Nectin proteins are expressed at early stages of nephrogenesis and play a role in renal epithelial cell morphogenesis

Abstract

Development of the nephron requires conversion of the metanephric mesenchyme into tubular epithelial structures with specifically organized intercellular junctions. The nectin proteins are a family of transmembrane proteins that dimerize to form intercellular junctional complexes between epithelial cells. In this study, we demonstrate that nectin junctions appear during the earliest stages of epithelial cell morphogenesis in the murine nephron concurrently with the transition of mesenchymal cells into epithelial cells. We have defined the role of nectin during epithelial cell morphogenesis by studying nectin in a three-dimensional culture of Madin-Darby canine kidney (MDCK) cells. In a three-dimensional culture of MDCK cells grown in purified type 1 collagen, expression of a dominant negative form of nectin causes disruption of the formation of cell polarity and disruption of tight junction (TJ) formation, as measured by zonula occludens-1 (ZO-1) localization. In MDCK cells cultured in Matrigel, exogenous expression of nectin-1 causes disruption of normal epithelial cell cyst formation and decreased apoptosis. These data demonstrate that nectins play an important role in normal epithelial cell morphogenesis and may play a role in mesenchymal-to-epithelial transition during nephrogenesis by providing an antiapoptotic signal and promoting the formation of TJs and cell polarity.

the nectins are a family of transmembrane proteins that contain three IgG domains, a single transmembrane domain, and a cytosolic terminus that contains a PDZ-domain binding sequence. Four family members have been identified which are widely distributed throughout the tissues of the body (23, 25, 31). Nectins 1–3 are found in varying amounts in the kidney by Northern blot analysis (25). In cell culture, nectins form homodimers on the surface of cells and trans dimers between cells to form a junctional complex in the apical region of the adherens junction (AJ) in epithelial cells (16, 29, 35). When the AJ first forms between two epithelial cells, a primordial spot junction forms which contains E-cadherin, nectins, α- and β-catenins, and zonula occludens-1 (ZO-1) (21, 30, 33). This spot junction matures into a line-like AJ. At the apical end of the AJ in epithelial cells, claudin, occludin, junctional adhesion molecule (JAM), and other proteins are recruited and a tight junction (TJ) is formed. Nectin dimers appear to stabilize early E-cadherin junctions in a cooperative fashion via the association of nectin with the actin binding protein afadin (13, 24). In addition, nectin can recruit ZO-1 and JAM and influence TJ assembly (7, 34, 36).

The effect of nectins on junctional formation has been documented in vitro and in vivo, but the role of nectins in the kidney during three-dimensional epithelial cell morphogenesis has not been studied. In the developing murine kidney at the tip of each terminal branch of the ureteric bud, there is a coinduction event between the metanephric mesenchyme and the ureteric bud tip. Induced mesenchymal cells undergo a mesenchymal-to-epithelial transition (MET) to become the epithelial cells of the renal vesicle with a classic polarized structure with TJs, AJs, and segregated apical and basolateral markers (5, 6, 12, 26). MET is accompanied by the upregulation of a variety of epithelial markers, including ion channels, E-cadherin, ZO-1, and occludin (6, 14, 38). The role of the nectin proteins in this MET has not been defined.

The development of epithelial cell polarity such as during MET requires a well-defined series of events, including the formation of the TJ through the coordinated action of several signaling complexes including the partitioning-defective protein (PAR) complex (27). The PAR complex consists of PAR-3, PAR-6, and atypical protein kinase C and is a master regulator of cellular polarity conserved across the animal kingdom. The PDZ domain consensus sequence of nectin can bind PAR-3 as well as afadin and may be important in the recruitment of the PAR complex to the region of the TJ and the formation of cell polarity (8, 19, 32). In Madin-Darby canine kidney (MDCK) cells in a two-dimensional culture, PAR-3, nectin, and afadin cooperatively regulate formation of AJs and TJs (3, 19). The role of PAR-3 in MET during renal development has not been investigated.

In the studies presented here, we demonstrate that nectins appear during nephrogenesis when the condensed mesenchyme trans-differentiates into epithelial cells and that nectins partially colocalize with PAR-3 in the region of the TJ in renal vesicles. In addition, we evaluate the role of nectin in epithelial morphogenesis using a three-dimensional culture of MDCK cells as a model system. Expression of a truncated dominant negative form of nectin-1 that is unable to bind to afadin in a three-dimensional culture of MDCK cells grown in purified type-1 collagen leads to a disruption of cell polarity and abnormal lumen formation. In Matrigel, exogenous expression of full-length nectin leads to recruitment of PAR-3 to sites of nectin junctions. In addition, in MDCK cells cultured in Matrigel exogenous expression of full-length nectin causes decreased apoptosis and results in abnormal epithelial cell morphogenesis. These data demonstrate that nectins may be important for the MET seen during nephrogenesis by providing an antiapoptotic signal and contributing to the formation of polarity via recruitment of PAR-3.

MATERIALS AND METHODS

Plasmid construction and cell lines.

The expression constructs [FLAG-tagged, full-length nectin expressing nectin-1α (FL-nectin), dominant negative nectin-1 (ΔC-nectin), and green fluorescent protein (GFP)-nectin] for human nectin-1α were previously described by the authors (8). For this study, the nectin-1α open reading frames were cloned into the pAD-tet expression construct under control of the tetracycline-regulated promoter (1). Clonal cell lines were generated by cotransfection of pAD-tet constructs and a blasticidin resistance plasmid (pcDNA6/V5-His) into the T23 clone of MDCK type 2 cells expressing the tTa transactivator previously generated by the authors (1). Cells were selected with blasticidin, and clones were chosen and screened for expression of nectin under control of doxycycline. The cell lines were tested for mycoplasma, and the GFP-nectin cell line was found to be infected with mycoplasma.

MDCK cell culture.

For propagation, MDCK type 2 cells were maintained in MEM containing Earle's balanced salt solution (Cellgro, Washington, DC) with added 5% fetal calf serum (Hyclone, Logan, UT) or DMEM (Invitrogen, Carlsbad, CA) with 10% fetal calf serum and antibiotics (17). For growth in Matrigel, MDCK cells were trypsinized 1 day before plating and plated at lower density. On the day of plating, plastic tissue culture wells were coated with a commercial preparation of the basement membrane-like material produced by Engelbreth-Holm-Swarm sarcoma tumor cells (Matrigel, BD Biosciences, San Jose, CA). Cells were trypsinized and resuspended into a single-cell suspension containing media and 2% Matrigel and then plated at a density of 30,000 cells/ml in media with or without doxycycline. Cultures were fed after 2 days with media supplemented with 2% Matrigel. For growth in collagen, cells were trypsinized 1 day before plating as above. On the day of plating, cells were resuspended as a single-cell suspension as above and then diluted in a type 1 collagen solution (66% Vitrogen, 3 mg/ml, Cohesion, Palo Alto, CA) to give a density of 40,000 cells/ml collagen. One hundred sixty microliters of the collagen mixture was plated onto a 10-mm filter. The collagen solution was allowed to gel at 37°C, and then media with or without doxycycline was added. Doxycycline induction was maintained throughout the growth of the cysts.

Immunohistochemistry and confocal microscopy.

Primary nectin antibodies were generated by the authors. PAR-3 (Millipore, Billerica, MA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA), and occludin (BD Biosciences) antibodies were purchased. Anti-ZO-1 antibody was a gift from Bruce Stevenson. DBA lectin was obtained from Vector Labs (Burlingame, CA). For immunofluorescence staining of cysts, cysts were rinsed with PBS containing 1 mM CaCl₂ and 0.5 mM MgCl₂, fixed for 30 min with 4% paraformaldehyde in PBS, and blocked and permeabilized using 0.7% fish skin gelatin in PBS with 0.025% saponin (PFS) for 1 h. For cysts grown in collagen, the collagen gel was digested for 10 min with collagenase type VII (Sigma-Aldrich) at 37°C before fixation. Primary antibodies were incubated in the PFS solution with 0.02% azide at 4°C overnight. After extensive washing with PFS, samples were incubated with fluorescent-conjugated secondary antibodies in PFS. Finally, samples were washed with PFS and PBS and then mounted using ProLong mounting media (Molecular Probes). For confocal microscopy, images were collected using a krypton-argon laser coupled to a Zeiss 510 confocal head and an Optiphot II Nikon Microscope. Cysts were imaged in the x-y plane of the sample at 0.5- to 2.0-μm increments. Images were analyzed using Adobe Photoshop. For mouse kidney immunohistochemistry, embryonic day 15 (e15) and 18 (e18) mouse kidneys were dissected out of euthanized fetuses and then fixed for 30–120 min with 4% paraformaldehyde in PBS, and embedded in OCT. Five-micrometer sections were permeabilized with PBS+0.5% Triton X-100 and then blocked with normal goat serum (NGS). Primary antibodies were incubated in PBS with NGS at 4°C for 18–24 h, and fluorescent-conjugated secondary antibodies (Molecular Probes) for 1–2 h. Sections were washed with PBS and then mounted using ProLong Gold mounting media, and imaging acquisition and processing were performed as above.

Anti-cleaved caspase-3 measurement of apoptosis.

MDCK cell cysts were grown for 5 days and then fixed and stained as described above. Anti-cleaved caspase-3 antibody (Cell Signaling) was used, and nuclei were costained using Hoechst 33342 (Molecular Probes). To measure the percentage of apoptotic cells in the lumen, a single 2-μm slice was obtained in the x-y plane at the largest point in a cyst and the number of nuclei was compared with the number of cells staining positive for cleaved caspase-3. For each experimental condition, 30–50 cysts were analyzed. For measurement of apoptosis in ΔC-nectin cysts, cells were grown in collagen as above and then analyzed by Western blot analysis using anti-cleaved caspase-3 antibody. Blots were scanned, digitized, and quantitated. The results shown represent three replicates for each condition.

Measurement of cell proliferation.

Five-day MDCK cell cysts were fixed and stained as described above using anti-human Ki-67 antigen antibody (Zymed). Nuclei were costained using Hoechst 33342. To measure the percentage of dividing cells in the lumen, a single 2-μm slice was obtained in the x-y plane at the largest point in a cyst and the number of nuclei in the luminal space was compared with the number of cells staining positive for Ki-67. For each experimental condition representing 100–200 cells, 30–50 cysts were analyzed.

Statistical analysis.

Data were analyzed using Microsoft Excel (Microsoft). P values were calculated using a two-tailed Student's t-test. Error bars represent SD.

RESULTS

Expression of nectin proteins in the embryonic kidney.

The localization of nectin-1, -2, and -3 proteins was determined by immunofluorescence microscopy in e15 (Fig. 1, A–C′) and e18 (Figs. 1, D and D′, 2, and 3) embryonic mouse kidneys. Nectin-4 was not evaluated because it is primarily expressed in the placenta (22). Nectin-2 and -3 were expressed in the ureteric bud (UB) in the region of the TJ as indicated by costaining with Dolichos biflorus agglutinin (DBA; open arrowheads, Fig. 1, B–D′), whereas nectin-1 is not detectably expressed in the ureteric bud (asterisks, Fig. 1, A and A′) as identified by a lack of costaining between nectin-1 and DBA, which stains all ureteric bud-derived structures. In contrast, nectins 1–3 were all expressed in early epithelial structures of the nephron generated from metanephric mesenchyme, such as the renal vesicle (filled arrowheads, Fig. 1, A–C′). Higher magnification images of nectin-2 demonstrate the apical subcellular localization of nectin in the region of the TJ (open arrowheads, Fig. 1, D and D′). This localization of nectin to the region of the TJ in epithelial structures has been previously described in intestinal cells using the same nectin-2 antibody (28). It is possible that some structures identified as renal vesicles may be comma-shaped bodies that have been sectioned so as to make them appear to be renal vesicles.

Fig. 1.

Localization of nectin proteins in ureteric bud and renal vesicles. Five-micrometer sections of embryonic day 15 (e15; A–C′) and e18 (D and D′) mouse kidney were stained with either monoclonal anti-nectin antibody (A, A′, B, B′, D, and D′) or polyclonal anti-nectin anti-body (C and C′), Dolichos biflorus agglutinin (DBA) and Hoechst 33342. A and A′: nectin-1 expression in the renal vesicle (filled arrowhead) but not ureteric bud (asterisks). B, B′, C, and C′: nectin-2 (B and B′) and -3 (C and C′) expression in the renal vesicle (filled arrowhead) and ureteric bud (open arrowhead). D and D′: higher power magnification views illustrate the localization of nectin-2 in the region of the tight junction (open arrowheads). The ureteric bud was identified and labeled based on staining with DBA, and mesenchyme-derived renal vesicles were identified by lack of DBA staining and morphology. Scale bars = 10 μm.

Fig. 2.

Partial colocalization of nectin and PAR-3. Five-micrometer sections of e18 mouse kidney were stained with monoclonal anti-nectin-2 antibody (A′ and A′′′), anti-partitioning-defective protein 3 (PAR-3) antibody (A and A′′′), and Hoechst 33342. Ureteric bud (UB) and renal vesicles (RV) are labeled using arrows. B and C: higher magnification views of UB and RV that demonstrate regions where nectin and PAR-3 colocalize (filled arrowheads) and regions where nectin and PAR-3 are coexpressed but not colocalized (open arrowheads). It is possible that structures labeled as RV may be comma-shaped bodies that appear to be RV due to artifacts of sectioning. Scale bars = 10 μm.

Fig. 3.

Nectin-2, but not occludin, is expressed in RV, comma-shaped bodies, and S-shaped bodies. Five-micrometer sections of e18 mouse kidney were stained with monoclonal anti-nectin-2 antibody (A′, A′′′, and B–E) and polyclonal anti-occludin antibody (A, A′′′, and B–E), Hoechst 33342 (A′′, A′′′, and B–D), and lotus lectin (LTL; blue staining, E). UB and RV are labeled using arrows. Open arrowheads demonstrate punctate nectin-2 and occludin staining in UB (A–A′′′). In B at high magnification, yellow indicates colocalization of nectin-2 and occludin, and green and red indicate areas where nectin-2 and occludin did not colocalize (open arrowheads and filled arrowheads, respectively). Asterisks demonstrate RV, comma-shaped body, and S-shaped body where nectin-2 is expressed, but occludin is not (A–A′′′, C, and D). Scale bars = 10 μm.

Nectin partially colocalizes with PAR-3 in renal vesicles.

Because the PAR complex of proteins is known to be critical for the development of cell polarity, we examined the localization of PAR-3 and individual nectin proteins in the renal vesicles. Nectins-1, -2, and -3 partially colocalized with PAR-3 in punctuate structures at the apical region of the cells of the renal vesicles (data shown for nectin-2: yellow staining marked by filled arrowheads, Fig. 2, B and C). PAR-3 was also found in the apical region of the epithelial cells of the renal vesicle not associated with nectins (green staining marked by open arrowheads, Fig. 2, B and C). Because of limited availability of different species of PAR-3 antibody, we were unable to colocalize PAR-3, nectins, and a TJ marker directly; however, the punctuate regions of colocalization appear to be in the apical region, where TJs are found. In addition, nectins-2 and -3 also colocalized with PAR-3 in the ureteric bud (data shown for nectin-2: filled arrowhead, Fig. 2C). In reviewing more than 300 early nephrons, we were unable to identify any structures in which PAR-3 was not, at least, partially colocalized with nectins. The direct binding of PAR-3 to nectins-1 and -3 has been demonstrated in neurons (32); however, we were unable to demonstrate a biochemical interaction between nectin and PAR-3 in embryonic kidneys by coimmunoprecipitation (data not shown). These data suggest that in vivo during nephrogenesis nectins and PAR-3 may interact as has been demonstrated in neurons.

Nectin appears before occludin in renal vesicles.

Since it is known that nectins can influence the accumulation of occludin at nascent AJs in vitro, we examined the localization of occludin in nascent epithelia of the renal vesicle. In renal vesicles expressing nectin-2, there was no detectable expression of occludin (asterisks, Fig. 3, A–A′′′), whereas in the ureteric bud nectin-2 and occludin were coexpressed (open arrowheads, Fig. 3, A–A′′′). In the ureteric bud where both nectin-2 and occludin were expressed, there was a consistent pattern of nectin-2 being present in a subapical area just basal to the TJ as labeled by occludin. There was some colocalization of the two proteins, as indicated by the yellow color (Fig. 3B), and there was an area at the apical end of the TJ where occludin alone was present (green staining at open arrowheads, Fig. 3B), and an area just basal to the area of colocalization where nectin-2 alone was present (red staining at filled arrowheads, Fig. 3B). This staining is similar to the previously described localization of nectin-2 in intestine based on electron microscopy (28). In comma-shaped bodies and S-shaped bodies, occludin remained absent while nectin was present, as indicated by asterisks in Fig. 3, C and D (filled arrowheads indicate ureteric bud where occludin and nectin were coexpressed as a positive control for occludin staining, Fig. 3, C and D). In proximal tubules which are later mesenchyme-derived structures, nectin and occludin are coexpressed, as indicated by staining for nectin and occludin (arrowheads mark nectin/occludin costaining, Fig. 3E) in tubules that stain positive for lotus lectin (LTL; arrow marking blue LTL staining, Fig. 3E). While the appearance of nectin in the nascent renal vesicles, comma-shaped bodies, and S-shaped bodies in the absence of occludin and the coexpression of nectin and occludin in later mesenchyme-derived proximal tubules are not direct evidence that nectin recruits occludin to TJs in developing mesenchyme-derived tubules, the appearance of nectin before occludin in mesenchyme-derived tubules suggests that in vivo as in vitro, nectins may recruit occludin to the area of the TJ and contribute to TJ formation.

Expression of dominant negative nectin in MDCK cell cysts disrupts epithelial cell polarity.

To study the role of nectin in epithelial morphogenesis and polarity we used a three-dimensional culture of MDCK cells. When MDCK cells are cultured as a single-cell suspension in purified type I collagen or Matrigel, over the course of 7–10 days (type I collagen) or 3–4 days (Matrigel), the cells develop into three-dimensional cyst structures with a polarized monolayer of epithelial cells surrounding a fluid-filled lumen (18). This process is similar to MET during nephrogenesis, as the MDCK cells must generate de novo cell polarity by sensing the surrounding extracellular matrix.

We exogenously expressed a dominant negative form of human nectin-1α in MDCK cells under control of the Tet-trans-activator (ΔC-nectin). This dominant negative nectin lacks the terminal four amino acids that have been shown previously to be required for nectin binding to the PDZ domains of afadin and PAR-3 (Fig. 4A) (28, 32). In MDCK cell cysts grown in purified type I collagen from cells containing the ΔC-nectin plasmid, removal of doxycycline from the media led to expression of ΔC-nectin and disruption of cyst morphology. Cysts expressing ΔC-nectin demonstrated disorganized epithelial polarity at early time points and showed abnormal localization of ZO-1 (asterisks, Fig. 4B′′) on the exterior of the cysts. Cysts expressing ΔC-nectin also showed abnormal localization of other markers of polarity including β-catenin and podocalyxin/gp135 (Supplementary Fig. 1F; all supplementary material for this article is accessible on the journal website). In addition, these abnormal cysts did not form a single central lumen even after 10 days in culture (Fig. 4C′′). There did appear to be some rescue of epithelial cell morphology over time. By 10 days in culture, multiple small lumens were able to form surrounded by punctate ZO-1 staining (arrow, Fig. 4C′′). In contrast, by 10 days in control cysts a single central lumen was formed with punctate ZO-1 localization surrounding the lumen (arrowhead, Fig. 4C′′). Quantification of the percentages of cysts with a single central lumen demonstrated that 71 ± 4.8% of control cysts had a single central lumen, whereas only 18 ± 1.4% of ΔC-nectin cysts (P = 0.02) had a single central lumen (Fig. 4D).

Fig. 4.

ΔC-nectin disrupts Madin-Darby canine kidney (MDCK) cell cystogenesis. A: expression constructs used in this paper are illustrated with expression demonstrated by Western blotting using anti-green fluorescent protein (GFP) antibody (Roche) and anti-FLAG antibody (Sigma). B and C: MDCK cell cysts were grown in purified type I collagen in the presence (Control) or absence of doxycycline (ΔC-nectin). Cysts were fixed and stained with anti-FLAG antibody (Sigma), anti-zonula occludens (ZO)-1 antibody (red), and Hoechst 33342 (blue; Molecular Probes), and ∼1.5-μm optical sections were obtained using a Zeiss 510 confocal microscope. Expression of ΔC-nectin causes disorganized cyst morphology with no central lumen (B′′ and C′′). Note the normal lumen formed in the absence of exogenous ΔC-nectin (C′ compared with C′′). In the absence of ΔC-nectin expression, ZO-1 shows punctate staining in the region of the tight junction surrounding the lumen (filled arrowhead, B′ and C′). At 5 days, ZO-1 localization is disrupted in ΔC-nectin cysts, including having ZO-1 localized on the outside of cysts (asterisks, B′′), whereas, at 10 days some small lumens are formed in ΔC-nectin cysts with punctate ZO-1 staining (arrow, C′′). D: for quantification of cyst morphology, cysts were harvested at 10 days and processed as described above. Individual cysts were then scored as normal if they had a single central lumen and abnormal if they had no lumen or multiple lumens. Values shown are the means ± SD for 3 separate experiments, and a minimum of 50 cysts were evaluated for each condition for each experiment.

Interestingly, when ΔC-nectin cells were grown in Matrigel in the absence of doxycycline, they formed normal cysts with normal polarity and a single normal central lumen despite expression of ΔC-nectin (Fig. 5, B–B′′ and Supplementary Fig. 1C). Matrigel contains multiple extracellular matrix components including collagen IV, entactin, laminin, and growth factors. Our laboratory has previously demonstrated that addition of laminin to type I collagen has been able to rescue inversion of polarity caused by DN-Rac 1 or β1-integrin blocking antibody (37); however, exogenous addition of laminin to type I collagen was unable to restore normal cyst formation in ΔC-nectin cysts (data not shown).

Fig. 5.

FLAG-tagged, full-length nectin expressing nectin-1α (FL-nectin), but not ΔC-nectin, recruits PAR-3. MDCK cell cysts were grown in Matrigel in the presence (Control, C–C′′) or absence [FL-nectin (A–A′′) and ΔC-nectin (B–B′′)] of doxycycline and stained with anti-FLAG antibody, anti-PAR-3, and Hoechst 33342. Approximately 1-μm optical sections were obtained using a Zeiss 510 confocal microscope. Note colocalization of PAR-3 and FL-nectin in A–A′′ (arrows) and the absence of colocalization with ΔC-nectin in B–B′′ (open arrowheads). PAR-3 was also found not localized with FL-nectin in some areas (filled arrowheads, A–A′′).

Recruitment of PAR-3 to sites of nectin localization.

We hypothesized that ΔC-nectin affects polarity by disrupting the recruitment of PAR-3 to AJs and TJs in the developing cysts since the COOH-terminal four amino acids are known to be required for interaction with PAR-3. We evaluated PAR-3 localization in ΔC-nectin cysts compared with FL-nectin under control of the Tet-trans-activator in Matrigel. Expression of FL-nectin led to recruitment of PAR-3 to some of the cell-cell junctions where FL-nectin is localized (arrows, Fig. 5, A–A′′). PAR-3 also appeared to localize at the cell surface in the region of the TJ in the absence of FL-nectin (filled arrowheads, Fig. 5, A–A′′). In contrast, in ΔC-nectin cysts, there was no recruitment of PAR-3 to cell-cell junctions where ΔC-nectin was localized (open arrowheads, Fig. 5, B′ and B′′).

Exogenous expression of wild-type nectin causes decreased apoptosis and lumen filling in MDCK cell cysts.

Another feature of the cysts expressing FL-nectin is that they demonstrated filling of the lumen, which was not seen in either ΔC-nectin cysts or parent cell cysts when plated in Matrigel. To further understand this phenotype of lumen filling seen in the FL-nectin cell line, we evaluated the effects of expressing a GFP-tagged full-length nectin (GFP-nectin) as well as FL-nectin on cyst morphology. Similar to FL-nectin, exogenous expression of GFP-nectin disrupted the formation of a normal lumen in MDCK cells grown in Matrigel (Fig. 6A compared with Fig. 6B). While the cysts appeared to develop normal polarity, instead of having a lumen clear of cells, cysts expressing exogenous FL-nectin and GFP-nectin had lumens filled with cells. These appeared to be polyplike structures with an apical surface facing what remained of the luminal space and with normal orientation of β-catenin and podocalyxin/gp135 (Supplementary Fig. 1B).

Fig. 6.

Expression of exogenous FL-nectin and GFP-nectin decreases luminal apoptosis. A and B: MDCK cell cysts were grown in Matrigel in the presence (Control, A) or absence of doxycycline (GFP-nectin, B). Cysts were harvested at 5 days, fixed with paraformaldehyde, and stained for active caspase-3 (Cell Signaling) and nuclei using Hoechst 33342. Optical sections (1.5 μm) were obtained using a Zeiss 510 confocal microscope. C: for quantification, individual cells within the lumen were then scored for apoptosis based on positive staining for cleaved-caspase 3. Values shown are means ± SD for 3 separate experiments, and a minimum of 30 cysts were evaluated for each condition for each experiment. Results were normalized to cells cultured in doxycycline. Values are means ± SD for 3 separate experiments, and a minimum of 250 cells were evaluated for each condition for each experiment. D: Western blot analysis of MDCK cell cysts grown in collagen in the presence (Control) or absence of doxycycline (ΔC-nectin). Blots were probed with anti-active caspase 3 antibody (Cell Signaling) and then digitized and quantitated. Quantitation shown (right) represents n = 3 for each condition.

In wild-type cysts grown in Matrigel, apoptosis occurs to generate a lumen free of cells in cysts plated at high density (15). In addition, cells that are shed into the lumen at later stages also undergo apoptosis to maintain a clear lumen. In the developing kidney, many metanephric cells that do not undergo MET instead die via apoptosis (10, 12, 20). Caspase 3 is activated during apoptosis and has been used to detect apoptotic cells in three-dimensional epithelial structures (4). To evaluate the amount of apoptosis in cysts expressing exogenous nectin, we stained cysts with anti-active-caspase 3 antibody and counted the number of cells in the lumen that stained positive for anti-active-caspase 3. With expression of exogenous nectin, the percentage of cells in the lumen that stained positive for anti-cleaved-caspase 3 decreased to 56 ± 13% (P = 0.03) in FL-nectin cysts and 44 ± 12% (P = 0.004) in GFP-nectin cysts compared with a small insignificant increase in luminal cells staining positive for anti-active-caspase-3 in the parent line grown in the absence of doxycycline (114 ± 8%, P < 0.05). GFP-nectin cells tested positive for mycoplasma; however, it is unlikely that this impacted these results as the control group for GFP-nectin-expressing cells was the identical cell line grown in the absence of doxycycline. Increased cell proliferation might also contribute to an increase in the number of intraluminal cells. To evaluate cell proliferation in nectin-expressing cysts, we stained for Ki-67 (4). The percentage of cells staining positive for Ki-67 was not statistically different in cysts grown with or without the expression of exogenous nectin; although, in the parent cell line, culturing in the absence of doxycycline did cause a small, statistically insignificant increase in the number of cells staining positive for Ki-67 (data not shown). Because cells expressing ΔC-nectin do not form normal lumens in collagen and thus cannot be evaluated for intraluminal apoptotic cells, we evaluated the effect of ΔC-nectin on apoptosis by Western blot analysis for expression of active caspase 3. Expression of ΔC-nectin caused a 130 ± 56% increase (P < 0.02) in the expression of active caspase 3 compared with control cysts cultured in the presence of doxycycline (Fig. 6D).

DISCUSSION

Renal development requires a complex series of events, from the penetration of the ureteric bud into the metanephric mesenchyme through the trans-differentiation of mesenchyme into epithelia, and culminating in the generation of many thousands of nephrons. During MET, metanephric mesenchyme must develop epithelial polarity and generate a lumen to form renal vesicles. It is known that cell adhesion molecules such as cadherins are both regulated during this process and are required for normal MET (12). Here, we present data that normal nectin function is required for normal epithelial morphogenesis. In vivo, nectin proteins are present at the earliest stages of nephrogenesis in both ureteric bud and renal vesicles. In vitro, normal nectin function is required for normal lumen formation. Taken together, these data suggest that nectin proteins are important for nephrogenesis.

In vivo localization of nectin proteins indicates that nectin cell junctions are present in the ureteric bud and appear concurrently with the trans-differentiation of mesenchyme into epithelial cells. Previous localization of nectin in embryos and intestinal epithelia has demonstrated that nectins are localized to the region of the TJ in epithelial organs (28), at the region of the puncta adherens in neurons (32), and at intercellular junctions in sperm (2). Here, we present the localization of nectin in the developing kidney. Nectin cell junctions are present in polarized epithelia in the developing kidney, similar to cadherins. This confirms that nectins may play a critical role in the polarized epithelia in kidneys as in other organs. In addition, structures derived from metanephric mesenchyme appear to develop nectin cell junctions as a very early event during MET, consistent with the previously proposed early role for nectins in formation of AJs and TJs. This suggests that nectin may play an important role in lumen formation and epithelial polarity during nephrogenesis.

One of the proteins known to be critical for normal epithelial polarity is PAR-3 (27). Nectins-1 and -3 are known to interact with PAR-3 in neurons (32), and colocalization of nectin and PAR-3 has been demonstrated in MDCK cells grown in two-dimensional cultures (19). Here, we demonstrate that PAR-3 partially colocalizes with nectins in the early epithelial structures derived from metanephric mesenchyme. Early published studies of nectins and PAR-3 in embryonic neurons demonstrated nearly complete colocalization of nectins-1 and -3 with PAR-3 (32). Complete colocalization between nectins and PAR-3 was not demonstrated in our studies, which may be due to limitations in antibody or fixation conditions. The colocalization of PAR-3 and nectins in early nephron structures and the known interaction of nectins-1 and -3 with PAR-3 suggest a role for nectins in PAR-3 complex formation in the developing nephron.

It has been demonstrated that nectin and PAR-3 function is required for normal formation of TJs, including localization of occludin (19). We have demonstrated in vivo that nectin appears in the region of the TJ in the absence of occludin in nascent renal vesicles, comma-shaped bodies, and S-shaped bodies but that the two are coexpressed at later stages in the mesenchyme-derived proximal tubule, thus suggesting that nectin-nectin junctions may be important in the recruitment of occludin to TJs following MET. Occludin expression and localization are known to vary both developmentally and in different segments of the kidney (9). It is also possible that this developmental regulation may explain the absence of occludin staining in renal vesicles that demonstrate nectin staining. As in the case of nectin and PAR-3, occludin and nectin only partially colocalize in the ureteric bud and proximal tubule, which may be due to fixation or the nature of the junctions in these structures.

One of the roles that nectin might play during the transition from metanephric mesenchyme to epithelia is in establishing the polarity of the newly derived epithelia, possibly via its interaction with PAR-3. MDCK cells are a well-characterized epithelial cell line that has been used extensively to study epithelial polarity (27). The disruption of polarity in MDCK cell cysts by ΔC-nectin indicates that nectin may play a role in establishing polarity during MET in the kidney. With the available antibodies for PAR-3, we were unable to determine whether PAR-3-nectin colocalization is disrupted in ΔC-nectin cysts grown in collagen. In addition, we were unable to demonstrate a biochemical interaction between PAR-3 and nectins in either kidney or wild-type MDCK cells; thus it remains unclear whether the disruption of polarity by ΔC-nectin is accompanied by a disruption in PAR-3 localization or a nectin-PAR-3 interaction. However, we have demonstrated that PAR-3 does colocalize with exogenously expressed FL-nectin but not ΔC-nectin in cysts grown in Matrigel, thus indicating in a three-dimensional culture system that nectin may recruit PAR-3 to AJs and TJs and that this recruitment is dependent on the COOH-terminal four amino acids of nectin.

Disrupting MET in the developing kidney leads to increased apoptosis of metanephric mesenchyme cells (10, 12, 20). Expression of ΔC-nectin increases apoptosis in MDCK cell cysts. In addition, exogenous expression of nectin-1 in MDCK cells reduces apoptosis and causes the formation of budlike structures in the lumens in MDCK cell cysts grown in Matrigel. While there may be no in vivo correlate of these budlike structures, the antiapoptotic effect of exogenous nectin expression and the proapoptotic effect of expression of ΔC-nectin suggest that nectin-nectin interactions may provide an antiapoptotic signal in nascent renal epithelia and contribute to the survival of metanephric mesenchyme cells that undergo MET. Previous work with cadherins has shown that E-cadherin protects against anoikis via antiapoptotic signaling through β-catenin, c-Src, and phosphatidylinositol 3-kinase/Akt (11). Nectins are known to promote cadherin-cadherin junction formation via c-Src (24), and thus the exogenous expression of nectin may lead to reduced apoptosis by promoting cadherin-cadherin junctions and c-Src activity.

In the developing kidney, cell-cell interactions are critical for ureteric bud branching morphogenesis and MET. We have shown that nectins appear during nephrogenesis after the condensed mesenchyme trans-differentiates into epithelial cells, and nectins colocalize with PAR-3 in the region of the TJ in nascent renal epithelia. Nectins provide important signals for epithelial cell morphogenesis in vitro. In MDCK cells cultured in three dimensions, exogenous expression of full-length nectin causes decreased apoptosis and results in abnormal epithelial cell morphogenesis. Expression of a truncated dominant negative form of nectin-1 that cannot bind afadin or PAR-3 in MDCK cell cysts leads to a disruption of cell polarity and lumen formation as well as increased apoptosis. Nectins may be important for the MET seen during nephrogenesis by providing an antiapoptotic signal, and the binding of nectin to PAR-3 may be important for the recruitment of PAR-3 to nascent TJs and the establishment of apical-basal polarity in mesenchymal cells undergoing MET.

GRANTS

This study was supported by National Institutes of Health Grants K12-HD-00850 and K08-DK-068358 (P. R. Brakeman) and R01-DK-074398 (K. E. Mostov).