Nephrology Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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Clinically, a
decrease in blood pH may be due to either a reduction in bicarbonate
concentration ([HCO3], metabolic acidosis) or an increase in
PCO2 (respiratory acidosis). In
mammals, metabolic acidosis induces a far greater increase in urine
calcium excretion than respiratory acidosis. In cultured bone,
metabolic acidosis induces a marked increase in calcium efflux and a
decrease in osteoblastic collagen synthesis, whereas isohydric
respiratory acidosis has little effect on either parameter. We have
shown that metabolic acidosis prevents the normal developmental
increase in the expression of RNA for matrix Gla protein
and osteopontin in chronic cultures of primary murine calvarial bone
cells (predominantly osteoblasts) but does not alter expression of
osteonectin. To compare the effects of isohydric metabolic and
respiratory acidosis on expression of these genes, bone cell cultures
were incubated in medium at pH ~7.2 to model metabolic
([HCO
3], ~13 mM) or
respiratory (PCO2, ~80 mmHg)
acidosis or at pH ~7.4 as a control. Cells were sampled at
weeks 4,
5, and
6 to assess specific RNA content. At
all time periods studied, both metabolic and respiratory acidosis
inhibited the expression of RNA for matrix Gla protein and osteopontin
to a similar extent, whereas there was no change in osteonectin
expression. In contrast to the significant difference in the effects of
metabolic and respiratory acidosis on bone calcium efflux and
osteoblastic collagen synthesis, these two forms of acidosis have a
similar effect on osteoblastic RNA expression of both matrix Gla
protein and osteopontin. Thus, although several aspects of bone cell
function are dependent on the type of acidosis, expression of these two matrix genes appears to be regulated by extracellular pH, independently of the type of acidosis.
bone; matrix Gla protein; osteonectin; osteopontin
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INTRODUCTION |
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IN HUMANS AND EXPERIMENTAL animals, chronic metabolic acidosis, a decrease in systemic pH induced by a decrease in serum bicarbonate concentration, increases urine calcium excretion (9, 30, 32) without increasing intestinal calcium absorption (31, 32), resulting in a negative calcium balance (9, 31, 32). Because >98% of total body calcium is contained within the bone mineral (43), this negative calcium balance implies a loss of bone calcium. Indeed, in humans, dietary intake of acid precursors causes an apparent decrease in bone mineral content, which is reversed by the provision of alkali (31, 40).
In contrast, chronic respiratory acidosis, a decrease in systemic pH induced by an increase in the PCO2, appears to have little (17) to no (29, 38, 39) effect on urine calcium excretion. Clearly, any effect on urine calcium excretion is far less than that induced by metabolic acidosis (17). Serum calcium may increase slightly during respiratory acidosis (29). The lack of an appreciable change in urine calcium excretion during respiratory, compared with metabolic, acidosis suggests a marked difference in the osseous response to these two types of acidosis (6).
We have extensively compared the response of cultured bone to a similar degree of metabolic and respiratory acidosis. During short-term (3 h) incubations, both types of acidosis cause a net calcium efflux from bone; however, isohydric metabolic acidosis causes a far greater net efflux than respiratory acidosis (12, 14, 15, 18). Respiratory acidosis not only causes less unidirectional calcium efflux but has been shown to cause deposition of medium calcium onto bone (15). Over this short time period, these changes are due to alterations in the physicochemical driving forces for bone mineralization and dissolution (10, 13). The low total CO2 concentration during metabolic acidosis favors dissolution, whereas high total CO2 concentration during respiratory acidosis favors formation of the carbonated apatite in bone (14).
During longer-term incubations, there is a cell-mediated component of
calcium efflux from bone during models of metabolic, but not
respiratory, acidosis (5, 7, 28). Although we have shown that metabolic
acidosis increases the resorptive activity of osteoclasts, as measured
by -glucuronidase release, and inhibits osteoid synthesis by
osteoblasts, as measured by collagen synthesis and alkaline phosphatase
activity, isohydric respiratory acidosis has little effect on these
parameters of bone cell activity (7, 28). Primary bone cells,
principally osteoblasts and osteoblast precursors, differentiate and
mineralize in culture (1, 2, 20, 41); metabolic acidosis inhibits
mineralization to a greater extent than respiratory acidosis (42).
We have examined the effects of metabolic acidosis on the RNA levels of
several genes known to be expressed in osteoblasts. After acute
stimulation with serum, metabolic acidosis inhibits expression of
Egr-1 and type 1 collagen RNA compared
with stimulation at neutral pH medium (23). In contrast, expression of
c-fos, c-jun,
junB, and
junD RNA was not affected by a similar
decrement in medium pH. In chronic bone cell cultures maintained up to
6 wk, metabolic acidosis inhibited expression of matrix Gla protein and
osteopontin RNA relative to expression in neutral medium (22). Expression of osteonectin, transforming growth factor-1, and GAPDH
was not affected by acidosis. With restoration of neutral pH medium,
the acidosis-induced inhibition of matrix gene expression was reversed.
Given the marked differences in the osseous response to metabolic, compared with respiratory, acidosis, we hypothesized that these differences would also be reflected in the relative inhibitory effects of metabolic and respiratory acidosis on matrix gene expression. We found, contrary to our initial expectation, that RNA levels for osteopontin and matrix Gla protein were dramatically inhibited to a similar extent by both chronic metabolic and respiratory acidosis. RNA levels for osteonectin were not affected by either type of acidosis. These results suggest that a decrement in extracellular pH, irrespective of how it was achieved, influences the expression of genes important for osteoblastic function and that the cellular signals promulgated by acidosis are dependent on the increased H+ concentration itself.
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METHODS |
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Cell culture. Bone cells were obtained from calvariae (frontal and parietal bones of the skull) of 4- to 6-day-old CD-1 mice. Mice were killed by cervical dislocation, and the calvariae were immediately dissected and placed in chilled HEPES. After accumulation of 20-50 calvariae, depending on the number of cells required, the bones were washed in saline-EDTA and then subjected to collagenase (Wako Pure Chemicals, Dallas, TX) digestion (25). The cells released by collagenase were plated on Primaria plates (Becton Dickinson, Lincoln Park, NJ) in DMEM + 15% heat-inactivated horse serum at a density of 5 × 105 cells/100-mm dish and cultured at 37°C in a CO2 incubator at a physiological PCO2 of 40 mmHg. After 8-10 days, with medium changed every 3-4 days, the cells reached confluence.
Cells were then divided into three groups. Cells were incubated in differentiation medium (DMEM + 15% heat-inactivated horse serum + 10 mMGene probes and labeling.
Probes used for analysis of RNA include osteopontin (OP), mouse, cDNA
(generous gift of Gideon Rodan, Merck, Rahway, NJ) (36); matrix Gla
protein (MGP), mouse, cDNA (generous gift of Gerard Karsenty, Baylor
College of Medicine, Houston, TX) (27); osteonectin (ON), bovine, cDNA
(generous gift of Marian Young, National Institute of Dental Health)
(44); and GAPDH, mouse, cDNA (Ambion, Austin, TX) (21). In each case,
the inserts were removed from the vector by digestion with the
appropriate restriction enzyme(s) and separated by electrophoresis on
low-melting-point agarose. Ethidium bromide-stained fragments were
identified by ultraviolet transillumination, excised with a razor
blade, and DNA purified from the gel with Wizard PCR Preps (Promega,
Madison, WI). Radioactive probes were prepared by random-primer
extension with the use of the Decaprime II system (Ambion) and
[-32P]dCTP (NEN,
Boston, MA). Unincorporated nucleotides were removed by use of
CentriSep spin columns (Princeton Separations, Adelphia, NJ).
RNA extraction and analysis. Cells were harvested for RNA before differentiation (day 8) and at weeks 4, 5, and 6. The cells were quickly scraped into a chaotropic solution (TRI-LS; Molecular Research, Cincinnati, OH) that dissociates RNA from protein complexes. RNA was purified from TRI-LS following the manufacturer's modification of the Chomczynski protocol (19). After ethanol precipitation, the RNA was dissolved in sterile water at a concentration of 10 µg/µl. Despite extensive mineralization in older control cultures, no consistent differences in bulk RNA recovery were observed (data not shown). Aliquots (20 µg) were denatured in 50% formamide-6% formaldehyde by heating to 65°C for 15 min. They were then electrophoresed on 1% agarose in MOPS-formaldehyde buffer. Samples were routinely stained with ethidium bromide during electrophoresis to ensure the integrity of the rRNA bands. After electrophoresis, the RNA was transferred to a charged nylon membrane (Zeta Probe; Bio-Rad, Richmond, CA) by capillary blotting with 10× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). After blotting, the nucleic acid was fixed to the membrane by ultraviolet cross-linking (Stratalinker; Stratagene, La Jolla, CA). Filters were hybridized and washed according to the manufacturer's recommendations; prehybridization (at least 1 h) and hybridization (18-22 h) were conducted in 250 mM sodium phosphate, pH 7.2, 7% SDS, and 1 mM EDTA at 65°C. After hybridization, the spent solution was removed, and the filter(s) was washed in 40 mM sodium phosphate, pH 7.2, 5% SDS, and 1 mM EDTA at 65°C twice; it was then washed twice in 40 mM sodium phosphate, pH 7.2, 1% SDS, and 1 mM EDTA at 65°C. The washed filters were visualized, and the signal was quantified by use of a phosphorimager (Molecular Dynamics, Sunnyvale, CA). To minimize variability, the same filters were sequentially hybridized to each probe in turn. Filters were routinely stripped of probe by two 20-min washes in 0.1× SSC-0.5% SDS heated to 100°C, and the stripped filters were then reprobed with the next labeled cDNA. The signal was quantitated as above, and the process was repeated.
Statistical analysis. Hybridization signal intensities among the three groups, normalized to the level of the housekeeping gene GAPDH, were compared by using ANOVA, with a Bonferroni correction for multiple comparisons with the BMDP statistical package (Los Angeles, CA) on a digital computer.
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RESULTS |
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Medium pH,
PCO2, and
[HCO3].
Compared with Ctl medium, by design the medium used to incubate cells
in the MA group had a significantly lower pH and
[HCO
3] (Fig.
1; values are means ± SE for
weeks 2-6 from 3 of 4 separate, parallel studies for which complete data are available). There was no
difference in the PCO2 between these
two groups. Compared with Ctl medium, by design the medium used to
incubate cells in the RA group had a significantly lower pH and higher PCO2. Compared with MA, there was no
difference in medium pH with RA; however, both
PCO2 and
[HCO
3] were
higher with RA.
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MGP, OP, and ON expression.
With Ctl, RNA levels for both MGP and OP increased dramatically from
predifferentiation (day 8) to
week 4 (Fig.
2). However, compared with Ctl, there was
no increase in the RNA levels for MGP or OP from day
8 to week 4 with MA
or RA.
|
Quantitation of MGP, OP, and ON RNA
levels.
To determine whether the levels of RNA were stable during chronic
acidosis, hybridization intensity was quantitated for each experiment
for weeks 4,
5, and
6 (Fig. 3
represents a single, typical experiment). In all four experiments
performed, the levels of MGP, OP, and ON for Ctl, MA, and RA were
consistent during weeks 4,
5, and
6.
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DISCUSSION |
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Metabolic acidosis, whether studied in vivo or in vitro, has
significant effects on the bone mineral (6, 7, 16, 28, 40). In cultured
bone and in bone cells, metabolic acidosis not only causes a marked
increase in net calcium efflux but stimulates osteoclastic bone
resorption, inhibits osteoblastic bone formation, and decreases the
formation of mineralized bone nodules (10, 11, 13, 14, 28, 42).
Isohydric respiratory acidosis causes far less calcium efflux, does not
appear to affect parameters of bone cell function, and has less effect
on formation of bone nodules (4, 5, 7, 12, 15, 42). We have previously shown that when bone cells are incubated in medium simulating metabolic
acidosis, there is a marked decline in the accumulation of RNA for
matrix Gla protein and osteopontin, genes that encode components of the
bone extracellular matrix, but not osteonectin or GAPDH (22). The
purpose of the current study was to test the hypothesis that metabolic
acidosis would have a greater effect on the expression of matrix Gla
protein and osteopontin than would respiratory acidosis. Such a
differential response could arise if a hypothetical pH sensor on the
cell surface does not respond solely to proton concentration but is
modulated by changes in [HCO3]. We found,
contrary to initial expectations, that both metabolic and respiratory
acidosis had a similar suppressive effect on RNA accumulation of these genes.
It is unclear why metabolic and respiratory acidosis have a similar
effect on matrix Gla protein and osteopontin RNA accumulation but
differing effects on net calcium release and cell function in cultured
calvariae. There are several possibilities for these differences. The
calvarial studies were conducted over 3-96 h (4, 5, 7, 12, 15),
whereas the current studies took place over 4-6 wk. In bone cell
cultures used in this study, the increase in matrix Gla protein and
osteopontin in control medium is not yet evident by
day 8; thus it is possible that these
late markers were not yet affected by acidosis in the relatively
short-term studies using cultured calvariae. Additionally, osteoblasts
present in intact calvariae may represent a wider range of maturational states than collagenase-released bone cells. Calvariae were cultured in
an HCO3-buffered medium similar to
that used for the bone cells in this study; however, the
bone culture medium does not routinely include the
-glycerophosphate
and ascorbate that are present in the mineralization medium used in the
current experiment (4, 5, 7, 12, 15). In addition, cultured calvariae
include resorbing osteoclasts and mature osteoblasts, whereas the
isolated bone cells used in this study are predominantly osteoblasts
and osteoblast precursors (20). Because osteoclasts and osteoblasts are
believed to influence each other's activity, primarily through
cytokines, it is possible that the differential effects of metabolic
and respiratory acidosis observed in the calvarial studies lie in a
specific response of the osteoclast to the different types of acidosis.
Future studies on the comparative response of isolated osteoclasts to
metabolic and respiratory acidosis should shed light on this hypothesis.
We have previously shown that both metabolic and respiratory acidosis
inhibit the development of apatite-containing nodules that form when
primary cultures of bone cells are incubated for several weeks in the
presence of -glycerophosphate and ascorbate (42). Although both
types of acidosis caused a significant decrease in the number and
calcium content of the nodules compared with cells incubated at a
physiologically neutral pH, there was greater suppression by metabolic,
compared with respiratory, acidosis. Although the previous study and
the current study both determined the effects of metabolic and
respiratory acidosis on differentiated function of osteoblasts, it is
not clear that the gene expression examined in the current study
directly influences the formation of bone nodules. Neither matrix Gla
protein nor osteopontin has been shown to initiate bone formation (3,
24, 26), and osteopontin has actually been shown to retard
hydroxyapatite crystal growth (3, 26). Null mutants for each of these
genes have been constructed in mice, and each mutant shows apparently
normal bone growth (33, 34). It is possible that matrix Gla protein and/or osteopontin are important in determining the three-dimensional structure of bone and its consequent static and dynamic properties (24,
35), qualities that cannot be assessed through numbers of bone nodules.
Previously, we have shown that metabolic acidosis selectively inhibits
expression of matrix Gla protein and osteopontin RNA (22); however, this study did not allow us to determine
whether the suppression of expression was due to the low pH or the low [HCO3]. That metabolic
and respiratory acidosis have a similar inhibitory effect on matrix Gla
protein and osteopontin suggests that external cell pH, and not
PCO2 or
[HCO
3], modulates
expression of these genes. It is unclear whether the reduced
extracellular pH has direct effects on cell surface components that
would then alter intracellular signaling pathways or whether intracellular pH (pHi) itself
must be decreased for acidosis to affect bone cell function.
Respiratory acidosis might be expected to lower cytosolic pH more
rapidly than metabolic acidosis, as CO2 is far more permeable across
cell membranes than HCO
3. We have
previously determined the effects of models of metabolic and
respiratory acidosis on the pHi of
UMR106 rat osteosarcoma cells (37). Within 1 min after exposure to
acidosis, there is indeed a more pronounced fall in
pHi with respiratory, compared with metabolic, acidosis; however, after 24 and 48 h, the
pHi increased to normal with
metabolic acidosis but continued to be suppressed with respiratory
acidosis. That pHi is reduced in
chronic respiratory but not in chronic metabolic acidosis suggests that the similar suppression of specific gene expression found during both
types of acidosis in the present study cannot be explained by
pHi.
pHi was not measured in the
current study, as it is not clear that accurate measurements of
pHi can be made in mineralizing bone cells.
The sustained levels of osteonectin and GAPDH RNA indicate that prolonged acidosis does not have generalized toxic effects on the bone cells used in this study. Previously, we have shown that the suppression of matrix Gla protein and osteopontin, due to a more severe acidosis (pH 7.1) than that used in this study, was fully reversible on restoration of a physiologically normal pH even after 2 wk (22). The reversibility of suppression provides further evidence for the lack of generalized cytotoxicity. That both matrix Gla protein and osteopontin RNA levels are suppressed by acidosis suggests a common regulatory mechanism, which could be reflected in the presence of similar regulatory motifs in each gene. Previously, we have concluded that there are several shared domains with lengths of up to 43 nucleotides in the published upstream regions of these two genes, some of which could be proton-modulated sequence elements (22). At present, we do not know whether any of these shared sequences play a role in proton-modulated gene expression.
We have shown that acidosis, whether produced by a decrease in
[HCO3] or by an
increase in PCO2, suppresses the
expression of RNA for the two bone matrix proteins, matrix Gla protein
and osteopontin. In contrast, expression of osteonectin and GAPDH RNA
was not affected by either form of acidosis, indicating that the
suppression was a specific response to increased extracellular proton
concentration and not a generalized cellular toxicity. The
acidosis-induced inhibition of matrix Gla protein and osteopontin RNA
suggests that the perturbation of extracellular pH, however obtained,
may be detrimental to normal bone cell physiology.
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ACKNOWLEDGEMENTS |
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We thank Krystof J. Neumann for technical assistance.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants AR-39906 and DK-47631 (both to D. A. Bushinsky) and by a grant from the National Kidney Foundation of Upstate New York (to K. K. Frick).
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 and other correspondence: K. K. Frick, Univ. of Rochester School of Medicine, Nephrology Unit, Dept. of Medicine, 601 Elmwood Ave., Box 675, Rochester, NY 14642 (E-mail: Kevin_Frick{at}URMC.Rochester.edu).
Received 14 January 1999; accepted in final form 30 June 1999.
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