Cystine dimethylester loading promotes oxidative stress and a reduction in ATP independent of lysosomal cystine accumulation in a human proximal tubular epithelial cell line

Abstract

New findingsr What is the central question of this study?Are the molecular and cellular consequences of cystine dimethylester loading different from an alternative and more specific model of the cystinotic kidney proximal tubule epithelial cell, obtained by CTNS gene silencing. r What is the main finding and its importance?Using a human-derived kidney proximal tubular cell line, HK-2, we demonstrated that cystine dimethylester loading induces detrimental effects independent of lysosomal cystine accumulation. CTNS gene silencing, which resulted in comparable levels of cystine accumulation, evidence of oxidative stress and reduced ATP concentration but unaltered ATP generation capacity, represents a more useful model for investigating biochemical alterations in cystinosis.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 C

Lysosomal cystine accumulation promotes mitochondrial depolarization and induction of redox‐sensitive genes in human kidney proximal tubular cells

“…HK‐2 cells, a PTEC line derived from a normal adult human kidney, were cultured as described previously (Sumayao et al . ). Unless otherwise stated, all reagents were purchased from Sigma‐Aldrich (St Louis, MO, USA).…”

“…siRNA transfection was performed as described previously (Sumayao et al . ). The assessment of CTNS gene silencing and intracellular cystine accumulation was also described in Sumayao et al .…”

“…The assessment of CTNS gene silencing and intracellular cystine accumulation was also described in Sumayao et al . (). Succeeding experiments were performed 72 h following transfection.…”

“…The preparation of cell suspensions for ATP assay was performed as previously described (Sumayao et al . ). Intracellular ATP content was measured using the ATP Bioluminescence Assay Kit HSII (Roche Diagnostics, Mannheim, Germany) following the instructions of the manufacturer.…”

“…Western blot analysis was performed following the procedures described previously (Sumayao et al . ). The following concentrations of primary antibodies in 5% non‐fat milk/Tris‐base saline buffer (pH 7.4) containing 0.1% Tween‐20 (TBST) were used: anti‐nitrotyrosine (1:2000; Cell Signaling), anti‐iNOS (1:1000; Sigma), anti‐p47(phox) (1:1000; Cell Signaling), anti‐NF‐κB (p65) (1:1000; Cell Signaling), Phospho‐NF‐κB (p65) (1:1000; Cell Signaling), anti‐Nrf2 (1:1000; Cell Signaling), anti‐Hsp32 and ‐Hsp70 (1:1000; Cell Signaling), anti‐Beclin (1:1000; Cell Signaling), anti‐LC3 I/II (1:1000; Cell Signaling), β‐actin (1:2000; Thermo Scientific, Waltham, MA, USA) and GAPDH (1:2000; Abnova, Walnut, CA, USA).…”