FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (γ−/−) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between γ−/− and AT1−/− mice, we found that glomerular injury in γ−/− mice was associated with CD4+ T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-γ-inducible protein-10 and macrophage-inflammatory protein 1α) expression was markedly reduced in mesangial cells from AT1−/− mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-γ-inducible protein-10 was NF-κB-dependent, macrophage-inflammatory protein 1α was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.

The inflammatory cascade mediated by the Ab/immune complex (IC)3 has been redefined by intensive studies using targeted gene disruption of IgR (FcR) (1, 2). These studies have clarified that FcR is a critical molecule to initiate cellular response in type II and III inflammation. Studies with mouse strains lacking functional FcRs (γ−/−) also demonstrated their crucial roles in the acute phase of anti-glomerular basement membrane (GBM) Ab-induced glomerulonephritis (GN) (3, 4), which is one of the most important models available for assessing inflammatory mediators in both humoral and cellular immunity (5). Although γ−/− mice with anti-GBM disease were protected from acute polymorphonuclear cell (PMN) influx and subsequent lethal endothelial damage observed in their wild-type (WT) littermates (3), they still developed glomerular injury characterized by mesangial proliferation with monocyte/macrophage accumulation in the later phase of this disease (3).

In contrast, Holdsworth and Tipping and coworker (6) have convincingly demonstrated that glomerular-accumulated T cells are responsible for the major component of glomerular injury, mainly glomerular crescents, independent of the presence of autologous Ab in this disease (7). Those T cells have a Th1 phenotype (6) and require MHC class II expression by renal resident cells (RRC) for their sufficient effector response (8). Moreover, the crescentic formation is associated with mesangial proliferation and macrophage infiltration (6), indicating that glomerular delayed-type hypersensitivity (DTH) injury closely resembles the lesions observed in the γ−/− mice.

Angiotensin (Ang)II is a growth factor that regulates cell proliferation and extracellular matrix synthesis beyond its hemodynamic effect (9, 10, 11). Some studies have also revealed that AngII participates in cellular recruitment and adhesion (11, 12). Indeed, studies with Ang-converting enzyme (ACE) inhibitors or Ang type 1 receptor (AT1) blocker suggest that the renin-Ang system (RAS) contributes to the pathogenesis of inflammatory diseases, such as immune-mediated GN and allograft rejection (13, 14). However, the mechanisms of the beneficial effects of RAS blockade in those diseases are still unclear.

Although the precise mechanisms remain undefined, previous studies have demonstrated that Ab deposition onto GBM strongly activates the intrarenal (15) and systemic RAS (16, 17), inducing hemodynamic changes in a dose-dependent manner. Surprisingly, the AT1 blocker drastically attenuated glomerular injury and accumulation of macrophages in γ−/− GN (3). Furthermore, the activated RAS may participate early in the pathogenesis of this disease. Those findings highlighted a certain role of RAS in immune renal injury.

We hypothesized that RAS activation plays an essential role in the susceptibility of local cellular immune response. In the kidney and lymphocytes, AngII exerts its biological effects mainly via AT1 (18). In rodents, AT1 exists in two isoforms, AT1A and AT1B, regulated by two different genes. The murine AT1A is the isoform predominantly expressed in most tissues (19). AngII via AT1A triggers the proliferation of splenic lymphocytes following systemic cellular immune responses in mice (20). A recent study has shown that renal AT1 may play a role in the progression of this immune disease (21). However, the implication of AngII on T cell recruitment in the kidney as well as the molecular mechanisms involved is not yet defined. To circumvent the potential contribution of FcR in this process, some particular experiments were designed. For analyzing relevant cell types (resident or inflammatory cells) and the RAS system (local or systemic), we generated bone marrow chimeras between γ−/− and AT1A-deficient (AT1−/−) mice and induced anti-GBM GN. Based on the in vivo findings, we also examined the potential mechanisms coupling RAS activation and the cellular immune response, such as chemokine expression and T cell recruitment. In addition, we further studied intracellular events involved in cell signaling with special attention to transcriptional factors as mediators of the AngII-induced inflammatory process (11, 12), including NF-κB (22, 23) and NF-AT (24, 25). Our present findings show a novel mechanism in the pathogenesis of IC disease and propose the potential therapeutical interest of RAS blockade in immune renal diseases.

FcR γ-chain-deficient (γ−/−) and AT1AR-deficient (AT1−/−) mice were generated by a homologous recombination method. Construction of targeting vectors and generation of these knockout mice have been previously described in detail (3, 4, 16). In all in vivo experiments, we used female animals weighing 18–23 g. Both γ−/− and AT1−/− mice have a C57BL/6 background. Although WT littermates of each strain (γ+/+, AT1+/+) and C57BL/6 mice were analyzed in all experiments, no significant differences were found in the kinetics of proteinuria and glomerular/interstitium damage, as noted in our previous studies (3, 16). Thus, results of C57BL/6 mice matched for age were shown as representative controls of WT.

Bone marrow transplantation (BMT) was performed to 6- to 8-wk-old female γ−/−, AT1−/−, and WT mice. Bone marrows were collected from each mouse strain and treated with Gay’s solution to exclude RBC contamination for protection against vascular (thrombotic) injury, and then were transplanted i.v. (3–5 × 107 bone marrow cells) to mice which had been irradiated at 800 rad x-ray with renal protection by lead plates. Because all mice had a C57BL/6 background, there were no symptoms of graft-vs-host-disease in any of them. For an additional 4 wk, transplanted animals were kept in air-conditioned clean cages. We generated three different bone marrow chimeras as follows: γA (bone marrow from γ−/− to irradiated AT1−/−), γW (γ−/− to WT), and WA (WT to AT1−/−). In a pilot study, WW (WT to WT), γγ (γ−/− to γ−/−), and AA (AT1−/− to AT1−/−) mice were also generated as controls.

Genotype exchange in peripheral blood of each bone marrow chimera (γA and γW) was determined by PCR with purified genomic DNA from peripheral blood before and 5 wk after BMT (QIAamp Blood kit; Qiagen, Hilden, Germany). Primers used for murine FcR γ-chain were as follows: specific primer for exon 3 (5′-GGAATTCGCTGCCTTTCGGACCTGGAT-3′) and exon 2 (5′-GGAATTCGATGCTGTCCTGTTTTTGTA-3′) and for the created neo γ-chain (4) of which exon 2 was replaced (5′-GCCAACGCTATGTCCTGATAG-3′). PCR was simultaneously performed with these primers under the following conditions: 94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min with 33 cycles. After the confirmation of genotype exchange, these chimeras were subjected to the experiments.

The method for preparation of nephrotoxic serum (NTS) was previously described (3). In the present study, we used batches of NTS different from those of our previous study (3) to avoid the possibility that RAS-related injury in γ−/− mice was dependent on the batch. Anti-GBM GN was induced by i.v. injection of NTS through the tail vein in mice which were preimmunized with rabbit IgG and IFA (Difco, Detroit, MI) 4 days before the administration of NTS, and followed until day 150. Because a preliminary study showed that NTS at a dose of 20 μl/20 g body weight was sufficient to cause proteinuria and severe renal damage in WT mice, we used this dose of NTS (1× NTS) for general experiments and a 3-fold higher dose (60 μl/20 g) for the excess NTS model (3× NTS model) in γ−/− mice and their chimeras. No mice developed anaphylactic symptoms after the injection of NTS.

Urinary protein was determined at days 1, 3, 5, 7, and 10 and once a week after day 14 until day 50, and every 10 days after day 50 by Knight’s method, as previously described (3). To be sure of the disease kinetics in acute phase (before day 50), we also checked their spontaneous urine production when we moved them for cleaning cages and therefore confirmed that urinary protein, depicted in figures, well-represented the outcome of the disease. Kidneys were perfused with cold saline and removed under general anesthesia. For the evaluation of the effect of RAS blockade, γ−/− mice were treated with the AT1 blocker valsartan (Novartis Pharmaceuticals, Tokyo, Japan; 10 mg/kg/day orally) 24 h before the injection of NTS.

Kidney sections, fixed in 10% formaldehyde, were stained with periodic acid Schiff’s reagent in 4-μm-thick sections to assess histological changes by light microscopy. Frozen renal sections were used for immunofluorescence for rabbit and murine IgG, C3, CD4+ T cells, and then stained with FITC-labeled Abs (ICN Pharmaceuticals, Frankfurt, Germany; DAKO, Barcelona, Spain; and BD PharMingen, San Diego, CA). Mesangial proliferation was evaluated by the numbers of mesangial cells (MC) in one glomerular tuft (score 0, 0–2 cells; 1, 3–4; 2, 5–6; 3, 7–8; 4, >8). Glomerular endothelial damage was scored by the percentage of fibrin deposition occupancy in one glomerulus (score 0, 0%; 1, 0–25; 2, 25–50; 3, 50–75; 4, >75). At least 25 glomeruli of one animal and five animals of each group were examined. The mean scores of each group were expressed in Table I as follows: 0–1, (−); 1–2, (+); 2–3, (++); 3–4, (+++). For the evaluation of CD4+ T cells, at least 30 glomeruli per section were examined using a blinded protocol as previously described (26). The results were expressed as cells per glomerular section.

Table I.

Receptor phenotypes and glomerular lesions in each mouse straina

Phenotype of FcR and AT1Phenotype of Glomerular Lesion
FcRAT11× NTS3× NTS
BMCRRCBMCRRCend.damagemes.prolifmes.prolifCrescents
WT +++ − nd nd 
γ−/− − − − +++ ++ 
AT1−/− − − +++ − nd nd 
γA − − − − − − 
γW − − +++ ++ 
WA − +++ − nd nd 
Phenotype of FcR and AT1Phenotype of Glomerular Lesion
FcRAT11× NTS3× NTS
BMCRRCBMCRRCend.damagemes.prolifmes.prolifCrescents
WT +++ − nd nd 
γ−/− − − − +++ ++ 
AT1−/− − − +++ − nd nd 
γA − − − − − − 
γW − − +++ ++ 
WA − +++ − nd nd 
a

3× NTS, 3-fold higher amounts of NTS; nd, not done, because most of them died; end.damage, glomerular endothelial damage; mes.prolif, mesangial proliferation; crescents, glomerular crescents.

Murine MC (WT and AT1−/−) were cultured from isolated glomeruli by several sieving techniques and different centrifugation as previously described (23), and maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FCS, 1 mM L-glutamine, and 100 μg/ml penicillin/streptomycin. MC were characterized by phase contrast microscopy and immunohistochemistry (positive staining for desmin and vimentin, and negative staining for keratin and factor VIII Ag) (23, 27). Confluent cells between the first and third passages were used for assays.

After a 48-h starvation, both WT and AT1−/− MC were stimulated with AngII 10−6 M in serum-free medium for 3, 6, 12, and 24 h. Supernatants from stimulated MC were collected for chemotaxis assays. For inhibition assays, MC were preincubated with a NF-κB inhibitor, 10 μM parthenolide (Sigma-Aldrich, Madrid, Spain) for 1.5 h (28), or with calcineurin (CaN)/NF-AT inhibitors, 1 μM cyclosporin A (CsA; Sigma-Aldrich), or 10 μM CaN autoinhibitory peptide 457–482 (29) (Calbiochem, Darmstadt, Germany) for 2 h. After exposure to these inhibitors, the culture medium was removed and cells were washed with serum-free medium and subjected to the experiments.

The chemotactic activity of MC supernatants was evaluated in 24-well Transwell chemotaxis chambers (Costar, High Wycombe, U.K.) as previously described (30). The lower wells were loaded in triplicate with 600 μl of the supernatants and covered with a 5-μm pore-size polycarbonate membrane. Upper compartments were loaded with 100 μl of the cell suspension containing 5 × 105 T cells (Jurkat cell; ATCC TIB-152; American Type Culture Collection, Manassas, VA). The chambers were incubated at 37°C for 4 h to assess chemotaxis of T cells. Migrating cells in the lower compartment were counted by flow cytometry. Specific chemotaxis data represent the fold-increase of the average number of migrated cells with each MC supernatant vs the stimulation medium alone (serum-free medium with AngII 10−6 M).

Total mesangial RNA was obtained by the TRIzol method (Life Technologies). One microgram RNA from stimulated MC was reverse-transcribed and then amplified with a commercial kit (Promega, Buckinghamshire, U.K.), with the use of 0.5 μCi [α-32P]dCTP (3000 Ci/mmol, Amersham, Arlington Heights, IL) and 20 pmol specific primers for mouse IFN-γ-inducible protein (IP)-10 (sense, 5′-CAACCCAAGTGCTGCC-3′; antisense, 5′-GGGAATTCACCATGCTTGACCA-3′; fragment, 475 bp, ref. AF 227743) (31), mouse macrophage-inflammatory protein (MIP) 1α (sense, 5′-GCTGTCCTCC-TCTGCACCAT-3′; antisense, 5′-CTGCCGGCTTCGCTTGGTTA-3′; fragment, 189 bp, ref. NM 002983) (32), and mouse GAPDH (sense, 5′-CCGGTGCTGAGTATGTAGTG-3′; antisense, 5′-CAGTCTTCTGAGTGGCAGTG-3′; fragment, 289 bp, ref. AK 013857). The amplifications were conducted with annealing temperatures of 61°C (IP-10), 62°C (MIP1α), or 59°C (GAPDH). The optimum number of amplification cycles used for semiquantitative RT-PCR (30, 32, and 25, respectively) was chosen on the basis of pilot experiments (data not shown). In some cases, PCR products of IP-10 and MIP1α were purified from low-melting temperature agarose gel, radiolabeled with Random Primed DNA Labeling kit (Roche, Indianapolis, IN), and used as cDNA probes for hybridization in Northern blot analysis. The expression of GAPDH was used as internal control. Aliquots of each reaction were run on 4% acrylamide-bisacrylamide gels. The gels were dried and exposed to X-OMAT AS films (Eastman Kodak, Madrid, Spain). Autoradiograms were quantified by the Image Quant scanning densitometry (Molecular Dynamics, Sunnyvale, CA). The density of each gene was compared after the individual correction by density of GAPDH.

Twenty-five micrograms of denatured RNA were electrophoresed and transferred to nylon membranes (Genescreen; New England Nuclear, Boston, MA). The membranes were prehybridized for 6 h at 42°C in 50% formamide, 1% SDS, 5× SSC, 1× Denhardt’s solution, 0.1 mg/ml denatured salmon sperm DNA, and 50 mM PBS, pH 6.5. The hybridization was performed at 55°C for 20 h with 10% dextran sulfate and 1 × 106 cpm/ml of labeled denatured cDNA probe. Membranes were washed, autoradiographed, and films were scanned using the scanning densitometry (Molecular Dynamics). Relative amounts of mRNA were established in relation to 28S rRNA.

Nuclear extracts were obtained as previously described (33) and the activity of transcription factors was evaluated by EMSA. Briefly, frozen kidney pieces were pulverized in a metallic chamber and resuspended in a cold extraction buffer (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, 1 μg/ml pepstatin A). The homogenate was vigorously shaken, and the insoluble materials were precipitated by centrifugation at 12,000 rpm for 30 min at 4°C. Supernatants were dialyzed overnight against a binding buffer containing 20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF. These dialysates were cleared by centrifugation at 10,000 × g for 15 min at 4°C and stored in aliquots at −80°C until use. Protein concentration was quantified by the bicinchoninic acid method (Pierce, Rockford, IL).

NF-AT consensus oligonucleotides (5′-CGCCCAAAGAGGAAAATTTGTTTCATA-3′) (Santa Cruz Biotechnology, Santa Cruz, CA) were [32P]-end-labeled by incubation for 10 min at 37°C with 10 U T4 polynucleotide kinase (Promega) in a reaction containing 10 μCi [γ-32P]ATP (3000 Ci/mmol; Amersham), 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 proteins (10 μg) were 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 2 μg poly(dI-dC) for a 20-μl final volume. When competition and supershift assays were performed, the cold probe and Abs (anti-NF-ATc4; 0.5 μg)(Santa Cruz Biotechnology) were added to this buffer 30 and 60 min before the addition of the labeled probe. Labeled probe (0.035 pmol) was added to the reaction and incubated for 30 min at room temperature. The reaction was stopped by the addition of gel loading buffer (250 mM Tris-HCl, 0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) and run on a nondenaturing, 4% acrylamide gel at 100 V at room temperature in 89 mM Tris-borate, 2 mM EDTA (pH 8.0; TBE) (22).

This technique was developed to detect the in situ distribution and DNA-binding activity of transcriptional factors (34). NF-AT consensus oligonucleotide was digoxigenin-labeled with a 3′-terminal transferase (Boehringer Mannheim, Mannheim, Germany). Paraffin-embedded tissue sections were fixed in 0.5% paraformaldehyde and incubated with 0.1 mg/ml DNase I. The DNA binding reaction was performed by incubation with 50 pmol of the labeled DNA probe in buffer containing 0.25% BSA and 1 μg/ml poly(dI-dC). The sections were then incubated with alkaline phosphatase-conjugated anti-digoxigenin Ab, and colorimetric detection was performed as described. Preparations without probe were used as negative controls, and mutant-labeled probe and excess of unlabeled probe were used to test the specificity of the technique.

Mice were immunized i.p. with Ag (250 μg goat Ig) emulsified in CFA (8, 35). After 7 days, immunized mice were challenged with the same Ag (250 μg) in the hind footpad. For both induction of anti-GBM GN and DTH in γ−/− mice, those mice were immunized with rabbit IgG 3 days after DTH preimmunization and were injected with 3-fold higher NTS at the same time of the Ag challenge. DTH responsiveness was determined 24-h postchallenge by measuring the dorsal-ventral thickness difference of the Ag-injected left footpad and the saline-injected right footpad, as a control, using a micrometer (Mitutoyo, Kanagawa, Japan).

Results are expressed as mean ± SD and were analyzed by ANOVA (see Fig. 1) and Mann-Whitney test (see Figs. 3–5) for comparison of quantitative variables. Statistical significance was established as p < 0.05 (two-tailed curve).

FIGURE 1.

FcR on BMCs and tissue AT1 are critical for the induction of anti-GBM GN. Renal injury in mice was assessed by urinary protein excretion until day 150. Acute glomerular damage peaking at day 7 was dependent on FcR on BMCs (A). Although higher amounts of NTS (3× NTS) were required, mice strains lacking FcR on BMCs also showed glomerular damage in a later phase peaking at day 14, which was dependent on tissue AT1 (B). ∗, p < 0.01, ∗∗, p < 0.05, (A) vs γ−/−, (B) vs γA.

FIGURE 1.

FcR on BMCs and tissue AT1 are critical for the induction of anti-GBM GN. Renal injury in mice was assessed by urinary protein excretion until day 150. Acute glomerular damage peaking at day 7 was dependent on FcR on BMCs (A). Although higher amounts of NTS (3× NTS) were required, mice strains lacking FcR on BMCs also showed glomerular damage in a later phase peaking at day 14, which was dependent on tissue AT1 (B). ∗, p < 0.01, ∗∗, p < 0.05, (A) vs γ−/−, (B) vs γA.

Close modal
FIGURE 3.

AngII enhances the chemotactic activity for T cells and the mRNA expression of Th1-associated chemokines in MC through AT1. Supernatants from AngII (10−6 M) -stimulated WT MC (○) showed significantly higher chemotactic activity for T cells than those from AT1−/− MC (•) (A). In WT MC stimulated with AngII (10−6M) (□), mRNA expression of Th1-associated chemokines (IP-10 and MIP1α) was higher than those in AT1−/− MC (▪), as determined by RT-PCR (B) and Northern blotting (C). Data are presented as the mean ± SD (n = 4–5 experiments). ∗, p < 0.05 (B), ∗, p < 0.01 (C) vs AT1−/− MC.

FIGURE 3.

AngII enhances the chemotactic activity for T cells and the mRNA expression of Th1-associated chemokines in MC through AT1. Supernatants from AngII (10−6 M) -stimulated WT MC (○) showed significantly higher chemotactic activity for T cells than those from AT1−/− MC (•) (A). In WT MC stimulated with AngII (10−6M) (□), mRNA expression of Th1-associated chemokines (IP-10 and MIP1α) was higher than those in AT1−/− MC (▪), as determined by RT-PCR (B) and Northern blotting (C). Data are presented as the mean ± SD (n = 4–5 experiments). ∗, p < 0.05 (B), ∗, p < 0.01 (C) vs AT1−/− MC.

Close modal
FIGURE 4.

AngII-induced chemotactic activity and chemokine expressions in MC involve CaN/NF-AT and NF-κB pathways. A, Chemotactic activity for T cells in AngII-induced WT MC (10−6 M, at 6 and 12 h, Fig. 3 A), was markedly attenuated by preincubation with CaN/NF-AT inhibitors, CsA and CaN autoinhibitory peptide (CaN inhibitory P.), whereas NF-κB inhibitors (parthenolide) had less effect. AT1−/− MC also showed a similar response at 6 h. ∗, p < 0.05 vs without inhibitors. (M, control medium; TM, medium with AngII 10−6 M). B, Th1-associated chemokines were transcriptionally regulated in a different manner. Thus, mRNA expression of IP-10 in WT MC was drastically attenuated by pretreatment with parthenolide, but not with CsA. In contrast, CsA attenuated MIP1α mRNA expression more markedly than parthenolide. Data are presented as the mean ± SD (n = 3–4 experiments).

FIGURE 4.

AngII-induced chemotactic activity and chemokine expressions in MC involve CaN/NF-AT and NF-κB pathways. A, Chemotactic activity for T cells in AngII-induced WT MC (10−6 M, at 6 and 12 h, Fig. 3 A), was markedly attenuated by preincubation with CaN/NF-AT inhibitors, CsA and CaN autoinhibitory peptide (CaN inhibitory P.), whereas NF-κB inhibitors (parthenolide) had less effect. AT1−/− MC also showed a similar response at 6 h. ∗, p < 0.05 vs without inhibitors. (M, control medium; TM, medium with AngII 10−6 M). B, Th1-associated chemokines were transcriptionally regulated in a different manner. Thus, mRNA expression of IP-10 in WT MC was drastically attenuated by pretreatment with parthenolide, but not with CsA. In contrast, CsA attenuated MIP1α mRNA expression more markedly than parthenolide. Data are presented as the mean ± SD (n = 3–4 experiments).

Close modal
FIGURE 5.

γ−/− mice with anti-GBM GN exhibit NF-AT activation that is attenuated by an AT1 blocker. EMSA of nuclear proteins from γ−/− renal cortex showed NF-AT activation with two peaks at 3 and 24 h. This kinetics was similar to that of WT. Furthermore, the activation peaks in γ−/− mice were significantly attenuated by valsartan, an AT1 blocker (AT1A) (∗, p < 0.01, ∗∗, p < 0.05). Lane C denotes coincubation with a sample of γ−/− mice at 3 h and NF-AT cold oligos. Data are presented as the mean ± SD (n = 4–6 experiments).

FIGURE 5.

γ−/− mice with anti-GBM GN exhibit NF-AT activation that is attenuated by an AT1 blocker. EMSA of nuclear proteins from γ−/− renal cortex showed NF-AT activation with two peaks at 3 and 24 h. This kinetics was similar to that of WT. Furthermore, the activation peaks in γ−/− mice were significantly attenuated by valsartan, an AT1 blocker (AT1A) (∗, p < 0.01, ∗∗, p < 0.05). Lane C denotes coincubation with a sample of γ−/− mice at 3 h and NF-AT cold oligos. Data are presented as the mean ± SD (n = 4–6 experiments).

Close modal

We have previously demonstrated that FcR and AT1 are critical molecules in the induction of this disease (3). However, the relevant cell types expressing these receptors remain unknown. Therefore, to clarify this feature, we generated bone marrow chimeras between mice strains lacking each receptor. Their receptor phenotypes (FcR and AT1) of BMCs and RRCs are summarized in Table I. We also generated control mice which were transplanted with bone marrow from the same mice strain (WW, γγ, and AA) under same irradiation conditions, and induced this disease. We could not find any significant difference in their disease phenotypes (urinary protein and renal pathology) from mice without transplantation (WT, γ−/−, and AT1−/−, respectively; data not shown), indicating that bone marrows in recipient animals were functionally reconstituted by transplantation and the irradiation condition may not elicit significant alteration of this disease.

Next, we analyzed the evolution of anti-GBM GN in these animals. During the acute phase of the disease, WT, AT1−/−, and WA mice showed severe proteinuria peaking at day 7 (Fig. 1,A) with glomerular endothelial damage associated with fibrin deposits (Fig. 2,A, a–c) (Table I). However, proteinuria peak in AT1−/− and WA mice was significantly less than in WT (p < 0.05) mice. All WT and most AT1−/− (62%) and WA (67%) mice died with massive ascites before day 35, while γ−/−, γW, and γA mice were completely protected from proteinuria (Fig. 1,A) and endothelial damage (Fig. 2A, D–f). These data further confirm a critical implication of FcR on BMCs in the acute glomerular damage of this disease.

FIGURE 2.

FcR on BMCs and tissue AT1 are responsible for distinct glomerular damages. A, Severe endothelial damage with fibrin deposits in acute phase was observed in mice strains having FcR on BMCs (at day 7, a, WT; b, AT1−/−; c, WA), but not in mice strains lacking them (d, γ−/−; e, γW; f, γA). However, higher amounts of NTS (3× NTS) induced mesangial proliferative GN depending on tissue AT1 (at day 14, g, γ−/−; h, γW; i, γA). B, Although γA did not present morphological lesions, no obvious differences in the deposition of heterologous IgG (rabbit IgG) and autologous IgG and C3 (mouse IgG/C3) were noted between γ−/− and γA mice. (Original magnification in each panel, ×100).

FIGURE 2.

FcR on BMCs and tissue AT1 are responsible for distinct glomerular damages. A, Severe endothelial damage with fibrin deposits in acute phase was observed in mice strains having FcR on BMCs (at day 7, a, WT; b, AT1−/−; c, WA), but not in mice strains lacking them (d, γ−/−; e, γW; f, γA). However, higher amounts of NTS (3× NTS) induced mesangial proliferative GN depending on tissue AT1 (at day 14, g, γ−/−; h, γW; i, γA). B, Although γA did not present morphological lesions, no obvious differences in the deposition of heterologous IgG (rabbit IgG) and autologous IgG and C3 (mouse IgG/C3) were noted between γ−/− and γA mice. (Original magnification in each panel, ×100).

Close modal

In contrast to that shown above, γ−/− and γW mice injected with higher amounts of NTS (3× NTS) developed moderate proteinuria (Fig. 1,B) and glomerular injury characterized by mesangial proliferation, cellular infiltration, and glomerular enlargement with occasional crescents (Fig. 2,A, g and h) (Table I). Glomerular injury in γ−/− mice was associated with CD4+ T cell infiltration in a dose-dependent manner (Table II). Interestingly, γA mice were drastically protected from proteinuria (Fig. 1) and glomerular injury (Fig. 2,Ai) with absence of T cell infiltration (Table II) in the 3× NTS model, even though the heterologous (rabbit IgG), autologous (mouse IgG) Ab, and C3 depositions in γA mice were similarly noted in γ−/− (Fig. 2 B) or WT (data not shown) mice. These data indicate that tissue AT1 is responsible for T cell-associated glomerular injury in this disease.

Table II.

Glomerular T cell infiltration

GroupnCD4+ Cells (c/gcs)
γ−/− 1× NTS 0.44 ± 0.11 
γ−/− 3× NTS 1.24 ± 0.37a 
γA 3× NTS 0.36 ± 0.16 
GroupnCD4+ Cells (c/gcs)
γ−/− 1× NTS 0.44 ± 0.11 
γ−/− 3× NTS 1.24 ± 0.37a 
γA 3× NTS 0.36 ± 0.16 
a

p < 0.05, vs γ−/− 1× NTS or γA 3× NTS; c/gcs: cells/30 glomeruli cross-sections.

To investigate whether FcR or AT1 deficiency may affect systemic cell-mediated immune responses, we induced cutaneous DTH, a classical T cell-dependent inflammatory lesion. No difference in DTH responsiveness was noted in WT, γ−/−, and AT1−/− mice (Table III). This finding is consistent with the data of the autologous IgG deposition (Fig. 2 B), and suggests that cutaneous DTH response is independent of FcR and AT1.

Table III.

DTH responsiveness of each mouse

GroupnDTH Responsivenessa
Ag challengeControl saline
WT 29.3 ± 4.9 8.3 ± 5.9 
AT1−/− 27.7 ± 3.5 6.3 ± 4.2 
γ−/− 26.6 ± 2.1 4.0 ± 2.0 
γ−/−+ nephritis 27.3 ± 4.9 6.7 ± 2.1 
GroupnDTH Responsivenessa
Ag challengeControl saline
WT 29.3 ± 4.9 8.3 ± 5.9 
AT1−/− 27.7 ± 3.5 6.3 ± 4.2 
γ−/− 26.6 ± 2.1 4.0 ± 2.0 
γ−/−+ nephritis 27.3 ± 4.9 6.7 ± 2.1 
a

DTH responsiveness was determined 24-h postchallenge by measuring the increase of footpad size (×0.01 mm).

In certain conditions, AngII participates in the regulation of systemic cellular immune response (20). Therefore, to investigate whether the systemic RAS activation in anti-GBM GN (16, 17) is sufficient to nonspecifically enhance the systemic cellular immune response, we simultaneously induced anti-GBM GN (3× NTS model) and cutaneous DTH response in γ−/− mice. We failed to find any difference in systemic DTH response in γ−/− mice with or without GN (Table III), suggesting that systemic RAS activation in this disease may have no significant role in systemic T cell function.

Based on the above-mentioned in vivo findings from bone marrow chimeras and systemic DTH responses, we next postulated that glomerular T cell infiltration may be regulated by RRCs activated by AngII. We especially focused on glomerular MC, because they possess both AT1 and AT2, regulate the glomerular blood flow, and release proinflammatory cytokines in response to AngII (11, 12, 33). In addition, mesangial proliferation is abolished in γA mice with 3× NTS. In this study, we examined AngII-induced chemotactic activity for T cells, as a possible mechanism involved in the recruitment of those cells. In the chemotactic assays with T cells, supernatants from WT MC treated with AngII had significantly higher activity (∼3.5-fold) than treatment medium alone, reaching a plateau after 12 h (Fig. 3,A). By contrast, supernatants from AngII-stimulated AT1−/− MC showed significantly less activity (around basal at 12 h) (Fig. 3 A), indicating that the chemotactic activity induced by AngII in MC occurred mainly through AT1. We also noted that AngII by itself presented a low chemotactic activity for T cells (average of migration in control medium vs medium with AngII; 354 ± 90 vs 743 ± 92 cells), consistently with a previous study (36).

Recent data have convincingly demonstrated that nephritogenic T cells associated with crescent GN in this disease are mainly Th1 cells (6). Functional diversity between Th1 and Th2 is partly due to the difference the chemokine receptor phenotypes (37, 38). CXCR3 and CCR5 are preferentially expressed in Th1 cells (37). Therefore, we also studied the regulation of their corresponding ligands (CXCR3, IP-10; CCR5, MIP1α) in MC stimulated by AngII. As noted in Fig. 3,B, AngII (10−6 M) significantly up-regulated the mRNA expression of IP-10 and MIP1α in WT MC, peaking at 6 h, as determined by semiquantitative RT-PCR. These data were confirmed by Northern blot analyses. As shown in Fig. 3,C, AngII induced mRNA expression of both chemokines in MC with similar kinetics. By both methods (Fig. 3, B and C), AT1−/− MC showed significantly less mRNA expression in IP-10 and MIP1α than WT MC, indicating that the expressions of both chemokine genes are mainly elicited through AT1 stimulation.

Emerging data reveal that the AngII/NF-κB pathway contributes to the pathogenesis of inflammatory diseases via regulation of chemokine production (11). In contrast, although CaN/NF-AT pathways were firstly reported in T cells (39), their importance has been recently highlighted in other organs, such as the heart, vascular system, neurons, and muscles (24, 25, 40, 41, 42, 43). Special attention has been paid to the AngII/NF-AT pathway in the pathogenesis of certain diseases (24, 25). The activity of NF-AT proteins is tightly regulated by the calcium/calmodulin-dependent phosphatase CaN (39). Recent studies suggested the implication of the CaN-mediated activation of NF-AT in chemokine production (44, 45, 46). Therefore, to approach possible transcriptional regulations in this mechanism, we pretreated MC with inhibitors of NF-κB (parthenolide) and CaN/NF-AT (CsA and CaN autoinhibitory peptide), and then we analyzed the T cell chemotaxis. Surprisingly, both CaN inhibitors showed marked attenuation in WT MC (CsA, 97% inhibition at 6 h, 72% at 12 h; CaN autoinhibitory peptide, 98% at 12 h) (Fig. 4,A), even though CsA itself slightly induced chemotactic activation to MC (around 1.2- to 1.4-fold increase vs medium alone at 6 and 12 h). By contrast, parthenolide showed only 24 and 15% inhibition at 6 and 12 h, respectively (Fig. 4 A), suggesting that the CaN-dependent pathway plays a predominant role in the AngII-induced chemotaxis by MC.

Next, we examined the implication of both pathways in Th1-associated chemokine production. AngII-induced IP-10 mRNA was markedly attenuated by pretreatment with parthenolide (75% inhibition at 6 h, 85% at 12 h), but not with CsA (24% at 6 h, 4% at 12 h) (Fig. 4 B). In contrast, MIP1α mRNA expression was inhibited around 40–50% by CsA at 6 and 12 h. The data are consistent with previous studies that showed the presence of functional NF-AT sites in the MIP1α promoter-enhancer region (47). Accordingly, these data suggest that AngII did enhance mRNA expression of both Th1-associated chemokines (IP-10 and MIP1α), mainly via AT1 on MC, though their transcriptional regulation may be different.

To confirm the implication of the renal CaN/NF-AT pathway in this disease, we performed EMSA with nuclear proteins from the renal cortex. WT mice showed an early peak of NF-AT activation at 3 h after the injection of the Ab, and reactivation at 24 h (Fig. 5). γ−/− mice showed basically the same kinetics of NF-AT activation. There was no significant difference in the peak amplitude of NF-AT activation in WT and γ−/− mice. Preincubation with an anti-NF-ATc4 Ab attenuated the NF-AT peak signals, indicating that activated renal NF-AT in this disease involves NF-ATc4 (NF-AT3).

To clarify the relevant cell types of this activation, Southwestern histochemistry with NF-AT oligo probes was done in γ−/− mice. In the acute phase of this disease, NF-AT activation was observed in glomeruli, mainly at MC (Fig. 6, upper panels). Interestingly, in the chronic phase of this disease, γ−/− mice showed activation signals not only in glomeruli, but also in tubuli and interstitial infiltrating cells (Fig. 6, lower panels).

FIGURE 6.

NF-AT activation in RRCs is implicated in the pathogenesis of tissue injury in anti-GBM GN. Southwestern histochemistry with NF-AT-specific oligo probes revealed NF-AT activation in glomerular resident cells of γ−/− mice at 3 h, which was drastically attenuated by valsartan, an AT1 blocker (upper panels). Interestingly, in the chronic phase of this disease, this activation was detected not only in glomerular resident cells, but also in the tubular and infiltrating cells (lower panels). Arrows and open arrowheads denote representative activated nuclei of RRCs (glomerular and tubular) and infiltrating cells, respectively. (Original magnification in each panel, ×100)

FIGURE 6.

NF-AT activation in RRCs is implicated in the pathogenesis of tissue injury in anti-GBM GN. Southwestern histochemistry with NF-AT-specific oligo probes revealed NF-AT activation in glomerular resident cells of γ−/− mice at 3 h, which was drastically attenuated by valsartan, an AT1 blocker (upper panels). Interestingly, in the chronic phase of this disease, this activation was detected not only in glomerular resident cells, but also in the tubular and infiltrating cells (lower panels). Arrows and open arrowheads denote representative activated nuclei of RRCs (glomerular and tubular) and infiltrating cells, respectively. (Original magnification in each panel, ×100)

Close modal

Next, we investigated whether RAS blockade may affect the NF-AT activation in the acute phase of this disease. Surprisingly, treatment with valsartan, an AT1 blocker, drastically attenuated the NF-AT activation at 3 and 24 h in γ−/− mice (Fig. 5), consistently with the decrement of glomerular activation (Fig. 6, upper panels). These data suggest that AngII-induced NF-AT activation in RRCs may contribute to the initiation of this disease. Furthermore, the NF-AT pathway in resident and infiltrating cells could also be involved in the development of this disease.

The role of activating FcR in providing a critical link between ligands and effector cells in Ab/IC-mediated inflammation has been well-established (1, 2), but the significance of these receptors on each effector cell type along the disease still remains unclear. In the present study, even though γW and γA mice have FcR on RRCs, acute lethal damage observed in WT and AT1−/− mice was completely abolished in these chimeric mice. These findings further confirm the critical implication of FcR in the acute phase of anti-GBM GN (3) and indicate that FcRs, especially on BMCs, are essentially required for an initial inflammatory response after Ab deposition. Consistently, the injury can be induced in Wγ chimeras (BMCs; WT, RRCs; γ−/−) (48). Imasawa et al. (49) recently have suggested that BMCs may have the potential to differentiate into glomerular resident cells. However, in contrast, Mayadas and coworkers demonstrated that FcγRIII on PMN is essentially required for initial recruitment of PMN in anti-GBM GN (50) and, following interaction between FcγR and CD11b/CD18 (Mac 1) on PMN, is also necessary for sufficient PMN spreading on the glomerular capillary wall (51). In fact, the absence of acute glomerular damage in γ−/− mice was associated with the lack of PMN influx (3). Although we need to examine the contribution of bone marrow-derived glomerular resident cells in the acute inflammatory settings, our present data further support the idea that FcγR on PMN, but not on RRCs, may play a major role for acute endothelial damage.

Interestingly, γ−/− mice developed GN persisting for >5 mo and its severity was dependent on the amount of Ab injected and the number of glomerular-infiltrating CD4+ T cells. In addition, the morphological lesions are highly analogous to those seen in studies demonstrating T cell-dependent injury of this disease (6). These data indicate that CD4+ T cell (Th1)-dependent response is pivotal for the development of mesangial proliferative GN in γ−/− mice. This hypothesis is further supported by recent findings of our group using mouse strain overexpressing Smad7 (an inhibitory molecule of TGF-β signaling) (26), in which the CD4+ T cells cannot migrate into the inflammatory sites due to the disregulation of CD62 ligand (L-selectin) expression. In anti-GBM disease, the development of GN, including macrophage infiltration, in these animals was drastically attenuated, suggesting that the development and the persistence of this disease essentially require glomerular-infiltrating CD4+ T cells.

T cell-dependent injury in γ−/− and γW mice required three times higher amounts of anti-GBM Ab than FcR-mediated endothelial injury, indicating different thresholds for their activation by the same Ab. However, γA chimeras were protected from the glomerular T cell response even in the high dose model, emphasizing that AngII action via AT1 on recipients could be responsible for the threshold of the T cell-mediated mechanism. Besides, different T cell responses between γW and γA chimeras indicate that their bone marrow-derived glomerular resident cells (49) (presumably FcR but AT1+) may not play an important role for T cell recruitment. In this disease, dose-dependent activation of intrarenal and systemic RAS has been demonstrated (15, 16, 17). AngII has some cellular effects on most tissues, mainly via AT1, that may contribute to the disease pathogenesis (11, 19), and also regulates cellular immunity by acting on the proliferation of splenic lymphocytes (20). However, in a cutaneous DTH, AT1 deficiency did not alter the responsiveness, in accordance with a previous study (21), and we failed to find any difference between γ−/− mice with or without nephritis, suggesting that systemic RAS activation in this disease may not play a significant role in general T cell function. Accordingly, the amplitude of intrarenal RAS activation would dominantly conduct the glomerular DTH response in this disease, though we must carefully elucidate the alteration of AT1 expression on T cells by elevated plasma AngII.

It is already known that AngII itself is chemotactic for T cells (36). Besides confirming this feature, we noted that supernatants of MC treated with AngII elicited a marked chemotactic activity for T cells, indicating a predominant role of second mediators (presumably chemokines) induced by AngII. The experiments with AT1−/− MC revealed that those AngII actions were exerted mainly via AT1. These findings are consistent with previous studies showing that AngII, acting through both AT1 and AT2, induces T cell-chemokine production (12, 33, 52).

Enhanced expressions of IP-10, MIP1α, and their receptors in kidney have been previously shown in this disease (53), as well as in human mesangial proliferative GN (e.g., IgA nephropathy) (54). The present study shows that the expression of these Th1-associated chemokines in MC is up-regulated by AngII mainly through AT1. MIP1α redundantly cross-reacts with CCR5 and CCR1 as well as other chemokines (53), whereas IP-10 is more selective to CXCR3 (37). Moreover, CCR1 is expressed equally in Th1 and Th2, while CCR5 is not (37, 38). In this regard, interestingly, CCR1-deficient mice with anti-GBM GN showed enhanced Th1 response and glomerular crescents, where not only both chemokines, but also CXCR3 and CCR5, were up-regulated in association with higher CD4+ T cell and macrophage infiltration (53). This evidence suggests that Th1-deviated immune response of this disease may be partially enhanced by chemokine phenotypes produced by RRCs.

Importantly, once T cells and subsequent macrophages are, even if nonspecifically, recruited into the inflamed kidney by local RAS activation, they may orchestrate the autocrine/paracrine-acceleration loop accompanied by locally elevated AngII because they are equipped with all RAS components (55). In fact, significant sources of tissue ACE in human atherosclerotic plaques are regions of clustered macrophages (56). In addition, IL-12, a key cytokine for Th1 response, from mononuclear cells is suppressed by ACE inhibitors (57). In this regard, the role of immunocompetent cells in nonimmune renal diseases further supports this notion (55). Salt-sensitive hypertension after AngII infusion was associated with tubulointerstitial accumulation of AngII-producing lymphocytes and was prevented by the immunosuppressor mycophenolate mofetil coincidentally with a reduction of those cells (58).

Elevated local AngII in this disease may result from physiological responses to the alterations elicited by the specific Ab deposition. Therefore, MC could be one of the major targets of the AngII effect. Indeed, as a consequence of mesangial contraction by AngII, a significant decrease in glomerular plasma flow and single nephron glomerular filtration rate, followed by increased renal vascular resistance, was observed in this model in a dose-dependent manner (15, 17). Consequently, one can postulate that excessively elevated AngII may elicit increased intracellular calcium levels in MC and subsequently a wide variety of cellular responses by a Ca2+-dependent pathway. Some parts of RAS influence on immunological function may be due to such indirect outcome (20, 36, 59). In this sense, it is noteworthy that the chemotactic activity for T cells in AngII-treated MC was largely attenuated by CaN-specific inhibitors in the present study. Although NF-AT3 (NF-ATc4) mRNA was previously detected in the kidney (60) and endothelin 1 activates cyclooxygenase 2 expression via NF-AT in cultured MC (61), there are still no studies demonstrating the functional or pathological contribution of NF-AT during kidney disease. NF-AT activity requires the sustained Ca2+ stimulus provided by the Ca2+ release-activated Ca2+ influx channel and Ca-dependent phosphatase CaN (39, 43). Therefore, there is considerable evidence that the Ca2+ release-activated Ca2+ influx in MC is under the control of both protein kinase C and calmodulin, and thus represents a key mechanism for the control of Ca2+-regulated mesangial function (62).

Because a study with synthetic peptides blocking NF-AT activation by CaN postulates CsA-sensitive (presumably CaN-dependent) gene expressions that are not controlled by NF-AT (47), we must carefully elucidate the mesangial CaN/NF-AT pathway with AngII stimulation in future studies. However, our present data in EMSA and Southwestern histochemistry strongly support the notion that AT1-stimulated NF-AT activation may be involved in the pathogenesis of this immune-mediated disease, as it occurs in myocardial hypertrophy (24). It is interesting to note that CsA could directly prevent mesangial proliferative GN independently of its immunosuppressive action (63). AngII regulates cellular immune responses through the CaN-dependent pathway within the lymphoid tissue (20). The present data show for the first time the activation of the local CaN/NF-AT pathway early in this renal disease, and its attenuation by valsartan, an AT1 blocker, suggesting that locally elevated AngII, probably together with inflammatory cytokines (25), may regulate cellular immune responses partly via the local CaN/NF-AT pathway. Furthermore, the distribution of the activated NF-AT, changing from glomeruli to tubulo-interstitium and infiltrating cells along the disease course, suggests its implication in the different stages of the disease and supports the idea of the potential interest in targeting this pathway (24, 47).

Although we and others have already reported the contribution of AngII/NF-κB (11, 33) and IC/NF-κB pathways in the pathogenesis of GN (27, 64) through the chemokine release, NF-κB inhibitors had less effects on the chemotactic activity. Our data indicate that IP-10 expression was mainly regulated by the AngII/NF-κB pathway, while AngII-enhanced MIP1α expression was mainly through CaN/NF-AT. Interestingly, although MIP 1α can be active to resting T cells (65), IP-10 mainly influences on activated T cells (66). Tight regulation of the chemokine receptor expression in T cells can be the reason for the difference (38, 67). Because this cell clone behaves more like “naive” than “activated” T cells (68, 69), it could be one of the reasons why the good inhibitor of IP-10, parthenolide, had less effects on chemotaxis. In fact, CaN/NF-AT inhibitors showed marked attenuation of chemotaxis, suggesting that AngII-induced NF-AT activation may preferentially contribute to the chemotaxis of inactivated T cells. However, in vivo T cell chemotaxis may be regulated in a more complicated manner (38). Because chemokine/chemokine receptor interaction, for example, contributes to position effector T cells (38, 67), the infiltrating T cell population would shift from more like “naive” to “activated” cells along the disease course, as well it occurs in other immune diseases. Indeed, acute activation of NF-AT, peaking at 3 h, and delayed NF-κB activation at 24 h in γ−/− mice (our unpublished data) may support this idea. Therefore, our present data indicate that AngII-activated transcriptional factors and subsequent chemotactic mediators, including MIP1α and IP-10, play roles in a process of multistep navigation to T cells in this disease.

In conclusion, the current studies provide evidence for AngII-dependent CD4+ T cell-directed injury in γ−/− mice with anti-GBM GN. In addition, they demonstrate that local RAS activation, via AT1, facilitates glomerular T cell recruitment by Th1 chemokine release from RRCs activated with AngII. They also provide the first demonstration of the potential implication of the local AngII-dependent NF-AT pathway in the pathogenesis of IC nephritis. Finally, these results afford a rational basis for the use of RAS antagonists in patients with renal immune diseases.

We thank Drs. M. V. Alvarez Arroyo and Y. Hisada for helpful discussion, Drs. K. Hattori and Y. Kanamaru for their help with BMT, Dr. Juan José Granizo Martínez for helping us with statistical analyses, T. Shibata for excellent technical assistance, and Lise Lotte Gulliksen for secretarial assistance.

1

This work was supported by research grants from Comunidad Autónoma de Madrid (08.4/0019/2000, 08.4/0017, and 2000, 08.9/0002/2000), Fondo de Investigación Sanitaria (99/0425, 00/0111), European Union Concerted Action (BMH4-CT98-3631, DG12-SSMI), and Spanish Society of Nephrology. Y.S. is supported by funds from Japan Health Science Foundation and Alumni Association of Juntendo University. O.L.-F. is a fellow from Fondo de Investigación Saniteria.

3

Abbreviations used in this paper: IC, immune complex; GBM, glomerular basement membrane; GN, glomerulonephritis; anti-GBM GN, glomerular basement membrane Ab-induced GN; PMN, polymorphonuclear cell; WT, wild type; RRC, renal resident cell; DTH, delayed-type hypersensitivity; Ang, angiotensin; ACE, Ang-converting enzyme; AT1, Ang type 1 receptor; RAS, renin-Ang system; NTS, nephrotoxic serum; MC, mesangial cell; CaN, calcineurin; CsA, cyclosporin A; IP, IFN-γ-inducible protein; MIP, macrophage-inflammatory protein; BMC, bone marrow-derived cell.

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