Uremic Toxin Indoxyl Sulfate Increases Osteocyte Wnt Inhibitor Signaling and Decreases RANKL and Mineralization, Effects Negated by PTH

Background: In CKD, fractures can occur at both high and low PTH levels, indicating non-PTH mediated pathways. We hypothesized that uremic toxin indoxyl sulfate (IS), a potent ligand for the aryl hydrocarbon receptor (AhR), may be a factor in low bone turnover when PTH is not elevated. Methods: We cultured IDG-SW3 osteocytes that differentiate from osteoblasts to early osteocytes (day 14) and mature osteocytes (day 35). We examined the effect of IS in short term 24 hour exposure, long term (14 or 35 day) exposure, and with and without PTH or CH223191 (an inhibitor of AhR nuclear translocation). We assessed osteocyte gene expression by RT-qPCR, alkaline phosphatase (ALP) activity, and mineralization. AhR canonical signaling was assessed by upregulation of the genes CYP1A1, CYP1B1, and AhRR. Results: Short term exposure to IS for 24 hours dose dependently increased expression of the Wnt inhibitors SOST, Dkk1, and decreased RANKL in both early and mature osteocytes. IS dose dependently increased canonical AhR activation of CYP genes. Long term incubation of IS in early (IS for 14 days) and mature (IS for 35 days) osteocytes led to similar upregulation of Wnt inhibitors and decreased RANKL expression, ALP, and mineralization. CH223191 reduced AhR canonical CYP gene activation and reversed the IS inhibition of mineralization and alkaline phosphatase activity but did not reverse the IS induced expression SOST, Dkk1, or RANKL in osteocytes. Co-culture with both PTH and IS reversed the IS induced upregulation of SOST, Dkk1, and suppression of RANKL. In both early and mature osteocytes, AhR CYP genes were activated by IS but not PTH, but there was an additive effect of AhR CYP gene expression in the presence of both IS and PTH. Conclusion: In summary, IS in the absence of PTH, activated canonical and non-canonical AhR signaling to increase osteocyte Wnt inhibitor signaling and decrease RANKL osteoclast activation and mineralization, which would translate to low remodeling. However, in the presence of PTH, these effects were reversed, suggesting IS is likely an important factor in the low turnover bone disease observed in CKD. Funding: Commercial Support - Dialysis Clinic, Inc.