Post on 02-Feb-2023
Histopathological analysis of renal cystic epithelia in the
Pkd2WS25/- mouse model of ADPKD
R. Brent Thomson,1 SueAnn Mentone, 1 Robert Kim, 1 Karen Earle, 1 Eric Delpire,2 Stefan
Somlo, 1 and Peter S. Aronson1
1 Section of Nephrology, Department of Internal Medicine, Yale University School of
Medicine, PO Box 208029, New Haven, Connecticut 06520-8029
2 Department of Anesthesiology and Center for Molecular Neuroscience, Vanderbilt
University Medical Center, Nashville, Tennessee 37232
Running head: Histopathological analysis of renal cystic epithelia
Corresponding author: Peter S. Aronson
Section of Nephrology
Department of Internal Medicine
Yale University School of Medicine
1 Gilbert Street, CAB S-255C
P.O. Box 208029
New Haven, Connecticut 06520-8029
peter.aronson@yale.edu
phone: 203-785-4902 fax: 203-785-4904
Copyright (c) 2003 by the American Physiological Society.
Articles in PresS. Am J Physiol Renal Physiol (July 8, 2003). 10.1152/ajprenal.00153.2003
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ABSTRACT
It has been proposed that ADPKD-affected renal epithelial cells undergo a phenotypic
transition from a highly differentiated absorptive state to a much less differentiated
secretory state during cystogenesis and that this transition is accompanied by loss of
epithelial cell polarity and mistargeting of specific membrane proteins. We have
conducted a detailed evaluation of this hypothesis in the Pkd2WS25/- mouse model of
ADPKD. Ultrastructural analysis of Pkd2WS25/- cysts by electron microscopy confirmed
that cystic epithelial cells progressively dedifferentiate with cyst enlargement.
Immunocytochemical analysis of both early and late stage cysts with antibodies directed
against the Na+/K+-ATPase, Ksp-cadherin, and E-cadherin failed to detect evidence of
altered cyst cell polarity. The Na+/K+-ATPase and Ksp-cadherin were expressed
exclusively on the basolateral membranes (BLM) of epithelial cells in all early cysts.
Expression levels of both the Na+/K+-ATPase and Ksp-cadherin decreased progressively
with the degree of cyst cell dedifferentiation, but neither protein was ever mislocalized.
Highly dedifferentiated cysts did not express immunodetectable levels of either the
Na+/K+-ATPase or Ksp-cadherin. E-cadherin was expressed prominently on the
basolateral membranes of all cysts. Cysts were subsequently stained with an antibody
directed against the secretory isoform of the Na+-K+-Cl- cotransporter NKCC1. NKCC1
expression was detected on the BLM of advanced cysts only. Our data are consistent
with a model of progressive cystic epithelial cell dedifferentiation in which fluid
accumulation in late stage cysts is mediated by transepithelial secretion of chloride rather
than secretion of sodium by apical Na+/K+-ATPase.
Keywords: polarity, Na+/K+-ATPase, NKCC1, Ksp-cadherin, E-cadherin
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INTRODUCTION:
The physiological mechanisms involved in renal cyst formation in patients
affected by autosomal dominant polycystic kidney disease (ADPKD) are still largely
unknown. Intensive genetic analyses of families affected by ADPKD have revealed that
the majority of reported cases are directly attributable to mutations in the genes that
encode either polycystin-1 or polycystin-2. The functions of polycystin-1 and polycystin-
2 are not yet completely understood, but it is generally believed that their functions are
related and that a disruption in the function of either protein initiates a common
cystogenic pathway (for review see 18). In the currently accepted model of cystogenesis,
single progenitor cells are believed to enter a dedifferentiating hyperplastic pathway that
ultimately gives rise to the expansive fluid-filled cysts that are the hallmark of ADPKD.
Implicit in this model is the notion that the cystic epithelium undergoes a phenotypic
transition from a highly differentiated absorptive state to a much less differentiated
secretory state.
It has been suggested that alterations in epithelial cell polarity due to mistargeting
of specific membrane proteins may play a significant role in this transition. Charron et al
(8) reported that cultured human ADPKD cyst cells have a disrupted cytoarchitecture and
coupled with mislocalization of E-cadherin may have a generalized basolateral sorting
defect. Wilson et al (34; 35) proposed that the mistargeting of a fully functional Na+/K+-
ATPase to the apical membrane of cystic epithelial cells could result in net basal-to-
apical Na+ flux which in turn could drive luminal fluid accumulation and cyst expansion.
Although apical mislocalization of the Na+/K+-ATPase has been reported in several
different models of polycystic kidney disease (for example see 1 and 26), it has not been
universally accepted as the principal mechanism underlying either fluid secretion or cyst
expansion in ADPKD.
Grantham et al (13) have proposed that cyst luminal fluid accumulation may be
driven by net basal-to-apical Cl- movements in a manner similar to that observed in
classical secretory epithelia such as sweat duct or airway (see 30 for review) rather than
by apical secretion of Na+. In support of this hypothesis, Brill et al (6) reported that they
could find no evidence of mislocalization of the Na+/K+-ATPase in either excised human
ADPKD cysts or human ADPKD cyst cells in culture. Moreover, studies by Hanaoka et
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al (14) and Brill et al (6) indicated that apical Cl- exit could be facilitated by the CFTR
protein, and inhibitor studies by Mangoo-Karim et al (23) suggested that the basolateral
entry step may be mediated by the Na+-K+-2Cl- cotransporter NKCC1.
To address this controversy and to determine if altered cell polarity plays a role in
cyst development in ADPKD, we have conducted an extensive ultrastructural and
immunocytochemical characterization of renal cysts in the Pkd2WS25/- adult mouse model
of ADPKD. Cyst initiation in this model is due to a somatic inactivation of the second
Pkd2 allele and cystogenesis has been shown to proceed in a manner similar to that
reported for human ADPKD (36; 37). Importantly, the use of an animal model permitted
us to perform rapid in situ perfusion fixation of the kidneys, eliminating the potential for
ischemia-induced mistargeting of the Na+/K+-ATPase (see 7 and 24) and affording
optimal preservation of both cellular ultrastructure and protein localization.
Ultrastructural analysis by both transmission and scanning electron microscopy
confirmed that cyst epithelial cells undergo a dramatic dedifferentiation as cystogenesis
proceeds. Immunocytochemical analysis of both early- and late-stage cysts indicate that
neither altered cell polarity nor mislocalization of the Na+/K+-ATPase appears to play a
role in cyst expansion and fluid secretion in ADPKD. Consistent with the model of
progressive cystic epithelial cell dedifferentiation in which fluid secretion is mediated by
transepithelial Cl- movements rather than apical secretion of Na+, we detected expression
of the Na+-K+-2Cl- cotransporter, NKCC1, on the basolateral membranes of advanced
cyst epithelial cells.
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METHODS:
Animals and reagents. Pkd2WS25/- animals were generated by crossing Pkd2+/- and
Pkd2WS25/+ mice as described by Wu et al (36). To facilitate tissue preparation for
microscopy, all animals used in this study were one to two months of age. Kidneys from
animals of this age contained large numbers of cysts in all stages of development, but
retained enough non-cystic tissue to permit adequate perfusion fixation of both the cysts
and the unaffected renal parenchyma.
Two different mouse monoclonal antibodies directed against the |1 subunit of the
Na+/K+-ATPase were used in this study: antibody C464.6 (Upstate Biotechnology, Lake
Placid, NY) was used at a dilution of 1:50 and antibody Alpha-5 (Developmental Studies
Hybridoma Bank, Iowa City, IA; 22) was used at a dilution of 1:100. The anti-E-
cadherin mouse monoclonal antibody Clone 36 (Transduction Laboratories, Lexington,
KY) was used at a dilution of 1:100.
The anti-Ksp-cadherin antibody is an affinity purified rabbit polyclonal antibody
that we generated against the human isoform of Ksp-cadherin. Rabbits were immunized
with a maltose-binding protein (MBP; New England Biolabs, Beverly, MA) fusion
protein containing the C-terminal 267 amino acids (KspFP563-829) of the human
isoform of Ksp-cadherin. Sera was negatively purified against bacterial lysates
containing only the MBP and then positively purified on a column containing the MBP-
KspFP563-829 fusion protein. Specific reactivity was confirmed by Western analysis of
Cos-7 cells transiently transfected with full-length rabbit Ksp-cadherin. Specificity was
further verified by Western analysis of human, mouse, rabbit, and dog kidney
microsomes and immunocytochemical analysis of human and mouse kidney cryosections.
The purified serum was used for immunocytochemistry in the present study at a dilution
of 1:100.
The anti-NKCC1 antibody (19) is an affinity purified rabbit polyclonal antibody
directed against the mouse isoform of NKCC1 and was used at a dilution of 1:200. A
rabbit anti-mouse Tamm-Horsfall antibody (THP; a generous gift of Dr. John Hoyer) was
used at a dilution of 1:200 for identification of cysts of cortical thick ascending limb
origin (16). A rabbit anti-thiazide-sensitive Na-Cl cotransporter (NCC, TSC) antibody (a
generous gift of Dr. Steven Hebert) was used at a dilution of 1:200 for identification of
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cysts of distal convoluted tubule origin (28). The fluorescein-conjugated lectin Lotus
tetragonolobus (LTA; Vector Laboratories, Burlingame, CA) was used at a dilution of
1:10 for identification of cysts of proximal tubule origin (20). Rhodamine-conjugated
Dolichos biflorus agglutinin (DBA; Vector Laboratories, Burlingame, CA) was used at a
dilution of 1:100 for identification of cysts of collecting duct origin (20). Alexa Fluor
anti-mouse IgG (488 and 594) and anti-rabbit IgG (488) secondary antibodies were used
at a dilution of 1:200 (Molecular Probes, Eugene, OR).
Electron microscopy. Mice were anesthetized by intraperitoneal injection of
sodium pentobarbital. The kidneys were cleared (PBS) and fixed (3%
paraformaldehyde/3% glutaraldehyde) by cardiac perfusion. The kidneys were then
removed, cut into 2-4 mm thick sagittal sections and post-fixed in the same fixative for 1
h at 4°C. Tissue destined for transmission electron microscopy (TEM) was further cut
into 2-4 mm blocks for subsequent processing. Cortical tissue only was used for the
TEM study. The tissue sections were washed in 0.1 M cocodylate buffer containing
7.5% sucrose (pH 7.4; 3 changes; 1 h; 4°C) and then incubated in veronal acetate-
buffered 1% osmium tetroxide for 2 h at 4°C (1 h for TEM sections). Tissue used for
scanning electron microscopy (SEM) was again washed in cocodylate buffer (3 changes;
1 h; 4°C), dehydrated through a graded ethanol series, critical point dried, mounted on
stubs, sputter coated with PdAu, and then visualized on an International Scientific
Instruments SS-40 scanning electron microscope. Tissue used for TEM was washed and
incubated in a Kellenberger veronal-acetate buffer containing 0.5% uranyl acetate for 2 h
at room temperature, rinsed in H2O, dehydrated through a graded ethanol series, and
embedded in Epon 812. Ultrathin 80 nm sections were cut on a Reichert Ultracut E
ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Zeiss
EM910 electron microscope.
Immunocytochemistry. Kidneys from six non-sibling mice were perfused and
cleared in the same manner as above, except that PLP (2% paraformaldehyde, 750 mM
lysine, and 10 mM sodium periodate in a phosphate buffer, pH 7.4) was used as the
fixative. The cortices were separated from the kidneys, cut into 2-4 mm blocks and
postfixed in the same fixative for an additional 4 h. To maintain the structural integrity
of the fragile cyst walls, all tissue blocks were embedded in Epon 812 as above except
that the tissue blocks were not exposed to osmium tetroxide or uranyl acetate. One
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micron sections were cut on the Reichert Ultracut E ultramicrotome and mounted on
Superfrost Plus glass slides (Electron Microscopy Sciences, Fort Washington, PA).
Sections were etched for 5 minutes in a solution containing 2 g KOH, 10 ml 100%
methanol, and 5 ml propylene oxide, washed for 5 min in 100% methanol (2 changes),
rinsed in TBS (50 mM Tris-HCl, 100 mM NaCl, pH 7.4), and then subjected to antigen
retrieval as outlined in Biemesderfer et al (5). Briefly, sections were immersed in a
boiling 10 mM sodium citrate buffer (pH 6) and then heated for an additional 20 minutes
at the 40% power setting of a 700 watt microwave oven, allowed to cool, washed in TBS,
quenched in 0.5 M ammonium chloride with 0.1% BSA for 15 min, rinsed in TBS,
incubated in 1% SDS in TBS for 5 min, and finally washed in TBS. Sections to be
stained with mouse primary antibodies were incubated with unconjugated anti-mouse IgG
diluted 1:5 in TBS/0.1% BSA/ 10% goat serum for 1 h at room temperature. All other
sections were blocked with 0.1% BSA and 10% goat serum in TBS for 1 h at room
temperature. All sections were washed with TBS and then incubated with the respective
primary antibodies diluted in TBS/ 0.1% BSA/10% goat serum overnight at 4°C. The
next morning sections were washed in high salt TBS (527 mM NaCl) with 0.1% BSA
(5X; 5 minutes each wash) and then incubated with the appropriate fluorochrome-labeled
secondary antibody diluted in TBS/0.1% BSA/10% goat serum for 1 h at room
temperature. Sections were washed for 5 minutes in high salt TBS, 20 minutes in
standard TBS (4 changes), mounted in VectaShield (Vector Laboratories, Burlingame,
CA), and then visualized on a Zeiss Axiophot phase-contrast microscope. Antibodies and
lectins used for double labeling experiments were tested in control pilot studies to verify
lack of cross reactivity between reagents. Sequential serial sections were labeled with
specific cortical tubule segment markers to determine cyst tubule segments of origin.
Cyst staging. The criteria used to developmentally stage cysts were based on
cyst size, preliminary TEM ultrastructural surveys of PKD2WS25/- ADPKD cysts, and
recommendations by Devuyst et al (10). Generally, cysts were considered early-stage if
on cross-section there were less than 50 cyst-lining epithelial cells, intermediate-stage if
there were 51-200 cyst-lining epithelial cells, and advanced-stage if there were greater
than 200 cyst-lining epithelial cells. The relative position of a section within a cyst was
determined by examination of adjacent serial sections spanning approximately 40 µm in
each direction.
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RESULTS:
Ultrastructural analysis of PKD2WS25/- ADPKD cysts. In small cysts that are
presumably in the early stages of cyst expansion, the ultrastructure of the cyst cells is
highly reminiscent of unaffected tubular epithelial cells (Fig. 1a). The cells are cuboidal
to columnar, have approximately normal numbers of mitochondria, have well developed
lateral junctions, well organized basement membranes, and prominent cilia. None of the
early cyst cells that we examined retained sufficient “normal” ultrastructural details to
unequivocally assess the tubule segment of origin. As cyst expansion proceeds, the cells
take on a more squamoid appearance (Fig. 1b) and lose all resemblance to unaffected
tubular epithelial cells. The mitochondria become much less prominent, but the cells
continue to retain well-defined lateral junctions and a well-organized basement
membrane. Significant deposits of collagen begin to appear in the region just below the
basement membrane. In very large cysts that presumably represent the final stages of
cyst expansion, the cyst cells are highly attenuated and acquire a pronounced endothelial-
like appearance (Fig. 1c). Mitochondria are extremely rare, but again the cells continue
to retain well-defined lateral junctions (Fig. 1c inset). The basement membrane loses
much of its organization and becomes intimately associated with large deposits of striated
collagen. We did not observe thickening or lamination of the basement membrane per se
at any stage of cyst progression.
Scanning electron microscopy of the lumens of similarly staged ADPKD cysts
provide further support for the cellular dedifferentiation hypothesis. In the initial stages
of cyst growth the cyst epithelial cells appear to be highly proliferative and form
elaborate chords, ridges, and labyrinths within the cyst lumens (Fig. 2a). Early cyst cells
maintain regular well-defined lateral borders and have prominent cilia (Fig. 2b). The
degree of secondary cell-surface ultrastructure (eg. microvilli) varies from cyst to cyst
and presumably reflects the cyst tubule segment of origin. As cyst size increases the cell
borders become less regular and the cilia less prominent (Fig. 2c). In many late-
intermediate and early-advanced stage cysts we observed more than one cell phenotype
(see for example Fig. 2C arrow). These atypical cyst cells were not present in large
numbers (1-5% of the total cyst cell population) and appeared to be randomly
interspersed throughout the cyst walls. It is unclear if these cells are differentially staged
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progeny of the original cyst progenitor cell or if they are derived from outlying cell
populations. Cellular heterogeneity was never observed in extremely advanced cysts (Fig.
2d). The cells in advanced cysts appeared to be much larger than those seen in early
cysts, and they have irregular cell borders and inconspicuous cilia. The cyst walls are
very uniform and show little evidence of the cellular proliferation that was evident in the
earlier stages of cyst growth.
Immunocytochemical analysis of PKD2WS25/- ADPKD cysts. To determine the
localization of the Na+/K+-ATPase in PKD2WS25/- cysts and to facilitate comparison with
previously published reports, both Alpha-5 and C464.6 anti-Na+/K+-ATPase antibodies
were used in this study. Using antibody Alpha-5, Wilson et al (35) reported that the |-1
subunit of the Na+/K+-ATPase was localized to the apical membrane of ADPKD cyst
cells in immersion-fixed human nephrectomy samples and human ADPKD cyst cells in
culture. Using antibody 6H (clone C464.6), Brill et al (6) reported that the |-1 subunit of
the Na+/K+-ATPase was localized to the basolateral membrane of immersion-fixed
excised human ADPKD cysts and human ADPKD cyst cells in culture. Under the
conditions used in our study, the staining patterns for the two antibodies were identical
(Fig. 3). They each labeled the basolateral membranes of both normal and cystic
epithelia. Sixty cysts from six mice were positively labeled with the Na+/K+-ATPase
antibodies (see Table 1) and in no instance was apical localization ever detected.
In addition to assessing cyst Na+/K+-ATPase distribution, we also performed
double-labeling experiments with an antibody directed against Ksp-cadherin. Ksp-
cadherin is a kidney-specific member of the cadherin superfamily of cell adhesion
molecules and is expressed exclusively on the basolateral membrane of all tubular
segments of the mammalian kidney (33). If cystic epithelial cells truly have a
generalized basolateral sorting defect one would predict that other basolateral proteins,
such as Ksp-cadherin, would also be missorted. This proved not to be the case. As with
the Na+/K+-ATPase, apical localization of Ksp-cadherin in ADPKD cysts was never
detected (see Fig. 4 and Table 1).
Given our inability to detect evidence of a generalized basolateral sorting defect
or of altered cyst cell polarity, we suspected that E-cadherin may actually not be
mislocalized in native ADPKD cyst cells. Using the anti-E-cadherin antibody Clone 36,
Charron et al (8) reported that E-cadherin was not expressed on the lateral membranes of
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ADPKD cells in culture, but rather that it was sequestered in perinuclear vesicular
structures. Using the same antibody in the PKD2WS25/- ADPKD native cysts, we found no
evidence of cystic epithelial intracellular sequestration of E-cadherin. Rather, we
observed prominent E-cadherin staining on the lateral membranes of all cystic epithelial
cells (see Fig. 5 and Table 1).
While neither the Na+/K+-ATPase nor Ksp-cadherin was ever detected on the
apical membrane of PKD2WS25/- native cysts, we did observe a progressive decrease in the
level of expression of both proteins that appeared to be concomitant with the extent of
cyst expansion and the degree of cyst cell dedifferentiation from a cuboidal to a squamoid
phenotype. Na+/K+-ATPase immunostaining was never observed in cysts with either a
highly attenuated squamoid epithelium or extensive surrounding fibrotic tissue (Fig. 6a).
Ksp-cadherin immunostaining was also typically absent in advanced cysts, but was
occasionally detected in squamoid epithelial cysts that were not surrounded by extensive
fibrotic tissue. In Figure 6b, for example, both cysts have a highly attenuated squamoid
epithelium, but only the upper cyst was totally surrounded by fibrotic tissue. In the region
below the lower cyst (not included in the field shown in Fig. 6b), normal appearing
tubules were in direct apposition with the cyst wall and there was no evidence of
surrounding fibrotic tissue. We interpret this to indicate that the lower cyst is less
advanced than the upper cyst. The decrease in Na+/K+-ATPase and Ksp-cadherin
expression levels could not be correlated with the cyst tubule segment of origin because
advanced cysts also tended to lose expression of the tubule origin markers used in this
study (see Table 1). In the field shown in Figure 6a and 6b, the lower cyst was THP
positive whereas the upper cyst was negative for all tubule segment markers. This
observation is consistent with the notion that the upper cyst is more highly advanced and
dedifferentiated than the lower cyst. E-cadherin immunostaining, on the other hand, was
detected at high levels on the lateral membranes of all cysts regardless of the degree of
cyst expansion or dedifferentiation. In Figure 6c, for example, the anti-E-cadherin
antibody is shown labeling cyst epithelial cells that were negative for the Na+/K+-ATPase,
Ksp-cadherin, and all tubule segment markers.
If the Na+/K+-ATPase is not mislocalized to the apical membrane of ADPKD
cysts, primary active luminal Na+ extrusion is unlikely to account for cyst fluid secretion.
Sullivan et al (32) suggest that ADPKD cyst fluid accumulation is not driven by apical
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sodium transport, but rather by net basal to apical chloride flux. They propose that the
cystic epithelium undergoes a transition to a secretory phenotype similar to that observed
in airway epithelia and that the transepithelial chloride flux is mediated by the basolateral
bumetanide-sensitive Na+-K+-2Cl- cotransporter NKCC1 and the apical chloride channel
CFTR. Hanaoka et al (14) and Brill et al (6) have provided compelling evidence for
CFTR involvement in the apical exit step, but to date there has not been a clear
demonstration of NKCC1 localization on the basolateral membranes of native ADPKD
cyst cells.
In normal kidneys, NKCC1 expression is confined to the basolateral membrane of
the inner medullary collecting duct (IMCD), the glomerular and extraglomerular
mesangium, the glomerular afferent arterioles, and the papillary epithelium (19).
Consistent with these findings, NKCC1 expression in the non-cystic regions of the
cortices of PKD2WS25/- ADPKD cystic kidneys was restricted to structures associated with
glomeruli (Figure 7a). The vast majority of the cortical cysts that we observed did not
express immunodetectable levels of NKCC1 (see Table 1). In particular, anti-NKCC1
antibody labeling was never observed in cyst cells that expressed immunodetectable
levels of the Na+/K+-ATPase. For example, the tissue section illustrated in Figure 7a
depicting NKCC1-negative cysts is a serial section subsequent to that labeled with the
anti- Na+/K+-ATPase antibody shown in Figure 3a. Moderate levels of NKCC1
expression were detected on the basolateral membrane of squamoid-cell cysts whose
epithelia had typically dedifferentiated to the point of no longer expressing detectable
levels of the tubule segment-specific markers (see Figure 7b-c and Table 1). NKCC1
expression was occasionally detected in squamoid-cell cysts that were positive for
segment-specific marker labeling, but in the two observed cases the segment-specific
marker labeling was weak and inconsistent. Co-expression of NKCC1 with Ksp-cadherin
was rare and occurred in less than 10% of the cysts that expressed NKCC1.
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DISCUSSION:
The ultrastructural morphology of the mouse PKD2WS25/- ADPKD cysts is virtually
indistinguishable from that of human ADPKD cysts (see for example 9, 12, 11, and 7).
As reported for human ADPKD cystic kidneys, we observed two predominant cyst types:
those with highly differentiated cuboidal or columnar epithelial cells and those with
attenuated dedifferentiated squamoid epithelial cells. Our data suggest that the two cyst
cell populations are related and that they represent different stages of cyst development.
Small early cysts were predominantly lined by cuboidal or columnar epithelial cells, and
large advanced cysts were predominantly lined by dedifferentiated squamoid epithelial
cells. Intermediate cysts had less clearly demarcated cell types and frequently appeared
to have both cuboidal and squamoid cells in the same cyst. Carone et al (7) observed a
similar cellular phenotypic heterogeneity in a subpopulation of human ADPKD cysts.
The cyst cell phenotype appears to directly reflect the degree of cyst progression. In the
early stages of cystogenesis the cyst cells are well-differentiated and resemble normal
tubular epithelial cells. The tissue surrounding the cyst at this stage shows little or no
evidence of fibrosis or abnormal collagen deposition. As cyst expansion proceeds the
cyst cells gradually dedifferentiate to the attenuated squamoid phenotype seen in
advanced macro-cysts. The surrounding tissue becomes extremely fibrotic and large
deposits of collagen become intimately associated with the highly disorganized cyst
basement membrane.
Early studies of human ADPKD cystic kidneys also identified two cyst types, but
it was largely believed that the two cyst types were unrelated and that the phenotypic
differences reflected the respective cyst tubule segments of origin (for review see 32).
Cysts lined by dedifferentiated squamoid epithelial cells were reported to contain a fluid
with a high sodium content that was similar to that observed in serum and proximal
nephron segments. These cysts were therefore believed to be derived from proximal
tubules and were referred to as “proximal” or “non-gradient” cysts. Cysts lined with
cuboidal or columnar cells had sodium concentrations that were low relative to serum
levels and very similar to that observed in distal nephron segments. These cysts were
considered to be derived from the distal nephron and were referred to as “distal” or
“gradient” cysts. The cyst tubule segment of origin was determined solely on the basis of
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luminal sodium concentrations and an ability to maintain a transepithelial sodium
gradient. The identification of the tubule segment of origin was rarely corroborated by
cyst cell ultrastructure since the vast majority of the sampled cysts had typically
dedifferentiated to the point of no longer expressing recognizable segment-specific
characteristics (see for example 12). The ultrastructure of the “distal” cyst cells was
reminiscent of cells observed in distal segments of unaffected nephrons, but the
“proximal” cyst cells had no resemblance to normal proximal tubule cells. Statistical
analyses of human ADPKD cyst fluid compositions suggested that roughly 67% of the
sampled cysts were of proximal origin and that the remaining 33% were of distal origin
(17; 11). The ratio of “proximal” cysts to “distal” cysts closely reflected the expected
ratio of proximal nephron mass to distal nephron mass and was therefore taken as
additional supporting evidence that the two cyst types reflected the nephron segment of
origin. This conclusion implies that cystogenesis is a totally random event without a
clear segment-specific predilection for the site of cyst formation.
Our data (see Table 1) and that of Wu et al (36) argue strongly against a simple
stochastic model for the site of cyst formation in ADPKD-affected kidneys. Seventy to
eighty percent of the cysts that were observed in the PKD2WS25/- mouse renal cortex could
be unequivocally identified as being of either distal tubule or collecting duct origin. It is
possible that this correlation is specific to the PKD2WS25/- model, but recent
immunocytochemical studies on human ADPKD cystic kidneys suggest that this is not
the case. In two studies examining immunolocalization of aquaporin-1 (10) and of gp330
(2) only 30-44% of over one thousand cysts were considered to be of proximal origin.
Similar studies of aquaporin-2 immunolocalization (10;15) suggested that 30-33% of the
cysts were of collecting duct origin. Presumably the bulk of the remaining 33% of the
cysts are derived from other distal segments arguing that 66% and not 33% of the cysts
are of distal origin, and 33% and not 67% of the cysts are of proximal origin. If this is
true, the cyst fluid sodium concentration and hence the cyst cell ultrastructure would be
unlikely to correlate with the cyst segment of origin in the studies of human ADPKD
kidneys described above.
Like Grantham et al (12), we observed cellular phenotypic heterogeneity in a
significant subpopulation of ADPKD cysts. The source of the secondary cyst cell types
is unclear. The data presented by Grantham et al (12) suggest that the cysts in question
14
in their study had a collecting tubule origin and that the secondary cells were most likely
intercalated cells. The same may be true to some extent in our study, but the
ultrastructural evidence for a collecting tubule cyst origin is less compelling. Regardless
of the cyst tubule segment of origin, it is difficult to reconcile the cyst cellular
heterogeneity with the hypothesis of Qian et al’ (29) for cyst cell monoclonality. Using a
PCR-based cell clonality assay with human ADPKD cysts, Qian et al demonstrated that
at least 82% of the cysts (and quite probably more) were derived from single cyst
progenitor cells. If only collecting tubule-derived cysts showed cellular heterogeneity
one could postulate that both the intercalated- and principal-like cells were derived from
a single cyst progenitor cell. The exact origin of intercalated cells in vivo is unclear, but
it appears that both they and principal cells may be derived from a common ureteric bud
cell lineage during nephrogenesis (31). Perhaps ADPKD-induced cellular
dedifferentiation places collecting tubule-derived cyst progenitor cells into a limited
pleuripotent state that allows the subsequent transition to either an intercalated- or
principal-like cyst cell. A greater difficulty comes with cysts that do not appear to be of
collecting duct origin. In these instances one could postulate a polyclonal cyst origin or
the recruitment of cyst cells from an outlying cell population. The strongest argument
against either of those possibilities in our study is that the secondary cells were always
isolated and randomly dispersed throughout the cyst wall. If there truly was an
independently-derived mitotically-active subpopulation of cells within a cyst one would
expect to see clustering of phenotypically-identical cells. Moreover, the cellular
phenotypic heterogeneity was not preserved in all cyst stages. Heterogeneity was only
observed in intermediate or early advanced cysts coincident with the general timing of
the transition from a cuboidal to a squamoid cyst cell phenotype. This suggests that the
apparent “atypical” cells may actually be normal cyst cells in a slightly delayed state of
dedifferentiation and that they would eventually be phenotypically indistinguishable from
their neighbors upon completion of the transition to the squamoid dedifferentiated
phenotype.
The role of the primary cilium in the etiology of polycystic kidney disease is
currently receiving considerable attention. Studies in mouse models of PKD suggest that
defects in primary cilia may directly induce formation of renal cysts (for review see 18).
In orpk mice, for example, a mutation in the polaris gene has been shown to lead to
15
severe stunting of the primary cilia in the collecting tubule and concomitant cyst
formation (27). In the PKD2WS25/- model of ADPKD all cells in both early and
intermediate stage cysts, regardless of the cyst tubule segment of origin, have well-
developed normal appearing cilia. Cells in advanced cysts also have primary cilia, but
they appear to be much less prominent. Grantham et al (12) reported similar findings in
human ADPKD renal cysts. These observations suggest that if a ciliary defect is
involved in cyst formation in ADPKD, it likely does not have an ultrastructural
component. This is not wholly unexpected given that mutations in the C. elegans
orthologs of the PKD1 and PKD2 genes induced functional disruptions of the sensory
cilia without producing grossly obvious ciliary ultrastructural defects (3; 4). Nauli et al
(25) have recently proposed that polycystin-1 and polycystin-2 form heterodimeric
complexes on the surface of the primary cilium and that these complexes act as
mechanotransductive elements to convey information on luminal fluid flow and lumen
diameter to the morphoregulatory centers of renal tubular epithelial cells. They suggest
that a functional disruption of this complex could lead to cellular dedifferentiation and
tissue remodeling, and that cyst formation due to mutations in PKD1 or PKD2 is likely
due to a dysfunction in this signaling pathway rather than an overt alteration of ciliary
ultrastructure.
The only ultrastructural detail that appears to be significantly different between
PKD2WS25/- ADPKD cysts and human ADPKD cysts is the degree of basement membrane
perturbation. Cuppage et al (9) reported that there were abnormalities in human
ADPKD cyst basement membranes, but that there was not a consistent pattern in the
defect. Carone et al (7), however, reported that all of the human ADPKD cysts observed
in their study had thickened reticulated basement membranes. We did not observe either
thickening or reticulation of cyst basement membranes at any stage of cyst growth. The
basement membranes of all early and intermediate cysts were not noticeably different
from those of unaffected tubule segments. It was not until the cyst epithelial cells were
highly dedifferentiated that disorganization of the cyst basement membrane became
apparent. Consistent with the human ADPKD studies, we observed a significant increase
in interstitial fibrosis and collagen deposition in regions in direct apposition with
advanced cysts. The factors responsible for the fibrosis and the collagen deposition in
both the PKD2WS25/- and human ADPKD cystic kidneys remain uncertain.
16
It is clear that cyst cells undergo a dramatic dedifferentiation as cyst expansion
proceeds. However, from an ultrastructural perspective we could detect no evidence to
suggest that the cystic epithelium ever dedifferentiates to the point of losing cellular
polarity. Well-defined lateral junctions were preserved in even the most advanced cysts.
Consistent with this observation, the Na+/K+-ATPase, Ksp-cadherin, and E-cadherin were
all correctly targeted in cysts derived from every segment of the tubular nephron and the
collecting system. Moreover, we detected significant levels of anti-E-cadherin
immunostaining on the lateral membranes of all cyst cells regardless of the stage of cyst
expansion or cyst cell dedifferentiation. It is possible that some proteins may truly be
mislocalized in ADPKD cystic epithelia, but our observations in the PKD2WS25/- mice
suggest that this would be an uncommon event and that it would likely be due to a
perturbation in a processing pathway that is specific for that particular protein and not
due to a generalized basolateral sorting defect.
Our immunolocalization data do not support the model of apical Na+/K+-ATPase -
mediated, sodium-driven cyst fluid secretion. In advanced cysts at least, the data are
consistent with the model of chloride-driven cyst fluid accumulation proposed by
Sullivan et al (32) in which chloride uptake is mediated by the basolateral Na+/K+/2Cl-
cotransporter NKCC1. In a preliminary study of NKCC1 expression in human ADPKD
cystic kidneys, Lebeau et al (21) report that approximately 36% of all cysts examined
expressed immunodetectable levels of NKCC1 on their basolateral membranes. This
number compares favorably with our observations in PKD2WS25/- cystic mouse kidneys
(see Table 1), but it is unclear if they observed a similar correlation of NKCC1
expression with cyst expansion and dedifferentiation. Because we specifically focused
on cortical cysts in our study, the NKCC1 expression that we observed must have been a
direct result of an ADPKD-induced phenotypic transformation rather than the
consequence of cystic expansion of the inner medullary nephron segments that normally
express NKCC1. If advanced ADPKD cyst cells are using basolateral NKCC1 to drive
luminal fluid secretion in a manner similar to that proposed for airway epithelial cells,
one would presume that Na+/K+-ATPase activity would also be required on the
basolateral membrane of the same fluid secreting cells. Grantham et al (13) clearly
demonstrated that basolateral ouabain inhibited chloride-dependent cyst fluid secretion in
excised human ADPKD cysts. In our study the Na+/K+-ATPase was never detected in the
17
advanced cyst cells that expressed NKCC1. We suspect that the Na+/K+-ATPase may
have been present on the basolateral membrane, but that it was expressed at such low
levels that it was below the level of detection with the antibodies that were used in our
study. Given the extraordinarily low rate of fluid secretion that must occur in cysts in
vivo, it is possible that very few copies of the Na+/K+-ATPase are actually required to
maintain the necessary ionic gradients. The disparity of NKCC1 immunostaining
between early- and late-stage cysts suggests that the mechanism of cyst fluid
accumulation evolves as cystogenesis proceeds. It is likely that the earliest cysts retain
connections with the nephron segment of origin and that the cyst fluid is initially
glomerular filtrate. As the cyst expands and the connection with the parent nephron is
lost, fluid accumulation likely occurs by NKCC1-mediated NaCl and fluid secretion.
In summary, we have confirmed that ADPKD cyst cells undergo a dramatic
dedifferentiation as cystogenesis proceeds. We could detect no evidence of a generalized
basolateral sorting defect or of mislocalization of either the Na+/K+-ATPase, E-cadherin,
or Ksp-cadherin, and we therefore conclude that altered cyst cell polarity does not play a
role in either cyst expansion or cyst fluid secretion in ADPKD. We have directly
demonstrated the expression of the Na+/K+2Cl- cotransporter isoform NKCC1 on the
basolateral membrane of late cysts, supporting the model of chloride and fluid secretion
proposed by Grantham and colleagues.
18
ACKNOWLEDGEMENTS:
We thank Mr. Barry Piekos and Mr. Thomas Ardito for expert technical
assistance with the scanning and transmission electron microscopy.
This work was supported by National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-57328 (Yale PKD Center).
19
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23
FIGURE LEGENDS:
Figure 1. Representative transmission electron micrographs of early- through
late-stage PKD2WS25/- ADPKD cysts. Panel A. Early cyst (magnification
x8595). Panel B. Late intermediate cyst (magnification x6440). Panel C.
Extremely advanced cyst (magnification x14291). Panel C inset. Lateral
junction of cystic epithelial cells in extremely advanced cyst (magnification
x54351). A total of 48 cysts were analyzed by TEM; 29 were considered to
be early stage, 13 were considered to be intermediate stage, and 6 were
considered to be advanced. arrows indicate basement membranes; asterisks
indicate collagen deposits.
Figure 2. Representative scanning electron micrographs of PKD2WS25/- ADPKD
cysts. Panel A. Luminal surface of a comparatively small 123 µm diameter
advanced stage cyst illustrating the extent to which cyst cells are capable of
proliferating (magnification x544). Panel B. High magnification of an
early cyst cell. Note the regular cell borders and the prominent
cilia.(magnification x2367) Panel C. Luminal surface of a late intermediate
cyst. The arrow indicates an atypical cyst cell of the type frequently
observed in cysts of this size class. (magnification x1878) Panel D.
Luminal surface of an extremely advanced 3.2 mm diameter cyst.
(magnification x1801). Total number of cysts analyzed = 64.
Figure 3. Immunolocalization of the Na+/K+-ATPase in PKD2WS25/- ADPKD cystic
epithelia. Panel A. Overview of antibody C464.6 staining depicting
basolateral labeling in both normal and cystic epithelia. Cysts 1 and 2 were
DBA positive indicating collecting duct origin and Cyst 3 was NCC (TSC)
positive indicating a distal convoluted tubule origin. (magnification x98).
Panel B. Higher magnification of Cyst 3 labeled with antibody C464.6
(magnification x258). Panel C. Basolateral immunostaining of both normal
and cystic epithelia with anti-Na+/K+-ATPase antibody Alpha-5. The cyst
24
depicted in this panel was positively labeled with the anti-THP antibody
indicating a thick ascending limb origin. (magnification x707).
Figure 4. Immunolocalization of Ksp-cadherin in PKD2WS25/- ADPKD cystic
epithelia. Panel A. Overview of basolateral Ksp-cadherin staining in both
normal and cystic epithelia. The immunolabeled section depicted in this
panel is the anti-Ksp-cadherin portion of a double-labeling experiment done
with the anti-Na+/K+-ATPase antibody C464.6. The anti-Na+/K+-ATPase
portion of the experiment was presented in Panels A and B of Figure 3.
(magnification x98). Panel B. High magnification of the cyst labeled with
the asterisk in Panel A (magnification x258).
Figure 5. Immunolocalization of E-cadherin in PKD2WS25/- ADPKD cystic
epithelia. The anti-E-cadherin antibody Clone 36 clearly labels the
basolateral membranes of cystic epithelia. The cyst depicted in this panel
was DBA positive indicating a collecting tubule origin. (magnification
x354).
Figure 6. Immunolocalization of Na+/K+-ATPase, Ksp-cadherin and E-cadherin
in advanced PKD2WS25/- ADPKD cysts. Panel A. The tissue section
depicted in this panel was labeled with the anti-Na+/K+-ATPase antibody
C464.6. Positive immunostaining was not detected in either the highly
dedifferentiated cystic epithelium or the surrounding fibrotic tissue. Areas of
the section outside the field depicted in this panel showed strong basolateral
staining in both unaffected tubule segments and early cysts. The lower cyst
was THP positive indicating a thick ascending limb origin, but the upper
cyst was not positively labeled by any of the tubule segment markers used in
this study. (magnification x327). Panel B. Double-labeling of the same
tissue section depicted in Panel A with the anti-Ksp-cadherin antibody.
Note the prominent basal immunostaining in the lower THP positive cyst.
(magnification x327). Panel C. Prominent lateral E-cadherin
immunostaining of cystic epithelial cells that were not positively labeled by
25
anti-Na+/K+-ATPase or anti-Ksp-cadherin antibodies or tubule segment-
specific markers. (magnification x211).
Figure 7. Immunolocalization of NKCC1 in PKD2WS25/- ADPKD cysts. Panel A.
Immunolabeling of the cystic renal cortex with the anti-NKCC1 antibody.
Note the positive staining in the glomerular associated structures indicated
by the arrowheads and the lack of staining in the early cysts indicated by the
asterisks. See Figures 3a and 4a for sequential serial-section labeling with
anti-Na+/K+-ATPase and anti-Ksp-cadherin antibodies. (magnification x92)
Panel B. Anti-NKCC1 immunolabeling of an advanced cyst that does not
express detectable levels of the Na+/K+-ATPase, Ksp-cadherin, or tubule
segment-specific markers. The arrowhead indicates glomerular mesangial
staining. (magnification x278). Panel C. Heterogeneous anti-NKCC1 cyst
staining. Cyst 1 is a DBA /Ksp-cadherin positive, Na+/K+-ATPase negative
cyst (data not shown) that stains moderately with the anti-NKCC1 antibody.
Cyst 2 is a NKCC1-negative glomerular cyst that is also negative for all
tubule segment markers, the Na+/K+-ATPase and Ksp-cadherin. Note the
lack of anti-NKCC1 mesangial staining in the glomerular rudiment
indicated by the asterisk. Cyst 3 expressed high levels of basolateral
NKCC1, but was negative for all tubule segment markers, the Na+/K+-
ATPase and Ksp-cadherin. (magnification x740).
26
Table 1. Nephron segment of origin of Na+/K+-ATPase-, Ksp-cadherin-, E-cadherin-, and NKCC1-
positive cysts.
Cyst Segment of Origin
PT TAL DCT CT Indeterminate Total # of Total # of(+/tot) (+/tot) (+/tot) (+/tot) (+/tot) positive cysts cysts examined
Na+/K+-ATPase 3/3 21/21 2/3 34/36 0/19 60 82
Ksp-cadherin 3/3 21/21 3/3 36/36 3/19 66 82
E-cadherin 2/2 20/20 5/5 32/32 14/14 73 73
NKCC1 0/2 1/17 0/1 1/28 16/17 18 65
Cyst segment of origin was determined by lectin and antibody labeling as described in the Methods.
Cysts not labeled with the segment-specific markers used in this study were considered to be of an
indeterminate origin. PT, proximal tubule; TAL, thick ascending limb; DCT, distal convoluted tubule;
CT, collecting tubule; +/tot, number of positively labeled cysts/total number of cysts observed in each
cyst segment category.