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Knockdown of Lysosomal Cystine Transporter Causes the Fanconi Syndrome in Primary Renal Cells
Author(s) -
Taub Mary,
Springate James E,
Cutuli Facundo
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.663.3
Subject(s) - chemistry , fanconi syndrome , apoptosis , cystine , cystinosis , kidney , cancer research , microbiology and biotechnology , biochemistry , endocrinology , biology , enzyme , cysteine
The autosomal recessive disorder cystinosis is due to a defect in cystinosin, a lysosomal cystine transporter. The Fanconi Syndrome emerges in the kidney in this disorder, due to reduced reabsorption of phosphate (Pi) and other solutes by the renal proximal tubule (RPT). The hypothesis was examined that in RPT cells the defect in cystinosin not only causes a reduction in the activity of apical Na + solute/cotransporters, but also results in increased apoptosis. In order to examine this hypothesis, siRNA was used to knock‐down cystinosin in primary rabbit RPT cells. Na + dependent Pi and alpha methyl‐D‐glucoside uptake was reduced by more than 60% following a 3‐day incubation with cystinosin siRNA (and a 80% knock‐down of cystinosin) vs. scrambled (scr) controls. Na + independent 3‐0‐methyl‐D‐glucoside uptake was unaffected. The sensitivity of primary RPT cells to apoptotic stimuli was examined. TNFα caused a 10.8 +/− 0.7 fold increase in caspase 3 activity in RPTs treated with cystinosin siRNA (2.4 fold higher than scr controls), and 20 μM cisplatin resulted in the death of 31 +/− 3% primary RPTs treated with cystinosin siRNA after 12 hr, a value 2.2 fold higher than in scr controls. Cystinosin siRNA also reduced cell growth by 45 +/− 10%. Further studies are in progress to determine whether these alterations can be explained by a change in the cellular redox state caused by reduced transport of cystine from lysosomes.