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Bushinsky, David A., Walter R. Parker, Kristen M. Alexander, and Nancy S. Krieger. Metabolic, but not respiratory, acidosis increases bone PG[E.sub.2] levels and calcium release. Am J Physiol Renal Physiol 281: F1058-F1066, 2001. First published July 12, 2001; 10.1152/ajprenal.00355.2001.-- A decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HC[O.sup.-.sub.3]; metabolic acidosis) or to an increase in P[CO.sub.2] (respiratory acidosis). In mammals, metabolic, but not respiratory, acidosis increases urine calcium excretion without altering intestinal calcium absorption, indicating that the additional urinary calcium is derived from bone. In cultured bone, chronic metabolic, but not respiratory, acidosis increases net calcium efflux ([J.sub.Ca]), decreases osteoblastic collagen synthesis, and increases osteoclastic bone resorption. Metabolic acidosis increases bone PG[E.sub.2] production, which is correlated with [J.sub.Ca], and inhibition of PG[E.sub.2] production inhibits this acid-induced [J.sub.Ca]. Given the marked differences in the osseous response to metabolic and respiratory acidosis, we hypothesized that incubation of neonatal mouse calvariae in medium simulating respiratory acidosis would not increase medium PG[E.sub.2] levels, as observed during metabolic acidosis. To test this hypothesis, we determined medium PG[E.sub.2] levels and [J.sub.Ca] from calvariae incubated at pH ~7.1 to model either metabolic (Met; [HC[O.sup.-.sub.3]] ~11 mM) or respiratory (Resp; P[CO.sub.2] ~83 Torr) acidosis, or at pH ~7.5 as a control (Ntl). We found that after 24-48 and 48-51 h in culture, periods when cell-mediated [J.sub.Ca] predominates, medium PG[E.sub.2] levels and [J.sub.Ca] were increased with Met, but not Resp, compared with Ntl, and there was a direct correlation between medium PG[E.sub.2] levels and [J.sub.Ca]. Thus metabolic, but not respiratory, acidosis induces the release of bone PG[E.sub.2], which mediates [J.sub.Ca] from bone. calvariae; osteoblasts; protons; pH Received 1 December 2000; accepted in final form 10 July 2001

The effects of reduced medium potassium concentration on the release of bone calcium was analyzed to characterize the mechanisms that mediate the induction of net calcium efflux by metabolic acidosis. Analysis of neonatal mouse calvariae that were cultured in a medium with low potassium concentrations indicated the presence of reduced osteoblastic collagen synthesis and increased levels of osteoclastic enzyme release. Furthermore, reduced medium potassium concentration increased net calcium efflux in the culture neonatal rat calvariae.

Parathyroid hormone-induced osteoclast cell differentiation in the primary cells of neonatal mouse was cultured in bovine cortical bone to evaluate the changes in bone ion composition during resorption. Osteoclast-induced bone resorption reduces the sodium to calcium ion ration by increasing the efflux of bone calcium. Underlying bone minerals were also exposed due to the reduction of calcium and potassium ion levels on the bone surface during osteoclast-induced resorption.

Hypercalciuric patients have been suggested to show increased intestinal calcium resorption that leads to enhanced bone resorption. In a model of human hypercalciuria using hypercalciuric Sprague-Dawley rats, the increase in intestinal calcium absorption enhanced bone resorption by acting directly or indirectly through an increase in intestinal 1,25(OH)2D3 receptors (VDR). Exogenous VDR stimulated calcium efflux from hypercalciuric rats by increasing urine calcium excretion.

Chronic metabolic acidosis stimulates cell-mediated net Ca2+ efflux from bone mediated by increased osteoblastic cyclooxygenase 2, leading to prostaglandin E2-induced stimulation of receptor activator of NF-κB ligand-induced osteoclastic bone resorption. Ovarian cancer G protein-coupled receptor-1 (OGR1), an osteoblastic H+-sensing G protein-coupled receptor, is activated by acidosis and leads to increased bone resorption. As regulator of G protein signaling (RGS) proteins limit GPCR signaling, we tested whether RGS proteins themselves are regulated by metabolic acidosis. Primary osteoblasts were isolated from neonatal mouse calvariae and incubated in physiological neutral or acidic (MET) medium. Cells were collected, and RNA was extracted for real-time PCR analysis with mRNA levels normalized to ribosomal protein L13a. RGS1, RGS2, RGS3, RGS4, RGS10, RGS11, and RGS18 mRNA did not differ between MET and neutral medium; however, by 30 min, MET decreased RGS16, which persisted for 60 min and 3 h. Incubation of osteoblasts with the OGR1 inhibitor CuCl2 inhibited the MET-induced increase in RGS16 mRNA. Gallein, a specific inhibitor of Gβγ signaling, was used to determine if downstream signaling by the βγ-subunit was critical for the response to acidosis. Gallein decreased net Ca2+ efflux from calvariae and cyclooxygenase 2 and receptor activator of NF-κB ligand gene expression from isolated osteoblasts. These results indicate that regulation of RGS16 plays an important role in modulating the response of the osteoblastic GPCR OGR1 to metabolic acidosis and subsequent stimulation of osteoclastic bone resorption. [ABSTRACT FROM AUTHOR]

Changes in bone formation in vitro due to aluminum were investigated. Experiments involved the utilization scanning electron microscopy, analytical measurements and statistical analysis on a model of isolated neonatal mouse calvarial cells. The inhibition of in vitro calcification and nodule formation by aluminum concentrations common to dialysis patients was demonstrated.

Krieger, Nancy S., Walter R. Parker, Kristen M. Alexander, and David A. Bushinsky. Prostaglandins regulate acid-induced cell-mediated bone resorption. Am J Physiol Renal Physiol 279: F1077-F1082, 2000.--Metabolic acidosis induces bone calcium efflux initially by physicochemical dissolution and subsequently by cell-mediated mechanisms involving inhibition of osteoblasts and stimulation of osteoclasts. In rat kidney, acidosis increases endogenous prostaglandin synthesis, and in bone, prostaglandins are important mediators of resorption. To test the hypothesis that acid-induced bone resorption is mediated by prostaglandins, we cultured neonatal mouse calvariae in neutral or physiologically acidic medium with or without 0.56 [micro]M indomethacin to inhibit prostaglandin synthesis. We measured net calcium efflux and medium [PGE.sub.2] levels. Compared with neutral pH medium, acid medium led to an increase in net calcium flux and [PGE.sub.2] levels after both 48 h and 51 h, a time at which acid-induced net calcium flux is predominantly cell mediated. Indomethacin inhibited the acid-induced increase in both net calcium flux and [PGE.sub.2]. Net calcium flux was correlated directly with medium [PGE.sub.2] (r = 0.879, n = 29, P [is less than] 0.001). Exogenous [PGE.sub.2], at a level similar to that found after acid incubation, induced net calcium flux in bones cultured in neutral medium. Acid medium also stimulated an increase in [PGE.sub.2] levels in isolated bone cells (principally osteoblasts), which was again inhibited by indomethacin. Thus acid-induced stimulation of cell-mediated bone resorption appears to be mediated by endogenous osteoblastic [PGE.sub.2] synthesis. metabolic acidosis; prostaglandin E; osteoblasts; calcium

Serum fibroblast growth factor 23 (FGF23) increases progressively in chronic kidney disease (CKD) and is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the factors regulating its production are not clear. Patients with CKD have decreased renal acid excretion leading to metabolic acidosis (MET). During MET, acid is buffered by bone with release of mineral calcium (Ca) and phosphate (P). MET increases intracellular Ca signaling and cyclooxygenase 2 (COX2)-induced prostaglandin production in the osteoblast, leading to decreased bone formation and increased bone resorption. We found that MET directly stimulates FGF23 in mouse bone organ cultures and primary osteoblasts. We hypothesized that MET increases FGF23 through similar pathways that lead to bone resorption. Neonatal mouse calvariae were incubated in neutral (NTL, pH = 7.44, PCO2 = 38 mmHg, [HCO3 ¯] = 27 mM) or acid (MET, pH = 7.18, PCO2 = 37 mmHg, [HCO3 ¯] = 13 mM) medium without or with 2-APB (50 μM), an inhibitor of intracellular Ca signaling or NS-398 (1 μM), an inhibitor of COX2. Each agent significantly inhibited MET stimulation of medium FGF23 protein and calvarial FGF23 RNA as well as bone resorption at 48 h. To exclude the potential contribution of MET-induced bone P release, we utilized primary calvarial osteoblasts. In these cells each agent inhibited MET stimulation of FGF23 RNA expression at 6 h. Thus stimulation of FGF23 by MET in mouse osteoblasts utilizes the same initial signaling pathways as MET-induced bone resorption. Therapeutic interventions directed toward correction of MET, especially in CKD, have the potential to not only prevent bone resorption but also lower FGF23 and perhaps decrease mortality. [ABSTRACT FROM AUTHOR]