Feline chronic kidney disease is associated with upregulation of transglutaminase 2: a collagen cross-linking enzyme.

Chronic kidney disease is a major cause of morbidity and mortality in cats. Transglutaminase 2 (TG2) is a calcium-dependent enzyme proposed to mediate tubulointerstitial fibrosis in the kidney by cross-linking collagen fibrils. Postmortem kidney tissue was obtained from primary renal azotemic (n = 10) and nonazotemic (n = 5) cats (14 domestic short hair, 1 Burmese; aged 9-23.7 years). Extracellular matrix protein deposition was determined by Masson's trichrome staining and collagen immunofluorescence. Total kidney transglutaminase (TG) enzyme activity and TG2 protein were measured in tissue homogenates by putrescine incorporation and Western blotting. Extracellular TG enzyme activity and TG2 protein were determined in situ by immunofluorescence, quantified by multiphase image analysis. Results were compared using the unpaired Student's t-test with Welch's correction. Elevated plasma creatinine, urea, and phosphate concentrations were associated with tubulointerstitial fibrosis but not glomerular fibrosis. Kidney homogenates from azotemic cats showed a 3-fold higher total TG enzyme activity and TG2 protein compared with kidneys from nonazotemic cats. Immunofluorescent studies performed in situ confirmed a 3-fold higher extracellular TG enzyme activity and TG2 protein in cats with azotemia. Tubulointerstitial TG2 showed a positive linear correlation with both renal function and tubulointerstitial fibrosis. In conclusion, for cats with azotemia, both filtration failure and tubulointerstitial fibrosis were associated with the upregulation of TG2, a collagen cross-linking enzyme and the major isoform of transglutaminase in the kidney. TG2 may provide a new therapeutic target for drugs designed to slow the progression of feline chronic kidney disease.

Seliciclib inhibits renal hypertrophy but not fibrosis in the rat following subtotal nephrectomy.

BACKGROUND: 5/6 subtotal nephrectomy (SNx) is a non-immune stimulus used to induce renal fibrosis. The ability of seliciclib, a cyclin-dependent kinase inhibitor, to reduce kidney hypertrophy and extracellular matrix (ECM) deposition has been examined in the SNx rat. METHODS: Wistar rats were subjected to SNx under isoflurane anaesthesia. The acute effect of seliciclib 28 mg/kg (5 days) on compensatory renal growth (CRG), kidney protein and DNA was determined. In chronic studies albuminuria, hypertension and GFR were monitored. Ki67, apoptag and α-smooth muscle actin were determined by immunohistochemistry together with Masson's trichrome staining. The effect of a maximum non-hypotensive dose of seliciclib 28 mg/kg (8 weeks) was determined. RESULTS: Acutely, the remnant kidney developed CRG. Seliciclib 28 mg/kg inhibited both CRG by 45% and increased kidney protein by 48% without affecting increased kidney DNA. Chronically, SNx rats developed albuminuria, hypertension, low GFR with increased tubulointerstitial cell proliferation, apoptosis, myofibroblast accumulation and enhanced ECM deposition. Seliciclib 28 mg/kg (8 weeks) had no effect on either renal function or renal pathology. Plasma concentrations of seliciclib exceeded 5 µM throughout the study. CONCLUSIONS: Despite inhibition of early renal hypertrophy, a maximum non-hypotensive dose of seliciclib 28 mg/kg had no impact on the progression of kidney fibrosis in the SNx rat.

Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide.

The N-terminal sequence of a novel sheep-derived peptide with growth inhibitory activity has been obtained. The N-terminal fragment was chemically synthesised and designated EPL001. The kidney was chosen as the first mammalian system in which to study EPL001 since kidney growth can be accurately quantified following a surgical reduction in renal mass. Cell proliferation was measured in mouse collecting duct kidney (MCDK) cells stimulated with insulin-like growth factor I (IGF-I). Compensatory renal growth (CRG) was induced in Wistar rats and either EPL001 or an EPL001 antibody delivered by continuous renal tissue infusion. Mouse monoclonal antibodies to EPL001 were generated for immunoneutralisation, rabbit polyclonal antibodies were generated for immunohistochemistry. EPL001 had no apparent effect on IGF-I stimulated cell proliferation in MCDK cells in vitro, yet provoked a dose-dependent inhibition of CRG in vivo. An EPL001 antibody potentiated CRG, in the absence of exogenous EPL001, consistent with an inhibitory role in kidney growth for an endogenous peptide containing the EPL001 sequence. Tubular staining for epitopes to the EPL001 sequence was detected in normal human kidney sections and enhanced in renal cell carcinoma. Results support the presence of growth inhibitory activity in the N-terminus of a sheep-derived peptide with evidence for both its presence and endogenous activity in the kidney. Attempts to further characterise its structure and activity are ongoing.

Stem cell factor in a rat model of serum nephrotoxic nephritis.

BACKGROUND: Stem cell factor (SCF) has been implicated in many disease processes characterized by tissue remodelling and fibrosis. The growth factor (SCF) was evaluated in a rat model of nephrotoxic serum nephritis (NTN), characterized by early inflammation followed by later tissue fibrosis. METHODS: NTN was induced in male Wistar Kyoto rats using rabbit anti-rat glomerular basement membrane antibodies. Animals were sacrificed at days 7, 15, 30 and 45 (n = 4-10 per group). Rats' kidneys were immunostained for ED1 as marker of inflammation, CD34, SCF, c-kit, mast cell tryptase and markers of fibrosis; collagens III and IV and alpha-SMA. Changes in SCF protein and mRNA content were evaluated by Western blotting and Northern blotting, respectively. RESULTS: In the NTN kidney, levels of immunoreactive SCF and SCF receptor (c-kit) were significantly higher in glomerular, tubular and interstitial compartments. Mast cells were barely detectable in NTN and control rat sections. Double immunostaining showed the co-localization of SCF with alpha-SMA and of the SCF receptor with CD34 and ED1 positive cells. Immunostainable SCF protein in each of the 3 compartments, glomerular, tubular and interstitial, showed a positive linear correlation with serum creatinine, proteinuria, glomerulosclerosis score and interstitial fibrosis scores. Using multivariate analysis, immunostainable tubular SCF was a predictor of glomerular sclerosis and immunostainable glomerular SCF predicted tubular atrophy. Increased SCF immunostain was not a consequence of altered transcription as there was a fall in SCF mRNA determined by Northern blotting. Western blotting of NTN kidney homogenates revealed two bands for SCF, a 43-kDa band which decreased, and a 19-kDa band which increased throughout the study. CONCLUSION: These results highlight the potential role of SCF and its receptor in the remodelling process of the NTN kidney. Upregulation of SCF may involve a translational mechanism, with the soluble SCF protein KL-S1 (19 kDa) being derived from the transmembrane SCF protein KL-1 (43 kD) by proteolytic cleavage. The immunohistochemical staining of few CD34+ cells in NTN kidneys warrants further evaluation of the nature of these cells in the context of the inflammatory as well as the fibrotic processes.

The effect of body temperature in a rat model of renal ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is an unavoidable event in renal transplantation; the effects of IRI can be seen in both the acute and long-term function of the transplanted organ. For this reason, research into the pathophysiology of ischemia-reperfusion is at the forefront of transplantation research. Animal models, particularly in the rat, provide a useful research tool in studying the intricacies of IRI and in evaluating new treatments. We describe a model of right nephrectomy and left renal clamping for 45 minutes and demonstrate the effects of temperature variation during the ischemic period. METHODS: Male Sprague-Dawley rats (under isoflurane anesthesia) underwent bilateral flank incision with removal of the right kidney and clamping of the left renal hilum for 45 minutes. The animals were divided into 3 groups (n=6): group 1 had the procedure performed on a heating mat with no temperature control facilities, group 2 used no heating mat, and group 3 used a rectal temperature-controlled homeothermic blanket system (Harvard Medical, United Kingdom). Temperature was taken every 5 minutes throughout the procedure and blood samples were taken on a daily postoperative basis via tail vein venepuncture. RESULTS: The average temperature at the end of the procedure in group 1 was 39.67 degrees C and the creatinine level at day 3 was 574+/-17.84, in group 2 the temperature was 32.6 degrees C and the creatinine level was 115+/-4.06, and in group 3 the temperature was maintained between 36.5 degrees C-37 degrees C and the serum creatinine level was 329+/-19.18. The temperature of the animal during the ischemia phase of IRI significantly affects the severity of injury. Relative hyperthermia resulted in more severe renal injury and unrecoverable acute renal failure, no source of heat led to a relative hypothermia, and reduction of renal injury. Use of the homeothermic heating blanket led to an increase in creatinine level by day 3, indicating a significant ischemic stimulus; however, by day 10 the creatinine level had returned to normal. CONCLUSION: This illustrates the importance of temperature as a variable in animal models of IRI and thus should be clearly stated in all experimental methods to ensure an appropriate ischemic stimulus and for adequate comparisons between various therapeutic interventions.

FTY720 reduces extracellular matrix expansion associated with ischemia-reperfusion induced injury.

BACKGROUND: Ischemia-reperfusion (IR) is one of the strongest nonimmune factors associated with the development of chronic allograft nephropathy (CAN). This effect is often exacerbated by immunosuppressive medications, most notably cyclosporine. Although traditionally the macrophage was thought to stimulate fibroblast activity in CAN, recent evidence supports a role for lymphocytes. FTY720 is a new immunosuppressant that promotes lymphocyte sequestration into lymph nodes and Peyer's patches. This study investigated the effect of FTY720 on renal fibrosis in the rat following an IR insult (IRI). METHODS: A rat model of IRI was used in which male Sprague-Dawley rats (under isoflurane anaesthesia) underwent bilateral flank incision with removal of the right kidney and clamping of the left renal hilum for 45 minutes. Five groups of animals were studied (n=4): nephrectomy only, IRI only, IRI+FTY720 (1 mg/kg/d), IRI+cyclosporine (15 mg/kg/d), and IRI+FTY 720 (1 mg/kg/d) and cyclosporine (15 mg/kg/d). Animals were humanely killed at 30 days. RESULTS: Serum creatinine (SCr) level was significantly reduced in the FTY720-treated animals. IRI alone produced a significant increase in SCr level compared with neprectomized animals (138 micromol/L vs 55 micromol/L; P<.05). This effect was potentiated by treatment with cyclosporine (173 micromol/L vs 55 micromol/L; P<.05). Treatment with FTY720 significantly reduced SCr level in rats following IRI alone (81 micromol/L vs 138 micromol/L; P<.01) and in rats following IRI + cyclosporine (98 micromol/L vs 173 micromol/L; P<.014). Parallel changes were seen in the levels of proteinuria. Fibrosis was assessed using Masson's trichrome (MT) staining. IRI alone produced a significant increase in MT staining compared with nephrectomized animals (0.92 vs 0.03; P<.05). This effect was potentiated by treatment with cyclosporine (1.12 vs 0.92; P=.022). Treatment with FTY720 reduced the level of MT staining in rats following IRI alone (0.34 vs 0.92; P<.05) and in rats following IRI+cyclosporine (70.34 vs 1.12; P<.05). Levels of TGF-beta1 were considerably reduced in FTY720-treated animals (compared with cyclosporine+IRI and IRI only), either alone (196+/-31 pg/mL vs 1105+/-59 pg/mL and 611+/-38; P<.05) or in conjunction with cyclosporine (423+/-26 pg/mL vs 1105+/-59 pg/mL and 611+/-38; P<.05). CONCLUSION: Our study shows that treatment with FTY720 can reduce renal fibrosis as a result of IRI, both alone and in conjunction with cyclosporine. This provides promising evidence for using FTY720 in a calcineurin-free or reduced-dose immunosuppression protocol in an effort to reduce the incidence of CAN.

Localization of matrix metalloproteinases and their inhibitors in experimental progressive kidney scarring.

The extracellular matrix (ECM) is in a continual state of turnover with homeostasis maintained by balancing synthesis and degradation rates. During progressive kidney scarring an imbalance occurs leading to ECM accumulation. Reduced matrix metalloproteinase (MMP) activity is believed to central to this imbalance. However, most of the data relating to MMPs and their natural inhibitors (tissue inhibitors of matrix metalloproteinase (TIMP)) is based on homogenate studies where in situ compartmentalization is lost and thus changes in MMP activity may be artificial. To address this we have developed a sensitive, high-resolution in situ zymography technique and applied it, along with immunohistochemistry, to the 5/6th subtotal nephrectomy model of kidney scarring. ECM proteolytic activity in kidney homogenates progressively declined post-SNx against both gelatin (-82%) and collagen I (-78%) substrates. In situ zymography revealed higher activity with both substrates within the cytoplasm of normal tubular cells compared to the SNx. In contrast, there was 96% greater activity in the SNx glomeruli than normal. Immunohistochemistry confirmed a predominantly intracellular tubular location of all MMPs and TIMPs. Tubules showed reduced MMP-3 and elevated TIMP-2, whereas MMP-1 increased significantly in the glomeruli, especially in the mesangial matrix. TIMP-1 showed a fourfold increase in the remnant kidney by Western blot analysis, but could not be localized. Lowered MMP activity in homogenates results from reduced intracellular activity in the tubules, indicating that reduced MMP activity may not play a direct role in the expansion of the tubular ECM in scarring. However, elevated MMP-1 activity in the glomeruli may play a significant role in initiating glomerular remodelling.

Factors influencing long-term outcomes following renal transplantation: a review.

Despite continuing advances in immunosuppressive and supportive therapies, the success of renal transplantation is impacted by factors present in the donor and recipient pre- and post-transplantation. The pre-transplant factors influencing the long-term graft function in the donor include source, age, sex, and HLA mismatches; and in the recipient include age, duration of dialysis and sensitisation. After transplantation, a number of events may lead to progressive deterioration of renal function and graft loss, which include delayed graft function, acute rejection, viral infections, recurrent disease, drug nephrotoxicity, non-compliance and chronic allograft nephropathy. Modulation of individual factor is mandatory to preserve satisfactory renal function in long-term. In this review, each factor is discussed in the context of current transplant practice and an up to date review of literature is presented.

Caspase-3 and apoptosis in experimental chronic renal scarring.

BACKGROUND: Caspase-3 is a member of the caspase enzyme family, having a central role in the execution of apoptosis. However, the significance of Caspase-3 in the inappropriate and excessive apoptosis that contributes to the progression of non-immune-mediated renal scarring has not been established. METHODS: Kidneys from sham-operated and subtotal nephrectomized (SNx) rats were harvested on days 7, 15, 30, 60, 90 and 120 post-surgery. These were analyzed for apoptosis (in situ end labeling of DNA, light and electron microscopy), Caspase-3 activity (fluorometric substrate cleavage assay), protein and mRNA (Western and Northern blotting), as well as distribution (immunohistochemistry), inflammation (ED-1 immunohistochemistry) and fibrosis (Masson's Trichrome staining). RESULTS: Apoptosis, inflammation and fibrosis gradually increased in glomeruli, tubules and interstitium of SNx rats. Caspase-3 was mainly located in damaged tubules, but also was found in some glomerular and interstitial cells. Little or no staining was noted in sham-operated kidneys. In SNx kidneys, Caspase-3 activity was significantly increased from day 30 and peaked on day 120 (2.5-fold). This resulted from increases in the 17 and 24 kD active protein subunits. The 32 kD precursor was increased at all time points (1861% on day 120, P < 0.01). Caspase-3 changes were transcription-dependent with the 2.7 kb caspase-3 mRNA significantly increased at all time points (287% on day 120). Caspase-3 activity was a better predictor of apoptosis (Std beta coefficient = 0.347, P < 0.05) than Caspase-3 proteins or mRNA; however, Caspase-3 at all levels correlated with apoptosis, inflammation and fibrosis (all P < 0.01). CONCLUSIONS: Up-regulation of apoptosis in remnant kidneys is likely to be Caspase-3-dependent as it is associated with increases in Caspase-3 at the activity, protein and mRNA levels. Therefore, Caspase-3 is a potential therapeutic target for the modification of renal cell apoptosis and subsequently renal fibrosis.

Increases in renal epsilon-(gamma-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy: pathologic implications.

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, epsilon-(gamma-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney epsilon-(gamma-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p < 0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p < 0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular epsilon-(gamma-glutamyl) lysine (+361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular epsilon-(gamma-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca(2+) and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Effects of adenosine receptor antagonists on the responses to contrast media in the isolated rat kidney.

Contrast media can induce both a decrease in renal blood flow and a reduction in glomerular filtration rate (GFR) when administered to both experimental animals and humans. In the present study we have examined the role of adenosine in mediating these effects using the isolated perfused rat kidney. Kidneys were perfused with a 6. 7%-(w/v)-albumin-based perfusate supplemented with glucose and amino acids (n=6 per group). They were exposed to diatrizoate [20 mg of iodine (mgI)/ml; osmolality 1650 mOsm/kg of water] or iotrolan (20 mgI/ml; osmolality 320 mOsm/kg of water) in the presence or absence of theophylline (10.8 microg/ml), or to diatrizoate in the presence or absence of a specific adenosine A(1) receptor antagonist (KW-3902; 2 microg/ml) or a specific A(2) receptor antagonist (KF17837; 6 microg/ml). Diatrizoate (n=6) produced a fall in GFR from 0.65+/-0.04 to 0.42+/-0.03 ml.min(-1).g(-1) (P<0.05); renal perfusate flow (RPF) also declined, from 36.5+/-3.8 to 22.0+/-3.2 ml. min(-1).g(-1) (P<0.05). Iotrolan (n=6) produced a fall in GFR from 0. 64+/-0.02 to 0.48+/-0.04 ml.min(-1).g(-1) (P<0.05) and in RPF from 33.3+/-3.8 to 24.0+/-3.0 ml.min(-1).g(-1) (P<0.05). Theophylline (10.8 microg/ml) prevented the fall in GFR caused by either diatrizoate (baseline, 0.63+/-0.05 ml.min(-1).g(-1); diatrizoate+theophylline, 0. 60+/-0.04 ml.min(-1).g(-1)) or iotrolan (baseline, 0.64+/-0.04 ml. min(-1).g(-1); iotrolan+theophylline, 0.67+/-0.05 ml.min(-1).g(-1)), but did not affect the decreases in RPF caused by either agent. KW-3902 (2 microg/ml) also prevented the fall in GFR produced by diatrizoate (baseline, 0.66+/-0.05 ml.min(-1).g(-1); diatrizoate+KW-3902, 0.61+/-0.05 ml.min(-1).g(-1)), while the fall in RPF remained unaffected. KF17837 (6 microg/ml) had no effect on the decreases in either GFR or RPF induced by diatrizoate (n=6 per group). The results suggest a role for adenosine acting at the A(1) receptor in mediating the decrease in GFR induced by contrast media. This effect is independent of a change in renal vascular resistance, and possibly secondary to mesangial cell contraction causing a decrease in the ultrafiltration coefficient.

IGF-I inhibitors reduce compensatory hyperfiltration in the isolated rat kidney following unilateral nephrectomy.

BACKGROUND: A role for insulin-like growth factor-I (IGF-I) as a mediator of renal hyperfiltration and hyperperfusion following unilateral nephrectomy (UNx) has been examined. METHODS: Adult male Wistar rats were subjected to either left UNx or sham operation. Twenty one days after surgery, the right kidney was removed under barbiturate anaesthesia, and renal function was measured ex vivo using an isolated rat kidney perfusion system. The glomerular filtration rate was assessed from the renal clearance of [(14)C]inulin. RESULTS: UNx stimulated renal growth as shown by a significantly higher (P<0.02) tissue dry weight in kidneys from UNx (0.45+/-0.02 g) than from sham-operated rats (0.31+/-0.02 g). Compensatory hyperfiltration could be detected ex vivo; kidneys obtained from UNx rats having a significantly higher (P<0.05) [(14)C]inulin clearance (0.75+/-0.08 ml/min, n=8) than kidneys obtained from sham-operated animals (0.39+/-0.05 ml/min, n=8). Compensatory hyperperfusion was also detected ex vivo; kidneys obtained from UNx rats having a significantly higher (P<0.05) renal perfusate flow (28.2+/-2.7 ml/min) than kidneys obtained from sham-operated rats (22.5+/-0.8 ml/min). Following perfusion with either 50 microg monoclonal IGF-I antibody (n=4) or 6.5 microM genistein (n=4), an inhibitor of tyrosine kinase, no significant difference in [(14)C]inulin was observed between kidneys obtained from either UNx or sham-operated rats. In contrast to hyperfiltration, renal hyperperfusion remained unaffected by the IGF-I antibody and was only reduced by 30% following genistein administration. CONCLUSIONS: The results suggest a role for renal IGF-I as a mediator of compensatory hyperfiltration in the rat.

Effects of transforming growth factor-beta1 on renal extracellular matrix components and their regulating proteins.

Transforming growth factor-beta1 (TGF-beta1) is widely regarded as a potent fibrogenic renal growth factor. In cell culture, TGF-beta1 has been shown to increase various extracellular matrix (ECM) proteins and tissue inhibitors of metalloproteinases (TIMP), while decreasing matrix metalloproteinases (MMP), providing the optimum environment for progressive ECM accumulation. This study, which uses the isolated perfused rat kidney (IPRK), describes for the first time in a whole kidney preparation the action of TGF-beta1 on factors associated with ECM processing. This model allows the study of the intact rat kidney with physiologic cell-cell interactions in the absence of confounding systemic influences. Left kidneys were removed from male Wistar rats by a nonischemic technique and perfused with a sterile, apyrogenic, endotoxin-free perfusate, based on the plasma volume expander Hemaccel (polygeline), at constant pressure in a recirculating IPRK system. Kidneys were perfused for 1 h either with (n = 3) or without (n = 3) recombinant human TGF-beta1 (20 ng/ml). The effects of perfusion were controlled by comparison with the nonperfused contralateral kidney (n = 6). TGF-beta1 was measured in the perfusate and urine, at the start and end of the experiment using an enzyme-linked immunosorbent assay to its biologically active form. After perfusion, sections of the kidneys were analyzed for changes in mRNA by Northern blotting. Significant increases in mRNA for fibronectin (7.5-fold, P < 0.01), heparan sulfate proteoglycan core protein (53-fold, P < 0.001), laminin beta1 (12-fold, P < 0.001), collagen alpha1(IV) (17-fold, P < 0.001), collagen alpha1(III) (fourfold, P < 0.001), and MMP9 (twofold, P < 0.05) were observed after perfusion with TGF-beta1. Measurement of TIMP1, TIMP2, TIMP3, MMP1, and MMP2 mRNA demonstrated no detectable change, whereas determination of mRNA for tissue transglutaminase, an enzyme capable of cross-linking many ECM components, showed an eightfold increase (P < 0.01). This study suggests that in the IPRK and in the absence of other exogenous growth factors, TGF-beta1 selectively increases the synthesis of ECM and tissue transglutaminase without changes that would result in the reduction of ECM degradation.