Expression and localisation of the pyrophosphate transporter, ANK, in murine kidney cells.
OBJECTIVE
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.


CONCLUSIONS
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.
Carr
Georgina
G
Epithelial Transport Research Group, Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, UK.
Sayer
John A
JA
Simmons
Nicholas L
NL
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D007668:Kidney; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D028061:Phosphate Transport Proteins; C410982:ank protein, mouse