Dent’s disease, characterised by proteinuria, hypercalciuria, and renal stone formation, results from mutation of the Cl^– channel CLC-5. We have previously demonstrated that mCLC-5 is expressed in inner medullary collecting duct cells (mIMCD-3 cell line) within acidic endosomes (Sayer et al. 2001). Here we report the consequence of ablation of mCLC-5 expression upon endocytosis of wheatgerm agglutin (WGA) and upon binding of Ca-oxalate crystals.

mIMCD-3 cells were transiently transfected with expression vector alone (control) (pCDNA3.1/CT-GFP, Invitrogen) or combined with vector containing sense full-length mCLC-5 (with C-terminal stop codon) or antisense mCLC-5, using Lipofectamine 2000 (Life Technologies). Positive transfectants were identified using a Leica confocal laser imaging microscope equipped (CLSM) with a Kr-Ar laser by their GFP fluorescence and analysed 24-48 h post-transfection. Binding and endocytosis of TRITC-wheatgerm agglutinin (WGA) was followed over a 1 h time course in phosphate-buffered saline (PBS). Optical sections of positive transfectants were collected to allow identification of membrane-bound and internalised WGA. In 3-5 separate experiments, after 1 h, internalisation of WGA occurred in 17/21 of control transfectants and 13/17 of sense mCLC-5 transfectants. With antisense mCLC-5 transfectants endocytosis was disrupted with the majority of cells showing only membrane-bound WGA; only 1/13 of cells showed WGA internalisation (P < 0.001 antisense vs. combined control/sense, Fisher’s exact test).

Calcium oxalate monohydrate crystals were grown in a high calcium (50 mM CaCl₂) medium exposed to diethyloxalate vapour. Crystals were harvested and overlaid onto mIMCD-3 cultures for 30 min prior to a brief wash in PBS and CLSM imaging of unfixed cells. In 4-5 separate experiments, in the majority of control or sense transfectants (34/44 and 38/44 cells, respectively), no crystal adhesion was observed. The remaining cells (5/44 or 3/44) showed adhesion of single crystals (< 10 mM size) or agglomerates (5/44 and 3/44 >10 mM size). For antisense mCLC-5 transfectants the majority of cells were associated (33/50, P < 0.001 vs. control/sense cells, x² test) with crystal agglomerates, whilst 1/50 showed adhesion of single crystals and 16/50 showed no crystal adhesion.

Transfection of antisense mCLC-5 is therefore associated with disruption of endocytosis and aggregation of agglomerates of Ca-oxalate crystals on inner medullary collecting duct cells. Crystal retention and agglomeration at the point of maximal urinary concentration are likely to be key factors in renal stone formation.

This work was supported by the NCKRF and the NKRF.