Tenofovir Causes Bone Loss via Decreased Bone Formation and Increased Bone Resorption, Which Can Be Counteracted by Dipyridamole in Mice

Abstract

Osteopenia and fragility fractures have been associated with human immunodeficiency virus (HIV) infection. Tenofovir, a common antiviral in HIV treatment, also leads to increases in bone catabolism markers and decreased BMD in children and young adults. In murine models and human cell lines, tenofovir inhibits adenosine triphosphate release and decreases extracellular adenosine levels. Adenosine and adenosine A2A receptor inhibit osteoclast formation, and increase local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptake and stimulates new bone formation as well as bone morphogenic protein 2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and questioned whether dipyridamole may be a useful treatment to counteract the deleterous bone effects of tenofovir. Primary murine osteoclasts were induced by M-CSF/RANKL, and the number of TRAP-positive-cells was studied after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were studied by RT-PCR and MAPK/NFkB expression by Western blot. Male C57Bl/6 mice were treated as follows: saline 0.9% (control), tenofovir 75 mg/kg/day, dipyridamole 25 mg/kg/day, combination tenofovir/dipyridamole (n = 10, 4 weeks). Calcein/Alizarin Red-labeling of newly formed bone was used, and long bones were prepared for micro-computed tomography (μCT)/histology. Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC50= 44.5nM) that was reversed by dipyridamole (IC50= 0.3 μM). Tenofovir increased cathepsin K and NFATc1 mRNA levels and dipyridamole reversed the effect. Dipyridamole reversed the effect of tenofovir on pERK1/2, pp38, and NFkB nuclear translocation. Mice treated with tenofovir lost nearly 10% of their body weight (p < 0.001). μCT revealed decreased BMD and altered trabecular bone in tenofovir-treated mice, reversed by dipyridamole. TRAP-staining showed increased osteoclasts in tenofovir-treated mice (p < 0.005), an effect reversed by dipyridamole. Similar results were obtained for cathepsin K and CD68. RANKL-positive cells were increased in tenofovir-treated mice, whereas osteoprotegerin-positive cells were decreased; both effects were reversed by dipyridamole. These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment. © 2019 American Society for Bone and Mineral Research.