Lysophosphatidic acid-induced calcium mobilization and
proliferation in kidney proximal tubular cells
Richard J.
Dixon1,
Ken
Young1, and
Nigel J.
Brunskill1,2
1 Department of Cell Physiology
and Pharmacology, Leicester University School of Medicine; and
2 Department of Nephrology,
Leicester General Hospital, Leicester LE1 9HN, United
Kingdom
 |
ABSTRACT |
Patients with
proteinuria tend to develop progressive renal disease with proximal
tubular cell atrophy and interstitial scarring. It has been suggested
that the nephrotoxicity of albuminuric states may be due to the protein
molecule itself or by lipids, such as lysophosphatidic acid (LPA), that
albumin carries. LPA was found to cause a transient increase in
intracytoplasmic free Ca2+
([Ca2+]i)
in opossum kidney proximal tubule cells (OK) that was
maximal at 100 µM LPA and was dose dependent with an
EC50 of 2.6 × 10
6 M. This
Ca2+ mobilization was from both
internal stores and across the plasma membrane and was pertussis toxin
(PTX) insensitive. Treatment of OK cells with 100 µM LPA for 5 min
was found to cause a twofold increase in
[3H]thymidine
incorporation and a three- to fivefold increase over control after 24 h. This was highly PTX sensitive and insensitive to pretreatment with
the tyrosine kinase inhibitors genistein and herbimycin A. These
findings may be of significance in the progression of renal disease and
indicate the potential importance of lipids in modulating proximal
tubule cell function and growth.
OK cells; proteinuria; lipids; signaling
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INTRODUCTION |
ONCE INITIATED, renal failure tends to be relentlessly
progressive (33), and therapeutic strategies designed to slow or stop
this progression have so far been largely ineffective. It is recognized
both in animals with experimental renal disease and in humans that
declining renal function correlates most strongly with the pathological
changes seen in the tubulointerstitium of the kidney (1, 2). These
pathological changes are manifest as interstitial fibrosis and scarring
together with tubular atrophy (15). The presence of protein, most
notably albumin, in the urine of patients with renal disease has
conventionally been regarded simply as a marker of the severity of the
disease state. Nonetheless, it is recognized that those patients with
proteinuria are more likely to develop progressive renal failure than
those without proteinuria (6), and recently it has been hypothesized
that albuminuria may exert a toxic effect on proximal tubular
epithelial cells (PTEC) in its own right, thereby damaging the cells
and initiating the process of interstitial fibrosis and scarring (5, 8,
9, 30). Indeed, there is good evidence that proteinuria may result in
tubular injury (5), although the mechanisms whereby such injury may
occur are unclear. Furthermore, enhanced cellular proliferation has
been observed in PTEC cultured in the presence of albumin or nephrotic
urine (4). Again, however, the mechanism of this effect has not yet
been elucidated.
One current theory suggests that the nephrotoxicity of albuminuric
states is not directly determined by the protein molecule itself but
that the toxicity resides in other molecules, particularly lipids,
carried by albumin. Albumin contains many fatty acid binding sites
(14), and it has been shown that in PTEC which are exposed to nephrotic
levels of fatty acid-replete albumin, the resting cellular lipid pools
suffer major perturbations (29). Furthermore, when proximal tubule
segments are exposed to fatty acid-bearing albumin, but not fatty
acid-free albumin, they are able to produce a lipid chemoattractant
that may have an important role in the development of
tubulointerstitial inflammation (17).
Lysophosphatidic acid (LPA) is an intercellular lipid mediator with
growth factor-like activities (23, 24). LPA is rapidly produced and
released from activated platelets and influences target cells by
activating a specific G protein-coupled receptor that is present in
numerous cell types (23). As a product of the blood clotting system,
LPA is an abundant constituent of serum (but not plasma), where it is
present in albumin-bound form. Albumin-bound LPA may account for much
of the biological activity of serum (23). Extracellular LPA can also be
generated through secretory phospholipase A2 acting on microvesicles shed
from blood cells challenged with inflammatory stimuli (10), suggesting
that one of the in vivo functions of LPA is to stimulate proliferative
responses at sites of injury and inflammation. Platelet aggregation is
commonly observed in the glomerular capillaries in many renal diseases
(7), and LPA released by activated platelets is likely to enter the
proximal tubule when liberated in the glomerulus either alone or
complexed to albumin. Therefore it is possible that LPA exerts
receptor-mediated effects on the proximal tubule cells, and it is
likely that this effect may be of considerable importance in PTEC pathophysiology.
Using opossum kidney (OK) cells, a kidney proximal tubule epithelial
cell line (19) that has many characteristics of the proximal tubule
(26), we investigated whether LPA may have a cell signaling function in
PTEC by examining its effects on intracellular calcium. In addition,
the effects of LPA on cell growth were studied, as renal cell
hyperplasia occurs in many renal diseases associated with proteinuria,
and it has been suggested that such growth represents a maladaptive
response that contributes to the progression of renal failure (34).
 |
MATERIALS AND METHODS |
Materials. OK cells are an
immortalized line derived from opossum kidney and were obtained from
Dr. J. Caverzasio (Geneva, Switzerland). All tissue culture media and
chemicals were obtained from Sigma UK unless otherwise stated.
Cell culture. OK cells were maintained
in DMEM-Ham's F-12 mix (DMEM-F12) (GIBCO), supplemented with 10% FCS
(GIBCO), 2 mmol/l L-glutamine
(GIBCO), 100 U/ml penicillin, and 100 µg/ml streptomycin (Flow
Laboratories), at 37°C in a humidified 95%
O2-5%
CO2 atmosphere.
LPA. A stock solution of LPA (oleoyl)
was prepared by dissolving it in a 1 mg/ml solution of
fatty acid-free bovine serum albumin (FAF-BSA) and distilled water.
Intracellular calcium measurements in
suspension. OK cells were grown to confluence in
plastic flasks. Cell monolayers were washed twice in cell harvesting
solution (0.54 mM sodium EDTA, 154 mM NaCl, and 10 mM HEPES; pH 7.3)
and then incubated for 20 min at 37°C in this solution. The cells
were gently removed by agitation, washed once in Krebs-Henseleit buffer
(in mM: 1 CaCl2, 118 NaCl, 469 KCl, 1.2 KH2PO4,
1.2 MgSO4 · 7H2O,
4.2 NaHCO3, 10 glucose, and 10 HEPES; pH 7.4), then incubated in this solution at 37°C for 30 min
prior to loading with the molecular probe. Measurements of
intracytoplasmic free Ca2+
([Ca2+]i)
were performed with fura 2-AM (Calbiochem).
For measurement of
[Ca2+]i,
cells were resuspended at 2.5 × 106 cells/ml in Krebs buffer with
a 5 µM final concentration of fura 2-AM and incubated at 37°C in
a water bath for 45 min (in the dark) with occasional mixing. After
incubation the cells were washed once in Krebs buffer and resuspended
at 1 × 106
cells/ml in Krebs buffer. The cells were kept in a water bath at
37°C before the experiments were started. Fluorescence of 2 ml of
this cellular suspension was monitored with a Perkin-Elmer LS-50B
luminescence spectrometer in cuvettes thermostatically controlled at
37°C with constant stirring. Fluorescence of the cellular
suspension was first determined using unlabeled cells to correct
experimental measurements for autofluorescence. The cell suspension was
excited alternately at 340 and 380 nm, and the fluorescence was
measured at 510 nm. Ten-nanometer slit widths were used for both
excitation and emission. After stabilization of the baseline, agonists
were added in 20-µl volumes.
Graphic representations of
[Ca2+]i
were computed by using the equation
(13)
where
Kd is the
dissociation constant of Ca2+ for
fura 2 (224 nM at 37°C), Rmin
and Rmax are the minimal and
maximal fluorescent ratios obtained by perforating the cells with 0.1%
Triton X-100 for Rmax followed by
the addition of an excess of EGTA at 5 mM for
Rmin.
Fmin (380 nm) and
Fmax(380 nm) are the fluorescent
intensities after excitation at 380 nm, in the absence and presence of
Ca2+, respectively.
Single cell intracellular calcium
imaging. OK cells were grown for 24 h on sterilized
coverslips (22 mm diameter; Chance Proper) in culture dishes (35 × 10 mm) in DMEM-F12 medium (as above) at 37°C. The
coverslips were washed twice with Krebs buffer and then incubated in
the dark, at room temperature, for 1 h in Krebs buffer supplemented with 5 µM fura 2-AM. The coverslips were then washed twice in Krebs buffer and incubated for a further 30 min to allow for
complete de-esterification of the dye before being mounted on the stage
of a Nikon Diaphot inverted epifluorescence microscope. Krebs buffer
was continuously perfused over the cells at the rate of 5 ml/min.
Before application of the stimuli, the buffer was perfused away, and
the appropriate concentration of agonist was added to the cells after
which the buffer was perfused over the cells once again washing away
the stimuli. After subtraction of background fluorescence, images at
wavelengths above 510 nm, after excitation at 340 and 380 nm (40 ms at
each wavelength), were collected with an intensified charge-coupled
device camera (Photonic Science). Experiments were conducted on a
Quanticell 700 (Applied Imaging) system. Ratiometric values were
converted to approximate [Ca2+]i
values using the above equation, in which
Rmin and
Rmax are the minimal and maximal
fluorescent ratios obtained from a standard curve.
[3H]thymidine
proliferation assay.
OK cells were plated in 24-well plates and grown to 70-90%
confluence. They were then incubated in serum-free and thymidine-free DMEM for 24 h at 37°C. The medium was then replaced with fresh serum-free DMEM alone (control), serum-free DMEM supplemented with 10%
FCS, various concentrations of LPA, various concentrations of FAF-BSA,
or 10 ng/ml human recombinant epidermal growth factor (EGF)
(Calbiochem) as a control for pertussis toxin (PTX) sensitivity. After
an additional 24 h, 2 µCi of
[3H]thymidine
(Amersham Life Science, UK) was added to all wells. In time course
experiments, the media containing the agonist was washed out and
replaced with serum-free media and incubated at 37°C for the
remainder of the 24-h period. After 2-h incubation with
[3H]thymidine, the
cells were washed three times with DMEM, then incubated with 2 ml of
ice-cold 5% trichloroacetic acid (TCA) for 1 h at 4°C. The TCA was
removed, and the cells were washed once with fresh ice-cold TCA. Then 2 ml of ice-cold ethanol containing 200 µM potassium acetate was added
to each well for 5 min. The cells were then incubated twice in 2 ml of
3:1 mixture of ethanol:ether for 15 min per incubation. After allowing
the cell monolayers to air dry, cells were solubilized in 1 ml of 0.1 M
sodium hydroxide. [3H]thymidine counts
per min (cpm) were measured by adding samples to scintillation fluid
(Ultima Gold, Packard) and counted by using a Packard 1900CA Tri-Carb
liquid scintillation analyzer.
Statistics. Data are given as mean
values ± SE. For analyzing differences, unpaired two-tailed
Student's t-tests were performed. Differences were regarded significant at
P < 0.05.
 |
RESULTS |
Effect of LPA on
[Ca2+]i.
Basal values of
[Ca2+]i
averaged 50 ± 15 nM (n = 53) at 1 mM external calcium. As can be seen in Fig.
1A, LPA
was found to evoke a typical
[Ca2+]i
transient in fura 2-labeled OK cells. The calcium signal is initiated
within seconds and reaches its peak within 15-20 s of LPA addition
and, thereafter, declined to a plateau that was consistently observed
to be higher than basal
[Ca2+]i.
The maximal increase in
[Ca2+]i
was seen with 100 µM LPA as shown in Fig.
1A. It must be noted at this point
that the LPA applied to the cells is bound to FAF-BSA as a carrier;
however, the levels of FAF-BSA bound to LPA were found not to have any
effect on
[Ca2+]i
levels in this system. For example, the amount of FAF-BSA present in
administering a dose of 100 µM LPA is equivalent to 10 µg/ml FAF-BSA, which was found to have no effect on
[Ca2+]i
levels (data not shown). The effect of LPA was concentration dependent
in the range of 10
8 to
10
3 M (Fig.
1B). The dose response curve
calculates an EC50 of 2.6 ×10
6 M, with 95%
confidence intervals 9.7 × 10
7 to 6.9 × 10
6 M.

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Fig. 1.
A: effect of 100 µM lysophosphatidic
acid (LPA) on the intracellular calcium
([Ca2+]i)
levels of OK cells. This is a representative experiment of 5 replicate
experiments. B: dose-response
relationship of LPA-induced
[Ca2+]i
mobilization as determined from peak values. Data are presented as
means ± SE (n = 3).
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In the absence of external calcium (presence of 4 mM EGTA), the
LPA-induced rise in
[Ca2+]i
was found to be strongly reduced (Fig.
2A) and
averaged 24 ± 6% (n = 4) of that
observed in the presence of 1 mM external calcium. Under these
conditions, influx of calcium across the plasma membrane can no longer
take place, and any
[Ca2+]i
changes observed can be attributed to intracellular release. This can be seen clearly in Fig.
2A, as the LPA-induced
[Ca2+]i
response exhibits a sharp rise and fall without the plateau of
Ca2+ influx from the extracellular
medium shown in Figs. 1 and 3. Upon pretreatment of the cells with 10 µM thapsigargin, the endosomal Ca2+-ATPase inhibitor, the
LPA-induced rise in
[Ca2+]i
was attenuated, averaging 10 ± 4%
(n = 4) of control experiments in
which there was no thapsigargin pretreatment (Fig.
2B). Thus LPA-evoked
[Ca2+]i
rises reflect mobilization of Ca2+
from internal stores as well as transmembrane influx.

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Fig. 2.
A: effect of EGTA pretreatment on
LPA-induced
[Ca2+]i
mobilization in OK cells. Data presented as change in
Ca2+-dependent fura-2
fluorescence. This is a representative experiment of 4 replicate
experiments. B: effect of thapsigargin
pretreatment on LPA-induced
[Ca2+]i
mobilization in OK cells. This is a representative experiment of 4 replicate experiments
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Pretreatment with PTX (50 ng/ml, 18 h) was found to have no
effect on the LPA-induced
[Ca2+]i
signal (Fig. 3). This indicates
the possible involvement of a PTX-insensitive G protein-linked receptor
in the transduction of this response. The effect of LPA administration
on
[Ca2+]i
was instantaneous as is typically seen with
Ca2+-releasing agonists acting
through cell surface receptors. This observation makes it unlikely that
the LPA signaling occurs as a result of endocytosis of the albumin/LPA
complex, followed by intracellular release of the lipid agonist.

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Fig. 3.
Effect of pertussis toxin (PTX) pretreatment on LPA-induced
[Ca2+]i
mobilization in OK cells. This is a representative experiment of 3 replicate experiments.
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To compare this novel LPA-induced
[Ca2+]i
rise in OK cells with a known
[Ca2+]i-inducing
agent in OK cells, parathyroid hormone (PTH) was used, which has been
previously demonstrated to stimulate transient elevations in
[Ca2+]i
in OK cells (22). As can be seen in Fig. 4,
10
7 M PTH was found to
induce a
[Ca2+]i
transient of 30 ± 7 nM (n = 8),
which is comparable to published data (21). Thus the
[Ca2+]i
transients observed with LPA in OK cells are of comparable magnitude to
those observed with an established
[Ca2+]i-elevating
agonist, such as PTH in these cells. Also, it is worth noting that
addition of LPA shortly after stimulation with PTH (as shown in Fig. 4)
showed no effect on the LPA response and vice versa. This suggests that
LPA and PTH act through distinct receptors to cause
[Ca2+]i
transients within the cells.

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Fig. 4.
Comparison of effect of parathyroid hormone (PTH) and LPA on
[Ca2+]i
mobilization in OK cells. This is a representative experiment of 3 replicate experiments.
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These investigations were carried out using the Perkin-Elmer model
LS-50B fluorometer, in which large populations of cells (2 million per
experiment) were studied in suspension (see MATERIALS AND METHODS). Therefore, to ascertain what fraction
of the cells were responding to LPA, the same experiments were carried
out using fura 2 as the molecular probe again but using the
Quanticell-700 cell imaging system. This enabled the observation of
[Ca2+]i
responses at the single cell level (MATERIALS AND
METHODS). In Fig. 5 is a
representative experiment of 3 replicate experiments in which 17 adherent OK cells were stimulated with 10 µM LPA. The
results showed that 83 ± 11% (n = 3) of the cells responded with a
[Ca2+]i
transient of average 116 ± 42 nM in magnitude. This was evidence that the majority of the OK cells are responding to LPA with a [Ca2+]i
transient.

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Fig. 5.
Effect of 10 µM LPA on
[Ca2+]i
levels of 17 adherent OK cells as measured using the Quanticell-700
cell imaging system. This is a representative experiment of 3 replicate
experiments. Arrow, time point at which 10 µM LPA was administered to
the cells. Each line shown represents 1 of the 17 cells observed in
this experiment. Results show that 14 of 17 cells responded with an
increase in
[Ca2+]i
ranging from ~50 to 150 nM (average = 116 ± 42 nM).
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Effect of LPA on proliferation in OK
cells. The effect of LPA on the proliferation of OK
cells was assessed by measuring
[3H]thymidine
incorporation as an index of DNA synthesis (see
MATERIALS AND METHODS). As shown in
Fig. 6, incubation of OK cells with LPA for
24 h resulted in a dose-dependent increase in thymidine incorporation,
which was maximal at 100 µM LPA, causing an increase of 323 ± 16% (n = 4) over control cells. As a
positive control, the effect of 10% FCS on proliferation of OK cells
was studied and was found to stimulate proliferation comparably to 100 µM LPA [increase of 355 ± 15%
(n = 4) over control].

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Fig. 6.
Effect of LPA and its carrier BSA on proliferation of OK cells. This is
a representative experiment of 5 replicate experiments. Results are
presented as percentages (±SE, n = 4) of control, which was incubated with serum-free media.
* P < 0.05. *** P < 0.001.
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The method of preparation of LPA prior to its application to the cells
(see MATERIALS AND METHODS)
necessitates its reconstitution in a BSA-containing solution.
Therefore, the
[3H]thymidine
incorporation experiments involve the obligate exposure of the cells,
to not only LPA but also albumin. Shown in Fig. 6 are the effects of
the FAF-BSA carrier controls. For example, a dose of 100 µM LPA
includes FAF-BSA at a concentration of 10 µg/ml. As can be seen from
these data, FAF-BSA clearly has a significant effect on
[3H]thymidine
incorporation, causing an increase of 149.6 ± 15.1% (n = 4) at 10 µg/ml. Nevertheless,
LPA was found to have much more significant effects on thymidine
incorporation than FAF-BSA (Fig. 6). The effects of BSA are, however,
not significant (P < 0.05) at
concentrations less than 10 µg/ml. Similarly, 10 µM LPA causes an
increase of 130 ± 45% (n = 4)
over control, but the concentration of carrier FAF-BSA present in this
dose is 1 µg/ml, which exhibits no significant effect on
proliferation. Thus LPA is the predominant stimulator of proliferation
in this situation.
Figure 7 depicts the results of time course
experiments in which OK cells were transiently exposed to LPA. A 5-min
exposure to LPA is sufficient to cause significant subsequent
proliferation when measured 24 h later. The equivalent amount of
FAF-BSA (10 µg/ml) bound to 100 µM LPA had no significant effect on
proliferation from 5 min to 1 h in these experiments.

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Fig. 7.
Effect of transient exposure of LPA on proliferation of OK cells.
Exposure times of 5, 10, and 60 min and 24 h were used, after which
cells were washed and reimmersed in serum-free media for the rest of
the 24-h incubation time. This is a representative experiment of 3 replicate experiments. Results are presented as percentages (±SE,
n = 4) of control, which was incubated
with serum-free media. ** P < 0.01. *** P < 0.001.
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These effects of LPA (100 µM), FAF-BSA (10 µg/ml), and FCS (10%)
were inhibited by 415 ± 33%, 80 ± 12%, and 205 ± 25%
(n = 4), respectively, by PTX
pretreatment (50 ng/ml, 18 h) (Fig. 8). In
contrast, PTX pretreatment had no effect on the mitogenic response to
10 ng/ml EGF, which acts through a tyrosine kinase receptor. The
absence of a PTX effect on EGF-induced proliferation excludes nonspecific toxicity of this treatment as an explanation for the PTX
inhibition of LPA-induced proliferation. Hence, these data strongly
suggest the involvement of a PTX-sensitive G protein-linked receptor in
mediating the proliferative effects of LPA on OK cells.

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Fig. 8.
Effect of PTX pretreatment on LPA-, FCS-, and epidermal growth factor
(EGF)-induced proliferation in OK cells. Cells were pretreated for 18 h
with 50 ng/ml PTX and then stimulated with agonist for 24 h. This is a
representative experiment of 3 replicate experiments. Results are
presented as percentages (±SE, n = 4) of control, which was incubated with serum-free media.
** P < 0.01 and
*** P < 0.001, for comparison
between control cells (no PTX treatment) and PTX-pretreated
cells.
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Many growth factors (such as EGF) signal via activation of receptors
that possess intrinsic tyrosine kinase activity (31); we investigated
whether the mitogenic activity of LPA was mediated through a tyrosine
kinase-dependent mechanism. Tyrosine kinase inhibition was achieved by
24-h preincubation with either herbimycin A (0.25 µM) or genistein
(0.25 µM) (25). However, no significant inhibition of thymidine
incorporation by LPA, FCS, or FAF-BSA was observed in these experiments
(data not shown).
Cell viability as assessed by trypan blue exclusion was greater than
95% in all experiments and showed no significant difference between
the various conditions used, which therefore excluded the possibility
that LPA was acting as a mild detergent at the higher incubated concentrations.
 |
DISCUSSION |
The pathophysiology of progressive renal scarring in renal disease is
poorly understood, but it is likely to be multifactorial. The powerful
association between proteinuria, tubulointerstitial scarring, and renal
disease progression has led to the hypothesis that either proteinuria
per se, or some other unidentified bioactivity in nephrotic glomerular
ultrafiltrate, may play an important role in the development of renal
scarring and inflammation (3, 28). It is notable therefore that
associated with the filtered protein are large quantities of lipid
material that enter the proximal tubule in large part complexed with
albumin. Lipid molecules can be presented to proximal tubule cells at
concentrations far in excess of their maximum solubility by virtue of
their capacity to bind to albumin, and indeed intracellular lipid
droplets in proximal tubular epithelial cells are a prominent feature
of proteinuric states (15).
The potential pathophysiological effects of this filtered lipid
material have not been well studied, but it has been demonstrated that
incubation of proximal tubular cells with lipidated albumin has
profound effects on cell lipid metabolism (29). Furthermore, proximal
tubular cells exposed to lipidated albumin are able to produce a
monocyte chemoattractant. Exposure of these cells to delipidated
albumin, however, does not result in the production of this
chemoattractant substance (18). The precise lipid responsible for this
effect is unclear, but observations such as these clearly implicate
albumin-bound lipids as potential mediators of proximal tubular cell
stimulation and toxicity.
In view of the paucity of knowledge regarding lipid signaling in the
proximal tubule, the aim of this study was to investigate the effects
of potentially important lipid mediators on proximal tubular cell
function. LPA was a particularly attractive candidate to examine, since
it is likely to be liberated in substantial quantities in inflamed
glomeruli and subsequently is very likely to enter the proximal tubule.
Specifically, the study set out to investigate whether LPA could
stimulate
[Ca2+]i
changes, as this is a mechanism by which cells regulate
many of their activities and responses to extracellular stimuli. In addition, the effect of LPA on proximal tubule cell
growth was studied, since derangements of proximal tubule cell growth
have been implicated in the progression of renal disease (34).
LPA was found to stimulate a classic
[Ca2+]i
transient in OK cells that was dose dependent. The
EC50 of this response to LPA was
higher (2.6 × 10
6 M)
than has been observed in some other cell types. Furthermore, the
magnitude of the
[Ca2+]i
increase was found to be relatively small compared with the [Ca2+]i
responses observed in some cell types after LPA (24). The EC50 could be high due to this
being a pathophysiological response rather than a physiological
process. Nonetheless, the
[Ca2+]i
responses were found to be comparable to those observed in other
investigations in OK cells utilizing established
Ca2+-mobilizing agonists such as
PTH (22). Our results show the [Ca2+]i
response to LPA to be biphasic, reflecting an initial phase of
Ca2+ release from intracellular
stores followed by a more prolonged signal that is dependent on
Ca2+ entry into the cell. In the
absence of extracellular Ca2+
(i.e., in the presence of 4 mM EGTA), the response was limited to a
transient
[Ca2+]i
rise. This entry of Ca2+ into the
cell is a universal feature of nonexcitable cells and the most widely
accepted model for regulation of
Ca2+ entry, termed the
"capacitative" model, which argues that
entry is activated by prior depletion of the inositol
1,4,5-triphosphate-mediated intracellular
Ca2+ stores (27).
Classically, this capacitative
Ca2+ entry pathway is
activated for the duration of store emptying; once triggered,
it is independent of the presence or absence of agonist and is
inhibited on repletion of the stores (27).
LPA is commonly considered to exert its action via G protein-coupled
receptors (32). Results from many studies indicate that the LPA
receptor couples to at least three distinct G proteins (24):
Gq, which links the receptor to
phospholipase C; G12/13, which
mediates Rho activation; and Gi,
which triggers Ras-GTP accumulation and inhibition of adenylyl cyclase.
As we found the [Ca2+]i
response to be immediate upon administration of LPA and insensitive to
PTX pretreatment, it would seem that this effect of LPA is receptor
mediated and most likely occurs via a PTX-insensitive G protein-linked receptor.
Reports in the literature implicate at least three different G proteins
in the transduction of LPA responses in different systems. The obvious
question raised by these observations has been whether the responses to
LPA stimulation are mediated by unifunctional receptors or whether one
type of receptor can mediate its varied responses. A recent report (12)
has addressed this issue and has demonstrated that a single LPA
receptor can activate multiple LPA-dependent responses in cells from
distinct tissue lineages.
One problem of
[Ca2+]i
measurements in a cell suspension system is that the fluorescence
signal obtained is an integration of the signal obtained from many
individual cells. Therefore, as LPA stimulated modest
[Ca2+]i
transients in the suspended OK cells, we hypothesized that this may be
due to a fraction of the cells responding with a large [Ca2+]i
transient and this signal being diluted due to a proportion of cells
showing no response. Consequently, similar experiments were carried out
on small populations of adherent cells using the Quanticell-700 cell
imaging system. This showed that the great majority (>80%) of the OK
cells respond with a
[Ca2+]i
transient following stimulation with LPA. Also, this system produced
results similar to the fluorometer in terms of the magnitude of the
[Ca2+]i
responses, hence substantiating the initial data.
Both LPA and FAF-BSA were found to exert a mitogenic effect on OK
cells, with LPA causing a maximum 5-fold increase in
[3H]thymidine
incorporation at 100 µM and FAF-BSA causing a 1.5-fold increase at a
maximum concentration of 10 µg/ml. LPA was still significantly
mitogenic at a concentration of 10 µM, whereas the amount of carrier
FAF-BSA present at this dose (1 µg/ml) failed to cause any effect on
thymidine incorporation. Therefore, these data suggest that LPA is the
predominant stimulator. Although most biological effects of LPA on
other cell types are mediated by nanomolar concentrations of the
phospholipid, LPA-stimulated proliferation has been found to require
micromolar concentrations (16). This was true in our study as well, in
which stimulation of proliferation required micromolar concentrations
of LPA. The effect of 100 µM LPA in the current studies was found to
be equal to that of 10% FCS. The results of the present study are much more marked than the results documented recently with LPA and mouse
renal proximal tubule cells (21). These authors demonstrated only a
1.5-fold increase in thymidine incorporation with 100 µM LPA under
similar conditions. We have also demonstrated that, in common with many
mitogens, a transient 5-min exposure of LPA to the cells is sufficient
to produce subsequent proliferation.
The mitogenic effect of LPA was found to be highly PTX sensitive. As
discussed above, the LPA receptor couples to at least three distinct G
proteins (24). Our data suggest that LPA acts through a PTX-insensitive
receptor to cause subsequent
[Ca2+]i
mobilization; however, the mitogenic action of LPA would seem to occur
through a PTX-sensitive receptor system. This pattern has also been
reported previously, which suggests that LPA-stimulated mitogenesis
(PTX-sensitive) in fibroblasts is independent of the PTX-insensitive
Gq-mediated
[Ca2+]i
mobilization (32).
The role of tyrosine kinases in LPA-mediated proliferation of OK cells
in this study is still uncertain. Although we found no inhibition of
thymidine incorporation by preincubation of the cells with appropriate
concentrations (25) of genistein and herbimycin A, the possibility
remains that tyrosine kinases unaffected by these inhibitors are still involved.
A number of functions of proximal tubular cells have been identified
which suggest that they can take part in the process of inflammation
and scarring. These functions include the production of matrix
proteins, proinflammatory cytokines, and chemotactic substances (5).
This is not surprising, considering that embryologically they are
derived from mesenchymal cells, as are fibroblasts and the cells of the
immune system (20). Our evidence would suggest that lipids such as LPA
may directly modulate tubular cell function, altering both their growth
characteristics and possibly phenotype. It is interesting to speculate
that a consequence of LPA signaling within the proximal tubule
epithelium may be the production of proinflammatory substances such as
growth factors and cytokines (11). This issue is the subject of ongoing
research in our laboratory.
In summary, ours is the first report to demonstrate that LPA causes
[Ca2+]i
signaling within renal proximal tubule cells, and although it has been
recently reported that LPA is a mitogenic factor in mouse proximal
tubule cells, our evidence suggests that LPA is a considerably more
potent mitogenic factor than previously reported (21) and that its
action occurs through Gi or a
related PTX substrate.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Kevin Harris for critical reading of the manuscript.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. J. Dixon, Dept. of Cell Physiology and
Pharmacology, Medical Sciences Bldg., University Road, Leicester LE1
9HN, UK.
Received 1 June 1998; accepted in final form 25 September 1998.
 |
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