We have previously identified expression of the pyrophosphate transporter ANK in murine kidney (Carr et al, 2007). ANK is expressed in the cortical collecting duct in both principal and intercalated cells. Since pyrophosphate is a potent inhibitor of calcium crystal formation and since nephrolithiasis is most likely to occur in concentrated supersaturated urine, we hypothesised that control of ANK protein expression and function may be controlled by vasopressin. Inactivating mutations in the gene for the mouse ANK protein not only increase calcium deposition in joints, causing arthritis, but mutant ANK mice also exhibit renal calcification (Ho et al, 2000). RT-PCR confirmed ANKH expression in whole mouse kidney and two murine cortical collecting duct cell-lines, M1 murine epithelial cells and mpkCCDcl4 murine cortical collecting duct cells. mpkCCDcl4 cells were chosen for further study since they had previously been shown to respond to arginine vasopressin (AVP) treatment by increased expression of aquaporin 2 (AQP2) (Hasler et al, 2002). Confluent monolayers of mpkCCDcl4 cells were grown on Transwell filter inserts (Costar) in supplemented DMEM/F12 media for 6-10 days. By day 6 a significant transepithelial resistance (2.9 ± 0.3 kΩcm², n=140) and spontaneous p.d. (6-13 mV basal solution electropositive) were observed that was associated with transepithelial Na⁺ absorption. Incubations with vasopressin (10nM) were made from the basal epithelial surface for 24 h. Monolayers were then fixed for immunocytochemistry by methanol immersion (0^oC 15 min) and stored at 4^oC prior to block with horse serum and then rabbit anti-ANKH antibody (1 in 250, Ab3, Ho et al, 2000) and goat anti-AQP2 (1 in 250, Santa-Cruz). Following donkey serum incubation as a secondary block cells were incubated with donkey anti-rabbit 488 (Alexa Fluor, Molecular Probes), or donkey anti-goat 568. In the absence of vasopressin, ANK was localised both at the lateral and apical plasma membranes and within cytoplasmic vesicles, AQP2 staining was negative. After vasopressin treatment, a proportion of cells (6.6%) showed marked up-regulation of AQP2 with strong apical staining. In addition a proportion (1.8%) showed a markedly increased expression of ANK at the apical plasma membrane. The AQP2 positive cell population was distinct from the apical ANK population. Similar data were obtained after 24h treatment with 10μM forskolin. We conclude that ANK expression at the apical plasma membrane of mpkCCDcl4 cells is regulated by vasopressin via a cAMP-dependent pathway. ANK mediated pyrophosphate delivery to urine may be increased during urinary concentration helping to inhibit calcium crystal formation.