Extracellular nucleotides regulate cellular functions of
podocytes in culture
Karl-Georg
Fischer,
Ulrich
Saueressig,
Claudius
Jacobshagen,
Arndt
Wichelmann, and
Hermann
Pavenstädt
Division of Nephrology, Department of Medicine, University Hospital
Freiburg, D-79106 Freiburg, Germany
 |
ABSTRACT |
Extracellular nucleotides are assumed to be important
regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free
Ca2+ concentration of differentiated podocytes were
examined with the patch-clamp and fura 2 fluorescence techniques. mRNA
expression of purinergic receptors was investigated by RT-PCR.
Purinergic agonists depolarized podocytes. Purinergic agonists
similarly increased intracellular free Ca2+ concentration
of podocytes. The rank order of potency of various nucleotides on
membrane voltage and free cytosolic calcium concentration was UTP
UDP > [adenosine 5'-O-(3-thiotriphosphate)
(ATP-
-S)] > ATP > 2-methylthioadenosine 5'-triphosphate
(2-MeS-ATP) > 2'- and
3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate
(BzATP) > ADP-
-S.
,
-Me-ATP was without effect. In the
presence of UTP, BzATP did not cause an additional depolarization of
podocytes. Incubation of cells with ATP or BzATP did not induce lactate
dehydrogenase release. In RT-PCR studies, mRNAs of the
P2Y1, P2Y2, P2Y6, and P2X7 receptors were detected within glomeruli and
podocytes. The data indicate that extracellular nucleotides modulate
podocyte function mainly by an activation of both P2Y2 and
P2Y6 receptors.
adenosine 5'-triphosphate; purinoceptor; intracellular calcium
concentration; ion currents; nucleotides; P2X7 receptor
 |
INTRODUCTION |
EXTRACELLULAR ATP IS AN
IMPORTANT signaling molecule that regulates distinct cellular
functions via P2 receptors. These receptors have been divided into two
families of ligand-gated ion channels and G protein-coupled receptors,
termed P2X and P2Y receptors, respectively (23). Up until
now, seven subtypes of P2X receptors and eight subtypes of P2Y
receptors have been identified (11). Within the
glomerulus, ATP is involved in the regulation of glomerular hemodynamics and renal autoregulation. ATP is released by sympathetic nerve stimulation. Other sources of ATP are erythrocytes, platelets, mast cells, and endothelial cells (2). During glomerular
inflammation, ATP is released from damaged resident glomerular and
infiltrating cells, thereby modulating cellular responses during
glomerular injury (2). A P2Y receptor has been
pharmacologically characterized in cultured glomerular endothelial
cells (1), and P2Y receptors and a P2X7
receptor have been identified in cultured mesangial cells (25,
26). Knowledge about the expression of P2 receptors in podocytes
is limited. The glomerular basement membrane possesses ATP- and ADPase
activity, which is assumed to play an antiproteinuric role in the early
phase of anti-Thy1 nephritis. ATP possibly plays a role in podocyte
function and influences its barrier function (22). The
podocyte forms a crucial part of the glomerular filtration barrier. Its
foot processes possess contractile structures, which may respond to
vasoactive hormones and thereby regulate the ultrafiltration coefficient (Kf) (4, 12). Podocytes
thus contribute to size and charge selectivity of the glomerular
filtration barrier; their injury leads to proteinuria (10,
17). Here we investigate expression and functional properties of
P2 receptors in differentiated podocytes.
 |
METHODS |
Cell culture.
Immortalized mouse podocytes were derived from mice that have a
thermosensitive variant of the SV40-T antigen inserted into the mouse
genome. These mouse podocytes proliferate at 33°C in the presence of
-interferon (SV40-T antigen active). At 37°C and after removal of
-interferon, cells transform into the quiescent, differentiated
phenotype (14). Within the present study, cells then
stained positive for the podocyte markers Wilm's tumor protein (WT-1)
(15), synaptopodin (13), nephrin
(28), and p57 (16). In addition, mRNA
expression of the podocyte marker CD2AP (27) was detected
by RT-PCR. For experiments, cells between passages 15 and
25 were seeded at 37°C into sixwell plates and cultured in
standard RPMI media containing 1% FCS (Boehringer, Mannheim, Germany),
100 U/ml penicillin, and 100 mg/ml streptomycin (both GIBCO,
Eggenstein, Germany) for at least 7 days until cells were differentiated, showing an arborized morphology.
Patch-clamp experiments.
Most of the patch-clamp experiments were performed in the slow whole
cell configuration (SWC), which has been described in detail elsewhere
(5, 6). In brief, podocytes were placed in a bath chamber
on the stage of an inverted microscope, kept at 37°C, and
superperfused with a phosphate-buffered Ringer-like solution containing
(in mM) 145 NaCl, 1.6 K2HPO4, 0.4 KH2PO4, 1.3 CaCl2, 1.03 MgCl2, and 5 D-glucose, pH 7.4. The patch
pipettes were filled with a solution containing (in mM) 95 K-gluconate, 30 KCl, 4.8 Na2HPO4, 1.2 NaH2PO4, 0.73 CaCl2, 1.03 MgCl2, 1 EGTA, and 5 D-glucose, as well as
50-100 mg/l nystatin, pH 7.2 (10
7 M Ca2+
activity). The patch pipettes had an input resistance of 2-3 M
. A flowing (10 µl/h) KCl (2 M) electrode was used as a reference. The data were recorded using a patch-clamp amplifier (Fröbe and Busche, Physiologisches Institut, Freiburg, Germany) and were continuously displayed on a pen recorder. The access conductance (Ga) was monitored in most of the experiments by
the method recently described (6). Membrane voltage
(Vm) of the cells was continuously recorded
using the current-clamp mode of the patch-clamp amplifier. To obtain
the whole cell conductance (Gm), the voltage of
the respective cell was clamped in the voltage clamp mode
(Vc) to Vm. Starting from
this value, the whole cell current was measured by depolarizing or
hyperpolarizing Vc in steps of 10 to ± 40 mV. Gm was calculated from the measured whole
cell current (I), Ga, and
Vc using Kirchhoff's and Ohm's laws.
Measurements of intracellular calcium concentration.
Measurements of intracellular Ca2+ concentration
([Ca2+]i) were performed in single podocytes
with an inverted fluorescence microscope as recently described (bath
temperature: 37°C, bath solution changes within 1 s)
(24). In short, podocytes were incubated with the Ca2+-sensitive dye fura 2-acetoxymethyl ester (AM) (5 µM,
Sigma, Deisenhofen, Germany) for 30 min at 37°C. Thereafter, they
were mounted in a bath chamber on the stage of an inverted microscope
and perfused with a Ringer-like solution. The light from a 75-W xenon
lamp was directed through an infrared light filter (Tempax, Schott, Mainz, Germany) to avoid thermal damage of the three excitation filters
mounted in a motor-driven filter wheel (10 cycles/s). The excitation
filters were band-pass filters with transmission maxima at 340, 360, and 380 nm (Delta Light and Optics, Lyngby, Denmark). A dichroic mirror
(FT 425, Zeiss, Oberkochen, Germany) and a band-pass filter
(500-530 nm, Delta Light and Optics) were used in the emission
light pass. The fluorescence field of a phototube (Hamamatsu H3460,
Herrsching, Germany) could be chosen by means of an adjustable
rectangular diaphragm (50-150 × 30 µm). The photon counts
for the three excitation wavelengths were calculated for each turn of
the wheel. In 15 experiments, the calibration of the fura 2 fluorescence signal could be successfully performed at the end of the
protocol using the Ca2+ ionophore ionomycin (5 µM) and
low- and high-Ca2+ buffers. To vary the free
Ca2+ activity, solutions were prepared according to
established techniques with EGTA. [Ca2+]i was
calculated from the fluorescence ratio according to the equation
described by Grynkiewicz et al. (8). A dissociation constant for the fura 2-Ca2+ complex of 224 nM (37°C) was
assumed. The given concentrations for the
[Ca2+]i peak refer to the highest value of
the fluorescence ratio.
RT-PCR for evaluation of expression of purinergic receptor mRNA
in mouse glomeruli and podocytes.
The RNA preparation, reverse transcription, and PCR amplification were
performed according to the method recently described (7).
In brief, total RNA from mouse glomeruli, which were obtained with the
sieve technique and from cultured mouse podocytes, was isolated with
guanidinium-acid phenol-chloroform extraction, and the amount of RNA
was measured by spectrophotometry. For first-strand synthesis, total
RNA from podocytes and mouse glomeruli was mixed in 5× reverse
transcription buffer and completed with 0.5 mM dNTP, 10 µM random
hexanucleotide primer, 10 mM dithiothreitol, 0.02 U RNAse inhibitor/ng
RNA, and 100 U Moloney murine leukemia virus RT/µg RNA (RT was
omitted in some experiments to check for amplification of contaminating
DNA). The reverse transcription was performed at 42°C for 1 h,
followed by a denaturation at 95°C for 5 min. PCR was performed in
duplicate with a total volume of 20 µl, each containing 40 ng RNA/4
µl of reverse transcription template, 16 µl of PCR master mixture,
and 10 pmol each of sense and antisense primer. The mixture was
overlaid with mineral oil and heated for 2 min at 94°C. The samples
were kept at 80°C until 1 U Taq DNA polymerase and the
primer were added. The cycle profile consisted of 1 min of denaturation
at 94°C, annealing for 1 min for the P2Y1 at 62°C,
P2Y2 at 63°C, P2Y6 at 60°C, and for the
P2X7 receptor at 57°C, respectively, and extension for 1 min at 72°C. To amplify P2Y1 receptor mRNA, 37 cycles
were performed, 35 cycles for P2Y2, 40 cycles for
P2Y6, 35 cycles for P2X7 in glomeruli, and 40 cycles for P2X7 in podocytes, respectively. The
amplification products of 10 µl of each PCR reaction were separated
on a 1.5% agarose gel, stained with ethidium bromide, and visualized
by ultraviolet irradiation. The primer oligonucleotides were selected
from published cDNA sequences and are depicted in Table
1. All primers are noted in the
5'-3' direction.
Measurements of lactate dehydrogenase (LDH) release from
podocytes.
To assess cytotoxicity, LDH release was measured with a routine
autoanalyzer (Modular I, Hitachi). Total LDH content of the cells was
measured after incubation of cells in 1% Triton X-100. LDH release
from control and stimulated cells was expressed as a percentage of
total LDH release.
Chemicals.
Unless otherwise indicated, chemicals were purchased from Sigma.
Statistical analyses.
Data are given as means ± SE, where n refers to the
number of experiments. Student's t-test was used to compare
mean values within one experimental series. A P value
0.05 was considered statistically significant.
Concentration-response curves were calculated by nonlinear regression
analysis using the Hill formula with four parameters followed by
Durbin-Watson statistics.
 |
RESULTS |
Extracellular purinergic agonists depolarize differentiated
podocytes in culture.
The resting Vm of podocytes was
65 ± 1 mV (n = 39). ATP (100 µM) induced a depolarization of
podocytes from
64 ± 2 to
40 ± 2 mV (n = 16, P < 0.05). In the presence of ATP, an increase of
Gm from 2.7 ± 0.4 to 4.3 ± 0.7 nS in
the inward direction and from 2.3 ± 0.3 to 3.3 ± 0.6 nS in
the outward direction was detected (n = 16, P < 0.05 both for the inward and outward direction). After removal of ATP, the Vm response was
completely reversible. Figure
1A shows an original recording
of the effect of 100 µM ATP on Vm of a single
differentiated podocyte. UTP and, interestingly, 2'- and
3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; each 100 µM) similarly depolarized Vm from
58 ± 2 to
34 ± 2 mV (n = 12, P < 0.05) and from
63 ± 2 to
42 ± 2 mV
(n = 18, P < 0.05), respectively (Fig.
1, B and C). UTP (100 µM) led to a
Gm increase from 3.99 ± 1.16 to 4.36 ± 0.85 nS (inward direction) and from 4.25 ± 1.19 to 5.04 ± 1.05 nS (outward direction), respectively (n = 12, P < 0.05 both for the inward and outward direction). BzATP increased Gm from 2.21 ± 0.32 to
3.62 ± 0.64 nS (inward direction) and from 2.73 ± 0.46 to
4.41 ± 0.77 nS (outward direction), respectively
(n = 18, P < 0.05 both for the inward
and outward direction).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
Original recordings of the effect of the nucleotides ATP
(A), uridine 5'-triphosphate (UTP; B), and 2'-
and 3'-O-(4-benzoylbenzoyl)- (Bz)ATP (each 100 µM;
C) on membrane voltage (Vm) of podocytes.
|
|
To further pharmacologically characterize the P2 receptors involved,
the influence of additional P2 receptor agonists on the Vm response of podocytes was investigated.
Figure 2 shows the concentration-response
curves of the Vm response of podocytes to the
different purinergic agonists tested. On the basis of nonlinear regression analysis, the rank order of potency for the depolarizing effect of the purinergic agonists was UTP (EC50 1.8 * 10
6 M)
UDP (EC50 2 * 10
6 M) > ATP-
-S (EC50 3.8 * 10
6 M) > ATP (EC50 7.4 * 10
6 M) > 2-methylthioadenosine 5'-triphosphate
(2-MeS-ATP) (EC50 4.9 * 10
5 M) > BzATP (EC50 8.1 * 10
5 M) > ADP-
-S
(EC50 1.1 * 10
4 M) (n = 3-18).
,
-Me-ATP did not depolarize Vm
of podocytes (100 µM, n = 5).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration-response curves of the effect of
nucleotides on membrane voltage (Vm) of
podocytes as determined by nonlinear regression. The rank order of
potency in depolarizing Vm of podocytes
was UTP UDP > [adenosine
5'-O-(3-thiotriphosphate) (ATP- -S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > BzATP > [adenosine 5'-O-(2-thiodiphosphate) (ADP- -S)]
(n = 3-18).
|
|
ATP activates a Cl
conductance in podocytes.
The effect of ATP on Vm and
Gm was examined in the nominal absence of intra-
and extracellular Cl
. Pipettes were filled with 145 mM
Cs2SO4. After fast whole cell configuration was
achieved, cells were dialyzed with Cs2SO4 for ~5 min, and then extracellular Cl
was replaced by 145 mM Na+gluconate. The addition of ATP (100 µM) led to a
depolarization from
32 ± 5 to
21 ± 4 mV in this
setting, but no significant increase of Gm was
observed (n = 6). Figure
3 summarizes the data.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Summary of the effect of ATP (100 µM, n = 6) on whole cell conductance of podocytes in the nominal absence of
intra- and extracellular Cl . Cells were dialyzed with
Cs2SO4, and extracellular Cl was
replaced by sodium gluconate (Na+gluc ).
*P < 0.05. Go, outward
conductance; Gi, inward conductance.
|
|
Effect of the purinoceptor antagonists suramin and pyridoxal
phosphate 6-azophenyl-2', 4'-disulfonic acid on the Vm
response to ATP.
Pretreatment of podocytes with suramin (100 µM, 5 min) depolarized
resting Vm of cells from
62 ± 3 to
58 ± 3 mV. Suramin (100 µM) inhibited the depolarization
induced by 100 µM ATP by 50 ± 8% (n = 5).
Pyridoxal phosphate 6-azophenyl-2', 4'-disulfonic acid (PPADS; 100 µM, 5 min) did not change resting Vm of
podocytes. PPADS (100 µM) inhibited ATP-mediated depolarization by
88 ± 4% (n = 5). Figure
4 summarizes the data.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of suramin and pyridoxal phosphate 6-azophenyl-2',
4'-disulfonic acid (PPADS; each 100 µM, n = 5),
respectively, on ATP-induced (100 µM) depolarization of
Vm of podocytes. *P < 0.05.
|
|
Nucleotides increase
[Ca2+]i in podocytes.
To further characterize cellular responses of podocytes to
extracellular nucleotides, microfluorescence experiments using the
Ca2+-sensitive fluorescent dye fura 2 were performed.
Resting [Ca2+]i of podocytes was 72 ± 4 nM (n = 61). Apart from
,
-Me-ATP, all nucleotides
increased [Ca2+]i of podocytes (each 100 µM, Table 2). This
[Ca2+]i response varied between the different
agonists tested. Figure 5 shows an
original recording of the [Ca2+]i response of
a single podocyte to the extracellular nucleotides ATP
(n = 14), UTP (n = 12), and BzATP
(n = 11), respectively (each 100 µM). An additional
set of experiments addressed the question of whether this
[Ca2+]i increase was due either to the
Ca2+ release from intracellular stores or to a
Ca2+ influx from the extracellular space. Figure
6 shows original recordings of the effect
of ATP (6A), UTP (6B), and BzATP (6C; each 100 µM) on [Ca2+]i with normal or low
(from 1 mM to 1 µM) extracellular Ca2+ concentration. All
three agonists elicited a biphasic [Ca2+]i
response consisting of an initial Ca2+ peak followed by a
sustained Ca2+ plateau, the latter being dependent on
extracellular Ca2+. Ca2+ peaks were not
affected by lowering extracellular Ca2+. Repetitive
stimulation with 10 µM ATP in the presence of normal and reduced
extracellular Ca2+ resulted in Ca2+ peaks of
258 ± 42 (1 mM extracellular Ca2+), 267 ± 57 (1 µM extracellular Ca2+), and 284 ± 79 nM (1 mM
extracellular Ca2+), respectively (n = 6).
Similar results were obtained with UTP and BzATP (each 10 µM,
n = 6, data not shown). These results indicate initial
Ca2+ peaks to result from Ca2+ release from
intracellular stores.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
The nucleotides ATP, UTP, and BzATP increase the
cytosolic calcium concentration ([Ca2+]i) in
podocytes. Original fluorescence recording of 1 single podocyte is
shown.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Original fluorescence recordings of the effect of ATP
(A), UTP (B), and BzATP (C) on
[Ca2+]i in podocytes both in normal or low
(from 1 mM to 1 µM = Ca2+ 10 6)
extracellular Ca2+ concentration, respectively. The
sustained [Ca2+]i plateau induced by ATP,
UTP, and BzATP depends on extracellular Ca2+.
|
|
Podocytes and glomeruli express mRNA for P2Y1,
P2Y2, P2Y6, and P2X7 receptors.
Figure 7 shows ethidium bromide-stained
agarose gel electrophoresis of PCR products for the P2Y1,
P2Y2, P2Y6, and P2X7 receptors in
mouse glomeruli (top). Similar results were also found in
mouse podocytes, where PCR products for the P2Y1,
P2Y2, P2Y6, and P2X7 receptors were
also detected (bottom).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 7.
Analysis of RT-PCR products for P2Y1,
P2Y2, P2Y6, and P2X7 receptor mRNA,
respectively, in isolated mouse glomeruli and differentiated mouse
podocytes by agarose gel electrophoresis. To amplify P2Y1
receptor mRNA, 37 cycles were performed, 35 cycles for
P2Y2, 40 cycles for P2Y6, 35 cycles for
P2X7 in glomeruli, and 40 cycles for P2X7 in
podocytes, respectively.
|
|
Is there a functional role for the P2X7 receptor in
podocytes?
Among the P2 receptor family, the P2X7 receptor is unique
in that it constitutes a ligand-gated ion channel, which on sustained activation opens large- conductive nonselective pores, ultimately resulting in cell lysis (11, 23). P2X7
receptor mRNA expression was detected in podocytes (cf. Fig. 7). BzATP
is the most potent P2X7 receptor agonist presently
available. BzATP induced a Vm response in
podocytes that was comparable to the cellular responses initiated by
ATP and UTP, respectively (cf. Fig. 1). In addition, BzATP also induced
a biphasic Ca2+ transient in podocytes, its morphology
being similar to that elicited by ATP and UTP, respectively (cf. Fig.
6). To further elucidate whether the P2X7 receptor is
functionally present in podocytes, resulting in the aforementioned pore
formation, supplementary experiments were performed. In the presence of
UTP, the addition of BzATP did not result in an additional
depolarization of podocytes, indicating the lack of functional activity
of a P2X7 receptor (Fig. 8,
n = 5). On top of that, treatment of podocytes with ATP or BzATP (both 0.3 mM) for 1, 4, and 8 h, respectively, did not result in an increase in LDH release from podocytes (Fig. 8; 1, 4, 8 h: n = 9 each). Only after a prolonged exposure
over 24 h, a slight, albeit significant, increase in LDH release
was detected in podocytes (ATP: 14 ± 3%, BzATP: 25 ± 4%
vs. control: 17 ± 2%; Fig. 9,
24 h: n = 12).

View larger version (7K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of BzATP on Vm of podocytes
in the presence of UTP, original recording (n = 5). In
the presence of 100 µM UTP, BzATP (100 µM) did not evoke an
additional Vm response.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of ATP and BzATP on lactate dehydrogenase (LDH)
release of podocytes. Cells were treated with ATP, BzATP (both 0.3 mM),
or Triton X-100 (1%) for the time period indicated. Note that BzATP
did not induce significant LDH release in podocytes within 8 h (1, 4, 8 h: n = 9 each; 24 h: n = 12). *P < 0.05.
|
|
 |
DISCUSSION |
Extracellular ATP is considered to be an important regulator of
biological functions in glomerular cells. ATP might play a role in
mesangial cell injury during glomerulonephritis because it increases
[Ca2+]i and inositol phosphates in mesangial
cells, resulting in their depolarization and contraction (18,
21). It also stimulates proliferation of mesangial cells via a
P2 receptor (26). In addition, ATP concentrations >300
µM mediate apoptosis and necrosis of cultured mesangial cells
via the P2X7 receptor (25). P2
receptor-induced Ca2+ signaling has also been reported in
bovine glomerular endothelial cells and human glomerular epithelial
cells with a cobblestone appearance (1, 20).
In the past, it was not possible to identify cultured glomerular
epithelial cells as podocytes, and it was suggested that glomerular
epithelial cells in culture were not podocytes, but in fact were
glomerular parietal epithelial cells (14). Recently, successful cultivation of differentiated podocytes was
demonstrated; undifferentiated podocytes with a cobblestone
appearance developed into differentiated podocytes with cell processes
expressing podocyte-specific markers (14). In the present
study, podocytes showing a differentiated phenotype were used. Apart
from exhibiting an arborized morphology, cells stained positive for the
podocyte markers WT-1 (15), synaptopodin (13), nephrin (28), and p57
(16). In addition, mRNA expression of the podocyte marker
CD2AP (27) was detected by RT-PCR. However, although
cultured differentiated podocytes possess many in vivo properties of
podocytes, biological functions of the cells may change during culture,
and therefore results obtained from these cells have to be interpreted
with care.
We show that ATP regulates ion currents and
[Ca2+]i in differentiated podocytes. ATP
depolarized Vm and increased
Gm of podocytes. In addition, ATP led to an
increase in [Ca2+]i, which was due to the
release of Ca2+ from intracellular stores and to an influx
of Ca2+ from the extracellular space. An increase of
[Ca2+]i is known to activate a
Cl
current in podocytes (17). The
Cl
replacement experiments in this study showed that in
the nominal absence of Cl
, ATP failed to activate ion
currents. An ATP-induced activation of Cl
current has
been reported in mesangial cells, whereas in glomerular endothelial
cells ATP has been assumed to open nonselective ion currents (18,
19).
In this study, both the purines ATP and ATP-
-S, as well as the
pyrimidines UTP and UDP, were potent agonists in mediating Vm response and increase of
[Ca2+]i in podocytes. Cellular responses to
2-MeS-ATP were small;
,
-Me-ATP almost had no effect. The
pharmacological profile fits well to that of a P2Y2
receptor (23), whose mRNA expression was detected both in
glomeruli and podocytes by RT-PCR. In addition, the marked cellular
response of both Vm and
[Ca2+]i to extracellular uridine nucleotides
hints at a functional relevance of the uridine nucleotide-specific
receptor P2Y6 (see below). P2Y1 receptors seem
to play a minor functional role, because the P2Y1 receptor
has been reported to be strongly activated by 2-MeS-ATP, but not by UTP
or UDP (23). Moreover, adenine nucleotide diphosphates are
potent agonists to P2Y1 receptors (23). Here ADP-
-S elicited only weak responses of both
Vm and [Ca2+]i in
podocytes. Despite mRNA expression of the P2Y1 receptor found both in glomeruli and podocytes, our data clearly indicate that
it is functionally less active than its P2Y2 counterpart.
Apart from P2Y1 and P2Y2 receptor mRNA
expression, P2Y6 receptor mRNA expression similarly was
demonstrated in both glomeruli and podocytes. Coexpression of
P2Y6 mRNA, along with P2Y1 and P2Y2
mRNA, has also been demonstrated in adult rat cardiac myocytes (29). The P2Y6 receptor has also been detected
in other cell types, and it seems to be more widely distributed than
the P2Y4 receptor, for instance (23). In this
regard, the P2Y4 receptor was not detected in podocytes.
The P2Y6 receptor has been reported to be activated most
potently by UDP (3), and it was assumed that it accounts
for endogenous uridine nucleotide-specific responses (23).
Among the nucleotides tested in the present study, the marked cellular
responses of podocytes elicited by UDP and UTP clearly hint at a
functional role of P2Y6 receptors in podocytes.
Among the P2 receptor family, the P2X7 receptor is unique
in that it constitutes a ligand-gated ion channel, which, on sustained activation, opens large-conductive nonselective pores, ultimately resulting in cell lysis (11, 23). In our sets of
experiments, BzATP, the most potent agonist of the P2X7
receptor known so far (23), was shown to be less potent
than ATP and UTP in stimulating cellular responses of podocytes. In
this regard, in mouse tissue BzATP has been described to be 10-100
times more potent than ATP in activating P2X7 receptors
(23). To further clarify the role of the P2X7
receptor in podocytes, several sets of experiments were performed with
the following results. First, mRNA of the P2X7 receptor
could be detected in mouse glomeruli and podocytes. Second, in the
presence of UTP, BzATP did not induce an additional depolarization of
podocytes. An additional depolarization induced by BzATP would have
been expected in the case of a separate P2X7 receptor being
functionally active in podocytes. Third, BzATP released
Ca2+ from intracellular stores, suggesting that it might
act via a P2Y receptor. Apart from differences in magnitude, both the
time course and morphology of Ca2+ transients induced by
BzATP were almost similar to those elicited by ATP or UTP. Fourth,
P2X7 receptor-induced pore formation with subsequent cell
lysis has been reported to occur only after prolonged receptor
activation. One might thus argue that the cellular responses reported
here were only reversible due to short exposure to BzATP. To further
address this question, LDH release was tested in podocytes. Prolonged
exposure to high concentrations of ATP or BzATP did not cause LDH
release in podocytes within 8 h, indicating that in contrast to
mesangial cells (25), the P2X7 receptor did
not mediate cytotoxicity in podocytes. Therefore, although some mRNA expression of the P2X7 receptor could be detected in
podocytes, the present experiments do not support a functional role of
the P2X7 receptor in this cell type. This is in agreement
with a recent study by Harada et al. (9), in which weak
mRNA expression and very-low-intensity immunoreactivity of the
P2X7 receptor were detected in the glomerulus, including
mesangial cells and podocytes.
In summary, the data indicate that the nucleotide P2Y1,
P2Y2, P2Y6, and P2X7 receptors are
expressed in podocytes. The effects of extracellular nucleotides on
Vm and [Ca2+]i in
podocytes, however, mainly are mediated by P2Y2 and
P2Y6 receptors.
 |
ACKNOWLEDGEMENTS |
We thank Petra Dämisch, Monika von Hofer, Charlotte Hupfer,
and Temel Kilic for excellent technical assistance. We are indebted to
Dr. P. Mundel, Department of Medicine and Department of Anatomy and
Structural Biology, Albert Einstein College of Medicine, Bronx, New
York, for providing different podocyte cell lines.
 |
FOOTNOTES |
This work was supported by Deutsche Forschungsgemeinschaft Grant Fi
691/1-2.
Address for reprint requests and other correspondence: K.-G.
Fischer, University Hospital Freiburg, Dept. of Medicine, Div. of Nephrology, Hugstetter Str. 55, D-79106 Freiburg, Germany
(E-mail: fischer{at}med1.ukl.uni-freiburg.de).
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. Section 1734 solely to indicate this fact.
Received 10 July 2001; accepted in final form 8 August 2001.
 |
REFERENCES |
1.
Briner, VA,
and
Kern F.
ATP stimulates Ca2+ mobilization by a nucleotide receptor in glomerular endothelial cells.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F210-F217,
1994[Abstract/Free Full Text].
2.
Chan, CM,
Unwin RJ,
and
Burnstock G.
Potential functional roles of extracellular ATP in kidney and urinary tract.
Exp Nephrol
6:
200-207,
1998[ISI][Medline].
3.
Communi, D,
Parmantier M,
and
Boeynaems JM.
Cloning, functional expression and tissue distribution of the human P2Y6 receptor.
Biochem Biophys Res Commun
222:
303-308,
1996[ISI][Medline].
4.
Drenckhahn, D,
and
Franke RP.
Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.
Lab Invest
59:
673-682,
1988[ISI][Medline].
5.
Gloy, J,
Fischer KG,
Meyer TN,
Schollmeyer P,
Greger R,
and
Pavenstädt H.
Hydrogen peroxide activates ion currents in rat mesangial cells.
Kidney Int
56:
181-189,
1999[ISI][Medline].
6.
Greger, R,
and
Kunzelmann K.
Simultaneous recording of the cell membrane potential and properties of the cell attached membrane of HT29 colon carcinoma and CF-PAC cells.
Pflügers Arch
419:
209-211,
1991[ISI][Medline].
7.
Greiber, S,
Münzel T,
Kästner S,
Müller B,
Schollmeyer P,
and
Pavenstädt H.
NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate.
Kidney Int
53:
654-663,
1998[ISI][Medline].
8.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3340-3350,
1985.
9.
Harada, H,
Chan CM,
Loesch A,
Unwin R,
and
Burnstock G.
Induction of proliferation and apoptotic cell death via P2Y and P2X receptors, respectively, in rat glomerular mesangial cells.
Kidney Int
57:
949-958,
2000[ISI][Medline].
9a.
Kaiho, H,
Matsuoka I,
Kimura J,
and
Nakanishi H.
Identification of P2X7 (P2Z) receptor in N18TG-2 cells and NG108-15 cells.
J Neurochem
70:
951-957,
1998[ISI][Medline].
10.
Kerjaschki, D.
Dysfunction of cell biological mechanisms of visceral epithelial cells (podocytes) in glomerular diseases.
Kidney Int
45:
300-313,
1994[ISI][Medline].
11.
Khakh, BS,
and
Kennedy C.
Adenosine and ATP: progress in their receptors' structures and functions.
Trends Pharmacol Sci
19:
39-41,
1998[ISI][Medline].
12.
Kriz, W,
Hackenthal E,
Nobiling R,
Sakai T,
and
Elger M.
A role for podocytes to counteract capillary wall distension.
Kidney Int
45:
369-376,
1994[ISI][Medline].
13.
Mundel, P,
Heid HW,
Mundel TM,
Kruger M,
Reiser J,
and
Kriz W.
Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes.
J Cell Biol
139:
193-204,
1997[Abstract/Free Full Text].
14.
Mundel, P,
Reiser J,
Borja AZM,
Pavenstädt H,
Davidson GR,
Kriz W,
and
Zeller R.
Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.
Exp Cell Res
236:
248-258,
1997[ISI][Medline].
15.
Mundlos, S,
Pelletier J,
Darveau A,
Bachmann M,
Winterpracht A,
and
Zabel B.
Nuclear localization of the protein encoded by the Wilm's tumor gene WT1 in embryonic and adult tissues.
Development
119:
1329-1341,
1993[Abstract/Free Full Text].
16.
Nagata, M,
Nakayama K,
Terada Y,
Hoshi S,
and
Watanabe T.
Cell cycle regulation and differentiation in the human podocyte lineage.
Am J Pathol
153:
1511-1520,
1998[Abstract/Free Full Text].
17.
Pavenstädt, H.
Roles of the podocyte in glomerular function.
Am J Physiol Renal Physiol
278:
F173-F179,
2000[Abstract/Free Full Text].
18.
Pavenstädt, H,
Gloy J,
Leipziger J,
Klär B,
Pfeilschifter J,
Schollmeyer P,
and
Greger R.
Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture.
Br J Pharmacol
109:
953-959,
1993[Abstract].
19.
Pavenstädt, H,
Henger A,
Briner V,
Fischer KG,
Huber-Lang M,
Schollmeyer P,
and
Greger R.
Agonist-induced activation of a non-selective ion current in glomerular endothelial cells.
Kidney Int
52:
157-164,
1997[ISI][Medline].
20.
Pavenstädt, H,
Späth M,
Schlunck G,
Nauck M,
Fischer R,
Wanner C,
and
Schollmeyer P.
Effect of nucleotides on the cytosolic calcium activity and inositol phosphate formation in human glomerular epithelial cells.
Br J Pharmacol
107:
189-195,
1992[Abstract].
21.
Pfeilschifter, J.
Extracellular ATP stimulates polyphosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells. Involvement of a pertussis toxin-sensitive guanine nucleotide binding protein and feedback inhibition by protein kinase C.
Cell Signal
2:
129-138,
1990[ISI][Medline].
22.
Poelstra, K,
Heynen ER,
Baller JF,
Hardonk MJ,
and
Bakker WW.
Modulation of anti-Thy1 nephritis in the rat by adenine nucleotides. Evidence for an anti-inflammatory role for nucleotidases.
Lab Invest
66:
555-563,
1992[ISI][Medline].
23.
Ralevic, V,
and
Burnstock G.
Receptors for purines and pyrimidines.
Pharmacol Rev
50:
413-492,
1998[Abstract/Free Full Text].
24.
Rüdiger, R,
Greger R,
Nitschke R,
Henger A,
Mundel P,
and
Pavenstädt H.
Polycations induce calcium signaling in glomerular podocytes.
Kidney Int
56:
1700-1709,
1999[ISI][Medline].
25.
Schulze-Lohoff, E,
Hugo C,
Rost S,
Arnold S,
Gruber A,
Brune B,
and
Sterzel RB.
Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2z/P2x7 receptors.
Am J Physiol Renal Physiol
275:
F962-F971,
1998[Abstract/Free Full Text].
26.
Schulze-Lohoff, E,
Zanner S,
Ogilvie A,
and
Sterzel RB.
Extracellular ATP stimulates proliferation of cultured mesangial cells via P2 purinergic receptors.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F374-F383,
1992[Abstract/Free Full Text].
27.
Shih, NY,
Li J,
Karpitskii V,
Nguyen A,
Dustin ML,
Kanagawa O,
Miner JH,
and
Shaw AS.
Congenital nephrotic syndrome in mice lacking CD2-associated protein.
Science
286:
312-315,
1999[Abstract/Free Full Text].
28.
Tryggvason, K.
Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.
J Am Soc Nephrol
10:
2440-2445,
1999[Free Full Text].
29.
Webb, TE,
Boluyt MO,
and
Barnard EA.
Molecular biology of P2Y purinoceptors: expression in rat heart.
J Auton Pharmacol
16:
303-307,
1996[ISI][Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1075-F1081
0363-6127/01 $5.00
Copyright © 2001 the American Physiological Society