Mutations in CLCN5, encoding the voltage-dependent Cl^–/H⁺antiporter, CLC-5, cause Dent’s disease by impairment of endocytosis mainly in the proximal nephron. Dent’s is characterized by low-MW proteinuria, hypercalciuria, and nephrolithiasis. ClC-5 is expressed in acidic endosomes in collecting duct cells (mIMCD-3) where clc-5 ablation results in defective endocytosis and accumulation of annexin A2 at the membrane (Carr et al, 2006). Annexin A2 is implicated in the regulated expression of apical membrane channels TRPV5/6 (Van de Graaf et al, 2003). We have used mpkDCT cells as an appropriate model of a Ca²⁺-transporting cell-type since they express TRPV5/6 (Diepens et al, 2004) to investigate a possible role for CLC in control of endocytic membrane retrieval in DCT cells. RT-PCR confirmed expression of clc-5 mRNA in mpkDCT cells. Protein expression of clc-5 and trpv5 was demonstrated by immunocytochemistry and confocal microscopy using PEP5E1 antibody and anti-trpv5 (Santa Cruz). Confocal microscopy of clc-5-NT-GFP transfected mpkDCT cells with a 2 minute incubation with TRITC conjugated wheat germ agglutinin (WGA) lectin as a membrane marker revealed an intracellular location for clc-5-NT-GFP. LysoTracker Red (acidic endosome marker) or cotransfection with clc-5-CT-RFP and endosomal marker pEGFP-Endo showed substantial colocalisation of clc-5 with these markers. Using green fluorescent protein (GFP) as a transfection marker, internalisation of WGA lectin over 1h, as an endocytic marker, was determined in control GFP transfected cells and in cells where endogenous clc-5 was disrupted through cotransfection with antisense clc-5 and GFP (Carr et al, 2006). Endocytosis of WGA lectin in cells transfected with antisense clc-5 (n=18) was reduced in comparison to control cells (n=22) (quantification performed in mid-cell xy confocal sections, internalisation measured as a proportion of whole cell area, expressed relative to mean control values 11.08 ± 3.55% vs. 100 ± 15.18% respectively, p<0.0001). In addition, antisense clc-5 transfection also led to a marked reduction in intracellular acidic compartments labelled with LysoTracker Red (75nM, 30 min at 37^oC, Molecular Probes). Analysis of transfectants (quantification performed in mid-cell xy confocal sections as for lectin) revealed a reduction in the relative area of acidic compartments in antisense clc-5 (n=17) transfected cells when compared to GFP (n=22) transfected cells (30.99 ± 5.84% and 100.00 ± 13.89 % respectively, p<0.001). Antisense clc-5 treatment results in an acidification defect in endosomes that interrupts the endocytic retrieval of plasma membrane in mpkDCT cells similar to proximal tubule. Defects in CLC-family proteins may result in dysregulation of apical membrane TRPV5/6 proteins within the distal nephron.