1 Department of Paediatric Nephrology and 2 Laboratory of Paediatrics and Neurology, Radboud University Nijmegen Medical Centre, The Netherlands
Correspondence and offprint requests to: E. N. Levtchenko, Department of Paediatric Nephrology, Radboud University Nijmegen Medical Centre, PO 9101, 6500 HB Nijmegen, The Netherlands. Email: e.levtchenko{at}cukz.umcn.nl
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Abstract |
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Methods. Components of the -glutamyl cycle were measured in cultured skin fibroblasts (n = 9) and polymorphonuclear (PMN) leukocytes (n = 15) derived from patients with cystinosis and compared with the values in cultured fibroblasts (n = 9) and PMN cells (n = 18) of healthy controls.
Results. Cystine content in cystinotic fibroblasts and PMN cells was significantly elevated compared with the controls, consistent with the lysosomal cystine accumulation in these cells. Although no reduction of total intracellular GSH content was found in cystinotic cells, it inversely correlated with cystine levels. Furthermore, GSH disulfide (GSSG) was elevated in cystinotic cells, resulting in an increased GSSG/total GSH (%) ratio. No relationship between intracellular cystine and GSH was found in control fibroblasts and PMN cells.
Conclusion. An elevated GSSG/total GSH (%) ratio might indicate increased oxidative stress present in cystinotic cells. Inverse correlation between cystine accumulation and intracellular GSH content indicates that under stress conditions such as intensive energy demand or increased oxidative insult, cystinotic cells may be more prone to GSH depletion.
Keywords: cystinosis; fibroblasts; glutathione; glutathione disulfide; polymorphonuclear cells
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Introduction |
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Although the molecular basis of cystinosis is elucidated, the link between intralysosomal accumulation of cystine and cellular dysfunction is not yet clarified.
Altered ATP synthesis has been demonstrated in rabbit and rat proximal tubules loaded with cystine dimethyl ester, mimicking cystine accumulation in cystinosis [3]. However, this pathogenic mechanism has not been confirmed in cells of patients with cystinosis. Assuming that ATP deficiency is indeed the cause of cellular damage in cystinosis, it still has to be explained how cystine accumulation alters ATP synthesis. Several theoretical considerations and experimental findings indirectly point to disturbances in glutathione (GSH) metabolism. In normal cells, proteins are degraded within lysosomes to amino acids, which are transported to the cytosol. Cystine is carried by cystinosin to the cytosol where it is reduced to cysteine by cytosolic reducing systems [1]. Theoretically, the intralysosomal cystine accumulation in cystinosis can result in the cytosolic deficit of cysteine. Cysteine together with glutamate and glycine is needed for GSH synthesis (Figure 1), requiring two successive enzymatic reactions, both consuming one ATP molecule per enzymatic cycle. The first and rate-limiting reaction couples glutamate and cysteine and is catalysed by -glutamylcysteine synthetase (
-GCS), resulting in the formation of
-glutamylcysteine (
-GC). The second reaction couples
-GC with glycine and is catalysed by GSH synthetase (GSH-S). Humans with deficient GSH-S accumulate and excrete elevated amounts of 5-oxoproline, due to diminished conversion of 5-oxoproline to glutamate by 5-oxoprolinase (Figure 1) [4]. The elevated 5-oxoproline excretion has also been demonstrated in cystinotic patients with Fanconi syndrome, untreated with cysteamine, suggesting an alteration in GSH synthesis in these patients [5]. Additionally, the presence of aminoaciduria and metabolic acidosis in patients with a deficiency of
-GCS suggests that GSH deficiency might result in renal proximal tubular dysfunction [4].
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GSH depletion results in mitochondrial dysfunction in several cellular and animal models [7,8] and therefore might affect ATP synthesis in cystinosis. Interestingly, increased glutathionylation of the mitochondrial complex I due to elevated mitochondrial glutathione disulfide (GSSG) has been shown recently to increase superoxide production [9]. An increased superoxide production has been demonstrated by chemiluminescence assay in isolated polymorphonuclear (PMN) cells and mononuclear phagocytes from cystinotic children [10].
GSH has also a role in maintaining the intracellular pool of cysteine via the reaction catalysed by -glutamyl transpeptidase (
-GT), resulting in formation of cysteinyl-glycine (Figure 1).
Taking these considerations together, we suggested that GSH deficiency might link intralysosomal cystine accumulation and mitochondrial dysfunction in cystinosis. To test this hypothesis, we investigated total, reduced and oxidized intracellular GSH, cysteine and total -glutamyl-cysteine and cysteinyl-glycine levels in cultured fibroblasts and PMN leukocytes derived from patients with cystinosis and compared these results with control values.
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Subjects and methods |
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Cell preparations
Skin fibroblasts culture. Skin fibroblasts were obtained by skin biopsies after receiving informed consent from nine patients with cystinosis prior to the start of cysteamine treatment and in nine healthy controls, followed by culturing in custom-made M199 medium with Tween-20 (5 mg/l, Gibco) supplemented with fetal calf serum (10%), penicillin (100 U/ml, Gibco) and streptomycin (100 U/ml, Gibco) as described previously [12]. Approximately 5 x 106 cells were grown for each assay to confluency and detached using trypsin. After washing twice in phosphate-buffered saline (PBS; Gibco), cells were centrifuged and pellets were shock-frozen immediately in liquid N2 and stored at 80°C.
Polymorphonuclear leukocytes. PMN leukocytes of 15 cystinotic patients and 18 adult healthy controls were isolated immediately from 10 ml of freshly drawn blood as described previously, frozen in liquid N2 and stored at 80°C [12].
Determination of thiol-amino acids and small thiol-peptides
Preparation of cell extracts was performed as described previously [12]. Briefly, frozen cell pellets were resuspended on ice in sodium phosphate buffer (pH 7.2) with 1 mM N-ethylmaleimide (NEM) (oxidized thiols) or without NEM (total thiols) followed by sonication. Using NEM, binding all free -SH groups, does not allow us to distinguish between cytosolic disulfides and thiols bound to intracellular proteins. The homogenates were centrifuged at 14 000 g for 10 min at 4°C. The supernatants were used for measurements of protein concentrations by the Lowry method and for determination of intracellular cystine and total cysteine, GSSG and total GSH, total -glutamyl-cysteine and total cysteinyl-glycine by using high-performance liquid chromatography (HPLC) [11]. Free cysteine content was calculated as the difference between total cysteine and oxidized cysteine. Cystine was measured as cysteine (1/2 cystine) after addition of NEM and expressed as nmol cystine/mg protein (as some centres express cystine as 1/2 cystine/mg protein, we provide a conversion factor: 1/2 cystine/mg protein = 2 x cystine/mg ptotein). A modification of our method was using an elution buffer with pH 3.34 instead of pH 3.88 to obtain a better separation of the components. Values were expressed as median and range.
Statistical analysis
Non-parametrical MannWhitney U-test was used for the comparison of values from cystinotic and control cells. The correlations between two variables were estimated by a Spearman rank correlation method. The differences were considered statistically significant at P<0.05.
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Results |
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Intracellular cysteinyl-glycine content was elevated in cystinotic fibroblasts [median 0.5 (0.10.6) vs 0.1 (<0.10.2) nmol/mg protein] (Table 1).
Thiol compounds in polymorphonuclear cells
As all examined cystinosis patients were treated with the cystine-depleting drug cysteamine, the intracellular cystine content of PMN cells was elevated to a lesser extent compared with cystinotic fibroblasts, confirming the efficacy of cysteamine therapy. However, it was still significantly increased compared with the control subjects [0.5 (0.30.8) vs 0.1 (0.090.2) nmol/mg protein, P<0.001]. As in cystinotic fibroblasts, intracellular free cysteine and total GSH contents did not differ between patients and controls in PMN cells. GSSG was significantly elevated [median 0.9 (0.31.8) vs 0.3 (0.20.4) nmol/mg protein, P<0.05], resulting in an elevated GSSG/total GSH ratio (Table 2).
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Theoretically, the increased GSSG in PMN cells of the patients treated with cysteamine may have been influenced by cysteamine treatment. Therefore, we have measured GSSG in two cystinotic patients untreated with cysteamine, whose PMN cystine content was 1.58 and 1.97 nmol/mg protein, respectively. In these patients, GSSG was 1.1 (within the range of treated patients) and 2.8 nmol/mg protein (above the range of treated patients), respectively, opposing a possible role for cysteamine treatment in the elevation of GSSG in PMN cells.
Relationship between intracellular cystine accumulation and glutathione content
Although total GSH content was not different between cystinotic and control cells, we observed a significant inverse correlation between cystine accumulation and intracellular GSH content in cystinotic fibroblasts. In PMN cells, this correlation was less pronounced probably due to the lower cystine content in patients treated by cysteamine (Figure 2).
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Discussion |
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Cultured cystinotic fibroblasts accumulated cystine to a higher extent compared with PMN cells of the patients treated with cysteamine, and therefore fibroblasts may be a better model for studying the pathogenesis of the disease. However, the metabolism of cultured cells could be influenced by ex vivo growth, which is not the case for PMN cells immediately derived from patients with cystinosis or healthy controls. Interestingly, the trends found were comparable in both cell types.
The hypothesis of alterations in intracellular glutathione and cysteine levels in cystinosis was first tested >30 years ago. Comparably with our results, these early studies demonstrate no difference in absolute glutathione and cysteine contents between cystinotic and normal fibroblasts [13,14]. A direct and sensitive HPLC method with and without the use of NEM allowed us to determine not only total GSH, but also GSSG, which was elevated in cystinotic fibroblasts and PMN cells compared with the controls. Our data, however, are distinct from the results of States et al., who found decreased GSSG in cystinotic fibroblasts [14]. The possible reason for this discrepancy is the deproteinization of samples prior to thiol detection by States et al., which would eliminate the detection of GSH bound in disulfides to protein.
In contrast to the recent findings of Chol et al. who detected a modest decrease of GSH in three cystinotic conditionally immortalized fibroblast cell lines compared with three controls [15], we were not able to demonstrate any reduction of total intracellular GSH in a larger study population. Although in our opinion the data of Chol et al. should be interpreted with caution due to their rather small number of observations, the measurements of intracellular thiols during the different phases of the cell cycle could be an additional explanation of this difference. Chol et al. found decreased intracellular GSH content during the phase of exponential cell growth, while we determined intracellular thiol components in cells grown to confluency. This also explains the higher fibroblast cystine content in our study (4.3 vs 0.88 nmol cystine/mg protein, respectively), which is closer to cystine accumulation in tissues in vivo (for example in kidney: >25 nmol 1/2 cystine/mg wet weight) [1].
Deficient cytosolic cysteine resulting in impaired intracellular glutathione synthesis, due to the altered exit of cystine out of the lysosomes, was our starting hypothesis which could not be directly confirmed in this study. Equal free cysteine content in cystinotic and control cells was found in both cultured fibroblasts and PMN cells. Comparably with our results, no difference in cysteine content between cystinotic and control fibroblasts could be demonstrated earlier by Shulman et al. [13]. However, the intracellular cysteine content measured by their group was almost 30 times lower compared with our data. More recent studies, using direct determination by HPLC, in fibroblasts are in line with our measurements of intracellular cysteine content [16]. The cysteine content in the cells, however, do not necessary reflect the dynamic process of cysteine formation, consumption and transport into and out of the cells. The normal cysteine content in cystinotic cells might be maintained via increased import of this amino acid or from increased degradation of GSH. Although we do not have a clear explanation for the increased cysteinyl-glycine in cystinotic fibroblasts, it might be speculated that these cells use GSH degradation more extensively to replenish their cytosolic cysteine pool, especially as this amino acid is essential for the growth of human fibroblasts in culture [17]. The inverse correlation between cystine accumulation and total intracellular GSH content demonstrated in our study indicates that under stress conditions such as intensive cell proliferation or increased oxidative stress, cystinotic cells may become GSH depleted. In these situations, cysteine deficit due to a defective exit of cystine out of the lysosomes might limit GSH synthesis. As tissue cystine levels measured in patients with cystinosis are much higher compared with those detected in cultured fibroblasts [1], GSH deficiency might occur in vivo. To obtain more insights into GSH synthesis in cystinotic cells, studies with stable isotopes at rest and under stress conditions are required in order to investigate the activities of individual enzymes of the -glutamyl cycle.
Probably the most important finding of this study is significantly increased GSSG and concordantly GSSG/total GSH ratio, present in both cystinotic fibroblasts and PMN cells, which reflects a changed redox potential in the studied cells. The redox regulation of gene expression and intracellular communication is just emerging as a vital mechanism in health and disease [18]. As mitochondria are important sources of intracellular reactive oxygen species (ROS), it is tempting to speculate that cystinotic cells are less protected from mitochondrial free radical production. Mitochondrial superoxide is converted to H2O2 by inducible Mn-superoxide dismutase (Mn-SOD) and, when released to the cytoplasm, can change the redox state to the rest of the cell [18]. Increased free radical production can be responsible for increased apoptosis, demonstrated in cystinotic fibroblasts and renal tubular epithelial cells [19,20]. In agrrement with this hypothesis, increased activity of SOD, used as an index of superoxide overproduction, has been demonstrated in cystinotic fibroblasts by Chol et al. [15]. Increased oxidative stress might be the missing link between cystine accumulation within the lysosomes and two other proposed pathogenic mechanisms of cystinosis such as mitochondrial dysfunction and apoptosis.
In conclusion, although no depletion of absolute GSH and cysteine contents is found in cystinotic fibroblasts and PMN cells, we demonstrate an inverse correlation between cystine accumulation and intracellular GSH content. In addition, the elevated GSSG might indicate the presence of increased oxidative stress, which can be responsible for cell damage in cystinosis. An increased peroxidation in, for example, proximal tubular cells could have striking results on the signal transduction in these cells [20]. The study of the mitochondrial GSH content and ROS production in cystinotic cells in rest conditions and in conditions of intensive energy demand and increased oxidative stress are in our opinion promising directions for future research.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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