1 Physiologisches Institut and 2 Medizinische Poliklinik der Ludwig-Maximilians-Universität, 80336 Munich; and 3 Institut für Anatomie der Charité, 10098 Berlin, Germany
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ABSTRACT |
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The expression
patterns of plasma membrane transporters that specify the
epithelial cell type are acquired with ontogeny. To study this process
during metanephrogenic mesenchyme-to-epithelium transition, branching
ureteric buds with their adjacent mesenchymal blastema (mouse embryonic
day E14) were dissected and explanted on a collagen matrix.
In culture, induced mesenchymal cells condensed, aggregated, and
converted to the comma- and S-shaped body. During in vitro condensation
and aggregation, transcription factor Pax-2 protein was downregulated
while the epithelial markers E-cadherin and -catenin proteins were
upregulated. In addition, Wilms' tumor suppressor protein WT-1 was
detectable upon condensation and downregulated in the S stage, where
expression persisted in the long arm of the S. Patch-clamp, whole cell
conductance (G, in nS/10 pF) of pre-epithelial condensed
mesenchymal cells (n = 7) was compared with that of
tubular proximal S-shaped-body epithelium (n = 6). Both
stages expressed E-cadherin and WT-1 mRNA, as demonstrated by
single-cell RT-PCR, testifying further to the epithelial as well as the
nephrogenic commitment of the recorded cells. Mesenchymal cells
exhibited whole cell currents (G = 6.7 ± 1.3)
with reversal potentials (Vrev, in mV) near
equilibrium potential for Cl
(ECl)
(Vrev =
40 ± 7) suggestive of a
high fractional Cl
conductance. Currents of the
S-shaped-body cells (G = 4.0 ± 1.1), in sharp
contrast, had a Vrev at
EK (Vrev =
82 ± 6) indicating a high fractional K+ conductance. Further,
analysis of K+-selective whole cell tail currents and
single-channel recording revealed a change in K+ channel
expression. Also, Kir6.1 K+ channel mRNA and protein were
downregulated between both stages, whereas KvLQT
K+ channel mRNA was abundant throughout. In conclusion,
metanephrogenic mesenchyme-to-epithelium transition is accompanied by a
profound reorganization of plasma membrane ion channel conductance.
mesenchymal-epithelial transition; embryonic ion channels; single-cell reverse transcription-polymerase chain reaction; kidney developmental biology
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INTRODUCTION |
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THE METANEPHRIC KIDNEY ORIGINATES from two distinct embryonic precursor populations. The mesenchymal blastema generates the nephron from glomerulus to distal tubule, whereas the ureter gives rise to the collecting tubule system by branching morphogenesis. The nephron develops by a direct mesenchyme-to-epithelium transition when the ureteric buds invading the mesenchymal blastema induce the transition (9). The stages of this process can be defined in situ and have been separated into the following: induction, determination of the stem cell pathway, condensation, epithelialization, aggregation, cell polarization, and functional expression of the differentiated nephron cell type (reviewed in Ref. 12).
However, functional cell polarization and differentiation, i.e., the processes that lead to the specific absorptive and secretory functions of a nephron segment, have not been evaluated during mesenchyme-to-epithelium transition, most likely because direct access to defined epithelial cells beneath the renal capsule in embryonic organ cultures is limited. In the present work, therefore, the single nephrogenic unit, i.e., the most peripheral dichotomous ureteric branch with its buds and the adherent blastema, was dissected from the embryonic kidney and grown in coculture. The process of mesenchyme-to-epithelium transition in vitro was found to be phenotypically identical to that of the in situ organ culture, with respect to morphology and expression of transcription factors and morphoregulatory proteins.
Changes in plasma membrane ion channel expression during mesenchyme-to-epithelium transition and early tubulogenesis were studied by comparing in vitro pre-epithelial condensed mesenchyme cells with those of the epithelial S-shaped body applying patch-clamp techniques, single-cell RT-PCR, and immunohistochemistry. This work provides first evidence for a profound repatterning of plasma membrane ion channel expression during mesenchyme-to-epithelium transition.
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MATERIALS AND METHODS |
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Mesenchyme-to-epithelium transition in primary culture.
All chemicals were from Sigma (Deisenhofen, Germany) unless stated
otherwise. Ureteric buds with attached mesenchymal blastema were
microdissected from the most peripheral dichotomous branches of the
collecting duct system of embryonic mouse kidney (gestational day
E14, CD1 mouse; Charles River, Kisslegg, Germany) in
Ca2+- and Mg2+-free PBS (Seromed, Berlin,
Germany) at 4°C, explanted on coverslips coated with newborn mouse
tail collagen, and attached to the matrix at the ureteric trunk end.
The dissected tissues were grown in modified nephron culture medium
(11) containing DMEM/Ham's F-12 (1:1; Life Technologies,
Eggenstein, Germany), penicillin (100 U/ml), NaHCO3 (20 mM), streptomycin (10 µg/ml), bovine transferrin (5 µg/ml),
3,3',5-triiodo-L-thyronine (5 pM), L-glutamine
(20 µM), NaSe (50 nM), HEPES/NaOH (pH 7.4, 10 mM),
hydrocortisone (50 nM), prostaglandin E1 (25 ng/ml)
with fetal calf serum (3%, Seromed), and bovine pituitary extract (100 µg/ml) equilibrated with CO2 (5%) to pH 7.4. Medium was
exchanged daily and cells were analyzed after 3-4 days of culture.
For -catenin immunohistochemistry, tissue was explanted and cultured
in an identical manner on flexiPERM (In Vitro Systems and Services,
Osterode, Germany) mouse tail collagen-coated chamber slides (see below).
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Immunostaining of development-specific marker proteins and of
Kir6.1 K+ channels.
Immunostaining of Pax-2, WT-1, E-cadherin, and Kir6.1 were performed in
whole cocultures of ureteric bud and blastema. For immunostaining of
-catenin, cocultures were grown in flexiPERM chambers on glass
slides. After fixation (see below) cultures were stained with
hemalaun for 5 min and rinsed with PBS until the tissue could
be seen as a blue dot within the background. The chambers were filled
with 3% gelatin on top of the cultures and cooled for about 15 min at
4°C. Thereafter, chambers with cultures that were gelatin-embedded at
one side were removed from the glass slides, and gelatin was added to
other side of the cultures to prevent destruction of the tissue during
the paraffin embedding procedure. Cultures within gelatin blocks were
harvested from the chambers by a gentle push, embedded in paraffin, and
cut in 2-µm sections which were deparaffinized and rehydrated.
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Patch-clamp recordings. Patch-clamp recordings were performed at room temperature in voltage clamp mode as described previously (13). Applied voltages refer to the cytoplasmic face of the membrane with respect to the interstitial space. Inward currents, defined as flow of positive charge from the interstitial to the cytoplasmic membrane face, are negative currents and are depicted as downward deflections of the original current traces. Applied voltages were corrected by the liquid junction potential between pipette and bath solution that was set off by the patch clamp amplifier prior to sealing and that was estimated according to Ref. 3.
Cultures were rinsed with NaCl bath solution (in mM: 150 NaCl, 10 D-glucose, 10 HEPES, 5 KCl, 1.6 CaCl2, and 0.8 MgCl2, titrated with NaOH to pH 7.4) and constantly superfused (1 ml/min) with NaCl bath solution or with a solution containing 155 KCl, 10 D-glucose, 10 HEPES, 1.6 CaCl2, and 0.8 MgCl2 (titrated with KOH to pH 7.4). Patch-clamp experiments were performed using glass pipettes (2-5 MSingle-cell RT-PCR. Development-dependent mRNA expression of the electrophysiologically characterized in vitro nephrogenesis stages was studied by single-cell RT-PCR according to previous reports (14, 19). Cytoplasm that dialyzed into the patch pipette during whole cell recording was harvested by expelling the total pipette volume (6.5 µl) directly into a tube filled with 3.5 µl reverse transcriptase (RT) mixture (see below). Cytoplasm was harvested by whole cell recording mode in 65 cells. Because of either electrical coupling between the cells, or to loss of gigaohm seal during recording, or to an initially insufficient seal, the entire whole cell recording protocol (see Patch-clamp recordings) together with current analysis could be run in 13 cells only.
The RT mixture contained the following: 1 µl dithiothreitol (0.1 mM), 0.1 µl single-strand buffer (5-fold), 0.5 µl SuperScript RT (200 U/µl) (all Life Technologies), 0.5 µl RNase inhibitor (RNasin), 0.5 µl dATP/dCTP/dGTP/dTTP mixture (25 mM each), and 1 µl random hexamer oligonucleotide primer (Boehringer, Mannheim, Germany). The RNA was transcribed for 1 h at 37°C. From the total volume of about 10 µl cDNA, a 2-µl aliquot was placed in a second tube in some experiments, and both aliquots were stored at
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RESULTS |
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Protein expression of the transcription factor Pax-2 and the Wilms' tumor suppressor WT-1 was analyzed by immunofluorescence in metanephrogenic cultures to characterize the in vitro developmental stages of the nephron lineage. These stages as defined by light microscopy to represent mesenchymal blastema, condensed mesenchyme, globular aggregate, comma-shaped body, and S-shaped body differed in their WT-1 and Pax-2 protein expression.
WT-1 protein was apparent neither in ureteric bud tubules nor
monolayer, nor in blastemal cells (Fig.
4, A and B). Marked WT-1-specific staining that was confined to the nuclei occurred in all
cells of condensed mesenchyme (Fig. 4A), globular aggregate (Fig. 4B), and comma-shaped body (Fig.
4C). In S-shaped body, staining was restricted to the long
(proximal) arm of the S (Fig. 4D; see below), suggestive of
upregulation of WT-1 protein upon condensation and of downregulation in
most cells during comma-to-S conversion.
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In contrast to WT-1, rudimentary ureteric bud tubules (Fig. 4E) and blastema cell populations (Fig. 4G), as well as condensed mesenchyme cells (Fig. 4E), exhibited nuclear immunostaining for Pax-2. This staining was not apparent in globular aggregate (Fig. 4F), comma- (Fig. 4G), and S-shaped body (not shown), suggesting a rapid downregulation of Pax-2 protein before globular aggregation. In addition, nuclei of ureteric bud monolayer (Fig. 4H) were Pax-2 protein positive, similar to those of rudimentary ureteric bud tubules and of the early nephron lineage stages.
Progress of mesenchyme-to-epithelium transition during nephrogenesis
was monitored by E-cadherin and -catenin immunohistochemistry. Cell
adhesion molecule E-cadherin-specific immunostaining of the plasma
membrane occurred in ureteric bud monolayer between adjacent cells but
not in blastema (Fig. 5A),
confirming the mesenchymal phenotype of the latter. Condensed
mesenchyme (Fig. 5B), globular aggregate (Fig.
5C), and S-shaped body (Fig. 5D), similar to the ureteric bud, exhibited plasma membrane-localized staining, suggesting that E-cadherin-mediated cell adhesion starts upon condensation of the
mesenchyme.
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To further analyze the cell organization of the nascent epithelial
cells, protein expression of the plasma membrane-to-cytoskeleton linker
-catenin was studied in paraffin sections. Weak plasma membrane-associated
-catenin immunostaining was apparent in
condensed mesenchyme, further suggesting advanced epitheliogenesis in
the condensed state (Fig. 5G). Globular aggregate, ureteric
bud monolayer (Fig. 5H), and S-shaped body (Fig.
5I) showed regular sharp staining on both sides of the
lateral cell-cell junction. Blastema cells, by contrast, were
-catenin negative, emphasizing their mesenchymal phenotype. Negative
controls for E-cadherin and
-catenin immunohistochemistry were not
immunoreactive (Fig. 5, E, F, and J).
To study the changes in plasma membrane ion conductance during
mesenchyme-to-epithelium transition, patch-clamp whole cell recordings
were performed in two developmental stages, in the preepithelial
condensed mesenchyme (Fig. 6A)
and in the proximal S-shaped body (Fig. 6B). Specifically,
recordings were made from the region where the long, proximal arm
became the first bend of the S and where a lumen clearly was visible,
suggesting that this region represented the first stage of the nephron
lineage that was organized in a tubular epithelium (Fig. 6,
C and D).
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In cytosol that dialyzed into the recording pipette during whole cell recording, WT-1 and E-cadherin mRNA expression was analyzed by single-cell RT-PCR to directly identify the nephrogenic fate of the recorded cells (Fig. 6E). To ascertain that the single-cell RT-PCR method worked with similar yields in condensed mesenchyme and S-shaped-body cells, a cDNA fragment specific for the housekeeper GAPDH was amplified further from aliquots of the cytosol samples.
After one round of PCR (see MATERIALS AND METHODS and Fig. 2), about 40% of the cytosol samples from both developmental stages were positive for the housekeeper GAPDH, suggesting similar efficiency in applying the single-cell RT-PCR method to condensed mesenchyme and S-shaped-body cells (Fig. 6F). Using a strategy of "duplex" PCR combined with a second round of nested uniplex PCR, WT-1-specific PCR products were amplified in about 25% and 60% of the cytosol samples harvested from condensed mesenchyme and proximal S-shaped-body cells, respectively. In addition, E-cadherin-specific products were amplified in 20-25% of cytosol samples of both stages (Fig. 6F), thus demonstrating directly the nephrogenic commitment of both recorded cell populations.
Extracellular negative controls did not yield GAPDH-, WT-1-, or E-cadherin-specific PCR products (Fig. 6, E and F). Thus specific PCR products amplified from the cytosol samples were not due to mRNA spilled into the extracellular space as might occur, for instance, by cell death.
Whole cell currents of preepithelial condensed mesenchyme cells and of
tubular epithelial S-shaped body cells differed markedly (Fig.
7, A and B).
Condensed mesenchyme cells when recorded with potassium gluconate/KCl
pipette and NaCl bath solution exhibited large outward whole
cell currents at depolarizing, but only small inward currents at
hyperpolarizing voltages. Upon depolarizing voltage steps to greater
than or equal to +50 mV, outward currents activated time dependently in
five of a total of seven cells with a mean time constant of
T = 44 ± 10 ms at +90 mV voltage (Fig. 7A, left). This depolarization-dependent,
slow-activating current fraction rectified outwardly. Replacement of
bath NaCl by KCl (Fig. 7A, right) elicited an
increase in inward currents suggestive of a K+-selective
fractional whole cell K+ conductance
(GK). Mean current-voltage
(I/V) relation of capacitance-normalized whole cell currents (NaCl bath, potassium gluconate/KCl pipette solution) exhibited weak outward rectification and a conductance of
G = 6.7 ± 1.3 nS/10 pF (n = 7) as
calculated for the outward currents (Fig. 7C).
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The reversal potential (Vrev) of the
I/V curve (Vrev = 40 ± 7 mV) was between the equilibrium potential (E)
for Cl (ECl =
36 mV) and
EK (
85 mV). Replacement of bath NaCl by KCl in
paired experiments shifted Vrev by +29 mV along
the change of EK (EK = +3 mV) to Vrev =
10 ± 3 mV (Fig.
7C), further demonstrating a fractional
GK (Fig. 7C). A fractional
GCl was indicated by deflection of
Vrev in KCl bath solution from
EK (+3 mV) as well as from the equilibrium
potential for nonselective cations (NSC;
ENSC = +3 mV) toward
ECl by about
13 mV. This fractional
GCl, in addition to GK,
mainly contributed to the whole cell conductance of condensed mesenchyme cells. A further contribution of other cation conductances, e.g., of a nonselective cation conductance
(GNSC) to whole cell conductance could not be
deduced from these experiments (Fig. 7C).
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Proximal S-shaped-body cells, by contrast, did not show slowly
activating whole cell outward currents. Replacement of bath NaCl by KCl
evoked large inward currents suggestive of a very high fractional
GK (Fig. 7B). The mean
I/V curve when recorded in NaCl bath (potassium
gluconate/KCl pipette solution) did not rectify and exhibited a slope
of G = 4 ± 1.2 nS/10 pF (n = 6) and a Vrev at EK
(Vrev = 82 ± 6 mV). Upon
replacement of bath NaCl by KCl, Vrev shifted
with EK (Vrev =
1 ± 1 mV), indicating that whole cell conductance in proximal
S-shaped-body cells was almost exclusively generated by
GK (Fig. 7D).
GK was further characterized in whole cell tail
currents of both developmental stages (Fig. 7, E and
F). Tail currents were analyzed in NaCl bath solution
(potassium gluconate/KCl pipette solution) after repolarizing the
membrane potential from voltages between 110 and +90 mV to
10
mV holding potential. Three condensed mesenchyme cells that had
relatively high fractional GK exhibited fast
inactivating tail currents (Fig. 7E, left). The
amplitude of the tail currents increased with the degree of
hyperpolarization of the preceding square pulse, consistent with a
hyperpolarization-activated current that inactivated upon
repolarization. This current was K+ selective since
replacement of NaCl by KCl in the bath with consequent shift of
EK close to
10 mV holding potential abolished
the tail currents (Fig. 7E, right).
Tail currents of three proximal S-shaped-body cells, by contrast, activated fast (T in the range of 1 ms) upon repolarization from hyperpolarizing square pulses (Fig. 7F, left) and inactivated subsequently with larger time constants (not shown). These tail currents were extinguished when bath NaCl was replaced by KCl, indicating their K+ selectivity (Fig. 7F, right).
In addition to the differences in K+-selective whole cell
tail currents, condensed mesenchyme and proximal S-shaped body cells differed in their K+ channel activity (Fig. 8,
A-E). In three condensed
mesenchyme cells, channels with a mean unitary conductance of 35 pS, as
determined at 0 mV voltage with NaCl in the bath and potassium
gluconate/KCl in the pipette, were identified in outside-out excised
patches (Fig. 8, A and B). The
Vrev of the current amplitude extrapolated by
linear regression to 75 ± 5 mV voltage, indicating
K+ selectivity. In a single cell-attached experiment
(potassium gluconate/KCl pipette solution), channels with
characteristics identical to the 35-pS channel exhibited inwardly
rectifying I/V curves (Fig. 8B).
BaCl2 (1 mM) when added to the bath solution reversibly
blocked the 35-pS K+ channel in outside-out patches (Fig.
8C).
In the serosal membrane of proximal S-shaped body cells, by contrast, 35-pS K+ channels were not apparent. A further type of K+-selective channel was detected in the serosal outside-out patch of a proximal S-shaped-body cell (Fig. 8D). This type exhibited fast activation upon repolarization from hyperpolarizing voltages followed by a slow inactivation (Fig. 8D, inset), very similar to the kinetics of the macroscopic tail currents of the proximal S-shaped-body cells (Fig. 7F). The I/V curve of this channel type as recorded in outside-out and cell-attached mode was linear with a low conductance of ~10 pS (Fig. 8E).
To identify K+ channel molecules in both developmental stages, mRNA expression of the KvLQT, a voltage-activated K+ channel involved in epithelial Cl secretion, and Kir6.1, an inward rectifier which is highly expressed in the nephrogenic zone of embryonic kidney (unpublished observation), was investigated by single- cell RT-PCR. In addition, Kir6.1 protein expression was studied immunohistochemically.
KvLQT mRNA was detected in about 60% and 75% of condensed mesenchyme
and proximal S-shaped body cells, respectively, suggesting similar mRNA
abundance in both developmental stages (Fig.
9, A and B). Kir6.1
mRNA, by contrast, was identified in only 2 of 12 condensed mesenchyme
cells but not in proximal S-shaped-body cells (Fig. 9, A and
B).
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The low yield of the Kir6.1 PCR was not due to a low sensitivity of the PCR reaction since Kir6.1- and KvLQT-specific fragments were amplified with similar sensitivity from sequentially diluted cDNAs of whole cultures (i.e., mesenchyme-derived stages and ureteric bud epithelium) using the single cell PCR protocol (duplex combined with nested uniplex PCR; not shown). Rather, the inability to amplify Kir6.1-specific fragments from proximal S-shaped body cells might reflect a downregulation of Kir6.1 mRNA expression between both stages.
Similar downregulation of Kir6.1 was observed at the protein level. Kir6.1 protein was detected in the plasma membrane of condensed mesenchyme (Fig. 9C) and globular aggregate (Fig. 9D), but immunostaining was only weak or absent in that of S-shaped-body cells (Fig. 9E). In rudimentary tubules (Fig. 9D) and monolayers of the ureteric bud (Fig. 9F), Kir6.1 protein stained in the apical and lateral membrane and to a lesser extent in the basal membrane. Control cultures incubated with antigen-saturated antibodies yielded no staining (Fig. 9, G and H), indicating the specificity of the Kir6.1 immunohistochemistry.
Figure 10 summarizes the principal
findings of the present study.
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DISCUSSION |
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The present study has applied electrophysiological and RT-PCR
methods for the first time to single cells passing through
metanephrogenic mesenchyme-to-epithelium transition in vitro. The
developmental stages of this in vitro model, i.e., mesenchymal
blastema, condensed mesenchyme, globular aggregate, comma-, and
S-shaped body, were defined by their characteristic morphology and by
the expression of Pax-2, WT-1, E-cadherin, and -catenin.
Previous studies in vivo and on kidney rudiments in organ culture have shown that WT-1 which is weakly expressed in uninduced blastema cells is upregulated after induction in condensing cells, and expression continues through the comma- and S-shaped stage, while high levels in the terminal nephron persist in podocytes only (2, 8, 24, 25). The Pax-2 gene, by contrast, is activated in the mesenchymal blastema in response to induction and is subsequently downregulated rapidly at or soon after the S-shaped body stage (7, 8, 26). The in vitro model of the present study showed comparable time courses of protein expression and repression of both genes with an identical sequence. In addition, the time course of transition in the present study, from condensed mesenchyme morphology to the early tubular epithelium of the S-shaped body (1-2 days of culture; see Fig. 1, B and C), was similar to that occurring in renal rudiments in organ cultures (10), indicating the validity of in vitro model used in the present study.
Condensed mesenchyme, which formed epithelium-like arrays capping the
ureteric bud tip (e.g., in Fig. 6A), expressed E-cadherin and -catenin proteins. E-cadherin plays a crucial role in early epithelial polarization by mediating cell-cell adherens junctions, assembling basolateral plasma membrane-cytoskeleton complexes that
involve
-catenin, and by integration and retention of
Na+-K+-ATPase in these complexes
(21, 23, 28). The E-cadherin expression in the condensed mesenchyme stage of the present study suggests that this E-cadherin upregulation is a very early event in
mesenchyme-to-epithelium transition.
Expression changes of plasma membrane ion channels during this process were studied by comparing whole cell currents of an early, preepithelial (condensed mesenchyme cells) with those of a late developmental stage (tubular epithelium of the proximal S-shaped body). Both stages expressed WT-1 and E-cadherin mRNA as determined by single-cell RT-PCR, thus providing further proof for the nephrogenic commitment of the electrophysiologically characterized cells.
Whole cell currents of condensed mesenchyme and proximal S-shaped-body
cells differed in conductance, reversal potential, and
K+-selective tail currents. In addition, differing
K+ channel types were apparent in outside-out patches of
both developmental stages (summarized in Fig. 10). Whole cell currents
in condensed mesenchyme cells, by the high fractional Cl
currents, the depolarization- and time-dependently activating current
component, and by the low reversal potential resemble very closely
those of embryonic ureteric bud cells (17), the second
primordium of the kidney. These current properties were absent in the
proximal S-shaped-body cells (present study) and in the descendants of
the ureteric bud cells, the collecting duct epithelium
(13, 17).
Furthermore, in the present study, condensed mesenchyme but not proximal S-shaped body cells expressed Kir6.1 K+ channel mRNA and protein. Much like condensed mesenchyme, ureteric bud epithelium expresses Kir6.1 mRNA and protein that is downregulated during development of the collecting duct (Ref. 6 and unpublished observations). This further suggests that the same types of embryonic ion channels are expressed in branching morphogenesis of the collecting duct and mesenchyme-to-epithelium transition, which are simultaneously downregulated in both primordia-derived nascent nephron epithelia.
In the present study, KvLQT mRNA was abundant in condensed
mesenchyme and in proximal S-shaped body cells. KvLQT
interacts with IsK protein to form a low-conductance, cAMP-stimulated
K+ channel (5). These channels are expressed
in the basolateral membrane of cells at the colonic crypt base where
they maintain the driving force for Cl during
cAMP-stimulated colonic Cl
secretion (30).
The expression of secretory-type channel mRNA during
mesenchyme-to-epithelium transition might suggest that similar
secretory processes occur during tubulogenesis of the mesenchyme-derived nephron segments.
During branching morphogenesis of lung and collecting duct epithelium,
such Cl secretion is suggested by the
development-specific expression of secretory Cl channels
(15, 16, 22). In embryonic lung, a morphogenic action of secretion by the developing epithelium has been
demonstrated directly (1). Therefore, Cl
secretion might be a common feature of tubulogenesis and could contribute to build up and maintain the lumen of the embryonic tubular
epithelium against tissue pressure. Luminal volume expansion might
induce proliferation and longitudinal growth of the tubular epithelium.
It is of interest in this context that a reactivation of probably
embryonic programs of vectorial transport by mature renal epithelia is
involved in the pathogenesis of renal cysts (27).
In conclusion, dissected metanephrogenic units consisting of ureteric buds with adherent mesenchymal blastema, explanted in primary culture, provide an in vitro model that allows experimental access to the cell physiology of metanephrogenic mesenchyme-to-epithelium transition. During this process, a set of embryonic ion channels in the plasma membrane of condensed mesenchyme is replaced by other channel types that might be involved in secretory processes of the nascent epithelium.
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ACKNOWLEDGEMENTS |
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We thank Roswitha Maul for expert technical assistance.
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FOOTNOTES |
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This work was financially supported by the Deutsche Forschungsgemeinschaft (Ho 485/16-1 and 16/2).
A preliminary account of this work has been published in abstract form (J Am Soc Nephrol 9: 362, 1999).
Address for reprint requests and other correspondence: M. F. Horster, Physiologisches Institut, Universität München, Pettenkoferstr. 12, D-80336 München, Germany.
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.
Received 13 September 1999; accepted in final form 31 January 2000.
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