Thiazide diuretics inhibit the activity of the thiazide-sensitive sodium chloride co-transporter (NCC) in the kidney (Ellison et al. 1987). Thiazide therapy also increases bone mineral density and decreases fracture rate. Although the mechanism remains unknown, there is evidence that thiazides may have a direct effect on the bone tissue and recent studies have demonstrated the presence of NCC-related transcripts in a rat osteogenic cell line. However, the distribution of the protein in bone remains to be established. This study aims to provide immunohistochemical evidence for the presence of NCC in biochemically intact cryosections of rat bone and biopsies of human mandible. Informed consent was obtained from all the patients. Animals were killed humanely. Undecalcified, 9 µm thick sections from freshly frozen adult rat femora (n = 4) and freshly frozen human mandible (n = 2) were prepared using polyvinylpyrrolidone supporting films and a heavy-duty cryomicrotome. The sections were fixed in cold buffered formalin, briefly decalcified in EDTA and NCC-specific immunoreactivity was assessed using affinity-purified polyclonal antibodies raised against a 110 amino acid segment from the amino terminus of rat NCC. FITC-conjugated anti-rabbit immunoglobulins were used as secondary antibodies. For negative controls, primary antibodies were omitted and irrelevant antibodies against non-bone proteins were used. Comparisons were made with known distributions of the bone proteins osteopontin and bone sialoprotein. Our results show that there is clear NCC-specific immunofluorescence in bone. Staining was present in a characteristic punctate pattern in bone cells and was prominent in the osteocytes. In the extracellular matrix of mature lamellar bone, strong immunofluorescence was evident in the region of reversal lines and resorption cavities, while more general distribution was present in the matrix of primary woven bone. All control sections were unstained. This pattern of expression was confirmed in decalcified paraffin embedded sections of rat femur.

We conclude that NCC is present in the cells and extracellular matrix of bone. The extracellular distribution of the protein suggests that it is central to the reversal event and hence bone modelling and remodelling. Further studies characterising the role of NCC in bone metabolism and bone disease are in progress.

This work was funded by the MRC.