Hepatocyte Growth Factor-Mediated Renal Epithelial Branching Morphogenesis Is Regulated by Glypican-4 Expression

Abstract

The glypican (Gpc) family of cell surface heparan sulfate proteoglycans are expressed in a tissue-specific and developmentally regulated fashion. To determine if individual Gpcs can modulate heparin-binding growth factor signaling, we examined hepatocyte growth factor (HGF)-stimulated mitogenic, motogenic, and morphogenic responses of renal tubular cells expressing different Gpcs. Adult inner medullary collecting duct (IMCD) cells were found to express primarily Gpc4 and to proliferate, migrate, and form tubules with HGF, correlating with sustained extracellular signal-regulated kinase (ERK) activation. Embryonic IMCD cells expressing predominantly Gpc3 proliferated and migrated in response to HGF but activated ERK only transiently and failed to form tubules. Overexpressing Gpc-4 but not Gpc-3 or Gpc-1 led to sustained HGF-stimulated ERK activation and rescued the tubulogenic response in these cells. These results demonstrate that both signaling and phenotypic responses to HGF can be regulated by specific Gpc expression patterns.

Tubulogenesis or branching morphogenesis is a fundamental process involved in the development of several multitubular organs, e.g., lung, pancreas, and kidney. Studies using cell lines and genetically altered mice have implicated several growth factors, including hepatocyte growth factor (HGF), as important regulators of nephron development and ureteric bud branching morphogenesis (1-3, 28). For example, kidney organ explant cultures grown in the presence of a neutralizing antibody to HGF impair ureteric bud branching (29). However, mice deficient in HGF die by embryonic day 13 (E13) to E14 with no abnormalities in the earliest stages of renal branching morphogenesis.

The heparin-binding growth factor HGF signals through its low-abundance, high-affinity receptor c-met. In addition, heparan sulfate proteoglycans (HSPGs) have long been thought to serve as low-affinity receptors for HGF (22). While several studies have implicated cell surface proteoglycans in HGF-mediated mitogenic effects, their role in its morphogenic activity, in particular renal morphogenesis, has not been explored.

HSPGs are cell surface proteins that primarily belong to two families of molecules, the syndecans and the glypicans (Gpcs). Six members of the Gpc family (Gpc1 to Gpc6) have been identified in mammals, and two have been identified in Drosophila melanogaster (9). Gpc expression is differentially regulated during kidney development. Gpc5 is present during early stages of development, but it is markedly reduced in the adult kidney (31). Gpc3 is expressed most heavily during embryogenesis (including high levels of expression in the ureteric bud [11]) and is then down-regulated in the adult kidney (24, 32). Gpc4 expression is low during early tubule formation (E13) but is up-regulated in mature tubules (41). Thus, it is possible that specific Gpcs are required for heparin-binding growth factor signaling at defined points during kidney development.

Recently, our lab determined that adult inner medullary collecting duct cells (a-IMCD) express threefold-greater levels of Gpc4 mRNA but twofold less Gpc3 mRNA than embryonic IMCD cells (E18.5 IMCD, hereon called e-IMCD) (15). Gpc1 expression was similar in both cell types. Interestingly, when Gpc4 expression in the a-IMCD cells was diminished by using an antisense to Gpc4, these cells did not undergo branching morphogenesis in response to HGF (17). These observations led us to hypothesize that expression of specific Gpcs might regulate HGF-mediated morphogenesis in renal epithelial cells.

MATERIALS AND METHODS

Cell culture.

Immortalized a-IMCD (mIMCD-3 cells) were originally established by Rauchman et al. (25). e-IMCD from wild-type and Gpc3^−/ mice (Gpc3^−/ cells) were grown in Dulbecco's modified Eagle's medium (DMEM)-F12 (Gibco BRL) medium supplemented with 10% fetal calf serum (Gibco BRL). e-IMCD and Gpc3^−/ cells have been previously established and characterized by Grisaru et al. (11). All chemicals were purchased from Sigma Chemical Company unless otherwise mentioned.

Cell migration.

Cell migration assays were performed using collagen-coated trans-well filters. Briefly, 1.5 × 10⁴ cells suspended in DMEM-F12 were added to the top of each well, and the bottom wells were filled with DMEM-F12 with or without HGF (40 ng/ml) or epidermal growth factor (EGF; 20 ng/ml). Four hours later, the filters were removed and stained with Diff-Quik (Baxter Healthcare), and cells remaining on the top of the membrane were mechanically scraped off. Cells that had passed through the pores were counted to determine the number of cells per square millimeter of membrane. Each well represents an n of 1, and each experiment was repeated four times. P values were determined using Student's t test.

Cell proliferation.

An equal number of cells was plated into 96-well plates. Cells were serum starved overnight followed by stimulation with HGF or EGF for 48 h. At the end of 48 h, bromodeoxyuridine (Roche Diagnostics) incorporation into the cells was used to determine their proliferation index. Assays were performed as per the manufacturer's instructions.

Branching morphogenesis and tubulogenesis.

Cells were trypsinized, and isolated cells were resuspended in type I collagen and cultured in the presence or absence of the desired growth factor as previously described (15). After a 24-h period of incubation at 37°C, 40 single cells were scored for the number of branching tubular processes per cell. Each well represented an n of 1, and each experiment was repeated at least three times. For long-term tubulogenesis assays, cells were suspended in a 70:30 mixture of collagen and growth factor-reduced Matrigel (from BD Biosciences) as previously described (23).

Retroviral transduction.

The retroviral vector pLPXSHD², containing the Gpc1 or Gpc4 gene, was kindly provided by A. Karumanchi, Boston, Mass. The Gpc-3 cDNA (provided by N. D. Rosenblum) was subcloned into the pLPCX retroviral vector (Clontech). Virus was produced, and cells were transduced with the respective viruses, as described earlier (15, 17).

Immunocytochemistry.

To determine cell surface expression of Gpc3 and Gpc4, cells were cytocentrifuged on a slide and stained using the Vector Elite ABC staining kit (Vector Labs). Cells were fixed in 1% formalin, and staining was performed per the manufacturer's instructions. Cells were stained with antibody to Gpc3 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif.) or Gpc4 (1:5,000; a kind gift from M. Ford-Perris) (10) or preimmune serum (control). The slides were then washed in Tris-buffered saline (TBS), incubated in secondary antibody followed by incubation with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (DAKO Labs), and monitored for reaction color development. All slides were developed simultaneously. Quantification was performed using the NIH Image software by determining the individual cell staining intensity for 80 cells. The minimal background staining observed in the respective antibody control groups was subtracted from the values obtained from cells stained for either Gpc3 or Gpc4.

Immunocytochemistry for nuclear Erk detection.

Phosphorylated Erk in the nucleus was detected as described earlier (14). Briefly, cells were serum starved overnight and then stimulated with HGF for 0 to 240 min. Cells were then fixed in 4% paraformaldehyde, blocked in 5% goat serum, and stained at 4°C overnight with polyclonal anti-phospho Erk (1:500) antibody. Fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (Molecular Probes) was used for visualization. Images were acquired using a Zeiss epifluorescence confocal microscope.

Protein analysis.

Serum-starved cells were stimulated with either HGF (40 ng/ml; Sigma) or vehicle (phosphate-buffered saline) for 10 to 240 min at 37°C. Cell lysates prepared in modified RIPA buffer (1% Triton X-100, 1% deoxycholate, 20 mM Tris, 0.16 M NaCl, 1 mM EGTA, 1 mM EDTA, 15 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5 μg of leupeptin/ml, and 0.5 μg of pepstatin A/ml) were subjected to Western blot analysis as described previously (16). Activated ERK1/2 and Akt were probed with an antibody to phospho-ERK1/2 (New England BioLabs) and phospho-Akt (Santa Cruz Biotechnology). To ensure equal loading, membranes were reprobed for total extracellular signal-regulated kinase (ERK) or Akt.

c-met receptor activation.

For examining activation of the HGF receptor c-met, lysates were prepared as described above and 1 mg of protein was immunoprecipitated with anti-c-met antibody (Santa Cruz Biotechnology) (15). Protein was Western blotted with antiphosphotyrosine (α-pTyr) antibody (Upstate Biotechnology). To ensure equal loading, blots were reprobed for c-met.

RESULTS

e-IMCD cells have lower cell surface Gpc4 expression.

Consistent with differential Gpc3 and Gpc4 mRNA levels (15), cytopreps revealed a twofold-higher Gpc3 expression in e-IMCD cells (a-IMCD, 77 ± 3.73; e-IMCD, 143 ± 3.22 arbitrary units/cell) (Fig. 1A) and a threefold-higher Gpc4 expression in a-IMCD cells (a-IMCD, 157 ± 4; e-IMCD, 55 ± 3.63 arbitrary units/cell) (Fig. 1B). Similar observations have been made by others in developing kidney cells by in situ and microarray data analysis (11, 12, 32, 41).

FIG. 1.

Gpc3 and Gpc4 expression in embryonic and adult IMCD cells. Cytopreps from cells were stained for surface expression of Gpc3 (A) or Gpc4 (B). Staining density of 80 individual cells (visualized at higher magnification) was quantitated using NIH Image software. Values were corrected for any background staining.

e-IMCD cells migrate and proliferate normally in response to HGF but fail to undergo tubulogenesis.

The process of in vitro tubulogenesis begins with single cell process extension followed by cell-cell contact and eventually multicellular tubule formation. We therefore examined HGF-mediated single cell process formation in a three-dimensional (3D) collagen gel assay. After 24 h, HGF treatment induced process formation in the a-IMCD cells but not in e-IMCD cells (Fig. 2A and C and quantitation in panel E). This failure of HGF to induce branching morphogenesis was not rescued by increasing the concentration of HGF to 400 ng/ml (data not shown). However, EGF, a non-heparin-binding growth factor, induced significant branching process formation in both a-IMCD and e-IMCD cells (Fig. 2A, C, and E). The basal level of branching in e-IMCD cells was lower than in a-IMCD cells, but the fold increase with EGF was comparable. After 8 days of culture in a collagen-Matrigel mix, a-IMCD cells formed multicellular tubules in the presence of both HGF and EGF (Fig. 2B), whereas e-IMCD cells formed tubular structures only in the presence of EGF (Fig. 2D) and e-IMCD cells did so only with EGF (Fig. 2D). In response to HGF, e-IMCD cells formed either cystic structures or short stubby tubule-like structures. Serum, like EGF, was capable of promoting tubulogenesis in both a-IMCD and e-IMCD cells (data not shown). These data demonstrate that the e-IMCD cells are capable of undergoing tubulogenesis and suggest that the lack of such a response to HGF might be due to a change in the expression of specific Gpcs.

FIG. 2.

e-IMCD cells do not undergo tubulogenesis to HGF. a-IMCD and e-IMCD cells were cultured in a collagen I matrix for 24 h (A and C) or collagen mixed with Matrigel for 8 days (B and D) with or without HGF or EGF. Representative fields were photographed at 20× magnification. (E) Quantitation of the average number of processes induced by HGF or EGF. Data were pooled from three independent experiments. *, P < 0.05 versus control.

HGF can also induce epithelial cells to migrate and proliferate (7, 16). In contrast to single cell process formation and tubulogenesis, a-IMCD and e-IMCD cells migrated comparably towards both HGF and EGF (Fig. 3A). Additionally, both adult and e-IMCD cells showed a modest mitogenic response to HGF (Fig. 3B). Taken together, these data demonstrate that both a-IMCD and e-IMCD cells are capable of undergoing tubulogenesis, but only a-IMCD cells respond to HGF. The specificity of this effect suggests that signaling downstream of the c-met receptor may be altered in e-IMCD cells.

FIG. 3.

Growth factor-mediated cell migration and proliferation. (A) a-IMCD or e-IMCD cell migration was assayed using trans-well filters, and data were pooled from four different experiments. *, P < 0.05 versus control. (B) HGF-induced cell proliferation was measured in a-IMCD and e-IMCD cells, and data were pooled from four different experiments. *, P < 0.05 versus control. (C) c-met was immunoprecipitated and Western blotted for phosphotyrosine from cells stimulated with HGF for 10 min. (D and E) Lysates from a-IMCD and e-IMCD cells with or without HGF treatment for 10 min were blotted for phosphorylated ERK1/2 or phosphorylated Akt. Blots were reprobed for total ERK or Akt.

Sustained, but not initial, signaling by c-met is inhibited in e-IMCD cells.

HGF mediates branching morphogenesis in renal epithelial cells by activating c-met, which then sets into motion a series of downstream signaling pathways. Our laboratory and others have shown that HGF-induced cell migration and branching morphogenesis are dependent on activation of both the Erk and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. Inhibition of either pathway blocks these effects in multiple epithelial cell lines (7, 8, 16, 18). HGF-induced c-met activation and the downstream Erk and PI3-K signaling were therefore compared in a-IMCD and e-IMCD cells. Initial activation of the c-met receptor (as judged by antiphosphotyrosine blotting of the immunoprecipitated receptor [Fig. 3C]), of the Erk phosphorylation (as judged by phospho-specific antibody to Erk [Fig. 3D]), and of the PI-3K pathway (as judged by phospho-specific antibody to the downstream kinase Akt [Fig. 3E]) were all indistinguishable in the a-IMCD and e-IMCD cells.

Recent studies suggest that the phenotypic response to a growth factor can be regulated by the duration of signaling following receptor activation. Sustained ERK and PI3-K activation may be important for certain cellular responses in yeast, for integrin expression, and for branching process formation (19, 27, 38). The duration of c-met, ERK, and PI3-K activation in the a-IMCD and e-IMCD cells was therefore examined after HGF stimulation for 10 to 240 min.

Both the adult and e-IMCD cells showed a similar pattern of c-met receptor activation, with peak phosphorylation at 10 min and sustained phosphorylation for up to 240 min at ∼50% of peak levels (Fig. 4A). The pattern of Akt activation too was similar (Fig. 4B, quantitation in C), although densitometric analysis of five experiments revealed a statistically higher initial Akt activation in e-IMCD cells and then a faster return to baseline (Fig. 4C). In contrast, ERK activation was sustained in a-IMCD cells but was only transient in e-IMCD cells (Fig. 4D, densitometric analysis in E). Erk activation in e-IMCD cells was significantly lower at 60 and 120 min and returned to the baseline by 240 min.

FIG. 4.

Analysis of HGF-mediated signaling. (A to E) Lysates from cells treated with HGF for 10 to 240 min were immunoprecipitated for c-met and immunoblotted with anti-pTyr (A) or blotted for phosphorylated Akt or ERK (B and D). Data were normalized to total Akt (C) or total ERK (E). Data were pooled from five separate experiments. (F) a-IMCD cells were suspended in collagen plus HGF, with or without UO126 added 30 to 120 min after adding HGF. Process formation was quantitated at 24 h. Data were pooled from two independent experiments. *, P < 0.05 versus HGF alone.

To test the possibility that loss of sustained ERK activation is sufficient to prevent branching morphogenesis, we examined HGF-mediated process formation in a-IMCD cells with and without the MEK inhibitor UO126. Our lab and others have previously shown that adding UO126 simultaneously with HGF in a 3D collagen gel leads to complete inhibition of process formation in the a-IMCD cells (16). In the present study, we added 10 μM UO126 inhibitor at 30, 60, or 120 min (when Erk is still activated in a-IMCD cells) (Fig. 4F) after adding HGF to the a-IMCD cells suspended in collagen gel. Addition of UO126, even 2 h after adding HGF, significantly inhibited process formation at 24 h. UO126 was not removed after being added to the culture.

Thus, it appears that the initiation of tubulogenesis requires sustained ERK activation (as seen in a-IMCD cells), whereas transient ERK activation along with PI3-K activation may be sufficient to induce the motogenic and mitogenic responses seen in both a-IMCD and e-IMCD cells. These results suggested the novel idea that the expression of specific Gpcs may modulate the duration of heparin-binding growth factor-mediated downstream signaling.

Overexpression of Gpc4 in e-IMCD cells rescues the morphogenic effect of HGF.

Our group has previously demonstrated that expression of the antisense to Gpc4 results in loss of HGF-induced branching morphogenesis in a-IMCD cells (17). In the present study, HGF induced migration and proliferation but not branching morphogenesis in e-IMCD cells, which express high levels of Gpc3 but significantly lower levels of Gpc4 compared to a-IMCD cells. Taken together, these data support the hypothesis that the levels of Gpc4 expression may specifically modulate the cellular response to HGF. To test this, Gpc4 was overexpressed in e-IMCD cells by using a retroviral vector. Immunostaining of the cells revealed that e-IMCD cells overexpressing Gpc4 (hereon called e-IMCD-Gpc4) exhibited similar levels of Gpc4 on the surface as a-IMCD cells (e-IMCD, 55 ± 3.63; a-IMCD, 163 ± 3.84; e-IMCD-Gpc4, 142 ± 2.98 arbitrary units/cell) (Fig. 5A). In order to avoid any clonal specific responses, we utilized pooled retrovirally transduced cells rather than isolated single clones of cells.

FIG.5.

Overexpression of Gpc4 in e-IMCD cells rescues tubulogenesis. (A) Cytopreps were immunostained for surface expression of Gpc4. (B) Branching morphogenesis was assessed in e-IMCD and e-IMCD-Gpc4 cells grown in collagen for 24 h with and without HGF or EGF. (C) Cells were grown in a collagen-Matrigel mixture for 8 days. (D) Lysates from cells stimulated with HGF for 10 to 240 min were blotted for phospho-ERK1/2 and reprobed for total ERK. (E) Quantitative analysis of four separate experiments, as in panel D. *, P < 0.05 versus control. (F) Cells expressing high levels of Gpc4 showed sustained nuclear phospho-Erk. Cells were serum starved overnight and then stimulated with 40 ng of HGF/ml for 0, 10, or 240 min. Phosphorylated Erk1/2 was visualized under a confocal microscope. Representative fields were photographed at 40× magnification. (G and H) Branching process formation was assessed in cells lacking Gpc3 or cells overexpressing Gpc3 or Gpc4 after 24 h of treatment with or without HGF or EGF. *, P < 0.05 versus control.

e-IMCD-Gpc4 cells were then assayed for single cell process formation and tubulogenesis. As with parental e-IMCD cells (Fig. 2), cells expressing the vector alone underwent process formation with EGF but not HGF, while e-IMCD-Gpc4 cells responded to both ligands (Fig. 5B). Further, these cells now formed multicellular tubular structures in a fashion similar to the a-IMCD cells (Fig. 5C, right panel). Further, examination of ERK signaling revealed higher levels of ERK activation at all time points after 30 min in e-IMCD-Gpc4 cells (Fig. 5D, quantitation in E). Importantly, examination of nuclear Erk at the single-cell level using confocal microscopy revealed sustained levels of phosphorylated nuclear Erk in a-IMCD cells, whereas it was not detectable in e-IMCD cells after 4 h. However, e-IMCD cells overexpressing Gpc4 showed sustained nuclear phospho-Erk (Fig. 5F) similar to the a-IMCD cells. Thus, overexpression of Gpc4 led to sustained HGF-stimulated ERK activation and rescued HGF-stimulated tubulogenesis.

To determine if this effect was specific for Gpc4 or was simply due to a relatively high level of any Gpc expression, the morphogenic response was evaluated in e-IMCD cells lacking Gpc3 (Gpc3^−/) or overexpressing either Gpc3 or Gpc4. Overexpression of Gpc4 (Fig. 5H) but not of Gpc3 (Fig. 5G) led to branching morphogenesis and tubulogenesis in response to HGF. Our lab has previously demonstrated that Gpc3^−/ cells undergo branching morphogenesis in response to EGF and express low levels of Gpc4 compared to the a-IMCD cells (15). Of note, overexpression of Gpc1 also failed to rescue the tubulogenic response to HGF in these cells (data not shown). Taken together, these data indicate that HGF-mediated branching morphogenesis specifically requires surface expression of Gpc4 and that Gpc4 is capable of modulating the kinetics of HGF-induced ERK activation.

DISCUSSION

It has been previously demonstrated that global loss of heparan sulfate (HS) expression prevents the HGF-stimulated mitogenic response in individual cells (5, 6, 13, 33, 39) and HGF-stimulated branching of the embryonic kidney in culture (4). Several ligands are known to utilize the HSs decorating the core proteins (glypicans or syndecans) for their optimal biological function (35). To our knowledge, there are no reports in the literature correlating loss of HGF-induced responses to a specific HSPG. Utilizing a-IMCD and e-IMCD cells, we demonstrated that Gpc3 and Gpc4 are differentially expressed in cells derived from embryonic and adult kidney and that, specifically, a high level of Gpc4 expression correlates with sustained ERK activation and tubulogenic responses to HGF. The fact that high levels of Gpc4 are not required to initiate proliferative or motogenic responses in e-IMCD cells suggests that other HSPGs may be sufficient to regulate those responses.

Most known HSPG functions are thought to depend on the interaction between HS side chains and the protein ligands. Interaction of heparin-binding growth factors with HS can increase their stability and activity (30). Further, HSPGs may function as reservoirs for growth factors for their sustained released to interact with their high-affinity receptors (40) or target them to different intracellular compartments (26). In addition, the discrete sulfation pattern on the HS side chains may impart a certain degree of specificity to the ligand-HS interaction. Specific glycosaminoglycan modifications are critical in regulating the specific patterning process in Drosophila (34). In the context of the present study, this would suggest that the difference(s) in the HS side chains of Gpc1, Gpc3, and Gpc4 could potentially regulate the manner and/or site of HGF-c-met interactions. Interestingly, our group has previously demonstrated that endostatin (the proteolytic fragment of collagen XVIII) binds e-IMCD and a-IMCD cells via Gpcs that act as its functional coreceptor. In the same study, we demonstrated that cells lacking Gpc3 (Gpc3^−/) but expressing low levels of Gpc4 showed 50% less binding of endostatin, resulting in the lack of response to endostatin. Importantly, overexpressing either Gpc1 or Gpc4 rescued the binding and the biological activity of endostatin in those cells (15, 17). However, in the present study, the lack of tubulogenic response to HGF in e-IMCD cells could be rescued only by overexpressing Gpc4 and not by Gpc1 or Gpc3.

How exactly Gpc expression modulates Erk activation by HGF is not clear and will require further investigation. However, a few possibilities can be envisioned. Recently, ErbB receptors have been shown to discriminate between multiple ligands by using differential phosphorylation sites, leading to differential signaling (36, 37). In our studies, the overall pattern of c-met receptor tyrosine phosphorylation appeared to be unaffected by Gpc4 expression. However, we cannot rule out the possibility of differential phosphorylation of specific tyrosine residues on the c-met receptor in cells expressing high versus low levels of Gpc4. This in turn could alter the association of certain adaptor proteins with c-met that is required for mitogen-activated protein kinase (MAPK) activation, such as Gab or Grb2.

Alternatively, the presence of high levels of Gpc4 may target the HGF/c-met complex to specific compartments of the cell (as is the case with fibroblast growth factor [26]), thereby modulating the duration of signaling pathway activation by regulating the availability of interacting partners. In this regard, we have recently found that transient ERK activation at the membrane is necessary for HGF-stimulated migration and morphogenesis and that this membrane localization is mediated by the association of the MAPK proteins Raf, MEK, and ERK with the focal adhesion protein paxillin during the initial 30 min of c-met activation (14). In contrast, the sustained phase of ERK activation appears to be restricted to the nucleus and is therefore likely to be important for the regulation of transcription factors such as Ets1 and STAT-3, which are required for matrix-metalloproteinase induction and activation, process formation, and eventual tubulogenesis (19-21, 27). Indeed, IMCD cells expressing high levels of Gpc4 depict sustained levels of phosphorylated nuclear Erk compared to e-IMCD cells, which have lower levels of Gpc4. Thus, it is possible that the transient ERK activation in e-IMCD cells is sufficient for cell migration but that both transient membrane-associated ERK activation and sustained nuclear ERK activation (regulating transcriptional events) are required for the tubulogenic response seen in Gpc4-expressing IMCD cells.

In summary, the level of Gpc4 in renal IMCD cells regulates the duration of MAPK signaling in response to HGF and determines whether HGF acts only as a chemoattractant and mitogen or as a tubulogenic factor. Based on our data, one can speculate that early during kidney development, when Gpc4 expression is low, HGF may act as a mitogenic and/or motogenic stimulus, but it will not play a significant role in branching morphogenesis. This finding is consistent with the results seen in the c-met and HGF knockout mice, in which no abnormalities in ureteric bud branching were seen up to E12.5. However, in late embryonic and adult kidney (when Gpc4 expression increases), HGF is likely to exert a significant morphogenic effect on the tubule, thus potentially regulating the late stages of tubule formation and/or tubule repair following injury.