NO inhibits Na+-K+-2Cl
cotransport via a cytochrome P-450-dependent
pathway in renal epithelial cells (MMDD1)
Hao
He,
Tiina
Podymow,
Joseph
Zimpelmann, and
Kevin D.
Burns
Department of Medicine, Ottawa Hospital, and the Kidney
Research Centre, Ottawa Health Research Institute, University of
Ottawa, Ottawa, Ontario, Canada K1H 8L6
 |
ABSTRACT |
Nitric oxide (NO) exerts direct
effects on nephron transport. We determined the effect of NO on
Na+-K+-2Cl
cotransport in a cell
line (MMDD1) with properties of macula densa.
Na+-K+-2Cl
cotransport was
measured as bumetanide-sensitive 86Rb+ uptake
in the presence of ouabain. MMDD1 cells expressed mRNA for the neuronal
isoform of nitric oxide synthase, as well as NKCC1 and NKCC2(B)
isoforms of the Na+-K+-2Cl
cotransporter. Preincubation of cells with the NO donors sodium nitroprusside (SNP) or
S-nitroso-N-acetylpenicillamine (SNAP) caused
concentration-dependent inhibition of
Na+-K+-2Cl
cotransport. Both
apical and basolateral Na+-K+-2Cl
cotransport was inhibited by NO donors. SNP or SNAP had no significant effect on cellular levels of cGMP, cAMP, cytosolic calcium, or phosphorylation of ERK1 and ERK2. In contrast, the inhibitors of
cytochrome P-450, 1-aminobenzotriazole (ABT;
10
3 M) or ketoconazole (1.5 × 10
5 M),
completely reversed the inhibitory effect of SNAP on apical or
basolateral Na+-K+-2Cl
cotransport [apical: control 1.18 ± 0.15 vs. SNAP
(10
4 M) 0.41 ± 0.05 pmol · mg
1 · 5 min
1; P < 0.001; SNAP (10
4
M) + ABT 1.32 ± 0.10 pmol · mg
1 · 5 min
1; P = not significant vs. control;
n = 5]. The cytochrome P-450 epoxyeicosatrienoic acid (EET) metabolite 14,15-EET (5 × 10
7 M) inhibited both apical and basolateral cotransport,
whereas 8,9-EET and 11,12-EET had no significant effect. Although
20-hydroxyeicosatetraenoic acid inhibited apical cotransport, the
inhibitor of
-hydroxylase activity HET0016 did not reverse
SNAP-mediated inhibition of apical cotransport. These data indicate
that NO inhibits apical and basolateral Na+-K+-2Cl
cotransport in MMDD1
cells. The results suggest that the inhibitory pathway is independent
of cGMP and might involve stimulation of a cytochrome
P-450-dependent pathway.
NKCC1; NKCC2; nitric oxide synthase; epoxyeicosatrienoic acids
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INTRODUCTION |
THE LABILE GAS nitric
oxide (NO) is generated in the kidney and exerts direct effects on
transport in several nephron segments. In proximal tubule cells, NO has
been shown to inhibit both apical Na+-H+
exchange (32) and Na+-K+-ATPase
activity (22) via pathways involving generation of cGMP. Thick ascending limb apical and basolateral
Na+-H+ exchange is inhibited by NO, as is
cortical collecting duct transport of sodium and water (9,
35). The inhibitory actions of NO on sodium and water transport
along the nephron suggest that NO generated locally could promote
natriuresis and diuresis. Consistent with this view, recent studies in
the isolated perfused rat thick ascending limb showed that the NO donor
spermine NONOate blocks chloride reabsorption by selectively inhibiting
the apical Na+-K+-2Cl cotransporter, with no
effect on Na+-K+-ATPase activity or
apical K+ permeability (28). In isolated rat
cortical thick ascending limbs, inhibition of endogenous NO production
by the NO synthase (NOS) inhibitor
NG-nitro-L-arginine has been
observed to stimulate chloride transport, suggesting that thick limb NO
generation directly regulates cotransport (30).
Regulation of apical Na+-K+-2Cl
cotransport is of critical importance in cells of the macula densa,
which are involved in the tubuloglomerular feedback (TGF) response.
Macula densa cells express high levels of the neuronal isoform of NOS
(nNOS) (25), and several studies indicate that NO released
by macula densa nNOS inhibits TGF (13, 16, 40). In rabbit
afferent arterioles with attached macula densae, NO has been shown to
act directly on macula densa cells to block TGF responses in a
cGMP-dependent fashion (31, 39). Because NO donors
significantly inhibit the
Na+-K+-2Cl
cotransporter in rat
thick ascending limb (28), one possibility for the
attenuating effect of NO on TGF is via inhibition of the apical
Na+-K+-2Cl
cotransporter (NKCC2)
(41) in macula densa.
The purpose of the present studies was to investigate the effects
of NO on Na+-K+-2Cl
cotransport
and signaling pathways for NO in a renal epithelial cell line (MMDD1)
with properties of macula densa cells (42). We determined
that these cells express both NKCC1 and NKCC2 isoforms of the
Na+-K+-2Cl
cotransporter, and we
demonstrated that NO potently inhibits both apical and basolateral
cotransport. Furthermore, we uncovered a novel role for cytochrome
P-450 (also referred to as P-450) in mediating
responses to NO in these cells.
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MATERIALS AND METHODS |
Cell culture.
A renal epithelial cell line, MMDD1, with properties of macula densa
cells, was used for these studies, kindly provided by Dr. J. Schnermann
(National Institutes of Health, Bethesda, MD). These cells were derived
from SV40 transgenic mice, using fluorescent-activated cell sorting of
renal tubular cells labeled with segment-specific lectins
(42). The cell line has been shown to express well-known markers of macula densa, such as cyclooxygenase 2 (COX-2), nNOS, ROMK,
and NKCC2. In the present studies, MMDD1 cells at passages 5-20 were routinely trypsinized and suspended in DMEM
nutrient mixture-Ham's F-12 (DMEM/F-12) supplemented with 10% fetal
bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml).
The cells were plated onto six-well culture dishes and incubated at 37°C in a humidified atmosphere of 95% room air-5% CO2.
For experiments involving measurement of apical and basolateral
Na+-K+-2Cl
cotransport, cells
were plated onto six-well cell culture inserts (0.4-µm pore size,
Falcon, VWR Canlab, Mississauga, ON, Canada). The media was changed
every 2 days, and once the cells reached confluence (typically in
3-4 days), the cells were maintained in serum-free DMEM/F-12 for
24 h before experiments.
RT-PCR for nNOS, NKCC1, NKCC2, and cytochrome P-450 2J5.
Total RNA was isolated from MMDD1 cells, using a commercial kit
(RNeasy, Qiagen, Chatsworth, CA). RNA (1 µg) was treated with DNase I
and then reverse-transcribed using random hexamers (2.5 µM) and
murine leukemia reverse transcriptase (RT; 2.5 U/µl) (Gene Amp RNA
PCR core kit, Applied Biosystems Roche Molecular System, Branchburg,
NJ). To control for possible genomic DNA contamination, all experiments
included a reaction in which RT was omitted from the transcription
buffer. PCR amplification was carried out in 100 µl of a solution
containing 2.5 U ampliTaq DNA polymerase, 2 mM
MgCl2, 1× PCR buffer II (Gene Amp PCR core kit), and 1 µM of the sense and antisense oligonucleotide DNA primers for the cDNA of interest. For nNOS, the sense primer was
5'-ACTGCTCAAAGAGATAGAACC-3' and the antisense primer was
5'-GGTAATTCTCCAAACCGAG-3', corresponding to nucleotides 695-716
and 1201-1219 of the murine nNOS cDNA, respectively
(26), and predicting amplification of a 525-bp cDNA
product. For NKCC1, the sense primer was 5'-CAGAGACAATTTGAAGACC-3' and
the antisense primer was 5'-CCAACGTCAGCATGAGAT-3', corresponding to
nucleotides 5305-5323 and 5968-5985 of the murine NKCC1 cDNA, respectively, and predicting a PCR product of 681 bp (6).
For NKCC2, the sense primer was 5'-CCCGCTCTCTTGGATATATAAC-3' and the antisense primer was 5'-GCTCCGAACAAATTCTTCCG-3', corresponding to
nucleotides 2301-2321 and 2790-2809 of the murine NKCC2 cDNA, respectively (14), and predicting a PCR product of 509 bp.
RT-PCR specific for the B isoform of NKCC2 [found in macula densa and thick ascending limb (41)] was performed with sense
primer 5'-GTGTGATTATCATCGGCTTAGC-3' and antisense primer
5'-ATCAAGCCTATTGCACCACC-3', corresponding to nucleotides 853-874
and 977-1006 of the B isoform of murine NKCC2 and predicting a PCR
product of 154 bp (14). For cytochrome P-450
isoform CYP2J5, the sense primer was 5'-ATCAGAGAAGCGAAAAGAATGTAG-3' and
the antisense primer was 5'-TACTCAGTCTTAGTCTCCTTTACC-3', corresponding to nucleotides 1549-1572 and 1810-1833 of the murine
cytochrome P-450 2J5 cDNA, respectively, and predicting a
PCR product of 285 bp (21). PCR was routinely performed
for 35 cycles, with a hot-start at 95°C for 5 min, followed by cycles
at 95°C for 60 s, 58°C for 30 s, and 72°C for 60 s, followed by extension at 72°C for 10 min. DNA samples were
electrophoresed on 2% agarose gels stained with ethidium bromide. DNA
sequencing was performed by the DNA Sequencing Facility, University of Ottawa.
Measurement of
Na+-K+-2Cl
cotransport activity.
The activity of Na+-K+-2Cl
cotransport was measured by the uptake of
86Rb+, in the presence of ouabain and in the
presence or absence of the inhibitor bumetanide, essentially as
described (12). Briefly, confluent cells were rinsed twice
in Earle's salt solution consisting of (in mM) 140 NaCl, 5 KCl, 1 MgSO4, 1.8 CaCl2, 5 glucose, and 25 HEPES (pH
7.40 with Tris), and then incubated in this solution for 30 min at
37°C. Cells were then incubated for an additional 15 min in this
solution, supplemented with or without agonists/antagonists, at room
temperature. For assay of
Na+-K+-2Cl
cotransport, the
incubation solution was then changed to one supplemented with 1 µCi/ml 86Rb+ (Amersham, Oakville, ON,
Canada), with 2 mM ouabain, and with or without 6 µM bumetanide
(Sigma, St. Louis, MO), at room temperature. After 5 min, the uptake
was terminated by removal of the solution, followed by four rapid
washings with ice-cold 100 mM MgCl2, buffered with 10 mM
HEPES (pH 7.4 with Tris). Culture dishes were air-dried, and cells were
solubilized in 1 N NaOH with 0.1% SDS, followed by measurement of
radioactivity by liquid scintillation spectrometry. For cells grown on
six-well cell culture inserts, activity of apical or basolateral
Na+-K+-2Cl
cotransport was
determined by adding 86Rb+ to the apical or
basolateral surfaces, respectively, in the presence or absence of
bumetanide (6 µM). Bumetanide (6 µM) was added and maintained in
the media bathing the opposing cell membrane. Protein content was
measured by the Bradford assay method, using BSA as standard (Bio-Rad,
Montreal, QC, Canada). Activity of
Na+-K+-2Cl
cotransport is the
ouabain-insensitive, bumetanide-sensitive component of
86Rb+ uptake and is expressed as picomoles of
86Rb+ per milligram of protein per 5 min, with
all experiments performed in duplicate. In preliminary experiments,
Na+-K+-2Cl
cotransport activity
was linear for 30 min under these assay conditions.
Measurement of cAMP and cGMP.
MMDD1 cells were grown to confluence on 24-well plastic dishes and then
incubated for 24 h in serum-free DMEM/F-12 medium. For assays of
cAMP or cGMP, cells were incubated at 37°C for 15 min in DMEM/F-12,
supplemented with 3-isobutyl-1-methylxanthine (5 × 10
4 M), 0.5% BSA, in the presence or absence of
agonists. Medium was then aspirated and replaced with ice-cold 10%
trichloroacetic acid (TCA; vol/vol). After a further 30 min, samples
were extracted four times with 4 vol of water-saturated ether and
brought to pH 7.0 with Tris. Aliquots were assayed for cAMP or cGMP,
using radioligand competitive binding assay kits, containing
[3H]cAMP (Intermedico, Markham, ON, Canada) or
[3H]cGMP (Amersham), as we performed previously (5,
32).
Measurement of cytosolic calcium concentration.
Cytosolic calcium concentration ([Ca2+]i) was
measured in MMDD1 cells, essentially as we described (5).
Cells were grown to confluence on glass coverslips and loaded with 5 µM of fura 2-acetoxymethyl ester (fura 2-AM) in the presence of
0.005% Pluronic-F127 for 45 min at room temperature. Cells were then
continuously perfused at 37°C with a solution consisting of (in mM)
105 NaCl, 24 NaHCO3, 2 Na2HPO4, 5 KCl, 1.0 MgSO4, 1.5 CaCl2, 4 lactic acid, 5 glucose, 1 alanine, and 10 HEPES (pH 7.3), as well as 0.2% BSA, and NO donors were added at various times. Fluorescence was measured from dual
monochromators set at 340 and 380 nm, using a computer-linked analytic
system (Photon Technology International, South Brunswick, NJ).
Fluorescence emission was measured by photon counting, and the
corrected fluorescence emission intensity ratio, from 340- and 380-nm
excitation, was monitored continuously in selected cells and used as an
indicator of [Ca2+]i.
Western blotting of ERK1 and ERK2.
Phosphorylation of ERK1 (p44) and ERK2 (p42) was measured by Western
blotting. After incubation with or without donors of NO for 15 min,
cells were lysed in an ice-cold buffer consisting of 50 mM
Tris · HCl (pH 8.0), 150 mM NaCl, 1% Nonidet
P-40, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate, and 1 mM sodium fluoride. After quantification of
proteins, equal amounts of protein lysates (10 µg) were run on 15%
SDS-polyacrylamide gels and transferred to nitrocellulose membranes
(Bio-Rad). The membranes were incubated in TBS-T buffer [10 mM Tris
(pH 7.6), 150 mM NaCl, 0.05% Tween 20, 0.5% skim milk] for 1 h
at room temperature. The membranes were then incubated for 16 h at
4°C with 1:500 dilution polyclonal antibody to phosphorylated rat
ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by
incubation with 1:1,000 dilution of anti-rat secondary antibody
conjugated to horseradish peroxidase (Amersham). After membranes were
washed, phosphorylated proteins were detected by enhanced
chemiluminescence (Amersham) on Hyperfilm (Amersham). Prestained
standards were used as molecular weight markers (Bio-Rad), and to
control for protein loading, all membranes were stripped and reprobed
with polyclonal antibody to unphosphorylated rat ERK1 and ERK2 (Santa Cruz Biotechnology). Signals on Western blots were quantified by
densitometry and corrected for unphosphorylated ERK1 and ERK2 levels,
using an image analysis software program (Kodak Densitometer 1S440CF).
Eicosanoids.
The epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic
acid (20-HETE) were obtained from Sigma as HPLC-purified compounds.
Data analysis.
Results are presented as means ± SE of experiments performed in
duplicate. Significance was determined by Student's t-test and by ANOVA for cases with multiple comparisons. Significance is
considered as P < 0.05.
 |
RESULTS |
Characterization of MMDD1 cells.
The cell line used in these studies has previously been shown to
express a number of specific markers for macula densa, including nNOS,
NKCC2, and COX-2. In our laboratory, we confirmed expression of nNOS by
RT-PCR, with restriction endonuclease digestion with BglII
generating fragments of the expected sizes (400 and 125 bp) (Fig.
1A). RT-PCR also revealed the
presence of NKCC2 (Fig. 1B). DNA sequencing of the NKCC2 PCR
product confirmed its identity to the mouse NKCC2 isoform
(14). RT-PCR was also performed using primers that
generated a 154-bp product specific for the B isoform of murine NKCC2
(not shown) (14). DNA sequencing of this product confirmed
the presence of the B isoform in MMDD1 cells. In contrast, RT-PCR using
5'-primers specific for the A and F isoforms of NKCC2 did not generate
PCR products, although both products were amplified from mouse kidney
RNA (not shown). RT-PCR also demonstrated the presence of NKCC1 mRNA in
these cells (Fig. 1C), confirmed by DNA sequencing.

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Fig. 1.
Expression of neuronal nitric oxide synthase (nNOS),
NKCC1, and NKCC2 mRNA in MMDD1 cells by RT-PCR. A:
representative agarose gel showing DNA size ladder (lane 1),
525-bp nNOS PCR product (lane 2), and 400- and 125-bp nNOS
PCR products following digestion with BglII. B:
representative agarose gel depicting DNA size ladder (lane
1), 509-bp PCR product for NKCC2 in MMDD1 cells (lane
2), reaction lacking reverse transcriptase as negative control
(lane 3), 509-bp PCR product for NKCC2 in mouse kidney
cortex as positive control (lane 4), and corresponding
reaction lacking reverse transcriptase (lane 5).
C: representative gel showing DNA size ladder (lane
1), 681-bp product for NKCC1 in MMDD1 cells (lane 2),
corresponding reaction lacking reverse transcriptase (lane
3), 681-bp PCR product for NKCC1 in mouse kidney cortex (positive
control) (lane 4), and corresponding reaction lacking
reverse transcriptase (lane 5).
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Effect of NO on
Na+-K+-2Cl
cotransport.
NO has been shown to inhibit
Na+-K+-2Cl
activity in the rat
thick ascending limb (28) and to inhibit the
Na+H+ exchanger in proximal tubule and thick
ascending limb (9, 32). We first determined the effects of
NO donors on Na+-K+-2Cl
activity
in MMDD1 cells grown on plastic dishes. Both sodium nitroprusside (SNP)
and S-nitroso-N-acetylpenicillamine (SNAP) caused
concentration-dependent inhibition of ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake (Fig.
2). Significant inhibition was observed
at concentrations of SNP greater than or equal to 10
6 M
and at concentrations of SNAP greater than or equal to
10
7 M. SNP or SNAP had no significant effect on the
bumetanide-insensitive component of 86Rb+
uptake (not shown).

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Fig. 2.
Dose-dependent inhibition of
Na+-K+-2Cl cotransport by sodium
nitroprusside (SNP) and
S-nitroso-N-acetylpenicillamine (SNAP) in
MMDD1 cells. Cells were grown to confluence on plastic culture dishes
and incubated with varying concentrations of SNAP (A) or SNP
(B), followed by assay of bumetanide-sensitive
86Rb+ uptake, in the presence of ouabain.
Results are means ± SE of experiments performed in duplicate.
*P < 0.001 vs. control (C); **P < 0.05 vs. C; n = 6.
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These data suggested that NO inhibits
Na+-K+-2Cl
cotransport in MMDD1
cells. To indicate whether the effects of NO donors were indeed due to
release of NO, and not nonspecific, cells were preincubated with
hemoglobin (5 × 10
5 M), a scavenger of NO
(11), before incubation with SNP or SNAP. As shown in Fig.
3, the inhibitory effects of SNP or SNAP
on Na+-K+-2Cl
cotransport were
completely abolished by scavenging of NO [control 8.31 ± 0.40 vs. SNP (10
4 M) 2.50 ± 0.64 pmol · mg
protein
1 · 5 min
1;
P < 0.001; SNP (10
4 M) + hemoglobin
(5 × 10
5 M) 8.69 ± 0.77 pmol · mg
protein
1 · 5 min
1;
P = not significant (NS) vs. control, n = 4]. By contrast, incubation of cells with hemoglobin alone had no
significant effect on Na+-K+-2Cl
cotransport activity.

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Fig. 3.
Effect of scavenging of nitric oxide (NO) with hemoglobin (HB) on
SNP- and SNAP-mediated inhibition of cotransport. Bumetanide-sensitive
86Rb+ uptake, in the presence of ouabain, was
measured in cells grown on plastic dishes. A: effect of
preincubation with HB (5 × 10 5 M) on SNAP
(10 4 M)-mediated inhibition of cotransport. B:
effect of HB (5 × 10 5 M) on SNP (10 4
M)-mediated inhibition of cotransport. Results are means ± SE of
experiments performed in duplicate. *P < 0.005 vs. C;
n = 4.
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Because the MMDD1 cells express both NKCC1 and NKCC2, we determined
effects of NO on apical and basolateral
Na+-K+-2Cl
cotransport, using
measurements in cells grown on cell culture inserts. Basolateral
Na+-K+-2Cl
cotransport exceeded
apical cotransport by ~6- to 10-fold. Incubation of cells with SNAP
(10
4 M) significantly inhibited both apical and
basolateral Na+-K+-2Cl
cotransport (Fig. 4).

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Fig. 4.
Inhibition of apical and basolateral
Na+-K+-2Cl cotransport by SNAP in
MMDD1 cells. Cells were grown to confluence on cell culture inserts,
followed by measurement of apical or basolateral bumetanide-sensitive
86Rb+ uptake, in the presence of ouabain and in
the presence or absence of SNAP (10 4 M). A:
apical cotransport activity (n = 4). B:
basolateral cotransport activity (n = 5). Results are
means ± SE of experiments performed in duplicate.
*P < 0.03 vs. C.
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Because MMDD1 cells express nNOS, we determined the role of endogenous
NO production on Na+-K+-2Cl
cotransport. Incubation of cells with the inhibitor of nNOS, 7-nitroindazole (7-NI; 10
6 M), caused small but
significant increases in activity of both apical and basolateral
cotransport, suggesting that NO exerts autocrine, tonic inhibition of
cotransport in these cells (Fig. 5).

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Fig. 5.
Effect of the inhibitor of nNOS, 7-nitroindazole (7-NI), on apical
and basolateral Na+-K+-2Cl
cotransport. MMDD1 cells were grown on cell culture inserts and
incubated with the nNOS inhibitor 7-NI (10 6 M) before
measurement of bumetanide-sensitive 86Rb+
uptake. A: apical cotransport activity. B:
basolateral cotransport activity. Results are means ± SE of
experiments performed in duplicate. *P < 0.03 vs. C;
**P < 0.05 vs. C; n = 4.
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Signaling pathways for inhibition of
Na+-K+-2Cl
cotransport by NO.
In many cells, NO stimulates soluble guanylate cyclase, leading to
elevation of cGMP levels. In MMDD1 cells grown on regular plastic
dishes, SNP or SNAP had no significant effect on cGMP levels, at
concentrations up to 10
3 M. As a positive control, atrial
natriuretic peptide (ANP; 10
6 M) caused an eightfold
increase in cGMP release in these cells (Fig.
6A). Similarly, neither NO
donor had any significant effect on cellular levels of the second
messenger cAMP (Fig. 6B).

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Fig. 6.
Effect of NO donors on cGMP and cAMP in MMDD1 cells. A:
cells were incubated with varying concentrations of SNP or SNAP for 15 min, followed by assay of cGMP levels. As a positive control, cells
were incubated with atrial natriuretic peptide (ANP; 10 6
M). B: cells were incubated with SNP or SNAP
(10 3 and 10 4 M) for 15 min, followed by
assay of cAMP levels. As a positive control, cells were incubated with
forskolin, an activator of adenylate cyclase (10 5 M).
Results are means ± SE of experiments performed in duplicate
(n = 4). *P < 0.001 vs. C.
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In vascular smooth muscle cells, NO modulates the levels of cytosolic
calcium (43), thereby regulating signaling pathways linked
to calcium transients. In MMDD1 cells loaded with the calcium-sensitive dye fura 2-AM, however, we observed no significant effect of SNP or
SNAP (10
3 or 10
4 M) on cytosolic calcium
levels. By contrast, bradykinin (10
8 M) stimulated
significant increases in calcium (Fig.
7).

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Fig. 7.
SNAP has no effect on cytosolic calcium concentration in
MMDD1 cells. Cells were loaded with fura-2 AM as described in
MATERIALS AND METHODS, followed by administration of SNAP
(10 4 M) at the indicated point. As a positive control,
bradykinin (10 8 M) caused a significant increase in
cytosolic calcium. Shown is a representative tracing of experiments in
which either SNP or SNAP was administered (n = 4).
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Recent studies also suggested a role for NO in modulating the activity
of mitogen-activated protein kinase (MAP kinase) in mesangial cells
(15). To determine whether MAP kinase was affected by NO,
MMDD1 cells were incubated with NO donors, followed by immunoblot
analysis for the phosphorylated MAP kinase substrates ERK1 and ERK2
(p44 and p42, respectively). As shown in Fig.
8, SNP or SNAP had no significant effect
on phosphorylation of ERK or ERK2. Furthermore, immunoblot analysis
revealed no effect of NO on the levels of nonphosphorylated ERK1 and
ERK2 (not shown).

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Fig. 8.
SNP and SNAP do not affect phosphorylation of ERK1 and
ERK2. Shown is a representative immunoblot (n = 4) for
phosphorylated ERK1 (44 kDa) and ERK2 (42 kDa) from cell extracts
(10 5g) derived from cells incubated with SNP or SNAP
(10 4 M). No significant effect on phosphorylation was
observed, and there was also no effect on levels of nonphosphorylated
ERK1 or ERK2 by immunoblot analysis (not shown).
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Modulation of cytochrome P-450 by NO has been suggested by
recent in vivo studies in rats (20). To determine whether
the inhibition of Na+-K+-2Cl
cotransport activity in MMDD1 cells might involve this pathway, cells
were preincubated with the suicide inhibitor of cytochrome P-450, 1-aminobenzotriazole (ABT; 10
3 M)
(36), before determination of the effects of NO donors on cotransport activity. ABT completely reversed the inhibitory effects of
both SNP and SNAP on either apical or basolateral
Na+-K+-2Cl
cotransport [apical:
control 1.18 ± 0.15 vs. SNAP (10
4 M) 0.41 ± 0.05 pmol · mg
protein
1 · 5 min
1;
P < 0.001 vs. control; SNAP (10
4 M) + ABT (10
3 M) 1.32 ± 0.10 pmol · mg
protein
1 · 5 min
1;
P = NS vs. control, n = 5; Fig.
9A]. Incubation of cells with ABT alone did not significantly affect cotransporter activity. In MMDD1
cells grown on plastic dishes, ABT (10
3 M) completely
reversed the inhibitory effect of either SNP or SNAP (10
4
M) on total Na+-K+-2Cl
cotransport activity (not shown). The inhibitor of cytochrome P-450 ketoconazole (1.5 × 10
5 M)
(4) also completely blocked the inhibitory effect of SNAP on apical or basolateral
Na+-K+-2Cl
cotransport (Fig. 9,
C and D).

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Fig. 9.
Effect of inhibition of cytochrome P-450 on
SNAP-mediated inhibition of cotransport. Cells were preincubated with
the P-450 inhibitors 1-aminobenzotriazole (ABT;
10 3 M; A and B) or ketoconazole
(Keto; 1.5 × 10 5 M; C and D)
before determination of the effect of SNAP (10 4 M) on
cotransport. A and C: apical cotransport
activity. B and D: basolateral cotransport
activity. Results are means ± SE of experiments performed in
duplicate. *P < 0.001 vs. C; **P < 0.005 vs. C; n = 5 (ABT) or n = 3-4 (Keto).
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Effect of EET and 20-HETE on
Na+-K+-2Cl
cotransport.
To determine possible mediators of the effect of cytochrome
P-450 on inhibition of cotransport by NO, cells were
incubated for 15 min with metabolites of P-450. The EETs
8,9-EET and 11,12-EET (5 × 10
6 M) had no
significant effect on apical or basolateral cotransport activity (Fig.
10). In contrast, in cells grown on
plastic, incubation with 14,15-EET caused a concentration-dependent
inhibition of total Na+-K+-2Cl
cotransport (Fig. 11A). In
cells grown on cell culture inserts, both apical and basolateral
cotransport was also inhibited by preincubation with 14,15-EET (5 × 10
7 M) (Fig. 11, B and C).

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Fig. 10.
Effect of 8,9-epoxyeicosatrienoic acid (EET) and 11,12-EET on
Na+-K+-2Cl cotransport. MMDD1
cells were grown on cell culture inserts and were incubated for 30 min
with 8,9-EET or 11,12-EET (5 × 10 6 M) before assay
of cotransport activity. Cells were incubated with SNAP
(10 4 M) as a positive control. A: apical
cotransport activity. B: basolateral cotransport activity.
Results are means ± SE of experiments performed in duplicate.
*P < 0.005 vs. C; n = 3.
|
|

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Fig. 11.
Inhibition of
Na+-K+-2Cl cotransport by
14,15-EET. A: dose-dependent inhibition of cotransport in
MMDD1 cells grown on plastic dishes. Cells were incubated with varying
concentrations of 14,15-EET for 30 min before assay of cotransport.
B: effect of 14,15-EET (5 × 10 7 M) on
apical cotransport in cells grown on culture inserts. C:
effect of 14,15-EET (5 × 10 7 M) on basolateral
cotransport in cells grown on culture inserts. Results are means ± SE of experiments performed in duplicate. *P < 0.001 vs. C; **P < 0.05 vs. C; n = 3 (A) and n = 8 (B and
C).
|
|
The product of arachidonic acid
-hydroxylase activity, 20-HETE, has
been shown to inhibit apical
Na+-K+-2Cl
cotransport in thick
ascending limb (7). In MMDD1 cells grown on cell culture
inserts, 20-HETE caused a significant inhibition of apical
Na+-K+-2Cl
cotransport but had no
effect on basolateral cotransport (Fig. 12). However, the inhibitor of
-hydroxylase activity, HET0016 (10
5 M)
(23), had no significant effect on SNAP-mediated
inhibition of apical cotransport (Fig.
13).

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Fig. 12.
Effect of 20-HETE on
Na+-K+-2Cl cotransport in MMDD1
cells. Assay of Na+-K+-2Cl
cotransport was performed on confluent cells grown on cell culture
inserts following preincubation with 20-HETE (5 × 10 6 M). A: apical cotransport activity.
B: basolateral cotransport activity. Results are means ± SE of experiments performed in duplicate. *P < 0.005 vs. C; n = 8.
|
|

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Fig. 13.
Effect of the -hydroxylase inhibitor HET0016 on
SNAP-mediated inhibition of apical
Na+-K+-2Cl cotransport.
Bumetanide-sensitive 86Rb+ uptake was measured
in confluent cells grown on cell culture inserts after preincubation
with the selective -hydroxylase inhibitor HET0016 (10 5
M), with or without SNAP (10 4 M). Results are means ± SE of experiments performed in duplicate. There was no significant
difference between the effect of SNAP and SNAP + HET0016;
n = 8. *P < 0.001 vs. C;
**P < 0.01 vs. C.
|
|
To determine if MMDD1 cells express a cytochrome P-450
isoform that is abundant on mouse kidney, RT-PCR for the CYP2J5 isoform (21) was performed on RNA derived from these cells. As
shown in Fig. 14, the CYP2J5 isoform is
expressed in this cell line, with mouse kidney used as a positive
control.

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Fig. 14.
Expression of cytochrome P-450 (CYP) 2J5 in
MMDD1 cells by RT-PCR. Representative agarose gel showing DNA 100-bp
ladder (lane 1), 285-bp CYP 2J5 PCR product from MMDD1 cells
(lane 2), corresponding reaction lacking reverse
transcriptase (lane 3), 285-bp PCR product for CYP 2J5 from
mouse liver (lane 4), and corresponding reaction
lacking reverse transcriptase (lane 5).
|
|
 |
DISCUSSION |
Two isoforms of the electroneutral, loop diuretic-sensitive
Na+-K+-2Cl
cotransporter have
been identified and are encoded by separate genes. The NKCC1 isoform is
found on the basolateral membranes of secretory epithelia, and in the
kidney it is localized to outer and inner medullary collecting ducts,
glomerular and extraglomerular mesangium, and to afferent arteriole
(18). The NKCC2 isoform is found exclusively in the
kidney, and in the rabbit and mouse it consists of splice variants (A,
B, F), with the B variant expressed specifically on the apical surface
of cells of the cortical thick ascending limb and macula densa
(41). The mouse renal epithelial cell line (MMDD1) used in
the present study was derived from SV40 transgenic mice and retains
some well-known properties of macula densa, such as expression and
activity of NKCC2, COX-2, and nNOS (42). We confirmed
these properties, and we demonstrated expression of the B variant of
NKCC2 in these cells, and not the A or F variants, suggesting
derivation of these cells from macula densa or cortical thick ascending
limb (42). We also demonstrated that these cells express
NKCC1. When MMDD1 cells were grown on cell supports,
bumetanide-sensitive 86Rb+ transport was
present on apical and basolateral membranes, indeed with a relative
abundance of basolateral cotransport. Our major finding is that NO
inhibits apical and basolateral
Na+-K+-2Cl
cotransport activity
in these cells. The inhibitory mechanism does not involve generation of
cGMP, but our data suggest a novel pathway, with a requirement for
cytochrome P-450 epoxygenase activity.
In macula densa, transport through the apical membrane
Na+-K+-2Cl
cotransporter is
thought to be the initiator of the TGF response, resulting in afferent
arteriolar constriction. The nNOS isoform is highly expressed in macula
densa, and studies show that NO produced in the macula densa blunts TGF
(13, 16, 37, 40). Because our data show that the NKCC2 B
isoform is present in MMDD1 cells, the results are consistent with the
hypothesis that NO derived from macula densa or adjacent thick limb
cells in vivo could act in an autocrine or paracrine fashion to inhibit
apical cotransporter activity. In recent studies in rabbit afferent
arterioles with attached macula densae, inhibition of macula densa nNOS
with 7-NI increased TGF responses, and it was also suggested that NO derived from the thick ascending limb could act as a paracrine factor
to inhibit TGF at the macula densa (39). Similarly, our data in MMDD1 cells indicate that inhibition of nNOS with 7-NI caused a
small but significant increase in cotransporter activity. Autocrine
inhibition of apical cotransport by NO could therefore represent a
mechanism for precise modulation of the TGF mechanism, independently of
the direct inhibitory effects of NO on afferent arteriolar tone.
Although previous studies examined effects of NO donors and endogenous
NO on apical Na+-K+-2Cl
cotransport (NKCC2), there is little information on the effects of NO
on basolateral cotransport via NKCC1. In rat aortic smooth muscle, NO
inhibits basal NKCC1 activity and reduces its phosphorylation (1). In our studies, MMDD1 cells expressed abundant
basolateral cotransport activity. Earlier studies by Lapointe and
colleagues (3, 19) did not identify basolateral
cotransport activity in rabbit macula densa. Although the presence of
NKCC1 on the basolateral membrane of murine macula densa cells is
possible, it is surprising that basolateral cotransport activity far
exceeded apical cotransport in our study. Accordingly, it is possible
that the MMDD1 cells have undergone dedifferentiation, with new
expression of NKCC1, or they may contain nonepithelial cells or cells
from tubular segments known to express NKCC1.
In many cell types, including proximal tubule, thick ascending limb,
and cortical collecting duct, NO stimulates soluble guanylate cyclase,
leading to production of cGMP (8, 27, 32, 34). In these
segments, inhibition of transport pathways by NO is at least partly
dependent on generation of cGMP, and recent in vivo studies in rats
indicate that increases in renal interstitial cGMP levels promote
natriuresis (17). In thick ascending limb, NO inhibits
chloride reabsorption by activation of cGMP-stimulated phosphodiesterase, which decreases cAMP levels (27). In
contrast, our data indicate that NO had no effect on cGMP or cAMP
levels in MMDD1 cells. ANP significantly stimulated cGMP production. The effects of ANP on juxtaglomerular cell renin release have been
studied in animal models (10, 38), although there is presently no information on direct effects of ANP on macula densa function. Our data suggest that MMDD1 cells express ANP receptors and
also contain the particulate, but not the soluble, guanylate cyclase.
In contrast, studies in an isolated rabbit afferent arteriole preparation by Ren et al. (31, 39) showed that NO produced by the macula densa acts on macula densa cells to inhibit TGF, via a
pathway involving activation of guanylate cyclase and generation of
cGMP. It is possible that macula densa responses to NO may be species
specific or that MMDD1 cells may have reduced expression of guanylate
cyclase. However, the potent inhibition of cotransport by NO in the
present study does not exclude the possibility that in vivo cGMP
generation may also induce inhibition.
There has been recent interest in the effects of NO that are
independent of cGMP production, including effects of NO metabolites such as peroxynitrite. In the present studies, NO did not alter cell
levels of calcium. Of interest, previous studies suggest that the
B2 isoform of the bradykinin receptor is not expressed in
human macula densa (33). However, MMDD1 cells demonstrated reproducible calcium transients in response to bradykinin, suggesting expression of bradykinin B1 or B2 receptors in
these cells. In mesangial cells in culture, NO inhibits mechanical
stretch-induced ERK activity and nuclear translocation
(15). Although we did not measure ERK activity directly,
we observed no effect of NO on basal phosphorylation of ERK1 or ERK2 in
MMDD1 cells.
Our studies uncovered a unique signaling pathway for NO in MMDD1 cells.
ABT, an agent that blocks the renal metabolism of arachidonic acid to
EETs and 20-HETE by cytochrome P-450 (36), completely reversed the inhibitory effect of exogenous NO on apical and
basolateral Na+-K+-2Cl
cotransport, suggesting that cytochrome P-450 is involved in NO signaling. Similarly, the inhibitor of P-450 epoxygenase
ketoconazole reversed the inhibitory effect of SNAP. Neither ABT nor
ketoconazole alone affected cotransport activity. Because incubation
with 7-NI caused small but significant increases in apical and
basolateral cotransport, this suggests that either ABT or ketoconazole
does not completely inhibit P-450-mediated production of
EETs at baseline, induced by endogenous NO, but instead blocks
NO-stimulated P-450 activity. In this regard, incubation of
rat renal microsomes with ABT at concentrations of 10 mM or greater was
required to induce 80% inhibition of P-450 in vitro
(24). An alternate possibility is that basal NO production
in these cells inhibits cotransport activity via a pathway separate
from that involving activation of P-450 by exogenous NO.
Although both apical and basolateral cotransport activities were
inhibited by NO and this was reversed by inhibition of cytochrome P-450, and mimicked by 14,15-EET, only apical cotransport
was inhibited by the arachidonic acid
-hydroxylase product 20-HETE. In rabbit thick ascending limb, 20-HETE has also been shown to inhibit
Na+-K+-2Cl
cotransport
(7). However, in MMDD1 cells, the inhibitor of
-hydroxylase HET0016 did not have a significant effect on
SNAP-mediated inhibition of apical
Na+-K+-2Cl
cotransport, whereas
ABT and ketoconazole completely blocked the inhibitory effect of SNAP.
Taken together, these data suggest that the inhibitory effect of NO may
be mediated by selective activation of cytochrome P-450
epoxygenase activity in these cells, with no effect on the
-hydroxylase activity. The data also suggest that NKCC1 and NKCC2
have distinct regulatory mechanisms with regards to these arachidonic
acid metabolites.
Along the nephron, P-450-derived eicosanoids, including EETs
and 20-HETE, have been shown to influence sodium and water transport. NO has been shown to inhibit cytochrome P-450 isoenzymes of
the 1A, 2B1, 3C, and 4A classes by forming iron-nitrosyl complexes at
the heme binding sites (2, 29). Recent in vivo studies in
rats suggest that the renal vascular effects of NO are mediated via
inhibition of P-450 and activation of guanylate cyclase,
whereas the natriuretic effects are independent of P-450
(20). Our data suggest that in MMDD1 cells, NO may indeed
activate a cytochrome P-450 isozyme with selective
inhibition of the cotransporter by 14,15-EET.
Although kidney microsomal content of P-450 activity is
significant, knowledge of kidney P-450 isoforms is
incomplete. In mouse kidney, the CYP2J5 isoform is expressed in
abundance in proximal tubules and collecting ducts (21).
This isoform is associated with production of EETs, of which the
14,15-EET is predominant (21). Our studies demonstrate
that mRNA for this isoform is present in MMDD1 cells. Further studies
are required to determine whether the CYP2J5 isoform is expressed in
macula densa in vivo and whether NO activates this enzyme, leading to production of EETs.
In summary, we showed that NO inhibits apical and basolateral
Na+-K+-2Cl
cotransport activity
in MMDD1 cells, independently of guanylate cyclase activation. The
inhibitory mechanism may involve a cytochrome P-450-dependent pathway, possibly mediated by production of
14,15-EET.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from the Canadian Institutes of
Health Research and the Kidney Foundation of Canada (to K. D. Burns).
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
K. D. Burns, Division of Nephrology, The Ottawa Hospital and
Univ. of Ottawa, 1967 Riverside Dr., Rm. 535A, Ottawa, Ontario,
Canada K1H 7W9 (E-mail:
kburns{at}ottawahospital.on.ca).
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.
First published February 11, 2003;10.1152/ajprenal.00192.2002
Received 16 May 2002; accepted in final form 8 February 2003.
 |
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