Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, D-20246 Hamburg, Germany
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ABSTRACT |
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ANG II arrests LLC-PK1 cells in the G1 phase of the cell cycle and induces hypertrophy, an effect mediated by induction of p27Kip1. We studied whether atrial natriuretic peptide (ANP) may modulate ANG II-induced hypertrophy and p27Kip1 expression in tubular LLC-PK1 cells. ANP, through its fragments 3---28 and 4---27, prevented ANG II-induced cell cycle arrest. ANP inhibited >80% of ANG II-induced p27Kip1 protein expression (Western blots). ANP stimulated expression of MKP-1, a phosphatase involved in dephosphorylation of p44/42 mitogen-activated protein (MAP) kinase, up to 12 h. ANP prevented the ANG II-mediated phosphorylation peak of MAP kinase after 12 h of stimulation. 8-Bromo-cGMP mimicked all the effects of ANP. Transfection with MKP-1 antisense, but not sense, oligonucleotides abolished the modifying role of ANP on ANG II-mediated cell cycle arrest.. The effect of ANP on ANG II-mediated hypertrophy of LLC-PK1 cells is regulated on the level of MAP kinase phosphorylation, a key step in the induction of p27Kip1. Although ANP and ANG II both stimulate generation of reactive oxygen species, ANP additionally induces expression of MKP-1, leading to interference with ANG II-mediated MAP kinase phosphorylation.
tubular hypertrophy; cell cycle arrest; signal transduction; LLC-PK1 cells; mitogen-activated protein kinases
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INTRODUCTION |
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TUBULAR HYPERTROPHY IS AN early morphological feature of chronic renal disease (37, 41). Although thought to initially compensate for the loss of functioning renal tissue by increasing the work of surviving nephrons, there is accumulating evidence that hypertrophy of tubules is detrimental to the kidney in the long term (37). In fact, hypertrophy of tubular cells may be a precursor of the development of tubulointerstitial fibrosis as a common end point of many renal diseases with diverse etiologies (37). We and others have previously shown that ANG II as a single factor stimulates hypertrophy of cultured proximal tubular cells of various species (14, 34, 38, 40). ANG II may be particularly relevant as a hypertrophic factor because the renin-angiotensin system is activated in chronic renal injury, and interference with this system attenuates compensatory renal hypertrophy (37, 41). ANG II-treated proximal tubular cells actively reenter the cell cycle but remain arrested in the G1 phase, where they undergo hypertrophy (41). We have extensively investigated the mechanism of this G1-phase arrest and found that an inhibitor of cyclin-dependent kinases (CDK), p27Kip1, plays a pivotal role in this cell cycle arrest (10, 38). Furthermore, ANG II-mediated generation of reactive oxygen species, with subsequent activation of mitogen-activated protein (MAP) kinases, is necessary for the induction of p27Kip1 in cultured proximal tubular cells (10, 11).
On the other hand, the renal actions of atrial natriuretic peptide (ANP) are altered in chronic renal failure (20, 28). There is clear evidence that proximal tubular cells possess specific receptors for ANP. This vasorelaxant peptide influences proximal sodium, water, and bicarbonate reabsorption (7-9, 13). However, ANP itself likely exhibits no direct natriuretic effect but rather may inhibit ANG II-induced stimulation of sodium transport, probably by interaction with kinase activity (9, 12). In addition, many studies found that ANP exerts growth-inhibitory properties on several cell types, including renal mesangial cells (1, 3, 4, 16-18, 21, 39, 42). However, potential growth-modulatory effects of ANP on tubular cells and their mechanism are largely unknown. The present study was performed to investigate whether ANP and its bioactive fragments modulate ANG II-induced hypertrophy of tubular LLC-PK1 cells.
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METHODS |
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Cell culture.
LLC-PK1 cells are a well-characterized porcine cell line
with some properties of proximal tubular cells. We have previously demonstrated that these cells express specific receptors for ANG II and
that the vasopeptide induces hypertrophy of these cells (38,
40). Cells were grown in DMEM containing 450 mg/dl glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 × 103
mol/l supplemental glutamine, and 10% heat-inactivated FCS (GIBCO-BRL, Eggenstein, Germany) at 37°C in 5% CO2. Cells were
passaged every 96 h.
ANG II receptor binding studies.
Binding studies for ANG II were performed as previously described
(40). In brief, 104 cells/well of a 24-well
plate were plated (Nunc, Naperville, IL) and were rested for 24 h
in serum-free DMEM. Cells were then treated for another 24 h with
a single dose of 107 mol/l ANP (1-28;
Sigma, Deisenhofen, Germany). Binding studies were done in assay buffer
(phosphate-buffered saline supplemented with 5 × 10
3 mol/l MgCl2, 10
4 mol/l
-aminocaproic acid, 10
3 mol/l phenylmethylsulfonyl
fluoride, and 0.5% aprotinin) on a shaking platform at 0°C for
1 h. Previous experiments revealed that saturation of ANG II
binding sites occurred after 40 min under these conditions. For
saturation binding assays, increasing amounts (0.2-10 × 10
9 mol/l) of
125I-[Sar1,Ile8]ANG II (specific
activity 2,000 Ci/10
3 mol/l, Amersham, Braunschweig,
Germany) were added. Nonspecific binding was determined in the presence
of 10
3 mol/l cold ANG II (Sigma). After 1 h, cells
were gently washed four times with ice-cold binding buffer. Finally,
cells were lysed in 500 µl 0.5 mol/l NaOH with 0.5% Triton X-100,
and the amount of radioactivity was counted in a gamma scintillation
counter. Control wells without radioactivity were also lysed, and
protein content was measured with the Lowry method. Nonspecific biding was subtracted, and data were analyzed with the computer program Enzfitter (Elsevier-Biosoft, Cambridge, UK). Results are presented as
Scatchard plots of saturation binding data, and every point represents
the mean of four independent determinations.
RT-PCR and Southern blots.
To gain information as to whether LLC-PK1 cells express ANP
type A receptors, cDNA amplification was performed after reverse transcription of RNA. To this end, total RNA was isolated by repeated phenol-chloroform extractions, and 10 µg of total RNA were reverse transcribed by using 0.7 µg poly-d(T) primer (Pharmacia Diagnostics, Freiburg, Germany) in the presence of 500 U of Maloney murine leukemia
virus RT diluted in 50 µl of a buffer containing 5 × 102 mol/l Tris · HCl (pH 8.3), 7.5 × 10
2 mol/l KCl, 3 × 10
3 mol/l
MgCl2, 10
2 mol/l dithiothreitol (DTT), and
5 × 10
4 mol/l dNTP. After incubation for 2 h
at 37°C, 5 µl of the cDNA preparation were directly used for the
PCR amplification with 5 µl of 10× amplification buffer, 2.5 × 10
2 mol/l MgCl2, 10
2 mol/l
dNTPs, 1.5 µl of each primer (50 ng/µl), and 2.5 U Taq polymerase (Promega, Madison, WI). The following primers specific for the ANP receptor of the A type were used (22):
5'-CAAGCGCTCATGCTCTACGCCTAC-3' and 5'-GCTTTGCCCAAAGATATCC-3'. A total
of 35 amplification cycles (denaturing for 30 s at 94°C,
annealing for 90 s at 52°C, and extension for 90 s at
72°C) were performed. Fifteen microliters of the reaction product
were separated in a 1.8% agarose gel containing 0.5 µg/ml ethidium
bromide. After the correct size of 283 base pairs was revealed,
amplified cDNA was transferred to nylon membranes by Southern blotting
in alkaline buffer. Membranes were hybridized with a
[
-32P]ATP (3,000 Ci/10
3 mol,
Amersham)-endlabeled oligonucleotide, 5'-GAGTTATCTACATCTGCAGCT-3', obtained from the internal sequence between the two PCR primers (19, 22). Prehybridization, hybridization, and washes were done according to standard protocols using Denhardt's solution and
sodium chloride/sodium citrate (39).
Measurement of superoxide anion generation in intact cells.
Measurement of superoxide anion generation in intact cells was
performed exactly as previously described using the lucigenin method
(19, 11). LLC-PK1 cells were stimulated with
107 mol/l ANG II for 4 h with or without
preincubation for 1 h with 10
8 mol/l ANP,
10
8 mol/l ANP 4---27, 10
8 mol/l ANP
3-28, or 10
3 mol/l 8-bromoguanosine 3',5'-cyclic
monophosphate (8-BrcGMP). To test the involvement of membrane NADPH
oxidase, additional cells were also treated in the presence of
10
5 mol/l diphenylene iodinium (DIP). Cells were slightly
trypsinized and collected by centrifugation. The pellet was then washed
in a modified Krebs buffer containing 1.3 × 10
1
mol/l NaCl, 5 × 10
3 mol/l KCl, 10
3
mol/l MgCl2, 1.5 × 10
3 mol/l
CaCl2, 10
3 mol/l
K2HPO4, and 2 × 10
3 mol/l
HEPES (pH 7.4) and centrifuged. After being washed, cells were
resuspended in Krebs buffer containing 10
2 mol/l glucose
and 1 mg/ml bovine serum albumin. Measurement of superoxide anion
generation was performed in a luminometer (LB 9501, Berthold, Wildbad,
Germany) and started by addition of 100 µl lucigenin (Sigma; final
concentration 5 × 10
4 mol/l). For measurement of
superoxide anion generation, 5 × 106
LLC-PK1 cells were used. Photon emission was counted every
15 s for up to 15 min. A buffer blank (reaction buffer with
lucigenin, but without cells) was subtracted from each reading. To
calculate the amount of superoxide anion synthesized by intact cells, a standard curve was generated as previously described (10),
and total photon emissions at 10 min were converted into
10
9 mol/l superoxide anion.
Western blots.
A total of 106 LLC-PK1 cells were rested in
serum-free DMEM 24 h. Cells were stimulated for up to 24 h
with a single dose of 107 mol/l ANG II in the presence of
absence of 10
7-10
5 mol/l ANP. Additional
cells were also incubated with the ANP fragments, 10
7
mol/l ANP 3---28 and 10
7 mol/l ANP 4---27, or
10
3 mol/l 8-BrcGMP. For the detection of activated MAP
kinase, LLC-PK1 cells were stimulated for 15 min or 12 h with ANG II in the presence of absence of ANP and its fragments
because we have previously discovered that ANG II induces a biphasic
phosphorylation of MAP kinases in tubular cells with a maximal effect
at these time points (11). Cells were rinsed twice in
ice-cold PBS at the end of the incubation period. After removal of all
PBS, monolayers were directly lysed in 150 µl of lysis buffer (2%
SDS, 6 × 10
2 mol/l Tris · HCl, pH 6.8), and
the protein content was measured in supernatants after centrifugation
by a modification of the Lowry method that is insensitive to the used
concentrations of SDS (11). Protein concentrations were
adjusted to 80 µg/sample, 10
2 mol/ DTT, 5% glycerol,
and 0.03% bromophenol blue were added, and samples were boiled for 5 min. After centrifugation, supernatants were loaded onto a 12%
SDS-polyacrylamide gel. Low-molecular-weight Rainbow markers
(Amersham; comprising 2,350- 45,000 Da) served as
molecular weight standards. After completion of electrophoresis, proteins were electroblotted onto a polyvinylidene difluoride membrane
(Highbond-P, Amersham) in transfer buffer (5 × 10
2
mol/l Tris · HCl, pH 7.0; 3.8 × 10
1 mol/l
glycine, 20% methanol). Filters were stained with Ponceau S to control
for equal loading and transfer. Membranes were blocked for 1 h at
room temperature with 5% nonfat dry milk redissolved in PBS with 0.1%
Tween 20. For the detection of p27Kip1 protein, a 1:1,000
dilution of a mouse monoclonal anti-p27Kip1 antibody
(Transduction Laboratories, Lexington, MA) was used (38).
This antibody reacts with murine p27Kip1. Phosphorylated
p44/42 MAP kinases were detected with a polyclonal rabbit
phospho-specific p44/42 MAP kinase
(Thr202/Tyr204) antibody (1:1,000 dilution; New
England Biolabs, Beverly, MA). MAP kinase phosphatase (MKP-1)
expression was detected with a rabbit polyclonal antibody using a 1:200
dilution (Santa Cruz Biotechnology, Santa Cruz, CA). The specificity of
the anti-p27Kip1 and phospho-specific p44/42 antibodies has
been previously established (11, 38). To test the
specificity of the anti-MKP-1 antibody, selected blots were incubated
in the presence of a blocking peptide against which the antibody was
generated after incubation for another hour, membranes were washed in
PBS with 0.1% Tween 20 for 3 × 10 min, and horseradish
peroxidase-conjugated appropriate secondary antibodies (all from
Transduction Laboratories) were added at a 1:2,000 dilution. The
luminescence detection of peroxidase activity was performed with the
enhanced chemiluminesence system (Amersham) according to the
manufacturer's recommendations. To control for small variations in
protein loading and transfer, selected membranes were washed and
reincubated with a mouse monoclonal anti-
-actin antibody (Sigma).
Incubation with secondary antibody and detection were performed as
described above. Exposed films were scanned with a Fluor-S multi-imager
(Bio-Rad Laboratories, Hercules, CA), and data were analyzed with the
computer program Multi-Analyst from Bio-Rad. Western blots were
independently performed at least three times with qualitatively similar results.
[3H]leucine and [3H]thymidine
incorporation experiments.
Incorporation of [3H]leucine was used to assess de novo
protein synthesis (36, 39). LLC-PK1 cells were
plated (105/well) in 24-well plates and made quiescent for
12 h in serum-free DMEM. After an additional 12 h, cells were
treated with 107 mol/l ANG II in the presence or absence
of ANP, fragments, or 8-BrcGMP for another 48 h. Five microcuries
of [3H]leucine (142 Ci/10
3 mol, Amersham)
were included per well for the final 12 h. At the end of the
incubation period, cells were washed twice in ice-cold PBS, and
proteins were subsequently precipitated with ice-cold 10%
trichloroacetic acid. After the precipitates were redissolved in 0.5 mol/l NaOH containing 0.1% Triton X-100, 5 ml scintillation cocktail
(Roth, Karlsruhe, Germany) were added, and vials were measured by
liquid scintillation spectroscopy. [3H]leucine
incorporation experiments were repeated five times with duplicate
measurements for each experiment.
Statistical analysis. All values are presented as means ± SE. Statistical significance among multiple groups was tested with a nonparametric Kruskal-Wallis test. Individual groups were then tested by using a Wilcoxon-Mann-Whitney test. A P value of <0.05 was considered significant.
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RESULTS |
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Although it has been previously demonstrated that LLC-PK1 cells principally exhibit type A ANP receptors (24, 29), we initially tested whether our cells may indeed express transcripts for this guanylate cyclase-coupled receptor. Amplification of cDNA, constructed from LLC-PK1 mRNA, revealed a single band of the predicted size of 283 base pairs (22; data not shown). Southern blotting of amplification products and hybridizing with an endlabeled internal primer revealed that the amplified band is indeed the type A receptor for ANP (data not shown).
We were interested in potential growth-modulatory effects of ANP and
its analogs on ANG II-mediated hypertrophy. LLC-PK1 cells were treated with a single dose of 107 mol/l ANG II,
previously shown to stimulate hypertrophy of these cells. De novo
protein synthesis was measured by incorporation of
[3H]leucine, a parameter that has been previously found
to closely reflect hypertrophic growth of tubular cells (38, 39,
40). As shown in Fig.
1A, 10
7 M ANG II
readily stimulated de novo protein synthesis. This ANG II-stimulated
[3H]leucine incorporation was totally blocked by
10
8 mol/l ANP. Furthermore, the active analogs ANP 4---27
and ANP 3---28 that bind to type A receptors and activate guanylate
cyclase (2, 35) also attenuated ANG II-induced protein
synthesis (Fig. 1A). 8-BrcGMP (10
3 mol/l)
mimicked the effects of ANP on ANG II-mediated
[3H]leucine incorporation. ANP and its fragments as well
as 8-BrcGMP had no effect on baseline de novo protein synthesis.
LLC-PK1 cells undergoing ANG II-induced hypertrophy
significantly reduced their proliferation, suggesting cell cycle arrest
as previously demonstrated (Fig. 1B). ANP, its bioactive
fragments, and 8-BrcGMP alone had no significant effect on
[3H]thymidine incorporation into LLC-PK1
cells (Fig. 1B). However, these agents abolished the ANG
II-induced reduction in [3H]thymidine incorporation and
facilitated cell cycle progression (Fig. 1B).
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To rule out that the abolished ANG II-mediated hypertrophy in the
presence of ANP peptides may be simply a result of reduced ANG II
binding sites, formal receptor binding studies were performed in
LLC-PK1 cells. Figure 2 shows
a Scatchard transformation of binding data using whole
LLC-PK1 cells. The dissociation coefficient (KD) for ANG II is 0.4 × 109
mol/l, a value close to findings previously reported for
LLC-PK1 cells (40). Preincubation of
cells with 10
7 mol/l ANP for 24 h led to a
small increase in the KD
(KD = 0.56 × 10
9 mol/l,
n = 4). There was also no major change in the total
number of specific ANG II binding sites in LLC-PK1 cells
incubated for 24 h in control medium or medium supplemented with
ANP (control: Bmax = 380 × 10
15
mol/mg protein; ANP for 24 h: Bmax = 328 × 10
15 mol/mg protein, n = 4, where
Bmax is maximal binding).
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Because we have previously demonstrated that the ANG II-mediated
hypertrophy of tubular cells depends on the expression of p27Kip1, with a subsequent arrest of cells in the
G1-phase of the cell cycle (38), we
consequently investigated whether ANP may modulate ANG II-induced
expression of this cyclin-dependent kinase inhibitor. Figure
3 reveals that
107-10
5 mol/l ANP completely abolished ANG
II-induced expression of p27Kip1 protein. A single dose of
10
3 mol/l 8-BrcGMP had a similar effect (Fig. 3). In
addition, 10
7 mol/l of ANP 4---27 and ANP 3---28 also
significantly reduced ANG II-mediated p27Kip1 protein
expression in tubular LLC-PK1 cells but had no significant influence on baseline expression (Fig.
4). In accordance with previous findings
(38), ANG II-induced p27Kip1 protein
expression was principally regulated at a posttranscriptional level.
p27Kip1 transcripts as detected by RT-PCR were not
influenced by either ANG II or ANP (data not shown).
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ANG II stimulates the production of reactive oxygen species in tubular
cells, including LLC-PK1 cells (10). ANG
II-mediated generation of superoxide anions is an essential step for
the subsequent induction of p27Kip1 and hypertrophy
(10). Therefore, we tested whether ANP may interfere with
ANG II-induced superoxide anion formation. However, as shown in Table
1, ANP, ANP 3---28, ANP 4---27, and
8-BrGMP not only failed to attenuate the ANG II-mediated production of
superoxide anions in intact LLC-PK1 cells but themselves
additionally induced significant amounts of reactive superoxide
species. In accordance with previous findings that a ANG II-mediated
increase in reactive oxygen species depends on activation of
membrane-bound NADPH oxidase (10), the flavoprotein
inhibitor DIP prevented this induction. In addition, DIP also
attenuated the ANP-mediated increase in oxygen radicals, suggesting
that ANP utilizes enzymes similar to those for ANG II in this process
(Table 1).
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This ANG II-induced superoxide anion production activates
phosphorylation of MAP kinases (11). We found that ANG
II-treated LLC-PK1 exhibited a biphasic phosphorylation of
p44/42 MAP kinase, with a first peak starting after a few minuets and a
second phosphorylation maximum occurring after 12 h
(11). To further study whether ANP may modify
phosphorylation of these MAP kinases, ANG II-mediated p44/42 MAP kinase
phosphorylation was evaluated after 10-min and 12-h stimulation. Figure
5A shows that preincubation of
cells with 107 mol/l ANP for 24 h did not reduce
phosphorylation of p44/42 MAP kinase after 10 min stimulation with ANG
II. ANP had no influecne on total expression of p44/42 MAP kinase (Fig.
5A). However, 10
7-10
5 mol/l ANP
as well as 10
3 mol/l 8-BrcGMP partly inhibited the second
phosphorylation peak of p44/42 MAP kinase induced after 12 h of
treatment with ANG II without reducing total protein expression (Fig.
5B). It has previously been discovered that ANP activates
MKP-1, an enzyme mediating dephosphorylation of MAP kinase, in
glomerular mesangial cells (30). Figure
6A demonstrates that
incubation of LLC-PK1 cells with 10
7
mol/l ANP leads to a prolonged increase in MKP-1 protein expression starting after 10-30 min and declining after 12 h of
stimulation. The specificity of the anti-MKP-1 antibody was confirmed
by coincubation with the peptide against which this antibody was
generated (data not shown). In addition, the ANP analogs as well as
8-BrcGMP also stimulated MKP-1 protein expression (data not shown).
However, ANG II failed to significantly influence MKP-1 expression
(Fig. 6B). Furthermore, coincubation with ANP and ANG II did
not reduce the ANP-mediated increase in MKP-1 expression (data not
shown).
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To further investigate a functional role of ANP-stimulated MKP-1
expression, LLC-PK1 cells were transiently transfected with either MKP-1 sense or antisense phosphorothioate-modified
oligonucleotides. As shown in Fig. 7,
transfection with MKP-1 antisense, but not sense, oligonucleotides
reduced baseline as well as ANP-induced MKP-1 expression. Transfection
with MKP-1 antisense, but not sense, oligonucleotides attenuated the
inhibitory effect of ANP on ANG II-mediated hypertrophy
([3H]leucine incorporation) and also reversed the
influence of ANP on cell cycle progression ([3H]thymidine
incorporation; Table 2).
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DISCUSSION |
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It has been previously demonstrated that ANP inhibits mitogenic effects of growth factors in various cells (1, 18, 39, 42). For example, our group found that ANP attenuated proliferation of cultured mesangial cells induced by ANG II (38, 40). However, a potential effect of ANP on the growth of tubular cells is largely unknown. We were particularly interested in potential effects of ANP on ANG II-mediated tubular hypertrophy because of the known opposite actions of ANP and ANG II on tubular transport (12). In contrast to mesangial cells, ANG II induces tubular hypertrophy, an effect caused by arrest in the G1 phase of the cell cycle (38). We have previously demonstrated in a series of studies that ANG II stimulates superoxide anion production through activation of membrane-bound NADPH oxidase (10). This increase in reactive oxygen species led to the phosphorylation of p44/42 MAP kinase that, in turn, increases p27Kip1 expression, probably by direct phosphorylation of serine-threonine residues, enhancing the half-life (11). Finally, p27Kip1 inhibits G1 phase cyclin-CDK complexes and induces cell cycle arrest (11). Renal growth-promoting effects of ANG II, independent of concomitant hypertension, have also been found principally in vivo (23).
Our present study shows that ANP, its bioactive fragments that activate guanylate cyclase (2, 35), as well as 8-BrcGMP attenuate ANG II-induced hypertrophy and stimulate cell cycle progression of LLC-PK1 cells. This abolished G1 phase arrest is associated with an attenuated p27Kip1 expression in the presence of ANG II. However, these effects were not caused by a simple downregulation of ANG II receptors on the surface of tubular cells. In agreement with this finding, 8-BrcGMP failed to influence ANG II receptor expression in other cell types (15). Furthermore, ANP does not interfere with ANG II-mediated superoxide anion production. On the contrary, ANP, analogs, and 8-BrcGMP themselves stimulated reactive oxygen species by apparently using an enzyme machinery similar to that of ANG II, suggesting that the proximal parts of the signal transduction cascade leading finally to p27Kip1 expression are not influenced by ANP. Our data suggest that a decrease in ANG II-mediated p44/42 MAP kinase phosphorylation in the presence of ANP is responsible for the failure to induce p27Kip1. ANP and 8-BrcGMP induced protein expression of MKP-1, a dual tyrosine/threonine phosphatase with specificity and selectivity for p44/42 MAP kinase (31). The constitutive expression of MKP-1 was found to attenuate inducible p44/42 MAP kinase activation and blocked p44/42 MAP kinase-dependent gene expression as well as growth effects (31, 32). The central functional role of ANP-mediated MKP-1 expression is clearly shown by our MKP-1 antisense experiments that abolished the effects of ANP. An ANP-mediated induction of MKP-1 has been also described in mesangial cells (30).
Figure 8 summarizes the potential signal
transduction pathways. Both ANG II and ANP induce reactive oxygen
radicals that lead to phosphorylation and activation of p44/42 MAP
kinase. However, ANP additionally induces MKP-1 that keeps the MAP
kinase in an underphosphorylated state. Whether ANG II may also
directly further enhance MAP kinase activity in LLC-PK1
cells independently of reactive oxygen radicals is presently unclear.
The consequence of ANP treatment is an attenuation in
p27Kip1 protein expression, cell cycle arrest, and tubular
hypertrophy.
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Because the effects of ANP were mimicked by 8-BrcGMP, a cell-permeable analog of cGMP, it is very likely that the ANP-mediated inhibition of ANG II-induced hypertrophy requires binding to type A receptors that are coupled to guanylate cyclase. Previous studies provided clear evidence that LLC-PK1 cells express only the A-type receptor and not the C-type clearance receptor (24, 29). This may be partly explained by a subtype switching of ANP receptor expression on tubular cells under culture conditions with a loss of the C-type receptor (24). However, in specific neuronal cell systems, ANP inhibits MAP kinase through the C-type receptor via inhibition of upstream kinases such as MAP kinase kinase (MEK; 25). This mechanism in highly unlikely in our system because LLC-PK1 cells do not express the C-type receptor, and 8-BrcGMP exerted principally the same effect as ANP. Alternatively, in accordance with previous findings (30), we suggest that the ANP-mediated inhibition of ANG II-induced p44/42 MAP kinase phosphorylation is caused by induction of the phosphatase MKP-1. In fact, it has been clearly demonstrated in mesangial cells that ANP-mediated MKP-1 induction occurs through the A-type, and not the C-type, receptors and is also mimicked by 8-BrcGMP (30).
It has been previously described that ANP inhibits ANG II-mediated hypertrophy of vascular smooth muscle cells (16). The detailed molecular mechanism of the antihypertrophic effect of ANP on vascular smooth muscle cells is unknown but is apparently independent of protein kinase C (16). Interestingly, although ANP prevented the development of ANG II-induced hypertrophy in this system, it failed to prevent the induction of immediate early genes including c-fos and c-jun (17). Because these immediate early genes are expressed at the G0-to-G1 transit (27), these observation are in good agreement with our data that ANP prevents further progression through G1 but not reentry into the cell cycle.
However, other studies suggest that cAMP and cGMP primarily suppress vascular smooth muscle cell proliferation (6). In these studies, cAMP induces p27Kip1 expression whereas cGMP did not influence total cellular p27Kip1 levels (6). Thus the effects of 8-cGMP on p27Kip1 expression may vary with the cell type studied, indicating fundamental differences between epithelial and mesenchymal cells. In contrast to findings obtained from studies in mesenchymal cells, ANP alone did not influence baseline proliferation as measured with [3H]thymidine incorporation in epithelial LLC-PK1 cells. However, in the presence of ANG II, ANP favors cell cycle exit and progression into the S phase. These findings suggest that ANP has no effect on dormant cells resting in the G0 phase. However, cells reenter the cycle after ANG II exposure but do not progress into the S phase because of ANG II-induced p27Kip1 expression. By attenuating this ANG II-mediated p27Kip1 expression, ANP facilitates S phase progression.
ANP also suppresses compensatory renal hypertrophy induced in rats by nephrectomy (21). Although the target cell type of this antihypertrophic action of ANP has been not evaluated in this study, the findings nevertheless suggest that ANP inhibits renal hypertrophy in vivo. These results are in excellent agreement with our present observations because tubular cells constitute the bulk of renal mass.
Angiotensin-converting enzyme (ACE) inhibitors are presently the standard therapy to delay progression of chronic renal disease (26, 36). There is accumulating evidence that, at least to some extent, these effects are independent of antihypertensive effects and may involve inhibition of tubulointerstitial remodeling such as hypertrophy (36). Combined inhibitors of neutral endopeptidase and ACE that are presently undergoing clinical studies may produce a greater effect than those elicited by selective inhibition of either enzyme alone (33). This synergism likely stems from unmasking and potentiating vasorelaxant peptides such as ANP and bradykinin. In consideration of our findings that ANP prevents ANG II-mediated tubular hypertrophy, such dual peptidase inhibitors may better prevent tubulointerstitial remodeling than do ACE inhibitors alone. However, this intriguing hypothesis needs confirmation by clinical studies.
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ACKNOWLEDGEMENTS |
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This work was supported by the Deutsche Forschungsgemeinschaft (Wo 460/2-3, 2-4) and a Heisenberg Scholarship (to G. Wolfe).
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Wolf, Univ. of Hamburg, Univ. Hospital Eppendorf, Dept. of Medicine, Div. of Nephrology and Osteology, Pavilion 61, Martinstrasse 52, D-20246 Hamburg, Germany.
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.
Received 22 June 2000; accepted in final form 26 February 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Appel, RG.
Growth inhibitory activity of atrial natriuretic factor in rat glomerular mesangial cells.
FEBS Lett
238:
135-138,
1988[ISI][Medline].
2.
Atlas, SA,
Kleinert HD,
Camargo MJ,
Januszewics A,
Sealey JE,
Laragh JH,
Schilling JW,
Lewicki JA,
Johnson LK,
and
Maack T.
Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial peptide.
Nature
309:
717-719,
1984[ISI][Medline].
3.
Canaan-Kühl, S,
Ostendorf T,
Zander K,
Koch KM,
and
Floege J.
C-type natriuretic peptide inhibits mesangial cell proliferation and matrix accumulation in vivo.
Kidney Int
53:
1143-1151,
1998[ISI][Medline].
4.
Cao, L,
and
Gardner DG.
Natriuretic peptides inhibit DNA synthesis in cardiac fibroblasts.
Hypertension
25:
227-234,
1995
5.
Charles, CH,
Abler AS,
and
Lau LF.
cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein.
Oncogene
7:
187-190,
1992[ISI][Medline].
6.
Fukumoto, S,
Koyama H,
Hosoi M,
Yamakawa K,
Tanaka S,
Morii H,
and
Nishizawa Y.
Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells.
Cir Res
85:
985-991,
1999
7.
Garvin, JL.
ANF inhibits norepinephrine-stimulated fluid absorption in rat proximal straight tubules.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F581-F585,
1992
8.
Gomes, GN,
and
Aires MM.
Interaction of atrial natriuretic factor and angiotensin II in proximal HCO
9.
Gracia, NH,
and
Garvin JL.
ANF and angiotensin II interact via kinases in the proximal straight tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F730-F735,
1995
10.
Hannken, T,
Schroeder R,
Stahl RAK,
and
Wolf G.
Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals.
Kidney Int
54:
1923-1933,
1998[ISI][Medline].
11.
Hannken, T,
Schroeder R,
Zahner G,
Stahl RAK,
and
Wolf G.
Reactive oxygen species stimulate p44/42 MAP kinase and induce p27Kip1: role in angiotensin II-mediated hypertrophy of proximal tubular cells.
J Am Soc Nephrol
11:
1387-1397,
2000
12.
Harris, PJ,
and
Skinner SL.
Intra-renal interactions between angiotensin II and atrial natriuretic factor.
Kidney Int
38, Suppl30:
S87-S91,
1990[ISI].
13.
Harris, PJ,
Thomas D,
and
Morgan TO.
Atrial natriuretic peptide inhibits angiotensin-stimulated proximal tubular sodium and water reabsorption.
Nature
326:
697-698,
1987[ISI][Medline].
14.
Harris, RC.
Regulation of S6 kinase activity in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F127-F134,
1992
15.
Ichiki, T,
Usui M,
Kato M,
Funakoshi Y,
Ito K,
Egashira K,
and
Takeshita A.
Downregulation of angiotensin II type 1 receptor gene transcription by nitric oxide.
Hypertension
31:
342-348,
1998
16.
Itoh, H,
Pratt RE,
and
Dzau VH.
Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells.
J Clin Invest
86:
1690-1697,
1990[ISI][Medline].
17.
Itoh, H,
Pratt RE,
and
Dzau VJ.
Interaction of atrial natriuretic polypeptide and angiotensin II on protooncogene expression and vascular cell growth.
Biochem Biophys Res Commun
176:
1601-1609,
1991[ISI][Medline].
18.
Johnson, A,
Lermioglu F,
Garg UC,
Morgan-Boyd R,
and
Hassid A.
A novel biological effects of atrial natriuretic hormone: inhibition of mesangial cell mitogenesis.
Biochem Biophys Res Commun
152:
893-897,
1988[ISI][Medline].
19.
Katfuchi, T,
Mizuno T,
Hagiwara H,
Itakura M,
Ito T,
and
Hirose S.
Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.
J Biol Chem
267:
7624-7629,
1992
20.
Levin, ER,
Gardner DG,
and
Samson WK.
Natriuretic peptides.
N Engl J Med
339:
321-328,
1998
21.
Logan, JL,
and
Michael UF.
Atrial natriuretic peptide suppresses compensatory renal growth in rats.
J Am Soc Nephrol
4:
2016-2022,
1994[Abstract].
22.
Lowe, DG,
Chang MS,
Hellmi SR,
Chen E,
Singh S,
Garbers DL,
and
Goeddel DV.
Human atrial natriuretic receptor defines a new paradigm for second messenger signal transduction.
EMBO J
8:
1377-1384,
1989[Abstract].
23.
Mervaala, E,
Müller DN,
Schmidt F,
Park JK,
Gross V,
Bader M,
Breu V,
Ganten D,
Haller H,
and
Luft FC.
Blood pressure-independent effects in rats with human renin and angiotensinogen genes.
Hypertension
35:
587-594,
2000
24.
Mistry, SK,
Chatterjee PK,
Weerackody RP,
Hawksworth GM,
Knott RM,
and
McLay JS.
Evidence for atrial natriuretic factor induced natriuretic peptide receptor subtype switching in rat proximal tubular cells during culture.
Exp Nephrol
6:
104-111,
1998[ISI][Medline].
25.
Prins, BA,
Weber MJ,
Hu RM,
Pedram A,
Daniels M,
and
Levin ER.
Atrial natriuretic peptide inhibits mitogen-activated protein kinase through the clearance receptor.
J Biol Chem
271:
14156-14162,
1996
26.
Ruggenenti, P,
and
Remuzzi G.
Angiotensin-converting enzyme inhibitor therapy for non-diabetic progressive renal disease.
Curr Opin Nephrol Hypertens
6:
489-495,
1997[ISI][Medline].
27.
Shankland, SJ,
and
Wolf G.
Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation and apoptosis.
Am J Physiol Renal Physiol
278:
F515-F529,
2000
28.
Sterzel, RB,
Luft FC,
Lang RE,
and
Ganten D.
Effects of atrial natriuretic factor in rats with renal insufficiency.
J Lab Clin Med
110:
63-69,
1987[ISI][Medline].
29.
Suga, SI,
Nakao K,
Mukoyama M,
Arai H,
Hossoda K,
Ogawa Y,
and
Imura H.
Characterization of natriuretic peptide receptors in cultured cells.
Hypertension
19:
762-765,
1992[Abstract].
30.
Sugimoto, T,
Haneda M,
Togawa M,
Isono M,
Shikano T,
Araki SI,
Nakagawa T,
Kashiwagi A,
Guan KL,
and
Kikkawa R.
Atrial natriuretic peptide induces the expression of MKP-1, a mitogen-activated protein kinase phosphatase, in glomerular mesangial cells.
J Biol Chem
271:
544-547,
1996
31.
Sun, H,
Charles CH,
Lau LF,
and
Tonks NK.
MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo.
Cell
75:
487-483,
1993[ISI][Medline].
32.
Sun, H,
Tonks NK,
and
Bar-Sagi D.
Inhibition of ras-induced DNA synthesis by expression of the phosphatase MKP-1.
Science
266:
285-288,
1994[ISI][Medline].
33.
Trippodo, NC,
Robl JA,
Asaad MM,
Fox M,
Panchal BC,
and
Schaeffer TR.
Effects of omapatrilat in low, normal, and high renin experimental hypertension.
Am J Hypertens
11:
363-372,
1998[ISI][Medline].
34.
Weerackody, RP,
Chatterjee PK,
Mistry SK,
McLaren J,
Hawkswoorth GM,
and
McLay JS.
Selective antagonism of the AT1 receptor inhibits the effect of angiotensin II on DNA and protein synthesis of rat proximal tubular cells.
Exp Nephrol
5:
253-262,
1997[ISI][Medline].
35.
Winquist, RJ,
Faison EP,
and
Nutt RF.
Vasodilator profile of synthetic atrial natriuretic factor.
Eur J Pharmacol
102:
169-173,
1984[ISI][Medline].
36.
Wolf, G.
Molecular mechanisms of angiotensin II in the kidney: emerging role in the progression of renal disease beyond haemodynamcis.
Nephrol Dial Transplant
13:
1131-1142,
1998[Abstract].
37.
Wolf, G.
Vasoactive factors and tubulointerstitial injury.
Kidney Blood Press Res
22:
62-70,
1999[ISI][Medline].
38.
Wolf, G,
and
Stahl RAK
Angiotensin II-stimulated hypertrophy of LLC-PK1 cells depends on the induction of the cyclin-dependent kinase inhibitor p27Kip1.
Kidney Int
50:
2112-2119,
1996[ISI][Medline].
39.
Wolf, G,
Thaiss F,
Schoeppe W,
and
Stahl RAK
Angiotensin II-induced proliferation of cultured murine mesangial cells: inhibitory role of atrial natriuretic peptide.
J Am Soc Nephrol
3:
1270-1278,
1992[Abstract].
40.
Wolf, G,
Zahner G,
Mondorf U,
Schoeppe W,
and
Stahl RAK
Angiotensin II stimulates cellular hypertrophy of LLC-PK1 cells through the AT1 receptor.
Nephrol Dial Transplant
8:
128-133,
1993[Abstract].
41.
Wolf, G,
and
Ziyadeh FN.
Renal tubular hypertrophy induced by angiotensin II.
Sem Nephrol
17:
448-454,
1997[ISI][Medline].
42.
Wolf, G,
Ziyadeh FN,
and
Stahl RAK
Atrial natriuretic peptide stimulates the expression of transforming growth factor- in cultured murine mesangial cells. relationship to suppression of proliferation.
J Am Soc Nephrol
6:
225-233,
1995.