1 Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colorado 80262; 2 Department of Medicine, University of Southern California School of Medicine, Los Angeles, California 90033; and 3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The effect of hypoxia on the proliferation of cultured rat mesangial cells was examined. To evaluate the underlying signaling mechanisms, the roles of intracellular calcium ([Ca2+]i) and protein kinase C (PKC) were determined. Quiescent cultures were exposed to hypoxia (3% O2) or normoxia (18% O2), and [3H]thymidine incorporation, cell number, [Ca2+]i, and PKC were assessed. Mesangial cells exposed to 28 h of hypoxia exhibited a significant increase in [3H]thymidine incorporation followed by a significant increase in cell number at 72 h in comparison with respective normoxic controls. Hypoxia induced a biphasic activation of PKC, reflected by translocation of the enzyme activity from cytosol to membrane at 1 h, a return to baseline at 4 and 8 h, with subsequent reactivation from 16 to 48 h. In addition, hypoxia-induced proliferation was prevented by a PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Cells exposed to hypoxia produced progressive increases in resting [Ca2+]i from 15 to 60 min which remain sustained up to 24 h of examination. Verapamil significantly prevented the hypoxia-induced proliferation, and both verapamil treatment and incubations in a calcium-free medium for 1 h blocked the hypoxia-induced stimulation of [Ca2+]i as well as PKC. These results provide the first in vitro evidence that chronic hypoxia induces proliferation of cultured glomerular mesangial cells, which is mediated by the stimulation of [Ca2+]i and the subsequent activation of PKC.
mesangial cell growth; signal transduction
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INTRODUCTION |
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CHRONIC ISCHEMIA from atherosclerotic renovascular disease contributes significantly to end-stage renal disease (12). Studies in humans and in animal models suggest that chronic hypoxia plays an important pathogenetic role in the development of glomerulosclerosis, tubulointerstitial fibrosis, and potentially end-stage renal disease (7, 8, 20, 21, 24, 31). Both glomerulosclerosis and tubulointerstitial fibrosis are characterized by hypercellularity, deposition of extracellular matrix proteins, and the development of an undifferentiated phenotype.
Recent in vitro studies from our laboratory have provided important new insights concerning the mechanisms whereby chronic hypoxia alters cellular behavior. We have found that chronic hypoxia directly impairs the differentiation of both cultured LLC-PK1 renal proximal tubular epithelial cells as well as murine 3T3-L1 fibroblasts and also induces dedifferentiation of LLC-PK1 cells previously adapted to a normoxic environment (25-27). In addition to the inducement of dedifferentiation, our studies demonstrated for the first time that chronic hypoxia is mitogenic for LLC-PK1 renal epithelial cells (25). Chronic hypoxia also has been found to stimulate the proliferation and extracellular matrix production of pulmonary artery smooth muscle cells and dermal fibroblasts (5, 6, 19, 32, 33).
Despite the growing evidence that chronic hypoxia is a mitogenic and fibrogenic factor for multiple cell types, its role in the proliferation of mesangial cells is undefined. Furthermore, the underlying signaling mechanisms whereby hypoxia induces proliferation and extracellular matrix synthesis remain poorly defined. However, studies from our laboratory suggest that the activation of protein kinase C (PKC) is a critical step. Chronic hypoxia induces sustained activation of PKC in LLC-PK1 and 3T3-L1 cells, and studies with PKC inhibitors further confirm that its activation plays a key role in mediating alterations in cellular proliferation and differentiation by these cells (25-27). Besides PKC, hypoxia also stimulates intracellular calcium ([Ca2+]i) in cultured smooth muscle and endothelial cells, and increased [Ca2+]i is associated with the development of atherosclerosis as well as ischemic glomerulosclerosis and tubulointerstitial fibrosis (1, 4, 16, 29).
The present study examined whether chronic hypoxia effects the proliferation of cultured rat mesangial cells and, if so, whether the alterations in [Ca2+]i and/or PKC play a role in mediating this process.
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METHODS |
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Materials. Male Sprague-Dawley rats
weighing 200-250 g were obtained from Harlan
Laboratories.
[3H]thymidine and
[-32P]ATP were
purchased from ICN. The acetoxymethyl ester of fura 2 (fura 2-AM) was
obtained from Molecular Probes, Eugene, OR. All other reagents were of
high chemical grade from Sigma Chemical.
Cell culture. Primary cultures of glomerular mesangial cells were established from the kidneys of healthy male Sprague-Dawley rats as described by Kreisberg and Karnowsky (17). Kidneys were removed, washed with sterile phosphate-buffered saline (PBS), and encapsulated, and the cortex was separated from medulla. Cortex was cut into smaller fragments, washed, and passed through a series of steel sieves (Newark Wire Cloth) of progressively smaller sizes [pore size 425-µm pore (40 mesh), 125-µm pore (120 mesh), and 53-µm pore (270 mesh)]. The isolated glomeruli remaining on the top of 270-mesh sieve were rinsed with PBS containing penicillin (100 U/ml) and streptomycin (100 µg/ml). Glomeruli were transferred to a 15-ml tube, suspended with the same PBS containing 10 mg/ml collagenase (Sigma, type V), and incubated for 20-30 min in a 37°C water bath shaker. Digested glomeruli were centrifuged, washed with PBS, and resuspended in minimum essential medium (D-valine modification) containing 20% fetal bovine serum, 0.3 U/ml insulin, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml amikacin, and 2.5 µg/ml fungizone). Cells were kept in this medium for 3 passages, which permitted the proliferation of mesangial cells while the growth of fibroblasts was inhibited.
After passage 3, cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum, 0.3 U/ml insulin, and the antibiotics referred to as growth medium (GM). Cultures were maintained in 75-cm3 flasks with GM at 5% CO2-18% O2 environment under rocked conditions in a fashion similar to LLC-PK1 cells as previously described (27). Cells were passed by trypsinization after they reached 80% confluence and utilized between passages 4 and 12 for all the studies.
Characterization of mesangial cells.
Mesangial cell cultures in passage 4 were characterized by immunohistochemical methods. Cells were grown on
chamber slides (Nunc, Naperville, IL) until they reached 60-80%
confluence, rinsed with PBS, and fixed by immersing in Omnifix.
Cells were washed and permeabilized with 0.05% Triton
X-100 followed by incubation in 10% bovine serum albumin (BSA). After
blocking with BSA, cells were incubated overnight at 4°C with
polyclonal antibodies against either cytokeratin (1:1,000; Dako,
Carpinteria, CA), -smooth muscle actin (1:1,000, Sigma), factor VIII
(1:500; Synbiotics, San Diego, CA) or normal rabbit serum (1:500; Zymed
Labs, South San Francisco, CA). Subsequently, slides were processed
using a streptavidin-biotin kit (Zymed Labs) and examined by
phase-contrast microscopy.
Experimental protocol. All experimental maneuvers were performed under rocked conditions with cells exposed to either a hypoxic (3% O2, medium PO2 = 30-40 mmHg) or normoxic (18% O2, medium PO2 = 140-150 mmHg) environment. Preliminary studies indicated that exposure of normoxic cells to 3% oxygen environment for up to 72 h did not impair cell viability as assessed by trypan blue exclusion method. Therefore, this degree of hypoxia was used in all studies to assess the effects on cell proliferation, [Ca2+]i, and PKC activity. All experiments were carried out in paired fashion with parallel assessments of cells exposed to normoxia and hypoxia.
To assess the effect of hypoxia on various parameters (described below), mesangial cells were subcultured in GM until they reach to 60-70% confluence. At this time the GM was removed and cells were washed with insulin-free GM containing 0.5% serum (referred as serum-free medium) and incubated in the same medium for 48 h to achieve quiescence. Quiescent cultures were then exposed either to hypoxia (3% O2) or maintained normoxic (18% O2) in serum-free medium for varying periods of time to assess cell proliferation, [Ca2+]i, or PKC as described below.
Assessment of cell proliferation. To examine the effect of hypoxia on mesangial cell proliferation, normoxic cultures were subcultured in six-well plates. Quiescent cultures were then exposed either to hypoxia or maintained normoxic in serum-free medium for 28 or 72 h for the assessment of [3H]thymidine incorporation and cell number as indexes of cellular proliferation.
[3H]thymidine incorporation was assessed as described by Norman et al. (23). Cultures were exposed to hypoxia or normoxia for 28 h, and 1 µCi/ml of [3H]thymidine (specific activity 20 Ci/mmol) was added in the last 4 h of incubation. At the end of incubation, medium was removed, and cells were washed with 10% trichloroacetic acid and digested with 0.5 N NaOH. Radioactivity in the cell digest was counted in a Beckman liquid scintillation counter, and [3H]thymidine incorporation was expressed as counts per minute per well. For the assessment of cell number, cultures exposed to hypoxia and normoxia for 28 and 72 h were washed and trypsinized. Cells were then counted in a Coulter counter (Coulter Electronics).
Assessment of PKC. PKC activity was
assessed in cultures grown in 100-mm dishes. Cultures were exposed to
hypoxia or normoxia for 1, 4, 8, 16, 24, and 48 h followed by the
assessment of PKC activity as previously described (25). Briefly, at
the end of each incubation, cells were harvested, and cytosol and
membrane fractions were isolated. Experiments were also carried out in which cultures were exposed to hypoxia or normoxia for 1 h in the
absence or presence of verapamil
(106 M) or media calcium
(calcium-free Krebs buffer) followed by the isolation of cytosol and
membrane fractions. Both cytosolic and membrane fractions were applied
to 0.5-ml DEAE-cellulose columns. Columns were washed, and the bound
PKC activity was eluted with a buffer containing 100 mM NaCl and
collected in one fraction. PKC activity in the eluted fraction was
analyzed by measuring the transfer of
32P from
[
-32P]ATP to
histone as previously described (25).
Measurement of
[Ca2+]i.
Single-cell
[Ca2+]i
measurements were carried out by video imaging fluorescence microscopy
as previously described (18). Mesangial cells were grown on sterile
glass coverslips. Quiescent cultures were washed with serum-free medium
and labeled in the same medium with fura 2-AM (4 µM) for 45 min at
37°C. At the end of labeling, cells were washed and exposed to
hypoxia or normoxia for 15-60 min in the serum-free medium
previously equilibrated under hypoxic or normoxic conditions. Cells
were then immediately washed with Krebs buffer containing 1 mM
CaCl2, which was also previously equilibrated under hypoxic or normoxic conditions. The effect of 6 and
24 h of hypoxia on
[Ca2+]i
was assessed, cells were exposed to hypoxia or normoxia for 6 and 24 h,
and fura 2 was added in the last 45 min of incubations. Subsequently,
cells were processed in the same fashion. In some experiments cells
were exposed to hypoxia or normoxia for 60 min in the absence or
presence of verapamil (106
M), media calcium (calcium-free Krebs buffer), or
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 50 µM), and
[Ca2+]i
was assessed.
[Ca2+]i
was measured immediately, using the excitation at 340 and 380 nm and
emmision at 510 nm, as carried out previously (18). To estimate
[Ca2+]i,
the maximal ratio (Rmax) was
measured after the addition of 10 µM ionomycin, and the minimal ratio
(Rmin) was calculated after subsequent addition of 10 mM tris(hydroxymethyl)aminomethane + ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid. As many as 20 cells per condition were studied for each experiment.
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RESULTS |
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Identification of mesangial cells.
Mesangial cell cultures in passage 4 were characterized by immunostaining with cell marker antibodies as
described in METHODS. The cells
stained very heavily for -smooth muscle actin and negatively for
factor VIII, keratin, and normal rabbit serum. These results indicate
that cultures in passage 4 were 100%
mesangial cells. Furthermore, phase-contrast microscopy demonstrated
phenotypic characteristics of smooth muscle cells. These cultures were
maintained under rocked conditions at 5%
CO2-18%
O2 environment as described in
METHODS and referred to as normoxic
cells.
Effect of hypoxia on cell proliferation. Quiescent cultures of normoxic mesangial cells were exposed either to hypoxia (3% O2) or continuous normoxia (18% O2) for 28 to 72 h followed by the assessment of [3H]thymidine incorporation and cell number as indexes of cellular proliferation. Exposure of cells to hypoxia for 28 h in a serum-free medium resulted in a significant 112% increase in [3H]thymidine incorporation in comparison with the respective normoxic controls (Fig. 1A).
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Analysis of cell proliferation by cell counting demonstrated no significant changes in cell number between hypoxic and normoxic cells incubated for 28 h (data not shown). However, exposure to hypoxia for 72 h resulted in a significant 33% increase in cell number when compared with respective normoxic controls (Fig. 1B).
Effect of hypoxia on PKC. Our earlier studies demonstrated a role for PKC activation in hypoxia-induced alterations in differentiation and/or proliferation of LLC-PK1 and 3T3-L1 cells (25-27). Therefore, we first examined the effect of hypoxia on PKC activity in mesangial cells. Cultures were exposed to hypoxia from 1 to 48 h with control cultures maintained under normoxic conditions for the same duration of time. As shown in Table 1, hypoxia produced a significant decrease in cytosolic activity at 1 h followed by no difference at 4 and 8 h with a subsequent significant sustained decrease from 16 to 48 h when compared with respective normoxic controls. Parallel assessment of membrane PKC activities in response to hypoxia demonstrated a significant translocation from the cytosol to membrane pool with significant increases in membrane PKC activities at 1 and from 16 to 48 h in comparison with the activities observed under normoxic conditions (Table 1). Similar to the cytosolic data, cells exposed to hypoxia for 4 and 8 h exhibited no difference in membrane PKC in comparison with the activity observed in normoxic cells.
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Figure 2 shows the ratio of membrane to cytosolic PKC activity, which represents a better reflection of PKC activation. As shown in Fig. 2, normoxic cultures maintained a low membrane-to-cytosolic ratio of PKC during incubation from 1 to 48 h, consistent with a differentiated phenotype as observed in many other cell types (3, 22). In contrast, exposure of cells to hypoxia produced a significant increase in the membrane-to-cytosolic ratio at 1 h, followed by no significant changes at 4 and 8 h, with subsequent significant increases at 16 and 24 h that remained sustained for the duration of the experiment (Fig. 2). Thus hypoxia induces a biphasic (acute and subsequently sustained) activation of PKC in mesangial cells.
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Role of PKC activation in hypoxia-induced cell proliferation. We subsequently determined whether the activation of PKC plays a role in hypoxia-induced mesangial cell growth. Cultures were exposed to hypoxia or normoxia for 28 h in the absence or presence of the PKC inhibitor H-7 (50 µM) followed by the assessment of [3H]thymidine incorporation. H-7 rather than staurosporine or bisindolylmaleimide-like compound GF-109203X (GFX) was used as a PKC inhibitor because we found that both staurosporine and GFX substantially reduce thymidine incorporation in control normoxic cells. Similar to the observations in Fig. 1, hypoxia induced a significant increase in [3H]thymidine incorporation in comparison with cells maintained under normoxic conditions (Fig. 3). However, cells subjected to hypoxia in the presence of H-7 almost completely prevented the hypoxia-induced increase in [3H]thymidine incorporation (Fig. 3). H-7 alone had no significant effect on [3H]thymidine incorporation under control normoxic conditions (Fig. 3).
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Effect of hypoxia on
[Ca2+]i.
Since increased
[Ca2+]i
levels are associated with the development of glomerulosclerosis (16),
we examined whether hypoxia alters resting
[Ca2+]i
in mesangial cells. Normoxic mesangial cells exhibited basal levels of
[Ca2+]i
in the range of 146 ± 20 nM (Fig.
4). Exposure of mesangial cells to hypoxia
for 15-60 min induced progressive increases in [Ca2+]i,
with the maximal stimulation observed at 60 min (312 ± 19 nM, P < 0.025 vs.
normoxia). Hypoxia-induced increase in
[Ca2+]i
remained sustained up to 24 h of examination (Fig. 4).
Addition of 105 M
angiotensin II (ANG II) to 60 min normoxic or hypoxic cells significantly increased
[Ca2+]i
to similar levels (data not shown). However, ANG II response to
[Ca2+]i
was more pronounced in 6 h hypoxic than in normoxic cells (Fig. 5;
normoxia + ANG II and normoxia = 180 ± 22 nM, and
hypoxia + ANG II and
hypoxia = 400 ± 49 nM, P < 0.01).
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Role of
[Ca2+]i
in hypoxia-induced stimulation of PKC and cell
proliferation.
We first determined whether increased
[Ca2+]i,
due to the stimulation of calcium influx, participates in
hypoxia-induced proliferation of mesangial cells. Cultures were exposed
to normoxia and hypoxia for 28 h in the absence or presence of a
calcium channel blocker verapamil
(106 M), and
thymidine incorporation was assessed. As shown in Fig. 6, verapamil significantly prevented the
hypoxia-induced increase in
[3H]thymidine incorporation in
mesangial cells.
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DISCUSSION |
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We reported previously that chronic hypoxia induces proliferation and dedifferentiation of LLC-PK1 renal proximal tubular epithelial cells as well as impairs the differentiation of 3T3-L1 fibroblasts into mature adipocytes (25, 26). The present study provides the first direct evidence of the mitogenic effect of hypoxia in cultured mesangial cells and demonstrates a role for both [Ca2+]i and PKC in mediating hypoxia-induced proliferation of mesangial cells.
Results of the present study indicate that exposure of quiescent mesangial cells to a 3% oxygen environment induces cellular proliferation. Chronic hypoxia increased [3H]thymidine incorporation at 28 h, reflecting enhanced DNA synthesis, which was followed by an increase in cell number measured at 72 h, thereby clearly indicating that chronic hypoxia is mitogenic to cultured renal glomerular mesangial cells. This is consistent with our finding in LLC-PK1 cells, where chronic hypoxia also increased cellular proliferation (25). Of interest, a recent study found that intact normal mice exposed to chronic hypoxemia exhibited an increase in kidney weight, raising the possibility that chronic hypoxia may directly stimulate renal cell growth in vivo (2). Chronic hypoxia also has been reported to stimulate the proliferation and extracellular matrix production of cultured pulmonary artery smooth muscle cells and dermal fibroblasts (5, 6, 19, 32, 33). More recently chronic hypoxia was found to stimulate the extracellular matrix production in cultured rat mesangial cells (13). However, these studies neither examined the effect of hypoxia on mesangial cell proliferation nor did they explore the mechanisms of hypoxia-induced stimulation of matrix production. Studies in human and in animal models suggest that chronic hypoxia contributes to the development of glomerulosclerosis (7, 8, 20, 21, 24, 31). Taken together, these studies strongly suggest that chronic hypoxia is an important pathophysiological stimulus for mesangial cell growth and extracellular matrix synthesis both in vivo and in vitro. Despite the growing evidence that chronic hypoxia is mitogenic and fibrogenic to multiple cell types, the underlying cellular and molecular signaling mechanisms whereby hypoxia induces proliferation and matrix production are poorly defined.
The present study indicates that an increase in [Ca2+]i and the subsequent activation of PKC play a central role in mediating the hypoxia-induced mesangial cell growth. Two approaches were utilized to examine the role of both [Ca2+]i and PKC in hypoxia-induced proliferation of mesangial cells. In the first approach, the time courses of the effect of hypoxia on [Ca2+]i and PKC were assessed. We found that hypoxia induces an early rise in [Ca2+]i at 15-60 min that remains sustained up to 24 h of examination. Furthermore, hypoxia-induced increases in [Ca2+]i were accompanied by a biphasic activation of PKC. Hypoxia produced an activation of PKC at 1 h followed by a gradual return to baseline at 4 and 8 h with subsequent gradual activation at 16 h that was sustained up to 48 h of examination. Interestingly, this pattern of PKC activation by hypoxia was similar to the effect observed in LLC-PK1 cells (25). Therefore, this dual response appears to represent a general phenomenon with a transient acute activation followed by sustained PKC activation. This pattern of PKC activation raises the possibility that there may be two different PKC-activating mechanisms and more than one isoform of PKC involved in hypoxia-induced stimulation of cellular proliferation.
In the second approach, we assessed the effect of hypoxia on cell proliferation in the absence or presence of a calcium channel blocker verapamil and a PKC inhibitor H-7. The hypoxia-induced increases in [3H]thymidine incorporation were significantly prevented by both verapamil and H-7, suggesting a role for increased calcium influx and PKC activation in hypoxia-induced proliferation of mesangial cells. Present studies along with our earlier finding with both LLC-PK1 cells and 3T3-L1 fibroblasts indicate that sustained activation of PKC is crucial for chronic hypoxia-induced proliferation and dedifferentiation (25-27). Increased [Ca2+]i also is implicated in the smooth muscle cell growth as well as in the progression of chronic renal disease (16, 29). In addition, verapamil is reported to prevent the growth factor-induced proliferation of mesangial cells, suggesting a role for calcium influx in mesangial cell mitogenesis (30). Taken together, these studies strongly suggest that increased calcium influx and the subsequent activation of PKC play an important pathogenetic role in hypoxia-induced proliferation of mesangial cells and potentially in the development of glomerulosclerosis. Consistent with this notion, verapamil and other calcium channel blockers are also shown to inhibit mesangial cell proliferation and glomerulosclerosis in models of anti-Thy-1.1 glomerulonephritis and puromycin-induced focal segmental glomerulosclerosis (15, 28).
The mechanisms of acute and sustained activation of PKC by hypoxia are not completely defined by these studies. However, our results from studies utilizing verapamil and calcium-free medium strongly suggest that increased calcium influx accounts for hypoxia-induced stimulation of [Ca2+]i and subsequent acute activation of PKC. Whether the activation of L-type calcium channels causes an increase in calcium influx observed in our studies remains to be determined. Regardless of the mechanisms involved, the present study suggests that increased calcium influx mediates the rise in [Ca2+]i and the subsequent acute activation of PKC and cell growth induced by hypoxia.
The mechanisms of sustained activation of PKC by hypoxia remain
undefined. However, it is possible that acute PKC activation-mediated release of specific growth factors into the medium, in an autocrine fashion, may cause sustained increases in
[Ca2+]i
and PKC. Of potential relevance, hypoxia is shown to stimulate the
synthesis of specific growth factors in epithelial, endothelial, and
smooth muscle cells as well as dermal fibroblast (10). Studies in
experimental models of renal injury revealed that mesangial cell
proliferation occurs with concomitant upregulation of platelet-derived growth factor (PDGF) and PDGF -receptors, suggesting a role for PDGF
in mesangial cell proliferation in vivo (11). In addition to PDGF,
several other autocrine growth factors and cytokines including
endothelin-1, ANG II, fibroblast growth factor, transforming growth
factor-
, and interleukin-6 have been shown to stimulate the
proliferation and/or matrix production of mesangial cells (9).
Since in the present study ANG II response to
[Ca2+]i
was more pronounced in hypoxic than in normoxic cells, it is possible
that hypoxia not only may induce the synthesis of specific growth
factors but also can prime cells to respond more markedly to these
factors. Additional studies are required to confirm this speculation.
However, consistent with this notion, hypoxia is demonstrated to
upregulate the receptors for fibroblast growth factor and epidermal
growth factor in retinal pigment epithelial cells (14). Whether chronic
hypoxia increases the synthesis/release of specific growth factors
and/or their receptors in mesangial cells and whether the
induction of these factor(s) accounts for the sustained increases in
[Ca2+]i,
PKC, and cell proliferation, observed in our studies, remain to be
determined. Future studies to substantiate these speculations should be
of considerable interest.
In conclusion, the results of the present study indicate that chronic hypoxia induces proliferation of cultured glomerular mesangial cells. Furthermore, hypoxia increases [Ca2+]i and causes a biphasic activation of PKC, which plays an important role in hypoxia-induced proliferation of mesangial cells.
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ACKNOWLEDGEMENTS |
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This work was supported by Research Grant 971-GI from the American Heart Association of the Greater Los Angeles Affiliate and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-25248 and DK-35098.
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FOOTNOTES |
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Portions of this work have been published in an abstract form (J. Am. Soc. Nephrol. 5: 697, 1994).
Address for reprint requests: A. Sahai, Division of Renal Diseases and Hypertension, Univ. of Colorado Health Sciences Center, Campus Box C281, 4200 East Ninth Ave., Denver, CO 80262.
Received 31 January 1997; accepted in final form 26 August 1997.
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