Hormones, Growth Factors, Cell Biology and Structure

Angiotensin III Activates Nuclear Transcription Factor-κB in Cultured Mesangial Cells Mainly via AT2 Receptors: Studies with AT1 Receptor-Knockout Mice

Óscar Lorenzo, Marta Ruiz-Ortega, Yusuke Suzuki, Mónica Rupérez, Vanesa Esteban, Takeshi Sugaya and Jesús Egido
JASN May 2002, 13 (5) 1162-1171;

Abstract

ABSTRACT. Nuclear factor-κB (NF-κB) regulates many genes involved in renal pathophysiologic processes. It was previously demonstrated that angiotensin II (AngII) and its amino-terminal degradation product AngIII activate NF-κB in mesangial cells. However, which are the Ang receptor subtypes involved in the NF-κB pathway and whether these Ang peptides act through the same or different receptors in mesangial cells have not been evaluated. Under the culture conditions used, quiescent rat mesangial cells expressed both AT1 and AT2 receptors. To investigate the receptors involved in the NF-κB pathway, two different approaches were used, i.e., pharmacologic studies, using specific AT1 and AT2 receptor antagonists and agonists, and studies in AT1 receptor-knockout mice. In cultured rat mesangial cells, both AT1 and AT2 receptor antagonists inhibited AngII-induced NF-κB DNA binding activity, whereas NF-κB activation elicited by AngIII was mainly blocked by the AT2 receptor antagonist. Similar results were observed for cytosolic IκBα degradation. An AT2 receptor agonist also activated NF-κB. In AT1 receptor-knockout murine mesangial cells, AngIII and AngII increased NF-κB activity and degraded cytosolic IκBα; both processes were blocked by the AT2 receptor antagonist. These data demonstrate that, in mesangial cells, NF-κB activation is mediated by AT1 and AT2 receptors, suggesting a novel intracellular signaling mechanism for AT2 receptors in the kidney. Some differences in Ang peptide receptor-mediated responses were also observed. AngII activates NF-κB via AT1 and AT2 receptors, whereas AngIII acts mainly via AT2 receptors. These results suggest the potential involvement of the AngIII/AT2 receptor/NF-κB pathway in pathophysiologic processes in the kidney and provide a better understanding of the renin-angiotensin system.

All components of the renin-angiotensin system (RAS), including precursors and enzymes required for the formation and degradation of the biologically active forms of angiotensin (Ang), as well as different receptors, have been identified in the kidney (1). Although AngII has been considered the only effector of the RAS, other peptides, such as AngIII (or des[Asp1]AngII), AngIV, and Ang(1–7), also demonstrate biologic activities (1). AngIII plays an important role in brain and cardiovascular physiologic processes (1). Some actions originally attributed to AngII, such as vasopressin release, are attributable to AngIII (2). Recent data suggested that AngIII could be involved in renal pathophysiologic processes (3–5). In renal cells, AngIII increases angiotensinogen, transforming growth factor-β, fibronectin, and monocyte chemoattractant protein-1 (MCP-1) gene expression (4,5); therefore, AngII could contribute to inflammation and fibrosis in the kidney.

AngII and AngIII elicit cellular responses through their binding to specific receptors, namely AT1 and AT2 receptors (1,6). AngIII binds mainly to AT2 receptors and exhibits lower affinity for AT1 receptors, compared with that of AngII (6). However, whether AngII and AngIII act on the same or different receptors in renal cells is far from being understood. Most AngII effects, such as vasoconstriction, cell proliferation, and matrix production, are mediated by AT1 receptors (6,7). AT2 receptors are involved in cardiovascular and renal pathophysiologic processes, because they regulate BP control, diuresis/natriuresis, cell growth inhibition, and renal inflammatory cell infiltration (6–10). AT2 receptors are abundant in fetal tissues and are re-expressed in tissues undergoing remodeling (6). AT1 receptor antagonists are commonly used for the treatment of hypertension, heart failure, and renal diseases. When AT1 receptors are blocked, plasma AngII levels are increased and AT2 receptor expression is upregulated (6). AngII could then bind to AT2 receptors and/or be degraded to AngIII and other metabolites. The pathophysiologic and clinical consequences of these alternative pathways are unclear.

Nuclear factor-κB (NF-κB) plays a pivotal role in pathologic settings, because it regulates the expression of proinflammatory and profibrogenic genes (11). Recent data suggested a close relationship between the RAS and NF-κB. In experimental models of kidney or vascular injury, Ang-converting enzyme (ACE) inhibition decreased NF-κB tissue activity and the expression of NF-κB-regulated genes, such as cytokines, chemokines, and adhesion molecules, in association with a decrease in inflammatory cell infiltration (12–14). NF-κB inhibitors decreased AngII-induced proinflammatory events and ameliorated renal damage (15). These data suggest that blockade of the AngII/NF-κB pathway might be a therapeutic target in pathologic settings associated with tissue RAS activation. AngII activates NF-κB in vivo (10) and in cultured cells (12,13,16,17) and upregulates several genes under NF-κB control (12,13,17–19). However, the receptor involved in NF-κB/gene regulation has not been fully elucidated. In vascular smooth muscle cells, AngII induced NF-κB-mediated transcription via AT1 and AT2 receptors (19). In those cells, only AT1 receptor antagonists abolished AngII-induced interleukin-6 (IL-6), MCP-1, vascular cell adhesion molecule (VCAM-1), and angiotensinogen gene expression (17–19). In the kidney, both AT1 and AT2 receptors are involved in NF-κB activation (10). Moreover, only AT2 receptor antagonists decreased AngII-induced regulated upon activation, normal T cell expressed and secreted (RANTES) protein expression in glomerular endothelial cells (9). These data indicate the complexity of the NF-κB/gene pathway and suggest that the receptors involved in this process must be defined in each cell system.

In this study, we investigated the receptor subtype involved in AngIII-induced NF-κB activation in cultured mesangial cells, in comparison with the receptor of the AngII/NF-κB pathway. For this purpose, two different strategies were used: a pharmacologic approach, using selective nonpeptide antagonists for AT1 and AT2 receptors, and a genetic approach, using AT1 receptor-knockout mice.

Materials and Methods

Mesangial Cell Cultures

Rat and murine mesangial cells were cultured from isolated glomeruli by using several sieving techniques and differential centrifugation (12). Murine cells were obtained from AT1 receptor-knockout mice, which were generated via a homologous recombination method (20). Mesangial cells were characterized by phase-contrast microscopy and immunohistochemical analyses (positive staining for desmin and vimentin and negative staining for keratin and factor VIII antigen) (12). Confluent cells were serum-starved for 48 h and used between 0 and passage 3.

Determination of Ang Receptors in Mesangial Cells

Total RNA was extracted by using the Trizol method (Life Technologies, Grand Island, NY), and AT2 receptor mRNA expression was analyzed by reverse transcription (RT)-PCR (19). Control experiments were performed with RNA samples but without avian myeloblastosis virus reverse transcriptase. The DNA products were analyzed on 1.5% agarose gels, with ethidium bromide staining. AT1a and AT1b receptor gene expression was assessed by RT-PCR restriction fragment length polymorphism analysis, as described (21). Adrenal gland samples were used as positive controls. Glyceraldehyde-3-phosphate dehydrogenase (G3POH) was used as an internal loading control (12). Northern blotting was performed as described, using a rat AT1 receptor probe (22).

Protein levels were assessed by Western blotting (19). Total proteins were resolved on 12% sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes, blocked (in buffer containing 0.01 mM Tris, pH 7.5, 0.4 M NaCl, 0.1% Tween-20, 1% bovine serum albumin, and 5% milk), and incubated for 18 h at 4°C with AT1 and AT2 receptor-specific antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Detection was performed with peroxidase-conjugated secondary antibody, using an ECL chemiluminescence kit (Amersham, Arlington Heights, IL). Competition studies with blocking peptides were performed as controls (data not shown).

Determination of NF-κB DNA Binding Activity and Cytosolic IκBα Levels

Nuclear and cytosolic extracts from treated cells were prepared by homogenization and centrifugation (19). NF-κB activity was determined in assays of the binding of 5 μg of nuclear proteins to γ-32P-end-labeled NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′) (Promega Corp., Madison, WI), in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM ethylenediaminetetraacetate, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 50 μg/ml poly(dI-dC)] (Pharmacia LKB, Uppsala, Sweden), and then in electrophoretic mobility shift assays. Negative controls without extracts and competition assays with a 100-fold excess of unlabeled or mutant NF-κB or AP-1 (unrelated) oligonucleotides were used to establish specificity (19). The gels were dried and exposed to x-ray film. IκBα levels in cytosolic fractions were quantified by Western blot analysis (19), using a specific antibody against IκBα (Santa Cruz Biotechnology).

Statistical Analyses

Results are expressed as n-fold increases over control values (in arbitrary densitometric units, expressed as mean ± SEM). Significance was established by using GraphPAD Instat (GraphPAD Software, San Diego, CA) in t test and multigroup comparisons, and differences were considered significant at P < 0.05.

Results

Ang Receptors in Mesangial Cells

In adult kidney and cultured renal cells, the presence of AT2 receptors is still controversial, whereas that of AT1 receptors is well established (6,9,23,24). Only a single AT1 receptor gene is present in human subjects, but this receptor is encoded by at least two distinct genes (AT1a and AT1b) in rodents (25). These isoforms share 94% amino acid sequence identity and have indistinguishable binding properties. AT1a receptors are expressed in the same tissues as human AT1 receptors, including kidney, liver, heart, and other tissues, whereas AT1b receptors are detected only in adrenal gland, brain, and testis (21). Using Northern blotting, we and others previously demonstrated the presence of AT1 receptor mRNA in rat kidney and in cultured rat renal cells (24,25), including mesangial cells (Figure 1A). The AT1a receptor gene is expressed in the kidney and in mesangial cells, as demonstrated by PCR (Figure 1C) (21,25). Rat mesangial cells also expressed AT1 receptors at the protein level (Figure 1D) (6). As expected, mesangial cells from AT1a receptor-knockout mice did not express AT1a receptor mRNA or protein (Figure 1C).

Figure1

Figure 1. Angiotensin (Ang) receptors in mesangial cells. (A) Northern blot of AT1 receptors in rat mesangial cells (MC). (B and C) Reverse transcription (RT)-PCR assays. Rat mesangial cells expressed AT1a and AT2 receptor mRNA. AT1 receptor-knockout murine mesangial cells expressed only AT2 receptor mRNA. Positive, total RNA from adrenal glands; Negative, RNA samples without enzyme; WT, wild-type. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a loading control. Representative RT-PCR results of three experiments are presented. (D) Western blot. Mesangial cells from rats and AT1 receptor-knockout mice expressed AT2 receptor protein. Rat mesangial cells also exhibited AT1 receptors. A representative Western blot, of three experiments performed, is presented. Apparent molecular weight are shown on the left.

AT2 receptors have been observed in mesangial cells from Wistar rats but not hypertensive rats (24). Therefore, we first determined whether mesangial cells could express AT2 receptors under our experimental conditions (after 48 h of serum starvation). In RT-PCR experiments, AT2 receptor mRNA expression was observed in growth-arrested mesangial cells from Wistar rats and AT1 receptor-knockout mice (Figure 1B), as demonstrated by a band of 710 bp (19). Both cell types expressed AT2 receptor protein (Figure 1D), as indicated by a band with an apparent molecular mass of 42 kD, corresponding to the predicted AT2 receptor size (19).

Role of AT1 and AT2 Receptors in NF-κB Activation Elicited by AngIII and AngII in Cultured Rat Mesangial Cells

We previously demonstrated that both AngIII and AngII activated NF-κB in mesangial cells (5,12). Both peptides caused dose- and time-dependent increases in NF-κB DNA binding, with maximal responses at 10−9 M after 30 min (5.7- and 5.2-fold over control values for AngIII and AngII, respectively; n = 6, P < 0.05). Selective antagonists and agonists were used to investigate the receptors involved in this process. Growth-arrested cells were preincubated for 60 min with the AT1 receptor antagonist losartan (10−5 to 10−8 M) or the AT2 receptor antagonist PD123319 (10−5 to 10−7 M) and were then stimulated with AngIII or AngII (10−7 M) for 30 min. The AT1 receptor antagonist partially inhibited the NF-κB activation caused by AngIII or AngII (Figures 2 and 3), in a dose-dependent manner (with maximal effects at the concentration of 10−5 M losartan) (Figures 2A and 3A). However, some differences were observed. The AT1 receptor antagonist produced greater inhibition of NF-κB activation induced by AngII, compared with that induced by AngIII (85 and 62% inhibition for AngII and AngIII, respectively, with 10−5 M losartan; n = 6). The AT2 receptor antagonist also decreased the NF-κB activation induced by both peptides, in a dose-dependent manner (with maximal inhibition at the concentration of 10−5 M PD123319) (Figures 2 and 3). Interestingly, the AT2 receptor antagonist produced greater inhibition of NF-κB activation induced by AngIII, compared with that induced by AngII (86 and 78% inhibition for AngIII and AngII, respectively, with 10−5 M PD123319; n = 6). When losartan and PD123319 were added together, the activation of NF-κB elicited by AngIII or AngII was abolished (Figures 2 and 3). None of the AT1 or AT2 receptor antagonists (losartan, PD123319, or GCP42112) alone significantly affected NF-κB activation in control cells (Figure 2). These results suggest that the AngII/NF-κB pathway is mediated via both AT1 and AT2 receptors, whereas AngIII acts mainly via AT2 receptors/NF-κB.

Figure2

Figure 2. Role of AT1 and AT2 receptors in nuclear factor-κB (NF-κB) activation elicited by AngII in cultured mesangial cells. (A) Cells were preincubated for 1 h with AT1 (losartan) or AT2 (PD123319) receptor antagonists (10−5 to 10−8 M). Some cells were pretreated with another AT2 receptor antagonist, GCP42112 (10−5 M). Antagonists were also added together (10−5 M). After the preincubation period, cells were stimulated with 10−7 M AngII for 30 min. In addition, cells were treated with the AT2 receptor agonist p-aminophenylalanine6-AngII (pNH2FAII) (10−7 M) for 30 min. Results are expressed as n-fold increases versus control values, in arbitrary densitometric units (mean ± SEM of four to six experiments). *P < 0.05 versus control. #P < 0.05 versus AngII alone. @P = NS versus control. (B) Representative electrophoretic mobility shift assay. Cells were treated with AT receptor antagonists (10−5 M), AngII (10−7 M), or p-aminophenylalanine6-AngII (10−7 M). The specificity of the reaction for AngII-treated samples was established by using competition assays with a 100-fold excess of unlabeled (cold) NF-κB, mutant NF-κB, or unrelated AP-1 oligonucleotides. The positions of specific NF-κB binding and free oligonucleotide are indicated.

Figure3

Figure 3. AngIII activates of NF-κB mainly via AT2 receptors in cultured mesangial cells. (A) Cells were preincubated for 1 h with losartan or PD123319 (10−5 to 10−7 M) and were then treated with 10−7 M AngIII for 30 min. The two antagonists were also added together (10−5 M). Results are expressed as n-fold increases versus control values, in arbitrary densitometric units (median ± SEM of four to six experiments). *P < 0.05 versus control. #P < 0.05 versus AngIII alone. (B) Representative electrophoretic mobility shift assay. Cells were treated with AT receptor antagonists (10−5 M) and AngIII (10−7 M).

To further demonstrate the role of AT2 receptors in NF-κB activation, we used the AT2 receptor agonist p-aminophenylalanine6-AngII (19). When mesangial cells were treated with p-aminophenylalanine6-AngII (10−7 to 10−9 M) for 30 min, dose-dependent NF-κB activation was observed, with a maximal response at 10−7 M (2.7-fold over control values, n = 4; P < 0.05). Pretreatment of cells with GCP42112, another AT2 receptor antagonist, decreased NF-κB activation induced by AngII (84%, compared with AngII alone); in combination with losartan, GCP42112 abolished AngII-induced NF-κB activation (Figure 2). However, in control cells, GCP42112 also activated NF-κB, demonstrating an agonist effect, as previously reported (19). These data support the existence of the AT2 receptor/NF-κB pathway in mesangial cells.

AngIII and AngII Activation of NF-κB in Cultured Mesangial Cells from AT1 Receptor-Knockout Mice

Studies with AT1a receptor-knockout mice demonstrated a role for the AngII/AT1 pathway in immune nephritis and lymphocyte proliferation (26,27). To further investigate whether Ang peptides could activate NF-κB via AT2 receptors, we performed experiments in cultured mesangial cells obtained from AT1a receptor-knockout mice.

In AT1 receptor-knockout murine mesangial cells, AngIII and AngII increased NF-κB DNA binding activity (Figure 4). Pretreatment with the AT2 receptor antagonist PD123319 abolished NF-κB activation induced by AngIII or AngII, whereas the AT1 receptor antagonist losartan had no effect (Figure 4). The AT2 receptor agonist also activated NF-κB (threefold over control values) (Figure 4). All of these data confirm the existence of the AT2 receptor/NF-κB pathway in mesangial cells. Some similarities were observed; AngII and AngIII demonstrated similar kinetics, increasing NF-κB binding activity as early as 30 min and decreasing it after 120 min, with similar dose-dependent responses that were maximal at the dose of 10−7 M (Figure 4). Interestingly, AngIII had a greater effect on NF-κB activity than did AngII (3.3- and 2.6-fold over control values at 30 min with 10−7 M AngIII and AngII, respectively), suggesting that the AT2 receptor/NF-κB pathway could be more responsive to AngIII.

Figure4

Figure 4. AngIII and AngII activates of NF-κB in mesangial cells from AT1 receptor-knockout mice. (A) Binding assays. Cells were stimulated with Ang peptides (10−7 M for 30 to 120 min or 10−9 M for 60 min) and with the AT2 receptor agonist p-aminophenylalanine6-AngII (pNH2FAII) (10−7 M for 60 min). Some cells were preincubated for 1 h with AT2 (PD123319, 10−5 M) or AT1 (losartan, 10−6 M) receptor antagonists and were then stimulated with 10−7 M AngIII or AngII for 60 min. Results are expressed as n-fold increases versus control values, in arbitrary densitometric units. Mean ± SEM of three experiments: *P < 0.05 versus control; #P < 0.05 versus AngIII-clone; †P < 0.05 versus AngII-clone. (B) Representative electrophoretic mobility shift assay. The specificity of the reaction was established in competition assays with a 100-fold excess of unlabeled (cold) NF-κB.

Molecular Mechanisms of AngIII-Induced NF-κB Activation

We next investigated which intracellular signals elicited by AngIII, compared with AngII, could be involved in NF-κB activation in mesangial cells, and we attempted to elucidate the differences between AT1 and AT2 receptors. AngII, via AT1 receptors, activates several kinases, including protein kinase C (PKC) and phosphotyrosine kinases (PTK) (6). Cells were preincubated for 60 min with several PKC inhibitors, H-7, staurosporine (a serine-threonine kinase inhibitor, 10−5 M), and bisindolylmaleimide (a highly sensitive PKC blocker, 10−7 M), and were then stimulated for 60 min with 10−7 M AngIII or AngII. The PKC inhibitor bisindolylmaleimide did not affect the NF-κB activation induced by Ang peptides (Figure 5A); similar results were observed with staurosporine and H7 (data not shown). These data suggest that PKC is not involved in this process. Cells were pretreated with the PTK inhibitors genistein, erbstatin, and herbimycin A. Genistein caused marked reductions in NF-κB activity in response to AngIII or AngII (77 and 82% inhibition versus peptides alone, respectively, at 10−6 M, n = 3; P < 0.05) (Figure 5A). Similar results were observed with 10−6 M erbstatin and herbimycin A (data not shown), suggesting that activation of tyrosine phosphorylation could be involved in AngIII-induced NF-κB responses via AT1 receptors. NF-κB activation is also mediated by active oxygen radicals (6). We used the antioxidant superoxide dismutase (10 g/L) and the thiol agent pyrrolidine dithiocarbamate (10−4 M), which is a known NF-κB inhibitor with antioxidant properties. Pretreatment of cells with antioxidants markedly decreased the NF-κB activation elicited by AngIII or AngII (Figure 5A). In mesangial cells from AT1 receptor-knockout mice, antioxidants also decreased the NF-κB activation elicited by AngIII or AngII (Figure 5B). These data suggest that the AT2 receptor/NF-κB pathway is mediated by oxygen radicals acting as second messengers.

Figure5

Figure 5. Molecular mechanisms of AngIII- and AngII-induced NF-κB activation. (A) Rat mesangial cells were preincubated for 1 h with the following inhibitors: the protein kinase C (PKC) inhibitor bisindolylmaleimide (10−7 M), the phosphotyrosine kinase (PTK) inhibitor genistein (10−6 M), and the antioxidants superoxide dismutase (SOD) (10 g/L) and pyrrolidine dithiocarbamate (PDTC) (10−4 M). (B) AT1 receptor-knockout mesangial cells were preincubated for 1 h with the antioxidants SOD (10 g/L) and PDTC (10−4 M). The cells were then stimulated with 10−7 M AngII or AngIII for 60 min. Values are presented as means ± SEM of three to five experiments. *P < 0.05 versus control. #P < 0.05 versus AngIII. †P < 0.05 versus AngII.

Loss of IκBα in Response to AngIII Correlates with NF-κB Activation in Mesangial Cells from AT1 Receptor-Knockout Mice and the Role of AT2 Receptors

NF-κB activation occurs after dissociation of its inhibitory subunit IκB, by protein phosphorylation and proteasome-mediated degradation in the cytoplasm (11). Therefore, we evaluated the effects of AngIII and AngII on cytosolic IκBα protein levels.

In control AT1 receptor-knockout murine mesangial cells, IκBα was observed as a cytosolic protein of approximately 39 kD. After 60 min of AngIII stimulation, this band disappeared, suggesting degradation of IκBα (Figure 6A). This effect was maximal after 60 min and was closely correlated with the time course of the increase in NF-κB DNA binding activity (as assessed in electrophoretic mobility shift assays). The AngIII-induced IκBα degradation was abolished by the AT2 receptor antagonist, whereas losartan had no effect (Figure 6A). The effect of AngII on cytosolic IκBα levels was slightly different, with a faster weaker response (Figure 6A). These data suggest that AT2 receptors participate in IκBα degradation.

Figure6

Figure 6. IκBα levels in Ang-stimulated AT1 receptor-knockout murine (A) and rat (B) mesangial cells. (A) Mesangial cells from AT1 receptor-knockout mice were stimulated for 30 to 120 min with 10−7 M AngIII or AngII. (A and B) Cells were preincubated for 1 h with losartan (10−6 M) or PD123319 (10−5 M) and were then treated with 10−7 M AngIII or AngII for 60 min. Cytosolic extracts were subjected to electrophoresis under reducing and denaturing conditions, stained with an anti-IκBα antibody, and analyzed with enhanced chemiluminescence detection of IκBα protein levels. Ponceau staining and tubulin were used as loading controls. Results are presented as representative Western blots of three (A) or four (B) experiments (upper) and densitometric analyses (lower) of mean ± SEM: #P < 0.05 versus AngIII; †P < 0.05 versus AngII.

In rat mesangial cells, cytosolic IκBα was degraded after 30 min of stimulation with AngIII or AngII (Figure 6B), as previously demonstrated (5,19). Pretreatment with AT1 or AT2 receptor antagonists partially inhibited IκBα degradation induced by AngIII or AngII, suggesting that both AT1 and AT2 receptors are involved in this process (Figure 6B).

Discussion

We have observed that, although both AngII and AngIII activate the transcription factor NF-κB, the receptor subtypes stimulated by the two peptides are different. The AngII/NF-κB pathway is mediated by both AT1 and AT2 receptors, whereas the AngIII/NF-κB pathway is mediated mainly by AT2 receptors.

Many studies have investigated the renal effects of AngII (7). This peptide causes vasoconstriction, cell proliferation, extracellular matrix accumulation, and inflammatory cell infiltration (6–10). AngIII shares some of these effects, including vasoconstriction and induction of several genes, such as c-fos, matrix proteins, and chemokines (4,5,28). However, whether AngII and AngIII act through the same receptors in the same cellular responses requires further elucidation. Most of these common responses are AT1 receptor-mediated, such as vasoconstriction, perfusion pressure alteration, calcium mobilization, and c-Fos induction (1,28,29). Some other actions are opposite and are also mediated by AT1 receptors, such as antinociception (30). AngII and AngIII, via AT2 receptors, mediate the depressor phase of the biphasic pressure response (31) and reduce the activity of α2-adrenoreceptors (30). AT2 receptors seem to be involved in AngII-induced cell growth inhibition/apoptosis and mononuclear cell infiltration (6,9), although whether AngIII causes some of these effects has not been investigated. Interestingly, AngIII produces tonic inhibitory modulation of the baroreceptor reflex response by acting on both AT1 and AT2 receptors, whereas AngII engages only AT1 receptors (32). Both receptor types are also involved in collagen synthesis (33), nitric oxide (NO) release (34), and NF-κB activation (10,19). Our pharmacologic studies with specific receptor antagonists and agonists and our genetic studies with cells from AT1 receptor-knockout mice demonstrated that AngII activates NF-κB via both AT1 and AT2 receptors, whereas AngIII/NF-κB is mediated mainly by AT2 receptors.

The fact that Ang peptides cause NF-κB activation via different receptors could have important pathologic and clinical implications. Elevated NF-κB activity has been observed during tissue injury (11–13). NF-κB controls many proinflammatory genes (11), some of which are upregulated by AngII (12,13,17–19). However, the receptor subtypes involved in these processes are not completely defined. In cultured cells, AngII upregulates IL-6, VCAM-1, and MCP-1 via the AT1 receptor/NF-κB pathway (17–19). In vascular smooth muscle cells, both AT1 and AT2 receptors mediate AngII-induced NF-κB DNA binding and transcription of a NF-κB reporter gene (19). In glomerular endothelial cells, AngII upregulates gene expression of RANTES, a chemokine under NF-κB control (9). Moreover, in these cells and in COS-7 cells transfected with AT1 and AT2 receptors, AngII increases NF-κB DNA binding activity and stimulates the transcription of a NF-κB reporter gene (35). In mesangial and mononuclear cells, AngIII upregulates MCP-1 (5), although whether AngIII could regulate the expression of other proinflammatory genes and which receptors are involved have not been investigated. Other important candidates regulated by the AT2 receptor/NF-κB pathway are inducible NO synthase and cycloxygenase-2 (8). NO production is mediated by AT2 receptors in the kidney (36), although it is AT1 receptor-dependent in other tissues, such as rat carotid artery (37). AT1 and AT2 receptors mediate nitrite release caused by AngII and AngIII in large arteries and coronary microvessels in normal dog heart (34). The intracellular signals elicited after activation of the AT1 and AT2 receptors are different (6). We have demonstrated that, in mesangial cells and vascular smooth muscle cells (10,19), the two receptor types share a common molecular pathway: the activation of NF-κB. We observed some differences between the AT1 and AT2 receptor signaling systems. PTK inhibitors decreased AngII- and AngIII-induced NF-κB activation, demonstrating that PTK mediates the AT1 receptor/NF-κB pathway. In contrast, we observed that PKC is not involved in AngII- or AngIII-induced NF-κB activation. In addition, a variety of structurally diverse antioxidants blocked the activation of NF-κB elicited by AngII and AngIII in wild-type and AT1 receptor-knockout mesangial cells, suggesting that oxygen radicals act as intermediates for both AT1 and AT2 receptors, as occurs with other stimuli (such as okadaic acid, tumor necrosis factor-α, and IL-1β) (19). Although future studies are necessary to establish the receptors involved in the regulation of proinflammatory genes in different tissues, our results indicate that both peptides, via AT1 and/or AT2 receptors, activate NF-κB and thus could participate in the inflammatory process.

Several studies demonstrated that AT1 receptor antagonists were as efficient as ACE inhibitors in some renal diseases, although differences in the inflammatory responses were observed (38–40). In experimental models, ACE inhibitors decreased mononuclear cell infiltration, renal NF-κB activity, and related gene expression (such as MCP-1 and VCAM-1) (12,14). In a unilateral ureteric obstruction model, interstitial monocyte/macrophage infiltration and expression of MCP-1 and VCAM-1 were reduced only by ACE inhibitors but not by AT1 receptor blockers (39). In anti-thymocyte serum-induced nephritis, AT1 receptor antagonists only partially decreased glomerular monocyte/macrophage infiltration and MCP-1 expression (40). AngII induces monocyte binding to endothelial cells and the expression of adhesion molecules via AT1 receptors (17). In AngII-infused rats, only an AT2 receptor antagonist decreased glomerular monocyte infiltration and RANTES expression (9), suggesting that AT2 receptors could participate in renal inflammation. In models of renal injury and AngII infusion, both AT1 and AT2 receptor antagonists decreased renal NF-κB activity (10,41). All of these data indicate that the Ang receptor subtype involved in these inflammatory responses in vivo is controversial. In fact, clinical studies investigating AT1 receptor antagonists in renal diseases are ongoing. However, clinical studies in the cardiovascular field revealed that ACE inhibitors were more effective than AT1 receptor antagonists (42–44). The pharmacologic differences between ACE inhibitors and AT1 receptor blockers could be explained by their effects on substrates other than AngII, such as bradykinin or NO (7). However, there are other potential explanations. AT1 receptor blockade leads to more free ligand (AngII), which could bind to AT2 receptors and then produce some beneficial effects, including vasodilation and antiproliferative/apoptotic responses (6); there is, however, little evidence for this mechanism in renal tissue. In addition, AngII can be degraded to other metabolites, such as AngIII, which could bind to AT2 receptors and cause some of the effects described above.

Renal concentrations of AngII and its degradation products are higher than concentrations in blood (45). AngII is converted into AngIII in vivo by aminopeptidase A (APA) (1), which in the kidney is expressed in glomeruli, the brush border of proximal tubules, and along the nephron (45–47). In pathologic situations associated with RAS activation, such as hypertension, diabetes mellitus, and nephritis, renal APA mRNA and activity levels are increased (46,47), suggesting that local AngIII production could be elevated. Experimental evidence suggests that AngIII could play an active role in tissue injury. AngIII is a chemoattractant for polymorphonuclear leukocytes and monocytes and regulates chemokine production (5,48). AngIII causes proteinuria and increases expression of growth-related and profibrotic genes (3,4). Inasmuch as elevated renal APA and AT2 receptor expression and NF-κB activity levels have been observed during kidney injury (6,11,12,46,47), AngIII, acting via the AT2 receptor/NF-κB pathway, could participate in the progression of renal damage. However, AT1 receptor blockade stimulates APA activity (49), suggesting a potential role for AngIII via AT2 receptors. Although more studies are necessary, our data demonstrating that AngII and AngIII activate NF-κB via AT2 receptors could explain the observed differences between ACE inhibitors and AT1 receptor antagonists in inflammatory responses in various experimental models of kidney diseases.

Another important NF-κB-controlled gene induced by AngII and AngIII is angiotensinogen, which is upregulated by AT1 receptors/NF-κB in hepatocytes (50); this observation suggests the involvement of an autocrine reinforcing loop in local AngII (and AngIII) production, which could contribute to the perpetuation of tissue damage. These data suggest that a variety of genes associated with renal damage can be upregulated by the NF-κB pathway, via AT1 and/or AT2 receptors.

Our studies, using selective nonpeptide antagonists for AT1 and AT2 receptors and cells from AT1 receptor-knockout mice, demonstrate that in mesangial cells AngIII activates NF-κB mainly via AT2 receptors, whereas AngII acts via both AT1 and AT2 receptors. These data suggest the potential involvement of the AngIII/AT2 receptor/NF-κB pathway in renal diseases. In addition, they could explain some observed differences between ACE inhibitors and AT1 receptor antagonists in experimental renal diseases (mainly in the recruitment of mononuclear cells). Further studies are needed to determine whether these differences also have some implications in human diseases.

Acknowledgments

This work was supported by grants from Comunidad Autonoma de Madrid (Grant 08.4/0017/2000), Fondo de Investigacion Sanitaria (Grant 99/0425), and the Fundación Renal Iñigo Alvarez de Toledo. Drs. Lorenzo and Esteban are Fellows of the FIS, and Dr. Rupérez is a Fellow of the Fundación Conchita Rábago. We thank Professor Yasuhito Tomino (Juntendo University, Japan) for his support to Y. Suzuki.

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Angiotensin III Activates Nuclear Transcription Factor-κB in Cultured Mesangial Cells Mainly via AT2 Receptors: Studies with AT1 Receptor-Knockout Mice
Óscar Lorenzo, Marta Ruiz-Ortega, Yusuke Suzuki, Mónica Rupérez, Vanesa Esteban, Takeshi Sugaya, Jesús Egido
JASN May 2002, 13 (5) 1162-1171;
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