Atrial natriuretic peptide attenuates ANG II-induced hypertrophy of renal tubular cells

Tete Hannken, Regine Schroeder, Rolf A. K. Stahl, and Gunter Wolf

Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, D-20246 Hamburg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

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.


    METHODS
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INTRODUCTION
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 × 10-3 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 10-7 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 epsilon -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 × 10-2 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 [gamma -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).

To asses mRNA expression of p27Kip1, RNA was isolated from LLC-PK1 cells stimulated for 20 h with 10-7 mol/l ANG II in the presence or absence of 10-8 mol/l ANP or 10-8 mol/l ANP 4---27. cDNAs were prepared as described above. For cDNA amplification, the following primers were used (38): 5'-GTCTAACGGGAGCCCGAGCCTGG-3' and 5'-GAAGGCCGGGCTTCTTGGGCG-3' (p27Kip1) and 5'- GGCCAAGTCATCACTATTGG-3' and 5'-GGACTCATCGTACTCCTGC-3' (beta -actin). A total of 150 ng of primers was used. The complete amplification mix without the primers was equivalently distributed to separate tubes containing either p27Kip1 or beta -actin primers. Reactions were performed by using the GeneAmp kit (PerkinElmer Cetus, Überlingen, Germany). Reactions were performed for 30 cycles with an annealing temperature of 60°C for 1.5 min, an extension step at 72°C for 1.5 min, and a denaturation step at 92°C. Ten microliters of the reaction product were run out on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. Bands of the predicted size [560 bp (p27Kip1), 360 bp (beta -actin)] were photographed with Polaroid 55 negative film, and the reaction products on the film were scanned by laser densitometry. We have previously confirmed the identities of the amplified bands (36) by subcloning and sequencing. RT-PCR with separate stimulation of cells was independently performed three times.

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 10-7 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 10-7 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-beta -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 10-7 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.

The incorporation of [3H]thymidine into DNA was used as a measurement of proliferation (38, 42). Cells (104 cells/well) were transferred to a 96-well microtiter plate. After incubation for 12 h in serum-free DMEM, LLC-PK1 cells were subsequently incubated for another 48 h as described above. They were pulsed with 1 µCi [3H]thymidine (5 Ci/10-3 mol, Amersham) during the final 6 h of culture. At the end of the incubation period, cells were washed in PBS, trypsinized for 10 min a 37°C, and finally collected on glass-fiber paper with an automatic cell harvester. Radioactivity of dry filters was measured by liquid scintillation spectroscopy. [3H]thymidine experiments were independently performed four times with triplicate measurements.

To test the functional role of MKP-1 in [3H]leucine and [3H]thymidine incorporation, cells were transiently transfected with 10-6 mol/l of mouse MKP-1 sense (5'-GGCCATGGTGATGGAGGTGGG-3') or antisense (5'-CCCACCTCCATCACCATGGCC-3') phosphorothioate-modified oligonucleotides. The sequences were obtained from the published mouse sequence (5). For these experiments, cells were rested for 6 h in serum-free medium and then transfected by using lipofectin reagent (GIBCO) as recommended by the manufacturer. After another 12 h, cells were transferred to 24- or 96-well plates and stimulated as described above. In addition, Western blots were performed for MKP-1 expression to gain insight into whether protein expression was indeed downregulated by antisense oligonucleotide treatment.

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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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 10-7 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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Fig. 1.   A: de novo protein synthesis as a parameter of cell hypertrophy measured by [3H]leucine incorporation into LLC-PK1 cells. A single dose of 10-7 mol/l ANG II significantly stimulated protein synthesis. The increase in [3H]leucine incorporation was inhibited by atrial natriuretic peptide (ANP), ANP 3---28, ANP 4---27, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). However, ANP, its bioactive fragments, and 8-BrcGMP had no influence on baseline protein synthesis. B: [3H]thymidine incorporation as a parameter of proliferation. In contrast to de novo protein synthesis, ANG II inhibits proliferation, indicating cell cycle arrest. ANP, ANP 3---28, ANP 4---27, and 8-BrcGMP abolished this inhibitory action of ANG II and facilitated cell cycle progression but did not influence baseline [3H]thymidine incorporation (n = 10-12). CPM, counts/min.*P < 0.01 vs. control. #P < 0.05 vs. ANG II only.

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 × 10-9 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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Fig. 2.   Scatchard transformation of binding data of 125I-[Sar1,Ile8]ANG II to intact LLC-PK1 cells. Nonspecific binding was determined in the presence of 10-3 mol/l ANG II and was subtracted from all data points. Cells were either left in serum-free DMEM (control) or incubated for 24 h in 10-7 mol/l ANP. Incubation of LLC-PK1 cells in ANP did not lead to a downregulation of specific ANG II receptors [dissociation coefficient (KD) for control = 0.4, ANP for 24 h = 0.56 × 10-9 mol/l; maximal binding (Bmax) for control = 380, ANP for 24 h = 328 × 10-15 mol/mg protein]. Data are mean values of 4 independent binding experiments.

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 10-7-10-5 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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Fig. 3.   Top: Western blot of cell lysates from LLC-PK1 cells incubated with an antibody against p27Kip1 (p27Kip1). The blot was reincubated with an antibody against beta -actin. In accordance with previous findings, a single dose of 10-7 mol/l ANG II for 24 h induced a robust increase in p27Kip1 protein expression. This induction was significantly attenuated by 10-7-10-5 mol/l ANP or 10-3 mol/l 8-BrcGMP. Bottom: densitometry analysis of 7 independent blots. *P < 0.01 vs. control. +P < 0.05 vs. ANG II only.



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Fig. 4.   Top: Western blot of cell lysates from LLC-PK1 cells incubated with an antibody against p27Kip1 (p27Kip1); the blot was reincubated with an antibody against beta -actin. The ANG II-mediated induction of p27Kip1 was also significantly reduced by the bioactive ANP fragments ANP 3-28 and 4-27. Bottom: densitometry analysis of 7 independent blots. *P < 0.01 vs. control. +P < 0.05 vs. ANG II only.

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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Table 1.   Superoxide production in intact LLC-PK1 cells

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 10-7 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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Fig. 5.   Effect of ANP and analogs on ANG II-induced phosphorylation of p44/42 mitogen-activated protein (MAP) kinase in LLC-PK1 cells. Western blot of cell lysates probed with a phospho-specific anti-p44/42 MAP kinase antibody or an antibody against total p44/42 MAP kinase as a control for equal loading and protein transfer is shown. Cells were preincubated with ANP, its analogs, or 8-BrcGMP for 24 h. A: ANP did not reduce the first phosphorylation peak induced by ANG II after 10 min. Densitometry analysis of 4 independent blots is shown. *P < 0.05 vs. control. B: ANP as well as 8-BrcGMP abolished the 2nd phosphorylation peak induced after 12-h ANG II treatment. Densitometry analysis of 4 independent blots is shown. *P < 0.05 vs. control. +P < 0.05 vs. ANG II only.



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Fig. 6.   Western blot of cell lysates incubated with a specific antibody against a MAP kinase phosphatase (MKP-1). A: single doses of 10-7 mol/l ANP induced a strong increase in MKP-1 protein expression up to 12 h (n = 5). *P < 0.01 vs. control. B: ANG II (10-7 mol/l) failed to significantly modulate MKP-1 expression (n = 4).

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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Fig. 7.   Western blot for MKP-1 expression in untransfected LLC-PK1 cells, and cells transiently transfected with either MKP-1 antisense or sense phosphothioate-modified oligonucleotides. Cells were challenged for 1 h with 10-7 mol/ANP. Transfection with MKP-1 antisense, but not sense, oligonucleotides reduced baseline as well as ANP-induced MKP-1 expression, suggesting that the antisense oligonucleotides effectively inhibited MKP-1 expression. Blot is representative of 2 independent experiments with qualitatively similar changes.


                              
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Table 2.   Effect of MKP-1 antisense and sense oligonucleotides on [3H]leucine and [3H]thymidine incorporation


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 8.   Potential signal transduction pathways leading to the inhibitory effect of ANP on ANG II-mediated hypertrophy of tubular cells. ANG II and ANP both induce reactive oxygen radicals, likely by utilizing diphenylene iodinium (DIP)-inhibitable membrane-bound NAD(P)H-oxidase (10, 11). The increase in reactive oxygen species in turn leads to phosphorylation and activation of p44/42 MAP kinase. Whether ANG II may additionally activate MAP kinase in LLC-PK1 cells independently of reactive oxygen radicals is not known. However, ANP additionally induces MKP-1 that keeps this MAP kinase in an underphosphorylated state. The consequence is an attenuation of p27Kip1 protein expression, cell cycle arrest, and eventually tubular hypertrophy.

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.


    ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (Wo 460/2-3, 2-4) and a Heisenberg Scholarship (to G. Wolfe).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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