Background/Purpose:  Human Immunodeficiency Virus (HIV) infection devastates the immune system but also affects tissues and organs such as kidney, liver, central nervous system, heart and bone. Bone alterations have been observed in HIV disease for nearly two decades, in particular a higher risk of low bone mineral density (BMD) and fragility fractures. Treatment of patients with tenofovir alone or in combination (as part of HAART), leads to changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylated; tenofovir-phosphate inhibits HIV-reverse transcriptase by mimicking AMP. We have recently found that tenofovir inhibits Pannexin-1/Connexin-43-mediated ATP release from cells and decreases extracellular adenosine levels and fibrosis in murine models. As adenosine and ATP are key regulators of bone homeostasis, we determined whether tenofovir directly affects bone by an adenosine- or ATP-dependent mechanism.

Methods:  M-CSF/RANKL-induced osteoclast (OC) and stimulated osteoblast (OB) differentiation were studied in primary murine bone marrow culture as the number of TRAP-positive or Alizarin Red-positive cells, respectively, after challenge with tenofovir (1nM-100mM) alone or in combination with dipyridamole (1nM-100mM), an agent that increases extracellular adenosine by blocking cellular adenosine uptake. Pannexin-1 and Connexin-43 expression were permanently knocked down by lentiviral infection with appropriate shRNA or scrambled shRNA and these cells were induced to differentiate into OC by RANKL. Male C57Bl/6 (WT), A2AKO and A2BKO mice received tenofovir 75mg/Kg/day for 4 weeks. Double labelling of bone with calcein (15mg/Kg)/Alizarin Red (30mg/Kg) was performed and long bones prepared for mCT and histology.

Results:  There was a dose-dependent increase in OC differentiation after treatment with Tenofovir (EC50=44.5nM), that was reversed by dipyridamole (IC50=0.3µM). Moreover, tenofovir inhibited OB differentiation (IC50=0.4µM) which was also reversed by dipyridamole (EC50=10nM). When both Pannexin-1 and Connexin-43 were absent, tenofovir did not increase OC number. Tenofovir treatment reduced bone formation in WT-mice (49±8µm vs 110±7µm untreated p<0.0005) but not in A2AKO (72±6µm vs 71±5µm untreated, p=ns) and A2BKO mice (64±8µm vs 86±9µm untreated, p=ns). mCT revealed decreased BMD and both cortical and trabecular bones were affected. TRAP-staining showed increased OCs in vivo in tenofovir-treated WT mice (21±1 vs 16±1 OC/hpf in untreated, p<0.005). There are increased osteoclasts in A2AKO mice and this was unaffected by tenofovir treatment (24±1 OC/hpf, vs 22±1 OC/hpf in untreated p=ns) and osteoclast number in A2BKO mice was unchanged (19±1 OC/hpf, vs 18±1 OC/hpf in untreated, p=ns). Similar results were obtained for Cathepsin K.

Conclusion:  These results indicate that tenofovir enhances osteoclast differentiation and inhibits osteoblast differentiation by an adenosine-dependent mechanism and suggests that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment.

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/tenofovir-a-nucleoside-analog-reverse-transcriptase-inhibitor-for-treatment-of-hiv-promotes-osteoclast-differentiation-and-decreases-osteoblast-formation-by-a-mechanism-depending-on-atp-release-and/