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J. Biol. Chem., Vol. 275, Issue 48, 37922-37929, December 1, 2000
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From the
Received for publication, July 13, 2000, and in revised form, August 31, 2000
Reducing luminal NaCl concentration in the macula
densa region of the nephron stimulates renin secretion, and this
response is blocked by a specific inhibitor of cyclooxygenase-2 (COX-2) (Traynor, T. R., Smart, A., Briggs, J. P., and Schnermann, J. (1999) Am. J. Physiol. Renal Physiol. 277, F706-710). To study whether low NaCl activates COX-2 activity
or expression we clonally derived a macula densa cell line (MMDD1
cells) from SV-40 transgenic mice using fluorescence-activated cell
sorting of renal tubular cells labeled with segment-specific
fluorescent lectins. MMDD1 cells express COX-2, bNOS, NKCC2, and ROMK,
but not Tamm-Horsfall protein, and showed rapid
86Rb+ uptake that was inhibited by a reduction
in NaCl concentration and by bumetanide or furosemide. Isosmotic
exposure of MMDD1 cells to low NaCl (60 mM) caused a prompt
and time-dependent stimulation of prostaglandin
E2 (PGE2) release that was prevented by the
COX-2 specific inhibitor NS-398 (10 µM). Reducing NaCl to
60 and 6 mM for 16 h increased COX-2 expression in a
chloride-dependent fashion. Low NaCl phosphorylated p38
kinase within 30 min and ERK1/2 kinases within 15 min without changing
total MAP kinase levels. Low NaCl-stimulated PGE2 release
and COX-2 expression was inhibited by SB 203580 and PD 98059 (10 µM), inhibitors of p38 and ERK kinase pathways. We conclude that low chloride stimulates PGE2 release and
COX-2 expression in MMDD1 cells through activation of MAP kinases.
Activation of the renin-angiotensin system is one of the most
important regulatory responses to extracellular volume depletion. Both
the direct effects of angiotensin to cause vasoconstriction and to
enhance tubular Na+ absorption and its indirect actions to
regulate the production of aldosterone are major factors in renal
Na+ conservation and blood pressure maintenance. The
regulation of release and synthesis of renin by body salt content is
multifactorial. The macula densa mechanism for control of renin
secretion is an intrarenal control system in which a direct consequence
of salt depletion, a reduced NaCl concentration in the macula densa
segment of the nephron, elicits an activation of the renin-angiotensin system (2). In the mammalian kidney, the thick ascending limb of the
loop of Henle returns to its own glomerulus contacting the glomerular
vascular pole with a plaque of epithelial cells, the macula densa
(MD).1 The MD cells are in
close contact with the vascular smooth muscle cells of the afferent
arteriole and with the renin-producing granular cells. There is ample
evidence that the MD cells act as receptors of luminal NaCl
concentration and that a reduction in NaCl concentration results in
stimulation of renin release and renin synthesis (3).
Substantial experimental evidence indicates that prostaglandins
act as extracellular mediators of MD-dependent renin
secretion. Prostaglandins of the E and I series have been shown to
stimulate renin secretion in a number of preparations including in
cultured JG cells (4). In animals with denervated kidneys, the response of renin to salt depletion or furosemide is significantly suppressed by
non-steroidal anti-inflammatory drugs (5). In the perfused JGA
preparation, low NaCl-induced renin secretion is completely blocked by
non-steroidal anti-inflammatory drugs (6). The presence of the
inducible COX-2 isoform in macula densa cells and adjacent thick
ascending limb cells suggests that a reduction in luminal NaCl may
cause increased COX-2-mediated production of prostaglandins which upon
release interact with granular cells to stimulate renin release and
renin synthesis. This hypothesis is fully compatible with the
observation that in the perfused JGA the COX-2-specific inhibitor
NS-398 blocks low NaCl-stimulated renin release (1). Furthermore, COX-2
inhibition has been shown to inhibit renin release in various high
renin states such as salt depletion, ACE inhibition, and renal artery
stenosis (7-9).
Progress in the understanding of the cellular mechanisms responsible
for MD control of prostaglandin release has been hampered by the lack
of an appropriate cell model. In this paper we report the development
of a cell line with MD-like properties and its use in exploring the
mechanisms that may mediate the direct effect of a change in medium
NaCl concentration on prostaglandin release and COX-2 gene expression.
Our results indicate that a reduction in luminal NaCl concentration
causes the rapid phosphorylation of MAP kinases, and that MAP kinase
activation is causal in the acute release of PGE2 as well
as in the delayed rise in COX-2 mRNA and protein expression.
Materials--
Cell culture media and serum were from Life
Technologies, Inc. PD-98059, and antibodies against p38 and ERK1/2
kinases were from New England Biolabs Inc. (Beverly, MA). SB-203580 was
from Upstate Biotechnology Inc. (Lake Placid, NY). Murine COX-2
polyclonal antibody, NS-398, and PGE2 enzyme immunoassay
kit were from Cayman Chemical (Ann Arbor, MI). Rat bNOS antibody that
cross-reacts with mouse bNOS was from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). Polyclonal peptide antibodies against NKCC2 and ROMK were developed by Ecelbarger et al. (10, 11).
Isolation of Macula Densa Cells--
Mice transgenic for the
SV40 large T-antigen (6-TgN(SV)7Bri) were obtained from The Jackson
Laboratory (Bar Harbor, ME). After decapsulation, the kidney cortex was
minced and digested in a dissociation solution (0.25% trypsin, 0.15%
collagenase A, and 50 mg/ml DNase I) at 37 °C with vigorous shaking
for 1 h. The cells were further dissociated by pipetting the
mixture 2-3 times during incubation. The cell suspension was filtered
through a 20-µm mesh into a 50-ml centrifuge tube to remove any
undigested tissue. The cells were resuspended in cell suspension
solution (HBSS, 5% FBS/DNase I, 20 mg/ml) and washed twice with
the same suspension solution. The cell suspension was adjusted to
5-10 × 10 5 cells/ml and incubated on ice for 30 min
with FITC-conjugated Dolichos biflorus lectin recognizing
sugar residues in collecting duct cells and phycoerythrin-conjugated
Helix pomatia lectin recognizing sugar residues on macula
densa cells, collecting duct cells, and cells in S1 and S2 segments of
proximal tubules solution (50 µg/ml in cell suspension solution). The
cells were washed twice with cell suspension solution and analyzed by
flow cytometry on a Coulter Elite ESP. Unlabeled cells were used for
initial light scatter analysis. The cells were sorted based on their
FITC and phycoerythrin fluorescence (Fig. 1A). Cells that
were phycoerythrin-positive and FITC-negative (left upper
quadrant of the scatter diagram) were collected aseptically into
culture medium (Dulbecco's modified Eagle's medium, supplemented with
10% fetal calf serum, 2 mM glutamine, and 50 µg/ml
penicillin and streptomycin). The cells were cultured at 37 °C in a
humidified atmosphere of 95% air, 5% CO2 until confluent. The cells were then cloned by limiting dilution and 12 clones were
grown to confluence and frozen in 10% dimethyl sulfoxide in fetal
bovine serum. Four of these clones were further screened for expression
of macula densa cell markers by RT-PCR using positive and negative
selection criteria. mRNAs of COX-2, bNOS, NKCC2, ROMK, and oxytocin
receptors were chosen as positive selection markers while the
expression of Tamm-Horsfall protein (THP), vasopressin receptor type 2 (V2R), and glucose-6-phosphatase (Glu-6-Pase) was used as negative
selection criteria.
Experimental Protocols--
MMDD1 cells were grown to confluence
in 6-well plates in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum and antibiotics. Two protocols were used to
reduce extracellular NaCl concentration. In the first protocol,
confluent MMDD1 monolayers were exposed to serum-free Dulbecco's
modified Eagle's medium in a 1:1 mixture with isotonic saline
(control), 300 mM mannitol or raffinose (to reduce NaCl to
half; LS solution 1), 150 mM Na gluconate (to reduce
Cl 86Rb+ Uptake Assay--
MMDD1 cells were
grown to confluence in 6-well plates and assayed at room temperature.
As described previously, cells were preincubated in hypotonic low
Cl RT-PCR--
RT-PCR was performed as described previously (13).
Briefly, total RNA was isolated from the frozen cells using TRI
reagent, and cDNA was synthesized by Moloney murine leukemia
virus reverse transcriptase (Superscript; Life Technologies,
Inc., Gaithersburg, MD). Sequences of the oligonucleotide primers used
to detect COX-2, bNOS, and Western Blotting for COX-2--
Cells were lysed and
subsequently sonicated in phosphate-buffered saline containing 1%
Triton X-100, 250 µM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 5 mM dithiothreitol (pH 7.5).
Protein concentration was determined by Coomassie reagent. 40 µg of
protein from whole cell lysates was denatured in boiling water for 10 min, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline, followed by
incubation for 1 h with rabbit anti-murine polyclonal antiserum to
COX-2 at a dilution of 1:1000. After washing with Tris-buffered saline,
blots were incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody and visualized with ECL kits
(Amersham Pharmacia Biotech).
Phosphorylation of MAP Kinases--
The confluent MMDD1 cells in
6-well plate were lysed by sonication for 10 s in 300 µl of
1 × Laemmli sample buffer containing 10 mM Tris,
1.4% SDS, and 40 mM dithiothreitol (pH 6.8). The protein samples were heated at 60 °C for 15 min and electrophoresis was performed as described above. The blots were blocked in 5% non-fat dry
milk for 1 h. The primary antibodies against phospho-ERK1/2 and
phospho-p38 at a dilution of 1:1000 were incubated overnight a 4 °C.
The secondary antibody and ECL reaction were the same as described
above. The blots were stripped in 0.2 M NaOH for 4 min and
reprobed with antibodies against total ERK1/2 or total p38 with a
similar immunoblotting procedure.
PGE2 Enzyme Immunoassay--
PGE2 in the
culture media was measured with an enzyme immunoassay kit. The assay
was performed according to the manufacturer's instruction. Briefly, 25 or 50 µl of the medium, along with a serial dilution of
PGE2 standard samples were mixed with appropriate amounts
of acetylcholinesterase-labeled tracer and PGE2 antiserum, and incubated at room temperature for 18 h. After the wells were emptied and rinsed with wash buffer, 200 µl of Ellman's reagent containing substrate for acetylcholinesterase was added. The enzyme reaction was carried out on a slow shaker at room temperature for
1 h. The plates were read at 415 nm and the results were analyzed by KC4 software (Bio-Tek Instrument, Inc.).
Fluorescence Microscopy--
Confluent MMDD1 cells grown on
glass coverslips were placed in a perfusion chamber (40 µl volume)
and perfused at about 5 µl/s. The composition of the bath solution
was changed through a manifold at the entrance of the perfusion
chamber. Experiments were performed on an inverted microscope (Diaphot,
Nikon, Melville NY) equipped with differential interference contrast
combined with low light level fluorescence as described previously
(15). Preparations were imaged using a 100x/1.3 N.A lens, and an
intensified CCD camera (ICCD-1001, Video Scope, Sterling VA). The
normal salt solution (NS) contained (in mM): NaCl, 135;
KCl, 4.2; CaCl2, 1; Na2HPO4, 1;
Na2SO4, 2; HEPES, 15. The composition of the
low salt solution (LS) was the same except that NaCl was reduced to 20 and 230 mM mannitol was added to maintain isotonicity. For
calcium measurements cells were incubated for 15 min with the calcium fluorophore Fluo-4-AM (5 µM) at 37 °C (Molecular
Probes, Eugene, OR). After removing extracellular fluorophore by
washing and a 10-min de-esterification period the Fluo-4 signal was
monitored continuously at a sampling rate of 0.5 Hz during the change
of the perfusion medium from NS to LS (excitation at 477 nm, emission at 520 nm). The responsiveness of the system was checked by treatment of the cells with the Ca2+ ionophore A23187 (10 µM). For pH measurements cells were incubated for 10 min
with BCECF-AM (5 µM) at 37 °C (Molecular Probes,
Eugene, OR). Intracellular pH was determined as the excitation ratio
signal (488/458 nm) monitored continuously at a sampling rate of 0.1 Hz. During the medium change from NS to LS the sampling rate was increased to 0.5 Hz. The responsiveness of the system was checked by
the response of intracellular pH to an ammonium chloride pulse (30 mM).
Statistical Analysis--
Values shown represent mean ± S.D. Statistical analysis was performed by Student's t test
or ANOVA with Bonferroni correction with a p value of less
than 0.05 being considered statistically significant.
Characterization of Macula Densa-derived Cell Line (MMDD1
Cells)--
All experiments reported in this study were performed on
cells derived from a clone that fulfilled both positive and negative selection criteria by expressing mRNAs of COX-2, bNOS, NKCC2, ROMK,
and OT receptors (not shown), but not of THP, V2R, and Glu-6-Pase (Fig.
1B). THP was used as a marker
for TAL and DCT, V2R as a marker of TAL and collecting duct cells, and
Glu-6-Pase as a marker for PCT. In addition to the initial selection by
RT-PCR, expression of COX-2, bNOS, NKCC2, and ROMK was confirmed at the
protein level by Western blotting (Fig.
2). The bNOS antibody recognized a
100-kDa protein, smaller than the expected size of 155 kDa;
nevertheless, antibody binding was completely prevented by pretreatment
with bNOS peptide. Cells derived from this clone were designated as MMDD1 cells. After expansion and several initial passages, experiments were performed on cells at passages 5-10. Confluent MMDD1 cells have
an epithelial, cobblestone morphology in phase contrast light microscopy (Fig. 3). Electrical
resistance of confluent MMDD1 cells was in the order of 100
The existence of a functional NKCC2 transporter in MMDD1 cells was
confirmed by determining bumetanide-sensitive 86Rb uptake
in the presence of ouabain to prevent entry of 86Rb through
the Na/K pump. We found measurable uptake of 86Rb within
seconds and the uptake was linear for about 10 min (Fig. 4A). Bumetanide in the 10-100
µM concentration range significantly inhibited
86Rb uptake in a dose-dependent manner with
maximal inhibition being achieved at 60 µM (Fig.
4B). 86Rb uptake was also inhibited by
furosemide with maximal inhibition being produced by a concentration of
200 µM (data not shown). 86Rb uptake was
inversely related to the NaCl concentration of the medium (Fig.
4C).
Time Course of Low NaCl Stimulation of PGE2 Release and
COX-2 Expression in MMDD1 Cells--
To study the time course of the
effect of low NaCl on PGE2 release and COX-2 expression,
confluent MMDD1 cells were incubated in normal salt and low salt medium
for 1, 3, 6, and 16 h. Isosmotic normal and low salt medium was
made by mixing equal volumes of serum-free culture medium with saline
(control), or 300 mM mannitol (low NaCl, LS). Incubation in
LS medium induced a rapid and significant increase of PGE2
release rate that caused a statistically significant increase in medium
PGE2 concentration at 1 h (Fig.
5A). A significant stimulation
of COX-2 protein expression was observed at 3 h and further
induction was observed at the later time points (Fig. 5B).
PGE2 release by low salt was virtually abolished by the
COX-2-specific inhibitor NS-398 (10 µM), and by the
non-selective cyclooxygenase inhibitor indomethacin (10 µM) (Fig. 6).
Effect of Genistein on Low NaCl-stimulated PGE2
Release--
The effect of the nonspecific tyrosine kinase inhibitor
genistein on PGE2 release consisted of a complete
inhibition of the stimulatory effect of low NaCl (Fig.
7). In fact, genistein reduced PGE2 release below basal levels.
Low Cl
To further characterize the low Cl Mechanism for Low Salt-stimulated COX-2 Expression in MMDD1
Cells--
To study the role of MAP kinases in mediation of low salt
stimulation of COX-2 expression in MMDD1 cells, experiments were carried out to test whether exposure to low salt or low
Cl Measurements of Changes in Intracellular Ca2+ and
pH--
In the presence of the control NaCl solution (135 mM NaCl) Fluo-4 fluorescence intensity expressed in units
of gray level averaged 108.7 ± 28.1 (8 experiments with a total
number of 36 cells). Changing the extracellular medium from control to
low NaCl solution (20 mM NaCl) while maintaining
isotonicity did not affect intracellular Ca2+ concentration
in MMDD1 cells. Fluo-4 fluorescence intensity gradually declined to a
gray level of 93.4 ± 28.7, but this fall could not be
distinguished from the bleach rate of the probe. No Ca2+
transients were detected, nor was there a change in Ca2+
that developed with a slower time course. In MMDD1 cells exposed to the
ionophore A23187 (10 µM), Fluo-4 fluorescence intensity increased markedly. A slight and slowly developing acidification was
observed when the control NaCl solution was changed to the low NaCl
perfusate with BCECF fluorescence intensity in gray level units falling
from 2.56 ± .27 to 2.25 ± .22 (10 experiments with 38 cells; p < 0.05 by paired t test). The
measurements of pH were validated by the prompt and marked
alkalinization caused by exposure of the cells to an ammonium chloride pulse.
MD cells are specialized tubular epithelial cells that play a
central role in NaCl-dependent control of glomerular
arteriolar tone and renin release. Despite their paucity and
inaccessibility, substantial progress has been made using
electrophysiological and fluorescent techniques in defining their
membrane characteristics and transport functions (18, 19). However,
understanding of the cellular and molecular mechanisms responsible for
the generation and release of cell-specific signaling molecules
requires availability of MD cells in quantities sufficient for
biochemical analysis. In the present study we report the development of
a new cell line with MD characteristics and its use in determining the
role of MAP kinases in the stimulation of PGE2 release and
COX-2 expression by low medium NaCl.
A number of cell lines from different parts of the nephron have been
established in recent years using SV-40 transgenic mice (16, 17, 20).
Immortal tubular epithelial cell lines were derived from defined
microdissected tubular segments or from tissue enriched in the desired
tubule segment. Attempts in our laboratory to develop an MD cell line
from microdissected MD specimen have not been successful because of
overgrowth with other cells, presumably of mesangial origin. In the
present paper we have used a different strategy to obtain a cell clone
with MD-like properties. Differential labeling of H. pomatia
lectin with phycoerythrin and D. biflorus lectin with FITC
permitted separation of Helix and Dolichos
positive cells by FAC sorting. Since Helix binds
preferentially to MD cells and collecting ducts whereas
Dolichos is a rather specific marker of collecting duct
cells, cells sorted for high phycoerythrin (Helix)
fluorescence, but low FITC (Dolichos) fluorescence should represent an MD-enriched cell fraction (21, 22). Clonally derived cell
lines from this fraction were then screened for the presence of
MD-specific markers such as COX-2, bNOS, and others. MMDD1 cells were
finally established as a cell line that most closely mimics the known
molecular expression pattern of native MD cells. By phase-contrast
light microscopy, MMDD1 cells have an epithelial, cobblestone-like
appearance. Western blotting analysis established the presence of NKCC2
at about 160 kDa and ROMK at about 45 kDa, two transport proteins known
to be expressed in the MD in vivo (23, 24). The observation
that 86Rb+ uptake in MMDD1 cells was
bumetanide-sensitive provided evidence for the presence of a functional
Na-K-2Cl co-transporter. Inhibition of 86Rb+
uptake by bumetanide was found to be dose-dependent with
maximal inhibition of uptake being achieved with 60 µM
bumetanide. Furthermore, MMDD1 cells consistently expressed COX-2 and
bNOS proteins (7, 25, 26). The explanation for the finding that the
bNOS antibody recognized an about 100-kDa protein rather than the
expected size 160-kDa protein is unclear, but may possibly be
consistent with the reported predominant expression of the shorter
bNOS It has been shown previously that COX-2 expression in MD and adjacent
cTAL cells is increased by low salt intake and by a reduction in renal
perfusion pressure, two experimental conditions in which luminal NaCl
at the MD is likely to be reduced (7-9, 29). Our finding that an
isoosmotic lowering of medium NaCl concentration to 60 or 6 mM caused an increase in both PGE2 release and
COX-2 expression, is consistent with the notion that extracellular NaCl
concentration regulates prostanoid secretion from MD cells. A similar
response was not seen in IMCD cells, indicating some degree of cell
specificity. We believe that this observation provides some validation
of the MMDD1 cell model and justifies its use in an attempt to further
characterize the cellular and molecular responses that may be
responsible for MD-mediated regulation. Stimulation of COX-2 expression
by a low NaCl concentration has also been reported in preliminary form
in a primary culture of thick ascending limb cells (30). Furthermore,
stimulation of PGE2 release by a reduction in
Cl Previous work from our and other laboratories has shown that
MD-mediated changes in both renin secretion and vascular tone are
regulated by luminal Cl Exposure of collecting duct cells to hypertonicity has been shown in
our laboratory to cause induction of COX-2 expression through mediation
of MAP kinases (36). Since MAP kinase activation may be a common
terminal pathway of COX-2 induction, we examined the role of MAP
kinases in low salt stimulation of COX-2 expression in MMDD1 cells. We
observed that a reduction in NaCl caused a rapid phosphorylation of
ERK1/2 and p38 (36). While the known stimuli for ERK1/2 are mitogens,
and for p38 are osmotic shock, free radicals, and cytokines, the
present study adds a low Cl The initial signaling cascade resulting in
Cl Our results show that low NaCl treatment induces a rapid
PGE2 release causing a measurable increase in medium
PGE2 concentration within 1 h. Stimulation of
PGE2 release precedes measurable changes in COX-2 protein
expression. We speculate that activated MAP kinases cause
phosphorylation and activation of phospholipase A2 and that increased substrate availability is the main cause for the early increase in PGE2 production. MAP kinases including ERK and
p38 have been shown to activate cytosolic PLA2 and increase
arachidonic acid release in macrophages and platelets (44, 45). It has also been observed in endothelial cells that the activity of COX-2 can
be stimulated by direct tyrosine phosphorylation (46).
In summary, we have developed and characterized a MD-derived cell line
from SV-40 T antigen transgenic mice, designated as MMDD1 cells. In
MMDD1 cells a low medium NaCl concentration causes rapid
phosphorylation of ERK1/2 and p38 MAP kinases accompanied by an
increase in PGE2 release. Within 16 h of exposure to
low NaCl a significant increase in COX-2 protein expression is found that can be inhibited by ERK1/2 and p38 inhibitors and is apparently Cl We are grateful to Dr. Mark Knepper for
providing NKCC2 and ROMK antibodies.
*
This work was supported by NIDDK, National Institutes of
Health Grants DK-37448, DK-39255, and DK-40042 and by intramural funds
from NIDDK and NHLBI.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.
**
To whom correspondence should be addressed: NIDDK, National
Institutes of Health, Bldg. 10, Rm. 4D51, 10 Center Dr., MSC 1370, Bethesda, MD 20892. Tel.: 301-435-6580; Fax: 301-435-6587; E-mail: jurgens@intra.niddk.nih.gov.
Published, JBC Papers in Press, September 11, 2000, DOI 10.1074/jbc.M006218200
The abbreviations used are:
MD, macula densa;
COX-2, cyclooxygenase-2;
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated kinase;
MMDD1, mouse macula densa
derived cells;
JGA, juxtaglomerular apparatus;
NKCC2, Na+-K+-2Cl
Low Chloride Stimulation of Prostaglandin E2
Release and Cyclooxygenase-2 Expression in a Mouse Macula Densa Cell
Line*
,,,,
,,, and**
NIDDK and NHLBI, National Institutes
of Health, Bethesda, Maryland 20892, the § Department of
Urology, University of Michigan, Ann Arbor, Michigan 48109, and the
¶ Department of Pathology and Laboratory Medicine, University of
Rochester, Rochester, New York 14642
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to half), or 167 mM choline chloride (to
reduce Na+ to half). In the second protocol, solutions with
Cl concentrations of 73 (LS solution 2) and 6 mM (LS solution 3) were made by replacing NaCl with Na
gluconate. The composition of these solutions is given in Table
I.
Composition of low Cl solutions
concentration in LS Solution
2 and LS Solution 3 are 73 and 6 mM, respectively.
medium (in mM: 67 Na gluconate, 2.5 KCl,
0.5 CaCl2, 10.5 Na2HPO4, 1 Na2SO4, and 7.5 HEPES, pH 7.4) for 1 h
(12). Influx was measured during a timed exposure of cells to regular
influx medium (in mM: 135 NaCl, 5 RbCl, 1 CaCl2, 1 Na2HPO4, 2 Na2SO4, 15 HEPES, pH 7.4, and 0.1 ouabain)
containing 1 µCi/ml 86RbCl. Cells were washed five times
with an ice-cold high potassium solution. The cells were incubated in
H2O for 5 min, scraped, and passed 3-5 times through a
23-gauge needle with a 3-ml syringe. 86Rb content was
measured by scintillation counting, and protein content was determined
by Coomassie protein assay (Pierce, Rockford, IL).
-actin cDNA were as published
previously (13, 14). Primers used for vasopressin type 2 receptor
(V2R), glucose-6-phosphatase (Glu-6-Pase), and THP cDNA
amplification were THP sense, 5'-CTGGATGTCCATAGTGACTC-3' (bp
1161-1180), antisense, 5'-TGTGGCATAGCAGTTGGTCA-3' (bp 1541-1560) (accession number NM_017082); V2R sense, 5'-TTTCAAGTGCTACCCCAGCT-3' (bp
312-331), antisense, 5'-TTTGATTGCTGGGCCCGATT-3' (bp 609-628) (accession number Z11932); G-6-Pase, sense, 5'-GAACGCCTTCTATGTCCTCT-3' (bp 150-169), antisense, 5'-CACCGGAATCCATACGTTGA-3' (bp 460-479) (accession number NM_013098). PCR reactions were performed in the
presence of 1.5 µCi/50 µl of [32P]dCTP (Amersham
Pharmacia Biotech) and the product intensity was analyzed by phosphorimaging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/cm2, substantially lower than that of M1 or mIMCD-K2
cells, cell lines derived from cortical and medullary collecting ducts,
respectively (16, 17). MMDD1 cells were found to be cytokeratin
positive and vimentin negative confirming their epithelial nature (data not shown). Furthermore, MMDD1 cells retained the ability to bind H. pomatia lectin, and specificity of binding was confirmed
by the finding that preincubation with the competing sugar
N-acetyl-D-galactosamine (500 µg/ml)
eliminated binding of H. pomatia lectin to MMDD1 cells (data not shown).
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Fig. 1.
A, fluorescence activated sorting of a
suspension of renal cortical cells from SV-40 T antigen transgenic mice
after incubation with FITC-labeled D. biflorus lectin
(DB), phycoerythrin (PE)-labeled H. pomatia lectin (HP), or both (DB + HP).
Cells that were phycoerythrin-positive and FITC-negative (left
upper quadrant of the scatter diagram) were collected. Unstained
cells were used for initial light scatter analysis. B,
characterization of MMDD1 cells by RT-PCR of COX-2, bNOS, THP, V2R,
Glu-6-Pase, and -actin. PCR products were analyzed by
phosphorimaging. Positive controls include RT-PCR of THP and V2R on
microdissected mTAL segment, and RT-PCR of Glu-6-Pase on PCT.
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Fig. 2.
Immunoblotting on 40 µg of protein from whole cell lysates of MMDD1 cells
using antibodies against COX-2, bNOS, NKCC2, and K channel (ROMK).
Specificity of bNOS binding was confirmed by absence of binding after
preincubation with 30 µg of bNOS immunizing peptide. The protein
bands were visualized by ECL detection method.
View larger version (100K):
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Fig. 3.
Morphology of MMDD1 cells by phase-contrast
(left) or Hoffman (right) light
microscopy (× 200).
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Fig. 4.
86Rb+ uptake by MMDD1
cells. A, time dependence of
86Rb+ uptake (n = 3).
B, effect of increasing concentrations of bumetanide on
86Rb+ uptake (n = 7; expressed
as % of maximum). C, effect of increasing Cl
concentrations on 86Rb+ uptake
(n = 3; expressed as % of maximum); low, medium, and
high Cl solutions were as shown in Table I except for
replacing KCl with RbCl.
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Fig. 5.
Time course of low NaCl stimulation of
PGE2 production and COX-2 protein expression in MMDD1
cells. MMDD1 cells were treated by LS solution 1 (1:1 mixing of
serum free medium with mannitol) for the indicated period of time.
Medium PGE2 concentration was determined by enzyme
immunoassay and COX-2 protein by immunoblotting. Shown is time
dependence of PGE2 concentration (A)
(n = 3) and COX-2 protein (B)
(n = 5) following low NaCl treatment. NS,
normal salt; LS, low salt.
View larger version (19K):
[in a new window]
Fig. 6.
Effect of non-steroidal anti-inflammatory
drugs on low NaCl-stimulated PGE2 production. MMDD1
cells were treated by normal or low NaCl medium (LS solution 1) for
16 h in the presence or absence of 10 µM NS-398 or
indomethacin (n = 3).
View larger version (17K):
[in a new window]
Fig. 7.
Effect of genistein on low NaCl-induced
increase in medium PGE2 concentration. The cells were
treated normal or low NaCl medium (LS solution 1) for 3 h in the
presence or absence of 50 µM genistein (n = 3).
Stimulation of COX-2 Expression and
Prostaglandin Release in MMDD1 Cells--
To determine if a reduction
in Na+ or Cl was responsible for the low NaCl
effect on COX-2 expression, confluent MMDD1 cells were incubated in
normal salt and low salt medium for 16 h. Isosmotic normal and low
salt medium was made by mixing equal volumes of serum-free culture
medium with saline (control), 300 mM mannitol or raffinose
(to reduce NaCl), 150 mM Na gluconate (to reduce Cl), or
167 mM choline chloride (to reduce Na+). In
these experiments MMDD1 cells at passages 5-10 were used. Compared
with normal salt control, replacement of NaCl with mannitol or
raffinose caused a significant induction of COX-2 protein levels (Fig.
8A). Replacement of
Cl with gluconate had a similar stimulatory effect (Fig.
8A). In contrast, replacement of Na+ with
choline did not induce COX-2 expression (Fig. 8A).
Densitometric values are: 20.5 ± 5.4 in NS group, 48.3 ± 11.2 in low NaCl (with mannitol) group (n = 8, p = 0.002 versus NS), 48.5 ± 4.0 in
low NaCl (with raffinose) group (n = 8, p < 0.001 versus NS), 36.3 ± 5.8 in
low Cl group (n = 7, p = 0.001). To address the concern that the stimulation of COX-2 might be
due to a specific effect of the added carbohydrate, the effect of
glucose on COX-2 expression was examined. Glucose at 15 and 25 mM in serum-free medium was without an effect on COX-2
protein expression (Fig. 8B). The low salt stimulation of COX-2 expression in MMDD1 cells appeared to be a cell type-specific phenomenon because the same treatment had no effect on COX-2 expression in mIMCD-K2 cells, a cell line derived from the inner medullary collecting duct (Fig. 8C). Bumetanide (120 and 240 µM) and furosemide (220 and 420 µM)
augmented COX-2 expression (Fig. 8D).
View larger version (50K):
[in a new window]
Fig. 8.
Effect of isosmotic reduction of NaCl,
Na+, or Cl
concentrations on COX-2 protein expression in MMDD1 cells.
A, immunoblots of COX-2 protein following 16 h
incubation in normal NaCl (lanes 1 and 2) and low
NaCl medium (lanes 3 and 4; lanes 7 and
8), low Cl medium (lanes 5 and
6), and low Na+ medium (lanes 9 and
10). Densitometric values are shown under "Results."
Osmolalities are measured values for the experiment shown.
B, COX-2 protein in MMDD1 cells treated with glucose at the
indicated concentrations. Low NaCl treatment serves as a positive
control (last 2 lanes). C, COX-2 protein in
mIMCD-K2 cells treated with normal and low NaCl (1:1 mixing of
serum-free medium with mannitol). D, COX-2 protein
expression in MMDD1 cells treated with bumetanide or furosemide at the
indicated concentrations.
effect on COX-2
expression and PGE2 release, the dose response to varying
Cl concentrations was examined. COX-2 protein expression
(Fig. 9A) and PGE2
release (Fig. 9B) was found to increase in a
dose-dependent manner when medium Cl
concentration was reduced to 73 and 6 mM.
View larger version (41K):
[in a new window]
Fig. 9.
Effect of low Cl concentration
on COX-2 expression and PGE2 release. A,
average COX-2 protein expression in MMDD1 cells treated for 16 h
with 141, 73, or 6 mM Cl (solutions as given in Table I).
B, PGE2 release rate in MMDD1 cells treated for
16 h with 141, 73, or 6 mM Cl (n = 3).
stimulates MAP kinase activity and whether inhibition
of MAP kinases with pharmacological inhibitors suppresses the
stimulation of COX-2 expression by low salt. ERK1/2 and p38 activity
was assessed by phosphorylation of MAP kinases using antibodies against
the phosphorylated forms of ERK1/2 and p38. PD 98059 and SB 203580 were
used to interfere with MEK1/2 and p38, respectively. Low Cl (6 mM) treatment markedly increased
phosphorylation of both ERK1/2 and p38 in a time-dependent
manner while total ERK1/2 and p38 were not changed (Fig.
10). Low salt treatment (1:1 mixing
serum-free medium with isotonic mannitol) also increased
phosphorylation of the two kinases (data not shown). SB 203580 at 10 µM reduced low salt stimulated COX-2 expression by 60%
and PD 98059 at 10 µM abolished the stimulation (Fig.
11).
View larger version (51K):
[in a new window]
Fig. 10.
Phosphorylated and total p44/42
(upper) and p38 kinases (lower)
following treatment of MMDD1 cells with normal NaCl (time 0) or low
Cl solution (solution 3, 6 mM
Cl) for the indicated periods of time
(representative example of three experiments). Phosphorylation of
ERK1/2 and p38 was determined by immunoblotting using polyclonal
antibodies against phospho-ERK1/2 and phospho-p38. The blots were
stripped in alkaline solution and reprobed by antibodies against total
ERK1/2 and total p38.
View larger version (39K):
[in a new window]
Fig. 11.
Effect of the p38 inhibitor SB 203580 (at 10 and 20 µM) and of the p44/42
pathway inhibitor PD 98059 (at 10 µM) on low NaCl-induced COX-2
expression. Cells were treated for 16 h. A,
representative gel of three experiments. B, densitometric
analysis of COX-2 protein (n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform in the kidney (27, 28). Markers for other nephron
segments including Tamm-Horsfall protein for TAL, vasopressin type 2 receptor for TAL and collecting ducts, and glucose-6-phosphatase for
proximal tubules were not detectable in MMDD1 cells by RT-PCR. While it is not possible to prove that MMDD1 originated from native macula densa
cells, their properties are consistent with this notion. We believe
therefore that they are a valid model of MD cells and may be used to
test MD-specific signaling pathways.
concentration has been observed earlier in cultured
mesangial cells (31). Although MMDD1 cells express low levels of COX-1, more than 90% of the PGE2 release stimulated by low NaCl
was inhibited by NS-398, indicating that most of the released
prostanoids resulted from the action of COX-2.
concentration (32, 33).
Cl dependence has been related to the involvement of
NKCC2 in the initiation of a transmitted MD signal (34). The relative
affinities of NKCC2 for Na+ and Cl are such
that the Cl ion is predicted to act as the dominant
physiological regulator of NKCC2 transport rate (35). Our present
studies show that the increase in COX-2 expression in response to low
NaCl also appears to be Cl-dependent since
substitution of Na+ with gluconate did not affect the
stimulatory response whereas substitution of Cl with
choline prevented it. The mechanism underlying this effect is not quite
clear. It is conceivable that a reduction in NKCC2 transport rate is a
requirement for COX-2 up-regulation and that this was achieved only by
reducing Cl concentration, not by reducing
Na+ concentration. Presence or absence of a Na-K-2Cl
co-transporter may confer cell specificity to the COX-2 stimulating
effect of a reduction in Cl concentration. Whether the
apparent coupling of NKCC2 transport rate to COX-2 expression results
from direct protein interactions or reflects the effect of some change
in cytosolic ionic composition remains to be studied.
concentration to the list of
extracellular signals capable of activating ERK1/2 and p38. That ERK1/2
and p38 kinases play a critical role in the induction of COX-2 by low
extracellular NaCl was confirmed by the finding that SB 203580, an
inhibitor of p38, and PD 98059, an inhibitor of the ERK pathway,
abolished the stimulation of COX-2 expression by low NaCl. Thus our
findings demonstrate that low salt stimulation of PGE2
release and COX-2 expression is mediated through phosphorylation of
ERK1/2 and p38 and possibly other kinases.
-dependent activation of MAP kinases is
still unclear. It has been shown that an increase in cytosolic calcium
can activate MAP kinases (37-39). However, as shown here cytosolic
calcium did not change when MMDD1 cells were exposed to low
extracellular NaCl. Furthermore, previous studies in an isolated
perfused JGA preparation indicate that changes in cytosolic
Ca2+ concentration are directly, not inversely related to
luminal NaCl concentration (40). Cell acidification may be another
ionic signal associated with MAP kinase activation (41). Current
results as well as previous findings indicate that a low luminal NaCl concentration is associated with a decrease in cytosolic pH, most likely resulting from a reduction in NHE activity (42). Thus, the
predicted change of cytosolic pH is directionally consistent with a
possible role of pH in regulating MAP kinase activity. However, to the
extent that the pH change is mediated through NHE one would expect MAP
kinase activation to be apparently Na+-, not
Cl-dependent. Based on our observations with
genistein, we conclude that tyrosine kinases act as upstream activators
of MAP kinases. A role of membrane-associated tyrosine kinases in the
activation of ERK is well established (43).
-dependent. We conclude that MMDD1 cells
provide a useful in vitro model for the study of MD function
and that MAP kinase activation is a major downstream mechanism in the
stimulation of COX-2 expression by low extracellular NaCl.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
co-transporter;
bNOS, neuronal nitric oxide synthase;
ROMK, renal outer medullary
K+ channel;
BCECF, 2',7'-bis(2-carboxyethyl)-5'-carboxyfluorescein;
PGE2, prostaglandin E2;
FITC, fluorescein isothiocyanate;
RT-PCR, reverse transcriptase-polymerase chain reaction;
THP, Tamm-Horsfall
protein;
V2R, vasopressin type 2 receptor;
bp, base pair(s);
NS, normal
salt solution;
LS, low salt solution;
Glu-6-Pase, glucose-6-phosphatase;
IMCD, inner medullary collecting
duct.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Traynor, T. R.,
Smart, A.,
Briggs, J. P.,
and Schnermann, J.
(1999)
Am. J. Physiol. Renal Physiol.
277,
F706-F710
2.
Vander, A. J.
(1967)
Physiol. Rev.
47,
359-382
3.
Skott, O.,
and Briggs, J. P.
(1987)
Science
237,
1618-1620
4.
Jensen, B. L.,
Schmid, C.,
and Kurtz, A.
(1996)
Am. J. Physiol. Renal Physiol.
271,
F659-F669
5.
Francisco, L. L.,
Osborn, J. L.,
and DiBona, G. F.
(1982)
Am. J. Physiol. Renal Physiol.
243,
F537-F542
6.
Greenberg, S. G.,
Lorenz, J. N.,
He, X. R.,
Schnermann, J. B.,
and Briggs, J. P.
(1993)
Am. J. Physiol. Renal Physiol.
265,
F578-583
7.
Harris, R. C.,
McKanna, J. A.,
Akai, Y.,
Jacobson, H. R.,
Dubois, R. N.,
and Breyer, M. D.
(1994)
J. Clin. Invest.
94,
2504-2510
8.
Hartner, A.,
Goppelt-Struebe, M.,
and Hilgers, K. F.
(1998)
Hypertension
31,
201-205
9.
Yang, T.,
Singh, I.,
Pham, H.,
Sun, D.,
Smart, A.,
Schnermann, J. B.,
and Briggs, J. P.
(1998)
Am. J. Physiol. Renal Physiol.
274,
F481-F489
10.
Ecelbarger, C. A.,
Terris, J.,
Hoyer, J. R.,
Nielsen, S.,
Wade, J. B.,
and Knepper, M. A.
(1996)
Am. J. Physiol. Renal Physiol.
271,
F619-F628
11.
Ecelbarger, C. A., Kim, G.-H., Knepper, M. A., Liu, J., Tate,
M., Welling, P. A., and Wade, J. B. (2001) J. Am.
Soc. Nephrol., in press
12.
Gillen, C. M.,
and Forbush, B.
(1999)
Am. J. Physiol. Cell Physiol.
276,
C328-C336
13.
Singh, I.,
Grams, M.,
Wang, W. H.,
Yang, T.,
Killen, P.,
Smart, A.,
Schnermann, J.,
and Briggs, J. P.
(1996)
Am. J. Physiol. Renal Physiol.
270,
F1027-1037
14.
Yang, T.,
Schnermann, J. B.,
and Briggs, J. P.
(1999)
Am. J. Physiol. Renal Physiol.
277,
F1-9
15.
Xia, P.,
Bungay, P. M.,
Gibson, C. C.,
Kovbasnjuk, O. N.,
and Spring, K. R.
(1998)
Biophys. J.
74,
3302-3312
16.
Kizer, N. L.,
Lewis, B.,
and Stanton, B. A.
(1995)
Am. J. Physiol. Renal Physiol.
268,
F347-F355
17.
Stoos, B. A.,
Naray-Fejes-Toth, A.,
Carretero, O. A.,
Ito, S.,
and Fejes-Toth, G.
(1991)
Kidney Int.
39,
1168-1175
18.
Bell, P. D.,
and Lapointe, J. Y.
(1997)
Clin. Exp. Pharmacol. Physiol.
24,
541-547
19.
Lapointe, J. Y.,
Laamarti, A.,
and Bell, P. D.
(1998)
Kidney Int. Suppl.
67,
S58-64
20.
Rauchman, M. I.,
Nigam, S. K.,
Delpire, E.,
and Gullans, S. R.
(1993)
Am. J. Physiol. Renal Physiol.
265,
F416-F424
21.
Rielle, J. C.,
Brown, D.,
and Orci, L.
(1987)
Anat. Rec.
218,
243-248
22.
Schulte, B. A.,
and Spicer, S. S.
(1983)
Am. J. Anat.
168,
345-362
23.
Xu, J. Z.,
Hall, A. E.,
Peterson, L. N.,
Bienkowski, M. J.,
Eessalu, T. E.,
and Hebert, S. C.
(1997)
Am. J. Physiol. Renal Physiol.
273,
F739-F748
24.
Nielsen, S.,
Maunsbach, A. B.,
Ecelbarger, C. A.,
and Knepper, M. A.
(1998)
Am. J. Physiol. Renal Physiol.
275,
F885-893
25.
Mundel, P.,
Bachmann, S.,
Bader, M.,
Fischer, A.,
Kummer, W.,
Mayer, B.,
and Kriz, W.
(1992)
Kidney Int.
42,
1017-1019
26.
Wilcox, C. S.,
Welch, W. J.,
Murad, F.,
Gross, S. S.,
Taylor, G.,
Levi, R.,
and Schmidt, H. H.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
11993-11997
27.
Brenman, J. E.,
Chao, D. S.,
Gee, S. H.,
McGee, A. W.,
Craven, S. E.,
Santillano, D. R.,
Wu, Z.,
Huang, F.,
Xia, H.,
Peters, M. F.,
Froehner, S. C.,
and Bredt, D. S.
(1996)
Cell
84,
757-767
28.
Oberbaumer, I.,
Moser, D.,
and Bachmann, S.
(1998)
Biol. Chem.
379,
913-919
29.
Wang, J. L.,
Cheng, H. F.,
and Harris, R. C.
(1999)
Hypertension
34,
96-101
30.
Wang, J.-L.,
Cheng, H.-F.,
McKanna, J. A.,
and Harris, R. C.
(1999)
J. Am. Soc. Nephrol.
10,
464A (abstr.)
31.
Okuda, T.,
Kojima, I.,
Ogata, E.,
and Kurokawa, K.
(1989)
J. Clin. Invest.
84,
1866-1872
32.
Lorenz, J. N.,
Weihprecht, H.,
Schnermann, J.,
Skott, O.,
and Briggs, J. P.
(1991)
Am. J. Physiol. Renal Physiol.
260,
F486-493
33.
Schnermann, J.,
Ploth, D. W.,
and Hermle, M.
(1976)
Pfluegers Arch.
362,
229-240
34.
Schnermann, J.,
and Briggs, J. P.
(1982)
Kidney Int.
22 Suppl. 12,
S82-S89
35.
Greger, R.
(1985)
Physiol. Rev.
65,
760-797
36.
Yang, T.,
Huang, Y.,
Heasley, L. E.,
Berl, T.,
Schnermann, J. B.,
and Briggs, J. P.
(2000)
J. Biol. Chem.
275,
23281-23286
37.
Lee, S. A.,
Park, J. K.,
Kang, E. K.,
Bae, H. R.,
Bae, K. W.,
and Park, H. T.
(2000)
Brain Res. Mol. Brain Res.
10,
16-24
38.
Atherfold, P. A.,
Norris, M. S.,
Robinson, P. J.,
Gelfand, E. W.,
and Franklin, R. A.
(1999)
Mol. Immunol.
36,
543-549
39.
Naidu, P. S.,
Velarde, V.,
Kappler, C. S.,
Young, R. C.,
Mayfiled, R. K.,
and Jaffa, A. A.
(1999)
Am. J. Physiol. Heart Physiol.
277,
H1061-H1068
40.
Peti-Peterdi, J.,
and Bell, P. D.
(1999)
Am. J. Physiol. Renal Physiol.
277,
F472-476
41.
Parfenova, H.,
Haffner, J.,
and Leffler, C. W.
(1999)
Am. J. Physiol. Cell Physiol.
277,
C728-C738
42.
Fowler, B. C.,
Chang, Y. S.,
Laamarti, A.,
Higdon, M.,
Lapointe, J. Y.,
and Bell, P. D.
(1995)
Kidney Int.
47,
746-751
43.
Marais, R.,
and Marshall, C. J.
(1996)
Cancer Surv.
27,
101-125
44.
Hiller, G.,
and Sundler, R.
(1999)
Cell Signal.
11,
863-869
45.
Borsch-Haubold, A. G.,
Ghomashchi, F.,
Pasquet, S.,
Goedert, M.,
Cohen, P.,
Gelb, M. H.,
and Watson, S. P.
(1999)
Eur. J. Biochem.
265,
195-203
46.
Parfenova, H.,
Balabanova, L.,
and Leffler, C. W.
(1998)
Am. J. Physiol. Cell Physiol.
274,
C72-C81
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H. Castrop, J. N. Lorenz, P. B. Hansen, U. Friis, D. Mizel, M. Oppermann, B. L. Jensen, J. Briggs, O. Skott, and J. Schnermann Contribution of the basolateral isoform of the Na-K-2Cl- cotransporter (NKCC1/BSC2) to renin secretion Am J Physiol Renal Physiol, December 1, 2005; 289(6): F1185 - F1192. [Abstract] [Full Text] [PDF]
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U. G. Friis, J. Stubbe, T. R. Uhrenholt, P. Svenningsen, R. M. Nusing, O. Skott, and B. L. Jensen Prostaglandin E2 EP2 and EP4 receptor activation mediates cAMP-dependent hyperpolarization and exocytosis of renin in juxtaglomerular cells Am J Physiol Renal Physiol, November 1, 2005; 289(5): F989 - F997. [Abstract] [Full Text] [PDF]
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N. Jeck, K. P. Schlingmann, S. C. Reinalter, M. Komhoff, M. Peters, S. Waldegger, and H. W. Seyberth Salt handling in the distal nephron: lessons learned from inherited human disorders Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2005; 288(4): R782 - R795. [Abstract] [Full Text] [PDF]
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S. Fraser, P. Mount, R. Hill, V. Levidiotis, F. Katsis, D. Stapleton, B. E. Kemp, and D. A. Power Regulation of the energy sensor AMP-activated protein kinase in the kidney by dietary salt intake and osmolality Am J Physiol Renal Physiol, March 1, 2005; 288(3): F578 - F586. [Abstract] [Full Text] [PDF]
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N. Niisato, D. C. Eaton, and Y. Marunaka Involvement of cytosolic Cl- in osmoregulation of {alpha}-ENaC gene expression Am J Physiol Renal Physiol, November 1, 2004; 287(5): F932 - F939. [Abstract] [Full Text] [PDF]
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F. Schweda, J. Klar, S. Narumiya, R. M. Nusing, and A. Kurtz Stimulation of renin release by prostaglandin E2 is mediated by EP2 and EP4 receptors in mouse kidneys Am J Physiol Renal Physiol, September 1, 2004; 287(3): F427 - F433. [Abstract] [Full Text] [PDF]
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F. Schweda, M. Kammerl, C. Wagner, B. K. Kramer, and A. Kurtz Upregulation of macula densa cyclooxygenase-2 expression is not dependent on glomerular filtration Am J Physiol Renal Physiol, July 1, 2004; 287(1): F95 - F101. [Abstract] [Full Text] [PDF]
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A. Paliege, D. Mizel, C. Medina, A. Pasumarthy, Y. G. Huang, S. Bachmann, J. P. Briggs, J. B. Schnermann, and T. Yang Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E2 Am J Physiol Renal Physiol, July 1, 2004; 287(1): F152 - F159. [Abstract] [Full Text] [PDF]
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H.-F. Cheng and R. C. Harris Cyclooxygenases, the Kidney, and Hypertension Hypertension, March 1, 2004; 43(3): 525 - 530. [Abstract] [Full Text] [PDF]
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G. Kovacs, P. Komlosi, A. Fuson, J. Peti-Peterdi, L. Rosivall, and P. D. Bell Neuronal Nitric Oxide Synthase: Its Role and Regulation in Macula Densa Cells J. Am. Soc. Nephrol., October 1, 2003; 14(10): 2475 - 2483. [Abstract] [Full Text] [PDF]
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V. Campean, F. Theilig, A. Paliege, M. Breyer, and S. Bachmann Key enzymes for renal prostaglandin synthesis: site-specific expression in rodent kidney (rat, mouse) Am J Physiol Renal Physiol, July 1, 2003; 285(1): F19 - F32. [Abstract] [Full Text] [PDF]
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J. Schnermann The Juxtaglomerular Apparatus: From Anatomical Peculiarity to Physiological Relevance J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1681 - 1694. [Full Text] [PDF]
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H. He, T. Podymow, J. Zimpelmann, and K. D. Burns NO inhibits Na+-K+-2Cl- cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1) Am J Physiol Renal Physiol, June 1, 2003; 284(6): F1235 - F1244. [Abstract] [Full Text] [PDF]
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P. D. Bell, J.-Y. Lapointe, R. Sabirov, S. Hayashi, J. Peti-Peterdi, K.-i. Manabe, G. Kovacs, and Y. Okada Macula densa cell signaling involves ATP release through a maxi anion channel PNAS, April 1, 2003; 100(7): 4322 - 4327. [Abstract] [Full Text] [PDF]
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H. Castrop, J. Klar, C. Wagner, K. Hocherl, and A. Kurtz General inhibition of renocortical cyclooxygenase-2 expression by the renin-angiotensin system Am J Physiol Renal Physiol, March 1, 2003; 284(3): F518 - F524. [Abstract] [Full Text] [PDF]
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 H.-F. Cheng and R. C. Harris Cyclooxygenase-2 Expression in Cultured Cortical Thick Ascending Limb of Henle Increases in Response to Decreased Extracellular Ionic Content by Both Transcriptional and Post-transcriptional Mechanisms. ROLE OF p38-MEDIATED PATHWAYS J. Biol. Chem., November 15, 2002; 277(47): 45638 - 45643. [Abstract] [Full Text] [PDF]
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F. Theilig, V. Campean, A. Paliege, M. Breyer, J. P. Briggs, J. Schnermann, and S. Bachmann Epithelial COX-2 Expression Is Not Regulated By Nitric Oxide in Rodent Renal Cortex Hypertension, April 1, 2002; 39(4): 848 - 853. [Abstract] [Full Text] [PDF]
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 G. G. Goss, L. Jiang, D. H. Vandorpe, D. Kieller, M. N. Chernova, M. Robertson, and S. L. Alper Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1978 - C1990. [Abstract] [Full Text] [PDF]
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 J. Schnermann Cyclooxygenase-2 and macula densa control of renin secretion Nephrol. Dial. Transplant., September 1, 2001; 16(9): 1735 - 1738. [Full Text] [PDF]
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R. C. Harris and M. D. Breyer Physiological regulation of cyclooxygenase-2 in the kidney Am J Physiol Renal Physiol, July 1, 2001; 281(1): F1 - F11. [Abstract] [Full Text] [PDF]
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G. Kovacs, J. Peti-Peterdi, L. Rosivall, and P. D. Bell Angiotensin II directly stimulates macula densa Na-2Cl-K cotransport via apical AT1 receptors Am J Physiol Renal Physiol, February 1, 2002; 282(2): F301 - F306. [Abstract] [Full Text] [PDF]
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