Date Published: July 17, 2017
Publisher: Public Library of Science
Author(s): Donatella Granchi, Elena Torreggiani, Annamaria Massa, Renata Caudarella, Gemma Di Pompo, Nicola Baldini, Sakamuri V. Reddy.
The extracellular acidic milieu in bones results in activation of osteoclasts (OC) and inhibition of osteoblasts (OB) causing a net loss of calcium from the skeleton and the deterioration of bone microarchitecture. Alkalinization through supplementation with potassium citrate (K citrate) has been proposed to limit the osteopenia progression, even though its pharmacological activity in bone microenvironment is not well defined. We evaluated if K citrate was able to prevent the adverse effects that acidic milieu induces on bone cells. OC and OB were maintained in neutral (pH 7.4) versus acidic (pH 6.9) culture medium, and treated with different K citrate concentrations. We evaluated the OC differentiation at seven days, by counting of multinucleated cells expressing tartrate-resistant acid phosphatase, and the activity of mature OC at 14 days, by quantifying of collagen degradation. To evaluate the effects on OB, we analyzed proliferation, mineralization, and expression of bone-related genes. We found that the low pH increased OC differentiation and activity and decreased OB function. The osteoclastogenesis was also promoted by RANKL concentrations ineffective at pH 7.4. Non-cytotoxic K citrate concentrations were not sufficient to steadily neutralize the acidic medium, but a) inhibited the osteoclastogenesis, the collagen degradation, and the expression of genes involved in RANKL-mediated OC differentiation, b) enhanced OB proliferation and alkaline phosphatase expression, whereas it did not affect the in vitro mineralization, and c) were effective also in OC cultures resistant to alendronate, i.e. the positive control of osteoclastogenesis inhibition. In conclusion, K citrate prevents the increase in OC activity induced by the acidic microenvironment, and the effect does not depend exclusively on its alkalizing capacity. These data provide the biological basis for the use of K citrate in preventing the osteopenia progression resulting from low-grade acidosis.
The deterioration of bone microarchitecture is a common event in postmenopausal women with the consequent decrease in bone mass and increased susceptibility to fracture . The bone loss starts early and continues for many years depending on many causes, all noxious to the skeletal homeostasis since impair equilibrium between the removal of old bone by osteoclasts (OC) and the formation of bone matrix by osteoblasts (OB) . An imbalance between these processes, due to enhanced osteoclastogenesis or excessive OC activity, is implicated in several conditions characterized by the loss of bone mass and compromised bone strength, including osteopenia and osteoporosis . It is well recognized that skeletal homeostasis influences a variety of physiological functions, but the regulation of acid-base balance is undoubtedly one of the most important . The skeleton is an alkaline reservoir that is able to buffer systemic acidosis by modifying the composition of the bone mineral matrix, namely hydroxyapatite . In physiological conditions, the pH values of venous and arterial blood fluctuate from 7.36 to 7.40, respectively . Postmenopausal estrogen deficiency , diets rich in salt and meat proteins , the decreased renal function due to aging , are some of the conditions in which a high acid loading may exceed the physiological neutralization capacity, eventually leading to a latent or low-grade acidosis . The pH decrease can also arise locally in the bone tissue. Normal intra-bone pH has been determined in blood samples from experimental models and differs from metaphysis to bone marrow, i.e. from 7.3 to 7.4 , but lower values are expected in the interstitial fluid around bone cells . Even though bone is a well-perfused tissue, the amount of blood supply tends to decline with age, thus promoting an hypoxic status . The poor vascularization leads to an increase in CO2 and an excess of protons derived from cellular metabolism that accumulates in the tissue and decreases the interstitial pH .
The excessive acid load is physiologically balanced through metabolic adaptation that involves primarily kidney, lung, and bone . In bone tissue, the acidic microenvironment affects bone cells and favors the resorption of the mineralized matrix in order to recruit hydroxyl groups that neutralize the proton excess when the capacity of other buffer systems is limited . However, if the acidic overload persists, the bone becomes osteopenic due to the net loss of calcium and deterioration of microarchitecture. The oral administration of alkali compounds, i.e. K citrate in doses ranging from 30 to 90 mmol/day, has been proposed for opposing the low-grade acidosis and preventing bone loss [15,16,18–22]. Although preliminary clinical data are encouraging, it is still unclear whether the beneficial effects are exclusively due to the alkalizing function of K citrate, or if it directly influences bone cell activity. In this study, we aimed to investigate if K citrate is able to counteract the adverse effects induced by acidosis regarding the resorption activity of OC and the mineralization capability of OB.
In summary, this study has been planned to highlight the K citrate activity on biological epiphenomena regarding bone cells, i.e. the formation of new OC, the mineralized matrix resorption, and the mineralized matrix deposition. For the first time, we showed that K citrate inhibits osteoclastogenesis and enhance the anti-osteoclastogenic activity of alendronate, and that such effect does not depend exclusively on its alkalizing properties. Our results provide a biological basis for the use of K citrate in preventing the bone loss that occurs in low-grade acidosis, and offer a number of suggestions to explore deeply the anti-osteoclastogenic role of K citrate in clinical setting. Finally, the anti-resorptive properties of K citrate have been demonstrated also at low doses, but clinical studies are needed to define dosage schedule, timing and duration of supplementation, and to determine the effective benefits of this strategy in preventing the osteopenia progression.