was extracted, and mRNA accumulation was assessed by RT-PCR
using primer sets specific for COX-2, COX-1, and GAPDH.
B: relative intensities of bands were
determined by phosphorimaging and analysis with Imagequant software;
data were normalized based on the amounts of GAPDH mRNA detected in
each sample. * P 0.05;
n = 4.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Southern blot analysis. MTAL cells were quiesced for 18 h and then
challenged with PMA (1 µM) for 2 h. Total RNA was extracted, and mRNA
accumulation was assessed by Southern blot analysis using a
32P-labeled cDNA probe specific
for COX-2. This blot is representative of 3 experiments.
|
|
Effects of actinomycin D and cycloheximide on COX-2
mRNA accumulation. As mRNA accumulation is a function
of gene transcription and mRNA stability, we determined whether PMA-
and TNF-mediated increases in COX-2 mRNA were detectable either after
blocking gene transcription with actinomycin D (AcD) or protein
synthesis with cycloheximide (CHX). Interestingly, pretreatment for 30 min with AcD (1 µM) had no effect on COX-2 mRNA accumulation in
either unstimulated cells or cells challenged with either PMA or TNF (Fig. 6, A
and B). These data suggest that AcD
inhibits transcription of a repressor protein that contributes to the
decrease of COX-2 mRNA half-life. Moreover, since AcD inhibits gene
transcription, the failure to markedly inhibit COX-2 mRNA accumulation
in cells treated with either PMA or TNF in the presence of AcD
indicates an important role for posttranscriptional regulation of COX-2 in MTAL cells. Pretreatment of cells for 15 min with CHX (1 µM) caused a significant superinduction of COX-2 mRNA accumulation in
unstimulated cells and increased the stimulatory effects of PMA or TNF
(Fig. 7, A
and B). Thus, inhibition of putative
repressor proteins by CHX also promotes COX-2 mRNA accumulation by a
posttranscriptional mechanism(s).


View larger version (44K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of actinomycin D (AcD) on COX-2 mRNA accumulation.
A: MTAL cells were quiesced for 18 h
and then challenged with either PMA (1 µM) or TNF (1 nM) for 2 h
following pretreatment for 30 min with AcD (1 µM). Total RNA was
extracted, and mRNA accumulation was assessed by RT-PCR using primer
sets specific for COX-2 or GAPDH. B:
relative intensities of bands were determined by phosphorimaging and
analysis with Imagequant software; data were normalized based on the
amounts of GAPDH mRNA detected in each sample.
* P 0.01 and
** P 0.001;
n = 3.
|
|


View larger version (41K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of cycloheximide (CHX) on COX-2 mRNA accumulation.
A: MTAL cells were quiesced for 18 h
and then challenged with either PMA (1 µM) or TNF (1 nM) for 2 h
following pretreatment for 15 min with CHX (1 µM). Total RNA was
extracted, and mRNA accumulation was assessed by RT-PCR using primer
sets specific for COX-2 or GAPDH. B:
relative intensities of bands were determined by phosphorimaging and
analysis with Imagequant software; data were normalized based on the
amounts of GAPDH mRNA detected in each sample.
P 0.05 vs. control;
* P 0.01 vs. control;
** P 0.01 vs. PMA or TNF;
n = 3.
|
|
COX-2 protein expression in MTAL cells and tubules:
effects of Dex. The expression of COX-2 was determined
by Western blot analysis using a monospecific polyclonal antibody.
Thirty micrograms of MTAL cell lysate were prepared from untreated
control cells, and cells were incubated with either PMA or TNF for 2, 6, and 20 h. Incubation with PMA for either 6 or 20 h significantly
increased COX-2 expression compared with unstimulated cells (Fig.
8, A and B). COX-2 expression also was
increased significantly when MTAL cells were challenged with TNF for 6 h (Fig. 8, A and
B). These data indicate that MTAL
cells express COX-2 protein when challenged with either PMA or TNF, and
are consistent with the ability of NS-398 to inhibit production of
PGE2, as described in Fig. 1, A and
B, and Fig. 2. Variable amounts of
COX-2 protein expression also were detected in lysates (30 µg)
prepared from freshly isolated MTAL tubules. These data suggest that
the ability of MTAL cells in culture to express COX-2 is not merely an
artifact of the culture conditions but reflects the inherent capacity
of these cells to express this protein after appropriate stimulation
(Fig. 9). Pretreatment for 2 h with Dex (2 µM) had little or no effect on PMA-mediated COX-2 expression (Figs.
10, A
and B). Similar results were
obtained when cells were challenged with TNF in the presence of Dex
(data not shown). In contrast, Dex inhibited COX-2 expression in
unstimulated cells. COX-2 bands were not observed when the following
controls were performed to ensure the specificity of the antisera for
their respective antigens: 1)
isotype control for the primary antibody; 2) omission of the primary antibody;
and 3) addition of preimmune sera in
place of the primary antibody.


View larger version (39K):
[in this window]
[in a new window]
|
Fig. 8.
Kinetics of PMA- and TNF-mediated COX-2 protein expression.
A: MTAL cells were quiesced for 18 h,
then incubated for the indicated times in absence or presence of PMA (1 µM) or TNF (1 nM). Cell lysates (30 µg) were separated on a 10%
SDS-PAGE gel; COX-2 was detected by Western blot analysis. Molecular
weight of COX-2 (72/74 kDa) is indicated.
B: relative intensities of bands were
determined by phosphorimaging and analysis with Imagequant software..
* P 0.05 and
** P 0.01;
n = 3.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
COX-2 protein expression in MTAL tubules. Lysates from unstimulated
MTAL tubules freshly isolated from four individual rats were separated
on a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose
membrane and probed with anti-COX-2 antisera. Molecular mass of COX-2
(72/74 kDa) is indicated.
|
|


View larger version (32K):
[in this window]
[in a new window]
|
Fig. 10.
Effects of Dex on COX-2 protein expression.
A: MTAL cells were preincubated in
absence or presence of Dex (2 µM) for 2 h, then challenged with PMA
(1 µM) for 6 h. Analysis of protein was by Western blot and
phosphorimaging. B: relative
intensities of bands were determined by phosphorimaging and analysis
with Imagequant software. * P 0.05 and ** P 0.01;
n = 3.
|
|
COX-2-dependent inhibition of
86Rb uptake.
Previous work from our laboratory indicated that the ability of TNF to
inhibit ouabain-sensitive 86Rb
uptake was prostanoid-dependent and required a latency period of more
than 4 h (6). We determined whether this effect was dependent on
COX-2-mediated prostanoid formation. As shown in Fig. 8,
A and
B, expression of COX-2 protein was not
observed until ~6 h after challenge with TNF. Moreover, significant
increases in PGE2 levels were not
observed until cells had been exposed to the cytokine for ~20 h. The
functional considerations of these data were assessed by inhibiting
COX-2 activity with NS-398 and comparing the ability of TNF to inhibit
86Rb uptake in the absence or
presence of selective COX-2 inhibition. Ouabain-sensitive
86Rb uptake was inhibited by
~40% after addition of TNF for 24 h (Fig. 11). In contrast, TNF had
no effect when cells were pretreated with NS-398 and then challenged
with the cytokine (Fig. 11). These data
suggest that regulation of COX-2 by TNF may subserve a regulatory mechanism in the MTAL that is expressed after a period of several h.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 11.
NS-398 prevents TNF-mediated inhibition of
86Rb uptake. MTAL cells were
pretreated for 15 min with NS-398 (0.1 µM) and then incubated in
absence or presence of TNF (1 nM) for 20 h. Uptake was initiated by
adding 0.5-1.0 µg of 86Rb
for 10 min at 37°C, and was terminated by addition of a stop
solution, as described in METHODS.
Cells were lysed, and the radioactivity was determined in a gamma
counter. Ouabain-sensitive component of total
86Rb uptake was calculated by
subtracting 86Rb uptake in
presence of ouabain (1 mM) from uptake in absence of ouabain (6). Data
represent the means ± SD of triplicate determinations from three
similar experiments. * P 0.05.
|
|
 |
DISCUSSION |
We demonstrated that MTAL cells in primary culture express COX-2
protein after challenge with either PMA or TNF. Posttranscriptional regulatory mechanisms contributed to mRNA accumulation, and
pretreatment of cells with Dex attenuated expression of the protein in
unstimulated cells. Production of
PGE2, in response to either PMA or
TNF, was completely prevented by pretreatment with the COX-2-selective inhibitor, NS-398, only when COX-2 protein was expressed. The inhibitory effect of TNF on ouabain-sensitive
86Rb uptake was linked to
expression and activity of COX-2 protein and was consistent with the
latency period previously described for the effects of this cytokine on
86Rb uptake (6).
The amino acid sequences for COX-1 and COX-2 are similar (~75%
homology), and the residues that are important for the catalytic activities of these enzymes are highly conserved (12, 22). However,
COX-1 and COX-2 differ significantly at amino acids prior to residue
30, and COX-2 has an 18-amino acid insert close to the COOH terminus
that is not present in COX-1 (37). These two enzymes catalyze the same
reactions; i.e., AA is converted to PGG2 via the COX
reaction, followed by a peroxidase reaction in which the
15-hydroperoxyl group of PGG2 is
reduced to the 15-hydroxyl group of
PGH2. The latter is metabolized by
specific isomerases to prostanoids in a cell-specific manner.
Regulation of the two COX isoforms is quite different. COX-1 is
constitutively expressed in several renal cell types, including
interstitial cells, renal blood vessels, and several nephron segments.
Our recent finding that MTAL epithelial cells can produce TNF, a
cytokine shown to increase COX-2 expression, prompted us to determine
whether this cytokine increased COX-2 expression in the MTAL. We
suggested that the latency period observed for TNF-mediated inhibition
of 86Rb uptake in the MTAL was
related to the time required for gene transcription and subsequent
protein expression of this COX isoform. The possibility that the MTAL
expressed COX-2 also was addressed by stimulating cultured MTAL cells
for various times (2-20 h) with PMA, a known activator of COX-2 in
several cell types. Both TNF and PMA increased COX-2 protein expression
and PGE2 production. Increased
expression of COX-2 protein, concomitant with an increase in
NS-398-sensitive PGE2 production,
was observed after challenge with PMA for 6 and 20 h. However, although
TNF-mediated COX-2 protein expression was only evident at 6 h,
significant increases in NS-398-sensitive
PGE2 production were not observed
until ~10-20 h of exposure to the cytokine. This apparent
discrepancy probably reflected a combination of several factors
including differences in signaling pathways and the interposition of
additional steps such as a nitric oxide-dependent step, that has been
described (34). Thus the kinetics of COX-2 expression and the effects of TNF on 86Rb uptake suggest that
the time- and prostanoid-dependent components of these effects are
likely related to induction of the COX-2 gene, posttranscriptional
effects, and the subsequent increase of COX-2 activity, which accounted
for the eventual increase in PGE2 production.
In contrast to COX-1 knockout mice, COX-2 gene disruption in mice
causes a severe nephropathy that results in death by the third month
(29). COX-2 knockout mice present with small kidneys having few
functional nephrons with immature glomeruli and marked impairment of
nephrogenesis, which, in the rodent, normally continues for the first
several postnatal weeks. Constitutive expression of COX-2 has been
reported in the macula densa and in adjacent epithelial cells of the
cortical TAL (14). The MTAL expresses low levels of COX-1 and also
appears to express COX-2 protein constitutively in a subpopulation of
cells (43), as well as in unstimulated freshly isolated MTAL tubules
and primary cultures of MTAL cells. Thus the prostanoid-dependent
effects of TNF on MTAL function may be mediated via this COX isoform.
Moreover, local production of TNF by the MTAL is compatible with a role for this cytokine as an essential component of a regulatory mechanism that affects ion transport and operates in a microenvironment rather
than systemically as occurs in endotoxic shock.
The MTAL metabolizes AA via a CyP450-dependent pathway to several
products, including 20-HETE, which contribute importantly to MTAL
function by regulating ion transport mechanisms (5, 36).
Notwithstanding the relatively low levels of COX expression in the
MTAL, this nephron segment also metabolizes AA via a COX-dependent pathway to PGE2 and
PGF2
(11). Indeed, the levels
of PGE2 produced by various MTAL
preparations have been reported to range from
10
8 M (11, 35) to
10
5 M (24). Certainly, many
biochemical and functional effects mediated by
PGE2 can occur over this dose
range, or less. For instance, PGE2
inhibits oxygen consumption in rabbit MTAL at a concentration of
10
7 M (24). It is well
established that eicosanoids contribute importantly to the regulation
of function along the nephron, as PGE2, an important regulator of
ion transport in the MTAL, inhibits the activity of the
Na+-K+-ATPase
(Na+ pump) and
Na+-K+-2Cl
cotransporter, two important transepithelial ion transport mechanisms present in this nephron segment (18, 40, 44). Moreover, the capacity of
cytokines such as TNF and interleukin-1 (IL-1), to modulate function
within the kidney may require interactions with AA metabolites (1, 2,
21, 46), as TNF and IL-1 stimulate prostaglandin synthesis by
glomerular mesangial cells, MDCK cells, and papillary collecting duct
cells (3, 20, 25). Conversely, TNF production is regulated by
PGE2 in several cell types (10,
23). Thus a regulatory feedback system involving cytokines and
prostaglandins may determine the net effect exerted by these two
classes of potent biological mediators.
COX-2 gene transcription is induced by mitogens, growth factors,
cytokines, and tumor promoters and is glucocorticoid inhibitable, whereas expression of COX-1 mRNA is less responsive to these conditions (3, 31, 32). Accordingly, it is not surprising that COX-1 mRNA
accumulation did not change in MTAL cells challenged with either TNF or
PMA. Posttranscriptional mechanisms also are important in the sustained
induction of COX-2 mRNA (33, 39). For instance, Srivastava et al. (39)
demonstrated that IL-1 enhances the stability of COX-2 mRNA in the
absence of any further transcription in rat mesangial cells.
Superinduction of COX-2 mRNA in MTAL cells challenged with PMA or TNF
after pretreatment with CHX is consistent with posttranscriptional
regulation in these cells. Moreover, inhibition of gene transcription
with AcD had little effect on mRNA accumulation, suggesting that, in
the MTAL, a posttranscriptional mechanism(s) can have a major impact on
mRNA accumulation even in the absence of ongoing transcription. A
similar effect of AcD has recently been reported in human pulmonary
A549 cells (30). As previous studies demonstrated that TNF can increase
COX-2 by different signaling pathways within the same cell type (27,
28), it is likely that the signaling pathways used by TNF and PMA,
although potentially convergent, are distinct. These differences in
signaling pathways could contribute to molecular mechanisms that
account for the differences in mRNA accumulation and
PGE2 production in response to PMA
and TNF, reflecting changes in the rate of COX-2 gene transcription as
well as differences in mRNA half-life.
Expression of COX-2 in the macula densa and TAL may be an essential
component in renal mechanisms that affect salt and water excretion, as
changes in dietary salt intake have been shown to differentially
regulate renal COX-2 expression. Namely, a high-sodium diet increased
COX-2 in the medulla, and a low-sodium diet increased COX-2 in the
cortex (45). These findings are compatible with a COX-2-dependent
mechanism that participates in the regulation of extracellular fluid
volume. The findings in the present study also suggest an important
link of a critical nephron segment, the MTAL, to the regulation of
extracellular fluid volume via a COX-2-dependent mechanism evoked by
activation of the renin-angiotensin system. Thus we have shown that ANG
II stimulates TNF production by the MTAL, an effect that results in the
expression of COX-2 in this nephron segment.
 |
ACKNOWLEDGEMENTS |
We thank Melody Steinberg for editorial assistance.
 |
FOOTNOTES |
This work was supported, in part, by NIH grants RO1-HL-56423 (to N. R. Ferreri) and HL-34300 (to J. C. McGiff) and by a grant from the
American Heart Association Grant 9740001N (to N. R. Ferreri). N. R. Ferreri is an Established Investigator of the American Heart Association.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. R. Ferreri,
Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: nick_ferreri{at}nymc.edu).
Received 10 October 1998; accepted in final form 17 May 1999.
 |
REFERENCES |
1.
Beasley, D.,
C. A. Dinnarello,
and
J. G. Cannon.
Interleukin-1 induces natriuresis in conscious rats: role of renal prostaglandins.
Kidney Int.
33:
1059-1065,
1988[Medline].
2.
Caverzasio, J.,
R. Rizzoli,
J.-M. Dayer,
and
J.-P. Bonjour.
Interleukin-1 decreases renal sodium reabsorption: possible mechanism of endotoxin-induced natriuresis.
Am. J. Physiol.
252 (Renal Fluid Electrolyte Physiol. 21):
F943-F946,
1987[Abstract/Free Full Text].
3.
Coyne, D. W.,
M. Nickols,
W. Bertrand,
and
A. R. Morrison.
Regulation of mesangial cell cyclooxygenase synthesis by cytokines and glucocorticoids.
Am. J. Physiol.
263 (Renal Fluid Electrolyte Physiol. 32):
F97-F102,
1992[Abstract/Free Full Text].
4.
DeWitt, D. L.,
and
E. A. Meade.
Serum and glucocorticoid regulation of gene transcription and expression of the prostaglandin H synthase-1 and prostaglandin H synthase-2 isozymes.
Arch. Biochem. Biophys.
306:
94-102,
1993[Medline].
5.
Escalante, B.,
D. Erlij,
J. R. Falck,
and
J. C. McGiff.
Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle.
Science
251:
799-802,
1991[Medline].
6.
Escalante, B. A.,
N. R. Ferreri,
C. E. Dunn,
and
J. C. McGiff.
Cytokines affect ion transport in primary cultured thick ascending limb of Henle's loop cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1568-C1576,
1994[Abstract/Free Full Text].
7.
Evett, G. E.,
W. Xie,
J. G. Chipman,
D. L. Robertson,
and
D. L. Simmons.
Prostaglandin G/H synthase isoenzyme 2 expression in fibroblasts: regulation by dexamethasone, mitogens, and oncogenes.
Arch. Biochem. Biophys.
306:
169-177,
1993[Medline].
8.
Feng, L.,
Y. Xia,
G. E. Garcia,
D. Hwang,
and
C. B. Wilson.
Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-
, and lipopolysaccharide.
J. Clin. Invest.
95:
1669-1675,
1995[Medline].
9.
Ferreri, N. R.,
B. A. Escalante,
Y. Zhao,
S. An,
and
J. C. McGiff.
Angiotensin II induces TNF production by the thick ascending limb: functional implications.
Am. J. Physiol.
274 (Renal Physiol. 43):
F148-F155,
1998[Abstract/Free Full Text].
10.
Ferreri, N. R.,
T. Sarr,
P. W. Askenase,
and
N. H. Ruddle.
Molecular regulation of tumor necrosis factor-
and lymphotoxin production in T cells.
J. Biol. Chem.
267:
9443-9449,
1992[Abstract/Free Full Text].
11.
Ferreri, N. R.,
M. Schwartzman,
N. G. Ibraham,
P. N. Chander,
and
J. C. McGiff.
Arachidonic acid metabolism in a cell suspension isolated from rabbit renal outer medulla.
J. Pharmacol. Exp. Ther.
231:
441-448,
1984[Abstract].
12.
Fletcher, B. S.,
D. A. Kujubu,
D. M. Perrin,
and
H. R. Herschman.
Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase.
J. Biol. Chem.
267:
4338-4344,
1992[Abstract/Free Full Text].
13.
Futaki, N.,
S. Takahashi,
M. Yokoyama,
I. Arai,
S. Higuchi,
and
S. Otomo.
NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro.
Prostaglandins
47:
55-60,
1994[Medline].
14.
Harris, R. C.,
J. A. McKanna,
Y. Akai,
H. R. Jacobson,
R. N. Dubois,
and
M. D. Breyer.
Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction.
J. Clin. Invest.
94:
2504-2510,
1994[Medline].
15.
Hebert, S. C.,
and
T. E. Andreoli.
Control of NaCl transport in the thick ascending limb.
Am. J. Physiol.
246 (Renal Fluid Electrolyte Physiol. 15):
F745-F756,
1984[Abstract/Free Full Text].
16.
Hebert, S. C.,
R. M. Culpepper,
and
T. E. Andreoli.
NaCl transport in mouse medullary thick ascending limb. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport.
Am. J. Physiol.
241 (Renal Fluid Electrolyte Physiol. 10):
F412-F431,
1981[Abstract/Free Full Text].
17.
Hirano, T.,
K. Yasukawa,
H. Harada,
T. Taga,
Y. Watanabe,
T. Matsuda,
S. Kashiwamura,
K. Nakajima,
K. Koyama,
A. Iwamatsu,
S. Tsunasawa,
Y. Sakiyama,
H. Matsui,
Y. Takahara,
T. Taniguchi,
and
T. Kishimoto.
Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.
Nature
324:
73-76,
1986[Medline].
18.
Kaji, M., Jr.,
H. S. Chase,
J. P. Eng,
and
J. Diaz.
Prostaglandin E2 inhibits Na-K-2Cl cotransport in medullary thick ascending limb cells.
Am. J. Physiol.
271 (Cell Physiol. 40):
C354-C361,
1996[Abstract/Free Full Text].
19.
Kikeri, D.,
S. Azar,
A. Sun,
M. L. Zeidel,
and
S. C. Hebert.
Na+/H+ antiporter and Na+-(HCO3)n symporter regulate intracellular pH in mouse medullary thick limbs of Henle.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F445-F456,
1990[Abstract/Free Full Text].
20.
Kohan, D. E.
Interleukin-1 regulation of prostaglandin E2 synthesis by the papillary collecting duct.
J. Lab. Clin. Med.
114:
717-723,
1989[Medline].
21.
Kohan, D. E.,
C. A. Merli,
and
E. E. Simon.
Micropuncture localization of the natriuretic effect of interleukin-1.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F810-F813,
1989[Abstract/Free Full Text].
22.
Kraemer, S. A.,
E. A. Meade,
and
D. L. DeWitt.
Prostaglandin endoperoxide synthase gene structure: identification of the transcriptional start site and 5'-flanking regulatory sequences.
Arch. Biochem. Biophys.
293:
391-400,
1992[Medline].
23.
Kunkel, S. L.,
M. Spengler,
M. A. May,
R. Spengler,
J. Larrick,
and
D. Remick.
Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression.
J. Biol. Chem.
263:
5380-5384,
1988[Abstract/Free Full Text].
24.
Lear, S.,
P. Silva,
V. E. Kelley,
and
F. H. Epstein.
Prostaglandin E2 inhibits oxygen consumption in rabbit medullary thick ascending limb.
Am. J. Physiol.
258 (Renal Fluid Electrolyte Physiol. 27):
F1372-F1378,
1990[Abstract/Free Full Text].
25.
Leighton, J. D.,
and
J. Pfeilschifter.
Interleukin 1- and tumor necrosis factor-stimulation of prostaglandin E2 synthesis in MDCK cells, and potentiation of this effect by cycloheximide.
FEBS Lett.
259:
289-292,
1996.
26.
Macica, C.,
B. A. Escalante,
M. S. Conners,
and
N. R. Ferreri.
TNF production by the medullary thick ascending limb of Henle's loop.
Kidney Int.
46:
113-121,
1994[Medline].
27.
Mahboubi, K.,
W. Young,
and
N. R. Ferreri.
Induction of prostaglandin endoperoxide synthase-2 by serine-threonine phosphatase inhibition.
J. Pharmacol. Exp. Ther.
282:
452-458,
1997[Abstract/Free Full Text].
28.
Mahboubi, K.,
W. Young,
and
N. R. Ferreri.
Tyrosine phosphatase-dependent/tyrosine kinase-independent induction of nuclear factor-
B by tumor necrosis factor-
: effects on prostaglandin endoperoxide synthase-2 mRNA accumulation.
J. Pharmacol. Exp. Ther.
285:
862-868,
1998[Abstract/Free Full Text].
29.
Morham, S. G.,
R. Langenbach,
C. D. Loftin,
H. F. Tiano,
N. Vouloumanos,
J. C. Jennette,
J. F. Mahler,
K. D. Kluckman,
A. Ledford,
C. A. Lee,
and
O. Smithies.
Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.
Cell
83:
473-482,
1995[Medline].
30.
Newton, R.,
J. Seybold,
L. M. Kuitert,
M. Bergmann,
and
P. J. Barnes.
Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA.
J. Biol. Chem.
273:
32312-32321,
1998[Abstract/Free Full Text].
31.
O'Banion, M. K.,
V. D. Winn,
and
D. A. Young.
cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase.
Proc. Natl. Acad. Sci. USA
89:
4888-4892,
1992[Abstract].
32.
Raz, A.,
A. Wyche,
N. Seigel,
and
P. Needleman.
Regulation of fibroblast cyclooxygenase synthesis by interleukin-1.
J. Biol. Chem.
263:
3022-3028,
1988[Abstract/Free Full Text].
33.
Ristimaki, A.,
S. Garfinkel,
J. Wessendorf,
T. Maciag,
and
T. Hla.
Induction of cyclooxygenase-2 by interleukin-1
.
J. Biol. Chem.
269:
11769-11775,
1994[Abstract/Free Full Text].
34.
Salvemini, D.,
T. P. Misko,
J. L. Masferrer,
K. Seibert,
M. G. Currie,
and
P. Needleman.
Nitric oxide activates cyclooxygenase enzymes.
Proc. Natl. Acad. Sci. USA
90:
7240-7244,
1993[Abstract].
35.
Schlondorff, D.,
R. Zanger,
J. A. Satriano,
V. W. Folkert,
and
J. Eveloff.
Prostaglandin synthesis by isolated cells from the outer medulla and from the thick ascending loop of Henle of rabbit kidney.
J. Pharmacol. Exp. Ther.
223:
120-124,
1982[Medline].
36.
Schwartzman, M.,
N. R. Ferreri,
M. A. Carroll,
E. Songu-Mize,
and
J. C. McGiff.
Renal cytochrome P450-related arachidonate metabolite inhibits (Na+-K+-ATPase).
Nature
314:
620-622,
1985[Medline].
37.
Sirois, J.,
and
J. S. Richards.
Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.
J. Biol. Chem.
267:
6382-6388,
1993[Abstract/Free Full Text].
38.
Smith, W. L.,
and
T. G. Bell.
Immunohistochemical localization of the prostaglandin-forming cyclooxygenase in renal cortex.
Am. J. Physiol.
235 (Renal Fluid Electrolyte Physiol. 4):
F451-F457,
1978[Abstract/Free Full Text].
39.
Srivastava, S. K.,
T. Tetsuka,
D. Daphna-Iken,
and
A. R. Morrison.
IL-1
stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F504-F508,
1994[Abstract/Free Full Text].
40.
Stokes, J. B.,
and
J. P. Kokko.
Inhibition of sodium transport by prostaglandin E2 across the isolated, perfused rabbit collecting tubule.
J. Clin. Invest.
59:
1099-1104,
1977[Medline].
41.
Trinh-Trang-Tan, M.-M.,
N. Bouby,
C. Coutaud,
and
L. Bankir.
Quick isolation of rat medullary thick ascending limbs: enzymatic and metabolic characterization.
Pflügers Arch.
407:
228-234,
1986[Medline].
42.
van-Lanschot, J. J.,
K. Mealy,
D. O. Jacobs,
D. O. Evan,
and
D. W. Wilmore.
Splenectomy attenuates the inappropriate diuresis associated with tumor necrosis factor administration.
Surg. Gynecol. Obstet.
172:
293-297,
1991[Medline].
43.
Vio, C. P.,
C. Cespedes,
P. Gallardo,
and
J. L. Masferrer.
Renal identification of cyclooxygenase-2 in a subset of thick ascending limb cells.
Hypertension
30:
687-692,
1997[Abstract/Free Full Text].
44.
Wald, H.,
P. Scherzer,
D. Rubinger,
and
M. M. Popovtzer.
Effect of indomethacin in vivo and PGE2 in vitro on mTAL Na-K-ATPase of the rat kidney.
Pflügers Arch.
415:
648-650,
1990[Medline].
45.
Yang, T.,
I. Singh,
H. Pham,
D. Sun,
A. Smart,
J. B. Schnermann,
and
J. P. Briggs.
Regulation of cyclooxygenase expression in the kidney by dietary salt intake.
Am. J. Physiol.
274 (Renal Physiol. 43):
F481-F489,
1998[Abstract/Free Full Text].
46.
Zeidel, M. L.,
H. Brady,
and
D. E. Kohan.
Interleukin-1 inhibition of Na+-K+ATPase in inner medullary collecting ducts cells: role of PGE2.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F1013-F1016,
1991[Abstract/Free Full Text].
Am J Physiol Renal Physiol 277(3):F360-F368
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society