Matrix metalloproteinases and their inhibitors regulate in vitro ureteric bud branching morphogenesis

Martin Pohl*, Hiroyuki Sakurai*, Kevin T. Bush, and Sanjay K. Nigam

Departments of Pediatrics/Medicine, Division of Nephrology-Hypertension, University of California, San Diego, La Jolla, California 92093


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mammalian kidney development is initiated by the mutual interaction between embryonic metanephric mesenchyme (MM) and the ureteric bud (UB), leading to tightly controlled UB branching morphogenesis. In a three-dimensional cell culture model, which employs MM cell-derived conditioned medium (BSN-CM) to induce UB cell branching morphogenesis in extracellular matrix (ECM) gels (Sakurai H, Barros EJ, Tsukamoto T, Barasch J, and Nigam SK. Proc Natl Acad Sci USA 94: 6279-6284, 1997), branching morphogenesis was inhibited by both chemical agents (ilomastat and 1,10-orthophenanthroline) and a physiological protein factor [tissue inhibitor of metalloproteinases (TIMP)-2], known to act as matrix metalloproteinase (MMP) inhibitors. In addition, UB branching was inhibited in isolated UB culture (Qiao J, Sakurai H, and Nigam SK. Proc Natl Acad Sci USA 96: 7330-7335, 1999) by TIMP-2 and ilomastat, suggesting a direct role for MMPs in UB branching. Gelatin zymography and enzymatic measurement of MMP activity revealed that MMPs could originate from at least three different sources: the conditioned medium, the ECM, and the UB cells themselves. In the UB cells, transcription of several MMPs [gelatinase A (MMP2) and B (MMP9), stromelysin (MMP3), MT1-MMP] and TIMPs was altered by BSN-CM and changed as more complex branching structures formed. The ECM appeared to serve as both a reservoir for MMPs and modulated their expression because different ECM compositions altered the total MMP activity as well as specific subsets of MMPs expressed by the UB cells (as determined by zymography and Northern analysis). In the context of UB branching morphogenesis during kidney development, our data suggest a complex model in which soluble factors produced by the MM, in the context of specific ECM components, modulate the expression of specific subsets of MMPs and TIMPs in the UB, which alter as structures develop and the matrix environment changes. This suggests distinct roles for different subsets of MMPs and their inhibitors during different phases of branching morphogenesis.

kidney development; extracellular matrix; three-dimensional; cell culture; tissue inhibitor of metalloproteinases; MMP2; MMP3; MMP9; MT1-MMP; epithelial morphogenesis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN ORGANOGENESIS, during which developing organs expand into surrounding tissue, is characterized by extensive tissue growth and remodeling. Matrix metalloproteinases (MMPs) and their inhibitors have been implicated in these complex processes (5-7, 10, 16, 29). Several MMPs and tissue inhibitors of metalloproteinases (TIMPs) have been shown to be widely expressed during development (1, 4, 21, 30, 34). In the developing kidney, involvement of MMPs and TIMPs has been demonstrated in organ culture. For example, addition of TIMP-1, TIMP-2, or neutralizing antibodies to gelatinase B inhibited murine kidney development in organ culture (3, 14). In addition, synthetic proteinase inhibitors and TIMP-2 have been shown to inhibit in vitro branching morphogenesis of renal adult cell lines (9, 25), and growth factors appear to exert their morphogenetic effect, at least partially, by regulating the balance between MMPs and TIMPs (24, 28). The highly dynamic nature of the extracellular matrix (ECM) during development of the kidney and other epithelial tissues adds another level of complexity to the analysis, because matrix composition can either facilitate or inhibit branching morphogenesis in vitro (26). Because of the interdependent relationship of matrix and MMP expression, it is possible that the effect of ECM composition on branching morphogenesis is as much a function specific of MMPs and TIMPs expressed in that particular ECM context as of the structural and direct functional properties of the ECM proteins themselves. However, the way in which these MMPs are involved in morphogenetic processes is not clear. The analysis of branching morphogenesis in whole organ culture of epithelial tissues (such as the embryonic kidney) is too complex, as these tissues contain many cell types in various developmental stages. Therefore, we analyzed the role of MMPs and their inhibitors in two different in vitro models, which were designed to study a key morphogenetic process involved in kidney development: branching morphogenesis of the ureteric bud.

The development of the mature or metanephric kidney arises through mutual interactions between mesenchymal cells of the metanephric mesenchyme (MM) and an epithelial outgrowth of the Wolffian duct known as the ureteric bud (UB). The MM stimulates the UB to undergo extensive branching tubulogenesis when it develops into the kidney's collecting system and ureteric tree (24, 28). We have recently established a cell culture system to model this process (23). In this system, an epithelial cell line, derived from embryonic UB cells, which retains certain characteristics specific to the UB, can be induced to undergo branching tubulogenesis in the presence of conditioned medium (BSN-CM) secreted by an apparently embryonic MM-derived cell line (BSN cells) in an appropriate ECM. Both cell lines were derived from the UB and MM of mouse embryos at gestational day 11.5 (e11.5) around the time of UB induction. A second recently developed system utilizes explanted isolated UB, which is separated from the MM and observed to branch repetitively over several days in a defined three-dimensional ECM under the stimulation of soluble factors (20). To our knowledge, these are the simplest authentic cell culture- and explant-based systems available for analyzing UB branching tubulogenesis.

We demonstrate here not only that MMPs are required for in vitro branching morphogenesis of the UB cells and the isolated rat UB in three-dimensional culture but also that there are at least three potential sources for morphogenetic MMPs. They may be supplied by the mesenchymal cells but can also be produced by the UB cells themselves in response to soluble tubulogenic factors. In addition, the ECM both serves as a reservoir for MMPs and modulates the expression of MMPs in UB cells. As branching structures become more complex, the UB cells express different sets of MMPs and TIMPs, suggesting distinct roles for these molecules at different steps of branching morphogenesis.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Cell culture. Immortalized UB cells, derived from SV40 large T antigen transgenic murine embryos at e11.5, were maintained in culture with MEM (GIBCO) supplemented with 10% (vol/vol) FCS (Sigma) and 1% (vol/vol) antibiotic-antimycotic (GIBCO) at 32°C and 5% CO2. Cells derived from embryonic murine MM at e11.5 of gestation of SV40 large T antigen transgenic mice (BSN cells) were cultured in DMEM/F-12 (Mediatech) with 10% (vol/vol) FCS and 1% (vol/vol) antibiotic-antimycotic (GIBCO) at 37°C and 5% CO2. If not used for experiments, UB cells were passaged biweekly and BSN cells twice weekly. BSN-CM was produced by growing BSN cells to confluence over 3-4 days and then exposing them to serum-free DMEM/F-12 (10-ml/10-cm culture dish) after three thorough washes with Hanks' balanced salt solution (GIBCO). The BSN-CM was harvested after 3-4 days of serum-free culture. Maximally, two consecutive collections were made from a monolayer.

Three-dimensional cell culture. For three-dimensional morphogenetic evaluation, UB cells were trypsinized at confluence and dispersed in collagen type I gels with or without the addition of 20% (vol/vol) growth factor-reduced Matrigel (MG; Collaborative Biomedical) at ~100,000 cells/ml. Ten milliliters of collagen gel were produced by mixing 7.5 ml ice-cold rat tail collagen type I (~ 4.0 mg/ml, Collaborative Biomedical) with 1 ml 10× DMEM (Sigma), 1 ml 200 mM HEPES (GIBCO), and 0.5 ml 74 mg/ml NaHCO3 before the pH was adjusted to 7.4 with 1 M NaOH. For MG/collagen mixtures, 20% (vol/vol) ice-cold MG was added to the collagen preparation. One hundred-microliter aliquots of the prepared cell suspension in the gel were plated per well in 96-well plates (Falcon). After ~30 min in 32°C, the gels had solidified and were overlaid with 100 µl of medium. Unsupplemented DMEM/F-12 or BSN-CM with or without the proteinase inhibitors 1,10-orthophenanthroline (Sigma), TIMP-2 (Calbiochem), ilomastat (AMS Scientific), E64 (Sigma), or aprotinin (Sigma) was used as different media conditions. 1,10-Orthophenanthroline was kept as a 200 mM stock solution in methanol and used in a final concentration of 0.1 mM. Ilomastat and its control peptide (N-t-butyloxycarbonyl-L-leucyl-L-tryptophan methylamide; AMS Scientific) were dissolved as a 1 mM stock solution in 1% DMSO and used at 50 µM. TIMP-2, aprotinin, and E64 were dissolved in water or serum-free DMEM/F-12 and used in final concentrations of 1 µg/ml, 10 µg/ml, and 10 µM, respectively. For morphological evaluation, 20 consecutive randomly selected cells/well were counted 48 h after plating, and the percentage of cells with processes or branches was recorded. The average number of cells with processes in the wells treated with BSN-CM in the absence of inhibitors was defined as 100%, and other results were calculated accordingly. Wells were plated at least in triplicate, and the analyses were repeated at least three times. Statistical significance was tested by using Student's t-test for unpaired two-tailed samples, and the error bars represent the SE. Photomicrographs were taken with an inverted phase-contrast microscope (Nikon) at ×100 magnification.

Isolation and culture of UB epithelium. Embryonic kidneys were isolated from Sprague-Dawley rats at e13, and the UBs were separated from the MM as previously described (20). Isolated UBs were suspended within 200 µl of an ECM gel consisting of a 1:1 mixture of both Matrigel and collagen type I (produced as described above) and applied to the top of a polycarbonate Transwell filter (Corning Costar) in 12-well tissue culture plates. Isolated UBs were cultured in the presence of 800 µl BSN-CM, 125 ng/ml rat recombinant glial cell-derived neurotrophic factor (GDNF), 500 ng/ml FGF-1, and 10% FCS in 5% CO2 and 100% humidity at 37°C. As experimental conditions, 50 µM ilomastat (AMS Scientific), 50 µM of its control peptide (AMS Scientific), or 10 µg/ml TIMP-2 (Calbiochem) was added to the media. Phase-contrast photomicrographs of the developing UBs were taken by using a Kodak DC120 digital camera attached to a Nikon Eclipse TE300 inverted microscope. Some UBs were fixed in 4% paraformaldehyde (Sigma), stained with fluorescein-coupled Dolichus biflorus lectin (Vector Laboratories) for 1 h at room temperature, washed, postfixed for 10 min with 4% paraformaldeyde, and visualized with a Zeiss confocal microscope.

Zymography. BSN-CM, identically produced UB cell conditioned medium (UB-CM), and serum-free as well as BSN-CM-containing supernatant from gels with and without UB cells were subjected to substrate gel electrophoresis for detection of gelatinolytic activity. Equal volumes of the samples were mixed with nonreducing sample buffer and electrophoresed without boiling in 8% SDS-polyacrylamide (Sigma) gels containing 1% (wt/vol) gelatin type A (Sigma) at 4°C. To eliminate their SDS content, the gels were washed twice with 2.5% (vol/vol) Triton X-100 at room temperature for 30 min. Afterward, they were incubated for 36 h in 50 mM Tris · HCl, pH 8, 5 mM CaCl2, 1 µM ZnCl2, and 0.02% (vol/vol) NaN3, which allows gelatinolytic enzymes to act. To determine whether gelatinolysis result from metalloproteinase activity, zinc and calcium were omitted and 1,10-orthophenantroline was added in a control experiment. After a twofold rinse, the gels were stained with Coomassie blue (Gelcode blue stain, Pierce) according to the instructions of the manufacturer and scanned. For better clarity, the negative images are shown.

Immunoblotting. Supernatants (DMEM/F-12) from MG/collagen gels with and without cells were harvested after 48-72 h, concentrated 100-fold with spin columns (Millipore, cutoff 10 kDa), electrophoresed under reducing conditions in 8% polyacrylamide gels (Novex) after boiling, and transferred to nitrocellulose membranes (Micron Separation) For specific detection of gelatinase B, the nitrocellulose membranes were incubated with 50 µg/ml polyclonal goat anti-mouse MMP9 antibody (Santa Cruz Biotech) in 5% nonfat dry milk for 16 h at 4°C. The signal was detected by incubation with a horseradish peroxidase-coupled anti-goat IgG antibody (Santa Cruz Biotech) and exposure to a chemiluminescence reagent (Pierce).

MMP activity assays. For determining MMP activity, 1-ml aliquots of collagen gels and MG/collagen mixtures were plated with or without UB cells in 12-well plates (Falcon) and overlaid with an equal volume of either serum-free DMEM/F-12 or BSN-CM. UB-CM and BSN-CM were prepared identically as described. After harvesting, the supernatants were kept on ice until measured. For detection of MMP activity, a fluorogenic MMP2/MMP7 substrate (25 µM, Novobiochem) was added to the solutions after allowing them to warm to 37°C, and the increase in fluorescence was continuously measured with a luminescence spectrometer (excitation wavelength 323 nm; emission wavelength 393 nm; SLM Aminco Bowman Series 2). The slope of the increase was calculated in the linear range, and the amount of substrate cleaved per minute was determined by comparison with a standard of known concentration (MMP2/MMP7 control, Novobiochem). According to information from the manufacturer, all MMPs except MMP3 cleave the MMP2/MMP7 substrate, so that results can be considered to represent total MMP activity, with the exception of MMP3 activity. Assays were performed in triplicate, and representative measurements are shown.

Northern blotting. For isolation of total RNA, 3-5 ml of the cell suspension in the gel were plated in sterile culture dishes (Falcon) and covered with an equal volume of medium. Gels were harvested after 16-18 h, briefly dehydrated on Whatmann paper, and dissolved in Tri-Reagent LS (Molecular Research Center). Total RNA was extracted according to the instructions of the manufacturer. Total RNA (15-17.5 µg/lane) was separated by electrophoresis, blotted onto nylon membranes (Micron Separations), and then probed with radioactively labeled cDNA probes (Ready-to-Go labeling kit, Pharmacia; 32dCTP, DuPont). Equal loading was asserted by evaluation of ethidium bromide (Bio-Rad) staining of ribosomal RNA after transfer. The cDNA probes for TIMP-2 and -3 were purchased from the American Type Culture Collection. The cDNA probes for gelatinase A, stromelysin, and mouse interstitial collagenase were generous gifts from L. Matrisian (Vanderbilt Univ.). The cDNA probe for TIMP-1 was donated by Dr. G. P. Stricklin (VA Hospital, Nashville, TN) and for gelatinase B by Dr. B. Marmer (Univ. of Washington). A 611-bp cDNA strand was amplified by PCR from reverse-transcribed UB cell cDNA with designed primers (forward 5'-CCAGAAGCTGAAGGTAGGAGC-3', reverse 5'-CATTTGGCCGTTGCCTATAAGG-3', GIBCO-Life Sciences) specific for murine membrane type 1-MMP (MT1-MMP; accession no. gb X83536) and used as a probe for Northern analysis. Identity of the PCR product with the targeted coding region of the MT1-MMP gene was proven by sequence analysis. After hybridization at 42°C for 16-20 h and repeated washes of the membranes, the radioactive signals were evaluated by autoradiography (Kodak Biomax film). Up to three different membranes were used for the evaluation of mRNA expression of the different MMPs and their inhibitors. Autoradiographic bands were quantified with the aid of standard National Institutes of Health imaging software.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MMPs are necessary for in vitro morphogenesis. The outgrowth of the UB from the Wolffian duct through the surrounding ECM and into the MM is a critical step in nephrogenesis. To investigate this process in more detail, two in vitro models have been established. UB cells and MM cells (BSN cells) were isolated from SV40 large T transgenic murine embryos around the time of UB outgrowth on e11.5 as described (2, 23). The derived cell lines largely showed the expected features and markers for epithelial and mesenchymal cells. The UB cells are seeded into an appropriate three-dimensional matrix and exposed to serum-free BSN-CM. Stimulated with this medium, UB cells undergo branching morphogenesis and tubulogenesis resembling the development of the ureteric tree (23). It has been shown that the ECM and soluble factor requirements for UB cell branching morphogenesis in this model are very similar to those necessary for branching morphogenesis of the isolated UB (20). Here, we exploited both these models to gain insight into the role of MMPs in the process of renal collecting duct system development.

UB cells, grown in a 20%-80% mixture of growth factor-reduced MG and collagen, developed processes and then multicellular cords when stimulated by BSN-CM (Fig. 1A-C). After ~2 wk of culture, some of the multicellular cords formed tubules with lumens (Fig. 1, D and E). During this process, the developing structures needed to overcome the constraints imposed by the surrounding ECM, presumably through the action of MMPs. Consistent with this hypothesis, inhibition of MMPs with the broad-spectrum inhibitor 1,10-orthophenanthroline decreased cell process formation. Concentrations of 0.1 mM reduced the formation of processes by 75% (P < 0.00005). Although this agent has been widely used to inhibit MMPs, because it is a zinc chelator there are legitimate concerns about specificity. Therefore, we employed a specific synthetic MMP inhibitor (ilomastat, 50 µM) and a broad-spectrum physiological tissue inhibitor of metalloproteinases (TIMP-2, 1 µg/ml). These agents also inhibited the cell process formation significantly (P < 0.001 and P < 0.01) (Fig. 2, A, B, and D). In contrast, an inhibitor specific to a single MMP (stromelysin, 100 µM, MMP3-inhibitor, Calbiochem) did not significantly reduce the number of cell processes after 48 h of culture (P > 0.05). Inhibition of serine proteinases with aprotinin or cystine proteinases with E64 showed no effect on cellular outgrowth (Fig. 2, A and D). These results indicate that MMPs are required for process formation, an early step in in vitro branching morphogenesis and that serine proteinases and cystine proteinases either are not involved or can be functionally compensated for.


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Fig. 1.   Ureteric bud (UB) cells suspended in collagen type I supplemented with 20% (vol/vol) growth factor reduced Matrigel and nurtured with BSN cell-derived conditioned medium (BSN-CM) for 1 day (A), 3 days (B), and 5 days (C). Cells form processes and proceed to multicellular cords in the first 5 days of culture. After 2 wk, some multicellular tubular structures have developed (D, E). Cell membranes were stained with FITC-labeled lectin from Dolichus biflorus, and cell nuclei were counterstained with propidium iodine (E). Scale bars correspond to 50 µm.



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Fig. 2.   Cell process formation was quantified 48 h after cells were suspended in a 20%/80% MG/collagen type I mixture. Twenty adjacent cells were evaluated in randomly selected fields, and the percentage of cells with cellular process formation was recorded. Wells were seeded at least in duplicate, and assays were repeated at least 3 times. Exposure of UB cells to the cysteine proteinase inhibitor E-64 (10 µg/ml), the serine proteinase inhibitor aprotinin (Aprot; 100 µg/ml; E), a specific stromelysin inhibitor (MMP3-I; 100 µM), or the control peptide for Ilomastat (Ilo-cont; 50 µM) did not impair process formation (the earliest phase of in vitro tubulogenesis), whereas 1,10-orthophenanthroline (o-Phe; 0.1 mM, P < 0.00005; C), TIMP-2 (1 µg/ml; P < 0.01; D), and Ilomastat (ilo; 50 µM, P < 0.001) significantly inhibited cell process formation. The bars indicate SE, and P values were calculated with the Student's t-test for unpaired values (A). Morphologically, inhibition corresponded to round cells without processes in the conditions with 1,10-orthophenanthroline and TIMP-2, whereas addition of aprotinin showed no effect compared with culture in BSN-CM alone (B). Scale bars correspond to 50 µm.

In support of the importance of MMPs in branching morphogenesis, our data indicate that the isolated UB also appears to require MMPs for the completion of its morphogenetic program. When cultured in a three-dimensional matrix and stimulated with soluble factors in the presence of 10% FCS, the isolated UBs branched repetitively over the course of several days. In the presence of ilomastat (Fig. 3B), but not the control peptide (Fig. 3A) or TIMP-2 (Fig. 3D), the UBs survived but showed much less morphogenetic activity compared with control (Fig. 3, A and C). Thus the mechanism of structural growth appears dependent on local degradation of the ECM by MMPs rather than on creating space by pushing into the meshwork of surrounding ECM proteins, and this process seems to occur independently of local cell-cell contact of the MM with the UB.


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Fig. 3.   Kidneys were dissected from rat embryos on gestational day 13, and the UB was separated from the metanephric mesenchyme (MM.) The isolated UBs were then cultured for 7 days embedded in a mixture of Matrigel and collagen I on Transwell filters in the presence of medium conditioned by embryonic MM (BSN) cells, glial cell-derived neurotrophic factor (GDNF; 125 ng/ml), FGF-1 (500 ng/ml), and 10% FCS. The UB underwent branching morphogenesis with branch elongation in the control conditions, where a control peptide for Ilomastat (50 µM; A) or no inhibitory factor (C) were added. When either Ilomastat (50 µM; B) or physiological protein factor (TIMP-2; 1 µg/ml; D) were added to the medium, branching was decreased. This further supports the importance of MMPs in UB branching morphogenesis. Photographs in A and B were taken as phase-contrast pictures; the structures in C and D were stained with fluorescein-coupled D. biflorus lectin and visualized by confocal microscopy. Scale bars correspond to 100 µm.

There are multiple sources of potential MMPs: BSN cell conditioned medium, growth factor-reduced MG, and the cells themselves. In these two in vitro models for UB branching tubulogenesis, there are multiple potential sources of MMPs. These include the UB cells themselves, which, reacting to soluble tubulogenic factors and to the ECM environment, might secrete proteinases necessary for complex morphogenesis. In addition, BSN-CM, which contains growth factors such as hepatocyte growth factor, epidermal growth factor receptor ligands, and probably also other factors (18, 25), could also contain active proteinases or their inactive precursors that are necessary for branching tubulogenesis. Finally, the ECM itself may store trapped or bound MMPs, which can be released by invading cell structures.

To investigate the origin of the MMPs, BSN-CM, UB-CM, and supernatants from collagen and MG/collagen gels, with or without UB cells, were analyzed by zymography. Equal volumes of the conditioned media or supernatants were subjected to electrophoresis in gelatin substrate gels, and areas of gelatinolysis were documented after 36 h. In addition, the conditioned media and supernatants from the different conditions were tested for MMP activity by measuring their rate of MMP substrate cleavage. By substrate gel analysis, we found that the BSN cells produced significant amounts of gelatinolytic enzymes, which are present as either active enzymes or their precursors in the BSN-CM (Fig. 4A). On the basis of published data from others (13), the faint higher molecular mass band in Fig. 4A is likely to represent the activity of progelatinase B, whereas the area of intense lysis at ~70 kDa is likely to be progelatinase A. In comparison with the BSN-CM, the UB-CM, derived from a confluent UB cell monolayer after 3 days of culture in DMEM/F-12 alone, showed only weak gelatinolysis (Fig. 4A). Measurement of total MMP activity (except stromelysin activity) in the two media showed nearly fourfold stronger enzymatic activity in BSN-CM compared with UB-CM (Fig. 4B). These results support the possibility that the BSN-CM, the branching tubulogenesis-stimulating medium derived from cells that appear to originate in the MM, could supply at least part of the required MMPs during the morphogenetic process.


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Fig. 4.   Equal volumes of BSN-CM, UB cell conditioned medium (UB-CM; A), and supernatants [from serum-free DMEM/F-12 (C) or BSN-CM (E)] of gels with or without UB cells after 2-3 days of incubation were subjected to electrophoresis in gelatin-containing SDS-polyacrylamide gels, incubated, and stained for detection of regions of gelatinolysis. Lanes were loaded with equal volumes of CM or supernatants. In contrast to UB-CM, BSN-CM showed strong gelatinolysis mainly at ~70 kDa, likely representing the effect of gelatinase A (A). When the matrix metalloproteinase (MMP) activity was measured by the amount of substrate cleaved per minute, equal volumes of BSN-CM were found to contain ~3-fold more total MMP enzyme activity than identically produced UB-CM (B). Supernatants from MG/collagen-gels (MG/Col) without UB cells led to more gelatinolysis and contained more MMP activity than did supernatants from pure collagen gels (Col; C, D). When UB cells were added to the gels (+UB), the harvested supernatants showed clearly more overall gelatinolysis (C) and MMP activity (D) than did supernatants from gels without suspended UB cells in both collagen and MG/Col matrices. These results indicate that, in addition to the BSN-CM and the ECM, the UB cells also contribute to the supply of MMPs. In the MG/Col gel with UB cells, a strong band at ~92 kDa (*) appeared (C). The identity of this band was confirmed by the detection of increased gelatinase B levels in supernatants from MG/Col gels with UB cells compared with gels without UB cells by using a polyclonal anti-mouse MMP9 antibody (E). In addition, when MG/Col gels with UB cells are cultured with BSN-CM, the gelatinolysis at ~92 kDa (*), corresponding to gelatinase B activity, is further increased (F), supporting a role of this metalloproteinase in branching morphogenesis. The lower band, at ~65 kDa (**, C), which appeared in both collagen and MG/Col gels containing UB cells, corresponds to the lytic activity of either active gelatinase A or procollagenase. For better clarity, the zymographs are shown as negative images. MW, molecular wt.

To examine the potential contribution of the matrix, supernatants from collagen and 20%-80% MG/collagen gels were subjected to substrate gel analysis after ~48 h of incubation with unsupplemented DMEM/F-12 in 32°C. In the MG/collagen gel, UB cells undergo more complex and more sustained branching morphogenesis compared with the pure collagen gel. Greater than 50% MG content inhibits branching tubulogenesis in both Madin-Darby canine kidney cells and UB cells (23, 26). As shown in Fig. 4C, the MG/collagen preparation contained significant gelatinolytic activity (in the absence of cells), whereas collagen contained only small amounts of gelatinolytic enzymes (in the absence of cells). When UB cells were added to the ECM, the harvested supernatant clearly showed increased gelatinolysis in comparison with the cell-free preparation. Furthermore, this effect was more prominent in the case of the three-dimensional culture in the MG/collagen mixture than in collagen alone (Fig. 4C). When MMP enzymatic activity was quantified, supernatants of the MG/collagen mixture were shown to contain more active MMPs than the did supernatants of pure collagen gels, as shown by a higher cleavage rate. The addition of UB cells increased the MMP activity in both types of ECM (Fig. 4D). Thus the addition of UB cells adds MMPs to the supernatants, as shown by the increase in gelatinolysis in the substrate gels (which detect inactive as well as activated MMPs), and leads to stronger MMP activity, possibly by activating inactive MMPs already in BSN-CM or the ECM gel. After size separation on substrate gels, supernatants of the UB cell-containing MG/collagen mixture showed a major increase in gelatinolysis ~92 kDa, where the progelatinase B activity is expected (13). The increase in gelatinase B content, when cells were added to MG/collagen gel preparations, was confirmed by using a polyclonal anti-mouse MMP9 antibody in immunoblots of supernatants (Fig. 4E). A clear but less pronounced increase in gelatinolytic activity was found at ~65 kDa, which could represent either gelatinase A or procollagenase activity (Fig. 4C). These proteinases migrate in close proximity and are not easily distinguished by size differentiation alone (19). This activity, but not gelatinase B, seems to be secreted by UB cells suspended in pure collagen I, indicating a specific response of the UB cells to the surrounding ECM. The gelatinolytic activity detected at 57 kDa, which likely corresponds to prostromelysin (19), is mainly found in MG-containing preparations where it is unaltered by the addition of UB cells. Culture in the absence of zinc and calcium and in the presence of the MMP inhibitor 1,10-orthophenanthroline completely abolished gelatinolysis, supporting the presence and activity of MMPs in the model (data not shown). Thus, alone and with the UB cells, the MG/collagen gels contained and stimulated more gelatinolytic activity than did pure collagen gels. Nevertheless, although this is consistent with the morphogenetic potential of the ECM preparations (MG/collagen > collagen alone), it is important to emphasize that branching tubulogenesis requires soluble factors present in BSN-CM (23). These could be growth factors, or, on the basis of the results presented here, conceivably MMPs (in BSN-CM, MG/collagen, or both), perhaps acting synergistically with growth factors. Consistent with the increased morphogenesis in the presence of BSN-CM (which itself produced only a very faint 92-kDa band), the 92-kDa gelatinolysis band representing gelatinase B was further increased in supernatants, when cells were cultured in MG/collagen gels and stimulated by BSN-CM compared with gels incubated with DMEM/F-12 alone (Fig. 4F).

UB cell MMP expression is modulated by BSN-CM and the ECM composition. UB cells dispersed in the ECM and stimulated by BSN-CM initiate morphogenesis within 24 h by forming cell processes, which subsequently branch and divide or join with other cells to form multicellular cords and can ultimately proceed to tubular structures (Fig.1, A-E), similar to the in vitro development of the isolated UB in a three-dimensional gel. In the context of the functional inhibition data already discussed (1,10-orthophenanthroline, Ilomastat, TIMP-2 inhibition; Figs. 2, A-E, and 3, A-D), these obvious morphological changes suggest that the process of ECM degradation is crucial from the start of the three-dimensional culture. As serum-free BSN-CM is sufficient to stimulate morphogenesis in the cell culture model, and MMPs appear to be required for this process, we examined the effect of BSN-CM on the expression of those genes in UB cells. As shown in Fig. 5A, the transcription of the examined MMPs involved in degradation of ECM (gelatinase A and B, stromelysin, MT1-MMP, and TIMP-1-3) were upregulated by BSN-CM after ~18 h, compared with the expression in DMEM/F-12 under otherwise identical conditions. Murine interstitial collagenase was upregulated by BSN-CM as well but only in a pure collagen matrix and not in a MG/collagen mixture (not shown). This raises the possibility that, in the degradation of the ECM, multiple proteinases and their inhibitors act in concert but specific expression patterns depend on the matrix. By Northern analysis, the major difference in MMP expression between three-dimensional culture in collagen, with or without the addition of 20% MG (vol/vol), appears to be the increased expression of gelatinase B (and, to a much lesser extent, gelatinase A) and decreased expression of stromelysin and collagenase in the MG/collagen mixture (Fig. 5B). In general, these results appear consistent with gelatin zymography (Fig. 4B), which showed increased expression of 92- and 65-kDa gelatinolytic activities, highly likely corresponding to gelatinase B and A, respectively. Thus, even though early morphogenesis can occur in both collagen and MG/collagen (though much more impressively in the latter context), there is a different pattern of MMP expression. UB cells react to the surrounding matrix (and possibly the presence of gelatinolytic enzymes in the matrix itself).


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Fig. 5.   Total RNA was extracted from UB cell suspensions in MG/Col or Col gels incubated with or without BSN-CM for 18-24 h. Total RNA (17.5 µg) was loaded in each lane, and membranes were hybridized with cDNA probes for different MMPs and TIMPs. All analyses were performed at least twice, and autoradiographic bands were quantified by using standard National Institutes of Health imaging software. Transcription of MMPs and TIMPs was stimulated by BSN-CM in 3-dimensional gels (MG/Col) compared with culture in serum-free DMEM/F-12 (SF) alone (A). Different extracellular matrix compositions influenced the expression of MMPs. Seeding in Col (Col) in the presence of BSN-CM led to increased expression of collagenase and stromelysin, whereas seeding in MG (MG/Col) mainly stimulated the expression of gelatinase B. Gelatinase A is clearly expressed in both conditions, although more in the MG-containing ECM (B).

With increasing structural complexity, soluble MMP expression decreases and expression of MT1-MMP, TIMP-2, and TIMP-3 remains constant or rises. To evaluate the role of the MMPs and their inhibitors in the course of in vitro branching morphogenesis, we investigated their expression on days 1, 3, and 5 as cell processes (day 1) develop into branching multicellular cords (day 5) and are on the verge of forming more complex structures (tubules). The expression of all MMPs, with the exception of the membrane-bound MT1-MMP, decreased after the first day. TIMP-1 decreased considerably after the first day, whereas TIMP-2 and -3 increased in expression, with TIMP-3 showing the most significant rise. MT1-MMP was increased at days 3 and 5 (Fig. 6A). MT1-MMP is a membrane-bound gelatinase A-activating enzyme, which can form local complexes with TIMP-2 and progelatinase A (27). Quantification shows an increase in MT1-MMP by at least twofold over the soluble MMPs between days 1 and 5. Expression of TIMP-2 and -3 increased ~3-fold and ~10-fold, respectively, after 5 days relative to TIMP-1 (i.e., ratio of relative increases) (Fig. 6B). The observed expression pattern suggests that MT1-MMP, TIMP-2, and TIMP-3, perhaps acting in concert, might be important for the regulation of branching tubulogenesis as UB cells form more elaborate structures beyond the stage of initial process formation. The increasing expression of TIMP-3, which has been reported to bind to the ECM and inhibit MT1-MMP as effectively as TIMP-2 (32), suggests a potential regulatory role of this inhibitory factor in the formation of branching multicellular structures.


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Fig. 6.   Total RNA from UB cells grown in MG/Col gels and stimulated with BSN-CM was extracted after 1, 3, and 5 days and hybridized with cDNA probes for different MMPs and TIMPs to evaluate the course of their expression during the growth of increasingly complex morphological structures (A). All analyses were performed at least twice. Expression of MT1- MMP increased at least 2-fold in comparison with the soluble MMPs stromelysin, gelatinase A, and gelatinase B over 5 days of in vitro culture (B). In addition, the expression of TIMP-2 and -3 increased ~3- and 10-fold relative to TIMP-1 (ratio of relative increases), suggesting a role together with MT1-MMP in isolated UB cell branching morphogenesis (C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In summary, using two in vitro models for UB branching morphogenesis, we show that 1) multiple MMPs are likely to be involved; 2) the subset of MMPs expressed during morphogenesis is a function of the matrix environment; 3) the pattern of MMP and TIMP expression depends on the stage of morphogenesis (process formation vs. branching multicellular cords and more complex structures), and the ratio of soluble MMPs to TIMPs decreases with increasing structural complexity; and 4) potential sources of morphogenetic MMPs include the UB cells undergoing branching tubulogenesis, MMPs secreted by the metanephric mesenchyme (BSN) cells, and those bound to the surrounding ECM.

The involvement of MMPs in branching morphogenesis has been shown in different organs (6, 7, 14, 22, 29, 33). Local regulation of proteolytic activity should be important in this process (24). In UB cells undergoing in vitro branching morphogenesis, after an initial increase, the mRNA expression of soluble MMPs decreased, whereas MT1-MMP, TIMP-2, and TIMP-3 were increased over a longer term. The membrane-bound MT1-MMP concentrates gelatinase A locally and can activate the enzyme by proteolytic cleavage, whereas TIMP-2 inhibits this activation as part of a trimolecular complex (27). The main source of gelatinase A in the embryonic kidney is the MM adjacent to epithelial cells, as indicated by in situ hybridization (21). UB cells may therefore control local gelatinase A-dependent ECM degradation by localized expression of MT1-MMP and secretion of TIMP-2. Consistent with this hypothesis, it is reported that MT1-MMP, TIMP-2, and gelatinase A are colocalized to the plasmalemma of UB branches at day 13 and to the collecting tubules and tubular epithelia in medulla and cortex at day 17 in embryonic mouse kidney (17) and that antisense inhibition of MMP2 and MT1-MMP expression led to dysmorphic changes, whereas antisense inhibition of TIMP-2 induced a mild increase in the size of kidney explants in organ culture (11).

In addition, the expression of another MMP inhibitor, TIMP-3, was strongly increased over 5 days of culture, altering the MT1-MMP/TIMP-3 ratio toward inhibition. TIMP-3 inhibits soluble, activated MMPs and is equally effective as an inhibitor of the membrane-bound MT1-MMP as TIMP-2 (32). TIMP-3 is different from TIMP-2 in that it is not secreted into the medium but binds to the ECM (12). Thus TIMP-3 would be ideally suited to protect the ECM from degradation in clefts between the processes, which contribute to the formation of the branched structures. In situ hybridization of TIMP-3 showed transcripts in the epithelium of developing tubules in the prospective renal medulla at day 14.5 of murine embryogenesis, suggesting a role in renal development (1).

In mouse embryonic kidney organ culture, a neutralizing antibody against gelatinase B, but not gelatinase A, perturbed normal kidney development (14). We cannot assign any differential role to these gelatinases on the basis of our data, although gelatinase B seems to represent a large portion of the gelatinolytic activity secreted by UB cells in MG/collagen. However, both gelatinase B- and gelatinase A-deficient mouse models have been established, but no developmental renal defects have been published to date (8, 15, 31). This seems to argue in favor of a considerable in vivo plasticity of the MMP effector system in nephrogenesis. In general, our data support this notion, in that 1) on in vitro branching morphogenesis of UB cells, an inhibitor of a single MMP (MMP3-inhibitor) had no significant effect, even in high concentrations, whereas more broadly active inhibitors did (1,10-orthophenanthroline, ilomastat, TIMP-2); 2) the supply of MMPs can originate from several different sources; 3) the UB cells are able to continue branching morphogenesis in very different ECM gels and can markedly alter the subset of MMPs they express in these different matrices, suggesting considerable adaptability; and 4) the UB cells may alter their own MMP production in relation to the stage and complexity of the structures being formed.

We thus propose a model of branching morphogenesis of the UB that requires MMPs, which may be recruited from the secreted products of mesenchymal cells and existing ECM stores as well as produced and activated by the UB itself. The specific subset of MMPs and TIMPs expressed is highly dynamic and likely to be a function of local morphogenetic factors, the changing ECM environment, and the topology and complexity of developing structures.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO-1-DK-49517 (to S. K. Nigam). M. Pohl was supported by the Deutsche Forschungsgemeinschaft, H. Sakurai was supported in part by a fellowship grant from Uehara Memorial Life Science Foundation, and K. T. Bush was supported in part by an American Heart Association Scientist Development Award.


    FOOTNOTES

* M. Pohl and H. Sakurai contributed equally to this work.

Address for reprint requests and other correspondence: S. K. Nigam, Depts. of Pediatrics/Medicine, Division of Nephrology/Hypertension, Univ. of California, San Diego, 9500 Gilman Dr. 0693, La Jolla, CA 92093-0693.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 19 April 2000; accepted in final form 19 July 2000.


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