INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE EXTRACELLULAR MATRIX (ECM), which includes the glomerular basement membrane (GBM) and the mesangial matrix, fulfills an important structural and regulatory function in the renal glomerulus. In the numerous pathological processes involved in the glomerulus, the integrity of GBM and mesangial matrix is frequently disrupted. Proteinuria, partially due to proteolytic destruction of the basement membrane, and mesangial expansion, resulting from the abnormal accumulation of matrix molecules, are two major hallmarks of glomerular diseases. Thus knowledge of the mechanisms and processes regulating the synthesis and breakdown of GBM or mesangial ECM is critical for our understanding of the pathogenesis of glomerular diseases.

Located in the center of the glomerulus, the intrinsic mesangial cell (MC) plays a key role in both synthesis and degradation of the glomerular ECM (35). There have been extensive reports about the ability of cultured MCs to synthesize a broad variety of glomerular ECM proteins (18, 35). Recently, the expressions of proteolytic activity by MCs and its role in glomerular pathophysiology have been the focus of numerous studies (4, 12, 14, 20, 31, 35, 37, 39). Matrix metalloprotease-2 (MMP-2, also called gelatinase A or collagenase IV) is the predominant MMP synthesized by MCs (12, 13) and plays an essential role in the remolding of ECM under various physiological and pathological conditions (10). Altered expression and activation of MMP-2 in a variety of glomerular diseases, in both human beings and experimental animal models, have been reported (14, 31, 37). In addition to its collagen-degrading activity, it has been recently suggested that active MMP-2 may play a role in the proliferation and in the expression of an inflammatory phenotype of rat mesangial cells (39). A wide variety of compounds, such as inflammatory cytokines and mediators [interleukin-1 (IL-1), tumor necrosis factor- (TNF-), interferon- (IFN-), PGE₂, nitric oxide], growth factors (TGF-), and tumor promoting agent phorbol 12-myristate 13-acetate (PMA), as well as nonphysiological agents such as cytochalasin D and concanavalin A, have been demonstrated to be able to upregulate MMP-2 expression and/or activation in MCs (1, 2, 13, 26, 27, 29, 38, 42). However, factors involved in the downregulation of MMP-2 activity in MCs have not been studied in any depth.

Endothelin (ET) is one of the major pathogenic factors implicated in the abnormal accumulation of ECM in the kidney. Increased expressions of ET as well as ET receptors are found in a variety of glomerular diseases with matrix deposition (7, 8). Treatment with a specific ET_A receptor antagonist attenuates ECM accumulation and glomerulosclerosis in several models of kidney injury (5, 6). Furthermore, induction of the synthesis of multiple extracellular matrix components via ET_A receptor by ET in cultured MCs has been reported (18). Because matrix turnover represents both matrix synthesis and degradation, an alternative way of ET action on matrix accumulation might be the inhibition of matrix degradation, via regulation of the synthesis and/or activation of proteolytic enzymes, released by MCs or other cell types. In this respect, the suppressive effects of ET on the activity of fibrinolysis via upregulation of plasminogen activator inhibitor in cultured MCs have been documented (21). In addition, exogenous ET-1 was found to be able to inhibit the activity of collagenase in cultured cardiac fibroblast (19). Conceivably, ET might also be able to modulate the activity of MMP-2 in cultured MCs. This study was designed to test this hypothesis. Our results demonstrate that ET is a potent inhibitor of both MMP-2 secretion and activation in cultured rat MCs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Gelatin, lipopolysaccharide (LPS), polyclonal rabbit anti-MMP-2 antibody, anti-tissue inhibitor of metalloproteinase-2 (TIMP-2) antibody, prestained molecular weight markers for SDS-PAGE, DMEM, and synthetic human endothelin-1 were obtained from Sigma (St. Louis, MO). Monoclonal antibody to endothelin-1, -2, and -3 was purchased from Biogenesis. BQ-123 and IRL-1038 were from Alexis (San Diego, CA). FR139317 was a gift of Fujisawa Pharmaceutical (Osaka, Japan). Recombinant hTIMP-2 was purchased from Fuji Chemical (Toyama, Japan). An MMP-2 activity assay kit was obtained from Amersham Pharmacia (Piscataway, NJ). Microcon and Immobilon polyvinylidene difluoride membrane were supplied by Millipore (Bedford, MA). Enhanced chemiluminescence (ECL) reagents were obtained from Amersham (Arlington Heights, IL).

Rat MC culture. MC isolation and culture were performed as described previously (40, 41). In brief, the renal cortices of male Wistar rats (150 g) were homogenized under sterile conditions and passed over three sieves with pore sizes of 200, 100, and 75 µM. Glomeruli, which were retained on the 75-µm sieve, were seeded in DMEM containing 20% FCS, insulin (5 µg/ml), penicillin (100 U/ml), and streptomycin (100 U/ml). After three to four passages in DMEM containing 20% FCS, pure MC populations were obtained. MCs were characterized by the following criteria: positive immunocytochemical staining with antibodies against Thy-1.1 (32) and smooth muscle -actin, absence of staining for antigen factor VIII, and cytokeratins. MCs were used for experiments between passages 5 and 20.

Preparation of conditioned medium. MCs were seeded into a 24-well culture plate and allowed to grow in 20% FCS-DMEM until 90% confluence; then MCs were thoroughly washed with serum-free DMEM and starved in the same medium for 1 day. Afterward, the medium was replaced with fresh serum-free medium with or without the addition of particular test agents, and the culture was continued for the indicated time intervals. The conditioned medium was collected, centrifuged, aliquoted, and stored at 70°C until required.

Zymography. To analyze gelatinolytic activity, aliquots of MC conditioned medium (20 µl) were mixed with 5 µl 5× nonreducing sample buffer (0.5 M Tris · HCl, pH 6.8, 10% SDS, 50% glycerol, 0.5% bromphenol blue). Electrophoresis was performed in a 0.75-mm-thick 7.5% polyacrylamide/SDS gel containing 1 mg/ml gelatin as substrate. After electrophoresis the gels were incubated in 2.5% Triton X-100 and 50 mM Tris · HCl, pH 7.5, for 1 h at room temperature, followed by 16-20 h at 37°C in a collagenase buffer containing 50 mM Tris · HCl, pH 7.5, 100 mM NaCl, and 10 mM CaCl₂. Thereafter, the gels were stained with Coomassie blue, and zones of lysis were visualized. Standard protein markers were utilized for assignment of molecular mass.

Detection of MMP-2 activity by antibody capture assay. To quantitate the MMP-2 activity secreted by MCs, a specific MMP-2 activity assay was conducted by using an antibody capture method (11). For this assay, purified MMP-2 or MC culture supernatants were added to wells of a 96-well microliter plate previously coated with a monoclonal MMP-2 antibody (RPN2631; Amersham Pharmacia). After incubation overnight at 4°C, plates were washed vigorously with phosphate buffer solution containing 0.05% Tween 20. Next, p-aminophenylmercuric acetate (1 mM), an organomercurial, was added to activate any captured MMP-2, after which an enzyme substrate solution containing 50 mM Tris · HCL, 1.5 mM NaCl, 0.5 mM CaCl₂, 1 µM ZnCl₂, 0.01% BRIJ 35, and chromogenic peptide substrate S-2444 (Amersham Pharmacia Biotech) was introduced. The reaction was allowed to proceed at 37°C for 2 h, and then the absorbance at 405 nm was recorded. Cleavage of chromogenic substrate produced a linear increase in absorbance with increasing concentrations of MMP-2 standards. MMP-2 activity and concentration in MC culture supernatants were expressed as nanograms per milliliter based on the results obtained with purified MMP-2 standards.

Western blots. MCs were seeded onto 60-mm culture plates and allowed to grow in 20% FCS-DMEM until 90% confluence. MC were then starved in serum-free DMEM for 2 days, before being stimulated with different agents for various periods of time. The reaction was terminated by washing cells rapidly with cold PBS at 4°C. The cells were lysed with RIPA lysis buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS) containing 25 µg/ml aprotinin, 2 mM sodium orthovanadate, 25 µg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 50 mM sodium fluoride for 30 min on ice. Lysates were clarified by centrifugation at 13,000 rpm for 15 min at 4°C, and protein concentrations were determined by using a Bio-Rad protein assay kit. Equal amounts of cellular lysates or concentrated culture supernatants were separated in 7.5% SDS-polyacrylamide gels and electrotransferred to 0.4 µM polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA in PBS-0.1% Tween 20, pH 7.4, overnight at 4°C. After washing with PBS-0.1% Tween 20, membranes were incubated with either anti-MMP-2 antibody (1:1,000) or anti-TIMP-2 (1:1,000) antibody at room temperature for 1 h. After extensive washing with three changes of PBS-0.1% Tween 20, membranes were incubated for 1 h with horseradish peroxidase-conjugated sheep anti-rabbit IgG or rabbit anti-mouse IgG at 1:10,000 dilution in blocking buffer. After washing, immunoreactivity was detected by using the ECL system.

RT-PCR. Total RNA was extracted from mesangial cells by using a kit of RNA STAT from Tel-Test B (Friendswood, TX). First-strand cDNA was synthesized by a T-Primed First-Stand Kit from Amershan Pharmacia. PCRs were performed and optimized according to standard protocols by using a kit from TaKaRa Shuzo (Shiga, Japan). Primers used for PCR were custom synthesized (GIBCO-BRL), and the sequences of each primer were as follows: 1) MMP-2, forward, 5'-ATCTGGTGTCTCCCTTACGG and reverse, 5'-GTGCAGTGATGTCCGACAAC; 2) TIMP-2, forward, 5'-CAAAGGACCTGACAAGGAC and reverse, 5'-TTGATGCAGGCAAAGAAC; 3) MT1-MMP, forward, 5'-ATTGATGCTGCTCTCTTCTGG and reverse, 5'-GTGAAGACTTCATCGCTGCC; and 4) GAPDH, forward, 5'-TCCCTCAAGATTGTCAGCAA and reverse, 5'-AGATCCACAACGGATACATT. The predicted sizes of the amplification products are 150, 182, 348, and 308 bp for MMP-2, MT1-MMP, TIMP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively.

Statistical analysis. Statistical analyses were performed by the Student's t-test. Data are presented as means ± SD. P values of <0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ET is an endogenous inhibitor of MMP-2 secretion and activation. We first examined whether endogenous ET had any effect on the basal secretion of MMP-2 by cultured MCs. For this purpose, a neutralizing monoclonal antibody against ET-1, -2, and -3 was added to MC cultures, and this produced a dose-dependent enhancement of MMP-2 activity, as revealed by zymography (Fig. 1A). The effect was not observed after addition of an irrelevant, isotype-matched control IgG.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Effects of neutralizing anti-endothelin-1 (ET-1), -2, and -3 monoclonal antibody and ETA receptor antagonists on matrix metalloproteinase-2 (MMP-2) activity as revealed by zymography. Mesangial cells (MCs) were incubated with the indicated concentrations of neutralizing anti-ET antibody (A, C) or ETA receptor antagonists (B, C) for periods of up to 72 h. Culture supernatants were harvested and assayed for gelatinase activity as described in MATERIALS AND METHODS. Similar results were obtained in 2 additional experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of the neutralizing anti-ET-1, -2, and -3 monoclonal antibody and ETA receptor antagonist on MMP-2 protein secretion by Western blot analysis. MCs were incubated with the indicated concentrations of neutralizing anti-ET antibody (A) or ETA receptor antagonists (B) for 24 h. Equal amounts of supernatants were concentrated and analyzed for MMP-2 protein by using immunoblotting as described in MATERIALS AND METHODS. Blots shown are 1 representative experiment from a series of 3, with similar results.

Exogenous ET-1 inhibits the secretion of MMP-2. If ET is an endogenous inhibitor of MMP-2, addition of exogenous ET-1 into cultures should result in a reduction of MMP-2 activity. As shown in Fig. 3, the addition of exogenous ET-1 into MC culture slightly lowered the basal level of MMP-2 secretion as revealed by zymography (Fig. 3). To clearly demonstrate the inhibitory action of exogenous ET-1 on MMP-2 production, we examined the effects of ET in a system where the activity of MMP-2 was amplified by the addition of different combinations of TNF-, IFN-, or LPS into MC culture. As indicated in Fig. 4, in the presence of stimulants, the activity of MMP-2 was obviously enhanced, and this enhancement could be partially prevented by pretreatment of MC with exogenous ET-1 (10⁷ M) (Fig. 4). Because zymography is only a semi-quantitative assay for MMP-2, to further confirm the above results and to accurately quantitate the change of MMP-2 in the presence of exogenous ET-1, additional quantitative assays for MMP-2 were employed. By using an antibody capture assay for MMP-2 activity, we selectively examined the effects of ET-1 on cytokines (INF- and TNF-) plus LPS-stimulated releasing of MMP-2 by MCs. It was found that under the stimulated condition, MCs increased the secretion of MMP-2 more than twofold that of control, whereas treatment of MCs with ET-1 significantly inhibited both the basal and stimulated secretion of MMP-2 (Fig. 5). Very similar results were obtained in Western blot studies by using a specific antibody against MMP-2, as revealed in Fig. 6. Consistent with the reduction of MMP-2 activity and protein secretion, we also found a decreased expression of MMP-2 at mRNA level in the presence of ET under both basal and stimulated conditions. This is reflected by the decreased amount of the amplication product of MMP-2 gene, but not of the product of the housekeeping gene GAPDH in RT-PCR (Fig. 7).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of ET-1 on MMP-2 activity. A representative zymographic analysis of MMP-2 activity in culture supernatants of MCs after treatment for 24 h with increasing concentrations of ET is shown. Similar results were obtained from an additional experiment.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Suppressive action of ET-1 on stimulant-induced enhancement of MMP-2 activity. MCs either untreated or treated with 107 M of ET-1 for 45 min were exposed to different combinations of lipopolysaccharide (LPS) (10 µg/ml), tumor necrosis factor- (TNF-, 50 ng/ml), and interferon- (IFN-, 100 U) for 48 h. Culture supernatants were harvested and assayed for gelatinase activity by zymography. Similar results were obtained in 3 additional experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of ET-1 on basal and stimulant-induced activity of MMP-2 as revealed by antibody capture assay. MCs treated with or without 107 M of ET-1, were exposed to the mixed stimulant (10 µg/ml LPS, 50 ng/ml TNF-, and 100 U IFN-) for 48 h. Supernatants were collected and assayed for MMP-2 activity by using a commercial kit as described in MATERIALS AND METHODS. Data are expressed as means ± SD (n = 4). Similar results were obtained from an additional experiment. *P < 0.01.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Inhibitory effects of ET-1 on the basal and stimulant-induced protein secretion of MMP-2 as indicated by Western blot. A: MCs, with or without treatment with 107 M of ET-1, were exposed to the mixed stimulant (10 µg/ml LPS, 50 ng/ml TNF-, and 100 U IFN-) for 48 h. Equal amounts of culture supernatants were concentrated and analyzed for the presence of MMP-2 protein by using immunoblotting. B: densitometric analysis of data from A. Values represent the means ± SD of 3 separate experiments and are expressed as percent untreated controls. * P < 0.01.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   RT-PCR analysis of MMP-2. MCs, with or without treatment with 107 M of ET-1, were exposed to the mixed stimulant (10 µg/ml LPS, 50 ng/ml TNF-, and 100 U IFN-) for 24 h. RNA extraction and RT-PCR analysis were performed as described in MATERIALS AND METHODS. Result shown is a representative of the 3 separate experiments.

Exogenous ET-1 inhibits the activation of MMP-2. MMP-2 is known to be secreted in latent form and must be activated to exert catalytic action. Therefore, it is important to understand the effects and the mechanisms of endothelin on the activation of MMP-2. For these purposes, a well-established model of MMP-2 activation induction by cytochalasin D was employed (1, 2). Mesangial cells were exposed to cytochalasin D in the presence or absence of ET-1 for 18 h, and the culture supernatant was then subjected to gel zymography. As indicated in Fig. 8, cytochalasin D elicited the activation of MMP-2, as demonstrated by the appearance of one major band of ~62 kDa. This action of cytochalasin D was dose dependent (Fig. 8B). In the presence of ET-1, the conversion of MMP-2 from the latent to the active form by cytochalasin D was significantly inhibited as revealed by zymography (Fig. 8) and Western blot (Fig. 9). Desitometric analysis of data from four separate experiments by Western blot indicated that the percent active MMP-2 in total MMP-2 was significantly decreased, from 49.5 ± 8.8 in cytochalasin D-treated cells to 34.2 ± 8 in cells pretreated with ET-1 (Fig. 9B). This action of ET-1 could be observed in a wide range of concentrations tested (Fig. 8A) and was most probably mediated by ET_A receptors, because ET_A receptor antagonist FR139317 almost completely blocked this effect of ET-1 (Fig. 10).

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Suppressive effects of ET-1 on cytochalasin D (Cyto D)-elicited activation of MMP-2. A: MCs, with or without treatment with the indicated concentrations of ET-1, were exposed to 1 µg/ml of Cyto D for 18 h. Culture supernatants were harvested and assayed for MMP-2 activity by using zymography. B: MCs were pretreated with 107 M of ET-1 before stimulation with the indicated concentrations of Cyto D.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Western blot showing the suppressive effects of ET-1 on Cyto D-elicited activation of MMP-2. A: MCs were pretreated with 107 M of ET-1 before exposure to Cyto D (1 µg/ml) for 18 h. Equal amounts of supernatants were concentrated and analyzed for MMP-2 protein by using immunoblotting as described in MATERIALS AND METHODS. B: desitometric analysis of data from A. Values represent the means ± SD of 4 separate experiments and are expressed as percent active MMP-2 in total MMP-2. #P < 0.05.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Blocking of the suppressive effects of ET-1 on Cyto D-induced activation of MMP-2 by ETA receptor antagonist. MCs were either left untreated or pretreated with 5 × 105 M ETA receptor antagonists for 1 h before exposure to ET-1 (107 M), as well as Cyto D (1 µg/ml), for an additional 18 h. Culture supernatants were harvested and assayed for MMP-2 activity by using zymography. Similar results were obtained in 2 additional experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 11.   Effects of ET-1 on tissue inhibitor of metalloproteinase-2 (TIMP-2) protein distribution as analyzed by Western blot. MCs were either left untreated or pretreated with 107 M of ET-1 before the addition of the indicated concentrations of Cyto D (up to 1 µg/ml) for 18 h. Equal amounts of supernatants (top) and cellular lysates (bottom) were harvested and analyzed for TIMP-2 protein expression by using immunoblotting. Similar results were obtained in 2 additional experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 12.   Prevention of Cyto D-elicited activation of MMP-2 by the recombinant TIMP-2. MCs were exposed to Cyto D (1 µg/ml) with or without the simultaneous addition of the indicated concentrations of recombinant TIMP-2 (µg/ml) for 18 h. Culture supernatants were harvested and assayed for MMP-2 activity by using zymography. Result shown is a representative of the 3 separate experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 13.   RT-PCR analysis of MMP-2, membrane type 1 matrix metalloproteinase (MT1-MMP), and TIMP-2. MCs were either left untreated or pretreated with 107 M of ET-1 before the addition of Cyto D (1 µg/ml) for 18 h. RNA extraction and RT-PCR analysis were performed as described in MATERIALS AND METHODS. Result shown is a representative of the 3 separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The suppressive action of exogenous ET-1 on collagenase activity has been previously reported in cardiac fibroblasts (19). However, the role of endogenous ET on collagenase activity as well on the activation of collagenase and the underlying mechanisms implicated has not been fully examined so far. Here, we reported several novel findings related to the regulation of MMP-2 by ET in cultured MCs. First, ET was demonstrated to be an endogenous inhibitor of MMP-2 secretion and activation in cultured MCs. Second, ET was found to be able to inhibit the cytokines (IFN- and TNF-) plus LPS-elicited production of MMP-2. Third, ET had the ability to block the conversion of pro-MMP-2 to active MMP-2, possibly via suppressing the MT1-MMP expression and increasing the secreted form of TIMP-2.

Addition of ET_A receptor antagonists into MC cultures caused an obvious increase of MMP-2 activity into the medium. Similar effects were produced by neutralizing anti-ET monoclonal antibody. The results suggest that the action of ET_A receptor antagonists was through blocking of the function of endogenous ET, rather than a direct induction of MMP-2 expression. Besides the obviously augmented activity of pro-MMP-2 in zymography, the amount of activated MMP-2 was also increased by ET_A receptor antagonists or by the neutralizing anti-ET antibody. This was reflected by the earlier appearance as well as the enhanced intensity of the 62-kDa band, representing activated MMP-2. These results clearly indicate that ET is a potent endogenous inhibitor of both MMP-2 secretion and activation in rat MCs. This idea is further strengthened by the fact that exogenous ET was able to inhibit the synthesis and activation of MMP-2 in both basal and stimulated conditions. It should be noted that the accumulation of the main substrate of MMP-2, collagen IV, in cultured rat mesangial cells in the presence of ET, has been previously reported (18). Furthermore, production of ET by MC (36) and the autocrine actions of ET on MC behavior have also been well documented (23, 24).

The inhibitory action of exogenous ET on basal MMP-2 secretion as detected by zymography was marginal, whereas by using Western blot or quantitative MMP-2 activity assay, a 30% reduction of MMP-2 was found; the discrepancy of these results may reflect the different sensitivities of the assay systems employed. Zymography is only a simple, semi-quantitative assay for MMP-2. It is worth mentioning that the detection and comparison of MMP-2 activity in this study were based on the equal volume of culture supernatants. The possible influence of increased MC numbers under ET stimulation was not taken into account. Because ET is a potent mitogen for MCs and there was a constant increase of MC numbers in the presence of ET (data not shown), the actual suppressive effect of ET on MMP-2 activity is, on a cell-for-cell basis, greater than that expressed by the data.

The mechanisms by which ET exerts its effect on the activity of MMP-2 appear to be both complicated and multiple. ET could act by inhibiting the mRNA and protein expression of MMP-2, as indicated in this study by RT-PCR, Western blot, and the specific antibody capture assay. It could also control the activation of MMP-2 by regulating the expression and/or activation of molecules involved in MMP-2 activation. In this respect, we demonstrated that ET was able to suppress the mRNA expression of MT1-MMP and to enhance the proportion of the secreted form of TIMP-2. The increased level of TIMP-2 in the culture medium may contribute to the suppression of MMP-2 activation by ET. This is supported by the observation that the addition of recombinant TIMP-2 into medium concentration dependently inhibited the cytochalasin D-elicited activation of MMP-2. A previous study has reported that ET has the ability to upregulate plasminogen activator inhibitors and suppress the activity of fibrolysis in MCs (21). Several studies have suggested a close interaction between MMPs and the plasminogen/plasmin system. For example, a role of plasmin in the activation of latent MT1-MMP, which in turn could activate latent MMP-2, has been proposed (33). Addition of plasmin into MC cultures can lead to the conversion of latent MMP-2 into active MMP-2 (3, 4). Thus it is likely that part of the action of ET on MMP-2 activation could be a secondary phenomenon, resulting from its effects on the plasminogen/plasmin system.

Mesangial cells have both ET_A and ET_B receptors. The available data support the concept that ETs act on MC via two different receptors (23). In this study, we demonstrated that ET_A receptor antagonists potentiated the secretion and activation of MMP-2 in MCs, indicating that the ET_A receptor is responsible for the regulation of MMP-2 activity. The complete blockade of the suppressive effects of ET on cytochalasin D-elicited activation of MMP-2 by the ET_A receptor antagonist FR139317, provided additional evidence supporting this conclusion. Several investigators have reported that ET induces mesangial matrix synthesis via ET_A receptor (18). Furthermore, ET_A receptor antagonist treatment of rats with various kidney diseases reduces the glomerular deposition of ECM proteins compared with untreated controls (5, 6, 17). In addition, the suppressive action of exogenous ET-1 on collagenase activity in cultured cardiac fibroblasts has also been demonstrated to be mediated by ET_A receptors (19). Thus it seems likely that ET acts on matrix turnover via ET_A receptors.

It is worth noting that some of the actions of ET on matrix synthesis in MCs are reported to be mediated by TGF- (18). TGF- itself has proved to be a potent inhibitor in ECM degradation in cultured MCs (3). Therefore, it can be assumed that the effects of ET on MMP-2 activity might also be via TGF-. However, most of the previous studies have implicated TGF- as a promoter rather than an inhibitor of MMP-2, releasing in MCs as well as in other cell types (26, 30, 34). Thus the exact role of TGF- in ET-induced suppression of MMP-2 activity remains to be addressed.

What are the potential in vivo pathophysiological implications of this study? As a predominant MMP involved in the degradation of major components of the GBM and mesangial matrix, MMP-2 plays an important role in the turnover of ECM in the renal glomerulus. The production of MMP-2 under steady-state conditions has been reported. Studies on renal biopsies demonstrated the presence of small amounts of MMP-2 in the normal glomerulus (14). The expression of MMP-2 in quiescent mouse, rat, and human mesangial cell lines has also been shown (25, 27, 28). Physiological levels of ET, secreted by MCs as well as other cell types within the glomerulus, may negatively regulate the activity of MMP-2, keeping its proteolytic potential within acceptable limits. Interestingly, one of the substrates of MMP-2 is reported to be big ET-1. MMP-2 cleaves big ET-1 to yield a novel and potent vasoconstrictor, ET-1 (15). It is highly possible that the autoregulatory loop between MMP-2 and ET might play an important role in the regulation of vascular responses. Under inflammatory conditions, ET expression is upregulated by a variety of cytokines, including IL-1, TNF-, and IFN- (22, 24). The enhanced level of ET might, in turn, counteract the action of these cytokines on MMP-2 activity, thus lessening the abnormal degradation of ECM and damage to glomerular structures. On the other hand, in diseases characterized by the accumulation of glomerular extracellular matrix, such as diabetic glomerulosclerosis, the increased expression of ET and decreased activity of MMP-2 have been reported (8, 9, 16, 37). Suppression of the activity of the protein-degrading enzymes by ET could contribute to glomerulosclerosis. In this context, augmentation of MMP-2 activity by ET receptor antagonists may be one of the important mechanisms by which such agents act therapeutically to slow progressive glomerulosclerosis.

In conclusion, our results demonstrate that ET is a potent inhibitor of MMP-2 secretion and activation in MCs. This finding may help us understand the subtle regulation of the synthesis and activation of MMP-2 in MCs. It also provides us with further insight into the pathophysiological mechanisms involving ET in the regulation of matrix turnover in glomerulus.