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Nitric Oxide Stimulates the Activity of a 72-kDa Neutral Matrix Metalloproteinase in Cultured Rat Mesangial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
218, 704–708 (1996)
0125
Nitric Oxide Stimulates the Activity of a 72-kDa Neutral Matrix Metalloproteinase in Cultured Rat Mesangial Cells Howard Trachtman1, Stephen Futterweit, Puneet Garg, Krishna Reddy, and Pravin C. Singhal Division of Nephrology and Pediatric Research Center, Schneider Children’s Hospital and Division of Nephrology, Department of Medicine, Long Island Jewish Medical Center, Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11042 Received December 13, 1995 We recently demonstrated that stimulation of inducible nitric oxide synthase (iNOS) activity reduced the accumulation of collagen and fibronectin in cultured rat mesangial cells. Therefore, we examined whether nitric oxide (NO) influenced the activity of a 72 kDa neutral matrix metalloproteinase by these cells in vitro. Enzyme activity was assessed in a biotin-avidin ELISA and by zymography. Exposure of mesangial cells to the cytokines, interferon (IFN)-g and lipopolysaccharide (LPS), increased gelatinolytic activity by 325 ± 60% (P < 0.025). Co-incubation with 20 mM L-arginine caused a further increase in matrix metalloproteinase levels. Addition of L-NAME, an inhibitor of iNOS, reversed the IFN-g/LPS-induced rise in gelatinolytic activity. Incubation with the exogenous NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), resulted in a dose dependent increase in metalloproteinase activity (P < 0.01). The NO-induced changes in metalloproteinase activity were also demonstrable by zymography. These data indicate that NO modulates the activity of a 72 kDa neutral matrix metalloproteinase and suggest that altered NO production may contribute to the development of glomerulosclerosis and tubulointerstitial fibrosis in chronic renal disease states. © 1996 Academic Press, Inc.
Accumulation of extracellular matrix (ECM) proteins in the glomerular mesangium compromises filtration function and is an important feature of progressive renal diseases such as diabetic nephropathy (1). Numerous hemodynamic factors such as mechanical stress (2) and circulating substances, such as the cytokine, transforming growth factor-6 (3), modulate the synthesis of these materials and the development of glomerulosclerosis. ECM deposition in the mesangium represents a balance between the rate of synthesis and degradation by various matrix metalloproteinases (4). These enzymes break down ECM proteins and prevent their build up in the mesangial regions (5). Gelatinase is a 72 kDa neutral matrix metalloproteinase and its activity is altered in several disease states including diabetic nephropathy (5,6). Nitric oxide (NO) is a short-lived messenger molecule that is synthesized from L-arginine using the enzyme nitric oxide synthase (NOS) (7,8). NO serves many functions within the kidney including regulation of afferent arteriolar tone (9), tubular handling of sodium (10,11), and mesangial cell proliferation (12). Glomerular mesangial cells and renal tubular epithelial cells possess the inducible isoform of NOS and synthesize NO in response to numerous cytokines (13,14). We recently demonstrated that NO down regulates the synthesis of type IV collagen and fibronectin and stimulates the production of laminin by rat mesangial cells (15). In order to better understand the role of NO in modulating glomerular accumulation of ECM proteins, we conducted the following studies to examine the effect of NO on gelatinolytic activity in cultured rat mesangial cells. METHODS Culture of rat mesangial cells. Rat mesangial cells were isolated and characterized in accord with previously described methods (15). The cells were grown in Dulbecco’s modified Eagle medium (DME) supplemented with penicillin (50 U/ml), 1 Correspondence address: Howard Trachtman, MD, Schneider Children’s Hospital, Division of Nephrology, 269-01 76th Avenue, New Hyde Park, NY 11042. FAX: 718-470-0887.
704 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
streptomycin (50 mg/ml) and 10% fetal bovine serum. The plastic flasks (25 cm2) were kept in a 90% air-10% CO2 environment at 37°C. Experimental conditions. When the cells became confluent (within 3–5 days), plates were randomly assigned to one of the following four experimental conditions: (1) Control (n 4 5): biotin-free RPMI media 1640 containing L-glutamine mixed with lactalbumin hydrolysate in a 2:1 ratio and no further additives; (2) L-NAME (n 4 5): control media supplemented with 1 mM L-NAME (NG-nitro-L-arginine methylester); (3) IFN-g/LPS (n 4 5): control media + g-interferon, 50 U/ml + lipopolysaccharide, 10 mg/ml; (4) IFN-g/LPS + L-NAME (n 4 5): control media + g-interferon, 50 U/ml + lipopolysaccharide, 10 mg/ml + 1 mM L-NAME. Followup experiments were performed in which 20 mM L-arginine was added to the experimental media outlined above. The effect of the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), on gelatinolytic activity was examined. Cells were maintained in the test media for 24 h. Conditioned media were collected, centrifuged at 1,000 g for 15 min and supernatants were stored at −20°C. Before assay for gelatinolytic activity, samples were concentrated with membrane concentrators with a molecular mass cut-off of 30 kDa. Biotin–avidin assay. A nonradioactive assay for gelatin degradation was modified from a previously published method using type IV collagen (16). Gelatin (1 mg/ml in distilled water) was dialyzed overnight against 0.1 M NaHCO3 at 4°C. D-biotin-N-hydroxysuccinimide ester (BNHS) was dissolved in dimethyl sulfoxide to a final concentration of 1 mg/ml and allowed to react with the gelatin at 4°C (0.25 mg BNHS/mg gelatin). Biotinylated gelatin was exhaustively dialyzed against PBS, pH 7.4, and aliquots of 0.5 mg were stored at −20°C. Two 96 well flat-bottom ELISA plates were coated overnight with avidin (100 ml of 10 mg/ml in distilled water per well) and blocked with 0.3% bovine serum albumin in PBS. Biotinylated gelatin was diluted to 50 mg/ml and 100 ml was added to each well of one of the avidin-coated ELISA plates. After incubation for 1 h at 20°C, this plate was rinsed with PBS. DL-a-amino-3-hydroxy-5-methylisoxazole-propionic acid was added to the conditioned media (5 ml of 10 nmol/100 ml), and 100 ml of media were added per well. After 3 h incubation at 37°C, supernatants from this first plate were transferred to the second avidin-coated ELISA plate and incubated for 30 min at 20°C. The second plate was washed twice with PBS before incubation with biotinylated horseradish peroxidase (1:16,000 in distilled water) for 30 min. The o-phenylene-diamine (OPD) substrate (50 ml distilled water, 5 ml of 30% H2O2, 500 ml of 10 mg/ml OPD in methanol) was added after rinsing the plate. The reaction was stopped after 30 min incubation in the dark at 20°C by adding 8 M H2SO4. The optical density was read at 490 nm in an ELISA plate reader. Gelatinolytic activity was expressed as mg/mg protein/1–3 h. Zymography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on a vertical slab gel containing 7.5% acrylamide and 0.25 mg/ml gelatin under nonreducing conditions at 4°C. After excessive washing with at least 500 ml of 2.5% Triton X-100 over 1 h, the gels were incubated overnight at 37°C in the collagen assay buffer (0.05 M Tris-HCl, 0.2 M NaCl, 10 mM CaCl2, pH 7.4). For visualization of the gelatinolytic bands, the gels were stained for 2 h in 0.1% Coomassie Brilliant Blue (50% methanol, 10% acetic acid. and 0.1% Coomassie Blue in distilled water). Protein assay. Mesangial cells were scraped and dissolved in 0.2 N NaOH. Protein content in each sample was determined using the Bio-Rad assay with bovine serum albumin standards. Materials and reagents. All plasticware was obtained from Corning Costar (Cambridge, MA) or Fisher Scientific (Pittsburgh, PA). The tissue culture materials were purchased from GIBCO BRL Life Technologies (Grand Island, NY) while the g-interferon was obtained from Genzyme (Cambridge, MA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Statistical analysis. Each experimental condition was studied 4–5 times and the results are presented as mean ± SEM. Groups were compared using an analysis of variance and the student t-test with the Bonferroni correction and differences were considered significant if the P value was less than 0.05.
RESULTS In the absence of IFN-g/LPS, gelatinolytic content of mesangial cells was unchanged by the addition of L-NAME and/or L-arginine to the culture media. This suggests that enzyme activity is unaffected by the basal level of NO synthesis (Figure 1). In contrast, exposure of mesangial cells to IFN-g/LPS consistently increased metalloproteinase content from 4.31 ± 1.35 to 13.32 ± 4.52 mg/mg protein (n 4 5), a 325 ± 60% elevation above the basal level, P < 0.025 (Figure 1). Incubation of the rat mesangial cells with IFN-g/LPS resulted in a six-fold increase in nitrite accumulation in the conditioned media (data not shown). The enhanced matrix metalloproteinase content following exposure to the cytokines was fully reversed by incubation with L-NAME. Addition of 20 mM L-arginine to experimental media containing IFN-g/LPS resulted in a further 27 ± 7% increment in the amount of gelatinolytic activity in mesangial cells to 15.22 ± 7.50 mg/mg protein, P < 0.05. This elevation in proteinase content was also antagonized by the NOS inhibitor, L-NAME (Figure 1). Addition of the NO donor, SNAP, caused a dose-dependent increase in matrix metalloproteinase 705
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Gelatinolytic activity (mg/mg protein) in rat mesangial cells measured by biotin-avidin ELISA. The presence or absence of IFN-g + LPS and L-NAME is indicated by the column labels. Open bars represent control media (biotin-free RPMI media 1640 mixed with lactalbumin hydrolysate in a 2:1 ratio) while hatched bars represent control media supplemented with 20 mM L-arginine. * P < 0.025 versus paired control media.
content that paralleled the change achieved by cytokine-stimulation of iNOS activity, i.e., 6.40 ± 0.64 mg/mg protein, control, 7.80 ± 0.25 mg/mg protein, 10−4 M SNAP and 16.40 ± 0.95 mg/mg protein, 10−3 M SNAP, P < 0.01 (Figure 2). The alterations in gelatinolytic activity that were observed in the biotin-avidin ELISA were reproduced by zymography. Thus, exposure to the cytokine mixture, IFN-g/LPS, (Lane 2) stimulated synthesis and activation of the 72 kDa matrix metalloproteinase compared to control conditions (Lane 1) (Figure 3). L-NAME had no effect on basal matrix metalloproteinase production in standard conditions. However, addition of L-NAME to test media containing IFN-g/LPS reduced production and activation of the gelatinase (data not shown). These findings were noted in three separate experiments. DISCUSSION These data indicate that stimulation of iNOS activity in rat mesangial cells by exposure to IFN-g and LPS leads to increased activity of the neutral 72 kDa matrix metalloproteinase, gelatinase.
FIG. 2. Gelatinolytic activity (mg/mg protein) in rat mesangial cells measured by biotin-avidin ELISA. Cells were incubated in control media (biotin-free RPMI media 1640 mixed with lactalbumin hydrolysate in a 2:1 ratio), control media supplemented with 10−4 M SNAP (hatched bar), or control media containing 10−3 M SNAP (cross-hatched bar). * P < 0.01 versus control; ** P < 0.001 versus control. 706
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Gelatinolytic activity in rat mesangial cells assayed by zymography. The arrow indicates the position of the 72 kDa matrix metalloproteinase gelatinolytic activity. Lane 1, control media (biotin-free RPMI media 1640 mixed with lactalbumin hydrolysate in a 2:1 ratio); Lane 2, control media + IFN-g/LPS.
Elevated levels of the enzyme were documented in both a quantitative and a functional assay. NO has been shown to induce activation of metalloproteinase enzymes in bovine and human articular cartilage (17). When the findings from this study are combined with our recent report on the effect of NO on ECM protein synthesis by cultured rat mesangial cells (15), it is evident that this short-lived messenger molecule plays an important role in the accumulation of matrix components in the glomerular mesangium. The mechanism by which NO alters gelatinolytic activity cannot be discerned from these studies. NO may be acting at the transcriptional, translational or post-translational level to modify enzyme activity. In addition, NO may exert its effects through other mediators including reactive oxygen molecules or cytokines such as TGF-6 (18). The coordinated NO-mediated changes in mesangial cell ECM production—decreased synthesis and increased degradation—suggest that NO may act as an antifibrotic agent to prevent glomerular matrix build up in disease states. It is conceivable that the action of other agents such as TGF-6, which also increase matrix synthesis and suppress degradation, may be mediated via NO as a second messenger. The role of NO in the pathogenesis of renal disease is becoming increasingly complex. During circumstances in which there is acute immunologically-mediated glomerular injury, NO may act as a pro-inflammatory agent to foster glomerular damage. For example, in rats with glomerulonephritis induced by injection of anti-thymocyte serum, proteinuria, glomerular expression of TGF-6, and accumulation of ECM proteins is suppressed by treatment with the iNOS inhibitor, L-NMMA (19). In contrast, in rats with the remnant kidney model of chronic renal failure, administration of supplemental L-arginine ameliorates structural damage while L-NAME treatment exacerbates glomerulosclerosis and tubulointerstitial fibrosis (20,21). Our data are consistent with the later studies in which NO synthesis attenuated renal parenchymal scarring during the course of a chronic, non-inflammatory disease process. In summary, we have demonstrated that increased NO production by rat mesangial cells in vitro stimulates the activity of the 72 kDa neutral metalloproteinase, gelatinase. These findings suggest that NO may play an important role in regulating ECM accumulation in chronic renal disease states. ACKNOWLEDGMENTS This work was supported in part by grants from the Genentech Foundation for Growth and Development (HT) and the National Institute of Health Grant 2-RO1-DA06753-05 (PCS).
REFERENCES 1. Mauer, S. M., Steffes, M. W., Ellis, E. N., Sutherland, D. E. R., Brown, D. M., and Goetz, F. C. (1984) J. Clin. Invest. 74, 1143–1155. 2. Harris, R. C., Haralson, M. A., and Badr, K. F. (1992) Lab. Invest. 66, 548–556. 3. Border, W. A., and Ruoslahti, E. (1992) J. Clin. Invest. 90, 1–7. 4. Davies, M., Martin, J., Thomas, G. J., and Lovett, D. H. (1992) Kidney Int. 41, 671–678. 5. Lovett, D. H., Sterzel, R. B., Kashgarian, M, and Ryan, J. L. (1983) Kidney Int. 23, 342–349. 6. Sagar, S., Sorbi, D., Arbeit, L., and Singhal, P. C. (1994) Am. J. Physiol. 267, F654–F659. 707
Vol. 218, No. 3, 1996 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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Moncada, S., and Higgs, A. (1993) N. Engl. J. Med. 329, 2002–2012. Ignarro, L. J. (1990) Hypertension 16, 477–483. Ito, S., and Ren, Y. L. (1993) J. Clin. Invest. 92, 1093–1098. Radermacher, J., Klanke, B., Schurek, H. J., Stolte, H. F., and Frolich, J. C. (1992) Kidney Int. 41, 1549–1559. Schultz, P. J., and Tolins, J. P. (1993) J. Clin. Invest. 91, 642–650. Garg, U. C., and Hassid, A. (1989) Am. J. Physiol. 257, F60–F66. Shultz, P. J., Archer, S. L., and Rosenberg, M. E. (1994) Kidney Int. 46, 683–689. Markewitz, B. A., Michael, J. R., and Kohan, D. E. (1993) J. Clin. Invest. 91, 138–143. Trachtman, H., Futterweit, S., and Singhal, P. C. (1995) Biochem Biophys Res. Commun. 207, 120–125. Wilkinson, M. J., Cohen, R. L., and Shuman, M. A. (1990) Anal. Biochem. 185, 294–296. Murrell, G. A. C., Jang, D., and Williams, R. J. (1995) Biochem. Biophys. Res. Commun. 206, 15–21. Ketteler, M., Border, W. A., and Noble, N. A. (1994) Am. J. Physiol. 267, F197–F207. Narita, I., Borde, W. A., Ketteler, M., and Noble, N. A. (1995) Lab. Invest. 72, 17–24. Reyes, A. A., Purkerson, ,M. L., Karl, I., and Klahr, S. (1992) Am. J. Kid. Dis. 20, 168–176. Fujihara, C. K., DeNucci, G., and Zatz, R. (1995) J. Am. Soc. Nephrol. 5, 1498–1507.
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218, 704–708 (1996)
0125
Nitric Oxide Stimulates the Activity of a 72-kDa Neutral Matrix Metalloproteinase in Cultured Rat Mesangial Cells Howard Trachtman1, Stephen Futterweit, Puneet Garg, Krishna Reddy, and Pravin C. Singhal Division of Nephrology and Pediatric Research Center, Schneider Children’s Hospital and Division of Nephrology, Department of Medicine, Long Island Jewish Medical Center, Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11042 Received December 13, 1995 We recently demonstrated that stimulation of inducible nitric oxide synthase (iNOS) activity reduced the accumulation of collagen and fibronectin in cultured rat mesangial cells. Therefore, we examined whether nitric oxide (NO) influenced the activity of a 72 kDa neutral matrix metalloproteinase by these cells in vitro. Enzyme activity was assessed in a biotin-avidin ELISA and by zymography. Exposure of mesangial cells to the cytokines, interferon (IFN)-g and lipopolysaccharide (LPS), increased gelatinolytic activity by 325 ± 60% (P < 0.025). Co-incubation with 20 mM L-arginine caused a further increase in matrix metalloproteinase levels. Addition of L-NAME, an inhibitor of iNOS, reversed the IFN-g/LPS-induced rise in gelatinolytic activity. Incubation with the exogenous NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), resulted in a dose dependent increase in metalloproteinase activity (P < 0.01). The NO-induced changes in metalloproteinase activity were also demonstrable by zymography. These data indicate that NO modulates the activity of a 72 kDa neutral matrix metalloproteinase and suggest that altered NO production may contribute to the development of glomerulosclerosis and tubulointerstitial fibrosis in chronic renal disease states. © 1996 Academic Press, Inc.
Accumulation of extracellular matrix (ECM) proteins in the glomerular mesangium compromises filtration function and is an important feature of progressive renal diseases such as diabetic nephropathy (1). Numerous hemodynamic factors such as mechanical stress (2) and circulating substances, such as the cytokine, transforming growth factor-6 (3), modulate the synthesis of these materials and the development of glomerulosclerosis. ECM deposition in the mesangium represents a balance between the rate of synthesis and degradation by various matrix metalloproteinases (4). These enzymes break down ECM proteins and prevent their build up in the mesangial regions (5). Gelatinase is a 72 kDa neutral matrix metalloproteinase and its activity is altered in several disease states including diabetic nephropathy (5,6). Nitric oxide (NO) is a short-lived messenger molecule that is synthesized from L-arginine using the enzyme nitric oxide synthase (NOS) (7,8). NO serves many functions within the kidney including regulation of afferent arteriolar tone (9), tubular handling of sodium (10,11), and mesangial cell proliferation (12). Glomerular mesangial cells and renal tubular epithelial cells possess the inducible isoform of NOS and synthesize NO in response to numerous cytokines (13,14). We recently demonstrated that NO down regulates the synthesis of type IV collagen and fibronectin and stimulates the production of laminin by rat mesangial cells (15). In order to better understand the role of NO in modulating glomerular accumulation of ECM proteins, we conducted the following studies to examine the effect of NO on gelatinolytic activity in cultured rat mesangial cells. METHODS Culture of rat mesangial cells. Rat mesangial cells were isolated and characterized in accord with previously described methods (15). The cells were grown in Dulbecco’s modified Eagle medium (DME) supplemented with penicillin (50 U/ml), 1 Correspondence address: Howard Trachtman, MD, Schneider Children’s Hospital, Division of Nephrology, 269-01 76th Avenue, New Hyde Park, NY 11042. FAX: 718-470-0887.
704 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
streptomycin (50 mg/ml) and 10% fetal bovine serum. The plastic flasks (25 cm2) were kept in a 90% air-10% CO2 environment at 37°C. Experimental conditions. When the cells became confluent (within 3–5 days), plates were randomly assigned to one of the following four experimental conditions: (1) Control (n 4 5): biotin-free RPMI media 1640 containing L-glutamine mixed with lactalbumin hydrolysate in a 2:1 ratio and no further additives; (2) L-NAME (n 4 5): control media supplemented with 1 mM L-NAME (NG-nitro-L-arginine methylester); (3) IFN-g/LPS (n 4 5): control media + g-interferon, 50 U/ml + lipopolysaccharide, 10 mg/ml; (4) IFN-g/LPS + L-NAME (n 4 5): control media + g-interferon, 50 U/ml + lipopolysaccharide, 10 mg/ml + 1 mM L-NAME. Followup experiments were performed in which 20 mM L-arginine was added to the experimental media outlined above. The effect of the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), on gelatinolytic activity was examined. Cells were maintained in the test media for 24 h. Conditioned media were collected, centrifuged at 1,000 g for 15 min and supernatants were stored at −20°C. Before assay for gelatinolytic activity, samples were concentrated with membrane concentrators with a molecular mass cut-off of 30 kDa. Biotin–avidin assay. A nonradioactive assay for gelatin degradation was modified from a previously published method using type IV collagen (16). Gelatin (1 mg/ml in distilled water) was dialyzed overnight against 0.1 M NaHCO3 at 4°C. D-biotin-N-hydroxysuccinimide ester (BNHS) was dissolved in dimethyl sulfoxide to a final concentration of 1 mg/ml and allowed to react with the gelatin at 4°C (0.25 mg BNHS/mg gelatin). Biotinylated gelatin was exhaustively dialyzed against PBS, pH 7.4, and aliquots of 0.5 mg were stored at −20°C. Two 96 well flat-bottom ELISA plates were coated overnight with avidin (100 ml of 10 mg/ml in distilled water per well) and blocked with 0.3% bovine serum albumin in PBS. Biotinylated gelatin was diluted to 50 mg/ml and 100 ml was added to each well of one of the avidin-coated ELISA plates. After incubation for 1 h at 20°C, this plate was rinsed with PBS. DL-a-amino-3-hydroxy-5-methylisoxazole-propionic acid was added to the conditioned media (5 ml of 10 nmol/100 ml), and 100 ml of media were added per well. After 3 h incubation at 37°C, supernatants from this first plate were transferred to the second avidin-coated ELISA plate and incubated for 30 min at 20°C. The second plate was washed twice with PBS before incubation with biotinylated horseradish peroxidase (1:16,000 in distilled water) for 30 min. The o-phenylene-diamine (OPD) substrate (50 ml distilled water, 5 ml of 30% H2O2, 500 ml of 10 mg/ml OPD in methanol) was added after rinsing the plate. The reaction was stopped after 30 min incubation in the dark at 20°C by adding 8 M H2SO4. The optical density was read at 490 nm in an ELISA plate reader. Gelatinolytic activity was expressed as mg/mg protein/1–3 h. Zymography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on a vertical slab gel containing 7.5% acrylamide and 0.25 mg/ml gelatin under nonreducing conditions at 4°C. After excessive washing with at least 500 ml of 2.5% Triton X-100 over 1 h, the gels were incubated overnight at 37°C in the collagen assay buffer (0.05 M Tris-HCl, 0.2 M NaCl, 10 mM CaCl2, pH 7.4). For visualization of the gelatinolytic bands, the gels were stained for 2 h in 0.1% Coomassie Brilliant Blue (50% methanol, 10% acetic acid. and 0.1% Coomassie Blue in distilled water). Protein assay. Mesangial cells were scraped and dissolved in 0.2 N NaOH. Protein content in each sample was determined using the Bio-Rad assay with bovine serum albumin standards. Materials and reagents. All plasticware was obtained from Corning Costar (Cambridge, MA) or Fisher Scientific (Pittsburgh, PA). The tissue culture materials were purchased from GIBCO BRL Life Technologies (Grand Island, NY) while the g-interferon was obtained from Genzyme (Cambridge, MA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Statistical analysis. Each experimental condition was studied 4–5 times and the results are presented as mean ± SEM. Groups were compared using an analysis of variance and the student t-test with the Bonferroni correction and differences were considered significant if the P value was less than 0.05.
RESULTS In the absence of IFN-g/LPS, gelatinolytic content of mesangial cells was unchanged by the addition of L-NAME and/or L-arginine to the culture media. This suggests that enzyme activity is unaffected by the basal level of NO synthesis (Figure 1). In contrast, exposure of mesangial cells to IFN-g/LPS consistently increased metalloproteinase content from 4.31 ± 1.35 to 13.32 ± 4.52 mg/mg protein (n 4 5), a 325 ± 60% elevation above the basal level, P < 0.025 (Figure 1). Incubation of the rat mesangial cells with IFN-g/LPS resulted in a six-fold increase in nitrite accumulation in the conditioned media (data not shown). The enhanced matrix metalloproteinase content following exposure to the cytokines was fully reversed by incubation with L-NAME. Addition of 20 mM L-arginine to experimental media containing IFN-g/LPS resulted in a further 27 ± 7% increment in the amount of gelatinolytic activity in mesangial cells to 15.22 ± 7.50 mg/mg protein, P < 0.05. This elevation in proteinase content was also antagonized by the NOS inhibitor, L-NAME (Figure 1). Addition of the NO donor, SNAP, caused a dose-dependent increase in matrix metalloproteinase 705
Vol. 218, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Gelatinolytic activity (mg/mg protein) in rat mesangial cells measured by biotin-avidin ELISA. The presence or absence of IFN-g + LPS and L-NAME is indicated by the column labels. Open bars represent control media (biotin-free RPMI media 1640 mixed with lactalbumin hydrolysate in a 2:1 ratio) while hatched bars represent control media supplemented with 20 mM L-arginine. * P < 0.025 versus paired control media.
content that paralleled the change achieved by cytokine-stimulation of iNOS activity, i.e., 6.40 ± 0.64 mg/mg protein, control, 7.80 ± 0.25 mg/mg protein, 10−4 M SNAP and 16.40 ± 0.95 mg/mg protein, 10−3 M SNAP, P < 0.01 (Figure 2). The alterations in gelatinolytic activity that were observed in the biotin-avidin ELISA were reproduced by zymography. Thus, exposure to the cytokine mixture, IFN-g/LPS, (Lane 2) stimulated synthesis and activation of the 72 kDa matrix metalloproteinase compared to control conditions (Lane 1) (Figure 3). L-NAME had no effect on basal matrix metalloproteinase production in standard conditions. However, addition of L-NAME to test media containing IFN-g/LPS reduced production and activation of the gelatinase (data not shown). These findings were noted in three separate experiments. DISCUSSION These data indicate that stimulation of iNOS activity in rat mesangial cells by exposure to IFN-g and LPS leads to increased activity of the neutral 72 kDa matrix metalloproteinase, gelatinase.
FIG. 2. Gelatinolytic activity (mg/mg protein) in rat mesangial cells measured by biotin-avidin ELISA. Cells were incubated in control media (biotin-free RPMI media 1640 mixed with lactalbumin hydrolysate in a 2:1 ratio), control media supplemented with 10−4 M SNAP (hatched bar), or control media containing 10−3 M SNAP (cross-hatched bar). * P < 0.01 versus control; ** P < 0.001 versus control. 706
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FIG. 3. Gelatinolytic activity in rat mesangial cells assayed by zymography. The arrow indicates the position of the 72 kDa matrix metalloproteinase gelatinolytic activity. Lane 1, control media (biotin-free RPMI media 1640 mixed with lactalbumin hydrolysate in a 2:1 ratio); Lane 2, control media + IFN-g/LPS.
Elevated levels of the enzyme were documented in both a quantitative and a functional assay. NO has been shown to induce activation of metalloproteinase enzymes in bovine and human articular cartilage (17). When the findings from this study are combined with our recent report on the effect of NO on ECM protein synthesis by cultured rat mesangial cells (15), it is evident that this short-lived messenger molecule plays an important role in the accumulation of matrix components in the glomerular mesangium. The mechanism by which NO alters gelatinolytic activity cannot be discerned from these studies. NO may be acting at the transcriptional, translational or post-translational level to modify enzyme activity. In addition, NO may exert its effects through other mediators including reactive oxygen molecules or cytokines such as TGF-6 (18). The coordinated NO-mediated changes in mesangial cell ECM production—decreased synthesis and increased degradation—suggest that NO may act as an antifibrotic agent to prevent glomerular matrix build up in disease states. It is conceivable that the action of other agents such as TGF-6, which also increase matrix synthesis and suppress degradation, may be mediated via NO as a second messenger. The role of NO in the pathogenesis of renal disease is becoming increasingly complex. During circumstances in which there is acute immunologically-mediated glomerular injury, NO may act as a pro-inflammatory agent to foster glomerular damage. For example, in rats with glomerulonephritis induced by injection of anti-thymocyte serum, proteinuria, glomerular expression of TGF-6, and accumulation of ECM proteins is suppressed by treatment with the iNOS inhibitor, L-NMMA (19). In contrast, in rats with the remnant kidney model of chronic renal failure, administration of supplemental L-arginine ameliorates structural damage while L-NAME treatment exacerbates glomerulosclerosis and tubulointerstitial fibrosis (20,21). Our data are consistent with the later studies in which NO synthesis attenuated renal parenchymal scarring during the course of a chronic, non-inflammatory disease process. In summary, we have demonstrated that increased NO production by rat mesangial cells in vitro stimulates the activity of the 72 kDa neutral metalloproteinase, gelatinase. These findings suggest that NO may play an important role in regulating ECM accumulation in chronic renal disease states. ACKNOWLEDGMENTS This work was supported in part by grants from the Genentech Foundation for Growth and Development (HT) and the National Institute of Health Grant 2-RO1-DA06753-05 (PCS).
REFERENCES 1. Mauer, S. M., Steffes, M. W., Ellis, E. N., Sutherland, D. E. R., Brown, D. M., and Goetz, F. C. (1984) J. Clin. Invest. 74, 1143–1155. 2. Harris, R. C., Haralson, M. A., and Badr, K. F. (1992) Lab. Invest. 66, 548–556. 3. Border, W. A., and Ruoslahti, E. (1992) J. Clin. Invest. 90, 1–7. 4. Davies, M., Martin, J., Thomas, G. J., and Lovett, D. H. (1992) Kidney Int. 41, 671–678. 5. Lovett, D. H., Sterzel, R. B., Kashgarian, M, and Ryan, J. L. (1983) Kidney Int. 23, 342–349. 6. Sagar, S., Sorbi, D., Arbeit, L., and Singhal, P. C. (1994) Am. J. Physiol. 267, F654–F659. 707
Vol. 218, No. 3, 1996 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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