Effect of asymmetric dimethylarginine on osteoblastic differentiation.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) that accumulates in renal insufficiency and may be a uremic toxin. To determine whether ADMA inhibits bone metabolism, we investigated the in vitro effect of ADMA on osteoblastic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). METHODS: The effect of ADMA on nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit. The temporal sequence of osteoblastic differentiation in BMSCs was assessed in the presence and absence of ADMA by measuring alkaline phosphatase (ALP) activity, mineralization, and osteoblast gene expression at 0, 4, 8, 12 days of culture. RESULTS: ADMA (5, 50, 500 micromol. L-1) resulted in a dose-dependent decrease in nitrite formation in conditioned media of BMCS cultures, consistent with inhibition of NOS. ADMA treatment was associated with reduced ALP activity, calcium deposition and osteoblast-related gene expression in BMSCs cultures. Concurrent treatment with l-arginine (3600 micromol. L-1) reversed the ADMA (500 micromol. L-1)-mediated decrease in NO production, restored the differentiation potential of BMSCs, and significantly attenuated the down-regulation of Cbfa1 and osteocalcin gene expression by ADMA. CONCLUSIONS: ADMA inhibition of the NO-NOS pathway in BMSCs impairs osteoblastic differentiation of mouse BMSC cultures. These studies further support a role of NO in the local regulation of bone metabolism and the possibility that ADMA may act as uremic toxin on bone through its effect to inhibit NO actions in osteoblasts.