Osteoclast response to low extracellular sodium and the mechanism of hyponatremia-induced bone loss

Abstract

Our recent animal and human studies revealed that chronic hyponatremia is a previously unrecognized cause of osteoporosis that is associated with increased osteoclast numbers in a rat model of the human disease of the syndrome of inappropriate antidiuretic hormone secretion (SIADH). We used cellular and molecular approaches to demonstrate that sustained low extracellular sodium ion concentrations ([Na(+)]) directly stimulate osteoclastogenesis and resorptive activity and to explore the mechanisms underlying this effect. Assays on murine preosteoclastic RAW 264.7 cells and on primary bone marrow monocytes both indicated that lowering the medium [Na(+)] dose-dependently increased osteoclast formation and resorptive activity. Low [Na(+)], rather than low osmolality, triggered these effects. Chronic reduction of [Na(+)] dose-dependently decreased intracellular calcium without depleting endoplasmic reticulum calcium stores. Moreover, we found that reduction of [Na(+)] dose-dependently decreased cellular uptake of radiolabeled ascorbic acid, and reduction of ascorbic acid in the culture medium mimicked the osteoclastogenic effect of low [Na(+)]. We also detected downstream effects of reduced ascorbic acid uptake, namely evidence of hyponatremia-induced oxidative stress. This was manifested by increased intracellular free oxygen radical accumulation and proportional changes in protein expression and phosphorylation, as indicated by Western blot analysis from cellular extracts and by increased serum 8-hydroxy-2'-deoxyguanosine levels in vivo in rats. Our results therefore reveal novel sodium signaling mechanisms in osteoclasts that may serve to mobilize sodium from bone stores during prolonged hyponatremia, thereby leading to a resorptive osteoporosis in patients with SIADH.

Figures

FIGURE 1.

Lowering [Na+] proportionally stimulates osteoclastogenesis. A, murine RAW 264.7 monocytic cells were grown in medium with the indicated [Na+] and treated for 7 days with recombinant RANKL (50 ng/ml) and M-CSF (10 ng/ml) to induce osteoclast differentiation. After fixation, cells were stained for TRAP activity, and quantification of TRAP-positive multinucleated cells was performed in triplicate wells. The graph (top) shows that lowering the [Na+] in the medium dose-dependently increased osteoclast formation. This direct effect was significant even with mild changes in [Na+] (e.g. 129 mmol/liter), a concentration that is associated with no symptoms in most patients. Data represent means ± S.E. (error bars). *, p < 0.01 comparing samples with one-step lower [Na+]. Representative images of TRAP-positive osteoclast cultures at medium [Na+] = 136 mmol/liter and medium [Na+] = 112 mmol/liter show marked differences of TRAP-positive cell density. Scale bars, 50 μm. B, RAW 264.7 cells were adapted to grow in media with the desired [Na+]. Equal numbers of cells were transferred to 96-well plates in triplicates. Cell viability was measured using a LIVE/DEAD cell kit. Calcein fluorescence emission intensities are expressed as percentage of control (emission from cells killed by incubation with digitonin according to the manufacturer's instructions). Data are expressed as mean ± S.E. No statistically significant differences were detected in viability of cells upon lowering [Na+]. C, BMMs were prepared from femora of four Sprague-Dawley rats, and monocytes were enriched by Ficoll gradient centrifugation, plated into 12-well culture dishes, adapted, and differentiated in media with target [Na+], as depicted on the graph. After 14 days, cells were fixed and stained for TRAP and counted. Shown are representative images from cells grown in medium with [Na+] = 136 mmol/liter and [Na+] = 117 mmol/liter; TRAP activity appears as a dark brown signal. Scale bars, 10 μm. Data are expressed as means ± S.E. from triplicates. *, p < 0.01; **, p < 0.001 comparing samples with one-step lower [Na+]. D, BMMs from hyponatremic and normonatremic rats were differentiated into osteoclasts while maintaining normal or low [Na+] as appropriate. Both the [Na+] = 117 mmol/liter and the [Na+] = 136 mmol/liter media were supplemented with M-CSF and RANKL. Images from representative 10-cm culture plates and the graph indicate that low [Na+] promoted formation of more TRAP+ colonies than normal [Na+]. Data are means ± S.E.; *, p < 0.001.

FIGURE 2.

Lowering [Na+] stimulates resorptive activities of RAW 264.7 and bone marrow-derived osteoclastic cells. A, murine RAW 264.7 monocytic cells were grown in medium with the indicated [Na+] concentration in 24-well OAAS plates for 14 days with recombinant RANKL (50 ng/ml) and M-CSF (10 ng/ml). At the termination of the experiment, osteoclasts were removed, and the remaining calcium hydroxyapatite was stained using 5% silver nitrate under intense light exposure (dark). Resorbed surfaces (unstained areas) were assessed in triplicate wells, and the results show that the areas were inversely proportional to [Na+] in the extracellular medium. Data represent the means ± S.E. (error bars). *, p < 0.01 comparing samples with one-step higher [Na+]. Representative photomicrographs showing enlarged resorbed areas (gray) in wells where osteoclasts were cultured with normal or low [Na+] media. Scale bars, 50 μm. B, bone marrow macrophages were grown in differentiation media of decreasing [Na+], supplemented with M-CSF and RANKL for 5 days, and then overlaid onto whale dentin slices to differentiate another 9 days. Osteoclasts were removed, and dentin slices were stained with 1% toluidine blue to visualize resorption areas. The graph shows that resorption areas increased proportionally to lowered [Na+]. Average resorbed areas are expressed as a percentage of the total dentin area of two discs from each [Na+]. Data are mean ± S.D. *, p < 0.01; **, p < 0.001, compared with control samples ([Na+] = 136 mmol/liter). Representative images depicting [Na+]-dependent difference in resorbed areas (blue). Scale bars, 500 μm.

FIGURE 3.

Hyponatremia signaling associated with increased osteoclastogenesis. A, to test if osteoclastogenic effect of hyponatremia is related to sensing changes of osmotic pressure and cell swelling or to sensing changes in extracellular [Na+], RAW 264.7 cells were grown in medium with normal [Na+] (136 mmol/liter), in medium with both low [Na+] (112 mmol/liter) and uncorrected low osmolality (237 mosmol/kg H2O), or in medium with low [Na+] (112 mmol/liter) and corrected normal osmolality (290 mosmol/kg H2O) during differentiation. Cells were fixed and stained for TRAP activity, and TRAP+ multinucleated cells were counted. Data are mean ± S.E. (error bars) of triplicate samples. **, p < 0.001 compared with osteoclast counts in normonatremic samples. B, 2 × 106 RAW 264.7 cells adapted to grow in media with the indicated [Na+] were seeded into 96-well plates in triplicates and grown overnight. Cytosolic free [Ca2+] was measured using the Fluo-4 kit from Invitrogen, and calcium calibration was done using Fluo4 pentapotassium salt and standards from Invitrogen. Results show [Na+]-dependent decreases of cytosolic [Ca2+]. Data are mean ± S.E. (error bars). *, p < 0.01; **, p < 0.001 compared with [Ca2+] in control samples grown with medium [Na+] = 136 mmol/liter. C, RAW 264.7 cells grown in media with graded concentrations of [Na+] were subcultured into 24-well plates and then incubated with [14C]ascorbic acid for 30 min. After several washings with buffer containing unlabeled ascorbic acid, cells were solubilized, and radioactivity was counted. Values were corrected for protein content. Data represent means ± S.E. (error bars). *, p < 0.001. Experiments were in triplicate wells and repeated three times. The results show that lowering [Na+] in the media dose-dependently reduced ascorbic acid uptake into RAW 264.7 cells. D, RAW 264.7 cells were grown in minimal essential medium α without ascorbic acid or in the same medium supplemented with 50 mg/liter l-ascorbic acid and recombinant RANKL and M-CSF. TRAP-positive multinucleated cells were quantified after 7 days. Results show that the removal of ascorbic acid from the growth medium stimulates osteoclast formation. Similarly, cells were grown in OAAS resorption plates in media without and with ascorbic acid for 14 days, resorbed areas were quantified from triplicate wells, and the experiment was repeated three times. Data are means ± S.E. (error bars). *, p < 0.01. Results show that removal of ascorbic acid from the growth medium stimulates resorbing activity of RAW 264.7 osteoclastic cells.

FIGURE 4.

Lowering [Na+] increases accumulation of reactive oxygen species and elicits oxidative stress response in cellular and animal models of hyponatremia. A, RAW 264.7 cells were grown in media with the indicated [Na+] and normalized osmolality; cells were subcultured into 96-well plates and the next day were incubated with DCF with and without H2O2 for 30 min and the Hoescht 33342 dye for the last 5 min. DCF fluorescence was measured using a fluorometer plate reader (Tecan US Inc., model Ultra384) and corrected with values of Hoechst fluorescence (DNA content). Data represent means ± S.E. (error bars). *, p < 0.01. Results show that lowering the [Na+] concentration in the media increased base-line and peroxide-induced accumulation of reactive oxygen species, an indicator of oxidative stress. B, whole cell lysates were generated from RAW 264.7 cells grown for 48 h in media with the specified [Na+] without the correction of osmolality and probed for the abundance of key signaling molecules in multiple osteoclastogenesis pathways as described under “Experimental Procedures.” β-Actin was used to confirm equal protein loading. Blots on the left indicate no change in three of five key RANKL-induced pathways, whereas blots on the right indicate that lowering extracellular [Na+] proportionally increases the phosphorylation of key proteins in the Akt and NF-κB pathways common to both RANKL and ROS signaling. The abundance of additional key molecules belonging to the oxidative stress signaling cascade was proportionally increased by lowering [Na+]. C, sera from hyponatremic and normonatremic rats were collected as described under “Experimental Procedures.” 8-OHdG concentrations were determined in duplicates using an ELISA kit. Data represent means ± S.E. (error bars). **, p < 0.01.