Disordered calcium crystal handling in antisense CLC-5-treated collecting duct cells.
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.
Sayer
John A
JA
Department of Medicine, University of Newcastle Upon Tyne, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. j.a.sayer@ncl.ac.uk
Carr
Georgina
G
Pearce
Simon H S
SH
Goodship
Timothy H J
TH
Simmons
Nicholas L
NL
D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism; D003460:Crystallization; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009397:Nephrocalcinosis / Q000209:etiology; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D014162:Transfection
D018118:Chloride Channels; D016376:Oligonucleotides, Antisense; D002118:Calcium