Proceedings of The Physiological Society

University College London (2011) Proc Physiol Soc 24, PC20

Poster Communications

Disruption of clc-5 leads to a redistribution of annexin A2 in cells of the collecting duct and distal convoluted tubule of a Dent

R. Ngadze1, M. Harjanggi1, J. A. Sayer1,2, N. L. Simmons1, S. E. Guggino3, G. Carr1

1. Epithelial Research Group, Newcastle University, Newcastle, United Kingdom. 2. Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom. 3. Department of Medicine, Johns Hopkins University, Baltimore, Maryland, United States.

Mutations in CLCN5, encoding the voltage-dependent Cl-/H+antiporter, CLC-5, cause Dent’s disease (Type 1) characterised by low molecular weight proteinuria, hypercalciuria, and nephrolithiasis. Impairment of endocytosis occurs primarily in the proximal tubule, but CLC-5 is also expressed more distally in the collecting duct (CD) intercalated cells1. Expression of clc-5 occurs in several distal/collecting duct mouse cell lines such as mIMCD-3 and mpkDCT cells2,3. In mIMCD-3 cells, clc-5 ablation results in defective endocytosis and plasma membrane expression of the crystal adhesion molecule, annexin A2 together with crystal agglomeration3. Antisense clc-5 treatment of the mpkDCT cell model of the distal convoluted tubule (DCT) disrupts endocytosis2,4. Here we investigate the effect of clc-5 disruption on annexin A2 distribution in mpkDCT cells. We extend this to examine annexin A2 localisation in the CD and DCT of the Guggino clcn5 knockout mouse, a model that recapitulates the renal attributes of Dent's disease, including intra-tubular Ca2+-crystal deposition5. Using immunocytochemistry and confocal microscopy, annexin A2 distribution was determined in mpkDCT cells transfected with control GFP (transfection marker) and in cells where endogenous clc-5 was disrupted through cotransfection with antisense clc-5 and GFP2,3. Paraffin embedded sections of kidney tissue of wild type (WT) and clcn5 knockout (KO) mice (12 months, high citrate diet) were subjected to antigen retrieval with 0.8M urea and assessed for annexin A2 distribution. Annexin A2 was detected by a rabbit anti-annexin A2 antibody (H-50 raised against residues 1-50 of human protein, cross-reactive with mouse) at 1:100 (Santa Cruz). Goat anti-aquaporin 2 antibody (epitope mapping at C-terminus, human origin, cross-reactive with mouse, 1:250) and goat anti-calbindin antibody (epitope mapping at C-terminus of Calbindin D28K, human origin, cross-reactive with mouse,1:100) were used as markers of the CD and DCT respectively. Appropriate secondary antibodies were used. Control GFP transfected mpkDCT cells showed an intracellular perinuclear location for annexin A2 (n=6). Following transfection with antisense clc-5, there was a marked redistribution of cytoplasmic annexin A2 to the cell periphery (n=5). In the WT mouse, annexin A2 showed an intracellular, vesicular pattern in cell types within the DCT and CD. In the clcn5 knockout, annexin A2 relocalised to the apical (lumen) cell pole in cells within the DCT and CD. All experiments were carried out in material from 3 KO or WT animals. We speculate that abnormal expression of the crystal binding molecule annexin A2 at the cell surface, together with hypercalciuria, will facilitate intra-tubular Ca2+-crystal retention within the collecting ducts of KO animals.

Where applicable, experiments conform with Society ethical requirements