1Division of Nephrology, Department of Medicine, 2Department of Pathology, 7Department of Cancer Biology, Vanderbilt University Medical Center, and 6Veterans Affairs Hospital, Nashville, Tennessee 37232; 3Departments of Medicine and Pediatrics, University of California, La Jolla, California 92093; 4Department of Medicine, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115; and 5MediCity Research Laboratory and the Department of Medical Biochemistry, University of Turku, Turku, Finland 20520
Submitted 15 January 2004 ; accepted in final form 1 June 2004
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ABSTRACT |
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kidney development; branching morphogenesis; three-dimensional culture; basement membranes
The collecting system of the kidney is formed by iterative branching morphogenesis of the UB. Although this process has been studied in whole animal, organ, and cell culture models, the molecular cues for its control are still poorly understood. Cell culture models, which predominantly utilize Madin-Darby canine kidney (MDCK) cells grown in three-dimensional (3D) collagen-I (3D-CI) gels, recapitulate branching morphogenesis in its simplest form. These models suggest that branching morphogenesis is a multistep process that requires sequential cell adhesion to extracellular matrix (ECM), cell spreading, cell proliferation, and cell migration to ultimately form multicellular tubelike structures (14). A balance between proliferation and apoptosis is critical for the final formation of the lumen of the tubule (5).
Integrins, the predominant cell-surface receptors that mediate interactions between cells and ECM, play a crucial role in cellular functions such as cell adhesion, migration, proliferation, and apoptosis (7, 21). Both in vivo and in vitro data demonstrate the importance of integrins that interact with the laminin basement membrane components in normal UB development; however, there is no information on the role of the collagen receptors, 1
1- and
2
1-integrins. Mice deficient in the laminin receptor
3
1-integrin have fewer collecting ducts in the papilla, resulting in decreased branching morphogenesis (11). Surprisingly, mice lacking the
6-integrin subunit, which dimerizes to form the laminin receptors
6
1 and
6
4, do not show any anomalies in the developing UB (6). However, when
6- and
3-null mice are crossed, ureters fail to develop (4).
3
1- And
6-integrin subunits are required for UB branching morphogenesis in organ and cell culture model systems (24).
The spatiotemporal expression of laminin- and collagen-binding integrins in the development of the UB is poorly described and controversial. Studies performed in human tissues describe 3- and
6-integrin subunit (9, 10) expression in the developing collecting system, whereas
2-,
3-, and
6- containing integrin subunits are present in the adult collecting ducts (9, 10). Although inconclusive, these studies suggest that the expression pattern of integrins in the UB changes as it undergoes terminal differentiation to the collecting duct.
To further understand how integrin expression modulates UB development, we investigated the functional role of murine laminin- and collagen-binding integrins in cell culture models utilizing cells isolated from the UB at embryonic (E) day 10.5 and the adult IMCD. We show both in vivo and in vitro that the undifferentiated UB and the well-differentiated IMCD express the laminin receptors 3
1,
6
1, and
6
4, whereas expression of the collagen-binding integrins,
1
1 and
2
1, is restricted to the differentiated IMCD. Utilizing 3D tubulogenesis assays, we demonstrate that cells derived from the IMCD, but not the UB, can undergo tubulogenesis in CI gels in an
1
1- and
2
1-dependent manner. In contrast, both UB and IMCD cells undergo tubulogenesis in CI/Matrigel (MG) gels; however, UB cells primarily utilize
3
1- and
6-integrins, whereas IMCD cells mainly employ
1
1 for this process. These findings suggest that temporal and spatial changes in integrin expression could help organize the pattern of branching morphogenesis of the developing collecting system in vivo.
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MATERIALS AND METHODS |
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Constructs and cell culture.
Immortalized UB (a gift from J. Barasch, Columbia University, New York, NY) and IMCD (a gift from E. Delpire, Vanderbilt University, Nashville, TN) cells were cultured and maintained as previously described (18, 19). Renal papilla cells derived from E18 kidneys of 3-integrin-null mice and
3-integrin-null cells reconstituted with the human
3-integrin subunit were cultured and maintained as previously described (23). The human
2-integrin expression construct was prepared as described previously (16). A pure UB cell population expressing
2
1-integrin (
2-UB) was derived by transfecting UB cells with the
2-integrin expression construct, after which cells were sorted via fluorescence-activated cell sorting (FACS). An antibody directed to the extracellular domain of human
2-integrin was utilized to define
2-integrin expression. Control cells were transfected with the empty vector.
Inhibition of 1- and
2-integrin subunit expression by small interfering RNA.
The murine
1-integrin sequence 5'-GGTCACTGTAGCCTGCATT-3' and the murine
2 sequence 5'-GGAGCGAAAATATTTTCCG-3' were targeted for RNA interference. Double-strand small interfering (si)RNA were obtained from Ambion (Austin, TX). Subconfluent populations of IMCD cells were transfected using siPORT Amine Transfection Agent (Ambion) according to the manufacturer's instructions. Control cells were transfected with Silencer Negative Control No. 1 siRNA obtained from Ambion. Tubulogenesis assays were performed 48 h after transfection, and flow cytometry and migration experiments 7 days after transfection.
3D cell culture. Tubulogenesis of UB- and IMCD-derived cells was performed in 3D ECM gels as previously described (1, 18). The CI gels were composed of 0.1 mg/ml CI in DMEM containing 20 mM HEPES (pH 7.2). For the MG/CI gels, a 1:1 mixture of the collagen solution described above was mixed with growth factor-reduced MG, giving a final concentration of 0.5 mg/ml of CI and 0.5 mg/ml of MG (18). One hundred microliters of medium supplemented with 10% FCS were added to the gels after they had solidified. For quantification of the branches, cells that formed branching structures (defined as more than 1 branch) were counted in five randomly picked high-power fields. The image analysis was performed using the Metamorph cell-imaging program (Universal Imaging, Downingtown, PA). The degree of branching morphogenesis was quantified by using the number of end points as a correlative measure of the number of branching events. Assays were performed at least in triplicate, and error bars represent SE. P values were calculated with Student's t-test.
Flow cytometry. A suspension of UB or IMCD cells was incubated with monoclonal antibodies to the appropriate integrin (1:100 dilution), followed by incubation with the appropriate secondary antibodies (FITC-coupled rabbit anti-rat or hamster immunoglobulin; 1:100 dilution). Flow cytometry was performed with a FACScan instrument (Becton Dickinson). Cell suspensions incubated with secondary antibody only were used as a negative control for integrin expression.
Immunohistochemistry. Frozen sections of embryonic or adult mouse kidneys were utilized in these studies. Sections were incubated for 20 min with 2% H2O2 in methanol to quench the endogenous peroxidase. After an additional wash in PBS, sections were incubated for 1 h at room temperature with 3% bovine serum albumin, 3% normal goat serum in PBS (blocking solution), followed by incubation with the appropriate primary antibodies diluted in blocking solution overnight at 4°C. After being washed with PBS, the slides were incubated with horseradish peroxidase-conjugated secondary antibodies (1:100 dilution in blocking buffer) for 2 h at room temperature, and staining was evaluated by incubating the slides with 0.05 M Tris solution containing 0.05% DAB tetrahydrocloride Sigma Fast DAB tablets. Finally, the slides were counterstained in Harris hematoxylin and mounted using Permount.
Cell proliferation and apoptosis. UB or IMCD cells (5 x 103 cells/ml) were embedded in the 3D gels (100-µl final volume) in 96-well plates as described above and incubated in DMEM/F-12 containing 2% FCS in the presence or absence of an anti-integrin antibody (10 µg/ml final concentration). After 2 days in culture, cells were pulsed for an additional 48 h with [3H]thymidine (1 µCi/well). The gels were then removed from the plates and dialyzed against PBS for 24 h to remove free [3H]thymidine. The gels were then lysed in 10% SDS (100-µl final volume), and the lysates were measured with a beta counter.
For apoptosis studies, the cells were incubated in 3D gels as described above. After 4 days in culture, the gels were fixed in 4% paraformaldehyde for 30 min, followed by DMSO-methanol in a ratio of 1:1 for 30 min. Apoptosis was detected using the Apoptag Apoptosis Detection Kit, as described by the manufacturer. The gels were also stained with 46-diamidino-2-phenylindole to identify all the cell nuclei. Apoptotic cells were counted in five different microscopic fields of a fluorescence microscope, and the apoptotic index was expressed as follows: (no. of positive apoptotic cells/total no. of cells/microscopic field) x 100.
Cell adhesion. Microtiter plates (96-well) were coated with matrix proteins at the indicated concentrations in PBS for 1 h at 37°C. Plates were then washed with PBS and incubated with PBS containing 0.1% BSA for 60 min to block nonspecific adhesion. One hundred microliters of single-cell suspensions (106 cells/ml) in serum-free DMEM containing 0.1% BSA were added in triplicate to 96-well plates and incubated for 60 min at 37°C. In some experiments, cell suspensions were preincubated with anti-integrin antibodies (10 µg/ml final concentration) on ice for 30 min before the assay. Nonadherent cells were removed by washing the wells with PBS. Cells were then fixed with 1% formaldehyde, stained with 1% crystal violet, solublized in 2% SDS, and the cell lysates were then read at 570 nm. Cells bound to fetal calf serum were used as a positive control to indicate maximalcell adhesion, whereas cells bound to 1% BSA-coated wells were used as background, and this opitcal density was subtracted from that obtained in serum or extracellular matrix proteins.
Cell migration. Cell migration was assayed in Transwells consisting of polyvinylpyrolidone-free polycarbonate filters with 8-µm pores. The underside of each Transwell was precoated with ligand overnight at 4°C, and the filter was subsequently blocked with 1% BSA for 1 h at 37°C to inhibit nonspecific migration. One hundred microliters of a cell suspension (1 x 106 cells/ml) in serum-free medium containing 0.1% BSA were added to the wells, and the cells were allowed to migrate into the matrix coated on the underside of the Transwell for 4 h. Cells on the top of the filter were removed by wiping, and the filter was then fixed in 1% formaldehyde in PBS. Migrating cells were stained with 1% crystal violet, and nine randomly chosen fields from triplicate wells were counted at x200 magnification.
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RESULTS |
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To determine whether the expression pattern of integrins in the UB and IMCD cells was representative of the anatomic structures from which they were originally derived, we determined the integrin expression patterns in embryonic and adult mouse kidneys. As expected, 1
1- and
2
1-integrins were poorly expressed in the UB of E15 embryonic kidneys as determined by immunohistochemistry (Fig. 1C). In contrast, expression of these two collagen-binding receptors was detectable in the collecting ducts of adult kidneys (Fig. 1D).
3
1-,
6
1-, And
6
4-integrins were expressed in the UB and adult collecting ducts. Although these are not quantitative assays, these results suggest that the integrin expression patterns found in the cells are similar to those for the organelles from which they were originally derived. Therefore, these cell populations are potentially valid model systems with which to study the role of integrin-ECM interactions in the formation of tubules at different stages of collecting system development.
To determine which integrins mediated IMCD cell tubulogenesis in CI, cells were grown in 3D-CI gels in the presence of blocking antibodies or isotype-matched immunoglobulins to different integrin subunits. Only antibodies directed against the 1- and
2-subunits, added alone or in combination, resulted in a dramatic reduction and/or complete inhibition of tubulogenesis (Fig. 2). The role of
3
1-integrin in tubulogenesis was determined by comparing
3-null cells (isolated from mouse E18 renal papilla) with
3-null cells reconstituted with the
3-subunit, as there are no blocking antibodies to the mouse
3-integrin subunit available. These cells have an integrin profile similar to UB cells (with little or no expression of
1
1 or
2
1) (23) and behave like UB cells when grown in 3D gels (24). Both the
3-null and the reconstituted cells were unable to form tubules in CI gels, suggesting that
3
1-integrin does not play a significant role in tubulogenesis in CI gels (data not shown). Blocking antibodies to
1-integrin impeded tubulogenesis to an extent similar to that observed with a combination of anti-
1- and -
2-integrin antibodies, suggesting that only
1
1- and
2
1-integrins are required for IMCD tubulogenesis in 3D-CI gels (Fig. 2).
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Expression of 2
1-integrin reconstitutes the ability of UB cells to form tubules in 3D-CI gels.
Our data suggest that differentiation of IMCD structures from the undifferentiated UB is associated with increased expression of the collagen receptors,
1
1- and
2
1-integrin. Antibodies to
2
1-integrin resulted in more inhibition of IMCD cell tubulogenesis (85% inhibition) in 3D-CI gels than anti-
1-integrin antibodies (40% inhibition) (Fig. 2), suggesting that
2-integrin plays a more critical role than
1-integrin in regulating branching morphogenesis in 3D-CI gels. To determine whether transduction of
2
1-integrin into UB cells would facilitate their ability to undergo tubulogenesis in 3D-CI gels, we generated UB cells that expressed a full-length human
2-integrin (
2-UB). A pure
2-UB cell population was derived by sorting, via FACS, cells incubated with an antibody directed to the extracellular domain of the human
2-integrin (data not shown).
2-UB cells cultured in 3D-CI gels formed tubelike structures, similar to IMCD cells grown in the same matrix in the presence of antibodies to
1
1-integrin (Fig. 2B vs. Fig. 6A).
2-UB cells proliferated 3 times more (Fig. 6B), adhered 50 times more (Fig. 6C), and migrated 30 times more (Fig. 6D) than UB cells when cultured within or on CI. These results strongly suggest that
2
1-integrin is required for UB cells to undergo tubulogenesis in 3D-CI gels by mediating cell adhesion, migration, and proliferation on a CI substratum.
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Taken together, these results suggest that the major integrin that mediates cell proliferation is 3
1 for UB cell tubulogenesis, whereas anti-apoptotic properties are exerted by
3
1- and
6
4-integrins. In contrast, IMCD cell proliferation does not appear to be dependent on any specific
-integrin subunit, whereas inhibiting
1
1-integrin function induces apoptosis.
UB and IMCD cells utilize different integrins for migration.
As cell migration is dependent on the ability of cells to adhere to a substrate, we investigated the role of different integrin subunits in mediating adhesion and migration of UB and IMCD cells on the different components of 3D-CI/MG gels. UB cells neither adhered nor migrated on CI (data not shown), corroborating the finding that UB cells express few or none of the two major collagen-binding receptors (Fig. 1B). We therefore restricted our comparison between UB and IMCD cells to MG alone. UB cells adhere and migrate significantly better than IMCD cells on this ECM (Fig. 9, A and B). Adhesion by both cell types to MG is dependent on multiple -integrin subunits as only antibodies against
1 inhibited UB or IMCD cell adhesion (Fig. 9C). UB cell migration appeared to be dependent on
6-integrins,
3
1, and, to a lesser extent, on
1
1 (Fig. 9D). In contrast, IMCD cell migration was primarily dependent on
1
1-integrin and, to a lesser extent, on the
6-integrin subunits (Fig. 9D). Due to the lack of blocking antibodies, the importance of
3
1 in IMCD cell migration on MG could not be tested. Together, these results suggest that specific integrins do play a role in UB and IMCD cell migration on MG.
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DISCUSSION |
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The integrin expression pattern in UB and IMCD cells correlated with that observed in vivo. The developing UB expresses the laminin receptors 3
1,
6
1, and
6
4, and the IMCD expresses these integrins as well as the collagen receptors
1
1 and
2
1. The lack of collagen receptor expression in UB cells explains why these cells are unable to undergo branching morphogenesis in 3D-CI gels. Our data not only clarify the expression pattern of the laminin- and collagen-binding integrins in the developing UB and the IMCD system of the mouse but also parallel the finding described in humans (9, 10). The relatively late expression of both
1
1 and
2
1 in the collecting system and/or the ability of these collagen receptors to functionally compensate for each other may explain the benign renal phenotypes seen in the
1- and
2-integrin-null mice. Although neither of these mice types has been studied in detail, there is no evidence of severe branching morphogenesis abnormalities in the developing UB, and only subtle renal abnormalities are present (Zent R and Pozzi A, unpublished observations). These changes may be similar to the minor differentiation defects found in breast tissue in the
2-null mice (2).
IMCD cell branching morphogenesis in CI gels was only dependent on the collagen receptors 1
1 and
2
1, with
2
1 playing the predominant role. Inhibiting or downregulating the expression of either of these receptors inhibited migration and induced apoptosis. The increased effect of decreasing
2-function on blocking tubulogenesis was likely related to its added effect of inhibiting proliferation. The results with decreased
2-integrin function are similar to those found in MDCK cells grown in CI gels, where
2-integrin was shown to be critical for branching morphogenesis (17). The role of
1
1-integrin in MDCK cell tubulogenesis in CI has never been fully explored; however, it is likely that, like IMCD cells, both
1
1- and
2
1-integrins play a role. The branching studies in CI alone are in contrast to those performed in CI/MG, where
2 plays no role in tubulogenesis. This raises the concern of the physiological relevance of model systems that only utilize CI, which is not a constituent of epithelial basement membranes.
Transduction of 2-integrin into UB cells confirmed the importance of
2
1 in renal cell branching morphogenesis in CI gels. The
2-UB cell tubulogenesis was similar to IMCD cells pretreated with
1-blocking antibodies or transfected with siRNA against the
1-integrin subunit, and these cells were able to adhere, proliferate, and migrate on a CI substrate. This result demonstrates that the morphological changes that occur when UB cells become terminally differentiated IMCD cells are, at least in part, due to expression of
2
1-integrin. Although it is unclear what determines the spatial and temporal alterations of integrin expression in UB development, it may be related to expression of collagens and laminins that are ligands for
1
1- and
2
1-integrins in tubular basement membranes (12). The physiological relevance of the temporal changes in integrin expression is also unknown. It is interesting to speculate that the increased expression of collagen receptors may render IMCD cells less migratory on basement membranes rich in collagens and laminins. This increase in the adhesive strength of cells to the basement membrane may result in increased epithelial cell polarization and the formation of tight junctions necessary for tubules to withstand increased luminal pressure and transport fluids and solutes in a unidirectional manner.
In CI gels, IMCD cell tubulogenesis is predominantly dependent on the collagen receptor 2
1-integrin, and, to a less extent,
1
1. In contrast, the inhibition of
1
1-integrin function on IMCD cells in CI was similar to that seen in CI/MG gels. These differences are likely related to the fact that
1
1-integrin is a better receptor than
2
1 for laminin-1 and collagen IV, which are the primary constituents of MG.
1
1-Integrin preferentially binds to collagen IV and to a lesser extent to collagen I, whereas
2
1 is an excellent receptor for fibrilla-forming type I collagen gels and a relatively poor receptor for type IV collagen (8, 22). In addition, although both
1
1- and
2
1-integrins can also be laminin-1 receptors (3), IMCD cell migration and proliferation on laminin-1 are primarily dependent on
1
1-integrin, to a lesser extent on
3
1- and
6-integrin, but not on
2
1 (Zent R, unpublished observations). In contrast to IMCD cells, UB cells do not migrate or proliferate on collagen IV, and migration and proliferation on laminin-1 are predominantly
3
1- and
6-integrin dependent (Zent R, unpublished observations). Therefore, UB and IMCD cells use distinct integrins for their interactions with MG and laminin-1 specifically. Interestingly, unlike UB and IMCD cells, MDCK cells are unable to form tubules in CI/MG gels (20). This difference between these cell types may be related to disparities in integrin expression.
Little is known about which integrins mediate cellular functions, such as cell proliferation, apoptosis, adhesion, and migration in the context of tubule formation. We demonstrated that inhibiting many specific integrin-ligand interactions induced cell apoptosis, whereas cellular proliferation was affected in a much less restricted manner in CI/MG gels. Blocking antibodies against 6,
1, and
4 markedly induced apoptosis in UB cells,
3-null cells underwent apoptosis, and inhibition of
1 function decreased tubulogenesis of IMCD cells in CI/MG gels by inducing apoptosis. In contrast, only inhibiting
3
1-integrin appeared to affect proliferation of UB cells. Together, these results suggest that inhibiting a specific integrin function of cells grown in 3D-CI/MG gels produces a phenotype of decreased branching morphogenesis predominantly by inducing apoptosis rather than by decreasing proliferation. It is only when all integrin-dependent signals are blocked, as seen with the
1 antibody, that cell proliferation is affected to a significant extent.
Interestingly, the results of cell migration on MG correlate with the phenotype observed in 3D tubulogenesis. This suggests that inhibiting migration may be a cue for the induction of apoptosis in the tubulogenesis assays. The mechanism for this is not known; however, it has been postulated that cells in 3D tubular structures require three different plasma membrane surfaces to survive: a free apical surface that borders the lumen, a lateral surface that adheres to neighboring cells, and a basal surface that adheres to the ECM (14). As tubules grow, cells that lack one of these surfaces are forced to migrate into a position where they can attain these three surface types, and if the cells are unable to achieve this architecture, they undergo apoptosis. Migration of cells in 3D gels is dependent on integrins; therefore, it is possible that inhibiting this integrin function accounts for increased apoptosis and decreased tubulogenesis.
In conclusion, we have characterized UB and IMCD cell branching morphogenesis with respect to integrin expression and demonstrated that they are representative of the developing UB and IMCD in vivo. These cell systems provide an alternative murine model to MDCK cells with which to study kidney tubule formation. With the generation of many genetically mutated mice, as well as the availability of numerous reagents for mouse cells, we propose that the UB and IMCD cells are excellent models for the study of branching morphogenesis that will be useful adjuncts to determine the molecular mechanisms of different stages of UB development.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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