Angiotensin II receptor subtypes determine induced NO production in rat glomerular mesangial cells

Jörg Schwöbel1, Tina Fischer1, Bettina Lanz2, and Markus Mohaupt2

2 Division of Nephrology/Hypertension, University of Berne, 3010 Berne, Switzerland; and 1 Medizinische Klinik IV, University of Erlangen-Nuremberg, Erlangen 8520, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II (ANG II) and nitric oxide (NO) have contrasting vascular effects, yet both sustain inflammatory responses. We investigated the impact of ANG II on lipopolysaccharide (LPS)/interferon-gamma (IFN)-induced NO production in cultured rat mesangial cells (MCs). LPS/IFN-induced nitrite production, the inducible form of nitric oxide synthase (NOS-2) mRNA, and protein expression were dose dependently inhibited by ANG II on coincubation, which was abolished on ANG II type 2 (AT2) receptor blockade by PD-123319. Homology-based RT-PCR verified the presence of AT1A, AT1B, and AT2 receptors. To shift the AT receptor expression toward the type 1 receptor, two sets of experiments were performed: LPS/IFN preincubation for 24 h was followed by 8-h coincubation with ANG II; or during 24-h coincubation of LPS/IFN and ANG II, dexamethasone was added for the last 6-h period. Both led to an amplified overall expression of NOS-2 protein and NO production that was inhibitable by actinomycin D in the first setup. Induced NO production was enhanced via the AT1 receptor; however, it was diminished via the AT2 receptor. In conclusion, induced NO production is negatively controlled by the AT2, whereas AT1 receptor stimulation enhanced NO synthesis in MCs. The overall NO availability depended on the onset of the inflammatory stimuli with respect to ANG II exposure and the available AT receptors.

inducible nitric oxide synthase; glomerular inflammation; angiotensin II type 1 and type 2 receptor; dexamethasone


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IMMUNE COMPLEX FORMATION within glomeruli initiates chemical and cellular responses mediating inflammatory signals, such as NO.

NO is a radical gas and signaling molecule. Its production is catalyzed by NO synthase (NOS) isoforms converting L-arginine and molecular oxygen to L-citrulline and NO. If adequately stimulated by endotoxin or cytokine, mesangial cells (MCs) express the inducible NOS isoform (NOS-2) (35). Nanomolar concentrations of NO generated by NOS-2 in MCs are sufficient to sustain inflammatory responses, influence glomerular perfusion (3, 45), or even evoke substantial cell damage leading to apoptosis or cytotoxic cell death (42, 43).

Increased NO production is part of the inflammatory glomerular response of various glomerulonephritis models (4, 26, 37) and has been localized to MCs (5). This is further supported by an amelioration of experimental glomerulonephritis observed with inhibition of NO synthesis (49).

ANG II leads to renal vasoconstriction, regulating glomerular perfusion as well as filtration (25), and directly influences the mesangial constrictive response (44). ANG II is a potent regulator of MC phenotypical changes, such as migration (28), proliferation (19), or protein kinase C (PKC) expression (1). This supports its role as a growth factor, profibrogenic and proinflammatory mediator (50), and regulator of additional proinflammatory mediators, such as transforming growth factor-beta (TGF-beta ) (54) or monocyte-chemoattractant protein-1 (51). The effects of ANG II are transmitted via at least three different receptor subtypes in rats, the AT1A, the AT1B, and the AT2 receptors, with very diverse downstream signaling mechanisms and temporospatial distributions (reviewed in Ref. 23).

Animal models of experimental glomerulonephritis exploiting ANG I-converting enzyme inhibitors and ANG II receptor blockers support the concept of ANG II being an important proinflammatory mediator via the AT1 receptor (36, 41, 54).

MCs present an important regulatory element for vascular conductance as well as the ultrafiltration surface area within the glomerulus. They are critically controlled by vasodilators as well as vasoconstrictors. During periods of glomerular inflammation, ANG II- and NO-related phenotypical changes of MCs could determine glomerular function as well as inflammatory responses. ANG II and NO have been postulated to interact closely, at least in the regulation of renal hemodynamics (14, 32).

Conflicting results have been published regarding the influence of ANG II on induced NO production in conditions of sepsis or in glomerulonephritis. Some authors report a downregulation or inhibition of the induced NO production in cultured rat astroglial cells by certain proinflammatory stimuli (29) and similarly in renal cells such as mouse proximal tubular cells (52) and rat MCs (27, 40). Others were able to demonstrate an augmented lipopolysaccharide (LPS)- or cytokine-induced NO production in rat cardiac myocytes (21, 53).

The aim of the present study was to examine the influence of ANG II on LPS/interferon-gamma (IFN)-induced NO production in cultured rat MCs. We were able to demonstrate that the resulting NO response to ANG II depended on the activation of discrete ANG II receptor subtypes. The AT1 receptor subtypes transferred stimulation of NO production, whereas the AT2 receptor subtype inhibited induced NO production. The overall NO availability depended on the onset of the inflammatory stimuli with respect to ANG II exposure and the available ANG II receptors. Thus vascular effects of ANG II could be counteracted by a modulated NO production; however, proinflammatory properties of ANG II mediated via AT1 receptor activation could be potentiated via an enhanced NO production.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. DMEM, L-glutamate, insulin, penicillin, streptomycin, LPS (Escherichia coli serotype 026:B6), IFN, NG-monomethyl-L-arginine (L-NMMA), actinomycin D, and ANG II were all purchased from Sigma (Deisenhofen, Germany). DUP-753 and PD-123319 were from Biotrend (Köln, Germany). FCS was from Boehringer (Mannheim, Germany), and tissue culture plastic was from Falcon (Becton-Dickinson, Heidelberg, Germany).

Cell culture and isolation of MCs. For preparation and culture of glomerular MCs from male Sprague-Dawley rats, standard techniques were used as described previously (34, 35). Briefly, the kidneys were excised, and the cortex was separated from the medulla and homogenized using razor blades. Glomeruli were isolated from the homogenate by sequential sieving and collected on a 75-µm sieve. After treatment with 500 U/ml collagenase (type IV, Sigma) in PBS for 30 min, the glomerular remnants consisting predominantly of endothelial cells and MCs were seeded in tissue flasks (30,000 glomeruli/flask) containing DMEM supplemented with 20% FCS, 5 µg/ml bovine insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. After three to four passages using 0.05% trypsin-0.02% EDTA, stable and homogenous cultures of MCs were obtained. The outgrowing cells were characterized as MCs by positive immunocytochemical staining for Thy-1.1 (Serotec; Blackthorn, Bicester, UK), smooth muscle cell actin, and myosin showing typical MC morphology. MCs were further cultured in DMEM supplemented with 10% heat-inactivated FCS (56°C for 1 h), 2 mM glutamate, 5 ng/ml insulin, 100 U/ml penicillin, and 1 mg/ml streptomycin. Cells were used for experiments at subconfluence or passaged at confluence with trypsin/EDTA (0.05%-0.02% wt/vol). To obtain quiescent cells, MCs were maintained in medium containing 0.5% FCS for 3 days before cytokine treatment. MCs were used between passages 15 and 25. To stimulate NOS-2 induction, we used LPS (E. coli serotype 026:B6, 10 µg/ml medium) and IFN (100 U/ml medium). Subconfluent quiescent MCs were coincubated according to the experimental conditions with ANG II (10-6-10-8 M), DUP-753 (10-5-10-7 M), PD-123319 (10-5-10-7 M), dexamethasone (10-6 M), and/or TGF-beta (5 ng/ml; Calbiochem, Germany).

Measurement of NO production by detection of nitrite in supernatants of cultured MCs. MCs were grown to subconfluence either in 24-well plates or, to accommodate protein or RNA extraction, on 60- and 100-mm plastic culture plates. The cells were conditioned in standard culture medium between 24 h and 32 h, according to the experimental protocol. The nitrite content was measured using the Griess colorimetric method (35). Briefly, 200 µl of the supernatant as well as control medium supplemented with known NaNO2 concentrations serving as standard were mixed with 75 µl Griess reagent including HCl [50 µl of Griess reagent (25 mM sulfanilamide, and 25 mM naphthylethylenediamine), and 25 µl 6 M HCl], and incubated for 30 min in the dark. Optical density was measured at 550 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the nitrite content of the conditioned medium was calculated according to the NaNO2 standard curve obtained.

Preparation of radioactive cDNA probes (rat NOS-2, rat GAPDH). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was gained using a homology-based RT-PCR (30 cycles, annealing at 50°C) of rat MC cDNA using the following primers (synthesized by Life Technologies): sense, 5'-AATGCATCCTGCACCACCAA-3' (position 469-488 of M17701); and antisense, 5'-GTCATTGAGAGCAATGCCAGC-3' (position 919-939 of M17701).

We then used a 1,639-bp rat NOS-2 fragment which we obtained by homology-based RT-PCR (40 cycles, annealing at 60°C) of LPS/IFN-induced (24 h) cultured rat MCs, using the following primers (synthesized by Life Technologies): sense, 5'-CTGAATTCTGCATGGACCAGTATAAGGCAAGC-3' (position 1877-1900 of M84373); and antisense, 5'-CTGGATCCACCTGCTCCTCGCTCAA-3' (position 3479-3495 of M84373) (added restrictions sites are marked with underscore).

32P labeling was performed for all probes using the random prime labeling kit (Boehringer).

Northern blot hybridization analysis. Total RNA from rat MCs grown to subconfluence in 100-mm culture dishes was obtained as described earlier (35). Twenty micrograms of total RNA were electrophoretically size-fractionated under denaturing conditions, using 1% agarose including 1.8% formaldehyde. The separated RNA was capillarily transferred by 20× SSC to nylon membranes (Amersham), and fixed through ultraviolet cross-linking. Next, the RNA was prehybridized (5× Denhardt's solution, 5× SSC, 50 mM Na3PO4, 0.1% SDS, 250 mg herring sperm DNA, and 50% formamide) for at least 2 h at 42°C and then hybridized with the 32P-labeled specific NOS-2 probe for 24 h, using the same conditions. The membrane was washed at 42°C twice for 15 min with 2× SSC and 0.1% SDS, for 30 min with 0.1× SSC and 0.1% SDS, and exposed to X-ray film (Kodak, XAR-5 supplied by Sigma, Deisenhofen, Germany) at -80°C for at least 24 h. To rectify for RNA transfer and content, filters were stained with bromophenol blue (0.04% in 500 mM sodium acetate, pH 5.5), and rehybridized with a 32P-labeled rat GAPDH probe.

ANG II receptor subtype analysis by RT-PCR. One microgram total RNA of quiescent cultured rat MCs either grown under control conditions or exposed to LPS/IFN was subjected to an RT reaction using oligo-(dT)16 according to the protocol of the Perkin-Elmer Cetus GeneAmp RNA PCR kit. Specific primers for the homology-based PCR (36 cycles of 1 min of denaturation at 95°C, 30 s of annealing at 59°C, and 1 min of extension at 72°C) produced PCR products that were size separated on ethidium bromide-stained agarose gels, revealing bands at the expected respective sizes. Negative controls for the RT and the PCR reagents remained negative. Primer (synthesized by GIBCO) sequences were as follows for the AT1 receptors, sense, 5'-TCG AAT TCC ACC TAT GTA AGA TCG CTT C-3' (for the AT1A receptor position 554-573 of M74054, and for the AT1B receptor position 448-467 of X64052); and antisense, 5'-TCG GAT CCG CAC AAT CGC CAT AAT TAT CC-3' (for the AT1A receptor position 998-978 of M74054, and for the AT1B receptor position 892-872 of X64052); and for the AT2 receptor, sense, 5'-TCG AAT TCT TGC TGC CAC CAG CAG AAA C-3' (position 3070-3089 of RNU22663); and antisense, 5'-TCG GAT CCG TGT GGG CCT CCA AAC CAT TGC TA-3' (position 4172-4195 of RNU22663) as reported before (added restriction sites are underlined) (30, 38). Contamination of sample RNA by genomic DNA was excluded by directly subjecting the sample RNA to PCR amplification without RT step. A suitable EcoR I restriction site (30) within the AT1A receptor PCR product was used to distinguish between AT1A and AT1B PCR products.

Western blot analysis of NOS-2 protein. Preparation of MC lysates and immunoblot analysis were performed using time-matched 60-mm plates of MCs. They were incubated with complete medium containing 0.5% FCS, medium supplemented with LPS (10 µg/ml medium) and IFN (100 U/ml medium), or medium supplemented additionally with reagents according to the experimental protocol. At the end of the incubation period, the medium was removed and assayed for the nitrite content, as described above. The plates were washed twice with ice-cold PBS. Thereafter, 0.5 ml of boiling lysis buffer solution (1% SDS and 10 mM Tris, pH 7.4) was added to the cells. The cell lysates were collected in centrifuge tubes after boiling and spinning at 12,000 g for 5 min at 4°C. Protein contents were determined using the bicinchoninic acid protein assay reagent (BCA; Pierce, Munich, Germany). The samples (10 µg) were diluted in electrophoresis sample buffer (250 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% mercaptoethanol), boiled for 5 min, quenched on ice, and resolved by electrophoresis through 0.1% SDS-10% polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes (Amersham, Braunschweig, Germany). The transfer efficiency was determined by staining the membranes with Ponceau S. After destaining in distilled water, the membranes were quenched in blocking solution [5% powdered dried low-fat milk in washing solution (10 mM Tris, pH 7.5; 100 mM NaCl, 0.1% Tween 20)] for 1 h at room temperature. The blocking solution was decanted, and membranes were incubated for 1 h at room temperature with the primary antibody (1:1,000, anti-NOS-2, rabbit, polyclonal; Affiniti, Nottingham, UK). Membranes were washed for 30 min at room temperature with several changes of the washing solution. The secondary detecting antibody was horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000; Serva, Heidelberg, Germany) which was used with the enhanced chemiluminescence protocol (Amersham).

Statistical analysis. When suitable, means ± SE were determined. To test for statistically significant differences, we used Student's t-test or analysis of variance, if multiple comparisons were made against a single control, whenever applicable. Significance was assigned at P < 0.05.


    RESULTS
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MATERIALS AND METHODS
RESULTS
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LPS/IFN-induced nitrite production is inhibited by ANG II. The stable metabolite of NO, nitrite, was utilized to assess NO production resulting from NOS-2 induction in cultured rat MCs. Incubation with LPS/IFN for 24 h increased nitrite accumulation in the cell culture supernatant (27.2 ± 1.7 µmol/ml medium) compared with untreated controls (0.6 ± 0.3 µmol/ml medium). Coincubation with ANG II (10-7 M) inhibited nitrite accumulation (16.2 ± 1.0 µmol/ml medium) (Fig. 1A). The dose response for ANG II (10-8-10-6 M) revealed the inhibition to be at maximum with concentrations of ANG II of 10-7 M. This concentration was also applied in successive experiments (Fig. 1B). The NOS inhibitor L-NMMA (10-4 M) was unable to reverse the nitrite production in all experimental conditions (data not shown).


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Fig. 1.   Angiotensin II (ANG II) inhibited synergistic upregulation of nitrite formation by lipopolysaccharide (LPS)/interferon-gamma (IFN) in supernatant from cultured rat mesangial cells (MCs) using the Griess reaction. A: agents used are indicated as LPS (10 µg/ml medium), IFN (100 U/ml medium), and ANG II (10-7 M); results are expressed in µmol/ml medium. Data are means ± SE with assays performed in duplicate (n = 5). B: agents as in A; however, ANG II was added at 10-8-10-6 M. Results are expressed as percentage of standard LPS/IFN induction and are presented as means ± SE with assays performed in duplicate (n = 4).

Twenty-four-hour coincubation of ANG II with LPS/IFN reduced cumulative NOS-2 mRNA and protein expression. To assess regulatory mechanisms involved in the effects of the coincubational downregulation of LPS/IFN-induced NO synthesis by ANG II, total RNA was subjected to size fractionation. No cumulative NOS-2 mRNA expression was detected by Northern blot analysis in cultured MCs in basal conditions; however, 24-h incubation with LPS/IFN induced NOS-2 mRNA, which was markedly diminished in the presence of ANG II (10-7 M) (Fig. 2A). The inhibitory effect of TGF-beta on LPS/IFN-induced NOS-2 mRNA expression was chosen as control.


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Fig. 2.   Coincubation with ANG II reduced cumulative mRNA expression of the inducible form of nitric oxide synthase (NOS-2) on Northern blot analysis of 20 µg total RNA after 24 h of stimulation with LPS/IFN in cultured rat MCs. Cultured rat MCs were treated for 24 h with either vehicle, LPS/IFN, ANG II, LPS/IFN + ANG II, transforming growth factor-beta (TGF), or LPS/IFN + TGF [LPS (10 µg/ml medium), IFN (100 U/ml medium), ANG II (10-7 M), TGF (5 ng/ml medium)]. Top of A: after hybridization using a 32P-labeled NOS-2 cDNA probe, variation of the RNA content of each lane was controlled by rehybridization with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (one representative experiment is presented, n = 4). Bottom of A: densitometrical analysis of autoradiography; ratio of NOS-2 to GAPDH as percentage of standard LPS/IFN induction. B: coincubation with ANG II for 24 h diminished NOS-2 protein levels in cultured rat MCs stimulated with LPS/IFN as assessed by Western blot analysis of 10 µg protein. Treatment was with either vehicle, LPS/IFN, or LPS/IFN + ANG II [LPS (10 µg/ml medium), IFN (100 U/ml medium), ANG II (10-7 M)]. The primary antibody was an anti-NOS-2 mouse polyclonal antibody detected via a peroxidase-conjugated rabbit anti-mouse IgG. Top of B: enhanced chemiluminescence of detected bands (one representative experiment is presented, n = 4). Bottom of B: densitometrical analysis of autoradiography (densitometrical arbitrary units).

In parallel with these findings, incubation with LPS/IFN induced NOS-2 protein expression, which was diminished by ANG II (10-7 M) on coincubation (Fig. 2B).

NOS-2 inhibition is ANG II receptor subtype specific. Using nitrite accumulation to determine NOS-2-derived NO production, we evaluated ANG II receptor subtype specificity. Incubation with the AT1- and AT2-specific inhibitors DUP-753 and PD-123319, respectively, (both at 10-6 M) did not influence LPS/IFN-induced nitrite production. The inhibition by ANG II (10-7 M) was not abolished by DUP-753; however, PD-123319 completely reversed the ANG II-dependent inhibition of NO production, suggesting an AT2-mediated inhibitory effect under these experimental conditions (Fig. 3).


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Fig. 3.   ANG II receptor antagonists affected the nitrite accumulation in supernatant from cultured rat MCs on 24-h coincubation of LPS/IFN with ANG II. The AT1 receptor blocker DUP-753 (DUP) was unable to reverse ANG II-dependent inhibition of nitrite accumulation; however, the AT2 receptor blocker PD-123319 completely abolished the ANG II effect, thus indicating an AT2 receptor related inhibitory effect on induced nitrite production. Agents used are indicated as LPS (10 µg/ml medium), IFN (100 U/ml medium), ANG II (10-7 M), DUP-753 (10-6 M), and PD-123319 (PD, 10-6 M). Results are expressed as percentage of standard LPS/IFN induction and are means ± SE, with assays performed in duplicate (n = 3 - 5).

Presence of ANG II receptor subtypes in cultured rat MCs. Homology-based RT-PCR reactions for AT1 and AT2 receptors generated PCR products of the expected size on separation on ethidium bromide-stained agarose gels. Their identity was verified by restriction analysis. A suitable EcoR I restriction site within the AT1A PCR product (30) was used to identify AT1A and AT1B receptor subtypes (data not shown).

Twenty-four-hour preincubation with LPS/IFN followed by 8-h coincubation with ANG II enhanced NOS-2 protein expression and NO production in cultured rat MCs. The NOS-2 protein expression increased with 32 h of LPS/IFN stimulation compared with experiments lasting only 24 h. The addition of ANG II (10-7 M) for the final 8-h period following 24-h preincubation with LPS/IFN alone augmented the NOS-2 protein expression even further compared with 32-h incubation with LPS/IFN alone (Fig. 4).


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Fig. 4.   Twenty-four-hour preincubation with LPS/IFN followed by 8-h coincubation with ANG II enhanced NOS-2 protein levels in cultured rat MCs stimulated with LPS/IFN compared with 32-h incubation with LPS/IFN without ANG II (Western blot analysis of 10 µg protein). Treatment with either vehicle for 32 h, LPS/IFN for 24 h and 32 h, or preincubation with LPS/IFN for a 24-h period followed by coincubation with ANG II for 8 h [LPS (10 µg/ml medium), IFN (100 U/ml medium), and ANG II (10-7 M)]. As primary antibody, an anti-NOS-2 mouse polyclonal antibody was used, detected by an peroxidase-conjugated rabbit anti-mouse IgG. Top: enhanced chemiluminescence of detected bands (one representative experiment, n = 3). Bottom: densitometrical analysis of autoradiography (densitometrical arbitrary units).

To study whether NO production reflects protein expression, cultured rat MCs were incubated with LPS (10 µg/ml medium)/IFN (100 U/ml medium) for a total of 32 h. Following 24 h of preincubation, either solvent, ANG II (10-7 M), the AT1 receptor blocker DUP-753, or the AT2 receptor blocker PD-123319 were added for the concluding 8-h period. The nitrite accumulation increased between 24 h (61.4 ± 3.2%) and 32 h (100.0 ± 2.1%) of incubation with LPS/IFN alone. The profound cumulative nitrite production after 32 h was even enhanced by the addition of ANG II at 24 h for the remaining 8 h (147.7 ± 2.6% of 32 h LPS/IFN treatment without ANG II). Addition of both PD-123319 and DUP-753 (both at 10-6 M) in combination with ANG II inhibited the ANG II response completely (96.0 ± 3.3% of LPS/IFN alone) (Fig. 5A).


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Fig. 5.   A: 24-h preincubation with LPS/IFN followed by 8-h coincubation with ANG II enhanced nitrite accumulation in supernatants from cultured rat MCs. ANG II or the AT receptor blocker are coincubated with LPS/IFN for the remaining 8 h of 32-h experiments. The AT1 receptor blocker DUP-753 reversed the ANG II-dependent augmentation of nitrite accumulation, thus indicating an AT1 receptor-related stimulatory effect; however, the AT2 receptor blocker PD-123319 markedly increased nitrite synthesis, arguing for an AT2 receptor-related inhibitory effect on induced nitrite production similar to coincubation. Agents used are indicated as LPS (10 µg/ml medium), IFN (100 U/ml medium), ANG II (10-7 M), DUP (10-7-10-5 M), and PD (10-7-10-5 M). Results are expressed as percentage of 32-h LPS/IFN induction and are means ± SE with assays performed in duplicate (n = 3 - 14). B: time course experiments are demonstrated with and without the addition of ANG II. The exposure to LPS/IFN was continued throughout the experiments. Coincubation with actinomycin D almost completely inhibited the increase in nitrite accumulation indicating a transcriptional mechanism. Agents used are indicated as LPS (10 µg/ml medium), IFN (100 U/ml medium), ANG II (10-7 M), and actinomycin D (10 µg/ml medium). Results are expressed in % of 24-h LPS/IFN induction, and presented as means ± SE, with assays performed in duplicate (n = 4-6).

Blockade of the AT1 receptor by DUP-753 (10-5-10-7 M) inhibited the ANG II effect in a dose-dependent fashion. With concentrations of DUP-753 >=  10-6 M, an inhibition beyond the ANG II-induced augmentation of nitrite production was visible, suggesting an inhibitory effect via the AT2 receptor. In accordance with this finding, AT2 receptor blockade (PD-123319, 10-5-10-7 M) doubled the nitrite production seen with LPS/IFN and delayed ANG II addition, thus arguing for an AT1 receptor-mediated stimulation of NO production (Fig. 5A).

Figure 5B reveals the time course of nitrite accumulation during 32 h of LPS/IFN induction and during the addition of ANG II (10-7 M) after 24 h of LPS/IFN preincubation, indicating a similar shaped curve. The addition of actinomycin D (10 µg/ml medium) almost completely inhibited a further increase in nitrite production.

Dexamethasone converts coincubational inhibition by ANG II into stimulation of nitrite production via the AT1 receptor. Dexamethasone is known to enhance AT1A receptor expression and ANG II binding to AT1 receptors in several tissues including MCs (8, 33, 46). To verify our finding of an AT1-mediated potentiation of NO production, we stimulated cultured MCs for 24 h with LPS/IFN; however, dexamethasone was added only after 18 h for the remaining 6-h period to allow an induced nitrite production, since dexamethasone is known to inhibit NOS-2 expression on coincubation. The coincubation with ANG II (10-7 M) for the whole experimental period of 24 h led to a significant increase in induced nitrite production (161.5 ± 25% of LPS/IFN and dexamethasone). The inhibition of the AT1 receptor abolished the ANG II effect completely to 92.3 ± 15.4% of LPS/IFN and dexamethasone. In support of an AT1-mediated NO synthesis as well as a downregulatory action of AT2 activation, inhibition of the AT2 receptor resulted in a pronounced increase in nitrite production (461.3 ± 46.3% of LPS/IFN and dexamethasone) (Fig. 6).


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Fig. 6.   Dexamethasone converted coincubational inhibition by ANG II into a stimulation of nitrite production via an AT1 receptor-dependent response. Cultured rat MCs were preincubated for 18 h with LPS/IFN alone followed by a 6-h coincubation with dexamethasone (Dex). Addition of ANG II for the total experimental period resulted in enhanced nitrite accumulation. AT1 blockade by DUP inhibited this response; however, AT2 blockade by PD allowed for a pronounced stimulation of nitrite accumulation, supporting a stimulatory role for the AT1 receptor, but an inhibitory AT2-related response. Agents used are indicated as LPS (10 µg/ml medium), IFN (100 U/ml medium), ANG II (10-7 M), DUP (10-6 M), PD (10-6 M), and Dex (10-6 M). Results are expressed as percentage of 24-h LPS/IFN + Dex induction and are means ± SE, with assays performed in duplicate (n = 3-4).


    DISCUSSION
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ABSTRACT
INTRODUCTION
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In glomerulonephritis, MCs are exposed to several proinflammatory stimuli such as cytokines which are either secreted by invading inflammatory cells, by resident cells themselves, or by other mediators such as systemically or locally synthesized ANG II.

Glomerular MCs play a critical role in the perpetuation of glomerular diseases (47). ANG II takes part in the inflammatory response of MCs (51, 54). Therefore, we analyzed whether ANG II regulates induced NO production in the inflammatory range resulting from expression of active NOS-2 protein.

We chose a model of inflammation simulated by exposure of MCs to LPS and IFN. These mediators lead to PKC/mitogen-activated protein kinase-dependent and -independent activation of nuclear factor-kappa B (NF-kappa B) and IFN regulatory factors, resulting in induced NO levels within the inflammatory range (9, 10, 11). Our experiments suggest ANG II-dependent inhibition of LPS/IFN-induced production of NO in MCs during simultaneous exposure of the cells to LPS/IFN and ANG II. This effect of ANG II was dose dependent within a physiological concentration range. Analysis of cumulative NOS-2 mRNA as well as of NOS-2 protein expression indicated a transcriptional regulation.

In an evaluation of the ANG II receptor subtype involved, neither specific receptor blocker (DUP-753 for the AT1 subtype, and PD-123319 for the AT2 subtype) exerted nonspecific effects on nitrite production at concentrations of 10-6 M if incubated without ANG II. This finding also excluded a significant contamination by exogenous ANG II. The inhibitory effect of ANG II was unaffected by coincubation with the AT1 but was completely abolished with the AT2 receptor blocker. This suggests an inhibitory effect mediated via AT2 receptors. We were unable to demonstrate a significant direct stimulatory effect through AT1 receptors in this coincubation setup, although a slight tendency toward an increased nitrite production was visible. ANG II binding studies and the demonstration of AT1A and AT1B receptor subtype mRNA (6, 7, 8) proposed the predominance of AT1 receptors in humans and rats, preferentially of the AT1B subtype in rat MCs. Conversely, Ernsberger et al. (13) and Goto et al. (15) demonstrated by pharmacological and molecular biological means, respectively, the existence of AT2 receptors in rat MCs. Ardaillou et al. (2) found a marked expression of AT2 receptors in mouse MCs. To demonstrate the receptor expression in our MC cultures, we performed RT-PCR for the AT1 and the AT2 receptor subtypes, as well as restriction analysis for the AT1A and AT1B subtypes, in support of our functional data regarding receptor subtype activity and specificity.

In all studies performed so far addressing the potential interaction of ANG II and induced NO production, coincubation experiments were performed omitting the impact of a sequential occurrence of stimuli modulating not just the inflammatory response but also ANG II receptor subtype expression. ANG II may act differentially with respect to the AT receptors present. The AT receptor expression is subject to changes in the presence of inflammatory stimuli or glucocorticosteroids. We hypothesized that ANG II would act proinflammatory via the AT1 receptor by enhancing induced NO production in inflammatory conditions. Inflammation, as induced by LPS and cytokines, led to downregulation of the AT2 receptor, as was previously described in rat fibroblasts (48). An enhanced AT1 receptor-mediated signal transduction (24) and an LPS-mediated induction of the cyclooxygenase (COX-2) via the AT1 receptor has been demonstrated in rat vascular smooth muscle cells (39). COX-2 activity then stimulates a cAMP-dependent PKA activation of the NF-kappa B pathway, a factor which results in a profound production of induced NO (9, 10, 11).

ANG II added to cultured MCs preexposed to LPS/IFN further stimulated NOS-2 protein and NO production. This increase was abolished by AT1 blockade in a dose-dependent fashion, even below control inductions, indicative of a downregulatory AT2 effect. In contrast to the coincubation experiments, a stimulatory effect of AT1 receptor activation was now observed on blockade of the AT2 receptor. This supported our assumption of a potential AT1 receptor-related stimulation of induced NO production, however, controlled via AT2 receptors.

To further clarify the role of the AT1 receptor, dexamethasone was applied to stimulate AT1A receptor expression (8) and binding of ANG II and to reduce AT2 receptor expression (48). We exploited the fact that the AT1A receptor promotor, in contrast to the AT1B and the AT2 receptor, contains three putative glucocorticoid-responsive elements, one of which has already been found to be dexamethasone responsive at concentrations of 10-6 M (17). An AT1-related stimulatory effect of ANG II on LPS/IFN-induced NO production was present despite the inhibitory effect of dexamethasone on NOS-2 transcription via upregulated inhibitor-kappa Balpha activity and decreased nuclear p65 translocation (12). This is consistent with previous observations in a model of interleukin-1beta (IL-1beta )-dependent induced NO production with either alpha 1-adrenergic- or adenosine-related stimulation in rat vascular smooth muscle cells and cardiac myocytes (20, 22).

The reduction of NO synthesis on coincubation with LPS/IFN and ANG II is in agreement with studies performed by Kihara et al. (27) and Wolf et al. (52) in cultured MCs and tubular cells, respectively. Neither study demonstrated ANG II receptor expression. Kihara et al. (27) demonstrated an inhibition of IL-1beta -induced NO production on coincubation with ANG II. In contrast to our findings, the AT1 receptor antagonist CV-11974, but not the AT2 blocker PD-123319, was able to reverse the ANG II-dependent inhibition at concentrations of 10-5 M, which may already exert unspecific effects on AT2 receptors (18). No NOS-2 transcriptional downregulation was observed in either study (27, 52). Wolf et al. (52) suggested posttranscriptional mechanisms beyond mRNA stability. However, in our experiments the LPS/IFN-induced steady-state NOS-2 mRNA and protein expression were reduced by ANG II at concentrations of 10-7 M on coincubation, indicating mechanisms involving transcription or mRNA stability. We were able to demonstrate that actinomycin D was able to almost completely abolish the ANG II-dependent stimulation of nitrite production of MCs when preincubated with LPS/IFN for 24 h. This suggested a transcriptional regulation, although additional mechanisms may still be active.

In line with our findings, an IL-1beta - and LPS-mediated NOS-2 induction has been demonstrated in rat and rabbit cardiac myocytes in which AT1 receptor-mediated augmentation of NO and cGMP production led to contractile depression (21, 53). In further support of our own finding of an AT2 receptor-dependent downregulation of induced NO production is a preliminary report by Peters et al. (40), who found inhibition of LPS/IFN-mediated NOS-2 induction by ANG II and ANG II fragments in cultured rat MCs (fragments 1-7 and 3-8).

In conclusion, we describe a regulatory mechanism by which ANG II acts on glomerular mesangial NO production in a time- and ANG II receptor-dependent manner, and we provide evidence for a significant contribution of renal AT2 receptors. This supplements reports of AT2-related renal responses, such as pressure natriuresis (16, 31). ANG II interacts with induced NO production upon proinflammatory stimuli. An inflammatory response may be enhanced following AT1 receptor stimulation by an augmented NO production. In contrast, AT2 receptor activation restricts the amount of induced NO synthesis. Once the AT1/AT2 receptor balance is shifted toward the AT1 receptor, the NO response is augmented most likely transcriptionally. Further work will need to be directed toward elucidating the related downstream signals. The reported ANG II-dependent regulation of induced NO production opens new insight into understanding renal damage and functional deterioration in the presence of glomerular inflammation, yet the extent of its contribution still needs to be defined.


    ACKNOWLEDGEMENTS

We thank Dr. Michael Wiederkehr for critical review of this manuscript and C. Staiger for excellent technical assistance.


    FOOTNOTES

This work was supported in part by the Deutsche Forschungsgemeinschaft Klinische Forschergruppe "Molekulare Regulationsmechanismen in glomerulären Zellen der Niere" and by a grant from the Swiss National Foundation, both to M. G. Mohaupt.

Address for reprint requests and other correspondence: M. G. Mohaupt, Univ. Hospital, Berne, Division of Nephrology/Hypertension, 3010 Berne, Switzerland (E-mail: markus.mohaupt{at}insel.ch).

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 27 March 2000; accepted in final form 11 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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