Endocrine Abstracts (2013) 31 P7

Alterations of CLC-5 expression, function and trafficking in Dent's disease

Caroline Gorvin1, Martijn Wilmer2, Sian Piret1, Brian Harding1, Lambertus van den Heuvel3,4, Parmjit Jat5, Jonathan Lippiat6, Elena Levtchenko3,4 & Rajesh Thakker1


1Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Oxford, UK; 2Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Sciences, Nijmegen, The Netherlands; 3Laboratory of Genetic, Endocrine and Metabolic Disorders, Department of Paediatric Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 4Department of Development and Regeneration, Catholic University Leuven, Leuven, Belgium; 5Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK; 6Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.


Dent’s disease, due to mutations in the chloride/proton antiporter, CLC-5, represents one form of familial hypophosphataemic rickets. Dent’s disease patients also have: low-molecular-weight-proteinuria; hypercalciuria with nephrolithiasis and renal failure; and urinary loss of parathyroid hormone and vitamin D-binding protein, due to defective receptor-mediated endocytosis within the renal proximal tubule. However, there is variability in these clinical phenotypes such that only 25% of patients have rickets, while ~40% have phosphaturia, and we have previously demonstrated that this is also affected in the cellular phenotypes of human conditionally-immortalised proximal tubular epithelial cell-lines (ciPTECs) established from the urines of patients harbouring CLC-5 mutations. To further elucidate the mechanisms underlying the differences in the cell phenotypes and their structural-functional relationships we studied two ciPTECs harbouring CLC-5 mutations in different intracellular domains: an in-frame insertion (30:insHis) in the N-terminus that lies in a highly charged region; and a nonsense mutation (Arg637Stop) in the C-terminus, that causes loss of a region of CLC-5 known to interact with endocytic accessory proteins. Heterologous expression and whole-cell patch-clamp recordings within HEK293 cells revealed CLC-5 mutations to cause a reduction in chloride currents, to ~70% of wild-type for 30:insHis-CLC-5 (n=10), and ~30% of wild-type for Arg637Stop-CLC-5 (n=6) (P<0.05 in both). Confocal microscopy of ciPTECs demonstrated that 30:insHis-CLC-5 had reduced cell surface expression, while the Arg637Stop-CLC-5 was predominantly intracellular. Thus, the Arg637Stop-CLC-5 mutation may disturb interactions with endocytic proteins, thereby disrupting the trafficking of CLC-5 to the cell surface, and reducing the overall chloride conductance of the cell; whilst the 30:insHis-CLC-5 mutation which alters the amino acid charge balance of the N-terminus, likely affects chloride conductance, thereby impairing endosomal acidification and receptor-mediated endocytosis. These studies further demonstrate that different intracellular mechanisms give rise to Dent’s disease and these may account for the differences in patient phenotypes.

Declaration of funding: Yes.

Details: This work was supported by the Medical Research Council (Grants G9825289 and G1000467), Kidney Research UK, EuFP6-EuReGene, (05085) and the Fund for Scientific Research Flanders (Fundamental Clinical Investigatorship, 1801110N).

DOI: 10.1530/endoabs.31.P7

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