Related Experiment Videos
Disordered calcium crystal handling in antisense CLC-5-treated collecting duct cells
John A Sayer1, Georgina Carr, Simon H S Pearce
1Department of Medicine, University of Newcastle Upon Tyne, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. j.a.sayer@ncl.ac.uk
Abstract:
Dent's disease, an X-linked tubulopathy secondary to defects in chloride channel CLC-5, is characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Mechanisms leading to nephrocalcinosis are unknown. Using a murine collecting duct cell line (mIMCD-3), we confirm endogenous expression of mCLC-5. During transfection of antisense CLC-5, we observe a reduction in CLC-5 protein expression that correlates with a reduction in the number of acidic endosomal compartments, as determined by quantitative analysis of confocal microscope images using LysoTracker Red. Using wheat germ agglutinin-lectin as an endocytic marker, an arrest of endocytosis is observed in antisense CLC-5 treated cells. Exposure of the cell surface to calcium oxalate crystals results in crystal agglomeration in a minority of sense CLC-5 transfectants (45%) and all antisense CLC-5 transfectants. We conclude that expression of CLC-5 in mIMCD-3 cells allows acidification of endosomes and endocytosis, and that disruption of CLC-5 expression causes abnormal crystal agglomeration.
Insights
Dent's disease, caused by chloride channel CLC-5 defects, leads to kidney stones. Disrupting CLC-5 function impairs endocytosis and causes abnormal calcium oxalate crystal buildup in kidney cells.
Area of Science:
- Nephrology
- Molecular Biology
- Cell Biology
Background:
- Dent's disease is an X-linked tubulopathy linked to chloride channel CLC-5 defects.
- It presents with low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones.
- The mechanisms underlying nephrocalcinosis in Dent's disease remain unclear.
Purpose of the Study:
- To investigate the role of CLC-5 in endosomal acidification and endocytosis.
- To explore the impact of CLC-5 disruption on calcium oxalate crystal handling in kidney cells.
Main Methods:
- Utilized a murine collecting duct cell line (mIMCD-3) expressing endogenous CLC-5.
- Employed antisense CLC-5 transfection to reduce protein expression.
- Assessed endosomal acidity using LysoTracker Red and confocal microscopy.
- Evaluated endocytosis using wheat germ agglutinin-lectin.
- Exposed cells to calcium oxalate crystals to observe agglomeration.
Main Results:
- Reduced CLC-5 protein expression correlated with fewer acidic endosomal compartments.
- Disruption of CLC-5 led to an arrest in endocytosis.
- Cells with reduced CLC-5 exhibited abnormal calcium oxalate crystal agglomeration.
Conclusions:
- CLC-5 expression in mIMCD-3 cells is crucial for endosomal acidification and endocytosis.
- Disruption of CLC-5 function results in aberrant crystal agglomeration, potentially contributing to nephrocalcinosis in Dent's disease.