Abstract
Angiotensin-converting enzyme (ACE) inhibitors reduce macrophage infiltration in several models of renal injury. We approached the hypothesis that angiotensin II (AngII) could be involved in inflammatory cell recruitment during renal damage through the synthesis of monocyte chemoattractant protein-1 (MCP-1). In a model of immune complex nephritis, we observed an up-regulation of renal MCP-1 (mRNA and protein) coincidentally with mononuclear cell infiltration that were markedly reduced by treatment with the ACE inhibitor quinapril. Exposure of cultured rat mesangial cells to AngII increased MCP-1 mRNA expression (2.7-fold) and synthesis (3-fold), similar to that observed with TNF-α. Since NF-κB is involved in the regulation of MCP-1 gene, we explored whether the effects of AngII were mediated through NF-κB activation. Untreated nephritic rats showed increased renal NF-κB activity (3.5-fold) that decreased in response to ACE inhibition. In mesangial cells, AngII activated NF-κB (4.3-fold), and the NF-κB inhibitor pyrrolidine dithiocarbamate abolished the AngII-induced NF-κB activation and MCP-1 gene expression. Our results suggest that AngII could participate in the recruitment of mononuclear cells through NF-κB activation and MCP-1 expression by renal cells. This could be a novel mechanism that might further explain the beneficial effects of ACE inhibitors in progressive renal diseases.
Afeature of progressive renal diseases is the presence of mononuclear cells in the glomerulus and interstitium (1). In the past years, several works have demonstrated that the monocyte-macrophages play an important role in the onset and progression of various kidney diseases (2, 3, 4). One hypothesis that could explain the increase in cell recruitment during glomerular damage is the release of chemotactic factors by resident renal cells (5, 6). Among the chemoattractant cytokines, monocyte chemotactic protein-1 (MCP-1)3 has an active role in renal injury (7). MCP-1 is expressed by a variety of renal cell types, including mesangial cells, tubular epithelial cells, and interstitial fibroblasts, and is stimulated by cytokines (9, 10, 11, 12, 13). Moreover, an up-regulation of MCP-1 levels was noted in the glomerulus in experimental and human nephritis (14).
NF-κB is the main factor involved in the transcription of MCP-1 gene induced by LPS, TNF-α, IL-1β, and phorbol esters (15). MCP-1 gene expression is regulated mainly by proteins that bind to a κB region, responsible for the induction of transcription after stimulation and to an Sp1-binding site responsible for the maintenance of basal transcription (16). The NF-κB is a heterodimer of p50 and p65 subunits present in inactive form in the cytoplasm complexed with its inhibitor IκB (17). NF-κB activation consists of the dissociation of IκB and the translocation of the heterodimer into the nucleus, where it binds to a specific DNA sequence and activates the transcription (18, 19).
Recent data suggest that activation of local renin-angiotensin system participates in the pathogenesis of renal damage (20). Tissue levels of angiotensin II (AngII), the effector peptide of this system, are increased under pathologic conditions. AngII is considered a renal growth factor involved in cell proliferation and extracellular matrix accumulation (20, 21, 22), two features of renal damage. Some evidence suggest that AngII could be involved in cell recruitment. AngII receptors have been demonstrated on human monocytes (23), and AngII stimulates chemotaxis of human mononuclear cells (24, 25). Moreover, in different models of renal damage, angiotensin-converting enzyme (ACE) inhibitors diminished the number of infiltrating cells at glomerular and interstitial level (26, 27, 28).
In this work, we evaluated the hypothesis that AngII may participate in mononuclear cell accumulation during renal injury through the production of MCP-1 by resident renal cells. In an experimental model of immune complex nephritis in rats that present increased renal ACE activity (27), we determined renal MCP-1 expression, inflammatory cell infiltration, and the effect of ACE inhibition.
To determine a potential direct effect of AngII on MCP-1 synthesis we performed in vitro studies with cultured rat glomerular mesangial cells. To further investigate this process we evaluated whether the AngII effects were mediated through NF-κB activation.
Materials and Methods
Abs and reagents
All culture reagents were purchased from Life Technologies (Paisley, U.K.). AngII was obtained from Calbiochem. [α-32P]dCTP (3000 Ci/mmol) and [γ-32P]CTP (3000 Ci/mmol) were from Amersham (Amersham, Buckinghamshire, U.K.). Primers for RT-PCR studies were obtained from Genosys Biotechnology (Cambridge, U.K.). The mAbs used in immunohistochemistry were the following: 0X1 (leukocyte common Ag) and W3/13 (T lymphocytes) obtained from Seralab (Sussex, U.K.); ED1 (monocytes/macrophages) from Serotec (Oxford, U.K.). Recombinant MCP-1 and goat polyclonal anti-MCP-1 Ab were from Immugenex (Los Angeles, CA). Horseradish peroxidase-conjugated donkey anti-goat IgG was from The Binding Site (Birmingham, U.K.), and normal control rabbit IgG was from Sigma (St. Louis, MO). For supershift assays, we used anti-p65 and anti-c-Rel Abs from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-p50 Ab from Chemikon (Temecula, CA). NF-κB consensus oligonucleotide was from Promega (Madison, WI). All other chemicals were from Sigma. None of the compounds was cytotoxic for mesangial cells at the concentrations used, as determined by trypan blue staining (not shown).
Experimental design
Immune complex nephritis was induced in normotensive Wistar rats as previously described (27, 29). Briefly, rats received an initial s.c. injection of 5 mg of OVA in CFA (Difco, Detroit, MI), and 3 wk later, the same dose was given in IFA (Difco). One week later, daily i.p. administration of 10 mg of OVA was started. Proteinuria appears at the 9th week, and when reaching 20 to 50 mg/day animals were randomly distributed into two groups: untreated (proteinuria, 29 ± 11 mg/day), animals with spontaneous development of nephritis; and quinapril-treated (proteinuria, 30 ± 10 mg/day; p = NS vs untreated), animals treated with the ACE inhibitor quinapril (a gift from Parke Davis as powdered hydrochloride) at a concentration of 100 mg/L, added to the drinking water, and replaced every 48 h.
Studies were done 3 wk after the onset of proteinuria when untreated animals develop full-blown nephrotic syndrome and moderate renal failure (27, 29). A parallel control group of animals of the same age, with or without treatment, was also studied. At the time of sacrifice, animals were fasted overnight and anesthetized with 5 mg/100 g sodium pentobarbital. The kidneys were perfused in vivo via the abdominal aorta with 100 ml of normal saline at 4°C. Blood samples and kidneys were removed immediately and further processed for histologic studies, RNA extraction, and NF-κB activity.
We have previously described the effect of ACE inhibition treatment in this normotensive model of immune complex nephritis (27). In the group of animals studied in the present work, the administration of quinapril during 3 wk caused a significant reduction in proteinuria (80 ± 29 vs 510 ± 49 mg/day in untreated nephritic rats, n = 10, p < 0.01) and in glomerular and tubulointerstitial lesions (not shown). Nephritic rats remained normotensive along the study, and quinapril did not significantly modify systemic blood pressure (not shown). Renal ACE activity was elevated in untreated rats compared with controls and diminished around 60% in response to ACE inhibition, as previously shown (27).
Renal histopathologic studies
Immunohistochemistry.
Tissue for immunohistochemistry was embedded in OCT (Tissue-Tek, Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −80°C until its study. The quantification of the infiltrating glomerular and interstitial cells was performed as previously published (29, 30). The mean numbers of cells per glomerular cross-section were determined by evaluating 50 glomeruli in each renal section. In the interstitium, areas of 0.45 mm2 were counted.
Tissue localization of MCP-1 immunoreactivity.
Paraffin-embedded renal tissue was cut at 4 μm and mounted on poly-l-lysine-coated slides, and immunoperoxidase staining was performed. The slides were deparaffinized with graded concentrations of xylene and ethanol. The slides were quenched in methanol containing 3% H2O2/methanol at 25°C for 30 min. They were subsequently incubated in PBS with 6% horse serum in 4% BSA for 1 h at 37°C to reduce nonspecific background staining and then incubated overnight at 4°C with goat polyclonal anti-MCP-1 Ab, 70 μg/ml, in PBS containing 1% horse serum and 4% BSA. After being washed with PBS, the sections were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG, diluted 1:100 in 4% BSA for 30 min, and after washing, they were stained with 0.05% 3,3′-diaminobenzidine (Dako A/S, Glostrup, Denmark) in 0.01% H2O2 for 10 min. The sections were counterstained with Mayer’s hematoxylin and mounted in Pertex (Medite, Burgdorf, Germany). In each experiment, negative controls without the primary Ab or using an unrelated Ab were included to check for nonspecific staining. About 15 glomeruli from each animal were examined, and the immunostaining was graded from 0 to 4+ by a semiquantitative score according to the following criteria: 0, no staining; 1+, minimal staining; 2+, mild staining; 3+, moderate staining; 4+, marked staining.
Histologic studies were quantified by two independent observers without knowing to which group the sample belonged, and the mean value was calculated for each rat.
Mesangial cell culture
Rat mesangial cells were cultured from isolated glomeruli of healthy rats by sieving techniques and differential centrifugation (31). Mesangial cells were grown in RPMI 1640 medium buffered with 25 mM HEPES at pH 7.4 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine in the presence of 10% FCS and cultured at 37°C in 5% CO2 atmosphere. At confluence, cells grown in 75-cm2 flasks were made quiescent for 48 h in 0.5% FCS medium, and then different studies were performed. Glomerular mesangial cells were characterized by phase contrast microscopy, positive staining for desmin and vimentin, and negative staining for keratin and factor VIII Ag, excluding epithelial and endothelial cell contamination, respectively (32).
Molecular techniques
cDNA probes.
The cDNA probes for human MCP-1 (JE*/pGEM-hJE34) and glyceraldehyde-3′-phosphate dehydrogenase (G3PDH) were obtained from the American Type Culture Collection, Manassas, VA. Probes were radiolabeled by nick translation (Boehringer Mannheim, Mannheim, Germany) with [α-32P]dCTP.
Northern blot assay.
Total RNA from cells and renal tissue was extracted by the method of Chomczynski and Sacchi (33) and quantitated by absorbance at 260 nm. Equal amounts of RNA (10–30 μg) were denatured, electrophoresed in a l% agarose-formaldehyde gel, and transferred to nylon membranes (Genescreen, New England Nuclear, Boston, MA). The quality of RNA was determined by ethidium bromide staining (not shown). RNA was fixed to the nylon membrane by baking for 90 min at 80°C. The membranes were prehybridized for 4 h at 42°C in hybridization solution (50% formamide, 1% SDS, 5× SSC, 1× Denhardt’s, 0.1 mg/ml denatured salmon sperm DNA, and 50 mM sodium phosphate buffer, pH 6.5), and hybridization was conducted at 42°C for 16 to 18 h in fresh hybridization solution with 20% dextran sulfate and α-32P-denatured probe. The membranes were washed using 2× SSC, 0.1% SDS, for 30 min at room temperature and then twice with 0.2× SSC, 0.1% SDS, at 55°C for 15 min. Autoradiographic signals obtained with the G3PDH cDNA probe served as a control for equal loading of the gel. The ratio of mRNA vs G3PDH was set at unity for basals, and other lanes on the same gel were expressed as n-fold increases over this value.
Reverse transcription and semiquantitative PCR analysis.
To obtain cDNA for the PCR, 1 μg of RNA from each sample was transcribed in a final volume of 20 μl which contained 5 mM MgCl2, RT buffer (10 mM Tris-HCl, 50 mM KCl, and 0.1% Triton X-100), 1 mM deoxynucleotide mixture, 20 U of RNasin (an RNase inhibitor), 15 U of reverse transcriptase of the avian Moloney virus, and 50 ng of random primer. The reaction mixture was incubated at 42°C for 45 min. At the end of the incubation, samples were heated at 95°C to eliminate transcriptase activity and to denaturalize the RNA-cDNA hybrids. PCR was conducted in the presence of [α-32P]dCTP for 25, 30, 35, and 40 cycles under the same conditions as for MCP-1 and glyceraldehyde 3′-phosphate dehydrogenase (G3PDH), used as internal control (1 min at 54°C to allow annealing of the primers, 3 min at 72°C for primer extension, and 1 min at 94°C to denature the double-stranded DNA). The following primers were used for rat MCP-1 (34): (sense) 5′-TTCTGGGCCTGTTGTTCACA-3′ and (antisense) 5′-GGTCACTTCTACAGAAGTCC-3′, that yielded a product of 409 bp. G3PDH was used as internal control and the following primers were used (35): (sense) 5′-AATGCATCCTGCACCACCAA-3′ and (antisense) 5′-GTAGCCATATTCATTGTCATA-3′ that yielded products of 515 bp. The amplification of PCR was linear up to 35 cycles, both for MCP-1 and for G3PDH, and data for cycle 25 were used for calculations. In all experiments, the presence of possible contaminants was checked by control reactions in which amplification was conducted in complete reaction mixture lacking template DNA or with RNA samples from RT reactions done in the absence of avian Moloney virus reverse transcriptase. The DNA products from the PCR reactions were analyzed on a 4% polyacrylamide-urea gel in Tris-borate, EDTA buffer (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA). The polyacrylamide gels were dried, exposed to x-ray films, and scanned using the IQ densitometer (Image Quant Densitometer, Molecular Dynamics, Sunnyvale, CA).
Protein extraction
From tissue.
For protein extraction from tissue samples, the method of Negoro et al. (36) was used with some modifications. Briefly, frozen kidney cortex sections were pulverized in a metallic chamber and resuspended in 1 ml of cold extraction buffer containing 20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.35 M NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 1 μg/ml pepstatin A. The homogenate was vigorously shaken, insoluble materials were precipitated by centrifugation at 40,000 × g for 30 min at 4°C, and the supernatant was dialyzed overnight against a binding buffer containing 20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 M NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF. The dialysate was cleared by centrifugation at 10,000 × g for 15 min at 4°C and frozen at −80°C in aliquots until use. Protein concentration was quantified by the BCA method (Pierce, Rockford, IL).
From cells.
Quiescent mesangial cells were stimulated for different periods of time. Then, cells were trypsinized and resuspended in 5 cell pellet vol of buffer A (10 mM HEPES (pH 7.8), 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF). After 10 min on ice, the cells were pelleted, resuspended in 2 cell pellet vol of buffer A, and homogenized. Nuclei were centrifuged at 1000 × g for 10 min, washed twice in buffer A, and resuspended in 2 vol of this buffer. Then 3 M KCl was added drop by drop to reach 0.39 M KCl. Nuclei were extracted for 1 h at 4°C and centrifuged at 100,000 × g for 30 min. Supernatant was dialyzed in buffer C (50 mM HEPES (pH 7.8), 50 mM KCl, 10% glycerol, 1 mM PMSF, 0.1 mM EDTA, and 1 mM DTT) and then cleared by centrifugation and stored at −80°C. Protein concentration was determined by the BCA method.
Electrophoretic mobility shift assays (EMSA)
Gel shift assays were performed with a commercial kit following the instructions of the manufacturer (Promega). Briefly, NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was 32P-end-labeled by incubation for 10 min at 37°C with 10 U of T4 polynucleotide kinase (Promega) in a reaction containing 10 μCi of [γ-32P]ATP, 70 mM Tris-HCl, 10 mM MgCl2, and 5 mM DTT. The reaction was stopped by the addition of EDTA to a final concentration of 0.05 M. Nuclear or cellular protein (10 μg) was equilibrated for 10 min in a binding buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 50 μg/ml of poly(dI-dC) (Pharmacia, Uppsala, Sweden). NF-κB activity was quantified in 10 μg of total protein from each pool, and experiments were done in duplicate. HeLa cell nuclear extract was used as a known positive control because it has been shown to contain NF-κB (37). To assess the specificity of the reaction, the following controls were done: negative assay without cellular extracts; and competition assays with a 100-fold excess of unlabeled NF-κB, mutant NF-κB, and unspecific (activating protein 1 (AP-1)) oligonucleotides. When competition assays were done, the unlabeled probe was added to this buffer 10 min before the addition of the labeled probe. The labeled probe (0.35 pmol) was added to the reaction and incubated for 20 min at room temperature. For supershift assays, 1 μg of anti-p50, anti-p65, or anti-c-Rel Abs was added and incubated for 1 h before the addition of the labeled probe. The specificity of anti-p50 and anti-p65 Abs was tested by Western blot. Briefly, nuclear extracts were obtained from cells treated with TNF-α and AngII, analyzed by PAGE-SDS. After incubation with anti-p50 and anti-p65 Abs, a band of the expected size was found (not shown). Supershift assays were also done with HeLa cell nuclear extracts. The supershift band was competed by a 100-fold excess of unlabeled specific (NF-κB), but not by mutant NF-κB probe (not shown). The reaction was stopped by adding gel loading buffer (250 mM Tris-HCl, 0.2% bromphenol blue, 0.2% xylene cyanol, and 40% glycerol) and run on a nondenaturing, 4% acrylamide gel at 100 V at room temperature in TBE. The gel was dried and exposed to x-ray film and scanned using the IQ densitometer.
Western blot analysis
Confluent resting mesangial cells were incubated for 24 h with 10−7 M AngII and 100 U/ml TNF-α, used as positive control. Then, the conditioned medium was isolated, concentrated 10 times by centrifugation using ultrafree MC filters (Millipore, Bedford, MA), and kept at −20°C until analysis. Protein content was determined by the BCA method. The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked by a 30-min incubation in 0.01 mM Tris, pH 7.5, and 0.1 mM NaCl containing 0.1% Tween 20, 1% BSA, and 5% milk and then incubated in the same buffer with an anti-MCP-1 Ab for 18 h at 4°C. After washing, detection was made by incubation with peroxidase-conjugated anti-rabbit IgG and developed using an ECL chemiluminescence kit (Amersham). The films were scanned using the IQ densitometer. Recombinant human MCP-1 was used as positive control.
Statistical analysis
Autoradiographs were scanned by densitometry. Results are expressed as n-fold increase over control in densitometric arbitrary units and expressed as mean of the experiments made. Results of immunohistochemistry are expressed as the mean ± SEM. Comparisons between means of multiple groups were analyzed by ANOVA and Student’s t test. Significance was established using the GraphPAD Instat (GraphPAD Software, San Diego, CA), and differences were considered significant if the p value was <0.05.
Results
ACE inhibition treatment diminishes renal mononuclear cell infiltration
We have previously described that administration of the ACE inhibitor quinapril to rats with immune complex nephritis caused a significant decrease in glomerular and interstitial cell infiltration (27). By means of mAbs, we characterized the phenotype of the inflammatory cells in the animals used in the present work. Compared with the untreated nephritic rats, animals receiving quinapril for 3 wk had significantly less glomerular cells expressing the leukocyte common Ag (3.0 ± 0.5 vs 8.0 ± 0.4 untreated rats, cells/glomerulus), T lymphocytes (3.2 ± 0.7 vs 7.5 ± 1.1), and monocytes/macrophages (5.0 ± 1.9 vs 7.5 ± 0.1) (n = 9 for all groups studied; p < 0.05 for all parameters). More marked effects were noted on the interstitial cell infiltrates, with an important reduction in the number of cells expressing the leukocyte common Ag (91 ± 9 vs 283 ± 57 untreated rats, cells/mm2), T lymphocytes (78 ± 12 vs 201 ± 60), and macrophages (37 ± 10 vs 148 ± 14), reaching values similar to those of healthy control rats (n = 9; p < 0.01). The presence of infiltrating cells in renal tissue has been associated with proteinuria in several human and experimental nephritis (2, 3, 4, 5, 6). In quinapril-treated rats, proteinuria was significantly reduced vs untreated rats (80 ± 29 vs 510 ± 49 mg/day, n = 10, p < 0.05). In this model, we have observed that a good correlation existed between infiltrating cells and proteinuria (r2 = 0.99; p < 0.01).
Renal MCP-1 mRNA and protein expression are up-regulated in nephritic rats and are diminished by ACE inhibition
The recruitment of monocyte-macrophages in renal tissue, through the release of chemotactic factors by resident and infiltrating cells, is an important fact in the induction and progression of renal injury (2, 3, 4). MCP-1 expression has been detected in nephritic glomeruli (14). For this reason, in nephritic rats, we have studied renal MCP-1 mRNA levels and protein localization as well as its modulation by ACE inhibition.
MCP-1 gene expression was studied with a semiquantitative PCR technique by amplifying a fragment of MCP-1 gene in the same conditions as a fragment of the housekeeping gene G3PDH (see Materials and Methods). Samples of total RNA from renal cortex of four different animals of each group were pooled and analyzed in two RT-PCR experiments. In nephritic rats, there was a fourfold increase in MCP-1 mRNA levels with respect to healthy controls (Fig. 1⇓). Quinapril treatment induced a dramatic reduction in MCP-1 gene expression (Fig. 1⇓).
Gene expression of MCP-1 in cortex of rats with immune complex nephritis in response to ACE inhibition. RNA isolated from cortex from four rats of each group was pooled and used as a single sample in two RT-PCR; values are for a representative experiment. The expected size of each PCR product was: MCP-1 (409 bp); and G3PDH (515 bp). The amplification product of G3PDH served as internal standard for RT-PCR. The quality of RNA was determined by ethidium bromide staining (not shown).
MCP-1 protein levels were studied by immunohistochemistry. Kidney samples were paraffin embedded, and serial sections were stained with the anti-MCP-1 Ab. In control rats (untreated and quinapril-treated), there was a slight MCP-1 staining in glomerular capillaries and some proximal tubules (Fig. 2⇓, A and B). In untreated nephritic rats, an intense MCP-1 staining was seen in glomerular capillaries, mesangial area, proximal tubules, and interstitial mononuclear cell infiltrates (Fig. 2⇓C). In nephritic rats treated with quinapril, there was a marked diminution in MCP-1 staining (Fig. 2⇓D). No immunoperoxidase staining was observed when a nonimmune serum was used (negative controls; Fig. 2⇓E). The semiquantification of immunostaining for MCP-1, done in a double-blinded manner, is shown in Figure 3⇓.
Glomerular MCP-1 immunostaining of control (A), control-quinapril-treated (B), untreated nephritis (C), and quinapril-treated (D) rats. Photomicrographs show a representative immunostaining of kidney sections with a specific MCP-1 polyclonal antiserum. There was no staining in the negative controls included in each experiment (E). (Magnification ×100).
Semiquantitative score of MCP-1 protein in glomeruli (A) and tubules (B). The staining was scored as: −, no staining; 1+, mild; 2+, moderate; 3+, intense. The evaluation was done a in blind manner by two independent observers. Results are expressed as mean ± SEM of six to eight animals per group. *p < 0.05 vs control, #p < 0.05 vs untreated nephritis.
All these data suggested that in this model of renal injury, which presents increased renal ACE activity (27), the accumulation of inflammatory cells could be due to the augmentation in MCP-1 production caused by the elevated local AngII generation. This could represent a new potential mechanism to further explain the beneficial effect of ACE inhibitors.
Renal NF-κB is activated in nephritic rats and is diminished by ACE inhibition
Recent studies have shown the activation of transcription factors during tissue damage (38). Cellular extracts were obtained from samples of the renal cortex of three animals from each group and then pooled. NF-κB activity was quantified in 10 μg of total protein from each pool, and EMSA experiments were done in duplicate. In relation to controls, untreated nephritis showed a 3.5-fold increase in NF-κB activity that decreased to control values in quinapril-treated animals (Fig. 4⇓A). The reaction was proved to be specific since an excess of unlabeled NF-κB, but not mutant NF-κB or AP-1 (unrelated nuclear protein binding), decreased the signal of the retarded bands (Fig. 4⇓A).
NF-κB activity in renal cortex of rat with immune complex nephritis. A, Cellular extracts from three different rats of each group were collected, pooled, and used for gel mobility shift assays with the NF-κB probe. Negative control was done in the absence of cellular extract. The specificity of the reaction was demonstrated with a 100-fold excess of unrelated oligonucleotide (AP-1), mutant NF-κB, and unlabeled NF-κB consensus sequence (specific competitor). The position of the NF-κB complexes and free oligonucleotide is indicated. B, Identification of NF-κB subunits in nephritic rats. The cellular extract of untreated rats was incubated with Abs against the NF-κB subunits p50, p65, and c-Rel. A supershifted band was observed with the anti-p50 and anti-p65 Abs. A representative EMSA of four experiments. C, Competition assays were done with HeLa cell nuclear extracts, a positive NF-κB control.
To further characterize the protein binding to the NF-κB motif, cellular extracts were preincubated with Abs to the protein subunits of the NF-κB p50, p65, and c-Rel (39). Incubation of the extracts with anti-p50 and anti-p65 Abs yielded a supershifted band. In the presence of anti-p65 Ab, a slight reduction in the intensity of the bands was also noted. No effect was observed with anti-c-Rel Ab (Fig. 4⇑B).
Angiotensin II induces MCP-1 mRNA and protein expression in cultured mesangial cells
The above-mentioned experiments suggested that in situations of renal injury, associated to increased local AngII generation, this peptide could participate in cell recruitment. However, we cannot discard that other factors involved in cell recruitment such as cytokines and immune complexes, present in the cell microenvironment during renal damage (29, 40), could be responsible for the increase in MCP-1 expression and inflammatory cell accumulation. To further investigate this point, we studied whether AngII regulates MCP-1 production in cultured glomerular mesangial cells. These cells produce MCP-1 in response to several stimuli, like cytokines or immune complexes (10, 11, 41), and may play a key role in inflammatory reactions in different glomerular diseases (42).
Mesangial cells were stimulated with several concentrations of AngII (10−7 to 10−11 M) for 3, 6, and 24 h. After incubation, RNA was extracted, and MCP-1 mRNA expression was determined by Northern blot. Under basal conditions, a MCP-1 mRNA band (0.8 kb) was apparent (Fig. 5⇓A), as described (8, 9, 10, 11). In response to AngII stimulation, an increase in MCP-1 mRNA expression was found, being maximal at 6 h and returning to basal levels after 24 h (Fig. 5⇓A). This result showed a similar kinetics to that induced by 1 μg/ml LPS (Fig. 5⇓A) and TNF-α (not shown), as previously described (8, 11, 43). This effect was dose dependent with maximal responses at 10−7 to 10−9 M AngII (∼2.7-fold increase vs basal, p < 0.05) (Fig. 5⇓B).
AngII increases MCP-1 mRNA expression in rat mesangial cells. A, Time course evolution. Quiescent cells were incubated for 3, 6, and 24 h with 10−7 M AngII and 1 μg/ml LPS. B, Dose response. Cells were stimulated with AngII (dose of 10−5 M–10−11 M) for 6 h. C, Regulation of MCP-1 mRNA expression induced by AngII. To inhibit PKC activity, cells were preincubated for 1 h with 10−7 M staurosporine (Staur) and 10−7 M H7 and afterward stimulated with 10−7 M AngII for 6 h. PMA (100 mM) was used as a positive control of PKC activation. To inhibit protein synthesis, cells were pretreated with 1 μg/ml cycloheximide (CHX). A representative Northern blot of three to five experiments.
Previous studies have demonstrated that protein kinase C (PKC) agonists, such as phorbol esters, increase MCP-1 gene expression (8, 9, 10, 11); therefore, we studied the role of PKC activation in AngII-induced MCP-1 mRNA expression. Preincubation for 1 h with 10−7 M staurosporine and H7 (two PKC inhibitors) diminished the increase in MCP-1 mRNA induced by AngII (∼80%) (Fig. 5⇑C), suggesting a PKC-dependent mechanism. None of the two inhibitors had any effect on the MCP-1 gene expression in control cells, and 100 mM PMA, a known PKC activator, was used as a positive control (Fig. 5⇑C).
We also evaluated the role of the de novo protein synthesis in this process. Preincubation of mesangial cells with 1 μg/ml cycloheximide for 1 h increased MCP-1 mRNA in unstimulated and AngII-treated cells (Fig. 5⇑C), indicating that its induction occurs in the absence of new protein synthesis and may be under the control of a labile repressor protein.
To determine whether the increase in MCP-1 expression in response to AngII led to the release of protein to the extracellular medium, mesangial cells were incubated for 24 h with 10−7 M AngII and 100 U/ml TNF-α (positive control), and MCP-1 synthesis was determined by Western blot. Under control conditions, mesangial cells secreted small amounts of MCP-1, but after treatment with AngII or TNF-α, a marked increase was observed (threefold) (Fig. 6⇓). The MCP-1 was detected as a band of 12 to 14 kDa, similar to that of the recombinant MCP-1, used as standard, and similar to that demonstrated in murine and human mesangial cells after treatment with IgG and IgA aggregates (11, 41).
Secretion of MCP-1 by mesangial cells in response to AngII. Cells were incubated in medium alone or in the presence of 10−7 M AngII or 100 U/ml TNF-α for 24 h. The supernatant was concentrated 10-fold, and MCP-1 synthesis was determined by Western blot analysis. The position of the apparent m.w. of the standards is indicated in kDa. Human recombinant MCP-1 was used as a positive control. Results are from one of three comparable series of experiments.
Ang II activates NF-κB in mesangial cells
NF-κB has been identified as an important DNA-binding protein that initiates the transcription (17, 18). Growth-arrested mesangial cells were incubated with AngII (10−7, 10−9, and 10−11 M) for 30, 60, and 120 min. Nuclear extracts were obtained, and NF-κB activity was determined by binding assay of nuclear proteins to an oligoconsensus labeled with γ-32P. Optimal induction was found after 30 min of stimulation and declined over the next 2 h (Fig. 7⇓A). All subsequent experiments were done at 30 min. AngII caused induction of NF-κB DNA-binding activity, being maximal at 10−9 M (4.3-fold over basal, n = 5, p < 0.05) (Fig. 7⇓B). Two strong inducers of NF-κB activation, LPS (1 μg/ml) and TNF-α (100 U/ml) (15), were used as positive controls in all experiments. Different cell culture preparations of nuclear extracts yielded mainly identical data, with some differences in the relative intensity of basal and positive control. The NF-κB activated by AngII contains several DNA-protein complexes with different electrophoretic mobilities, shown by two bands (Fig. 7⇓). All these complexes were specific because an excess of the same unlabeled oligonucleotide efficiently competed for the formation of the complexes, whereas an oligonucleotide containing mutations, or nonrelated, did not (Fig. 7⇓, A and C).
NF-κB activity in response to AngII stimulation in mesangial cells. A, Time course evolution. Cells were incubated for 30, 60, and 120 min with 10−7 M AngII. B,Dose response. Cells were stimulated with AngII (dose 10−5 to 10−9 M) for 30 min. TNF-α (100 U/ml) was used as positive control. C,Competition assays. The specificity of the reaction was established in AngII-treated nuclear extracts by competition assays with a 100-fold excess mutant NF-κB unrelated (AP-1) and with an unlabeled NF-κB oligonucleotides. Negative control was done by incubation without nuclear extracts. D,Identification of NF-κB complexes. The nuclear extract of AngII-treated cells was preincubated with Abs against the NF-κB subunits p50, p65, and c-Rel. Supershifted bands are observed with anti-p50 and anti-p65 Abs (marked by arrows, longer exposition shown in the right part). The position of the NF-κB complexes and free oligonucleotide is also indicated. Values are representative of an EMSA of five to seven experiments.
Different members of the NF-κB-Rel family could potentially interact with the κB motif, and therefore we used specific Abs to determine the composition of the NF-κB. When AngII-treated nuclear extracts were preincubated for 1 h with 1 μg of anti-p50 and anti-p65 Abs, a supershifted band appeared, and a reduction in the intensity of the complexes was noted with the anti-p65 Ab (Fig. 7⇑D). None of the complexes were inhibited by the Ab against c-Rel (Fig. 7⇑D); thus, this subunit does not seem to be present in mesangial cells as is the case in human vascular smooth muscle cells (44). These results suggest that in mesangial cells, AngII-activated NF-κB complexes contain p50 and p65 subunits.
AngII increases MCP-1 mRNA expression through NF-κB activation in mesangial cells
The nuclear factor NF-κB is involved in the MCP-1 transcription induced by IL-1β, TNF-α, and phorbol esters (16, 45). One potential mechanism of inhibiting NF-κB activation is by reducing oxidant stress (46). The antioxidant PDTC has been shown to block cytokine- and phorbol ester-induced NF-κB activation in several cell lines (46, 47). The preincubation of mesangial cells with 200 μM PDTC for 1 h abolished the AngII-induced NF-κB activation (Fig. 8⇓C), which correlated with the inhibition of AngII-induced MCP-1 mRNA levels (Fig. 8⇓A). These data suggest that the increase in MCP-1 mRNA expression induced by AngII in mesangial cells is at least, in part, NF-κB mediated.
Effect of PDTC in AngII-induced MCP-1 mRNA expression in mesangial cells. Cells were preincubated with PDTC (200 μM) for 1 h and then stimulated with AngII (10−7 M) for 6 h. A, A representative Northern blot of two made, corresponding to hybridization with MCP-1. B, RNA quality was determined by ethidium bromide staining. C, Effect of PDTC in AngII-induced NF-κB activity. Cells were preincubated with PDTC (200 μM) for 1 h and then stimulated with 10−7 M AngII for 30 min. Values are representative of one EMSA of two made.
Discussion
The current study shows that in an experimental model of immune complex nephritis in rats, presenting increased local AngII production, the administration of the ACE inhibitor quinapril diminished renal mononuclear cell accumulation, MCP-1 expression (mRNA and protein), and NF-κB activity. In addition, in cultured glomerular mesangial cells, AngII elicited an up-regulation of MCP-1 gene expression and synthesis in part due to the activation of NF-κB.
Several studies have shown that activation of tissue renin-angiotensin system is involved in the pathogenesis of kidney diseases (20, 26). The fact that the administration of ACE inhibitors reduced the number of inflammatory cells in various models of renal injury (25, 26, 27), as well as the accumulation of mononuclear cells in the kidney of normal rats after 7 to 14 days of systemic AngII infusion (47, 48), suggests that this peptide may be involved in the recruitment of mononuclear cells. In this regard, AngII may participate at many points in the onset and progression of inflammation. AngII induces adhesion molecule expression in human endothelial cells (49) and activates human monocytes leading to increased adhesion to endothelial cells (50). AngII is a chemotactic factor for mononuclear cells (24, 25). Since monocyte-macrophages play an important role in the induction of renal injury (2, 3, 4), we wondered whether AngII may regulate the synthesis of chemoattractant proteins in the kidney. Recent studies have demonstrated that several renal cells can produce MCP-1 after appropriate stimulation (8, 9, 10, 11) and that MCP-1 is overexpressed in the kidney in experimental and human glomerulonephritis (7). Moreover, the administration of anti-MCP-1 Abs to rats with nephrotoxic nephritis decreased glomerular monocyte-macrophage infiltration (51).
In this article, we observe that rats with immune complex nephritis presented increased MCP-1 gene and protein expression. This up-regulation was chiefly located in resident renal cells (glomerular and tubular epithelial cells) and in infiltrating mononuclear cells. The administration of the ACE inhibitor quinapril to rats with established nephritis decreased MCP-1 gene expression and protein levels and the number of infiltrating cells in the glomerular and interstitial areas. In this normotensive model of renal injury, we have recently demonstrated an increase in ACE activity (27) and an up-regulation and redistribution of angiotensinogen, ACE, and AT1 receptor gene expression in renal tissue (52). In addition, in this article we demonstrate that in cultured glomerular mesangial cells AngII can trigger the expression and synthesis of MCP-1, in a manner similar to that of inflammatory cytokines. Therefore, all these results strongly suggest that local AngII through MCP-1 production by resident renal cells may play a central role in the regulation of monocyte recruitment in renal pathology.
We further investigated the signal transduction pathways in response to AngII stimulation in cultured mesangial cells. AngII increases protein tyrosine phosphorylation and activates several protein kinases, including protein kinase C (32, 53, 54). AngII also phosphorylates the STAT family of transcription factors (55), and in vascular smooth muscle cells AngII activates nuclear factors that bind to AP-1 and NF-κB sequences, promoters found in several genes such as TGF-β and MCP-1 (56, 57). The intracellular mechanisms leading to mesangial MCP-1 gene activation in response to different stimuli have not been fully characterized, although this effect appears to be transcriptionally regulated (39). In this paper, we note that preincubation of mesangial cells with PKC inhibitors blocked the MCP-1 gene expression elicited by AngII, suggesting that PKC activation is involved in this process. One feature of primary response genes, including MCP-1, is that in the presence of translation blockers, such as cycloheximide, cytokines cause mRNA superinduction (5), as we have observed in response to AngII. This effect could be due to an increase in mRNA stability or, as recently suggested, to enhanced transcription due to NF-κB superinduction induced by cycloheximide treatment (58).
Deletion analysis of the 5′-flanking region of MCP-1 transfected into tumor cell lines showed that an NF-κB element was required for the cytokine-mediated reporter gene activity (16). We have observed that NF-κB activation preceded up-regulation of MCP-1 gene expression yielded by AngII. The rapid rise of MCP-1 mRNA levels induced by AngII and the kinetics of NF-κB activation are consistent with a role for NF-κB in transcriptional activation of MCP-1 gene. Moreover, such kinetics has been described for MCP-1 in response to cytokines (5), and activation of NF-κB was well correlated with MCP-1 expression in mesangial cells (45). In this sense, we have found that in cultured mesangial cells, the blockade of NF-κB activation with the antioxidant PDTC inhibited AngII-induced up-regulation of MCP-1 mRNA expression, suggesting that this phenomenon may be at least partially NF-κB mediated.
Recently, it has been demonstrated that in murine mesangial cells AngII increases IL-6 synthesis (59) and that in renal interstitial fibroblasts it up-regulates angiotensinogen gene expression (22), these two proteins being regulated by NF-κB. We have observed that in nephritic animals, coinciding with the up-regulation of MCP-1, there was an increase in NF-κB activity in renal cortex that was normalized in response to ACE inhibition. Our results strongly suggest that in vivo activation of transcription factors can be important during kidney damage. Since NF-κB regulates the expression of several genes, including those of chemokines and cytokines, it is possible that AngII could participate in the regulation of the inflammatory process during renal injury through the triggering of other inflammatory genes, besides MCP-1.
Several lines of evidence indicate that reactive oxygen intermediates serve as messengers in NF-κB activation (60). In fact, PDTC decreased NF-κB activation and gene expression of several inflammatory genes (45, 46). In this work, we have observed that in cultured mesangial cells reactive oxygen intermediates may be involved in AngII response since the antioxidant PDTC blocked NF-κB activation. Recent data have shown that oxygen radicals participate in intracellular transduction signals elicited by AngII in myogenic cells, regulating the induction of early genes (61), that can lead to cell proliferation or hypertrophy depending on cell environment. Moreover, hypertension caused by AngII is in part mediated by free radical generation (62).
Several studies have demonstrated that the vasoactive peptide AngII elicits cell proliferation and matrix production, two key events in the progression of renal damage (20, 21, 22). The results reported in this article suggest a novel mechanism by which AngII could participate in this process. AngII could be responsible for the recruitment of mononuclear cells into kidney tissue through the activation of NF-κB and the synthesis of MCP-1. This mechanism might further explain the beneficial effects of ACE inhibitors in progressive renal diseases.
Acknowledgments
We thank Dr. Julia Blanco for technical help with the immunohistochemistry and L. Gulliksen for her secretarial assistance.
Footnotes
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↵1 This work was supported by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social (Spain) (96/2021) and Comision Interministerial de Ciencia y Tecnologia (PM 94/0211, PM 95/0093, PM 97-0085 and SAF 97/0055). M.R-O. and M.H-P. are fellows of Ministerio de Educacion y Ciencia.
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↵2 Address correspondence and reprint requests to Dr. Jesús Egido, Renal Research Laboratory, Fundación Jiménez Díaz, Avenida Reyes Católicos, 2, 28040 Madrid, Spain. E-mail address: Eglom{at}uni.Fjd.es
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↵3 Abbreviations used in this paper: MCP-1, monocyte chemotactic protein-1; AngII, angiotensin II; ACE, angiotensin-converting enzyme; EMSA, electrophoretic mobility assay; PDTC, pyrrolidine dithiocarbamate; PKC, protein kinase C; G3PDH, glyceraldehyde-3′-phosphate dehydrogenase; AP-1, activating protein 1.
- Received May 27, 1997.
- Accepted February 26, 1998.
- Copyright © 1998 by The American Association of Immunologists
References
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