Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.     

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

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.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Decrease of intracellular cystine content in cystinotic fibroblasts by inhibitors of gamma-glutamyl transpeptidase

Cystine content of skin fibroblasts derived from patients with cystinosis was decreased by inhibitors of gamma-glutamyl transpeptidase, the initial enzyme in glutathione catabolism. The addition of maleate or the gamma-glutamyl hydrazone of alpha-ketobutyric acid to culture medium (1-20 mM) resulted in dose-dependent decreases of up to 55% on intracellular cystine content of cystinotic cells in 24 h. L-Serine in sodium borate buffer (40 mM each) produced similar results and further decreased cystine levels to 14% of cystinotic control values after 10 days incubation. Analysis of intracellular amino acids showed that, in general, other amino acids remained unchanged following serine-borate treatment. These results suggest that cystine storage in cystinotic tissues may be related to metabolism of glutathione.     

Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions.

Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.     

Cystinotic and normal fibroblasts: Differential susceptibility to cysteine toxicity in vitro

Summary Extracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagle's minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [³H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.     

Cysteamine depletes cystinotic leucocyte granular fractions of cystine by the mechanism of disulphide interchange.

Cystinotic lysosome-rich leucocyte granular fractions, loaded with [35S]cystine, were exposed to different cystine-depleting agents. During a 30 min incubation at 37 degrees C, untreated cystinotic granular fractions lost negligible [35S]cystine when corrected for lysosome rupture. Granular fractions exposed to 0.1 mM-cysteamine lost 64% of their initial cystine, and hexosaminidase activity was decreased by 10%. This was accompanied by the formation of high concentrations of [35S]cysteine-cysteamine mixed disulphide within the granular-fraction pellet, and, in the presence of N-ethylmaleimide, increasing amounts of [35S]cysteine-N-ethylmaleimide adduct outside the granular fraction. In separate experiments, [35S]cystine exited cystinotic leucocyte lysosomes at a negligible rate (half-times 199 and 293 min), but [35S]cysteine-cysteamine mixed disulphide exhibited substantial egress (half-times 66 and 88 min) and was recovered intact outside the granular-fraction pellet. We conclude that cysteamine depletes lysosomes of cystine by participating in a thiol-disulphide interchange reaction to produce cysteine and cysteine-cysteamine mixed disulphide, both of which traverse the cystinotic leucocyte lysosomal membrane.     

Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

Renal cell culture using autopsy material from children with cystinosis

Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.     

Effect of selenium compounds on selenium content, growth and 35S-cystine metabolism of skin fibroblasts from normal and cystinotic individuals.

Kidney samples from children with the inborn metabolic disease cystinosis contain 4 times more selenium (Se) than do kidney samples from normal individuals (p = 0.1). However, when cultured skin fibroblasts from cystinotic patients and normal control individuals are incubated in Se-D,L-methionine, Se-D,L-cystine, Se-cystamine X HCl, Se-urea, selenite or in medium without added selenium, only the cystinotic fibroblasts grown in Se-urea or selenite (SeO3=) contain more selenium than do the corresponding normal cells (p less than 0.05). In both types of cultured fibroblasts, the order of descending toxicity per ppm selenium is: Se-urea greater than Se-cystamine greater than Se-cystine greater than or equal to SeO3= much greater than Se-methionine. High (apparently toxic) concentrations of Se-urea and Se-cystamine lower the elevated intracellular free (nonprotein) cystine content of cystinotic fibroblasts to less than 60% of control values; at lower concentrations, these compounds raise the cystine content of these cells to over 140% of control values. Appropriate concentrations of SeO3=, Se-cystine and Se-methionine also elevate the free cystine content of the cystinotic cells. During a 75 minute incubation in 35S-cystine, the incorporation of 35S into the acid precipitable (protein) fraction of both cell types is significantly inhibited by Se-cystamine (approximately 55% control; p less than 0.05). The incorporation of 35S-cystine into glutathione is inhibited by Se-cystine (approximately 40% control) in both fibroblast types (p less than 0.05). In cystinotic cells, Se-cystamine significantly reduces incorporation of 35S-cystine into the cystine pool (40% control) as does SeO3= (67% control; p less than 0.05). Protein and glutathione synthesis in cystinotic fibroblasts are more strongly inhibited by Se-cystine and SeO3=, respectively, than in normal fibroblasts (p less than 0.05). These studies demonstrate that selenium compounds exhibit a different sequence of toxicity in fibroblasts than in the intact animal and that some previously unreported metabolic effects (i.e. inhibition of glutathione synthesis) may contribute to their toxicity.     

Renal cell culture using autopsy material from children with cystinosis

Summary Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE₁, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations. This work was supported by Grants AM 01074-01, AM 18434, and GM 17702 from the National Institutes of Health, Bethesda, MD.     

Elevated temperature produces cystine depletion in cystinotic fibroblasts

Increasing the incubation temperature of cystinotic fibroblasts to 40 or 43 degrees C produces a 70-80% decrease in lysosomal cystine content within 24-48 h. This effect is probably mediated by an altered substrate affinity for another lysosomal transport protein.     

Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.     

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport,Alleviates Endoplasmic Reticulum Stress,and Reduces Lysosome Overload in Cystinosis

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases, including Rab3 and Rab7, was downregulated in kidneys from Ctns^−/− mice and in human PTCs from cystinotic patients. Using total internal reflection fluorescence microscopy, we found that lysosomal transport is impaired in Ctns^−/− cells. Ctns^−/− cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload, manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contributes to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.     

Cystine Uptake and Glutathione Level in Fetal Brain Cells in Primary Culture and in Suspension

Abstract— The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine-free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na⁺-independent, glutamate-sensitive route previously found in various types of cells and designated as system x⁻_c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is discussed.     

Cystine storage in cultured myotubes from patients with nephropathic cystinosis.

Sorted muscle cells, cultured from a patient with nephropathic cystinosis, stored 100 times normal amounts of cystine. Subcellular fractionation and density-gradient centrifugation confirmed that the cystine was located in a lysosomal compartment. 2. Myoblasts from cystinotic patients in culture underwent fusion to myotubes in a normal fashion. 3. The free thiol cysteamine effectively depleted cystinotic-muscle cells of cystine. 4. Cultured myoblast and myotubes offered a unique system for investigating the effects of lysosomal storage on differentiated cell functions.     

Abnormal mitochondrial autophagy in nephropathic cystinosis

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with three clinical variants of cystinosis: nephropathic, intermediate and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to renal Fanconi syndrome and progressive renal injury.Key words: cystinosis, autophagy, mitochondria, kidney, lysosome, apoptosis, cell death, mitophagyCystinosis is an autosomal recessive metabolic disorder caused by mutations in the CTNS gene, which encodes a 7-transmembrane domain protein, cystinosin, a lysosomal cystine transporter. Cystinosis belongs to the family of lysosomal storage disorders (LSDs) characterized by the tissue accumulation of cystine crystals leading to multiple organ dysfunction. The three types of cystinosis, i.e., nephropathic (classic renal and systemic disease), intermediate (a late-onset variant of nephropathic cystinosis) and non-nephropathic (clinically affecting only the cornea) are allelic disorders caused by CTNS mutations. Children affected with nephropathic cystinosis present with the Fanconi syndrome and usually develop progressive renal failure within the first decade of life. The mechanism linking lysosomal cystine storage to pathological manifestations, in particular to the prominent proximal tubular defect and renal injury, remains unclear. Renal injury in nephropathic cystinosis may not simply be caused just by cystine accumulation, as disruption of the ctns gene in mice induces cystine storage in many tissues but does not result in signs of tubulopathy or renal failure; renal injury is not seen in other human forms of cystinosis and progressive renal injury occurs despite cystine depletion therapy.The purpose of our study was to investigate the specific mechanism leading to tubulopathy and end stage renal injury in nephropathic cystinosis. We used primary fibroblast and renal proximal tubular epithelial (RPTE) cells derived from patients with three clinical phenotypes of cystinosis. Our data show an abnormal increase in macroautophagy (hereafter referred to as autophagy), specific to the nephropathic variant of cystinosis. We also demonstrate that specific inhibition of autophagy rescues cell death in nephropathic cystinotic RPTE cells. Our results indicate that mitochondrial autophagy may be a critical mechanism contributing to renal Fanconi syndrome and progressive renal injury in nephropathic cystinosis.Abnormal autophagy was also recently observed in other types of lysosomal storage diseases (LSD). However, our study provides the first evidence supporting the extensive involvement of autophagy in nephropathic cystinosis pathogenesis. Abundant vacuolization and abnormal mitochondria are detected by electron microscopy (EM) in nephropathic cystinotic cells. Additionally, elevated levels of LC3-II and Beclin 1 are also observed in nephropathic cystinotic RPTE cells, indicating a role of Beclin 1-mediated autophagy in cystinosis. These results altogether establish an abnormal increase in autophagy in nephropathic cystinotic cells.Renal biopsies from patients with nephropathic cystinosis can reveal abnormally large mitochondria, but the relevance of this finding and other ultrastructural abnormalities is unclear. Our study further demonstrates a significant decrease in mitochondrial ATP generation with an increase in reactive oxygen species (ROS) in cystinotic cells. To further dissect the association of abnormal mitochondria with increased autophagy in cystinosis, we carefully examined the electron micrographs at higher magnifications. We discovered various stages of degradation of mitochondria by autophagy (hereafter referred to as mitophagy). To further validate mitophagy in cystinosis, we used an immunofluorescence (IF) approach to capture colocalization images of LC3, LAMP-2 (lysosomal marker) and ATP5H (mitochondrial marker). Intriguingly, an increase in LAMP-2 perinuclear staining is detected by IF assay in cystinotic cells. This observation may also denote enhanced active autophagy as LAMP-2 is involved in lysosomal biogenesis and/or the fusion between autophagosomes and lysosomes. Alternatively, LAMP-2 accumulation could be a manifestation of retarded autophagic flux in cystinotic cells. A decreased ability of lysosomes to fuse with autophagosomes has been reported in various LSDs. However, the colocalization of LC3 and LAMP-2 in nephropathic cystinotic RPTE cells argues against this possibility. Nevertheless, the possibility of autophagic flux blockade after autophagosome-lysosome fusion leading to detrimental effects is yet to be investigated. Interestingly, previously published EM reports of the renal biopsies of patients with nephropathic cystinosis show only the nucleus and a thin rim of cytoplasm as remnants in a proximal tubular cell, while mitochondria and lysosomes completely disappear.Conventionally, autophagy has been suggested as a cytoprotective mechanism to ensure cell survival during starvation. In contrast, several forms of cell death have been associated with the appearance of autophagic vesicles. To gain insight into the role of autophagy as regards to cell death or cell survival in nephropathic cystinosis, we used 3-methyladenine (3MA), a specific inhibitor of autophagy and assayed cell viability and apoptosis in cystinotic cells. Increased apoptosis has been previously reported in cultured cystinotic fibroblasts and RPTE cells. Treatment with 3MA in cystinotic cells significantly rescues cell death, thus suggesting a synergistic role of apoptosis and autophagy in cystinosis.In conclusion, as illustrated in Figure 1, we speculate that there is a multifaceted impact of autophagy in nephropathic cystinosis as follows: (1) the mechanism linking autophagy to lysosomal cystine or apoptosis in cystinotic cells could potentially be related to lysosomal membrane permeabilization (LMP), proposed as an early step in apoptosis in cystinosis. We hypothesize that abnormal induction of autophagy besides providing more cargo to be digested in the lysosomes, leads to increased fusion of autophagosomes with cystine-laden lysosomes, rendering them more sensitive to membrane destabilization, and thus making them readily enter the apoptotic pathway; (2) the second most important question is the link between abnormal mitochondria and mitophagy in cystinosis. A decreased level of cytosolic glutathione in cystinotic cells is one of the known factors responsible for generating damaged mitochondria. Our data also indicate an impairment of complex I activity, an increase in ROS and a decrease in mitochondrial ATP generation in cystinotic cells. We hypothesize that the abnormal induction of autophagy leads to depletion of mitochondria, forcing cells to enter the ‘starvation mode,’ thereby leading to an uncontrolled autophagy and cell death; (3) the third key question yet to be answered is the link between autophagy and renal injury in nephropathic cystinosis. Skeletal muscles and neuronal tissues are the primary organs where autophagy is physiologically enhanced. Recently, it has been shown that mouse kidneys exert a high level of autophagy under basal conditions, influencing the susceptibility to glomerular disease and renal failure. Thus, we postulate an organ- and tissue-specific effect of abnormally induced autophagy in nephropathic cystinosis, causing severe injury to kidneys leading to loss of renal function, ultimately culminating in end-stage renal disease.Open in a separate windowFigure 1A schematic view of the interplay between autophagy, abnormal mitochondria and cell death in cystinosis. Abnormal induction of autophagy, typically mitophagy, forces cells into a starvation mode leading to cell death; and renders cystine-laden lysosomes sensitive to lysosomal membrane permeabilization (LMP) making it readily enter the apoptosis pathway. A potential block in autophagic flux, after autophagosome-lysosome fusion, remains to be elucidated. Preferential severe kidney damage in nephropathic cystinosis may be due to the tissue- and organ-specific injury effect of autophagy.The recent progress in autophagy research has increased the need for additional studies so that we can fully understand the underlying pathological mechanisms and the significance of the lysosomal cell death axis in lysosomal storage disorders.     

Accumulation of cystine from glutathione-cysteine mixed disulfide in cystinotic fibroblasts; blockade by an inhibitor of gamma-glutamyl transpeptidase

Cystinotic and normal skin fibroblasts in tissue culture were treated with varying concentrations of reduced glutathione, oxidized glutathione and glutathione-cysteine mixed disulfide, substrates of gamma-glutamyl transpeptidase, the catabolic enzyme of the gamma-glutamyl cycle. Cystine accumulated more rapidly and to a greater extent from the glutathione-cysteine mixed disulfide in cystinotic than in normal cells. Inhibition of gamma-glutamyl transpeptidase activity by serine in a borate buffer partially blocked this accumulation of cystine. Reduced glutathione and oxidized glutathione have lesser effects on cystine accumulation. Stored cystine in cystinotic tissues may derive in part from glutathione-cysteine mixed disulfide via transpeptidation.     

Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal?

The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. trans-Stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. In addition, we find cystine exodus from the lysosomal fraction of cystinotic human fibroblasts to be greatly retarded as compared to that of normal human fibroblasts with half-times of exodus similar to those reported for the lysosomes of cystinotic and normal human leukocytes (Gahl, W. A., Tietze, F., Bashan, N., Steinherz, R., and Schulman, J. D. (1982) J. Biol. Chem. 257, 9570-9575). In contrast, normal and cystinotic human fibroblasts did not show any differences with regard to lysine efflux or its trans-stimulation by cationic amino acids. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.