Using the
cystine dimethylester (CDME) loading technique to achieve elevated lysosomal
cystine levels,
ATP depletion has previously been postulated to be responsible for the renal dysfunction in
cystinosis, a
genetic disorder characterized by an excessive accumulation of
cystine in the lysosomes. However, this is unlikely to be the sole factor responsible for the complexity of cell stress associated with
cystinosis. Moreover, CDME has been shown to induce a direct toxic effect on mitochondrial
ATP generation. Using a human-derived proximal tubular epithelial cell line, we compared the effects of CDME loading with
small interfering RNA-mediated cystinosin, lysosomal
cystine transporter (CTNS) gene silencing on
glutathione redox status,
reactive oxygen species levels, oxidative stress index,
antioxidant enzyme activities and
ATP generating capacity. The CDME-loaded cells displayed increased total
glutathione content, extensive
superoxide depletion, augmented oxidative stress index, decreased
catalase activity, normal
superoxide dismutase activity and compromised
ATP generation. In contrast, cells subjected to CTNS gene inhibition demonstrated decreased total
glutathione content, increased
superoxide levels, unaltered oxidative stress index, unaltered
catalase activity, induction of
superoxide dismutase activity and normal
ATP generation. Our data indicate that many CDME-induced effects are independent of lysosomal
cystine accumulation, which further underscores the limited value of CDME loading for studying the pathogenesis of
cystinosis. CTNS gene inhibition, which results in intracellular
cystine accumulation, is a more realistic approach for investigating biochemical alterations in
cystinosis.