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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02777

Journal of Cell Science 119, 559-570 (2006)
Published by The Company of Biologists 2006
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Research Article

EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases

Kazushige Ogawa1,*, Hiroki Wada1, Noriyoshi Okada1, Itsuki Harada1, Takayuki Nakajima1, Elena B. Pasquale2 and Shingo Tsuyama3

1 Department of Veterinary Anatomy, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
2 The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
3 Department of Molecular and Cell Biology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

* Author for correspondence (e-mail: kogawa{at}vet.osakafu-u.ac.jp)

Accepted 2 November 2005


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined functions in development, but their expression patterns and functions in many adult tissues are still largely unknown. We have detected substantial levels of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult mouse kidney by RT-PCR amplification. Immunolocalization experiments revealed that EphB2 is localized in the tubules of the inner and outer medulla and EphB6 is in the tubules of the outer medulla and cortex. By contrast, ephrin-B1 was detected in tubules throughout the whole nephron. Consistent with the overlapping expression of the EphB2 receptor and the ephrin-B1 ligand in the medulla, EphB2 is tyrosine-phosphorylated, and therefore activated, in the kidney. In the outer medulla, however, EphB2 signaling may be attenuated by the co-expressed kinase-inactive EphB6 receptor. Interestingly, we found that EphB signaling induces RhoA activation and Rac1 inactivation as well as cell retraction, enlargement of focal adhesions and prominent stress fibers in primary cultures of medullary tubule cells. These results suggest that EphB receptor signaling through Rho family GTPases regulates the cytoarchitecture and spatial organization of the tubule cells in the adult kidney medulla and, therefore, may affect the reabsorption ability of the kidney.

Key words: EphB2, Ephrin-B1, Rho, Rac, Kidney, Distal tubules


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and methods
 References
 
The tissue organization of the kidney reflects its function to excrete the waste products of metabolism. To produce urine, an initial ultrafiltrate of the blood is modified by selective resorption and specific secretion by the tubule cells of the kidney. The kidney is a vascular organ consisting of an assembly of fundamental structural and functional units, the nephrons, together with the associated collecting duct system. The development of the kidney involves signaling through receptor tyrosine kinases. Several families of receptor tyrosine kinases are essential for precise and coordinated cell migration, proliferation, and differentiation during kidney development as well as for cell-cell recognition, formation of intercellular connections and morphogenesis (Davies and Fisher, 2002Go; Robert and Abrahamson, 2001Go; Takahashi et al., 1998Go). Receptor tyrosine kinases may also be important in the maintenance of tissue organization and filtration functions in the adult kidney, given their known roles in regulating the polarity and renewal of kidney epithelial cells (Balkovetz and Lipschutz, 1999Go; Miao et al., 2003Go; Rosario and Birchmeier, 2003Go; Vargas et al., 2000Go). However, these aspects have not been extensively investigated.

In this study, we have examined whether Eph receptor tyrosine kinases and ephrin ligands may regulate kidney cytoarchitecture once development is completed. The Eph receptor family, with 14 members in mammals, is divided into EphA and EphB subclasses based on sequence homology of the extracellular domain (Pasquale, 2005Go). The ligands, ephrins, are membrane-anchored and are also divided into two subclasses, ephrin-A with five members and ephrin-B with three members in mammals. Ephrin-A ligands are anchored to the plasma membrane through a glycosyl phosphatidylinositol (GPI) linkage, while ephrin-B ligands are transmembrane. In general, EphA receptors promiscuously bind ephrin-A ligands and EphB receptors promiscuously bind ephrin-B ligands, although EphB4 preferentially binds ephrin-B2. Because Eph receptors and ephrins are both membrane-bound proteins, their interactions require cell-cell contact. In addition, their signals propagate bidirectionally in both the Eph receptor and the ephrin expressing cells. The EphA10 and EphB6 receptors, however, have variations in their kinase domain that suggest lack of kinase activity (Gurniak and Berg, 1996Go; Manning et al., 2002Go; Matsuoka et al., 1997Go). Thus, these receptors probably modulate the signals of catalytically active Eph receptors by forming heterocomplexes with them (Freywald et al., 2002Go).

The roles of Eph receptors and ephrins have been extensively characterized in developing tissues, especially in the central nervous system and the vascular system. Many different biological functions have been attributed to these proteins, including regulation of tissue-border formation, axon guidance, cell migration and vascular development (Noren and Pasquale, 2004Go; Palmer and Klein, 2003Go; Pasquale, 2005Go; Poliakov et al., 2004Go). In adult tissues, Eph receptors and ephrins have been implicated in synaptic plasticity, nerve regeneration, cancer progression and pathological angiogenesis (Dodelet and Pasquale, 2000Go; Ogawa et al., 2000Go; Palmer and Klein, 2003Go; Yamaguchi and Pasquale, 2004Go). However, little is known about the localization and functions of these proteins in normal adult organs, except for the nervous system, blood cells, and the intestinal epithelium (Batlle et al., 2002Go; Luo et al., 2002Go; Prevost et al., 2002Go; Sharfe et al., 2002Go; Yamaguchi and Pasquale, 2004Go). Both Eph receptors and ephrins have been detected in the adult kidney (Andres et al., 1994Go; Bennett et al., 1994Go; Bohme et al., 1993Go; Ciossek et al., 1995Go; Gale et al., 2001Go; Ikegaki et al., 1995Go; Kiyokawa et al., 1994Go; Maru et al., 1988Go; Sajjadi and Pasquale, 1993Go; Shin et al., 2001Go; Takahashi et al., 2001Go), but their expression patterns and functions with respect to adult kidney physiology are largely unknown. An interesting exception is the EphA2 receptor, whose expression in the adult rat kidney medulla and papilla is increased in response to hypertonicity, suggesting that it may regulate water or ion fluxes through unknown mechanisms (Xu et al., 2005Go).

We have characterized the expression of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult kidney by RT-PCR and immunohistochemistry. The results show that ephrin-B1 is expressed in epithelial cells throughout the nephron, EphB2 expression is restricted to the tubules of the kidney medulla and EphB6 expression is restricted to the tubules of the cortex and the outer medulla. We also show that stimulation of EphB signaling by ephrin-B1 induces Rho family GTPases-dependent changes in the cytoarchitecture of primary cultures of medullary tubule cells.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Expression of ephrin-B ligands and EphB receptors in the adult kidney
To determine which EphB receptors and ephrin-B ligands are present in the kidney after tissue organization is completed, we screened the adult mouse kidney by reverse transcription PCR (RT-PCR). We detected transcripts for all mammalian EphB and ephrin-B molecules (EphB5 has only been identified in the chicken), although ephrin-B3 was barely detectable (Fig. 1A). The more prominent amplification products were those for ephrin-B1, EphB2 and EphB6. Therefore, we focused on these mRNAs for further examination. We obtained similar ephrin-B1 and EphB6 amplification levels from the cortex and the medulla of the kidney, while EphB2 was preferentially amplified from the medulla (Fig. 1B,C). We next examined whether the EphB2 receptor is phosphorylated on tyrosine in adult mouse kidney and is, therefore, likely to be activated by a ligand. By probing EphB2 immunoprecipitates with anti-phosphotyrosine antibodies, EphB2 was found to be tyrosine-phosphorylated (Fig. 1D). This suggests that EphB2-bearing cells are in contact with ephrin-B-bearing cells and that EphB2 is not only expressed but also activated in the adult kidney in vivo.


Figure 1
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  		Fig. 1. Expression of B-class Eph receptors and ephrins in the adult mouse kidney. (A,B) Amplification of ephrin-B and EphB mRNAs by RT-PCR. (A) Substantial levels of endogenous ephrin-B1, EphB2 and EphB6 are amplified from adult mouse kidney. Bands were not detected in controls in which the RT reaction was omitted (not shown). (B) Amplification of ephrin-B1, EphB2 and EphB6 from the cortex and the medulla of the kidney. (C) Densitometric quantification of the amplification levels of ephrin-B1, EphB2 and EphB6 mRNA in the cortex and medulla of the kidney. Data from three independent experiments, normalized to the levels of the GAPDH amplification products, are shown as mean ± s.d. Comparisons were performed using unpaired t-test. EphB2 expression in the medulla is significantly higher than in the cortex (P=0.002), whereas expression levels of ephrin-B1 and EphB6 are not significantly different between the two (P=0.407 and 0.692, respectively). (D) EphB2 tyrosine phosphorylation in adult mouse kidney. Lysates from the whole kidney were immunoprecipitated with anti-EphB2 antibodies. Immunoprecipitates were separated by SDS-PAGE and probed by immunoblotting with antibodies as indicated at the right.

 
Localization of ephrin-B1, EphB2, EphB4 and EphB6 in the adult kidney
To determine the cellular and subcellular localization of ephrin-B1, EphB2, EphB3, EphB4 and EphB6 in the adult mouse kidney, we stained tissue sections with an anti-ephrin-B1-specific antibody (ephrin-B1e) and an antibody that recognizes all three ephrin-B ligands (ephrin-B1p), two different EphB2 antibodies as well as anti-EphB3, anti-EphB4 and anti-EphB6 antibodies. Similar staining patterns were obtained when using different antibodies to the same protein. The results show that ephrin-B1 is expressed ubiquitously in the nephron – including the renal corpuscles, proximal tubules, the thin limb of the loop of Henle and the distal tubules – and is localized in the basolateral membrane of the epithelial tubule cells (Fig. 2A). By contrast, we detected a very distinctive expression pattern for EphB2. This receptor is expressed in the tubules of the inner and outer medulla, including the distal straight tubules and the thin limb of the loop of Henle (Fig. 2B). In the tubules, EphB2 is concentrated in the basal infolds. EphB4 is mainly expressed in capillaries in the medulla, but weak staining was also detected in collecting ducts of the cortex (Fig. 2C). EphB6 is preferentially expressed in the renal corpuscles and tubules of both the cortex and the outer medulla, including the proximal and distal tubules (Fig. 2D). In the distal tubules, EphB6 is localized in the basal infolds while in the proximal tubules this receptor is localized in granular structures of the cytoplasm. We could not detect EphB3 expression by immunohistochemistry. The immunostaining patterns in nephron show that: (1) the thin limb of the loop of Henle coexpresses ephrin-B1 and EphB2; (2) distal straight tubules coexpress ephrin-B1, EphB2 and EphB6; and (3) proximal and distal convoluted tubules coexpress ephrin-B1 and EphB6 (Fig. 2F).


Figure 2
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  		Fig. 2. Localization of ephrin-B1, EphB2, EphB4 and EphB6 in the adult mouse kidney. Immunoperoxidase labeling of frozen sections with anti-ephrin-B1 (A), anti-EphB2 (B), anti-EphB4 (C) and anti-EphB6 (D) antibodies. (A) Ephrin-B1 immunoreactivity is broadly expressed in the nephron. In the proximal tubules (PT) of cortex, ephrin-B1 is localized in the basolateral membranes. Upper panel, anti-ephrin-B1p antibody; lower panels, anti-ephrin-B1e antibody. (B) EphB2 immunoreactivity is distinctly restricted to the medulla and absent from the cortex. EphB2 is localized in medullary tubules, including the distal straight tubules (DST) and the thin limb of the loop of Henle (TL). In the distal straight tubules, EphB2 is concentrated in the basal infolds. Upper panel, anti-EphB2p antibody; lower panel, anti-EphB2e antibody. (C) EphB4 is primarily localized in capillaries of the medulla (arrows in the lower panels). Faint immunoreaction of EphB4 is present in collecting duct (CD) in the cortex. (D) EphB6 immunoreactivity is primarily localized in tubules of the cortex and the outer medulla, including proximal tubules and distal tubules. In the distal straight tubules (DST) EphB6 is concentrated in the basal infolds and in the proximal tubules (PT) EphB6 is concentrated in granular structures of the cytoplasm. (E) There is no immunoreaction in the control without the primary antibodies. C, cortex; IM, inner medulla; OM, outer medulla; CD, collecting duct; DT, distal tubule; DST, distal straight tubule; PT, proximal tubule; TL, thin limb of the loop of Henle. Sections were developed with NiCl2 (upper panel in A and B) or without NiCl2 (lower panels in A and B, and all panels in C, D and E) and the sections in the lower panels except for the lower left of C were counterstained with hematoxylin. (F) Schematic drawings showing the expression patterns of ephrin-B1 (yellow), EphB2 (green), and EphB6 (blue) and predicted EphB2 tyrosine phosphorylation levels (dark green, high; light green, low) in the nephron. CD, collecting duct; CT, connecting tubule; DCT, distal convoluted tubule; DST, distal straight tubule (also known as thick ascending limb of loop of Henle); PCT, proximal convoluted tubule; PST, proximal straight tubule; TL, the thin limb of loop of Henle.

 
EphB signaling induces cell retraction in primary cultures of medullary tubule cells
We confirmed by RT-PCR that medullary tubule cells express ephrin-B1, EphB2 and EphB6 in culture. EphB2 amplification products were higher in cultured medullary tubule cells than in cultured cortical tubule cells (Fig. 3), suggesting that the differential in vivo expression of EphB2 in the cortex and the medulla is preserved in culture. EphB6 amplification was also higher in the cultured medullary tubule cells, despite the similar levels of EphB6 amplification from kidney medulla and cortex (Fig. 1B). EphB2 immunoprecipitated from medullary tubule cells was weakly phosphorylated on tyrosine (Fig. 4A), consistent with the expression of endogenous ephrin-B1 in these cells. These data indicate that the expression and activation characteristics of EphB2 are well preserved in cultures of medullary tubule cells and, thus, these cultures are a good model to study EphB2 and ephrin-B1 function in distal straight tubules.


Figure 3
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  		Fig. 3. Expression of ephrin-B1, EphB2, EphB6 and the cytoplasmic adaptor protein Dishevelled in cortical and medullary tubule cells. (A) RT-PCR amplification of mRNA prepared from cultured cortical and medullary tubule cells shows that the cells in culture retain ephrin-B1, EphB2 and EphB6 expression patterns similar to those in the regions of the kidney from which the cells are derived (see Fig. 1B,C). (B) Densitometric quantification of ephrin-B1, EphB2 and EphB6 amplification products from cortical and medullary tubule cells. Data from three independent experiments, normalized to the levels of the GAPDH amplification products, are shown as mean ± s.d. Comparisons were performed using unpaired Student's t-test. The levels of the EphB2 and EphB6 amplification products are significantly higher in medullary than in cortical tubule cells (P=0.0001 and P=0.009, respectively), whereas the expression level of ephrin-B1 is not significantly different between the two (P=0.367).

 

Figure 4
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  		Fig. 4. Treatment with ephrin-B1–Fc induces retraction of medullary tubule cells plated on Matrigel-coated surfaces. (A) Cells were stimulated with 1 µg/ml ephrin-B1–Fc or 1 µg/ml human Fc for the indicated time periods. Cell lysates were immunoprecipitated with anti-EphB2 antibody. The immunoprecipitates were separated by SDS-PAGE, probed by immunoblotting with phosphotyrosine (PY) antibodies, and reprobed for EphB2. (B,C) Phase-contrast time-lapse microscopy pictures at a low (B) and high (C) magnification. Cells were stimulated with 1 µg/ml ephrin-B1–Fc and a series of phase-contrast images of the same field were obtained at the indicated times. Treatment with ephrin-B1–Fc leads to dramatic changes in cell morphology. Cell retraction is particularly prominent at the free edges of the cells (arrowheads in B) and partial separation between the adjacent cell surfaces of neighboring cells is also evident (arrow in C). (D) Morphological changes are induced by ephrin-B1–Fc but not EphB2-Fc treatment. Cells were stimulated with ephrin-B1–Fc (1 µg/ml) or EphB2-Fc (1 µg/ml) with or without crosslinking with anti-human Fc antibody (0.25 µg/ml). 1 µg/ml Fc with or without anti-human Fc antibody, or vehicle only, were used as controls. Phase-contrast images of the same field were obtained at 0 minute and 15 minutes after beginning the stimulation and the fraction of retracting cells at the 15 minutes time point was calculated. Only cells at the periphery of the epithelial sheets were quantitated for statistical analysis. Data from three independent experiments are shown as mean ± s.d. Comparisons were performed using one-way factorial ANOVA.

 
We examined the kinetics of EphB2 activation in cultured medullary tubule cells after treatment with soluble ephrin-B1–Fc ligand, in which the ephrin-B1 ectodomain is dimerized by fusion to the Fc portion of human IgG (Fig. 4A). In the control cells treated with Fc for 15 minutes, EphB2 was weakly tyrosine-phosphorylated at levels similar to the control cells not treated with Fc. Additional tyrosine-phosphorylation (activation) of EphB2 was detectable within 2 minutes after stimulation with ephrin-B1–Fc. EphB2 phosphorylation peaked between 15 and 30 minutes and persisted for over 1 hour. We then investigated the morphological changes induced by the addition of ephrin-B1–Fc in medullary tubule cells by using time-lapse microscopy (Fig. 4B,C). The cells, which are normally spread on the Matrigel surface, started to retract at their periphery within 5 minutes after addition of ephrin-B1–Fc. Cell retraction peaked at 30-60 minutes, and spreading began to recover within 60-120 minutes. None of the cells detached from the extracellular matrix surface during the stimulation. Retraction was particularly prominent in peripheral regions, at the free surfaces of the cells at the edges of the epithelial sheets, while partial separation between adjacent cells occurred within the sheets. Prominent stress fibers also became visible by phase-contrast microscopy in the free surfaces of the retracting cells (Fig. 4C).

We counted the retracting cells by comparing images before and after 15 minutes of stimulation with ephrin-B1–Fc. This determined that about 70% of the cells retracted following stimulation with ephrin-B1–Fc (Fig. 4D) and one-way ANOVA analysis showed a significant difference between the ephrin-B1–Fc stimulated cells and the control cells (P<0.0001). By contrast, treatment with EphB2-Fc had no detectable effect on cell morphology, even though medullary tubule cells express a considerable amount of endogenous ephrin-B1.

EphB activation promotes cell adhesion and induces focal adhesion enlargement
Because the retraction of the cell periphery induced by ephrin-B1–Fc suggests that cell adhesion may be affected, we examined the effects of ephrin-B1–Fc on the initial attachment of cells seeded on a Matrigel-coated surface. Treatment with ephrin-B1–Fc stimulated cell attachment compared with the Fc only control (Fig. 5). A similar effect on cell attachment was detected when ephrin-B1–Fc was presented immobilized on the substrate. However, no additional attachment was observed when cells were both exposed to soluble ephrin-B1–Fc and plated on a surface coated with ephrin-B1–Fc.


Figure 5
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  		Fig. 5. EphB2 activation promotes cell attachment to Matrigel-coated surfaces. Medullary tubule cells were plated on wells coated with Matrigel alone or together with ephrin-B1–Fc (0.3 µg/cm2) and allowed to adhere for 40 minutes. Ephrin-B1–Fc (2.0 µg/ml) or human Fc (2.0 µg/ml) with or without crosslinking with anti-human Fc antibodies (0.5 µg/ml) was also added in soluble form at the time of plating. Cell attachment measurements from three independent experiments are shown as mean ± s.d. Values were normalized to the control (stimulation with human Fc). Comparisons were performed using one-way factorial ANOVA. Exogenous ephrin-B1–Fc significantly stimulates cell attachment (P=0.004).

 
Cell retraction and enhanced cell adhesion could be viewed as opposite effects with regard to cell substrate adhesion. Therefore, we also examined whether ephrin-B1–Fc induces changes in the focal adhesions of medullary tubule cells, which are the structures through which the cells attach to the substrate. Focal adhesions were visualized by immunofluorescence labeling with anti-vinculin antibodies. Quiescent cells on Matrigel-coated glass coverslips contacted the extracellular matrix surface with fine focal adhesions (Fig. 6). Stimulation with ephrin-B1–Fc induced dramatic changes in the focal adhesions, which became much more prominent and enlarged. This was accompanied by the appearance of prominent stress fibers. These results show that EphB activation by ephrin-B1–Fc promotes increased cell substrate adhesion and remodeling of focal adhesions in primary cultures of medullary tubule cells. By contrast, stimulation with soluble EphB2-Fc had no detectable effect on the focal adhesions.


Figure 6
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  		Fig. 6. Rearrangement of focal adhesions in medullary tubule cells is induced by treatment with ephrin-B1–Fc but not EphB2-Fc. Cells plated on Matrigel were stimulated with 1 µg/ml ephrin-B1–Fc, EphB2-Fc or human Fc for 15 minutes and stained with an anti-vinculin antibody. Focal adhesions revealed by vinculin staining are much more prominent in the cells stimulated with ephrin-B1–Fc, but remain unchanged in the cells stimulated with EphB2-Fc. Left panels, lower magnification; right panels, higher magnification.

 
EphB2 involvement in cell retraction
To determine whether cell retraction induced by ephrin-B1–Fc is mediated by the EphB2 receptor, we used an antagonistic peptide (SNEW peptide) that selectively binds to EphB2 and blocks ephrin-binding (Koolpe et al., 2005Go). Upon treatment with ephrin-B1–Fc medullary tubule cells retracted at the periphery, leaving behind spike-like protrusions that are detectable by fluorescent phalloidin staining. By contrast, preincubation of the cells with SNEW peptide inhibited cell retraction induced by ephrin-B1–Fc (Fig. 7). A control peptide that does not bind any Eph receptor did not inhibit the cell retraction. These results indicate that EphB2 activation by ephrin-B1–Fc is responsible for the retraction of medullary tubule cells.


Figure 7
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  		Fig. 7. Ephrin-B1–Fc-induced retraction is antagonized by a peptide that selectively binds to EphB2 and blocks ephrin binding. (A) Representative examples of cell morphologies are shown. Upon treatment with 1.5 µg/ml ephrin-B1–Fc, medullary tubule cells retracted at the periphery, leaving behind spike-like protrusions (arrows). Preincubation of cells with the SNEW peptide inhibited cell retraction induced by ephrin-B1–Fc, whereas a control peptide that does not bind any Eph receptor did not inhibit cell retraction. (B) Quantitation of the percent of cells with spike-like protrusions with or without ephrin-B1–Fc and in the presence or absence of 360 µM SNEW peptide is shown. Data from four independent experiments are shown as mean ± s.d.

 

RhoA and Rac1 involvement in downstream of EphB receptor activation
The changes in medullary tubule cell morphology induced by ephrin-B1–Fc, such as cell retraction and the formation of prominent focal adhesions, suggest the involvement of Rho family GTPase. Therefore, we measured the levels of activated RhoA using a pull-down assay with GST-Rhotekin, which binds only activated GTP-bound RhoA. In the control cells treated with Fc for 15 minutes, RhoA was already somewhat activated (Fig. 8A), but RhoA activation increased significantly after stimulation with ephrin-B1–Fc. We also investigated whether inhibiting Rho kinase (ROCK), which functions downstream of RhoA, interferes with cell retraction and focal adhesion formation in medullary tubule cells (Fig. 8B-E). In the presence of the ROCK inhibitor Y27632, ephrin-B1–Fc did not induce substantial cell retraction in medullary tubule cells (Fig. 8B,D). Furthermore, addition of the ROCK inhibitor after cell retraction had begun reversed the effect of ephrin-B1–Fc and the retracting cells began to spread within 5 minutes of treatment with the inhibitor (Fig. 8C,D). Pretreatment with the ROCK inhibitor also suppressed the enlargement of focal adhesions induced by ephrin-B1–Fc (Fig. 8E). These observations suggest that activation of the RhoA-ROCK pathway mediates the morphological effects of EphB receptors in medullary tubule cells.


Figure 8
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  		Fig. 8. EphB activation by ephrin-B1–Fc increases RhoA activity and decreases Rac1 activity, and the RhoA kinase inhibitor Y27632 antagonizes the effects of ephrin-B1–Fc in primary cultures of medullary tubule cells. (A) In cells stimulated for 15 minutes with 1 µg/ml ephrin-B1–Fc, or 1 µg/ml Fc as a control, activated RhoA was isolated by pull-down with a GST fusion protein of the Rho-binding domain of Rhotekin (GST-RBD). Proteins bound to GST-Rhotekin were separated by SDS-PAGE and probed by immunoblotting with an anti-RhoA antibody. The levels of active RhoA in ephrin-B1–Fc-treated cells were normalized to those in Fc control-treated cells. Data from three independent experiments are shown as mean ± s.d. Ephrin-B1 Fc treatment increased the level of activated RhoA by 1.7 fold (P=0.002). (B,C) Phase-contrast time-lapse microscopy photographs. Cells were stimulated with 1 µg/ml ephrin-B1–Fc in the presence of 10 µM Y27632 and a series of phase-contrast images of the same field were obtained at the indicated times. Y27632 was added 10 minutes before (B) and 20 minutes after (C) the addition of ephrin-B1–Fc. Pretreatment with the Y27632 inhibitor essentially abolished the effects of ephrin-B1–Fc (B). Treatment with the inhibitor after cell retraction has begun reverses the effects within 5 minutes (B, arrowheads). (D) Quantification of the percentage of retracting cells stimulated with 1 µg/ml ephrin-B1–Fc under the presence of 10 µM Y27632. Y27632 and ephrin-B1–Fc are added at the same time courses as B and C. Phase-contrast images of the same field were obtained: for the Y27632 addition before the ephrin-B1–Fc stimulation at 0 minute, 10 minutes (after the addition of Y27632) and 30 minutes (20 minutes after the stimulation with ephrin-B1–Fc); for the Y27632 addition after the stimulation at 0 minute, 20 minutes (after the stimulation with ephrin-B1–Fc), and 30 minutes (10 minutes after the addition of Y27632). The fraction of retracting cells at the 10 and 30 minutes time point (open column), and 20 and 30 minutes (dotted column) was calculated, respectively. Only cells at the periphery of the epithelial sheets were quantitated for statistical analysis. Data from three independent experiments are shown as mean ± s.d. (E) Focal adhesion rearrangements induced by treatment with ephrin-B1–Fc are blocked by pretreatment with the Y27632 Rho kinase inhibitor. Medullary tubule cells plated on Matrigel were preincubated with 10 µM Y27632 for 5 minutes and then stimulated for 15 minutes with 1 µg/ml ephrin-B1–Fc, or human Fc as a control. The cells were then stained with an anti-vinculin antibody. (F) In cells stimulated for 5 minutes and 15 minutes with 1 µg/ml ephrin-B1–Fc, or for 15 minutes with 1 µg/ml Fc as a control, activated Rac1 was isolated by pull-down with a GST fusion protein of the Rho-binding domain of PAK1 (GST-PBD). Proteins bound to GST-PAK1 were separated by SDS-PAGE and probed by immunoblotting with an anti-Rac1 antibody. The levels of active Rac1 in ephrin-B1–Fc-treated cells were normalized to those in Fc control-treated cells. Data from three independent experiments are shown as mean ± s.d.

 

The retraction of lamellipodia induced by ephrin-B1–Fc in medullary tubule cells also suggests that another Rho family GTPase, Rac1, may be inactivated. Pull-down assays with GST-PAK1, which only binds to activated Rac1, showed that Rac1 activation was greatly reduced after stimulation with ephrin-B1–Fc compared with control stimulation with Fc (Fig. 8F). Taken together, these data demonstrate that RhoA activation and Rac1 inactivation are coordinately involved in the changes in medullary tubule cell morphology induced by ephrin-B1–Fc.


   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Both EphB receptors and ephrin-B ligands are transmembrane cell-cell communication molecules that play a key role in tissue organization by mediating repulsion or adhesion of neighboring cells and cellular processes (Noren and Pasquale, 2004Go; Palmer and Klein, 2003Go; Pasquale, 2005Go; Poliakov et al., 2004Go). Because epithelial cell-cell interactions are particularly important for kidney resorption function, we have examined the expression and activation of B-class Eph receptors and ephrins in adult kidney epithelial cells. Many studies have reported expression of EphB receptors and ephrin-B ligands in the adult kidney, but a comparative analysis of the different members of these families was not carried out (Andres et al., 1994Go; Bennett et al., 1994Go; Bennett et al., 1995Go; Bohme et al., 1993Go; Ciossek et al., 1995Go; Ikegaki et al., 1995Go; Kiyokawa et al., 1994Go; Sajjadi and Pasquale, 1993Go). Because our RT-PCR amplifications revealed prominent expression of EphB2, EphB6, and ephrin-B1 in the adult kidney, and because the expression patterns of EphB2 and EphB6 in the kidney were unknown, we focused on these molecules for further characterization.

Our immunolocalization experiments determined that ephrin-B1 is expressed throughout the whole nephron, EphB6 is expressed in proximal and distal tubules, and EphB2 is expressed in medullary tubules, including the distal straight tubules and the thin limb of loop of Henle. All cells in distal straight tubules of the outer medulla expressed EphB2. However, proximal straight tubules and collecting ducts located in the outer medulla are EphB2-negative. Interestingly, these patterns define boundaries of EphB expression in the kidney that correspond to the histological boundaries, with the inner medulla expressing EphB2, the outer medulla expressing both EphB2 and EphB6, and the cortex expressing EphB6 (Fig. 2F). Previous analyses of mice expressing ß-galactosidase under control of the ephrin-B2 promoter found expression of ephrin-B2 in the collecting ducts and connecting tubules of the adult kidney and, during development, in the embryonic ureteric bud epithelium and glomerular epithelial cells (Gale et al., 2001Go; Shin et al., 2001Go; Takahashi et al., 2001Go). EphB1 receptor immunoreactivity has also been detected in the epithelial cells of collecting ducts in the mouse embryo, but not in the adult kidney (Daniel et al., 1996Go).

Previous work has particularly focused on EphB and ephrin-B expression in the vasculature of the kidney. Indeed, ephrin-B2 is prominently expressed in both the developing and adult arterial vasculture (Gale et al., 2001Go; Shin et al., 2001Go; Takahashi et al., 2001Go). A receptor that interacts with ephrin-B2, EphB4, is expressed in a complementary pattern in the venous vasculature and remains present at lower levels in adult kidney vasculature (Andres et al., 2003Go; Gale et al., 2001Go; Shin et al., 2001Go; Takahashi et al., 2001Go), as we also found. The EphB1 receptor, which can interact with both ephrin-B1 and ephrin-B2, is co-expressed with these ligands in the developing and adult arterial vasculature of the kidney (Daniel et al., 1996Go). In a previous study, ephrin-B1 immunoreactivity has been primarily detected in the vasculature of the developing and adult kidney (Daniel et al., 1996Go). We do not know the reason for the discrepancy between our ephrin-B1 immunolocalization and that in the previous study. However, our RT-PCR analysis of primary cultures of kidey tubule cells confirmed the ephrin-B1 expression in epithelial cells. Taken together, the EphB and ephrin-B expression patterns suggest important roles for B-class Eph receptors and ephrins in the assembly of kidney vasculature during development through interactions between arterial and venous endothelial cells and between endothelial and epithelial cells. Furthermore, EphB receptors and ephrin-B ligands appear to be also important in both the vasculature and the tubules of the adult kidney.

We have detected EphB2 tyrosine-phosphorylation and, therefore, signaling in the adult kidney in vivo as well as in purified medullary tubule cells in vitro. Based on the co-localization with ephrin-B1, EphB2 signaling is likely to occur in the thin limb of the loop of Henle and in distal straight tubules (Fig. 2F). EphB2 signaling may be weaker in the distal straight tubules, where kinase-inactive EphB6 is also expressed, than in the tubules of the thin limb (Fig. 2F). EphB6 can form heterocomplexes with EphB1 and as a result is transphosphorylated by EphB1 (Freywald et al., 2002Go). A similar heterocomplex may form between EphB2 and EphB6 in distal straight tubules, thus partially attenuating and possibly modifying EphB2 signals.

To examine how EphB2 signaling may affect the properties of tubule epithelial cells in the adult kidney, we used primary cultures of medullary tubule cells, which retain levels of ephrin-B1 and EphB2 expression and phosphorylation similar to those found in vivo. Enhancement of EphB activity following stimulation with ephrin-B1–Fc induces membrane retraction and the appearance of gaps between cells, accompanied by the formation of prominent stress fibers and the rearrangement of focal adhesions. These morphological changes are accompanied by increased RhoA activity and decreased Rac1 activity. Moreover, the Rho kinase inhibitor, Y27632, reverses the morphological effects of EphB signaling. The functions of Rho family GTPases (RhoA, Rac1 and Cdc42) are well defined in controlling cell shape and movement. RhoA promotes the assembly of contractile actin-myosin filaments (stress fibers) and of associate focal adhesion complexes, Rac1 promotes the assembly of a meshwork of actin filaments at the cell periphery to produce lamellipodia and membrane ruffles, and Cdc42 promotes actin-rich surface protrusions called filopodia (Hall, 1998Go; Nobes and Hall, 1995Go). Interestingly, RhoA and Rac1 are known to regulate paracellular permeability of small solutes in the MDCK cell line, which is derived from dog kidney tubule cells (Benais-Pont et al., 2003Go; Hasegawa et al., 1999Go; Jou et al., 1998Go) as well as in other epithelial cell (Wojciak-Stothard et al., 2001Go; Wojciak-Stothard et al., 2005Go).

Rho family GTPases are known to be major downstream targets of Eph receptors, involved in regulating the actin dynamics underlying repulsion or adhesion of neighboring cells or cellular processes (Noren and Pasquale, 2004Go). For example, in neurons EphB receptors activate Rac1 and Cdc42 through the exchange factors kalirin and intersectin, respectively (Irie and Yamaguchi, 2002Go; Penzes et al., 2003Go). In addition, the adaptor protein Dishevelled has been reported to connect EphB receptors with RhoA through an unknown pathway (Tanaka et al., 2003Go). Because we detected Dishevelled mRNA in medullary tubule cells (Fig. 3A), the downstream signaling cascade responsible for the morphological effects of EphB signaling may involve Dishevelled, RhoA and Rho kinase. EphB signaling has also been shown to induce cell retraction and rearrangement of focal adhesions in a colon carcinoma cell line through a pathway involving Rac inactivation (Batlle et al., 2002Go). Decreased Rac activity or increased Rho activity can lead to similar repulsive responses downstream of Eph receptors (Miao et al., 2003Go) and in cultured kidney tubule cells they both coordinately occur following ephrin-B1–Fc stimulation.

With regard to the fact that the changes we observed are transient, the time course of the ephrin-B1 effects may be influenced by whether the ligand is soluble (as the ephrin-B1–Fc protein) or surface bound and by the concentration of the available ligand. Lower levels of endogenous ligand may lead to morphologically less pronounced but longer lasting effects, which may nevertheless substantially affect permeability between cells.

Ephrin-B `reverse' signaling has also been shown to regulate cell morphology though reorganization of the actin cytoskeleton (Cowan and Henkemeyer, 2001Go; Noren and Pasquale, 2004Go). Ephrin-B1 reverse signaling should occur in the thin limb of the loop of Henle and the distal straight tubules, where EphB2 is also expressed. EphB6 may also stimulate ephrin-B1 signaling in the proximal tubules, although the interaction between EphB6 and ephrin-B1 has been reported to be of low affinity (Munthe et al., 2000Go). However, we did not observe morphological changes following stimulation of primary kidney medullary cells with EphB2-Fc. Further investigation is be required to define the role of ephrin-B1 signaling in kidney tubule cells.

During urine production in the loop of Henle, water is reabsorbed exclusively in the thin limb. By contrast, cations are reabsorbed primarily in the distal straight tubules, which are formed by cuboidal epithelial cells that have deep basal infolds filled with mitochondria. A key element in solute reabsorption, the Na+-K+-ATPase, is exclusively located in the lateral membranes of these basal infolds. Cation reabsorption in the distal straight tubules occurs both through a transcellular pathway by active transport and through a paracellular pathway due to `leaky' tight junctions and a positive voltage in the lumen of the tubules (Hebert, 1992Go; Kaissling and Kriz, 1992Go; Koeppen and Stanton, 1996Go). Our results suggest a model where EphB2 signaling regulates the permeability of cell-cell junctions as well as the depth of infolds in the epithelium and the width of gaps between infolds through membrane retraction and rearrangement of the sites of adhesion to the underlying basal lamina (Fig. 9). It will be interesting to investigate whether defective EphB2 signaling may aberrantly regulate the permeability of tubule cells in the renal medulla and possibly underlie kidney diseases by causing inefficient reabsorption of water and cations.


Figure 9
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  		Fig. 9. Schematic drawings illustrating possible effects of EphB2 signaling in medullary kidney epithelial cells. On the basis of the effects of ephrin-B1–Fc treatment in cultured medullary tubule cells, we propose that, in the kidney, EphB2 might regulate the geometry of the basal infolds and the gaps between cells through membrane retraction and remodeling of cell-matrix adhesion sites.

 


   Materials and methods
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Animals
Balb/c mice of either sex, kept under standard housing and feeding conditions, were used for these experiments. The animals aged 8-12 weeks were for immunohistochemistry, RT-PCR and immunoblotting, and 5-8 weeks for primary culture of tubule cells. Under ether anaesthesia, the kidneys were intracardially perfused with Hanks' balanced salt solution (HBSS) and removed. Animal experimentation protocols were approved by the Osaka Prefecture University's Animal Research Committee.

Antibodies
Two different anti-ephrin-B1, three anti-EphB2, and one anti-EphB3, EphB4 and EphB6 antibodies were used. Affinity purified rabbit polyclonal anti-EphB2 antibodies, anti-EphB2p and anti-EphB2g were prepared using 10 and 99 C-terminal amino acids of the chicken receptor, respectively, as described (Pasquale, 1991Go; Soans et al., 1996Go). The former is 100% and the latter is 92% identical to the corresponding mouse amino acid sequence, respectively. Goat anti-ephrin-B1, anti-EphB2 (we named anti-ephrin-B1e and anti-EphB2e, respectively, in the present study), anti-EphB3, anti-EphB4 and anti-EphB6 polyclonal antibodies against mouse extracellular domains of the respective proteins were from R&D Systems, Inc. (Minneapolis, MN). Rabbit anti-ephrin-B1 polyclonal antibody against the carboxy terminus (anti-ephrin-B1p) and rabbit anti-RhoA were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-human Rac1 monoclonal antibody and horseradish peroxidase (HRP) conjugated anti-phosphotyrosine antibody, PY20 were from BD Transduction Laboratories (San Jose, CA). Anti-human vinculin monoclonal antibody (hVIN-1) was from Sigma-Aldrich Japan K.K. (Tokyo, Japan). HRP-conjugate mouse anti-goat IgG and fluorescein (FITC)-conjugated goat anti-mouse IgG were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Reverse Transcription PCR (RT-PCR) analysis
Total RNA was isolated from the kidney including cortex and/or medulla, or tubule cells by TRIZOL reagent (Invitrogen Japan K.K., Tokyo). 1 µg of the total RNA was transcribed into first-strand cDNA using M-MLV reverse transcriptase (RNase H minus, point mutant, Promega, Madison, WI) and oligo(dT)18 primer according to the manufacture's instruction. For the detection of endogenous ephrin-B ligands and EphB receptors, 1 µl of the reaction mix (out of 25 µl in total) was amplified by PCR conducted for 36 cycles with reverse transcribed DNA as template. The extract without RT reaction was used as a template for the negative control. After amplification, PCR products were separated on 1 or 1.2% agarose gels and visualized by ethidium bromide staining. Expression levels of ephrin-B1, EphB2 and EphB6 mRNAs were compared in (1) the cortex and medulla and (2) cortical and medullary tubule cells. They were determined from three independent experiments and normalized by reference to expression levels of GAPDH mRNA (PCR for 23 cycles). The following primer pairs was used: ephrin-B1, 5'-TGCTTGATCCCAATGTACTG-3' (forward), 5'-CGGAGCTTGAGTAGTAGGAC-3' (reverse); ephrin-B2, 5'-ACCCACAGATAGGAGACAAA-3' (forward), 5'-GGTTGATCCAGCAGAACTTG-3' (reverse); ephrin-B3, 5'-CCGCTTCACCATCAAGTTCC-3' (forward), 5'-TCACCGCTCACCTTCTCGTA-3' (reverse); EphB1, 5'-AATGGCATCATCCTGGACTA-3' (forward), 5'-TCAATCTCCTTGGCAAACTC-3' (reverse); EphB2, 5'-CGACGAGAACATGAACACTA-3' (forward), 5'-CCCGTTACAGTAGAGTTTGA-3' (reverse); EphB3, 5'-TGAGACCTCGCTAATCCTCG-3' (forward), 5'-TGTCCGTAACCCGCTACTGT-3' (reverse); EphB4, 5'-AGCCCCAAATAGGAGACGAG-3' (forward), 5'-GGATAGCCCATGACAGGATC-3' (reverse); EphB6, 5'-CCGAGAGACCTTCACCCTTT-3' (forward), 5'-CCTGCCTTCGCCATTACAGT-3' (reverse); Dishevelled, 5'-TAACCTCGCATCCCTGAACC-3' (forward), 5'-ACTGGCGGCTACCTGTAAGT-3' (reverse); GAPDH, 5'-GACTCCACTCACGGCAAATT-3' (forward), 5'-TCCTCAGTGTAGCCCAAGAT-3' (reverse).

Statistical analysis
Analyses were performed with the statistical software package, StatView (SAS Institute Inc., Cary, NC). Values were expressed as mean±s.d. One-way factorial ANOVA and unpaired Student's t-test were carried out for statistical analysis.

Immunoprecipitation and immunoblotting
The kidney or medullary tubule cells stimulated with 1 µg/ml ephrin-B1–Fc (R&D Systems) were homogenized in modified RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10% glycerol, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin). Supernatants were collected after high-speed centrifugation and protein concentrations were measured using a Protein Assay kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturer's protocol. For immunoprecipitations, 800 µg of tissue extracts and 500 µg of cell extracts in 1 ml RIPA buffer were precleared with protein G-sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The samples were incubated overnight at 4°C with 2 µg of anti-EphB2e, 4 µg of anti-EphB2g, or 2 µl of normal rabbit or goat serum, and then with additional 20 µl of protein G-sepharose for 60 minutes at 4°C. After washing, sample buffer with ß-mercaptoethanol was added and immunoprecipitates were boiled for 3 minutes, separated on 7.5 or 10% polyacrylamide gels, and transferred onto PVDF membrane. The PVDF membrane was incubated for 60 minutes at room temperature in 3% BSA in Tris-buffered saline containing 0.1% Triton X-100 (TBS-T), and then for 60 minutes at room temperature in 3% BSA in TBS-T containing 1:10,000 anti-phosphotyrosine antibody (PY20). After washing with TBS-T, immunoblots were developed using ECL chemiluminescence reagents (Amersham Biosciences, Uppsala, Sweden). For reprobing of the same blots, the membrane was incubated for 30 minutes at 50°C in Tris-HCl buffer (pH 6.7) containing 100 mM ß-mercaptoethanol and 2% SDS. Then, the membrane was blocked with 5% nonfat dry milk in TBS-T and reprobed with 0.1 µg/ml anti-EphB2e or 1 µg/ml anti-EphB2g in TBS-T containing 3% BSA and 0.2% nonfat dry milk. After incubation with 1:5000 protein-A peroxidase (ICN Pharmaceuticals, Inc., Aurora, OH) or 1:40,000 HRP-conjugate anti-goat IgG, immunoblots were developed again.

Immunohistochemistry
Kidney was transversally cut into 3-4 mm thick slices and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 3-4 hours at 4°C. After washing with PBS, the tissue slices were immersed in 30% sucrose in PBS overnight and mounted in OTC compound (Sakura Finetechnical Co., Ltd., Tokyo). Cryostat sections, 8 µm in thickness, were immersed in 0.3% hydrogen peroxide for 30 minutes, preincubated in a humid chamber with 3% normal goat or rabbit serum in PBS followed by incubation with primary antibodies at a concentration of 0.2-0.5 µg/ml (anti-ephrin-B1e and anti-ephrin-B1p) or 1-2 µg/ml (anti-EphB2e, anti-EphB2p, anti-EphB3, anti-EphB4, anti-EphB6) overnight at 4°C. To remove background due to endogenous avidin and/or biotin binding sites, avidin and biotin (Avidin/Biotin Blocking Kit, Vector Laboratories, Inc., Burlingame, CA) were added to the blocking and the primary antibody solution, respectively. The sections were incubated with biotinylated goat anti-rabbit or rabbit anti-goat IgG, followed by an avidin-biotin peroxidase complex, and developed by immersing in DAB substrate with or without NiCl2 according to the manufacturer's instruction (Vectastain Elite ABC kit, Vector Laboratories). Some sections developed without NiCl2 were counterstained with hematoxylin. The specificity of the staining was verified by incubations without the primary or secondary antibodies and by the comparison of the different antibodies to the same protein.

Primary culture of renal tubule cells
The isolation procedure for renal tubules was according to that of Richardson et al. (Richardson et al., 1982Go) with some modifications. Kidneys were minced and digested with collagenase (0.02%, Type II, Sigma-Aldrich Japan K.K.) in Ca/Mg-free Hanks' balanced salt solution (HBSS) for 30 minutes at 37°C under gentle stirring. After the digestion, the suspension was filtered through a nylon mesh (100 µm openings) and sedimented at 110 g (800 rpm) for 3 minutes. The material was resuspended in HBSS and fractionated on a discontinuous two-step Percoll gradient consisting of 90% and 41.9% Percoll, and HBSS and made isosmotic by the addition of concentrated (10x) Eagle's Minimum Essential medium (MEM). After centrifugation at 40 g (470 rpm) for 10 minutes at 4°C, fractions were collected from the 0:41.9% interface (Fraction I, rich in distal straight tubules) and the 41.9:90% interface (Fraction II, rich in proximal tubules). Fraction I mainly contained distal tubules, although the thin limbs of the loop of Henle were also present in 10-20% under the inverted microscope. Tubules from each fraction were plated on dishes or glass coverslips coated with Matrigel (growth factor reduced type, BD Biosciences, San Jose, CA) at 2 µg protein/cm2 in PBS for 2.5 hours at 37°C and cultured in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (DME/F12) containing 10% fetal bovine serum. The cultures were maintained in a humidified 5% CO2/95% air incubator at 37°C and medium was regularly changed every 2 days until cells were fully spread. Because of a considerable amount of glomeruli in Fraction I, we selected tubule cells as hyperosmolarity tolerant cells, indicative of Na pump-rich cells, according to the method of Sato and Ozawa (Sato and Ozawa, 1977Go): to remove fibroblastic cells and glomerular epithelial cells, cells from Fraction I were cultured in the medium with additional 12.6 g/l NaCl (to give a final osmolarity of 550 mOsmol/l) for two days and then transferred to normal medium. The purity of tubule cells, as judged by their typical epithelial appearance under the light microscope, was >99%. We call cells from the Fraction I and II medullary and cortical tubule cells, respectively in the present study. The first passage of the primary cells was used in all experiments except for the cell-adhesion and cell-retraction assays in which the second passage was used.

Ligand stimulation and adhesion analysis by time-lapse microscopy
Medullary tubule cells spread on glass coverslips coated with Matrigel (3.5 µg/cm2 protein) were serum-starved for 12 hours in DME/F12 15 mM HEPES and with 2% FBS, and then stimulated with ephrin-B1–Fc (1 µg/ml, R&D Systems, Inc.) or EphB2-Fc (1 µg/ml, R&D Systems, Inc.) with or without anti-human IgG Fc (0.25 µg/ml, ICN Pharmaceuticals, Inc., Aurora, OH) on a heating plate (maintained at 37°C, Tokai Hit Co., Ltd., Fujinomiya, Japan) installed to a inverted microscope stage (DMIRB, Leica Microsystems Ltd., Heerbrugg, Switzerland) covered by a handmade chamber. Phase-contrast images were obtained at 5-minute intervals. For the control, 1 µg/ml human IgG Fc (OEM Concepts, Inc., Toms River, NJ) or the vehicle (PBS) was used. For quantitative analysis of cell adhesional changes, we counted the frequency of retracted cells after a 15-minute stimulation by comparison with images of the corresponding cells at 0 minute. We defined cells in retraction more than 10% of the long or short axis as retracted cells. The cells at the periphery of the epithelial sheets were chosen for this analysis because cell retraction was much easy to assess at the free surface of cells at the periphery of the sheets. More than 300 cells were examined in each stimulation and the results from three independent experiments were summarized as mean±s.d. In some experiments to investigate an implication of RhoA in EphB signaling in medullary tubule cells, 10 µM of Y27632 (Calbiochem, La Jolla, CA) were added to the medium 10 minutes before or 20 minutes after the addition of ephrin-B1–Fc. For the quantitative analysis, we counted the frequency of retracted cells.

Visualization of adhesion complexes
Medullary tubule cells on glass coverslips coated with Matrigel (3.5 µg/cm2 protein) were serum-starved for 12 hours and stimulated with ephrin-B1–Fc, EphB2-Fc or human IgG Fc at a concentration of 1 µg/ml for 15 minutes at 37°C. In some experiments cells were preincubated with 10 µM Y27632 for 5 minutes at 37°C before the stimulation with ephrin-B1–Fc or IgG Fc. The cells were fixed with 2% paraformaldehyde in PBS containing 0.1% Triton X-100 for 15 minutes at room temperature, rinsed with PBS and then preincubated in a humid chamber with 3% NGS-PBS, followed by incubation with anti-vinculin monoclonal antibody at a concentration of 1:800 in 3% NGS-PBS for 30 minutes at 4°C. After washing with PBS, cells were incubated with FITC-conjugated goat anti-mouse IgG for 30 minutes at 4°C, followed by washing with PBS and mounted with Permafluor (Immunotech, Marseille, France). Cells were photographed with a microscope (IX-70, Olympus, Tokyo) equipped with a cooled CCD camera (UIC-QE; Molecular Devices Co., Sunnyvale, CA) controlled by MetaMorph software (Molecular Devices Co.).

Cell-adhesion assay
48-well plates (Falcon) were coated with Matrigel (6.5 µg/cm2 protein) or ephrin-B1–Fc (0.3 µg/cm2) together with Matrigel at 37°C for 2.5 hours. Nonspecific binding sites were blocked with 1% BSA in PBS for 1 hour at room temperature. Medullary tubule cells were serum-starved for 12 hours in DME/F12 with 2% FBS. After washing with PBS, cells were detached by incubation with 5 mM EDTA in Ca/Mg-free HBSS for 5-10 minutes and then with 0.025% trypsin/0.01% EDTA for 1 minute, plated in duplicate at a density of 1.8x104 cells per well and allowed to adhere for 40 minutes at 37°C. Ephrin-B1–Fc (2.0 µg/ml) or human IgG Fc (2.0 µg/ml) together with or without anti-human IgG Fc (0.5 µg/ml) was added at the time of plating. Nonadherent cells were dislodged by tapping the side of the plate until the cells plated on 1% BSA-coated wells (without Matrigel) were fully detached (several taps). After washing with PBS, adherent cells were fixed with 4% paraformaldehyde in PBS, stained with hematoxylin, and counted in an area of 1 mm2 of a central region of the wells. The results from three independent experiments were averaged. Values were normalized to the control (stimulation with human Fc).

Cell-retraction assay with EphB2 binding peptide
The cell-retraction assay was carried out according to the method of Koolpe et al. (Koolpe et al., 2005Go) with some modifications. Tissue culture chamber slide (10x10 mm well, 8 wells/slide; Asahi Techno Glass Co., Tokyo) were coated with Matrigel (3.5 µg/cm2 protein) at 37°C for 2.5 hours. After washing with PBS, medullary tubule cells were detached with 0.25% trypsin/0.01% EDTA and plated at a density of 1.0x104 cells per well. Cells were serum-starved for 12 hours in DME/F12 with 2% FBS and incubated for 15 minutes with 360 µM SNEW peptide that selectively binds to EphB2 and blocks ephrin-binding (Koolpe et al., 2005Go). In the control, cells were incubated with an equal volume of HBSS or a control peptide of the same length as SNEW but that does not bind any Eph receptor. Then cells were either left untreated or stimulated with 1.5 µg/ml ephrin-B1–Fc for 15 minutes. The cells were then fixed in 4% formaldehyde, permeabilized in 0.1% Triton X-100 in PBS, stained with Alexa 546-labeled phalloidin (Molecular Probes) and mounted with Permafluor.

RhoA and Rac1 activation assay
RhoA and Rac1 activity were determined by measurement of RhoA-GTP binding to glutathione S-transferase (GST)-RhoA-binding domain (RBD) and Rac1-GTP binding to GST-p21-binding domain (PBD) in a pull down assay, respectively, using a Rho assay reagent (GST-RBD of the RhoA effector Rhotekin in glutathione-agarose slurry; Upstat, Lake Placid) and Rac assay reagent (GST-PBD of PAK1 in glutathione-agarose slurry; Upstat) according to the manufacturer's instructions with a miner modification. Medullary tubule cells serum-starved for 12 hours and stimulated with ephrin-B1–Fc or human IgG Fc (for the control) at a concentration of 1 µg/ml for 5 minutes and/or 15 minutes at 37°C were washed with ice-cold TBS and lysed in RIPA buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2% glycerol, 10 mM sodium fluoride, 1 mM sodium orthovanadate) containing protease inhibitors. Supernatants were collected after high-speed centrifugation and protein concentrations were measured using a Protein Assay kit (Bio-Rad). 250 µg of cell extracts in 0.5 ml RIPA buffer were incubated with 25 µl of the Rho or 10 µl of the Rac assay reagent for 60 minutes at 4°C. The samples were washed three times with RIPA buffer without SDS, and RhoA and Rac1 protein were detected by western blotting (see immunobotting) with anti-RhoA (0.4 µg/ml) and anti-Rac1 (0.25 µg/ml), respectively. Activation levels were determined from three independent experiments and normalized by reference to band densities of the control.


   Acknowledgments
 
We are grateful to Y. Yamamoto (Nihon Molecular Devices Corp.) for technical assistance with fluorescence microscopy. This study was supported by a Special Research Grant from the Graduate School of Osaka Prefecture University (K.O.) and NIH grants (E.B.P.).


   References
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and methods
 References
 

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