Matrix metalloproteinase-9 (MMP-9) is one of the major components of the matrix proteolytic network, and its role in the pathogenesis of renal interstitial fibrosis remains largely unknown. Here, we demonstrate that ablation of MMP-9 attenuated renal interstitial fibrotic lesions in obstructive nephropathy. Mice lacking MMP-9 were less likely to develop morphological injury, which was characterized by a reduced disruption of tubular basement membrane (TBM) and expression of fibronectin as well as deposition of total tissue collagen in the kidneys after sustained ureteral obstruction compared with their wild-type counterparts. Deficiency of MMP-9 blocked tubular epithelial-to-myofibroblast transition (EMT) but did not alter the induction of transforming growth factor (TGF)-β1 axis expression in the obstructed kidneys. In vitro, TBM, which was digested by MMP-9 instead of MMP-9 itself, induces EMT and enhances migration of transformed cells. Thus increased MMP-9 is detrimental in renal interstitial fibrogenesis through a cascade of events that leads to TBM destruction and in turn to promotion of EMT. Our findings establish a crucial and definite importance of MMP-9 in the pathogenesis of renal interstitial fibrosis at the whole-animal level.
- tubular basement membrane
- epithelial-to-myofibroblast transition
regardless of the diverse disease process, renal interstitial fibrosis is considered to be the common pathological pathway and irreversible process that eventually leads to end-stage renal disease. Both human disease and animal models have been able to show that massive interstitial myofibroblast activation is the predominant source of matrix proteins, such as collagen types I, III, fibronectin, etc., which results in renal fibrosis. Numerous evidence suggested that the myofibroblasts may derive from tubular epithelial cells undergoing epithelial-to-myofibroblast transition (EMT). EMT is an orchestrated, highly regulated process consisting of four key steps, in which the mechanism of destruction of the tubular basement membrane (TBM) resulting from upregulation of matrix proteinases is poorly understood. Matrix metalloproteinases (MMPs) are a family of more than 28 members that were initially identified as enzymes to cleave elements of the extracellular matrix (ECM) (20), which subsequently have been found to be upregulated in tumors (18). As digestion of the ECM is essential for cell migration and invasion, MMPs have been studied for their role in tumor metastasis. Alternation in regulation of MMP activity is implicated in diseases such as cancer, fibrosis, arthritis, and atherosclerosis (2, 6, 11, 27). Among the MMP family, there is emerging evidence indicated that MMP-9 can stimulate processes associated with EMT, a developmental process that is activated in renal fibrosis (15, 31). Gelatin zymographic analysis of both whole-kidney lysate of ureteral-obstructed mice and conditioned media of transforming growth factor (TGF)-β1-treated tubular epithelial cells displayed noticeable induction of MMP-9, while a marked decrease in MMP-9 induction resulting from tissue-type plasminogen activator (tPA) knockout led to a dramatic preservation of the structural and functional integrity of the TBM and prevention of EMT (32). Given the significance of MMP-9 for matrix homeostasis in multicellular organisms, it is not difficult to comprehend that its expression and activity are tightly regulated through a multitude of controlling mechanisms. Dysregulation of MMP-9 may contribute to the pathogenesis of a wide variety of diseases ranging from tumor metastasis to tissue fibrogenesis.
While the importance of the MMP-9 in matrix proteolytic systems is well documented, its role in the pathogenesis of renal interstitial fibrosis, characterized by excess matrix accumulation and deposition, is poorly defined. Tubular interstitial fibrosis is often regarded as a final, common endpoint/outcome of many forms of chronic renal disease (5, 14, 24, 30, 33, 36). One of the key events during interstitial fibrogenesis is the de novo activation of α-smooth muscle actin (α-SMA)-positive myofibroblast cells (31). Because these cells are primarily responsible for interstitial matrix accumulation and deposition in chronically diseased states, and because they are essentially absent in the interstitial compartment of normal kidney, elucidation of their origin(s) and the activation process is of fundamental importance both for understanding the pathologic mechanism of renal interstitial fibrosis and for identifying therapeutic targets. Myofibroblasts are often presumed to originate from residential fibroblasts (21, 25); however, numerous pieces of evidence suggests that they may also derive from tubular epithelial cells via an epithelial-to-myofibroblast transition (EMT) (14). It is well accepted that renal EMT is an orchestrated, highly regulated, stepwise process in which destruction of the integrity of the tubular basement membrane (TBM) may be crucial. However, whether the molecules such as MMP-9, an upstream regulator of collagen and laminin, which are the major components of TBM (4, 29), are involved in the destruction of the TBM under pathological conditions at the whole-animal level remains unknown.
The availability of genetically engineered mice in which the endogenous MMP-9 gene is inactivated provides an invaluable, unprecedented model system for studying the function of MMP-9 in the pathogenesis of renal interstitial fibrosis in vivo. In this study, we investigated the progression of renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) in MMP-9+/+ and MMP-9−/− mice. We found that the null mutation of the MMP-9 gene protected the kidney from developing interstitial fibrotic lesions. The detrimental effect of endogenous MMP-9 was apparently not mediated by alterations in the expression and activation of profibrogenic TGF-β1 in the kidney. Instead, it was likely mediated by destruction of the TBM, which in turn resulted in myofibroblastic transition of tubular epithelial cells via EMT. Our results validate the hypothesis that TBM integrity plays an essential role in preventing tubular EMT, thereby protecting normal renal morphology against development of fibrotic lesions.
MATERIALS AND METHODS
Colonies of homozygous MMP-9 knockout (MMP-9−/−) and wild-type (MMP-9+/+) mice were raised from original breeding pairs obtained from the Jackson Laboratory. They were housed in the animal facilities at the National Model Animal Center (NMAC; Nanjing, Jiangsu Province, China) with free access to food and water. Animals were treated humanely in accordance with NMAC guidelines and approved procedures of the Institutional Animal Use and Care Committee at the Nanjing Medical University. Genotype was confirmed using PCR amplification of genomic DNA from the tail. The experiments were performed using male MMP-9−/− and MMP-9+/+ mice with identical genetic profiles and with an average age of 10 wk. There was no statistical difference in the body weight of MMP-9−/− and MMP-9+/+ mice at the time of the experiments. Both knockout (MMP-9−/−) and wild-type (MMP-9+/+) animals were randomly assigned to four groups with five mice per group: sham for 3 days, UUO for 3 days, sham for 7 days, and UUO for 7 days. UUO was performed using an established procedure (31). Briefly, under general anesthesia, complete ureteral obstruction was performed by double-ligating the left ureter using 4-0 suture after a midline abdominal incision. Sham-operated mice had their ureters exposed and manipulated but not ligated. Mice were killed at different time points as indicated after surgery, and the obstructed kidneys were removed. One part of the kidney was fixed in 10% phosphate-buffered formalin followed by paraffin embedding for histological and immunohistochemical studies. The remaining kidneys were snap-frozen in liquid nitrogen and stored at −80°C for protein and total RNA extractions.
Cell culture and treatment.
Rat proximal tubular epithelial cells (NRK-52E) were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium-F12 medium supplemented with 5% fetal bovine serum (Sigma, Sigma-Aldrich, St. Louis, MO). For MMP-9 treatment, NRK-52E cells were seeded at ∼80% confluence in complete medium containing 5% fetal bovine serum. Twenty-four hours later, the cells were changed to serum-free medium, and recombinant MMP-9 (R&D Systems, Minneapolis, MN) was added at a final concentration of 10 ng/ml except where otherwise indicated. The cells and conditioned media were collected at different time points for further characterization. GM6001 was purchased from Sigma (Sigma-Aldrich).
RNA isolation and RT-PCR.
Total RNA was extracted from the kidney tissue using an Ultraspec RNA isolation system according to the instructions specified by the manufacturer (Biotecx Laboratories, Houston, TX). RT-PCR was performed as described elsewhere (26). Briefly, the first-strand of cDNA synthesis was carried out by using a Reverse Transcription System kit according to the instructions of the manufacturer (Promega, Madison, WI). PCR amplification was performed with a HotStar TaqMaster Mix Kit (Qiagen, Valencia, CA). The primer sequences were as follows: MMP-9 (forward) 5′-CCCACATTTGACGTCCAGAGAAGAA and (reverse) 5′-GTTTTTGATGCTATTGCTGAGATCC, and β-actin (forward) 5′-CTCTTCCAGCCTTCCTTCCTG and (reverse) 5′-GAAGCATT TGCGGTGGACGAT. The PCR products were size fractionated on a 1.0% agarose gel and detected by ethidium bromide staining. No detectable signal was found in a parallel control tube without reverse transcriptase (data not shown). Expression levels of mRNA were calculated after normalizing with housekeeping gene β-actin.
Western blot analysis.
Cells were lysed with SDS sample buffer (62.5 mmol/l Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mmol/l dithiothreitol, and 0.1% bromophenol blue). Kidney tissue was homogenized by a polytron homogenizer (Brinkmann Instruments, Westbury, NY), and the supernatant was collected after centrifugation at 13,000 g at 4°C for 20 min, as described previously (31). After protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Sigma), the tissue lysate was mixed with an equal amount of 2× SDS sample buffer. Samples were heated at 100°C for 5–10 min before loading and separated on precasted 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins were electrotransferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL) in transfer buffer containing 48 mmol/l Tris·HCl, 39 mmol/l glycine, 0.037% SDS, and 20% methanol at 4°C for 1 h. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% Carnation nonfat milk in TBS buffer (20 mmol/l Tris·HCl, 150 mmol/l NaCl, and 0.1% Tween 20). The membranes were incubated for 16 h at 4°C with various primary antibodies in TBS buffer containing 5% milk at the dilutions specified by the manufacturers. The mouse monoclonal anti-α-SMA antibody (clone 1A4) was purchased from Sigma. The anti-TGF-β1 type I receptor (sc-398) and anti-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Binding of primary antibodies was followed by incubation for 1 h at room temperature with the secondary horseradish peroxidase-conjugated IgG in 1% nonfat milk. The signals were visualized with the enhanced chemiluminescence system (ECL, Amersham), as described previously.
Gelatin zymographic analysis of MMP proteolytic activity in kidney tissue homogenates was performed according to the method described previously (31). Kidney homogenates were prepared essentially according to the methods described by Kim et al. (12). Protein concentration was determined using a BCA protein assay kit with BSA as a standard (Sigma-Aldrich). A constant amount of protein from the kidney tissue homogenates (30 μg) was loaded onto a 10% SDS-polyacrylamide gel containing 1 mg/ml gelatin (Bio-Rad Laboratories). After electrophoresis, SDS was removed from the gel by incubation in 2.5% Triton X-100 at room temperature for 45 min with gentle shaking. The gel was washed thoroughly with distilled water to remove detergent and incubated at 37°C for 16 h in a developing buffer containing 50 mM Tris·HCl at pH 7.6, 0.2 M NaCl, 5 mM CaCl2, and 0.02% Brij 35 (Sigma-Aldrich). The gel was then stained with a solution of 30% methanol, 10% glacial acetic acid, and 0.5% Coomassie blue G250, followed by destaining in the same solution without dye. Proteinase activity was detected as unstained bands on a blue background representing areas of gelatin digestion.
Histology and immunostaining.
Paraffin-embedded kidney sections were prepared at 4-μm thickness by a routine procedure. Sections were stained with hematoxylin and eosin for general histology. Indirect immunofluorescence staining was performed on kidney cryosections using an established procedure. Cryosections were prepared at 5-μm thickness and fixed for 5 min in PBS containing 3% paraformaldehyde. After being blocked with 1% normal donkey serum in PBS for 30 min, the sections were incubated with primary antibodies against α-SMA (Sigma-Aldrich) and collagen (Southern Biotechnology Associates, Birmingham, AL), respectively, in PBS containing 1% BSA overnight at 4°C. Sections were then incubated for 1 h with fluorescein isothiocyanate-conjugated secondary antibodies (Sigma-Aldrich) at a dilution of 1:100 in PBS containing 1% BSA, before being washed extensively with PBS. As a negative control, the primary antibody was replaced with nonimmune IgG, and no staining occurred. Some slides were then stained with proximal tubular marker, fluorescein-conjugated lectin from Tetragonolobus purpureas (Sigma-Aldrich) for localizing the proximal tubules. Slides were mounted with Vectashield antifade mounting media (Vector Laboratories, Burlingame, CA) and viewed with an Eclipse E600 epifluorescence microscope equipped with a digital camera (Nikon, Melville, NY). Immunohistochemical staining was performed by use of the Vector M.O.M. immunodetection kit, as described above (Vector Laboratories). The primary antibody used was anti-fibronectin (Sigma, Sigma-Aldrich). As a negative control, the primary antibody was replaced with nonimmune normal IgG, and no staining occurred.
For quantitation of TBM lesions in the obstructed kidneys of MMP-9+/+ and MMP-9−/− mice, sections from kidney cortex of each mouse were double-stained with collagen/tubular marker. The percentages of renal tubules that had one or more breaks in their TBM were counted in a blinded fashion in randomly chosen, nonoverlapping high-power (×400) fields, and the data were calculated based on individual values that were determined in 10 fields/mouse (n = 5 mice/group).
Biochemical measurement of total kidney collagen content.
For quantitative measurement of collagen accumulation and deposition in the kidney, total tissue collagen content was determined by biochemical analysis of the hydroxyproline in the hydrolysates extracted from kidney samples. This assay is based on the observation that essentially all the hydroxyproline in animal tissues is found in collagen. Briefly, accurately weighed portions of the obstructed kidneys were homogenized in distilled H2O. The homogenates were hydrolyzed in 10 N HCl by incubation at 110°C for 18 h. The hydrolysates were dried by speed vacuum centrifugation over 3–5 h and redissolved in a buffer containing 0.2 M citric acid, 0.2 M glacial acetic acid, 0.4 M sodium acetate, and 0.85 M sodium hydroxide, pH 6.0. Hydroxyproline concentrations in the hydrolysates were chemically measured according to the techniques previously described (17). Total collagen was calculated on the assumption that collagen contains 12.7% hydroxyproline by weight. The results of total tissue collagen content were expressed as micrograms of collagen per milligram of kidney weight.
Determination of tissue TGF-β1 levels by ELISA.
For measurement of active TGF-β1 protein levels in tissue, kidneys were homogenized in extraction buffer containing 20 mM Tris·HCl, pH 7.5, 2 M NaCl, 0.1% Tween 80, 1 mM ethylenediamine tetraacetate, and 1 mM phenylmethylsulfonyl fluoride, and then the supernatant was recovered after centrifugation at 19,000 g for 20 min at 4°C. Kidney tissue TGF-β1 level was determined by using a commercial Quantikine TGF-β1 ELISA kit in accordance with the protocol specified by the manufacturer (R&D Systems). This kit measures the abundance of active TGF-β1 protein that binds to its soluble type II receptor, which is precoated on a microplate. The concentration of TGF-β1 in kidneys was expressed as picograms per milligram total protein.
Boyden chamber coated with Matrigel for motility and invasion assay.
Cell motility and migration were evaluated using a Boyden chamber motogenicity assay with tissue culture-treated Transwell filters (Costar). Matrigel (1.43 mg/cm2) was added onto the Transwell filters (8-μm pore size, 0.33-cm2 growth area) of the Boyden chamber to form Matrigels at 1.0-mm depth. NRK-52E cells (1 × 104) were seeded onto the filters (8-μm pore size, 0.33-cm2 growth area) in the top compartment of the chamber. After 3 days of incubation with or without MMP-9 plus GM6001 in the lower compartment at 37°C, filters were fixed with 3% paraformaldehyde in PBS and stained with 0.1% Coomassie blue in 10% methanol and 10% actic acid, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. Cells that invaded and migrated across the Matrigel and passed the Transwell filter pores toward the lower surface of the filters were counted in 5 nonoverlapping ×10 fields and photographed with a Nikon microscope. The experiments were performed in triplicate.
All the computed data were expressed as means ± SE. Each of the experiments was repeated three times independently. For Western blot analysis, quantitation was performed by scanning and analyzing the intensity of the hybridization signals using NIH Image software. Statistical analysis of the data was performed by the Student-Newman-Keuls test using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparisons between groups were made using one-way ANOVA, followed by the Student-Newman-Keuls test. P < 0.05 was considered significant.
TBM integrity is preserved in obstructed kidneys of MMP-9−/− mutant mice.
Age- and sex-matched wild-type (MMP-9+/+) and knockout (MMP-9−/−) mice were induced to develop chronic renal interstitial fibrosis by UUO. The expression and activity of intrarenal MMP-9 in MMP-9+/+ and MMP-9−/− mice were examined by both RT-PCR analysis and gelatin zymography. As shown in Fig. 1A, ureteral obstruction for 7 days induced a dramatic increase in MMP-9 mRNA expression in the kidneys of wild-type mice. As expected, no MMP-9 mRNA was detected in the kidneys of either sham-operated or obstructed MMP-9−/− mice. Similarly, no MMP-9 activity was found in the kidney homogenates of MMP-9 −/− mice whereas a clear band at ∼92 kDa was evident in the samples derived from wild-type counterparts after 3 and 7 days of obstruction (Fig. 1B).
Because one of the specific substrates for MMP-9 is collagen IV, which is also a major component of TBM, induction of MMP-9 could lead to destruction of renal TBM integrity, which is one of the key events during tubular EMT. We further directly examined the collagen IV expression of the obstructed kidneys in MMP-9+/+ and MMP-9−/− mice. Western blotting of whole-kidney lysates revealed that the level of collagen IV protein in the kidneys at 7 days after UUO in MMP-9−/− mice was significantly preserved compared with their wild-type controls (Fig. 1C). Immunofluorescence staining with collagen IV revealed a broken, discontinuous TBM in significant numbers of renal tubules from the kidneys of MMP-9+/+ mice at 7 days after UUO (Fig. 1E). Through the interrupted TBM, tubular epithelial cells tended to migrate toward the interstitial compartment. However, under identical conditions, TBM integrity was largely preserved with uninterrupted staining of TBM in MMP-9−/− mice (Fig. 1G). Quantitative determination revealed that ∼30% of renal tubules in the cortical regions displayed TBM lesions manifested by one or more breaks in the kidneys of MMP-9+/+ mice at 7 days after UUO, whereas <15% of renal tubules had similar TBM lesions in MMP-9−/− kidneys (Fig. 1H). These results suggest that deficiency of MMP-9 expression preserves TBM from destruction under pathological conditions.
Blockade of tubular EMT in MMP-9−/− mice after ureteral obstruction.
Because α-SMA-positive myofibroblasts are the principal cells responsible for interstitial matrix accumulation and deposition, we next investigated the magnitude of myofibroblast activation after ureteral obstruction in MMP-9+/+ and MMP-9−/− mice. As shown in Fig. 2A, the level of α-SMA protein in the kidneys at 7 days after ureteral obstruction in MMP-9−/− mice was significantly lower than that in their wild-type controls. Quantitative determination of the Western blotting of whole-kidney lysates revealed ∼70% inhibition of α-SMA protein in MMP-9−/− mice compared with the levels in MMP-9+/+ mice (Fig. 2B). A similar level of α-SMA protein was detected in the sham-operated kidneys in MMP-9+/+ and MMP-9−/− mice, indicating that MMP-9 null mutation did not affect α-SMA protein expression under basal conditions. These results suggest that null mutation of the MMP-9 gene attenuates myofibroblast activation by blocking tubular EMT in the diseased kidneys.
Attenuation of renal interstitial fibrosis in obstructive nephropathy in MMP-9−/− mice.
Figure 3 shows representative micrographs of the obstructed kidneys in MMP-9+/+ and MMP-9−/− mice at 7 days after surgery. As expected, disruption of the MMP-9 gene resulted in less morphological injury to the obstructed kidneys than those seen in the wild-type. In MMP-9+/+ mice with sustained, complete ureteral obstruction for 7 days, kidneys developed severe morphological lesions characterized by tubular dilation with epithelial atrophy, interstitial expansion with hypercellularity resulting from myofibroblast activation, and extracellular matrix accumulation. Under the same conditions, renal pathology was significantly improved in mice lacking MMP-9 (Fig. 3), suggesting that endogenous MMP-9 induces damage by promoting renal fibrotic lesions after chronic injury.
To quantitatively measure matrix accumulation, we determined the total tissue collagen content in the obstructed kidneys by a biochemical assay. Figure 4 shows the total kidney collagen content in the obstructed kidneys at day 7 after surgery in MMP-9+/+ and MMP-9−/− mice. About sixfold-higher levels of collagen deposition were observed in the obstructed kidneys compared with sham-operated controls at day 7 in MMP-9+/+ mice. However, deficiency of MMP-9 dramatically inhibited collagen accumulation in the kidneys after the same period (7 days) of ureteral obstruction. Thus disruption of the MMP-9 gene protects the kidney from developing interstitial fibrotic lesions after chronic, persistent injury.
To demonstrate ECM protein expression, the magnitude of fibronectin after ureteral obstruction in MMP-9+/+ and MMP-9−/− mice was investigated. As shown in Fig. 5A, the level of fibronectin protein in the kidneys at 7 days after ureteral obstruction in MMP-9−/− mice was significantly lower than that in their wild-type controls. Quantitative determination of the Western blotting of whole-kidney lysates revealed ∼80% inhibition of fibronectin protein in MMP-9−/− mice compared with the levels in MMP-9+/+ mice (Fig. 5B). A similar level of fibronectin protein was detected in the sham-operated kidneys in MMP-9+/+ and MMP-9−/− mice, suggesting that MMP-9 deficiency did not alter fibronectin expression under basal conditions. Furthermore, fibronectin deposition in the obstructed kidneys in MMP-9+/+ and MMP-9−/− mice was examined by immunohistochemical staining. As shown in Fig. 5, C–F, at 7 days after UUO, marked deposition of fibronectin protein was observed in the MMP-9+/+ mice (Fig. 5D). In contrast, significantly less fibronectin was stained in the obstructed kidney of MMP-9−/− mice at 7 days after surgery, and this was predominantly confined to the interstitial compartment (Fig. 5F). These results suggest that null mutation of the MMP-9 gene attenuates renal interstitial fibrosis in obstructive nephropathy.
Induction of TGF-β1 axis expression is not altered in obstructed kidneys in tPA−/− mice.
Since TGF-β1 is believed to play a critical role in initiating and promoting myofibroblast activation and fibrotic lesions in vivo, we tested the hypothesis that altered TGF-β1 expression may account for the difference in interstitial fibrosis after chronic injury in MMP-9+/+ and MMP-9−/− mice. TGF-β1 protein levels were determined by ELISA of whole-kidney lysates. As demonstrated in Fig. 6A, ureteral obstruction caused a marked increase in renal TGF-β1 expression in the kidneys in a time-dependent manner. However, no significant difference in TGF-β1 protein level was found in the obstructed kidneys in MMP-9+/+ and MMP-9−/− mice at 7 days after UUO, suggesting that the renal protection seen in MMP-9−/− mice is not attributable to altered TGF-β1 induction following persistent injury. Levels of TGF-β1 type I receptor were also examined by Western blot analysis of whole-kidney lysates in MMP-9+/+ and MMP-9−/− mice after ureteral obstruction. As shown in Fig. 6B, the protein level of TGF-β1 receptor type I was virtually identical between MMP-9+/+ and MMP-9−/− mice in the obstructed kidneys at 7 days after UUO. Hence, expression of the TGF-β1 axis does not account for the renal protection elicited by MMP-9 deficiency in obstructive nephropathy.
Decomposition product from MMP-9-digested TBM but not MMP-9 itself induces EMT.
We next examined whether MMP-9 directly induced EMT in vitro. As shown in Fig. 7A, Western blot analysis shows that neither 10 nor 100 ng/ml MMP-9 alone could induce de novo α-SMA expression in NRK-52E cells as 2 ng/ml of TGF-β1 did within 48 h of incubation. However, in the presence of Matrigels that essentially mimic native TBM, MMP-9 induced NRK-52E cells to express α-SMA in a MMP-9 dose-dependent manner (Fig. 7B), which suggested that MMP-9-induced EMT is TBM dependent. The decomposition product from MMP-9-digested TBM may contribute to the induction of EMT in vitro.
Disruption of Matrigels by MMP-9 can enhance migration and invasive capacity of transformed NRK-52E cells.
To understand whether the disruption of TBM by MMP-9 could enhance the capacity of cells to migrate and finally enter the interstitial compartment in vivo, we analyzed the invasive capacity of cells with a Matrigel invasion assay. MMP-9 (6.6 ng/ml) or MMP-9 (6.6 ng/ml) plus its inhibitor, GM6001 (10 μM), were added into the lower compartment to degrade Matrigels. As shown in Fig. 8B, incubation of MMP-9 markedly promoted cell invasion and migration across the Matrigel. Most pores in Transwell filters were filled with cell extensions after 3 days of incubation with MMP-9, which resulted from the cells invading and migrating across the Matrigels (Fig. 8B). Under the same conditions, control NRK-52E cells without MMP-9 or cells with MMP-9 plus GM6001 treatment displayed a much less migratory property in this assay (Fig. 8, A and C). These data suggest that disruption of the Matrigel by MMP-9 facilitates tubular epithelial cells to invade and migrate through the Matrigel, which presumably allows them to move across the TBM and ultimately to enter the interstitial compartments of the obstructed kidney in vivo.
Tubulointerstitial fibrosis is considered the most common destructive pathway associated with chronic nephropathies, which is characterized by remodeling of the interstitial ECM, resulting in excessive deposition of ECM including collagens type I, type III, and type IV. In view of their matrix-degrading capacity, MMP-9 as well as tPA(9), and MMP-2 (3) were originally hypothesized to be beneficial in chronic renal fibrogenesis due to its perceived potential to lessen interstitial matrix accumulation and deposition. Decreased expression of MMP-2 or MMP-9, sometimes owing to increased expression of their inhibitors TIMP-1, TIMP-2, and TIMP-3, was demonstrated in animal models of renal fibrosis and in human biopsies. Several studies of renal fibrosis demonstrated that an increase in MMPs can inhibit the deposition of ECM in the context of renal fibrosis (23, 34). It was proved in Alport's syndrome that before the onset of glomerular basement membrane (GBM) damage, enhanced MMP activity is responsible for disease induction owing to its capacity to degrade GBM more rapidly. However, after the onset of proteinuria, the MMPs turn out to be beneficial as to remove ECM scarring and attenuate renal fibrosis (35). In fact, it has already been proved that disruption of the tPA gene in mice reduces renal interstitial fibrosis in obstructive nephropathy (32). In this report, we present evidence that the null mutation of the MMP-9 gene protects the kidney from developing interstitial fibrotic lesions after sustained ureteral obstruction, suggesting that endogenous MMP-9 actually accelerates renal fibrogenesis. In this regard, our results obtained from MMP-9−/− mice are rather unanticipated. This beneficial outcome of MMP-9 deficiency is likely attributable to a decrease in TBM destruction that in turn blocks the progression of EMT. The preservation of the TBM ultimately leads to avoidance of renal fibrosis as seen in wild-type kidneys. These data underscored that TBM integrity may play a fundamental role in the prevention of tubular EMT, thereby attenuating renal interstitial fibrosis.
The TBM consists predominantly of collagen IV and laminin (7, 29). It provides a structural foundation for the proper function of tubular epithelial cells. There is growing evidence suggesting that the TBM is not only a static physical barrier that separates tubular epithelial and interstitial compartments but also a regulator of tubular epithelial cell phenotype and physiology in the kidney (28). For example, a report shows that alterations in TBM composition regulate tubular epithelial cell transdifferentiation in vitro (7, 13, 18). It is also reported that interstitial collagen type I tends to promote EMT, whereas the collagen type IV is likely to inhibit this phenotypic transition. It was previously demonstrated that EMT is an orchestrated, highly regulated process consisting of four key steps, in which destruction of the TBM plays a crucial role by clearing the path for transformed cells to ultimately migrate into the interstitial compartment of the kidney (31). The present study using MMP-9−/− mice implies that TBM integrity may play a greater role in EMT than we anticipated before. Preservation of TBM integrity in MMP-9−/− mice leads to fewer myofibroblast cells in renal interstitium, which is probably due to restraint of cell migration by the TBM barrier, meanwhile reducing overall α-SMA expression and myofibroblast activation in the obstructed kidneys. This suggests that TBM integrity per se may have the ability to block myofibroblast transdifferentiation from tubular epithelia in vivo by preventing the access of tubular epithelial cells to the interstitial matrix microenvironment after destruction of TBM. Hence, TBM integrity is likely to be one of the pivotal determinant factors in regulating tubular epithelial cell phenotypic transition under pathological conditions.
Renal EMT and interstitial fibrosis are regulated by several growth factors (1, 8, 10, 16, 37). Profibrogenic TGF-β1 initiates and completes the entire EMT course in cultured tubular epithelial cells in vitro. Renal tubular cells incubated with TGF-β1 lose the epithelial marker E-cadherin, induce de novo expression of α-SMA, and reorganize actin to form stress fibers. These cells also express abundant MMPs, which can specifically degrade TBM, and acquire an enhanced motility and invasive capacity. It is logical to speculate that altered TGF-β1 expression and activation may account for amelioration of obstructive nephropathy in MMP-9−/− mice. Upon examination, TGF-β1 induction and activity in the obstructed kidneys were found to be similar in mice either retaining or lacking MMP-9, suggesting that any role for MMP-9 in TGF-β1 activation is compensated for by another mechanism in MMP-9−/− mice. Furthermore, it is suggested that the attenuation of renal myofibroblast activation and interstitial fibrosis in MMP-9−/− mice appears to be independent from TGF-β1. Thus preservation of TBM integrity alone has major implications in preventing EMT and renal interstitial fibrosis at the whole-animal level.
Our finding on the destruction of TBM integrity by MMP-9 suggests that this protease may have much broader functions in the diseased kidney than we originally thought. Although not tested, we would not be surprised if the expression of other genes besides collagen could also be regulated by MMP-9. Consistent with this view, there are numerous studies demonstrating that components of the MMP systems, such as MMP-2 and TIMP-1, elicit a wide range of cellular activities by initiating specific gene expression in ways other than their conventional function in the proteolytic cascade (3, 18, 22). In particular, MMP-9 has been reported to impact cellular signaling pathways that stimulate cell growth at early stages of tumor progression (19). MMP-9 has also been found to cleave intracellular targets and induce mitotic abnormalities and genomic instability. The Harris group (38) has recently demonstrated that MMP-9 directly mediated Slug-dependent EMT in NRK-52E cells downstream of TGF-β1. However, in our study, we applied a condition of lower dose (10 or 100 ng/ml) for a shorter time duration (48 h) of incubation, in which MMP-9 alone couldn't induce de novo α-SMA expression in NRK-52E cells as TGF-β1 did. However, in the same condition with the presence of Matrigel, MMP-9 induced NRK-52E cells to express α-SMA in a MMP-9 dose-dependent manner. In view of its ability to digest TBM, although the exact decomposition product from MMP-9-digested TBM is unknown; it is plausible to speculate that MMP-9 may function to trigger a cascade of intracellular signal transduction, leading to expression of specific genes.
In summary, the studies presented in this paper demonstrate that deficiency of MMP-9 protects the kidney from developing fibrotic lesions in obstructive nephropathy. This beneficial effect is likely mediated by a cascade of events that include reduction of extracellular matrix expression, preservation of TBM integrity, and blockade of tubular EMT. In addition, we have uncovered a link between TBM destruction and epithelial-to-myofibroblast transition in which the MMP-9 digest TBM and destruct its integrity. We show here that without significant alteration of major pro-fibrogenic TGF-β1, blockade of tubular EMT alone in MMP-9−/− kidneys dramatically ameliorate renal interstitial fibrosis after obstructive injury. Our results establish a vital role and definite contribution of MMP-9 in EMT process of renal interstitial fibrogenesis at the whole-animal level. Hence, blockage of any one of the key steps in TBM destruction will offer unique opportunities for initializing myofibroblast activation from tubular epithelial cells and for ultimately mitigating renal interstitial fibrosis.
This work was supported by National Science Foundation of China Grants 30470800/ 30771010/30871201, the “973” Science Program of the Ministry of Science and Technology, China (2006CB503909), and Jiangsu Province's Outstanding Medical Academic Leader Program (SWKJ 2006-50) to J. W. Yang.
No conflicts of interest, financial or otherwise, are declared by the authors.
Part of this work was presented as an abstract at the annual meeting of the American Society of Nephrology, November 14–19, 2006, San Diego, CA.
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