In vitro metabolic and respiratory acidosis selectively inhibit osteoblastic matrix gene expression

Kevin K. Frick and David A. Bushinsky

Nephrology Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York 14642


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Clinically, a decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO-3], 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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-beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 mM beta -glycerophosphate + 50 µg/ml ascorbic acid) (1, 2, 20, 41) either at a control pH (7.4, Ctl) or an acidic pH (7.2) produced by a decrease in the bicarbonate concentration ([HCO-3]), to model metabolic acidosis (MA), or an increase in the PCO2, to model respiratory acidosis (RA). To closely replicate physiological conditions, only the HCO-3/CO2 buffer system was used (8). To model MA, concentrated HCl was added to the Ctl medium (1.07 ml of 11.6 M HCl to 500 ml of Ctl medium), resulting in a reduction of [HCO-3] and thus pH. To model RA, Ctl medium PCO2 was increased by increasing the CO2 concentration in the incubator to ~80 mmHg. Cells were continuously incubated under these conditions for a total of 6 wk, with medium changes every 3-4 days. All culture media were preincubated for at least 18 h to allow the incubator and medium CO2 to reach equilibrium; we have previously shown that equilibration is reached by 3 h (11). Medium pH and PCO2 were measured with the use of a blood gas monitor (Radiometer ABL 30, Copenhagen, Denmark). The [HCO-3] was calculated with the use of the Henderson-Hasselbalch equation (11).

Gene 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 [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Medium pH, PCO2, and [HCO-3]. 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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Fig. 1.   Medium pH, PCO2, and bicarbonate concentration ([HCO-3]) of culture medium. Cells were grown for 8 days in physiologically neutral pH medium before incubation in control (Ctl) differentiation medium or in differentiation medium simulating metabolic acidosis (MA) or respiratory acidosis (RA). Medium was sampled weekly (weeks 2-6), 3-4 days after prior medium change and immediately before RNA harvest. Data shown (means ± SE; n = 15 in each group) represent 3 of 4 separate, parallel studies for which complete data for weeks 2-6 are available. * P < 0.05 vs. Ctl. + P < 0.05 vs. MA.

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.


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Fig. 2.   Northern analysis of matrix Gla protein (MGP), osteopontin (OP), and osteonectin (ON) RNA expression. Cells were grown for 8 days in physiologically neutral pH medium before incubation in control differentiation medium or in differentiation medium simulating MA or RA. Cells were harvested for RNA at weeks 4, 5, and 6. Aliquots of RNA (20 µg) were electrophoresed, transferred to a single nylon membrane, and then hybridized to probes for MGP, OP, ON, and GAPDH. Images shown were acquired with phosphorimager beta -capture. Extraneous lanes were removed from image with the use of Adobe Photoshop. d8, day 8.

With Ctl, there was no change in the RNA levels for ON and GAPDH from day 8 to week 4. In contrast to MGP and OP, there was no change in ON or GAPDH RNA levels between Ctl and either MA or RA, indicating that the acidosis-induced changes in MGP and OP are not due to a generalized decrease in cellular RNA levels.

To determine the more chronic effects of MA and RA on RNA accumulation, we continued the incubation for a period of 6 wk. At weeks 5 and 6, compared with day 8, there was a marked increase in MGP and OP RNA levels in Ctl but not in MA or RA, similar to that observed at week 4. There was no difference in ON or GAPDH RNA levels between day 8 and weeks 5 and 6, again similar to the pattern seen at week 4.

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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Fig. 3.   Quantitation of Northern analysis data for MGP, OP, and ON RNA expression from an individual experiment. Cells were grown for 8 days in physiologically neutral pH medium before incubation in control differentiation medium or in differentiation medium simulating MA or RA. Cells were harvested for RNA at weeks 4, 5, and 6. Aliquots of RNA (20 µg) were electrophoresed, transferred to a single nylon membrane, and then hybridized to probes for MGP, OP, ON, and GAPDH. Data were acquired with phosphorimager beta -capture and normalized relative to GAPDH RNA content on filter. Data are expressed as percentage of signal on day 8 (%d8; predifferentiation) and are from a separate experiment than that shown in Fig. 2.

Data from four independent experiments, each measured at weeks 4, 5, and 6, were combined (n = 12 in each group) and analyzed (Fig. 4). With Ctl, MGP RNA accumulation increased ~18-fold over predifferentiation levels. Compared with Ctl, the accumulation was significantly less with either MA or RA (each P < 0.001 vs. Ctl), whereas accumulation with RA was less than with MA (P < 0.01). With Ctl, OP RNA accumulation increased approximately fourfold over predifferentiation levels. Compared with Ctl, the accumulation was significantly less with either MA or RA (each P < 0.01 vs. Ctl), and accumulation with RA was not different from that with MA. Levels of ON RNA did not increase from predifferentiation levels and were not significantly affected by either form of acidosis.


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Fig. 4.   Summary of Northern analysis data for MGP, OP, and ON RNA expression from 4 separate experiments. Cells were grown for 8 days in physiologically neutral pH medium before incubation in control differentiation medium or in differentiation medium simulating MA or RA. Cells were harvested for RNA at weeks 4, 5, and 6. Aliquots of RNA (20 µg) were electrophoresed, transferred to a single nylon membrane, and then hybridized to probes for MGP, OP, ON, and GAPDH. Data were acquired with phosphorimager beta -capture and normalized relative to GAPDH RNA content on filter. Data are expressed as percentage of signal on day 8 (predifferentiation) and are from a total of 4 separate, parallel experiments. Data are means ± SE; n = 12 in each group. * P < 0.01 vs. Ctl. + P < 0.01 vs. MA.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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 [HCO-3]. 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 HCO-3-buffered medium similar to that used for the bone cells in this study; however, the bone culture medium does not routinely include the beta -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 beta -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 [HCO-3]. 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 [HCO-3] 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.


    ACKNOWLEDGEMENTS

We thank Krystof J. Neumann for technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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