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J Am Soc Nephrol 13:937-945, 2002
© 2002 American Society of Nephrology

Effects of Mycophenolate Mofetil in Mercury-Induced Autoimmune Nephritis

Elena Nieto*, Esther Escudero*, Elena Navarro*, María Yáñez-Mo§, Ana Martín{dagger}, Guillermo Pérez de Lema{ddagger}, Francisco Sánchez-Madrid§ and Francisco Mampaso*

*Department of Pathology, Hospital Ramón y Cajal, Universidad de Alcalá, Madrid, Spain; {dagger}Department of Biology, Universidad SEK, Segovia, Spain; {ddagger}Klinische Biochemie, Medizinische Poliklinik der Ludwig-Maximiliams-Universitä, Munich, Germany; and §Department of Immunology, Hospital La Princesa, Universidad Autónoma, Madrid. Spain.

Correspondence to: Dr. Francisco Mampaso, Department of Pathology, Hospital Ramon y Cajal, Carretera Colmenar km 9.100, Madrid 28034, Spain. Phone: 3413368052; Fax: 3413369016; E-mail: fmampaso{at}hrc.insalud.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Mycophenolate mofetil (MMF) is a new immunosuppressive drug whose active metabolite, mycophenolic acid (MPA), blocks the action of inosine monophosphate dehydrogenase, resulting in the inhibition of the novo purine synthesis. Thus, MPA has an antiproliferative effect on T and B lymphocytes and also inhibits the glycosylation of cell surface adhesion proteins involved in cell-cell contact and in the recruitment of circulating leukocytes to sites of tissue damage and inflammation. In this study, the effect of MMF in the mercury model of nephritis was examined. Repeated exposure to HgCl2 induces an autoreactive Th2 cell subset-inducing polyclonal B cell activation in the Brown Norway (BN) rat. This leads to the development of an autoimmune syndrome characterized by synthesis of autoantibodies (mainly anti–glomerular basement membrane [GBM] Abs) with glomerular linear deposits of IgG, proteinuria, and tubulointerstitial nephritis. Results show that MMF has a preventive effect on mercury-induced disease as it blocks anti-GBM Ab synthesis, thus avoiding glomerular IgG deposits and proteinuria and the development of interstitial nephritis. However, the therapeutic effect of MMF seems to be restricted to its antiinflammatory properties blocking the extravasation of circulating leukocytes to renal interstitium by interfering with the very late activation antigen 4/vascular cell adhesion molecule–1 (VCAM-1) cell adhesion pathway. Also, MMF administration to mercury-injected rats reduces the secretion of the proinflammatory cytokine tumor necrosis factor–{alpha}. These findings confirm that MMF has a strong effect on the primary immune response in this model. Nevertheless, when the disease is in progress, MMF acts exclusively on the inflammatory response. MMF could be useful in the treatment of diseases associated with renal inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mercuric chloride-induced nephritis was used to examine the effects of mycophenolate mofetil (MMF) administration on the course of this autoimmune model of disease. MMF is the morpholinoethyl ester of mycophenolic acid (MPA), which is a potent, non competitive and reversible inhibitor of eukaryotic inosine monophosphate dehydrogenase, a key enzyme in the novo purine synthesis. This enzyme catalyzes the conversion of inosine monophosphate into GMP, and due to lymphocytes rely on the novo pathway for production of nucleotides, MPA has antiproliferative effects on these cells by depleting intracellular GTP pool. Thus, MPA is a relatively selective inhibitor of lymphocyte proliferation since other cells can obtain guanine nucleotides via the salvage pathway (1). Furthermore, MPA-mediated depletion of GTP inhibits the transfer of mannose and fucose to lymphocyte and monocyte glycoproteins, some of which are adhesion molecules that mediate attachment of such cells to endothelial and target cells, as well as the extravasation to sites of inflammation. The down-regulated expression of these adhesion molecules also confers to this immunomodulating drug an anti-inflammatory effect (2).

MMF has been used in the prevention of acute rejection after renal transplantation (3–7). Its low toxicity, in contrast with other drugs used like cyclosporine, tacrolimus, and azathioprine (8), could make of this new immunosuppressant not only an alternative therapy for organ rejection, but also a potential therapeutic agent for treating autoimmune inflammatory disorders. Thus, several autoimmune experimental studies have shown improvement after MMF treatment. MMF is able to inhibit the development of experimental autoimmune uveoretinitis in Lewis rats (9) and spontaneous diabetes in Biobreeding rats (10). In addition, it has been shown that this immunosuppressive agent is effective in other autoimmune experimental diseases with renal affectation, such us lupus erythematosus in MRLlpr/lpr mice prone (11), NZBxW lupus mice (12), and in the active Heymann nephritis model of disease (13).

Given the beneficial effects in immune-mediated models, MMF has also been used for the treatment of various human glomerulonephritis in small pilot trials on minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and lupus nephritis (14–20). Such results have shown short-term clinical efficacy of this drug, but additional controlled clinical trials are necessary to evaluate long-term benefits.

Mercuric chloride is a T cell–dependent polyclonal B cell activator that induces autoimmunity in susceptible rodent strains. In the Brown Norway (BN) rat, mercury induces a self-limiting autoimmune syndrome characterized by the presence of an autoreactive Th2 CD4+ cell subset (21,22), hypergammaglobulinemia, and a number of auto-Abs, mainly anti–glomerular basement membrane (GBM) Abs (23). This autoimmune response is accompanied by the development of nephritis with glomerular linear IgG deposits and proteinuria. The histologic renal lesions consist of a transient influx of lymphocytes and monocytes into the renal interstitium (24–26).

Taking into account that cellular activation, synthesis of anti-GBM Abs, and release of soluble inflammatory mediators are important steps in the renal tissue dysfunction observed in mercury-treated rats (27), we have examined whether MMF has the potential to be an adequate therapeutic agent to modulate this autoimmune renal disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
BN female rats weighing 150 to 200 g were obtained from IFFA CREDO (Paris, France), fed standard laboratory chow ad libitum, and treated according to the institutional guidelines that are in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Monoclonal Antibodies
To identify rat cell surface markers we used the following mAbs: the mouse anti-human HP2/4 and HP2/1 mAb directed toward the {alpha}-4 integrin that crossreact with rat {alpha}-4 integrin, which have been described elsewhere (27); mAb TA2, which recognizes the rat very late activation antigen 4 (VLA-4) molecule (a generous gift from Dr. Thomas B. Issekutz, Izaak Walton Killam Hospital, Halifax, Canada); and mAb WT.1, which recognizes the {alpha}-subunit of rat LFA-1 (CD11a, LFA-1a) (28). The mouse anti-rat OX1 mAb, specific for the panleukocyte CD45 antigen, was purchased from Serotec (Oxford, UK).

Experimental Design
Animals were separated into four different groups. Groups I, II, and III received five subcutaneous injections of HgCl2 (1 mg/kg body wt) to induce the disease over a period of 2 wk. Rats from group I did not receive any additional treatment and served as positive control of the disease. Rats from groups II and III were also orally given the immunosuppressant MMF (80 mg/kg body wt) dissolved in the vehicle once a day until sacrificed. Rats from group II started the treatment on the first day of the experiment, but the animals belonging to group III started treatment the day after the last injection of HgCl2 (day 9 of the experiment, after the disease was developed). Finally, group IV served as a normal control in which rats were only injected with H2O adjusted to the same pH (3.8) as the HgCl2 solution, following the same protocol of HgCl2 administration. The dosages and days were established on the basis of previous optimizing experiments of the disease kinetics (29). All animals were sequentially bled on different days of the experiment by tail artery puncture. To titrate the optimal dose of MMF, a pilot experiment was performed. Rats were treated with different doses of the immunosuppressant (from 20 to 100 mg/kg body wt). We decided to choose the dose (80 mg/kg body wt) that was appropriate for completely blocking anti-GBM antibody production without producing diarrhea or considerable weight loss in rats.

Proteinuria
Rats were housed in metabolic cages with free access to food and water to collect 24-h urine. Urine samples were taken at regular intervals starting on day 0. Protein concentration in urine was determined by the Bio-Rad assay (Bio-Rad, Richmond, CA) according to the manufacturer’s protocol. Samples were assayed in triplicate, and the optical density from each one was measured in a Titertek Multiskan Plus (Flow, Irvine, Scotland) at 595 nm.

Anti-GBM Abs Assay
Rat GBM was essentially isolated as described by Bowman et al. (25). Briefly, glomeruli were obtained from healthy BN rats by differential sieving and centrifugation of minced kidney cortices. The glomerular suspension was sonicated, washed, and lyophilized. The GBM was digested with type I collagenase (Sigma Chemical Co., St. Louis, MO) at 0.7% wt/wt at 37°C for 1 h. Anti-GBM Abs were measured by enzyme-linked immunosorbent assay (ELISA), as described previously (25). All the samples were assayed in triplicate. Samples of a serum pool from untreated BN rats and from mercury-treated BN rats (which were bled on day 13 of the disease) served as negative and positive controls, respectively. Results were expressed as the percentage of binding obtained with samples from positive control serum. Absorbance was measured at 492 nm by using the Titertek Multiskan Plus.

Ribonuclease Protection Assay (RPA)
Total RNA was isolated from whole rat kidneys belonging to groups I, II, and IV at day 13 (when renal damage was maximal) and at day 16 (when renal injury started to decline). RPA is a highly sensitive and specific method for the detection and quantification of mRNA species. Twenty micrograms of total RNA from each sample were tested for cytokine expression analysis by multiprobe RPA (RiboQuant template set, rCK-1; PharMingen, San Diego, CA). Assays were performed according to manufacturer’s instructions. The radiolabeled {alpha}-(32) P-UTP (3000 Ci/mmol) used was from PerkinElmer (Freiburg, Germany). The samples were run out on a 5% denaturing polyacrylamide gel, and the intensity of mRNA rat cytokines bands was analyzed by phosphoimaging (Storm 840; Phosphor-Imager Molecular Dynamics, Sunnyvale, CA). mRNA bands were quantified using the ImageQuant software (Molecular Dynamics, Eugene, OR), normalized to L32 constitutive gene expression, and averaged over the four animals per group used for this assay.

Tumor Necrosis Factor–{alpha} Assay
The levels of serum tumor necrosis factor–{alpha} (TNF-{alpha}) were determined in all the experimental groups of rats along the course of the disease by using a commercial rat ELISA TNF-{alpha} Kit (Endogen, MA). The assay was carried out according to the manufacturer’s instructions. All samples were assayed in triplicate, and the plates were read at 450 nm in Titertek Multiskan Plus.

Kidney Tissue Processing
On days 13 or 23 of the experiment, rats (n = 8) from each group were sacrificed. Kidneys were removed and processed for histologic and immunohistochemical studies. For light microscopy, a piece of kidney tissue was fixed in neutral-buffered formol saline, and 3-µm paraffin-embedded sections were stained with hematoxylin and eosin and periodic acid-Schiff. For immunohistochemical studies, another piece of kidney tissue was snap frozen in precooled isopentane in liquid nitrogen and stored at -70°C until used. An indirect immunoperoxidase-stained method, was used to characterize OX1+ cells in the renal interstitium (30). Quantification of interstitial infiltrating cells bearing OX1+ surface marker was performed by counting, in two kidney tissue sections per rat, the total number of positive labeled cells examined in ten randomly chosen areas of interstitial infiltrates. These studies were performed by using a conventional light microscopy objective (x63), as described previously (31). Immunofluorescence (IF) microscopy was performed on ether/ethanol-fixed serial cryostat sections (5 µm) by using FITC-conjugated rabbit anti-rat IgG Ab (Serotec), as described elsewhere (32).

Expression and Function of the VLA-4 Integrin
To perform flow cytometry and cell adhesion assays, a group of rats (n = 4) were orally administered MMF (80 mg/kg wt) once a day for 3 d while another group of rats (n = 4, control group) only received the vehicle following the same protocol of MMF administration. Rat lymphocytes were obtained from the spleen as described previously (33). Mononuclear cells were separated by Histopaque-1077 (Sigma Chemical Co.) density gradient centrifugation and suspended in RPMI 1640. Macrophages were then depleted by adherence on plastic dishes for 1 h at 37°C and 5% CO2 atmosphere.

Flow Cytometry Assay
Spleen rat lymphocytes suspended in RPMI 1640 were incubated with saturating concentration of the anti-{alpha}4 HP2/4, anti-{alpha}4 HP2/1, anti-rat VLA-4 TA2 or anti-rat LFA-1 WT1 mAbs for 10 min at room temperature. After washing, cells were incubated with FITC-conjugated goat anti-mouse IgG highly cross-adsorbed (AlexaFluor 488, Molecular Probes Inc., Eugene, OR) secondary antibody for 30 min at 4°C in the dark. Direct application of the secondary antibody was used as negative control. The samples were analyzed using a FACScan cytometer (Becton Dickinson, Mountain View, CA).

Cell Adhesion Experiments
Adhesion assays were performed as described previously (34) with slight modifications. Spleen lymphocytes were labeled with BCECF-AM (Molecular Probes Inc.) and pretreated with different mAbs (1:100 dilution of ascitic fluid) for 10 min, and 2.5 x 105 cells/well were seeded on 96-microwell plates (Nunc-ImmunoPlates Maxisorp; Nunc Inc., Naperville, IL) coated with 2.5 µg/ml of recombinant VCAM-1–4D-Fc (35) and blocked with 1% bovine serum albumin. After 20-min adhesion at 37°C, unbound cells were washed by inversion, lysed and fluorescence intensity measured in a microplate fluorescence reader (Bio-tek FL500).

Statistical Analyses
Proteinuria, adhesion assay, and ELISA results are given as mean ± SD. The statistical analysis was performed by using the Mann-Whitney Test. P < 0.001 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of MMF on Proteinuria
As shown in Figure 1, HgCl2-treated rats (group I) developed proteinuria in two different phases. A first short phase, which occurred immediately after the first injection of HgCl2,, was caused by the direct effect of mercury on tubular renal cells. A second phase began between 9 to 11 d, reached maximal values on day 13, and declined thereafter. By the third week, all animals reached background levels. When HgCl2-injected rats were treated with MMF after the first injection of mercury (group II), the urinary levels of protein excretion showed a drastic reduction (P < 0.001) compared with group I. Statistical differences were not observed between MMF-treated rats (group II) and rats from the normal control group (group IV). By contrary, when HgCl2-injected rats were treated with MMF after the disease was developed (group III), the levels of proteinuria were similar to those found in rats treated only with mercury (group I).



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  		Figure 1. Protein excretion levels. Time course of urine protein excretion in all tested groups. Results are expressed as mg protein/24 h (mean ± SD). n = 15 per group. *P < 0.001 difference with HgCl2-treated rats.

 
Effect of MMF on Anti-GBM Abs Production and Glomerular Anti-GBM Abs Deposition
MMF treatment prevents anti-GBM Ab production in HgCl2-treated rats, but it was not able to inhibit such production after the disease was in progression. An increased anti-GBM Ab concentration was detected by ELISA in the serum of rats that were treated solely with mercury (group I) from day 9, being the maximal concentration observed at day 13 of the disease. Thereafter, serum levels of anti-GBM Abs started to decline, as occurred with the levels of proteinuria. Also, HgCl2- injected rats (group I) showed, by direct immunofluorescence, a positive linear IgG rat deposition along the GBM, days 13 (Figure 3A) and 23 of the disease. By contrast, rats that in addition to mercury administration were treated with MMF starting on day 0 (group II) exhibited a drastic reduction (90%) in the serum levels of anti-GBM Abs, being said reduction maintained along the course of the experiment (Figure 2). Therefore, IgG glomerular deposition was not detected in this group of rats (Figure 3C). Nevertheless, when rats received MMF starting on day 9 (when the disease was developed, group III), significant reduction in neither anti-GBM Ab production nor the intensity of IgG deposition along the GBM (Figure 3B) was observed, compared with rats injected solely with HgCl2 starting on day 0.



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  		Figure 3. Immunofluorescence (IF) staining. Positive linear pattern deposition of rat IgG in rats treated with HgCl2 alone (group I) (A) and in rats treated with HgCl2 plus MMF starting on day 9 (group III) (B). Negative staining for rat IgG in rats treated with HgCl2 and also receiving MMF since day 0 (group II) (C). Magnification, x400.

 


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  		Figure 2. Levels of circulating anti–glomerular basement membrane (GBM) Abs. Percentage of positive binding of anti-GBM Ab in rats treated with HgCl2 alone (n = 20), in rats treated with HgCl2 plus mycophenolate mofetil (MMF) starting on day 0(n = 20) or on day 9 (n = 15) of the experiments and in normal control rats (n = 15). Values are expressed as mean ± SD. *P < 0.001, difference with mercury treated rats.

 
Effect of MMF on TNF-{alpha} Expression
Rats treated with HgCl2 alone (group I) showed a sevenfold increase of TNF-{alpha} renal expression, expressed as relative units (TNF-{alpha}/L32), at day 13, compared with normal control rats (group IV) (P < 0.001)(Figure 4).



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  		Figure 4. Tumor necrosis factor–{alpha} (TNF-{alpha}) renal expression. Analysis of mRNA TNF-{alpha} by ribonuclease protection assay (RPA) in renal tissue from n = 4 rats of groups I, II, and IV (two rats representative from each group are shown) at day 13 (when nephritis was highly developed) and at day 16 (when the renal disease started to decline). (A) Representative RPA gel. Samples were organized at two time points (day 16 and 13 of the experiment) separated by an empty lane. Inside of each time point, samples were loaded on the gel as follows: first and second lanes were normal control samples from group IV (C), samples in the next two lanes were from HgCl2-treated rats (group I) (H), and the last two lanes were from HgCl2-treated rats also receiving MMF starting on day 0 (group II) (HM). t represents a 20-µg tRNA control sample. (B) Densitometric analysis for TNF-{alpha} renal expression at days 13 and 16 of the disease. At day 13, mercury-treated rats (Group I) showed an increase in the TNF-{alpha} renal expression as compared with rats treated with mercury plus MMF (group II), which showed levels close to normal control rats (group IV). *P < 0.001, difference with mercury-treated rats. In contrast, TNF-{alpha} expression levels at day 16 were similar in all tested groups.

 
By contrast, mercury-treated rats that also received MMF (group II) showed a marked decrease in the levels of TNF-{alpha} mRNA expression, with no statistical differences having being found when compared with the normal control group.

On day 16, when the disease began to regress and proteinuria levels started to decline, TNF-{alpha} mRNA renal expression was similar in the three groups, with NS differences having been observed among them.

Effect of MMF on the Kinetics of TNF-{alpha} Serum Concentration
Serum TNF-{alpha} concentration was measured at different days of the experiment, using a commercial ELISA method. As shown in Figure 5, serum levels of TNF-{alpha} from HgCl2-injected rats (group I) began to increase before day 9, reached maximal values at day 13 (P < 0.001), and then declined, returning to normal levels at day 22. When we analyzed the kinetics of TNF-{alpha} secretion in HgCl2-injected rats, which also received MMF treatment (group II), TNF-{alpha} serum levels were similar to those found in rats from the normal control group (group IV). When HgCl2-injected rats were treated with MMF starting at day 9 of the disease (group III), significant reduction in TNF-{alpha} serum levels was observed with rats treated with mercury alone. These differences were also found with respect to normal control rats (group IV).



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  		Figure 5. Kinetics of serum TNF-{alpha}. Serum levels of TNF-{alpha} along the course of the experiment in all tested groups. Results are expressed in pg/ml mean ± SD. n = 8 per group. *P < 0.001 versus HgCl2 treated rats (0); P < 0.001 versus normal control rats.

 
Effect of MMF on Renal Interstitial Cell Infiltrates
Light microscopy examination of renal tissue sections from rats treated with mercury alone (group I, n = 8) showed, at day 13 of the disease (Figure 6A), a pronounced interstitial nephritis, being less intense but still persisting on day 23. The interstitial cell infiltrates were preferentially located in the perivascular regions of the renal interstitium. Quantification in the HgCl2-treated rats (group I) of interstitial inflammatory cells bearing the OX1+ surface molecule was 42 ± 6 cells/HPF (day 13) and 22 ± 4 cells/HPF (day 23), respectively. By contrast, in both groups of mercury-treated rats also receiving MMF at the beginning (group II, n = 8) or after the disease was developed (group III, n = 8) (Figure 6, B and C) as well as in normal control rats (group IV, n = 8), the number of interstitial infiltrating cells was practically absent on the same examined days. OX1+ cells were 3.5 ± 0.8 cells/HPF and 3 ± 0.9 cells/HPF (group II), 3.1 ± 0.7 cells/HPF and 2.6 ± 0.9 cells/HPF (group III), and 2.5 ± 0.5 cells/HPF and 2 ± 0.5 cells/HPF (group IV) on days 13 and 23 respectively.



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  		Figure 6. Immunoperoxidase (PO) staining with OX-1. (A) Prominent renal interstitial OX-1+ cell infiltrates in rats treated with HgCl2 alone (group I). (B and C) Absence of renal interstitial inflammatory OX-1+ cells in rats treated with HgCl2 plus MMF starting on day 0 (group II) and starting on day 9 (group III). (D) Negative staining for isotype IgG matched control. Magnification, x300.

 
Effect of MMF on VLA-4 Expression and Function
Results of FACS experiments showed that MMF did not induce significant changes in VLA-4 spleen lymphocyte surface expression when compared with lymphocytes from rats that did not receive the immunosuppressant, as shown in Figure 7.



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  		Figure 7. VLA-4 expression on spleen rat lymphocytes. Expression of VLA-4 molecule chain on rat lymphocytes from control (green line) and MMF-treated (red line) rats using three different antibodies (HP2/4, HP2/1, and TA2) against anti-{alpha}4 chain. Cells were incubated with saturating concentrations of WT1 (as positive control), HP2/4, HP2/1, or TA2 mAb. Lymphocytes were then washed and incubated with FITC-conjugated goat anti-mouse IgG. All samples were analyzed by flow cytometry.

 
Next we tested the effects of MMF on {alpha}-4 integrin function due to the relevance of VLA-4/VCAM-1 adhesion pathway in mercury-induced nephritis (27,29). As shown in Figure 8, MMF was able to reduce by 30% the binding of rat lymphocytes to immobilized VCAM-1. On the other hand, it has been shown that the mouse anti-human HP2/4 mAb (which crossreacts with rat {alpha}-4 integrin) has the ability to block the attachment of rat lymphocytes to VCAM-1 (27). When HP2/4 mAb was added to rat lymphocytes, a proportional increasing reduction in the percentages of adhesion was found in both MMF-treated and normal rat lymphocytes (Figure 8).



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  		Figure 8. Adhesion of rat spleen lymphocytes to vascular cell adhesion molecule–1 (VCAM-1). Adhesion of spleen lymphocytes from MMF-treated and normal rats to recombinant VCAM-1 4D-Fc (2.5 µg/ml). Results are given as mean ± SD of n = 4 rats per group. *P < 0.001 versus control group (0); P < 0.001 versus no Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated the protective effect of MMF in mercury-induced autoimmune nephritis. Rats exposed to HgCl2 develop a self-limiting autoimmune disorder characterized by hypergammaglobulinemia and synthesis of autoantibodies, mainly anti-GBM Ab (23). The renal lesions consist of linear rat IgG deposition on the glomerular basement membrane, severe proteinuria, and interstitial nephritis (24–26).

The development of autoimmunity in this model is not fully understood. It is known that HgCl2 induces B cell proliferation dependent on autoreactive anti-class II Th2-cell subset in BN rats (36,37). In our study, we have found that MMF is able to block the production of auto-Abs against GBM in animals that have received the drug prior the induction of the disease. However, MMF was not able to exert immunosuppressive effects after the disease was in progression.

Several in vitro and in vivo studies have confirmed the immunosuppressive properties of MMF. Jonsson et al. (11) have observed that MMF-treated lupus mice (MRL/lpr) showed little or no Ig deposits in renal glomeruli, in contrast to Van Bruggen et al. (38), who did not find these clear-cut immunomodulating properties in the same lupus model. It is possible that such discrepancy might depend on the different doses of MMF given to MRL/lpr mice in each study. In this regard, we found that, when low doses of MMF (20 to 30 mg/kg wt) were administered to rats with mercury-induced nephritis, only a 40% reduction in anti-GBM Abs production was obtained, with no significant changes having been observed in the levels of proteinuria (data not shown). Nevertheless, when the dose of MMF was increased (80 mg/kg wt), a higher reduction (90%) in anti-GBM Ab production was found, which paralleled the drastic decrease in the urinary protein excretion. Our results have shown that the immunosuppressive effect of MMF on mercury-induced nephritis is dose-dependent, as has also been observed in other clinical and experimental MMF-treated diseases (39,40). However, we have found that even when the well-tolerated maximal dose of MMF was given to mercury-treated rats after the disease was in progress, the drug had no preventive effects on anti-GBM Ab production.

Adhesion molecules and their counter-receptors are determinant in leukocyte migration and extravasation to sites of inflammation. One possible mechanism of action of MMF may involve the VLA-4/VCAM-1 cell adhesion pathway. VLA-4 integrin is expressed mostly on leukocytes (41–43) and binds to the extracellular matrix protein fibronectin (44) and to VCAM-1 adhesion protein (45). It has been shown that the interaction between circulating leukocytes bearing VLA-4 molecules and endothelial cells expressing VCAM-1 molecules is crucial in the development of mercury-induced nephritis in BN rats. Previous data from our laboratory have shown that the administration of either anti-VLA-4 or anti-VCAM-1 mAbs to HgCl2-injected rats completely blocked the influx of circulating leukocytes into the renal interstitium (27,29). It is well known that MMF inhibits the transfer of mannose and fucose to glycoproteins that function as adhesion molecules, including the VLA-4 molecule (2).

Our results have shown that MMF was able to reduce the adhesion of rat lymphocytes to VCAM-1; meanwhile, the surface expression of rat VLA-4 integrin, tested with three different Abs against VLA-4 molecule, was practically similar in both MMF-treated and untreated rat lymphocytes. Such observations, along with the lower basal adhesion and stronger HP2/4 mAb blockade of MMF-treated lymphocytes, strongly suggest that MMF could act by reducing the proportion of rat functional VLA-4 integrins. Therefore, it is feasible that MMF, by altering the VLA-4/VCAM-1 cell adhesion pathway, might interfere with extravasation of circulating leukocytes to the renal interstitium and could explain, at least in part, the abrogation of the renal inflammatory cell infiltrate, not only in the absence of renal lesions (which was expected) but even when anti-GBM Ab glomerular deposits and proteinuria were detected.

Unmodified renal expression and serum basal levels of TNF-{alpha} cytokine found in MMF-treated rats from the beginning of the disease indicate the absence of renal inflammatory damage and also support its protective effects on the development of this renal disease. By contrast, we have observed that MMF administration to mercury-injected rats had no therapeutic properties, as it is reflected by the lack of effects on anti-GBM Ab synthesis, IgG glomerular deposition, and proteinuria. On the other hand, the complete absence of interstitial cell infiltrates may decrease the amplification of renal inflammatory injury, consequently provoking the partial reduction found in TNF-{alpha} serum levels.

The mechanism(s) whereby MMF prevents this autoimmune renal disease might act by impeding auto-reactive Th2 cell subset activation, thus intercepting B cell stimulation for producing anti-GBMs Abs. This blockade of the initial immune response avoids the development of the autoimmune syndrome. Consequently, we can assume that MMF has an strong effect on the primary immune response in this model. On the other hand, when the disease is initiated and lymphocyte cell-cell interactions as well as polyclonal activation are triggered, MMF therapy improvement is restricted to the blockade of lymphocyte extravasation to the renal interstitium. It is conceivable that, in addition to altering the VLA-4/VCAM-1 pathway, MMF could also be involved in other lymphocyte adhesion mechanisms as well as in chemokine/cytokine expression. Also, a possible direct effect of the drug on parenchymal renal cells cannot be ruled out (13,46–48).

Taking all these data into account, we conclude that MMF could be useful in the treatment of renal diseases associated with inflammation. More controlled trials with MMF as monotherapy or combined with other agents are required to warrant the clinical efficacy of this new drug.


   Acknowledgments
 
This work was supported by Grants 00/0246 and 98/1211 from Fondo de Investigaciones Sanitarias and Grant 08.4/15/98 from Comunidad Autónoma de Madrid (Dr. Mampaso). We are grateful to Virginia Lynne Desbrow for her help in the manuscript correction. G.P.d.L. was supported by a postdoctoral fellowship from Spanish Ministerio de Educación y Cultura (Grant Ex 97/7230290).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication December 29, 2000. Accepted for publication November 8, 2001.




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