Cystine Dimethylester Model of Cystinosis: Still Reliable?

Abstract

ABSTRACT:The ability of cystine dimethylester (CDME) to load lysosomes with cystine has been used to establish the basic defect in cystinosis: defective cystine exodus from lysosomes. Using CDME loading, it has been postulated that cystine accumulation in cystinosis affects mitochondrial ATP production, resulting in defective renal tubular reabsorption. Recent studies in cystinotic fibroblasts, however, show normal adenosine triphosphate (ATP) generation capacity. To investigate the effect of CDME in more detail, mitochondrial ATP generation, reactive oxygen species production, and viability are compared in fibroblasts loaded with CDME with those of cystinotic cells with a defective cystine transporter. Intracellular cystine levels were comparable in fibroblasts loaded with CDME (1 mM, 30 min) and cystinotic fibroblasts. Intracellular ATP levels and mitochondrial ATP production were decreased in fibroblasts loaded with CDME, but normal in cystinotic fibroblasts. Superoxide production was increased with 300% after CDME loading, whereas no changes were observed in cystinotic fibroblasts. Exposure to CDME led to cell death in a time-and concentration-dependent manner. Our data demonstrate that CDME has a toxic effect on mitochondrial ATP production and cell viability. These effects are not observed in cystinotic cells, indicating that a more appropriate model is required for studying the pathogenesis of cystinosis.

Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

“…Many studies have used a CDME in vitro model to study cystinosis in the past, for its ability artificially to load lysosomes with cystine 30,40 -43 ; however, the use of a CDMEloading model in studying pathogenesis of cystinosis can be questioned, because loading with CDME has a direct and acute impact on the viability of the cells as a result of direct toxicity from CDME, independent of cystine accumulation. 44,45 CDME is rapidly (within minutes) converted to cystine in lysosomes and leaves fairly rapidly from normal cells; therefore, drawing conclusions on cystinosis cellular injury on the basis of CDME-loading models may not be accurate. Our results suggest that the mitophagy observed in cystinosis may be independent of the effect of simply cystine accumulation in cystinotic cells.…”

Proteinuria and events beyond the slit

“…Different mechanisms underlying the pathological manifestations in cystinosis have been presented, of which many have been based on cystine loading by cystine dimethyl ester. However, toxicity of the drug has now been documented, and the results obtained by this method need reevaluation [94,95]. The proteinuric component of Fanconi syndrome could be caused by decreased levels of megalin and cubilin in the renal proximal tubule.…”

Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry

“…The HPLC with fluorescence detection assay described by Graaf‐Hess et al is currently used to quantify cystine levels in cystinotic fibroblasts, leukocytes, zebrafish and ciPTEC (Wilmer et al, 2007, 2011; Elmonem et al, 2017). Nevertheless, the assay is hampered by its high limit of detection (0.3 μmol/L), low cystine recovery and tedious sample preparation procedure (de Graaf‐Hess et al, 1999).…”