The metanephros develops via reciprocal inductive interactions between the ureteric bud (UB) and the metanephrogenic mesenchyme (MM) (1,2). Signals from the MM induce UB outgrowth from the nephric duct and its elongation and entrance into the mesenchyme followed by repetitive branching to form the renal collecting system (ureter, pelvis, calyces, and collecting ducts). In turn, emerging UB tips induce surrounding mesenchymal cells to condense, aggregate, undergo mesenchymal-to-epithelial transition, and form nephrons (from the glomerulus to the distal tubule). Therefore, UB branching morphogenesis is critical in determining nephron number, and subtle defects in the efficiency and/or accuracy of this process potentially can have profound effects on the proper development of the metanephric kidney. Decreased nephron endowment is linked to hypertension and eventual progression to chronic renal failure (3,4). In addition, aberrant UB branching morphogenesis causes renal dysplasia, the leading cause of chronic renal failure in human infants.
Genetic inactivation of the renin-angiotensin system (RAS) genes in mice causes abnormalities in the development of the ureter, renal pelvis, and papilla (5–9). Angiotensinogen-, angiotensin-converting enzyme (ACE)-, or angiotensin II (Ang II) type 1 receptor (AT1R)-deficient mice exhibit pelvic dilation (hydronephrosis) and a small papilla mimicking urinary tract obstruction. Elegant studies from Ichikawa’s laboratory have suggested that absence of AT1R signaling in ureteral smooth muscle cells impairs pelvic-ureteral smooth muscle development and peristalsis (9). Mutations in the AT2R gene in mice and humans are associated with increased incidence of lower urinary tract anomalies, including double ureters and vesicoureteral reflux (10). These findings indicate that UB growth and development are a target for Ang II actions.
Work that has performed by several laboratories, including ours, has revealed that the fetal kidney expresses a local RAS. Quantitative analysis of murine Ang II receptors AT1R and AT2R gene expression indicate that AT1R undergo a progressive increase during fetal and neonatal life, whereas AT2R are high in the fetus and decline significantly with maturation (11). Immunolocalization studies demonstrated that AT1R and AT2R are expressed in the UB and its early branches of embryonic day 12 (E12) mouse embryos, whereas angiotensinogen and renin are expressed in the stromal mesenchymal cells that surround the UB branches (12–14). The localization of RAS components in the stroma and UB provided the initial suggestion that paracrine signaling through RAS-AT receptors may regulate the early stages of kidney development. In vitro studies have shown further that UB cells that are isolated from E11.5 mouse embryos express AT1R that are functionally coupled to the mitogen-activated protein kinase (MAPK) pathway (15). UB cells that are seeded in collagen gels form cellular cords and tubular structures, and treatment with Ang II increases the number and the complexity of these cellular branches (12). Ang II–induced UB cell branching is mediated in part via the AT1R because it is abrogated by the specific AT1R antagonist candesartan. However, there is no evidence that Ang II can directly stimulate UB growth and branching in the intact metanephros.
In this work, we tested the hypothesis that Ang II stimulates UB branching morphogenesis in the intact metanephric kidney cultured in vitro. Moreover, given the known interactions between AT1R and EGF receptor (EGFR) signaling pathways in other systems (16,17), we examined the cross-talk between Ang II and the EGFR in Ang II–induced UB branching morphogenesis. The results demonstrate that Ang II, when applied directly on the metanephric organ culture, stimulates the UB branching morphogenesis program. Furthermore, the stimulatory effects of Ang II on metanephric UB branching and tubulogenesis are mediated via an AT1R-EGFR signaling pathway.
Materials and Methods
Immunohistochemistry for AT1R
We previously demonstrated that AT1R protein is expressed in the UB as early as E12 of murine development (12). In this study, we examined the expression of AT1R in mouse kidneys on E14 to assess further its distribution along the UB branches and collecting ducts. Immunostaining was performed by the immunoperoxidase technique using the Vectastain Elite kit (Vector Laboratories, Burlingame, CA) as described previously (12). A polyclonal rabbit AT1R antibody directed against the NH2-terminal domain of the human receptor (identical sequence in the mouse; N-10, sc-1173; Santa Cruz Biotechnology, Santa Cruz, CA) was used at concentration of 1:200. This antibody detects both AT1A and AT1B receptor subtypes. The specificity of immunostaining was assessed by preadsorption of the primary antibody with a ×100 excess of its immunogenic peptide overnight at 4°C.
Cell Immunostaining
For examination of the effect of Ang II on subcellular localization and tyrosine phosphorylation of EGFR in UB cells, UB cells that were grown on coverslips were incubated with medium alone (control), Ang II (10−6 M), or EGF (40 ng/ml) for 30 min. After washings, the cells were incubated with FITC-conjugated anti–phospho-EGFR antibodies (Tyr-1173, sc-12351, Santa Cruz Biotechnology, Santa Cruz, CA; or Tyr-845, 2231, Cell Signaling, Danvers, MA) and propidium iodide to stain the nuclei.
Cell Lysate Extraction, Immunoprecipitation, and Immunoblotting
Mouse UB cells (provided by Dr. Jonathan Barasch, Columbia University, New York, NY) initially were obtained from microdissected ureteric buds of E11.5 mouse embryo that was transgenic for simian virus 40 large T antigen (Immorto-mouse) (18). UB cells were maintained in DMEM/F12 medium supplemented with 10% FBS at 37°C in a humidified 5% CO2 atmosphere. The cells were cultured in serum-free medium for 48 h before experiments. The cells were used at passages 10 to 15. Cell stimulation with Ang II (10−5 to 10−7 M) or EGF (40 ng/ml) was carried out at 37°C in serum-free medium. Western blot analysis was performed as described previously (12). After stimulation, the cells were washed in PBS and homogenized in cold lysis buffer (50 mM Tris/HCl [pH 8.0], 150 mM NaCl, 0.02% sodium azide, 1% Nonidet P-40, and 0.5% deoxycholate) that contained a cocktail of enzyme inhibitors (0.1 mg/ml PMSF, 0.001 mg/ml aprotinin, 0.001 mg/ml leupeptin, and 0.01 mg/ml Na3VO4). The samples were centrifuged for 10 min at 14,000 × g at 4°C, and the supernatants that contained proteins (40 μg/sample) were resolved on 10% Tris-glycine gels and transferred to nitrocellulose membranes.
For immunoprecipitation of endogenous EGFR, 2 mg of cell lysates was incubated with 8 μg of anti-EGFR antibody (SC-03; Santa Cruz) overnight at 4°C. The cells were lysed in a lysis buffer (9803; Cell Signaling) that contained phosphatase (P5726; Sigma) and protease (P8340; Sigma) inhibitor cocktails and PMSF (0.1 mg/ml). The lysates were incubated with protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz; 60 μl of slurry) for 3 h. Samples were centrifuged at 13,000 rpm for 30 s at 4°C, and the beads were washed with lysis buffer, solubilized in sample buffer (7722; Cell Signaling), and subjected to immunoblotting (0.15 mg/lane). The adequacy of transfer was assessed by Ponceau-S staining of the membranes. Nonspecific binding was blocked by incubating the membranes with PBS that contained 0.2% Tween and 3% BSA overnight at 4°C. The membranes were incubated with the following primary antibodies: (1) A polyclonal rabbit antibody directed against phosphorylated Tyr-1173 in the carboxy terminal of EGFR (sc-12351; Santa Cruz); this antibody was used for immunoblotting at a concentration of 1:500; and (2) membranes with immunoprecipitated EGFR were probed with anti-phosphotyrosine mAb (4G10; Upstate Biotechnology, Lake Placid, NY). After three washes in PBS/Tween, the nitrocellulose membrane was exposed to the secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (ECL; Amersham, Pittsburgh, PA). The blots then were exposed to Hyperfilm-ECL films.
In Vitro Tubulogenesis
UB cells that are cultured in gels form processes and branching tubular structures when exposed to various growth factors, providing a convenient experimental system to analyze mechanisms of epithelial branching morphogenesis (19,20). A principal advantage of this approach is the ability to examine the direct effect of a specific factor on UB cell growth that is independent from confounding soluble factors that are released by the mesenchyme. The in vitro tubulogenesis assay was performed as described previously (12). Briefly, the cells were used at passages 10 to 15. The cells were trypsinized and suspended in type I rat-tail collagen (Upstate Biotechnology), 10× DMEM, and 200 mM HEPES (pH 8.0) in 8:1:1 ratio at 150 × 103 cell/ml. Subsequently, the suspension was dispensed in aliquots into a 96-well culture plate (0.1 ml/well). After gelation, 0.1 ml/well DMEM/F12 medium with 0.5% FBS or medium with 0.5% FBS that contained (1) Ang II (10−5 or 10−6 M; Sigma), (2) Ang II and AT1R antagonist candesartan (10−6 M; Sigma), (3) Ang II and EGFR tyrosine kinase inhibitor (AG1478, 0.3 μM; Calbiochem, San Diego, CA), (4) EGF (40 ng/ml; Upstate Biotechnology), alone or combined with AG1478, 0.3 μM, was added to each well on top of the collagen gel. Candesartan and AG1478 were added on top of the gels 30 min before Ang II or EGF were added. After a 24-h incubation period at 37°C and 5% CO2, 40 individual cells/well were scored for the presence of cell processes directly from the plates using an Olympus IX70 inverted phase-contrast microscope (Olympus, Melville, NY), and the average number of processes per cell was calculated. Each condition was set up in triplicate (n = 4 separate experiments). Images were acquired directly from the plates via an Olympus MagnaFire FW camera and processed with Adobe PhotoShop 7.0 (Adobe Systems, Mountain View, CA).
Metanephric Organ Culture
To determine the role of Ang II in early stages of UB growth and development in the intact kidney, we used metanephroi that were isolated from Hoxb7–green fluorescence protein (GFP) transgenic mice (a gift of Dr. Frank Costantini, Columbia University). The kidneys of Hoxb7–GFP transgenic mice express GFP exclusively in the UB (21). This allows continuous monitoring of UB growth in real time. Embryos were dissected aseptically from the surrounding tissues on E11.5, and the metanephroi were isolated. The day when the vaginal plug was observed was considered to be E0.5. GFP-positive metanephroi were photographed immediately after isolation (time 0) and were grown on air-fluid interface on polycarbonate transwell filters (0.5 μm; Corning Costar, Acton, MA) that were inserted into six-well plates that contained DMEM/F12 medium (Life Technologies BRL, Grand Island, NY) alone or in the presence of Ang II (10−5 M) alone or Ang II combined with AG1478 (0.3 μM) for 48 h at 37°C and 5% CO2. The media that did or did not contain Ang II, candesartan, or AG1478 were changed every 24 h. Our recent findings demonstrate that 10−5 M Ang II stimulates cell process formation in UB cells (12). Importantly, endogenous Ang II contents are higher in the embryonic than in the adult mouse kidney (22). Therefore, higher kidney tissue Ang II concentrations are physiologically relevant during embryonic kidney development. Therefore, we used 10−5 M Ang II in this study. A higher dose of Ang II was chosen also because Ang II has a short half-life as a result of degradation by tissue proteases (see below). AG1478 (0.3 μM) and dominant-negative EGFR mutant have been shown to inhibit Ang II–induced EGFR tyrosine phosphorylation in vascular smooth muscle cells (16). Therefore, this concentration of AG was used in our study. Considering the variability in the initial kidney size and in UB branching patterns among littermates (23), the effect of treatments on UB branching was studied in the kidneys that were obtained from the same fetus (left kidney was incubated with medium and right kidney with Ang II, or left kidney was incubated with Ang II+AG1478 and right kidney with Ang II). Sequential images of branching UB were obtained at 24 and 48 h of incubation directly from the inserts via an Olympus IX70 inverted phase-contrast microscope and Olympus MagnaFire FW camera and processed with Adobe PhotoShop 7.0. Quantitative assessment of UB branching in each treatment group was performed by counting the number of UB tips and branch points (BP) at time 0 and at 24 and 48 h.
For examination of the role of AT1R in UB branching, E11.5 metanephroi that were obtained from wild-type C57B/6J mice were grown as described above in the presence of medium alone (control) or medium + AT1R antagonist candesartan (10−6 M) for 48 h. Candesartan at 10−6 M blocks predominantly AT1R (24). Also, 10−6 M candesartan abrogated Ang II–induced UB cell branching in collagen matrix gels (12). Therefore, this concentration of candesartan was used in our study. After 48 h in culture, kidney explants were stained with anti-pancytokeratin antibody (C2562, 1:200; Sigma) to label the UB as described above, and the number of UB tips and BP was compared between the two treatment groups. To determine the degradation rate of exogenous Ang II present in the culture media, we measured Ang II contents in the media at time 0 and at 6, 12, and 24 h by RIA with the use of rabbit anti-Ang II antiserum as described (25).
Statistical Analyses
Differences among the treatment groups in process-per-cell number were analyzed by one-way ANOVA. Differences in EGFR phosphorylation were analyzed by Mann-Whitney rank sum test. Differences in the number of UB tips and BP in Ang II–, candesartan-, or AG1478-treated metanephroi were analyzed by t test. P < 0.05 was considered statistically significant.
Results
Expression of AT1R in the UB Tree In Vivo
On E14, AT1 immunoreactivity is abundant in the UB, UB stalks and branches, and ampullae (Figure 1A). AT1R is expressed on both luminal and basolateral aspects of UB branches (Figure 1B). Glomeruli and the surrounding mesenchyme also express AT1R, albeit at relatively lower levels than the UB branches (Figure 1A). Control sections that were incubated with the primary antibody preadsorbed with its immunogen showed a marked decrease in staining (Figure 1C).
Effect of Ang II on UB Branching Morphogenesis in the Ex Vivo Cultured Intact Kidney
In a previous study, we demonstrated that Ang II can initiate a branching morphogenesis-like program in UB cells (12). In this study, we examined whether Ang II can induce similar effects in a physiologically more relevant system, in which mesenchymal–epithelial interactions are still maintained. Metanephric kidneys that are grown ex vivo proceed through the stages of UB branching and nephrogenesis over several days and closely recapitulate kidney development in vivo (23). To monitor the effects of Ang II on the sequential changes in UB morphogenetic events, we used Hoxb7-GFP transgenic mice (26), which express GFP exclusively in the UB and its derived epithelial structures.
Quantitative analysis of UB branching morphogenesis included counting the number of UB tips and BP. A tip is defined as the distal end of terminal UB branch. BP was defined as the point at which three or more segments connect. To measure the time-dependent changes in UB branching, we analyzed three images of each kidney: Immediately after dissection (time 0) and 24 and 48 h thereafter (Figure 2). The number of UB tips and BP increased progressively during the observation period. As described previously (26), three modes of branching were observed: Bifid, trifid, and lateral. Bifid branching results in two new segments joined to the parental segment at a single point. Trifid branching involved three new segments joined to the parental segment. Lateral branching involved the outgrowth of a new segment from the side of an existing segment.
Ang II increased the number of UB tips and BP at 24 h (tips: 24.3 ± 1.1 versus 18.3 ± 0.7, P < 0.01; BP: 14.4 ± 0.6 versus 11.7 ± 0.6, P < 0.01) and 48 h (tips: 30.2 ± 1.3 versus 22.9 ± 0.8, P < 0.01; BP: 21.3 ± 0.9 versus 15.7 ± 0.6, P < 0.01) compared with control (Figure 2). During the 24-h incubation period, Ang II contents in the culture media (fmol/ml) decreased by approximately two-fold (time 0 2.4 ± 0.27 × 106, at 6 h 1.9 ± 0.19 × 106, 12 h 1.7 ± 0.23 × 106, 24 h 1.1 ± 0.28 × 106) presumably as a result of internalization of Ang II and/or its degradation by tissue proteases.
Role of Endogenous Ang II/AT1R
To examine the role of endogenous Ang II and AT1R in UB branching in the metanephros, we used the AT1R antagonist candesartan. Treatment of E11.5 metanephroi with candesartan (10−6 M) for 48 h decreased the number of UB tips and BP compared with control (tips: 40 ± 2 versus 52 ± 2, P < 0.01; BP: 29 ± 2 versus 38 ± 2, P < 0.01; n = 11/treatment group; Figure 3). These findings are consistent with the results that were obtained in UB cells demonstrating that Ang II–induced effects on UB cell branching are mediated in part via AT1R (12). Collectively, these findings indicate that the stimulatory effect of Ang II on branching of UB cells that are cultured in three-dimensional gels is a physiologically relevant event because it can be recapitulated in the intact organ culture.
Role of EGFR
We next examined the role of EGFR in Ang II–induced UB branching morphogenesis in metanephric organ culture. Treatment with Ang II+AG1478 was associated with a significantly lower number of UB tips and BP at 24 h (tips: 17.2 ± 0.9 versus 24.5 ± 1.2, P < 0.01; BP: 12.5 ± 0.6 versus 18.5 ± 0.8, P < 0.01) and 48 h (tips: 24.4 ± 1.1 versus 34.8 ± 2.2, P < 0.01; BP: 15.8 ± 0.8 versus 24.1 ± 1.1, P < 0.01) compared with Ang II alone (Figure 4). These findings are consistent with the results that were obtained in UB cells (see below) demonstrating that tyrosine phosphorylation of EGFR is a key event in Ang II–mediated signaling to stimulate UB branching.
Effect of Ang II on UB Cell Branching In Vitro: Role of EGFR
To determine the functional importance of tyrosine phosphorylation of EGFR elicited by Ang II in UB cell branching, we examined the effect of the EGFR-specific tyrosine kinase inhibitor AG1478 on cell process formation in Ang II–treated UB cells that were cultured in three-dimensional collagen gels. Figure 5, A through F, shows phase-contrast photographs depicting the effects of Ang II and EGF (positive control) on cell process formation and branching of UB cells 24 h after cells were plated into collagen gels. Ang II or EGF caused extensive changes in cell process formation. In the presence of AG1478, Ang II–or EGF-treated cells formed fewer and shorter branches as compared with cells that were exposed to Ang II or EGF alone (Figure 5).
Quantitative analysis revealed that Ang II or EGF increased the number of processes per cell compared with untreated control (Figure 5G). The data are presented as percentage of control (media + 0.5% FCS = 100%). EGF induced a 2.7-fold increase in the number of processes (270 ± 27 versus 100 ± 16%; P < 0.001; n = 5 experiments). The effects of EGF on UB cells were completely prevented by pretreatment with AG1478 (59 ± 11 versus 270 ± 27%; P < 0.001). At 10−5 and 10−6 M, Ang II increased the number of processes compared with control (194 ± 28 and 185 ± 28 versus 100 ± 16%, respectively; P < 0.05). These effects were abrogated by pretreatment with either AG1478 (Ang II 10−5 M: 71 ± 12 versus 194 ± 28%, P < 0.01; Ang II 10−6 M: 47 ± 11 versus 185 ± 28%, P < 0.01) or the AT1R antagonist candesartan (Ang II 10−5 M: 72 ± 11 versus 194 ± 28%, P < 0.01; Ang II 10−6 M: 73 ± 14 versus 185 ± 28%, P < 0.05). AG1478 alone did not have any effect on UB cell branching compared with control (90 ± 15 versus 100 ± 16%).
Effect of Ang II on Tyrosine Phosphorylation of EGFR in UB Cells
To determine the role of EGFR in Ang II signal transduction, we first investigated the ability of Ang II to induce tyrosine phosphorylation of EGFR in UB cells. We used antibody directed against phosphorylated Tyr-1173 of the carboxy terminal of EGFR. The carboxy terminal tyrosine residue Tyr-1173 on EGFR is the major site of autophosphorylation, which occurs as a result of EGF binding (27). In addition, we used monoclonal anti-phosphotyrosine antibody. Positive controls included EGF-treated UB cells and cell extracts from EGF-stimulated A431 human carcinoma cells that contained highly phosphorylated EGFR. As expected, EGF stimulated a robust five-fold increase in EGFR tyrosine phosphorylation (Figure 6). In comparison, Ang II stimulated a 1.64-fold increase in tyrosine phosphorylation of endogenous EGFR compared with control (Ang II: 100 ± 12 versus 164 ± 15, P < 0.05; EGF: 100 ± 12 versus 522 ± 45, P < 0.001; n = 3; Figure 6, A, C, and E). In addition, Ang II caused a dose-dependent increase in tyrosine phosphorylation of EGFR during immunoblotting with anti-phosphotyrosine mAb (Figure 6, B and D). These results indicate that Ang II activates the tyrosine phosphorylation of the EGFR in UB cells.
We next investigated the effect of Ang II on the nuclear localization of the phosphorylated EGFR in UB cells using anti–phosphotyrosine-EGFR antibodies directed against two different phosphotyrosine residues in carboxyl terminal of EGFR, Tyr-1173 and Tyr-845. Tyr-845 resides in the activation loop of the EGFR kinase domain (16). Phosphorylation of Tyr-845 stabilizes the activation loop and is required for the mitogenic function of the EGFR (28). Treatment of UB cells with Ang II or EGF caused an increase in tyrosine phosphorylation of the nuclear EGFR (Figure 6F). Nuclear EGFR may act as transcription factor to activate promitogenic genes (29).
Discussion
The findings of this study demonstrate that Ang II stimulates UB branching morphogenesis in the intact metanephric kidney cultured in vitro and that tyrosine phosphorylation of the EGFR is a critical step in the signal transduction pathway downstream of AT1R leading to UB branching. Genetic inactivation of the RAS genes in mice (angiotensinogen, renin, ACE, and AT1R) causes thinning of the medulla, hypoplastic papilla, and hydronephrosis (5–9). Also, homozygous mutation of AT2R leads to ectopic UB budding, duplicated collecting system, and hydronephrosis (10). It is currently believed that the small papilla and hydronephrosis in RAS-mutant mice are the result of defective formation and function of the smooth muscle layer in the pelvic and ureteral wall, mimicking the findings seen in urinary obstruction (22). However, to date, the hypothesis that Ang II can stimulate UB growth and branching directly and therefore promote formation of the collecting system has not been tested rigorously. We previously provided two compelling lines of evidence to support this hypothesis. First, angiotensinogen and renin are expressed around the UB and its main branches, whereas AT receptors are expressed on the basolateral and lumenal aspects of the UB branches. Second, Ang II acting via AT1R stimulates branching morphogenesis in UB cells that are grown in three-dimensional collagen matrix gels (12).
These findings extend our previous data by showing that Ang II stimulates the growth of the metanephric explant as a result of enhanced UB branching and division into daughter tips. The mechanisms by which Ang II mediates its growth-promoting effects on UB branching are not yet known. However, there are several possibilities. By stimulating the Gq-coupled AT1R, Ang II activates phospholipase Cβ (PLCβ) and inositol triphosphate production, ultimately leading to increases in intracellular calcium and protein kinase C activation (30). Activation of the extracellular signal–regulated kinase–MAPK pathway by protein kinase C stimulates transcription of cell-cycle progression genes, such as cyclin D1, through activation of the transcription factor AP-1 (31). In another pathway, AT1R activates intracellular tyrosine kinases, such as Src, which in turn activate the EGFR in a ligand-independent manner (32). Alternatively, Ang II may stimulate the proteolytic cleavage of pro–heparin-binding epidermal growth factor (HBEGF) at the cell membrane (33).
In this study, we investigated the contribution of EGFR activation to Ang II–induced stimulation of UB branching. EGFR is expressed in renal collecting ducts (34), and its stimulation enhances branching morphogenesis in murine inner medullary collecting duct cells in vitro (15,35). EGFR-mutant mice have abnormal collecting ducts (36). It is interesting that reduced EGF mRNA levels are observed in the renal papilla of angiotensinogen and AT1 null mice (37). In addition to EGF and EGF-like ligands (HBEGF or amphiregulin), EGFR is activated by G protein–coupled receptors, including AT1R. For example, activation of AT1R can induce tyrosine phosphorylation and activation of the EGFR in cultured aortic smooth muscle and COS-7 cells (16,17). Consistent with these findings, we report here that Ang II enhances tyrosine phosphorylation of EGFR in UB cells and that inhibition of EGFR tyrosine kinase activity abrogates Ang II–induced branching in UB cells that are grown in three-dimensional gels. Of course, a more important question is whether the AT1R–EGFR signaling pathway contributes to the growth-promoting effects of Ang II in vivo. To address this question, we performed a real-time examination of UB growth in metanephric explants that expressed GFP in the UB and its derivatives. Exogenously added Ang II enhanced UB branching at 24 and 48 h. Furthermore, pretreatment of metanephric explants with an EGFR tyrosine kinase inhibitor prevented Ang II–induced UB branching. Collectively, these findings support the hypothesis that cooperation of AT1R and EGFR signaling promotes the growth and development of the renal collecting system.
The signaling events that link Ang II receptors to EGFR transactivation and UB branching morphogenesis remain to be determined. Several signaling pathways, including MAPK and phosphatidylinositol-3 kinase (PI3K)/Akt, have been shown to mediate the effects of AT1 on cell proliferation and hypertrophy in vascular smooth muscle, inner medullary collecting duct cells, and renal mesangial cells (15,38,39). In this regard, pharmacologic inhibition of extracellular signal–regulated kinase 1/2 or PI3K attenuates EGF-induced renal epithelial morphogenesis in vitro (15). Furthermore, inhibition of PI3K blocks global cell-line–derived neurotrophic factor (GDNF)—dependent UB branching in the metanephric kidney (40). Therefore, one of the possible mechanisms that lead to Ang II–stimulated EGFR signaling may involve EGFR-mediated stimulation of MAP kinase and PI3K/Akt pathways. It is interesting that EGF induces phosphorylation of AT1 and leads to formation of a multireceptor complex that contains AT1 and activated EGFR. Phosphorylation of AT1 is prevented by AG1478 (41). These findings suggest that cross-talk between the RAS and EGFR is bidirectional. A schematic summary of potential signaling pathways that are involved in AT1R-EGFR cross-talk in UB branching is presented in Figure 7.
Conclusion
Our study demonstrates that UB-derived epithelial cells express AT1R and that Ang II stimulates UB branching morphogenesis, a process that depends on tyrosine phosphorylation of the EGFR. Cooperation of tyrosine kinase and G protein–coupled receptor therefore is important in the control of normal kidney development.
;)
Figure 1: Immunolocalization of angiotensin II (Ang II) type 1 receptor (AT1R) protein in the fetal kidney on embryonic day 14 (E14). AT1R is detectable using antibody concentrations of 1:200. (A) On E14, AT1R is abundantly expressed in the ureteric bud (UB) branches and to a lesser extent in glomeruli and the surrounding mesenchyme. Amp, UB ampulla. (B) The UB is positive for AT1R immunostaining on both apical and basolateral membranes. (C) Control section incubated after preadsorption of the primary antibody with its immunogen demonstrates no specific staining.
Figure 2: Effect of medium alone (control) and Ang II on UB branching in Hoxb7–green fluorescence protein (GFP) metanephroi that were grown ex vivo for 48 h. (A) Representative sequential images of branching UB. Absolute number of UB tips and branch points (BP) is shown. (B) Bar graph shows the effect of medium or Ang II on the number of UB tips and BP at 24 and 48 h.
Figure 3: Effect of medium alone (control) or candesartan on UB branching in mouse metanephroi that were grown ex vivo for 48 h. After 48 h in culture, kidney explants were stained with anti-pancytokeratin antibody to label the UB, and the number of UB tips and BP was compared between the two treatment groups. (A) Representative images of branching UB. Absolute number of UB tips and BP is shown. (B) Bar graphs show the effect of medium or candesartan on the number of UB tips and BP.
Figure 4: Effect of Ang II alone or Ang II with EGF receptor (EGFR)-specific tyrosine kinase inhibitor AG1478 (AG) on UB branching in Hoxb7-GFP metanephroi that were grown ex vivo for 48 h. (A) Representative sequential images of branching UB. Absolute number of UB tips and BP is shown. (B) Bar graph shows the effect of AG combined with Ang II or Ang II alone on the number of UB tips and BP.
Figure 5: Effect of Ang II and EGF in UB cell branching in three-dimensional collagen gels. (A) Control (medium + 0.5% FBS). (B) Ang II. (C) Ang II with candesartan. (D) EGF. (E) EGF with AG1478. (F) AG1478 alone. (G) Effect of Ang II, EGF, and AG1478 alone or combined with candesartan or AG1478 on cell process formation in UB cells 24 h after plating. Values are presented as percentage change relative to control (medium + 0.5% FBS) with control equaling 100%. *P < 0.05 versus other groups.
Figure 6: Ang II and EGF induce tyrosine phosphorylation of EGFR in UB cells. Quiescent mouse UB cells were treated or not with Ang II (10−5 to 10−7 M) or EGF for 5 min. Cell lysates were subjected either to immunoblotting with anti-phosphotyrosine EGFR antibody (A) or to immunoprecipitation with anti-EGFR antibody followed by immunoblotting with anti-phosphotyrosine mAb (B). After stripping, membrane “A” was reprobed with β-actin antibody (C) and membrane “B” with anti-EGFR antibody (D). (E and F) Bar graphs represent densitometric analysis of the results that were obtained in three independent experiments using anti-phosphotyrosine EGFR antibody (“A”; E) and anti-phosphotyrosine mAb (“B”; F). Bars represent ratio of EGFR protein/β-actin protein (“A”) or ratio of phosphorylated/total EGFR (“B”). Positive control, EGF-stimulated A431 cell lysate (Santa Cruz). *P < 0.01 versus other groups (F). (G) UB cells that were grown on coverslips were incubated with media (control), Ang II (10−6 M), or EGF (40 ng/ml) for 30 min and then immunostained with two different FITC-conjugated anti-phosphotyrosine EGFR antibodies (green) and propidium iodide (red). The yellow signals indicate localization of tyrosine phosphorylated EGFR in the nucleus.
Figure 7: Proposed signaling pathways involved in mediating the effects of Ang II on UB branching morphogenesis. AT
1-mediated activation of multiple signaling pathways involving Jak2/STAT, Ras/extracellular signal–regulated kinase 1/2 (ERK1/2), phosphatidylinositol-3 kinase/Akt, and EGF transactivation may stimulate UB cell proliferation, migration, tubulogenesis, and survival. Upon ligand binding, EGFR dimerize and autophosphorylate several tyrosine residues to generate docking sites for signaling and adaptor proteins. The mechanisms of EGFR activation by AT
1 and AT
1-dependent effects on UB branching may involve phosphorylation of Tyr-319 on carboxyl terminal of AT
1; activation of tyrosine kinases Src and Pyk2; and recruitment of SHP2, Shc, Grb2, and SOS with subsequent activation of the renin-angiotensin system. AT
1 also can induce autocrine generation of Ang II from angiotensinogen (AGT)
via Jak2/STAT3/STAT6 as shown in cardiomyocytes (
42). This creates an autocrine loop that amplifies the effects of Ang II on UB branching/stromal signaling. AT
2-dependent activation of MAPK phosphatase (MAPKP) leads to ERK1/2 inactivation and may induce death-related signaling. The growth-promoting effects of AT
2 may be mediated by Jak2/STAT-dependent activation of Pax2 in the UB. The ultimate effect of Ang II on UB branching and EGFR transactivation may depend on the balance between AT
1- and AT
2-mediated actions.
This work was supported by National Institutes of Health grants P20 RR17659, DK-56264, and DK-62250.
UB cells were a kind gift from Dr. Jonathan Barash (Columbia University). Hoxb7-GFP mice were a kind gift from Dr. Frank Costantini (Columbia University).
Published online ahead of print. Publication date available at www.jasn.org.
References
1. Aufderheide E, Chiquet-Ehrismann R, Ekblom P: Epithelial-mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme. J Cell Biol 105 : 599 –608, 1987
2. Ekblom P: Developmentally regulated conversion of mesenchyme to epithelium. FASEB J 3 : 2141 –2150, 1989
3. Brenner BM, Garcia DL, Anderson S: Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens 1 : 335 –347, 1988
4. Lisle SJ, Lewis RM, Petry CJ, Ozanne SE, Hales CN, Forhead AJ: Effect of maternal iron restriction during pregnancy on renal morphology in the adult rat offspring. Br J Nutr 90 : 33 –39, 2003
5. Nagata M, Tanimoto K, Fukamizu A, Kon Y, Sugiyama F, Yagami K, Murakami K, Watanabe T: Nephrogenesis and renovascular development in angiotensinogen-deficient mice. Lab Invest 75 : 745 –753, 1996
6. Takahashi N, Lopez ML, Cowhig JE Jr, Taylor MA, Hatada T, Riggs E, Lee G, Gomez RA, Kim HS, Smithies O: Ren1c homozygous null mice are hypotensive and polyuric, but heterozygotes are indistinguishable from wild-type. J Am Soc Nephrol 16 : 125 –132, 2005
7. Esther CR Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, Bernstein KE: Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 7 : 953 –965, 1996
8. Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, Coffman TM: Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci U S A 95 : 15496 –15501, 1998
9. Tsuchida S, Matsusaka T, Chen X, Okubo S, Niimura F, Nishimura H, Fogo A, Utsunomiya H, Inagami T, Ichikawa I: Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes. J Clin Invest 101 : 755 –760, 1998
10. Oshima K, Miyazaki Y, Brock JW, Adams MC, Ichikawa I, Pope JC 4th: Angiotensin type II receptor expression and ureteral budding. J Urol 166 : 1848 –1852, 2001
11. Garcia-Villalba P, Denkers ND, Wittwer CT, Hoff C, Nelson RD, Mauch TJ: Real-time PCR quantification of AT1 and AT2 angiotensin receptor mRNA expression in the developing rat kidney. Exp Nephrol 94 : e154 –e159, 2003
12. Iosipiv IV, Schroeder M: A role for angiotensin II AT1 receptors in ureteric bud cell branching. Am J Physiol 285 : F199 –F207, 2003
13. Iosipiv IV: Cellular expression of the angiotensin type 2 receptor (AT2) during murine organogenesis [Abstract]. J Invest Med 50 : 134A , 2002
14. Yosypiv IV, Schroeder M: Role of angiotensin type 2 (AT2) receptor in ureteric bud cell branching morphogenesis in vitro [Abstract]. J Am Soc Nephrol 15 : 419A , 2004
15. Karihaloo A, O’Rourke DA, Nickel C, Spokes K, Cantley LG: Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis. J Biol Chem 276 : 9166 –9173, 2001
16. Voisin L, Foisy S, Giasson E, Lambert C, Moreau P, Meloche S: EGF receptor transactivation is obligatory for protein synthesis stimulation by G protein-coupled receptors. Am J Physiol Cell Physiol 283 : C446 –C455, 2002
17. Seta K, Sadoshima J: Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor. J Biol Chem 278 : 9019 –9026, 2003
18. Barasch J, Pressler L, Connor J, Malik A: A ureteric bud cell line induces nephrogenesis in two steps by distinct signals. Am J Physiol 271 : F50 –F61, 1996
19. Piscione TD, Phan T, Rosenblum ND: BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathways. Am J Physiol 280 : F19 –F33, 2001
20. Zent R, Bush KT, Pohl ML, Quaranta V, Koshikawa N, Wang Z, Kreidberg JA, Sakurai H, Stuart RO, Nigam SK: Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development. Dev Biol 238 : 289 –302, 2001
21. Srinivas S, Goldberg MR, Watanabe T, Dagati V, Al-Awqati Q, Costantini F: Expression of green fluorescent protein in the ureteric bud of transgenic mice: A new tool for the analysis of the ureteric bud morphogenesis. Dev Genet 24 : 241 –251, 1999
22. Miyazaki Y, Tsuchida S, Fogo A, Ichikawa I: The renal lesions that develop in neonatal mice during angiotensin inhibition mimic obstructive nephropathy. Kidney Int 55 : 1683 –1695, 1999
23. Gupta IR, Lapointe M, Yu OH: Morphogenesis during mouse embryonic kidney explant culture. Kidney Int 63 : 365 –376, 2003
24. Berellini G, Gruciani G, Mannhold R: Pharmacophore, drug metabolism, and pharmacokinetics models on non-peptide AT1, AT2, and AT1/AT2 angiotensin II receptor antagonists. J Med Chem 48 : 4389 –4399, 2005
25. Yosipiv IV, El-Dahr SS: Activation of angiotensin-generating systems in the developing rat kidney. Hypertension 27 : 281 –286, 1996
26. Watanabe T, Costantini F: Real-time analysis of ureteric bud branching morphogenesis in vitro. Dev Biol 271 : 98 –108, 2004
27. Sakaguchi K, Okabayashi Y, Kido Y, Kimura S, Matsumura Y, Inushima K, Kasuga M: Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells. Mol Endocrinol 12 : 536 –543, 1998
28. Tice DA, Biscardi JS, Nickles AL, Parsons SJ: Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl Acad Sci U S A 96 : 1415 –1420, 1999
29. Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY, Bourguignon L, Hung MC: Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 3 : 802 –808, 2001
30. Berry C, Touyz R, Dominiczak AF, Webb RC, Johns DG: Angiotensin receptors: Signaling, vascular pathophysiology, and interactions with ceramide. Am J Physiol 281 : H2337 –H2365, 2001
31. Watanabe G, Lee RJ, Albanese C, Rainey WE, Batle D, Pestell RG: Angiotensin II activation of cyclinD1-dependent kinase activity. J Biol Chem 271 : 22570 –22577, 1996
32. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T: Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273 : 8890 –8896, 1998
33. Schafer B, Gschwind A, Ullrich A: Multiple G-protein-coupled receptor signals converge on the epidermal growth factor receptor to promote migration and invasion. Oncogene 23 : 991 –999, 2004
34. Bernardini N, Bianchi F, Lupetti M, Dolfi A: Immunohistochemical localization of the epidermal growth factor, transforming growth factor alpha, and their receptor in the human mesonephros and metanephros. Dev Dyn 206 : 231 –238, 1996
35. Sakurai H, Tsukamoto T, Kjelsberg CA, Cantley LG, Nigam SK: EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by embryonic kidney. Am J Physiol 273 : F463 –F472, 1997
36. Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D, LaMantia C, Mourton T, Herrup K, Harris RC: Targeted disruption of mouse EGF receptor: Effect of genetic background on mutant phenotype. Science 269 : 230 –234, 1995
37. Niimura F, Labosky PA, Kakuchi J, Okubo S, Yoshida H, Oikawa T, Ichiki T, Naftilan AJ, Fogo A, Inagami T: Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation. J Clin Invest 96 : 2947 –2954, 1995
38. Huwiler A, Stabel S, Fabbro D, Pfeilschifter J: Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: Comparison of hypertrophic and hyperplastic agonists. Biochem J 305 : 777 –784, 1995
39. Saward L, Zahradka P: Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells. Circ Res 81 : 249 –257, 1997
40. Tang MJ, Cai Y, Tsai SJ, Wang YK, Dressler GR: Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase. Dev Biol 243 : 128 –136, 2002
41. Olivares-Reyes JA, Shah BH, Hernandez-Aranda J, Garcia-Gaballero A, Farshori MP, Gatcia-Sainz JA, Catt KJ: Agonist-induced interactions between angiotensin AT1 and epidermal growth factor receptors. Mol Pharmacol 68 : 356 –364, 2005
42. Fukizawa J, Booz GW, Hunt RA, Shimizu N, Karoor V, Baker KM, Dostal DE: Cardiotrophin-1 increases angiotensinogen mRNA in rat cardiac myocytes through STAT3: An autocrine loop for hypertrophy. Hypertension 35 : 1191 –1196, 2000
