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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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).
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RESULTS |
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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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
1-and
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 |
1.
Berndorff, D.,
R. Gessner,
B. Kreft,
N. Schnoy,
A. Lajous-Petter,
N. Loch,
W. Reutter,
M. Hortsch,
and
R. Tauber.
Liver-intestine cadherin: molecular cloning and characterization of a novel Ca2+-dependent cell adhesion molecule expressed in liver and intestine.
J. Cell Biol.
125:
1353-1369,
1994[Abstract].
2.
Boller, K.,
D. Vestweber,
and
R. Kemler.
Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells.
J. Cell Biol.
100:
327-332,
1985[Abstract].
3.
Earle, K. E.,
R. C. Kim,
C. L. Yang,
R. B. Thomson,
and
P. S. Aronson.
Developmental regulation of Ksp-cadherin expression in the human kidney (Abstract).
J. Investig. Med.
44:
242A,
1996.
4.
Evan, A. P.,
and
L. Larsson.
Morphologic development of the nephron.
In: Pediatric Kidney Disease, edited by C. M. Edelmann. Boston, MA: Little, Brown, 1992, p. 19-48.
5.
Geiger, B.,
and
O. Ayalon.
Cadherins.
Annu. Rev. Cell Biol.
8:
307-332,
1992.
6.
Gumbiner, B.,
and
K. Simons.
A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of an uvomorulin-like polypeptide.
J. Cell Biol.
102:
457-468,
1986[Abstract].
7.
Hermiston, M. L.,
and
J. I. Gordon.
In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death.
J. Cell Biol.
129:
489-506,
1995[Abstract].
8.
Hoffmann, I.,
and
R. Balling.
Cloning and expression analysis of a novel mesodermally expressed cadherin.
Dev. Biol.
169:
337-346,
1995[Medline].
9.
Kaissling, B.,
and
W. Kriz.
Structural analysis of the rabbit kidney.
Adv. Anat. Embryol. Cell Biol.
56:
1-121,
1979[Medline].
10.
Klein, G.,
M. Langegger,
C. Goridis,
and
P. Ekblom.
Neural cell adhesion molecules during embryonic induction and development of the kidney.
Development
102:
749-761,
1988[Abstract].
11.
Larsson, L.
The ultrastructure of the developing proximal tubule in the rat kidney.
J. Ultrastruct. Res.
51:
119-139,
1975[Medline].
12.
Larsson, L.,
and
A. B. Maunsbach.
Differentiation of the vacuolar apparatus in cells of the developing proximal tubule in the rat kidney.
J. Ultrastruct. Res.
53:
254-270,
1975[Medline].
13.
Larue, L.,
M. Ohsugi,
J. Hirchenhain,
and
R. Kemler.
E-cadherin null mutant embryos fail to form a trophectoderm epithelium.
Proc. Natl. Acad. Sci. USA
91:
8263-8267,
1994[Abstract].
14.
Mars, J. A.,
C. Andersson-Fisone,
M. C. Jeong,
L. Cohen-Gould,
C. Zurzolo,
I. R. Nabi,
E. Rodriguez-Boulan,
and
W. J. Nelson.
Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules.
J. Cell Biol.
129:
507-519,
1995[Abstract].
15.
McNeill, H.,
M. Ozawa,
R. Kemler,
and
W. J. Nelson.
Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity.
Cell
62:
309-316,
1990[Medline].
16.
Monier-Gavelle, F.,
and
J. Duband.
Cross talk between adhesion molecules: control of N-cadherin activity by intracellular signals elicited by
1 and
3 integrins in migrating neural crest cells.
J. Cell Biol.
137:
1663-1681,
1997[Abstract/Free Full Text].
17.
Nelson, W. J.
Regulation of cell surface polarity from bacteria to mammals.
Science
258:
948-955,
1992[Medline].
18.
Nouwen, E. J.,
S. Dauwe,
I. van der Biest,
and
M. E. de Broe.
Stage- and segment-specific expression of cell-adhesion molecules N-CAM, A-CAM, and L-CAM in the kidney.
Kidney Int.
44:
147-158,
1993[Medline].
19.
Paul, R.,
C. M. Ewing,
J. C. Robinson,
F. F. Marshall,
K. R. Johnson,
M. J. Wheelock,
and
W. B. Isaacs.
Cadherin-6, a cell adhesion molecule specifically expressed in the proximal renal tubule and renal cell carcinoma.
Cancer Res.
57:
2741-2748,
1997[Abstract].
20.
Piepenhagen, P. A.,
L. L. Peters,
S. E. Lux,
and
W. J. Nelson.
Differential expression of Na+-K+-ATPase, ankyrin, fodrin, and E-cadherin along the kidney nephron.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1417-C1432,
1995[Abstract/Free Full Text].
21.
Ranscht, B.,
and
M. T. Dours-Zimmermann.
T-cadherin, a novel cadherin cell adhesion molecule in the nervous system lacks the conserved cytoplasmic region.
Neuron
7:
391-402,
1991[Medline].
22.
Redies, C.,
and
M. Takeichi.
Cadherins in the developing central nervous system: an adhesive code for segmental and functional subdivisions.
Dev. Biol.
180:
413-423,
1996[Medline].
23.
Riethmacher, D.,
V. Brinkmann,
and
C. Birchmeier.
A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development.
Proc. Natl. Acad. Sci. USA
92:
855-859,
1995[Abstract].
24.
Tassin, M. T.,
A. Beziau,
M. C. Gubler,
and
B. Boyer.
Spatiotemporal expression of molecules associated with junctional complexes during the in vivo maturation of renal podocytes.
Int. J. Dev. Biol.
38:
45-54,
1994[Medline].
25.
Thomson, R. B.,
P. Igarashi,
D. Biemesderfer,
R. Kim,
A. Abu-Alfa,
M. Soleimani,
and
P. S. Aronson.
Isolation and cDNA cloning of Ksp-cadherin, a novel kidney-specific member of the cadherin multigene family.
J. Biol. Chem.
270:
17594-17601,
1995[Abstract/Free Full Text].
26.
Tokuyasu, K. T.
Application of cryoultramicrotomy to immunocytochemistry.
J. Microsc.
143:
139-149,
1986[Medline].
27.
Tokuyasu, K. T.
Use of polyvinylpyrrolidone and polyvinyl alcohol for cryoultramicrotomy.
Histochem. J.
21:
163-171,
1991.
28.
Vestweber, D.,
R. Kemler,
and
P. Ekblom.
Cell-adhesion molecule uvomorulin during kidney development.
Dev. Biol.
112:
213-221,
1985[Medline].
29.
Xiang, Y.,
M. Tanaka,
M. Suzuki,
H. Igarashi,
E. Kiyokawa,
Y. Naito,
Y. Ohtawara,
Q. Shen,
H. Sugimura,
and
I. Kino.
Isolation of complementary DNA encoding K-cadherin, a novel rat cadherin preferentially expressed in fetal kidney and kidney carcinoma.
Cancer Res.
54:
3034-3041,
1994[Abstract].
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