Georgina Carr1, John A Sayer, Nicholas L Simmons. 1. Epithelial Transport Research Group, Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, UK.
Abstract
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. Copyright (c) 2007 S. Karger AG, Basel.
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 murineank, 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 Met48ThrANK-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. Copyright (c) 2007 S. Karger AG, Basel.
Authors: Eva Morava; Jirko Kühnisch; Jefte M Drijvers; Joris H Robben; Cor Cremers; Petra van Setten; Amanda Branten; Sabine Stumpp; Alphons de Jong; Krysta Voesenek; Sascha Vermeer; Angelien Heister; Hedi L Claahsen-van der Grinten; Charles W O'Neill; Michèl A Willemsen; Dirk Lefeber; Peter M T Deen; Uwe Kornak; Hannie Kremer; Ron A Wevers Journal: J Clin Endocrinol Metab Date: 2010-10-13 Impact factor: 5.958