Immunolocalization of Ksp-cadherin in the adult and developing rabbit kidney

R. Brent Thomson and Peter S. Aronson

Section of Nephrology, Departments of Internal Medicine and of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8029


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The potential for Ksp-cadherin involvement in either the development or maintenance of the metanephric kidney was assessed by immunocytochemical localization of a monoclonal antibody directed against the rabbit isoform of Ksp-cadherin in both neonatal and adult rabbit kidneys. In the adult kidney Ksp-cadherin expression was detected on the basolateral membrane of all cell types in both the tubular nephron and the collecting system. Immunoelectron microscopy indicated that Ksp-cadherin was expressed at uniform levels along the entire length of both the lateral membranes and the basal infoldings of all tubular epithelial cell types. In the nephrogenic zone of the neonatal rabbit kidney Ksp-cadherin expression was detected exclusively on the basolateral membranes of epithelial cells in the more highly differentiated regions of the expanding ureteric duct. In the highly differentiated corticomedullary and medullary regions of the neonatal kidney, distinct basolateral staining was observed in all segments of the tubular nephron and the collecting system. The relatively late appearance of Ksp-cadherin expression in the developing metanephros indicates that Ksp-cadherin probably does not participate in the direction of renal morphogenesis. However, the high levels of Ksp-cadherin expression observed in all segments of the tubular nephron and the collecting system in the adult kidney suggests that it may play a role in the maintenance of the terminally differentiated tubular epithelial phenotype.

renal development; cell adhesion; membrane polarity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

WE RECENTLY REPORTED THE identification of Ksp-cadherin, a novel kidney-specific member of the cadherin superfamily of cell adhesion molecules (25). cDNA cloning and molecular analysis of Ksp-cadherin indicate that it is a structurally unique member of the cadherin superfamily and that it most closely resembles members of the LI-cadherin/HPT-1 subgroup. Unlike other cadherins, Ksp-cadherin has a distinct organ-specific tissue distribution. Extensive Northern analysis and preliminary immunolocalization studies indicate that expression of Ksp-cadherin is kidney-specific and confined to the basolateral membranes of tubular epithelial cells.

The cadherin superfamily is a large, structurally diverse group of calcium-dependent, membrane-associated cell-adhesion molecules. They are known to be present in virtually every vertebrate tissue type and are regarded as the principal mediators of homotypic cellular recognition and cell-cell adhesion (5). They have been shown to play a crucial role in the early stages of embryogenesis (13, 23) and the morphogenesis of the vertebrate nervous system (22). The nature of their role in the development of epithelial tissues is less clear, but they are known to be capable of directing the formation of epithelial structures in vitro (15) and they are believed to play a critical role in the maintenance of the terminally differentiated epithelial phenotype (6). Cadherin-mediated interactions have also been shown to play a significant role in the maintenance of controlled epithelial cell growth (7). Geiger and Ayalon (5) have suggested that formation of cell-cell junctions via cadherin interactions may actually downregulate substrate adhesion and suppress responsiveness to growth-activating factors. Finally, it has been suggested that cadherins may play a central role in the establishment of a polarized epithelial phenotype (15). Mars et al. (14) have directly demonstrated that E-cadherin expression in cultured cells can induce a redistribution of constitutively expressed proteins to specific membrane domains and the generation of a structurally and functionally distinct epithelial phenotype.

As a first step in the evaluation of the potential for Ksp-cadherin involvement in either the morphogenesis or maintenance of the metanephric kidney, we performed a detailed immunolocalization study of Ksp-cadherin expression in both adult and developing rabbit kidneys. In both the adult kidney and the highly differentiated regions of the developing neonatal kidney, Ksp-cadherin expression was detected on the basolateral membranes of all cell types in both the tubular nephron and the collecting system. Detailed cellular localization studies of Ksp-cadherin expression in adult kidneys indicate that Ksp-cadherin is expressed at relatively uniform levels along the entire length of both the lateral membranes and the basal infoldings of all tubular epithelial cell types. In the nephrogenic zone of the developing kidney, the onset of Ksp-cadherin expression correlates directly with the onset of glomerular filtration and the acquisition of tubular epithelial cell polarity. The relatively late expression of Ksp-cadherin in the developing kidney indicates that it is likely not involved in the initial stages of metanephrogenesis. However, the high levels of Ksp-cadherin expression observed in all segments of the tubular nephron and the collecting system of both the adult kidney and the differentiated regions of the neonatal kidney suggest that Ksp-cadherin may play a role in the stabilization of the terminally differentiated tubular epithelial phenotype.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies. All immunocytochemical analyses performed in this study were conducted with the anti-Ksp-cadherin monoclonal antibody C575-5I. The production and characterization of this antibody were described previously (25). Briefly, the antibody was generated against a COOH-terminal 71-kDa proteolytic fragment of rabbit Ksp-cadherin. C575-5I (10 mg IgG/ml) was used at a dilution of 1:100 for both immunofluorescence staining and immunoperoxidase labeling and at a dilution of 1:10 for immunogold labeling. The anti-E-cadherin antibody (clone 6F9; ICN Pharmaceuticals, Aurora, OH) is a mouse monoclonal antibody directed against the human isoform of E-cadherin and was used at a dilution of 1:10 for immunofluorescence staining. The anti-N-CAM antibody (clone NCAM-OB11; Sigma Chemical, St. Louis, MO) is a mouse monoclonal antibody directed against the rat isoform of N-CAM and was used at a dilution of 1:100 for immunofluorescence staining.

Tissue preparation. Male New Zealand White rabbits were anesthetized by intravenous injection of pentobarbital sodium. Kidneys were cleared (PBS, pH 7.4, 37°C) and fixed (PLP: 2% paraformaldehyde, 750 mM lysine, and 10 mM sodium periodate in phosphate buffer, pH 7.4, 22°C) by retrograde perfusion from a cannula inserted into the descending aorta distal to the renal arteries. The kidneys were removed from the animals, cut into 2- to 4-mm blocks, and postfixed in the same fixative for an additional 6 h at room temperature. Representative blocks of tissue were cut from all regions of the kidney, and the relative orientation of each block was maintained. For immunofluorescence and immunogold labeling the tissue was cryoprotected by incubation in a phosphate buffer (pH 7.2) containing 2.3 M sucrose and 50% polyvinylpyrrolidone (27), mounted on aluminum nails, frozen, and stored in liquid nitrogen. For the immunoperoxidase studies, the tissue was cryoprotected in 10% dimethyl sulfoxide in PBS for 1 h, frozen in liquid nitrogen-cooled isopentane, and stored in liquid nitrogen.

Immunofluorescence staining. Semithin cryosections (0.5 µm) were cut with a Reichert Ultracut E ultramicrotome fitted with an FC-4E cryoattachment and then mounted on gelatin-coated slides. The slides were washed in PBS and then incubated with 1% BSA in PBS for 15 min at 20°C in a humidified chamber to block nonspecific antibody labeling. Primary antibody diluted to the appropriate concentration in blocking buffer was then added, and the sections were incubated for an additional 1 h at 20°C. Sections were washed five times (10 min each wash) and then incubated for 1 h with an FITC-conjugated secondary antibody (FITC-conjugated goat anti-mouse IgG, Zymed Laboratories) diluted to 1:100 with blocking buffer. The slides were washed five times (10 min each wash) and then mounted in VectaShield (Vector, Burlingame, CA) to inhibit fading of the immunofluorescent signal. The slides were then visualized on a Zeiss Axiophot phase-contrast microscope.

Immunoperoxidase labeling. Cryosections (15 µm) were cut on a Jung Frigocut 2800N cryostat, washed in PBS, and then incubated with 1% BSA in PBS for 15 min at 20°C. C575-5I diluted 1:100 with blocking buffer was added, and the sections were incubated for an additional 2 h. The sections were washed ten times (5 min each wash) with blocking buffer and then incubated with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Zymed) for 2 h at 20°C. The sections were washed ten times (5 min each wash) with blocking buffer, fixed in 3% glutaraldehyde, reacted with diaminobenzidene, postfixed in OsO4 reduced with potassium ferrocyanide, and embedded in Epon. Sections were cut, stained with lead citrate, and examined with a Zeiss EM910 electron microscope.

Immunogold labeling. Ultrathin cryosections were cut on a Reichert Ultracut E ultramicrotome fitted with an FC-4E cryoattachment as described by Tokuyasu (26). Sections were labeled with C575-5I diluted 1:10 in 1% BSA in PBS followed by incubation with a goat anti-mouse 10-nm gold-conjugated secondary antibody (Goldmark Biologicals, Phillipsburg, NJ). The labeled sections were postfixed in 2% glutaraldehyde in PBS and absorption stained with 2% uranyl acetate and 0.002% lead citrate in 2.2% polyvinyl alcohol (27).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Immunofluorescence localization of Ksp-cadherin in adult kidney. Localization of Ksp-cadherin expression was determined by immunofluorescence labeling of PLP-fixed semithin cryosections with the anti-Ksp-cadherin monoclonal antibody C575-5I. Anti-Ksp-cadherin antibody labeling was observed on the basolateral membrane of all segments of the tubular nephron and the collecting system. Labeling was not observed in glomeruli, blood vessels, papillary epithelium, or nonepithelial regions of the kidney. Levels of Ksp-cadherin expression were very high in all regions of the cortex (Fig. 1), but were especially pronounced in the thick ascending limb (TAL; Fig. 2) and the distal convoluted tubule (Fig. 3). All cell types of the tubular cortical nephron, including the juxtaglomerular cells of the macula densa (Fig. 4) showed distinct basolateral C575-5I labeling. As reported previously (25) the basal surface of a subpopulation of intercalated cells in both the connecting tubule (Fig. 5) and the cortical collecting duct (Fig. 6) did not appear to be labeled by the anti-Ksp-cadherin antibody. A distinct immunofluorescent signal was, however, associated with the lateral membranes of all intercalated cell types in both the connecting tubule and the collecting duct. Unfortunately, because intercalated cells in the cortex are always interspersed with either connecting tubule cells or principal cells (9), we could not conclusively determine if this immunofluorescent signal was due to antibody labeling of the lateral membranes of the intercalated cells per se or of their adjacent cell partners.


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Fig. 1.   Immunofluorescence localization of Ksp-cadherin in adult rabbit kidney cortex. A: semithin cryosection of adult rabbit kidney cortex labeled with MAb (monoclonal antibody) C575-5I. G, glomerulus; PT, proximal tubule. B: phase-contrast image of A. Magnification ×125.



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Fig. 2.   Immunofluorescence localization of Ksp-cadherin in cortical thick ascending limb of adult rabbit kidney. A: semithin cryosection of adult rabbit kidney cortex labeled with MAb C575-5I. TAL, thick ascending limb; PT, proximal tubule. B: phase-contrast image of A. Magnification ×820.



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Fig. 3.   Immunofluorescence localization of Ksp-cadherin in distal convoluted tubule of adult rabbit kidney. A: semithin cryosection of adult rabbit kidney cortex labeled with MAb C575-5I. DCT, distal convoluted tubule. B: phase-contrast image of A. Magnification ×570.



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Fig. 4.   Immunofluorescence localization of Ksp-cadherin in juxtaglomerular apparatus of adult rabbit kidney. A: semithin cryosection of adult rabbit kidney cortex labeled with MAb C575-5I. MD, macula densa; G, glomerulus. B: phase-contrast image of A. Magnification ×280.



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Fig. 5.   Immunofluorescence localization of Ksp-cadherin in connecting tubule of adult rabbit kidney. A: semithin cryosection of adult rabbit kidney cortex labeled with MAb C575-5I. CT, connecting tubule. B: phase-contrast image of A. Arrows indicate intercalated cells lacking basal C575-5I labeling. Magnification ×850.



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Fig. 6.   Immunofluorescence localization of Ksp-cadherin in cortical collecting duct of adult rabbit kidney. A: semithin cryosection of adult rabbit kidney cortex labeled with MAb C575-5I. CD, collecting duct. B: phase-contrast image of A. Arrows indicate intercalated cells lacking basal C575-5I labeling. Magnification ×800.

In the outer medulla, the TAL continues to be the site of highest Ksp-cadherin expression (Fig. 7). The intensity of the fluorescent signal associated with C575-5I labeling of the TAL was dramatically higher than that observed in all adjacent tubular cell types. To correctly convey the membranous nature of C575-5I labeling in the TAL, the photomicrograph used to prepare Fig. 7 was intentionally underexposed. The apparent low level of collecting duct labeling seen in Fig. 7 is a direct result of this manipulation. In actuality, the levels of Ksp-cadherin expression observed in both the outer (Fig. 7) and inner (Fig. 8) medullary collecting ducts are similar to that observed in the cortical collecting duct (Fig. 6). Likewise, the thin limbs of the loop of Henle were uniformly labeled with moderate levels of anti-Ksp-cadherin antibody staining throughout all regions of the medulla (Figs. 7 and 8). Ksp-cadherin was not detected in the ureter or the urinary bladder.


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Fig. 7.   Immunofluorescence localization of Ksp-cadherin in inner stripe of outer medulla of adult rabbit kidney. A: semithin cryosection of inner stripe of outer medulla of adult rabbit kidney labeled with MAb C575-5I. Print used to prepare A was intentionally underexposed to convey the membranous nature of C575-5I staining in the TAL. CD, collecting duct; TL, thin limb. B: phase-contrast image of A. Magnification ×320.



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Fig. 8.   Immunofluorescence localization of Ksp-cadherin in inner medulla of adult rabbit kidney. A: semithin cryosection of inner medulla of adult rabbit kidney labeled with MAb C575-5I. CD, collecting duct; T, thin limb. B: phase-contrast image of A. Magnification ×815.

Cellular localization of Ksp-cadherin. Electron microscopy of C575-5I peroxidase labeled adult rabbit kidney cortex and outer medulla sections corroborated the tubular distribution of Ksp-cadherin observed in the light-level immunofluorescence experiments. The pattern of antibody labeling seen in the electron micrograph of a C575-5I peroxidase-labeled proximal tubule cell in Fig. 9 is essentially identical to that observed in all cell types in both the tubular nephron and the collecting system. Uniform antibody labeling was always observed along the entire length of both the lateral membranes and the basal infoldings. High levels of antibody labeling at discrete sites of cell-cell contact as reported for E-cadherin in the mouse intestine (2) were never seen. Antibody labeling was also never observed on the regions of the basal membrane that are in direct apposition with the tubular basement membrane (see, for example, Fig. 10A).


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Fig. 9.   Cellular localization of Ksp-cadherin in proximal tubule of adult rabbit kidney cortex. Electron micrograph depicts immunoperoxidase localization of Ksp-cadherin in rabbit proximal tubule cell with MAb C575-5I. Arrows indicate sites of C575-5I labeling. Magnification ×13,600.




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Fig. 10.   Cellular localization of Ksp-cadherin in a connecting tubule intercalated cell in adult rabbit kidney. A: electron micrograph depicting immunoperoxidase localization of Ksp-cadherin in intercalated cell of rabbit kidney connecting tubule with MAb C575-5I. Arrows indicate sites of C575-5I labeling. Magnification ×9,730. B: electron micrograph depicting immunogold localization of Ksp-cadherin at lateral cell border between intercalated cell and adjacent connecting tubule cell. IC, intercalated cell; CNT, connecting tubule cell. Arrows indicate intercellular plasma membrane structures visible in plane of sectioning. Magnification ×107,194.

The immunofluorescence data previously described suggested that Ksp-cadherin might not be expressed in all intercalated cell types in the cortical collecting duct and the connecting tubule. Electron microscopy of C575-5I peroxidase labeled kidney cortex sections suggested that the lack of significant basal immunofluorescent staining in many of the intercalated cells may simply be due to the extremely low levels of basal infolding observed in intercalated cells in this region of the kidney (Fig. 10A). The observation in the light-level immunofluorescence experiments that some intercalated cells had basal labeling and some did not (Figs. 5 and 6) could be a direct consequence of this limited basal infolding and the narrow plane of tissue sectioning for each particular specimen. At the electron microscopy level, intercalated cell immunoperoxidase basal labeling was observed in all intercalated cells in which basal infolding could be detected. Regardless of the presence or absence of detectable basal immunostaining, extensive lateral peroxidase labeling was observed in every intercalated cell type in all tissue sections examined (Fig. 10A). Specific immunogold labeling with C575-5I confirmed that Ksp-cadherin was present along the entire length of the lateral membranes of all intercalated cells in the rabbit kidney (Fig. 10B).

Immunofluorescence localization of Ksp-cadherin in neonatal kidney. The potential for Ksp-cadherin involvement in the development of the metanephric kidney was assessed by examination of Ksp-cadherin expression patterns in kidneys from 1-day-old neonatal rabbits. Kidneys from rabbits of this age are in the final stages of metanephrogenesis and display a marked centrifugal pattern of tubule development in which highly differentiated early nephrons in the corticomedullary region gradually give way to undifferentiated nephron rudiments and uninduced metanephrogenic mesenchyme in a subcapsular cortical nephrogenic zone. This feature allows the investigator to monitor the expression of developmental markers in each stage of metanephrogenesis in a single specimen. For comparative purposes the coexpression of E-cadherin and N-CAM was examined at similar time points.

In the nephrogenic zone of the neonatal rabbit kidney cortex, Ksp-cadherin antibody labeling was confined to the epithelial cells of the more highly differentiated regions of the expanding ureteric duct (Fig. 11A). Staining was not observed in the metanephrogenic mesenchyme, the terminal ampulla of the ureteric duct, nephrogenic vesicles, comma-shaped bodies, or S-shaped bodies (Figs. 11A and 12A). E-cadherin antibody labeling was observed along the entire length of the ureteric duct (Fig. 11B). Like Ksp-cadherin, E-cadherin was not detected in either nephrogenic vesicles or comma-shaped bodies. E-cadherin was, however, detected in the distal and proximal anlagen of the S-shaped body and the region of the glomerular anlage that gives rise to the parietal epithelial cells of Bowman's capsule (Fig. 12B). E-cadherin antibody labeling of proximal and glomerular anlagen of S-shaped bodies was extremely weak in all specimens examined. N-CAM was detected in the uninduced mesenchyme of the nephrogenic zone, nephrogenic vesicles, S-shaped bodies, and the terminal ampulla of the ureteric duct (Figs. 11C and 12C).


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Fig. 11.   Immunofluorescence localization of Ksp-cadherin, E-cadherin, and N-CAM in outer nephrogenic zone of neonatal rabbit kidney. Semithin cryosections of neonatal rabbit kidney cortex were labeled with MAb C575-5I (anti-Ksp-cadherin), MAb 6F9 (anti-E-cadherin), or MAb NCAM-OB11 (anti-N-CAM) as indicated. A: Ksp-cadherin localization. B: E-cadherin localization. C: N-CAM localization. D: phase-contrast image of A. E: phase-contrast image of B. F: phase-contrast image of C. CB, comma-shaped body; U, ureteric duct. Arrow in A indicates location of terminal ampulla of ureteric duct. Magnification of A, C, D, and F ×130. Magnification of B and E ×260.



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Fig. 12.   Immunofluorescence localization of Ksp-cadherin, E-cadherin, and N-CAM in inner nephrogenic zone of neonatal rabbit kidney. Semithin cryosections of S-shaped bodies from neonatal rabbit kidney cortex were labeled with MAb C575-5I (anti-Ksp-cadherin), MAb 6F9 (anti-E-cadherin), or MAb NCAM-OB11 (anti-N-CAM) as indicated. A: Ksp-cadherin localization. B: E-cadherin localization. C: N-CAM localization. D: phase-contrast image of A. E: phase-contrast image of B. F: phase-contrast image of C. DA, distal anlage; PA, proximal anlage; GA, glomerular anlage; U, ureteric duct. Magnification of A, C, D, and F ×185. Magnification of B and E ×260.

In the highly differentiated corticomedullary and medullary regions of the neonatal kidney, distinct basolateral Ksp-cadherin antibody staining was observed in all segments of the nephron and the collecting system (Fig. 13A). Antibody labeling was not observed in the glomerulus or nonepithelial regions of the kidney. E-cadherin was also detected on the basolateral membranes of all tubular segments of the nephron and collecting system in this region of the neonatal kidney (Fig. 13B). Like Ksp-cadherin, E-cadherin was not detected in the glomerulus or nonepithelial structures of the neonatal kidney. N-CAM, on the other hand, was not detected in the epithelial cells of either the nephron or the collecting system in this region of the neonatal kidney (Fig. 13C). N-CAM distribution was limited to the basement membrane region of the tubular epithelial structures, portions of Bowman's capsule, and the stroma between the tubular elements.


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Fig. 13.   Immunofluorescence localization of Ksp-cadherin, E-cadherin, and N-CAM in differentiated cortical regions of neonatal rabbit kidney. Semithin cryosections of midcortical region of neonatal rabbit kidneys were labeled with MAb C575-5I (anti-Ksp-cadherin), MAb 6F9 (anti-E-cadherin), or MAb NCAM-OB11 (anti-N-CAM) as indicated. A: Ksp-cadherin localization. B: E-cadherin localization. C: N-CAM localization. D: phase-contrast image of A. E: phase-contrast image of B. F: phase-contrast image of C. G, glomerulus; P, proximal tubule; D, distal tubule. Magnification ×130.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ksp-cadherin expression in adult kidney. We have performed a detailed evaluation of Ksp-cadherin expression in every cell type in the nephron and the collecting system by both indirect immunofluorescence light microscopy and immunoelectron microscopy to unambiguously identify the exact sites of Ksp-cadherin expression in the adult kidney. The results of this study indicate that not only is Ksp-cadherin expressed in every segment of the tubular nephron and the collecting system but also that it is expressed on the basolateral membrane of every cell type in each of these segments.

To date, Ksp-cadherin and E-cadherin are the only cadherins known to be expressed along the entire length of the tubular nephron and the collecting system in the adult mammalian kidney. This ubiquitous renal expression pattern is well conserved for Ksp-cadherin in kidneys from rabbit, mouse, rat, and human (personal observation and Ref. 3), but the renal expression pattern for E-cadherin appears to be species specific. E-cadherin is expressed in all tubular segments of the mouse (20) and rabbit kidney (personal observation) but is notably absent in the proximal segments of human, monkey, and dog kidney (18). In the rabbit kidney we observed that expression levels for both Ksp-cadherin and E-cadherin were highest in the distal segments of the nephron. In contrast, Piepenhagen et al. (20) report that E-cadherin is expressed at uniform levels along the entire length of the nephron and the collecting system in the mouse kidney.

Cadherin-6, cadherin-11, N-cadherin, P-cadherin, K-cadherin, and T-cadherin have also all been detected in the mammalian kidney (8, 19, 21, 24, 29). Of these, detailed localizations in adult kidney have only been reported for cadherin-6 and N-cadherin. In the adult human kidney cadherin-6 is expressed in the proximal tubule (19) and N-cadherin is expressed in Bowman's capsule, the proximal tubule, and the thin limbs of the loop of Henle (18). Cadherin-6 and N-cadherin expression patterns in the rabbit kidney are unknown. Insufficient studies have been conducted to determine if other cadherins have species-specific differences in their renal distributions as reported for E-cadherin.

The results of the immunoelectron microscopy studies with the anti-Ksp-cadherin antibody indicate that Ksp-cadherin is expressed at relatively uniform levels along the entire length of the lateral membranes and the basal infoldings of every cell type in the rabbit nephron and the collecting system. This distribution is not consistent with the generally accepted notion that cadherins are predominantly expressed at junctional complexes in epithelial cells, but similar cellular distributions have also been reported for LI-cadherin in the rat intestine (1), Cadherin-6 in the human kidney (19), E-cadherin in Madin-Darby canine kidney (MDCK) cells (6), and E-cadherin in the mouse kidney (20).

It is unclear why Ksp-cadherin is expressed at such uniformly high levels over the entire surface of the lateral membranes and the basal infoldings of all tubular epithelial cells. If the primary functions of Ksp-cadherin are homotypic cellular recognition and cell-cell adhesion, as has been suggested for other members of the cadherin superfamily, one would predict that Ksp-cadherin should be expressed at significantly higher levels at sites of cell-cell contact. This is clearly not the case. It is not known if Ksp-cadherin participates in homotypic recognition, but given its ubiquitous cellular distribution it is unlikely that cell-cell adhesion is its primary function.

Ksp-cadherin is conspicuously absent from the basal regions of the plasma membrane in direct apposition with the tubular basement membrane. This implies that Ksp-cadherin does not interact directly with either the components of the extracellular matrix or their respective receptors on the basal surfaces of the tubular epithelial cells. It does not, however, rule out the possibility that Ksp-cadherin may interact indirectly with basal proteins through some unidentified pathway such as that reported for N-cadherin and the beta 1-and beta 3-integrin receptors in migrating neural crest cells (16).

The ubiquitous expression of Ksp-cadherin throughout the tubular nephron and the collecting system indicates that it is unlikely that Ksp-cadherin functions in a segment-specific capacity similar to that reported for other cadherins. In the vertebrate nervous system, for example, the segment-specific expression of individual cadherins is believed to play a prominent role in the genesis of functionally distinct neuronal phenotypes (22). Likewise, Nouwen et al. (18) have proposed that in the human kidney the segment-specific expression of N-cadherin and E-cadherin may help to establish the sharp boundaries between nephron segments and prevent the intermingling of different cell types at the transition between the segments. Rather than mediating segment-specific differences, Ksp-cadherin may be important for maintaining the integrity of the tubular phenotype of the epithelium as a whole. It may accomplish this by providing a consistent basolateral positional cue for the formation and maintenance of the tubular architecture.

Ksp-cadherin expression in developing kidney. It is clear that Ksp-cadherin expression is under tight developmental control. It is expressed relatively late in the development of the metanephric kidney, and its expression is preceded by the expression of both N-CAM and E-cadherin. In the subcapsular nephrogenic zone, Ksp-cadherin was detected in only the more highly differentiated regions of the ureteric duct. The lack of expression of Ksp-cadherin in comma- and S-shaped bodies was particularly surprising given the extremely high levels of Ksp-cadherin expression observed in all segments of the tubular nephron in the adult kidney. We fully expected that like E-cadherin, Ksp-cadherin would be expressed in both the distal and proximal anlagen of expanding S-shaped bodies. Ksp-cadherin was not detected in even the most advanced S-shaped structures. The onset of Ksp-cadherin expression in the developing nephron is very rapid. It is expressed at uniformly high levels in all segments of the developing tubular nephron as soon as a clearly recognizable capillary-loop-stage glomerulus is evident (developmental stage III, Ref. 11). At this early stage of development it is difficult to conclusively differentiate between proximal and distal nephron segments at the light microscopy level without the aid of specific segmental markers. Nevertheless, the uniform labeling of all tubules in the vicinity of stage III glomeruli strongly suggests that Ksp-cadherin is expressed at similar levels in both proximal and distal tubules. In the vicinity of stage IV glomeruli, distal tubules exhibited significantly higher levels of anti-Ksp-cadherin antibody staining than adjacent proximal tubules (see Fig. 13A). It is unclear if this reflects differences in basolateral membrane surface areas between the two segments or actual differences in expression densities of Ksp-cadherin.

The onset of Ksp-cadherin expression in the developing nephron appears to correspond directly with the establishment of tubular epithelial cell polarity and the onset of glomerular filtration (12). At stage II of nephron development, the S-shaped body stage, the presumptive tubular epithelial cells are aligned on a thin basement membrane and collectively form a tubular lumen that is continuous with the ureteric duct (see Ref. 4 for review). At this stage of tubule development N-CAM (Fig. 12C; see also Ref. 10) and E-cadherin (Fig. 12B; see also Ref. 28) are expressed in a nonpolarized fashion on the plasma membranes of the presumptive tubular epithelial cells. At stage III of nephron development, corresponding to the onset of Ksp-cadherin expression, N-CAM expression is lost and E-cadherin is now expressed in a polarized fashion on the basolateral membranes of both proximal and distal tubule cells.

In the ureteric duct Ksp-cadherin expression again appears to correspond directly with the aquisition of cell polarity and the final stages of cellular differentiation. The cells in the terminal ampulla of the ureteric duct are histologically uniform and undifferentiated, express E-cadherin in a nonpolarized manner (Fig. 11B; see also Refs. 18 and 28), and do not express Ksp-cadherin (Fig. 11A). In the segment of the ureteric duct distal to the terminal ampulla, the epithelial cells begin to actively differentiate, and the tubular epithelium is now composed of at least two different cell types. It is in this region that Ksp-cadherin expression is first detected and that E-cadherin begins to be expressed exclusively on the basolateral membranes of the tubular epithelial cells.

It is almost certain that Ksp-cadherin does not play a significant role in the early stages of the morphogenesis of either the tubular nephron or the collecting system. In both regions the developing epithelial cells are already at an advanced stage of differentiation before Ksp-cadherin expression is detected. In both the branching tip of the ureteric bud and the developing comma- and S-shaped bodies, the cells are largely undifferentiated and are undergoing active rearrangement. Nelson (17) has suggested that cadherins play a central role in both the direction and maintenance of epithelial cell polarity, and it is well known that cadherins are involved in cell rearrangement and tissue morphogenesis (5). E-cadherin and/or other cadherins that are expressed in the early stages of nephrogenesis likely establish and maintain the orientation of cell-cell contacts throughout this plastic period. The expression of Ksp-cadherin and the coincident development of recognizable epithelial cell polarity after the cell rearrangement phase suggests that Ksp-cadherin may provide the stable positional cue necessary for the polarized assembly of the cytoskeleton and the directed delivery of basolateral membrane proteins.

In summary, we have performed a detailed morphological evaluation of Ksp-cadherin expression in both adult and developing rabbit kidneys. In the adult kidney Ksp-cadherin is expressed on the basolateral membrane of all cell types in both the tubular nephron and the collecting system. In the developing metanephric kidney Ksp-cadherin is not expressed until the tubular epithelial cells have reached an advanced stage of differentiation. Given the relatively late stage of expression in the developing kidney and the ubiquitous distribution in mature nephrons and collecting ducts, it is possible that Ksp-cadherin may function to stabilize both the terminally differentiated phenotype of the individual epithelial cells and the integrity of the tubular epithelial architecture as a whole.


    ACKNOWLEDGEMENTS

We thank Dr. Daniel Biemesderfer for many helpful discussions and Sue Ann Mentone for expert technical assistance.


    FOOTNOTES

This work was supported by a fellowship from the Patrick and Catherin Weldon Donaghue Medical Research Foundation to R. B. Thomson and by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-17433 to P. S. Aronson.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. B. Thomson, Sect. of Nephrology, Dept. of Internal Medicine, Yale Univ. School of Medicine, LMP 2095, P. O. Box 208020, New Haven, CT 06520-8029 (E-mail: thomson{at}biomed.med.yale.edu).

Received 30 September 1998; accepted in final form 7 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Physiol 277(1):F146-F156
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