Expression and Localisation of the Pyrophosphate Transporter, ANK, in Murine Kidney Cells
Background/Aims: Mutation of the pyrophosphate transporter, ANK, results in progressive arthritis in mice. ANK is expressed in non-skeletal tissues including kidney. The aim was therefore to investigate ANK location at the cellular and subcellular level in renal cells. Methods: RT-PCR identified a murine cell-line, mIMCD3, expressing ANK. The intra-renal distribution of ANK was determined by immunohistochemistry and the subcellular distribution in mIMCD3 cells by transfection of an ANK-NT-GFP fusion protein. Furthermore, an inactivating mutation of murine ank, Glu440X, and a gain of function mutation, Met48Thr, were tested to determine whether membrane traffic contributed to a transport defect. Results: ANK is expressed in cells of the cortical collecting duct, as assessed by colocalisation with aquaporin 2 and at the lateral and apical plasma membranes of mIMCD-3 epithelial cells, as assessed by colocalisation with wheat germ agglutinin lectin (WGA). ANK-NT-GFP was also present in endoplasmic reticulum, Golgi, acidic endosomes and mitochondria. mIMCD3 expression of Glu440X ANK-NT-GFP shows evidence of Golgi retention whereas Met48Thr ANK-NT-GFP is unaltered at the plasma membrane compared to wild type. Conclusion: The intra-renal and subcellular localisation of ANK is consistent with pyrophosphate export from collecting duct cells and supports a role for ANK in limiting intra-renal calcium-crystal formation.