Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines

doi:10.1016/j.bbrc.2004.09.033

Biochemical and Biophysical Research Communications

Volume 324, Issue 1, 5 November 2004, Pages 231-235

https://doi.org/10.1016/j.bbrc.2004.09.033 Get rights and content

Abstract

Cystinosis is an inherited disorder due to mutations in the CTNS gene which encodes cystinosin, a lysosomal transmembrane protein involved in cystine export to the cytosol. Both accumulation of cystine in the lysosome and decreased cystine in the cytosol may participate in the pathogenic mechanism underlying the disease. We observed that cystinotic cell lines have moderate decrease of glutathione content during exponential growth phase. This resulted in increased solicitation of oxidative defences of the cell denoted by concurrent superoxide dismutase induction, although without major oxidative insult under our experimental conditions. Finally, decreased glutathione content in cystinotic cell lines could be counterbalanced by a series of exogenous precursors of cysteine, denoting that lysosomal cystine export is a natural source of cellular cysteine in the studied cell lines.

Section snippets

Materials and methods

Cell line production. Fibroblasts were derived from a skin biopsy from 3 patients affected by infantile cystinosis harboring different mutations in the CTNS gene previously reported [2], [18], [19]. One of these patients was homozygous for the common 57 kb deletion of the CTNS gene. The second patient was heterozygous for this deletion and an in-frame insertion (1386–1387 ins 12). The third patient was heterozygous for a splicing mutation (564 + 1 G > A) and the deleterious substitution 1354 G > A.

Results

We first determine the cystine content of the various cell lines. As predicted from the deleterious deletion/mutations identified in the CTNS gene in the three patients, a huge accumulation of cystine was measured in the CTNS−/− cell lines (1.76 ± 0.73 1/2 cystine nmol/mg protein) as compared to CTNS+/+ cell lines (0.11 ± 0.05 1/2 cystine nmol/mg protein).

Because glutathione is possibly synthesized in part from cytosolic cysteine originating from cystine delivered by lysosomes (Fig. 1), we first

Discussion

Oxidative stress and mitochondrial dysfunction resulting in decreased ATP are elusive features of cystinosis, which might participate in the unexplained tubulopathy observed in patients and account for abnormal mitochondrial morphology occasionally observed in mouse model kidney [5], [7], [8], [10]. The present study establishes that, under controlled conditions, exponentially growing cystinotic cell lines have lower glutathione content and increased superoxide dismutase activity as compared to

Acknowledgments

We thank Philippe Moulier and Frédéric Broucque for transforming the fibroblasts. This work was supported by Vaincre les Maladies Lysosomales, Association Française contre les Myopathies, and the Cystinosis Foundation. M.C. is the recipient of a Ph.D. grant from Association pour l’Information et la Recherche sur les Maladies Rénales Génétiques.

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GSH is a tripeptide of glutamate, cysteine, and glycine and is synthesized in a process involving two enzymatic reactions, each requiring one ATP molecule [18]. Studies suggest that cystinotic cells are more vulnerable to oxidative stress as a result of impaired GSH synthesis and a compromised gamma glutamyl cycle, possibly due to altered mitochondrial function and depleted ATP levels [7,19–22]. Reduced levels of GSH result in the inability of the cell to scavenge ROS, which puts the cells under oxidative stress, particularly in the mitochondria where most cellular ROS are produced.
Nephropathic cystinosis is a severe, monogenic systemic disorder that presents early in life and leads to progressive organ damage, particularly affecting the kidneys. It is caused by mutations in the CTNS gene, which encodes the lysosomal transporter cystinosin, resulting in intralysosomal accumulation of cystine. Recent studies demonstrated that the loss of cystinosin is associated with disrupted autophagy dynamics, accumulation of distorted mitochondria, and increased oxidative stress, leading to abnormal proliferation and dysfunction of kidney cells. We discuss these molecular mechanisms driving nephropathic cystinosis. Further, we consider how unravelling molecular mechanisms supports the identification and development of new strategies for cystinosis by the use of small molecules, biologicals, and genetic rescue of the disease in vitro and in vivo.
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Nephropathic cystinosis is an autosomal recessive metabolic, lifelong disease characterized by lysosomal cystine accumulation throughout the body that commonly presents in infancy with a renal Fanconi syndrome and, if untreated, leads to end-stage kidney disease (ESKD) in the later childhood years. The molecular basis is due to mutations in CTNS, the gene encoding for the lysosomal cystine-proton cotransporter, cystinosin. During adolescence and adulthood, extrarenal manifestations of cystinosis develop and require multidisciplinary care. Despite substantial improvement in prognosis due to cystine-depleting therapy with cysteamine, no cure of the disease is currently available. Kidney Disease: Improving Global Outcomes (KDIGO) convened a Controversies Conference on cystinosis to review the state-of-the-art knowledge and to address areas of controversies in pathophysiology, diagnostics, monitoring, and treatment in different age groups. More importantly, promising areas of investigation that may lead to optimal outcomes for patients afflicted with this lifelong, systemic disease were discussed with a research agenda proposed for the future.
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On the other hand, recent findings in a ctns−/− mouse model suggest that cystine accumulation itself is not sufficient for the development of renal aberrations [6]. Several mechanisms have been postulated to link lysosomal cystine accumulation in cystinosis with renal tubular defects, such as impaired ATP synthesis [7,8], involvement of altered glutathione (GSH) metabolism [9,10] and increased apoptosis rate [11,12]. Decreased levels of total GSH in cystinotic fibroblasts during exponential growth were first reported by Chol et al. [9].
Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.
Kidney preservation by bone marrow cell transplantation in hereditary nephropathy
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The prospect of cell-based therapy for kidney disease remains controversial despite its immense promise. We had previously shown that transplanting bone marrow and hematopoietic stem cells could generate renal cells and lead to the preservation of kidney function in a mouse model for cystinosis (Ctns^−/−) that develops chronic kidney injury, 4 months post transplantation. Here, we determined the long-term effects of bone marrow stem cell transplantation on the kidney disease of Ctns^−/− mice 7 to 15 months post transplantation. Transfer of bone marrow stem cells expressing a functional Ctns gene provided long-term protection to the kidney. Effective therapy, however, depended on achieving a relatively high level of donor-derived blood cell engraftment of Ctns-expressing cells, which was directly linked to the quantity of these cells within the kidney. In contrast, kidney preservation was dependent neither on renal cystine content nor on the age of the mice at the time of transplant. Most of the bone marrow-derived cells within the kidney were interstitial and not epithelial, suggesting that the mechanism involved an indirect protection of the tubules. Thus, our model may help in developing strategies to enhance the potential success of cell-based therapy for kidney injury and in understanding some of the discrepancies currently existing in the field.
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Thus, cystine inside cystinotic lysosomes has no means of communication with the cytosol, and hence, no means to damage cellular metabolism. In recent times, several authors have expanded on an idea first proposed by Patrick in 1965 [3], that cystine could alter the intracellular redox balance, or “poison” critical thiol moieties of varies enzymes, thus inhibiting them and damaging metabolic pathways [4–7]. Since it is now known that cystine is isolated inside lysosomes, these hypotheses must also explain how cystine can interact with cytosolic components to lead to the observed pathologic changes.
Nephropathic cystinosis results from lysosomal cystine storage and, if untreated with cysteamine, results in end-stage renal disease by 10 years of age. The renal Fanconi syndrome occurs in the first year of life and is accompanied by a characteristic “swan neck” deformity of the proximal renal tubule. The linkage between cystine storage, Fanconi syndrome, and renal failure has not been understood. This study reports the presence of substantial numbers of atubular glomeruli (ATG) in end-stage cystinotic renal tissue. Compared to normal renal tissue, cystinotic kidneys at end stage had 69% atubular glomeruli and 30% atrophic glomeruli. Normal renal tissue had 4% ATG and 0% atrophic glomeruli (p < 0.0001 for both comparisons). These nonfunctioning nephrons may be the end result of cell loss from the tubules and represent the final stage of the swan neck deformity. The process is consistent with the previously reported increased apoptosis in renal tubule cells due to lysosomal cystine storage.
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A key unresolved question in the pathogenesis of phenotype development in nephropathic cystinosis is whether intralysosomal cystine, the hallmark of this lethal inborn error of metabolism, alters cytoplasmic redox potential. Variable findings on this issue have been reported. This study of fetal and non-fetal skin and lung-derived cystinotic fibroblasts compared to origin and age-matched normal control fibroblasts reveals that cystinotic cells do not exhibit redox perturbations. We find that the steady-state redox status as assessed by the [GSH]/[GSSG] ratio, an indicator of the intracellular redox poise, is unchanged in cystinotic cells. Furthermore, the dependence of the intracellular GSH and cysteine pool sizes and the [GSH]/[GSSG] ratio are similarly dependent on the two major sources of cysteine, i.e. the transsulfuration pathway and the plasma membrane cystine transporter, xc⁻, in both cystinotic and control cells, and the presence of lysosomal cystine has no measurable effect on the redox status of these cells. Hence, mechanisms other than cytosolic redox perturbations are involved in the etiology of nephropathic cystinosis.

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Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Exp. Cell Res.

FEBS Lett.

Clin. Chim. Acta

Life Sci.

Am. J. Hum. Genet.

Kidney Int.

J. Biol. Chem.