SFEBES2009 Poster Presentations Bone (25 abstracts)
Transient receptor potential cation channel, subfamily Vanilloid, member 5 (Trpv5) mutation (Ser682Pro) results in loss of apical membrane expression in the distal convoluted tubule, thereby resulting in hypercalciuria
Nellie Y Loh 1 , Henrik Dimke 2 , Liz Bentley 3 , Paolo Tammaro 4 , Tertius Hough 5 , Roger D Cox 3 , Steve D M Brown 3 , Frances M Ashcroft 4 , Joost Hoenderop 2 , René Bindels 2 & Rajesh V Thakker 1
1Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 2Centrale Ontvangst Goederen, UMC St Radboud, Geert Grooteplein Zuid 30, 6525 GA, Nijmegen, The Netherlands; 3MRC Mammalian Genetics Unit, Medical Research Council, Harwell, UK; 4Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; 5Mary Lyon Centre, Medical Research Council, Harwell, UK.
Transient receptor potential cation channel, subfamily Vanilloid, member 5 (TRPV5) is a member of the TRP superfamily. TRPV5, which functions as a tetramer, is localized to apical membranes of distal convoluted tubules (DCT) and connecting tubules (CNT) of the kidney, and is involved in vitamin D-regulated calcium reabsorption. Mice with a targeted deletion of Trpv5 (Trpv5−/−) develop severe hypercalciuria, compensatory hyperabsorption of dietary calcium and osteopaenia. We recently identified an N-ethyl-N-nitrosourea-derived mouse model with autosomal dominant hypercalciuria, Hcalc1, due to a Trpv5 missense mutation, Ser682Pro. To investigate the molecular/functional consequences of this mutation, we assessed wild-type and mutant Trpv5 function in vitro by electrophysiology and examined kidneys from wild-type (Trpv5+/+), heterozygous (Trpv5+/682P) and homozygous (Trpv5682P/682P) mutant mice by immunofluorescence and western blot analyses. Mice were kept in accordance with UK Home Office welfare guidelines and project licence restrictions. Whole-cell patch-clamp analysis of wild-type and mutant Trpv5 channels in transiently transfected human embryonic kidney (HEK) 293 cells showed calcium currents of similar amplitudes (at −80 mV, wild-type: −915.0±82.26 pA/pF, n=7; mutant: −1000.0±87.33 pA/pF, n=10). However, immunofluorescence studies using antibodies to Trpv5 and markers to the DCT (anti-sodium/chloride cotransporter) and CNT (anti-aquaporin 2), revealed a dramatic loss/reduction of Trpv5 expression from apical membranes of the DCT in the kidneys of the mutant mice. Furthermore, semi-quantitative immunofluorescence and western blot analyses revealed a ~50–90% reduction in expression of the intracellular calcium binding protein, calbindin-D28K, in Trpv5682P/682P kidneys. Similar downregulation of calbindin-D28K abundance has also been observed in Trpv5 and Klotho deficient mice, both serve as models of impaired Trpv5 transport. Thus, these findings show that the Ser682Pro Trpv5 mutation identified in the Hcalc1 mouse model is associated with a loss of apical membrane expression in the DCT, and this likely contributes to the defect in renal calcium reabsorption in the Hcalc1 mutant mice.
Article tools
My recent searches
My recently viewed abstracts
Authors
Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
© Bioscientifica 2024 |
Privacy policy |
Cookie settings
BiosciAbstracts
Bioscientifica Abstracts is the gateway to a series of products that provide a permanent, citable record of abstracts for biomedical and life science conferences.
Find out more