Renal and Extra Renal Manifestations in Adult Zebrafish Model of Cystinosis.

Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.

Impaired autophagy: The collateral damage of lysosomal storage disorders.

Lysosomal storage disorders (LSDs), which number over fifty, are monogenically inherited and caused by mutations in genes encoding proteins that are involved in lysosomal function. Lack of the functional protein results in storage of a distinctive material within the lysosomes, which for years was thought to determine the pathophysiology of the disorder. However, our current view posits that the primary storage material disrupts the normal role of the lysosome in the autophagic pathway resulting in the secondary storage of autophagic debris. It is this "collateral damage" which is common to the LSDs but nonetheless intricately nuanced in each. We have selected five LSDs resulting from defective proteins that govern widely different lysosomal functions including glycogen degradation (Pompe), lysosomal transport (Cystinosis), lysosomal trafficking (Danon), glycolipid degradation (Gaucher) and an unidentified function (Batten) and argue that despite the disparate functions, these proteins, when mutant, all impair the autophagic process uniquely.

Protection of Cystinotic Mice by Kidney-Specific Megalin Ablation Supports an Endocytosis-Based Mechanism for Nephropathic Cystinosis Progression.

BACKGROUND: Deletions or inactivating mutations of the cystinosin gene CTNS lead to cystine accumulation and crystals at acidic pH in patients with nephropathic cystinosis, a rare lysosomal storage disease and the main cause of hereditary renal Fanconi syndrome. Early use of oral cysteamine to prevent cystine accumulation slows progression of nephropathic cystinosis but it is a demanding treatment and not a cure. The source of cystine accumulating in kidney proximal tubular cells and cystine's role in disease progression are unknown. METHODS: To investigate whether receptor-mediated endocytosis by the megalin/LRP2 pathway of ultrafiltrated, disulfide-rich plasma proteins could be a source of cystine in proximal tubular cells, we used a mouse model of cystinosis in which conditional excision of floxed megalin/LRP2 alleles in proximal tubular cells of cystinotic mice was achieved by a Cre-LoxP strategy using Wnt4-CRE. We evaluated mice aged 6-9 months for kidney cystine levels and crystals; histopathology, with emphasis on swan-neck lesions and proximal-tubular-cell apoptosis and proliferation (turnover); and proximal-tubular-cell expression of the major apical transporters sodium-phosphate cotransporter 2A (NaPi-IIa) and sodium-glucose cotransporter-2 (SGLT-2). RESULTS: Wnt4-CRE-driven megalin/LRP2 ablation in cystinotic mice efficiently blocked kidney cystine accumulation, thereby preventing lysosomal deformations and crystal deposition in proximal tubular cells. Swan-neck lesions were largely prevented and proximal-tubular-cell turnover was normalized. Apical expression of the two cotransporters was also preserved. CONCLUSIONS: These observations support a key role of the megalin/LRP2 pathway in the progression of nephropathic cystinosis and provide a proof of concept for the pathway as a therapeutic target.

Intrinsic Bone Defects in Cystinotic Mice.

Cystinosis is a rare lysosomal storage disorder caused by loss-of-function mutations of the CTNS gene, encoding cystinosin, a symporter that mediates cystine efflux from lysosomes. Approximately 95% of patients with cystinosis display renal Fanconi syndrome, short stature, osteopenia, and rickets. In this study, we investigated whether the absence of cystinosin primarily affects bone remodeling activity, apart from the influences of the Fanconi syndrome on bone mineral metabolism. Using micro-computed tomography and histomorphometric and bone serum biomarker analysis, we evaluated the bone phenotype of 1-month-old Ctns-/- knockout (KO) male mice without tubulopathy. An in vitro study was performed to characterize the effects of cystinosin deficiency on osteoblasts and osteoclasts. Micro-computed tomography analysis showed a reduction of trabecular bone volume, bone mineral density, and number and thickness in KO mice compared with wild-type animals; histomorphometric analysis revealed a reduction of osteoblast and osteoclast parameters in tibiae of cystinotic mice. Decreased levels of serum procollagen type 1 amino-terminal propeptide and tartrate-resistant acid phosphatase in KO mice confirmed reduced bone remodeling activity. In vitro experiments showed an impairment of Ctns-/- osteoblasts and osteoclasts. In conclusion, cystinosin deficiency primarily affects bone cells, leading to a bone loss phenotype of KO mice, independent from renal failure.

Switching from immediate- to extended-release cysteamine in nephropathic cystinosis patients: a retrospective real-life single-center study.

BACKGROUND: Nephropathic cystinosis is a rare lysosomal storage disease which is characterized by the accumulation of free cystine in lysosomes and subsequent intracellular crystal formation of cystine throughout the body. If not treated with cysteamine, a cystine-depleting agent, end-stage renal disease will develop early, followed by multiple organ failure as the disease progresses. The established cysteamine formulation requires a strict dosing regimen at 6-h intervals. An extended release (ER) twice-daily formulation has recently been developed. The aim of our study was to evaluate the implementation and outcomes of this option in routine care. METHODS: All pediatric cystinosis patients' records in Hannover Medical School were screened, and data on cysteamine therapy, tolerability, dosing, estimated glomerular filtration rates (eGFR), white blood cell cystine levels, and proton pump inhibitor (PPI) use were extracted for the period January 2014 to January 2016. RESULTS: The median age of the 12 patients enrolled in the study was 12.5 (range 1-18) years. At the end of the study period ten of these patients received ER-cysteamine. There were no additional side effects. Halitosis/bad breath was often subjectively judged as improved or eliminated, and PPI use could be stopped in one of three patients. The main reasons for switching to the ER formulation were difficult night-time administration and uncontrolled disease. Mean eGFR values remained stable with a median of 67 ml/min/1.73 m2 before and after the transition. White blood cell (WBC) cystine values remained low after the switch (1 nmol/mg protein before and after transition; p = 0.64). CONCLUSIONS: In this single-center cohort, the switch from IR- to ER-cysteamine was safe and effective over the short term and provided advantages in terms of frequency of administration and less halitosis/bad breath. The long-term benefit of this option needs to be evaluated in future studies.

Controversies and research agenda in nephropathic cystinosis: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference.

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.

Cystinosis: a review.

Cystinosis is the most common hereditary cause of renal Fanconi syndrome in children. It is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding for the carrier protein cystinosin, transporting cystine out of the lysosomal compartment. Defective cystinosin function leads to intra-lysosomal cystine accumulation in all body cells and organs. The kidneys are initially affected during the first year of life through proximal tubular damage followed by progressive glomerular damage and end stage renal failure during mid-childhood if not treated. Other affected organs include eyes, thyroid, pancreas, gonads, muscles and CNS. Leucocyte cystine assay is the cornerstone for both diagnosis and therapeutic monitoring of the disease. Several lines of treatment are available for cystinosis including the cystine depleting agent cysteamine, renal replacement therapy, hormonal therapy and others; however, no curative treatment is yet available. In the current review we will discuss the most important clinical features of the disease, advantages and disadvantages of the current diagnostic and therapeutic options and the main topics of future research in cystinosis.

Cystinosin is a Component of the Vacuolar H+-ATPase-Ragulator-Rag Complex Controlling Mammalian Target of Rapamycin Complex 1 Signaling.

Cystinosis is a rare autosomal recessive storage disorder characterized by defective lysosomal efflux of cystine due to mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin. Lysosomal cystine accumulation leads to crystal formation and functional impairment of multiple organs. Moreover, cystinosis is the most common inherited cause of renal Fanconi syndrome in children. Oral cysteamine therapy delays disease progression by reducing intracellular cystine levels. However, because cysteamine does not correct all complications of cystinosis, including Fanconi syndrome, we hypothesized that cystinosin could have novel roles in addition to transporting cystine out of the lysosome. By coimmunoprecipitation experiments and mass spectrometry, we found cystinosin interacts with almost all components of vacuolar H(+)-ATPase and the Ragulator complex and with the small GTPases Ras-related GTP-binding protein A (RagA) and RagC. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) pathway was downregulated in proximal tubular cell lines derived from Ctns(-/-) mice. Decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in these cells, suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine. Our results show a dual role for cystinosin as a cystine transporter and as a component of the mTORC1 pathway, and provide an explanation for the appearance of Fanconi syndrome in cystinosis. Furthermore, this study highlights the need to develop new treatments not dependent on lysosomal cystine depletion alone for this devastating disease.

Cystinosis: clinical presentation, pathogenesis and treatment.

Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in the CTNS gene ncoding the lysosomal cystine transporter cystinosin. Cystinosin deficiency leads to accumulation of cystine in the lysosomes of cells throughout the body and deregulation of endocytosis, trafficking of intracellular vesicles and related cell signalling processes. One of the early features of the disease is renal Fanconi syndrome characterized by polyuria, proteinuria and urinary loss of various solutes. Later in life, extrarenal complications become apparent, and decline of kidney function leads to the development of end-stage renal disease. Modern therapy of the disease is based on treatment with cystine-lowering drug cysteamine, which helps to postpone the disease progression and development of extra-renal pathologies, but offers no cure for the Fanconi syndrome. Besides the improvement of cystine-lowering therapy based on new formulations of cysteamine, further development of therapy is necessary. Some steps forward were done in the recent years, including studies of cell signalling abnormalities in cystinosis and development of stem cell and gene therapy approaches.

Inflammasome activation by cystine crystals: implications for the pathogenesis of cystinosis.

Intralysosomal cystine crystal accumulation, due to mutations in the CTNS gene, is a hallmark of nephropathic cystinosis, but the role of these crystals in disease pathogenesis remains unclear. We hypothesized that, similar to other host-derived crystalline moieties, cystine crystals can induce IL-1ß production through inflammasome activation. Thus, we investigated the proinflammatory effects of cystine crystals in primary human PBMCs. LPS-primed PBMCs stimulated with cystine crystals secreted IL-1ß in a dose-dependent manner. Similarly to IL-1ß secretion induced by other crystalline inflammasome activators, cystine crystal-induced IL-1ß secretion required activation of caspase-1. Additionally, exogenous cystine crystals were internalized by monocytes, and inhibition of phagocytosis, cathepsin B leakage, generation of reactive oxygen species, and potassium efflux reduced cystine crystal-induced IL-1ß secretion. Patients with cystinosis had higher levels of circulating IL-1ß and IL-18 compared with controls. Analysis of inflammasome-related gene expression in PBMCs from patients with cystinosis revealed a significant increase in IL-1ß and CASP-1 transcript levels compared with controls. Moreover, knockout of cystinosin in mice led to significant increases in serum IL-18 levels and kidney expression of inflammasome-related genes (Casp-1, Pycard, Il-18, Il18r1, Il1r1, and Il1rl2). Taken together, these data demonstrate that cystine crystals are endogenous inflammasome-activating stimuli, suggesting a novel role for cystine crystals in the pathogenesis of nephropathic cystinosis.

New-onset lupus nephritis in a kidney transplant recipient with cystinosis-differential diagnosis with cysteamine-induced lupus: case report.

A case of lupus nephritis in an adult female kidney transplant recipient with cystinosis under cysteamine therapy is reported. Previous reports of new-onset lupus in cystinotic patients have focused in a possible relationship of lupus with cysteamine therapy, but no obvious pathophysiological association has been disclosed. The authors present a case of a 19-year-old female kidney transplant recipient with cystinosis admitted for acute allograft dysfunction, with clinical and immunologic manifestations of lupus nephritis. Cysteamine was considered as a potential cause of drug-induced lupus, and we temporarily interrupted this drug. The clinical picture, the negativity of antihistone antibodies, the nondisappearance of antinuclear antibodies after discontinuation of the drug, and the clinical stability after resuming cysteamine therapy suggested that the underlying mechanism of lupus was unrelated to the drug. This may be the first report of new-onset lupus in a kidney transplant recipient with cystinosis. Clinicians should be aware of the association of autoimmune abnormalities in patients with cystinosis.

The pathogenesis of cystinosis: mechanisms beyond cystine accumulation.

Renal proximal tubules are highly sensitive to ischemic and toxic insults and are affected in diverse genetic disorders, of which nephropathic cystinosis is the most common. The disease is caused by mutations in the CTNS gene, encoding the lysosomal cystine transporter cystinosin, and is characterized by accumulation of cystine in the lysosomes throughout the body. In the majority of the patients, this leads to generalized proximal tubular dysfunction (also called DeToni-Debré-Fanconi syndrome) in the first year and progressive renal failure during the first decade. Extrarenal organs are affected by cystinosis as well, with clinical symptoms manifesting mostly after 10 yr of age. The cystine-depleting agent cysteamine significantly improves life expectancy of patients with cystinosis, but offers no cure, pointing to the complexity of the disease mechanism. In this review, current knowledge on the pathogenesis of cystinosis is described and placed in perspective of future research.

Renal phenotype of the cystinosis mouse model is dependent upon genetic background.

BACKGROUND: Cystinosis is caused by mutations in CTNS that encodes cystinosin, the lysosomal cystine transporter. The most severe and frequent form is characterized by a proximal tubulopathy that appears around 6 to 12 months of age. In the absence of treatment, end-stage renal disease is reached by 10 years. Ctns(-/-) mice of a mixed 129Sv x C57BL/6 genetic background show elevated renal cystine levels; however, proximal tubulopathy or end-stage renal disease is not observed. METHODS: As renal phenotype can be influenced by genetic background, we generated congenic C57BL/6 and FVB/N Ctns(-/-) mice and assayed renal lesions and function by histological and biochemical studies. RESULTS: C57BL/6 Ctns(-/-) mice showed significantly higher renal cystine levels than the FVB/N strain. Moreover, C57BL/6 mice presented with pronounced histological lesions of the proximal tubules as well as a tubulopathy and progressively developed chronic renal failure. In contrast, renal dysfunction was not observed in the FVB/N strain. CONCLUSIONS: Thus, the C57BL/6 strain represents the first Ctns(-/-) mouse model to show clear renal defects. In addition to highlighting the influence of genetic background on phenotype, the C57BL/6 Ctns(-/-) mice represent a useful model for further understanding cystinosin function in the kidney and, specifically, in the proximal tubules.

Maternal uniparental heterodisomy of chromosome 17 in a patient with nephropathic cystinosis.

We report maternal uniparental disomy of chromosome 17 (mat UPD17) in a 2.5-year-old girl presenting infantile cystinosis. This patient was homozygous for the 57 kb deletion encompassing the CTNS gene, frequently found in patients from the European origin. The proband's mother was heterozygous for the deletion and the father did not carry the deletion. We carried out haplotype analysis with polymorphic markers spanning the whole chromosome 17. Informative markers showed the presence of two maternal alleles but no paternal allele for regions spanning the 17q arm and the proximal half of 17p, and only one maternal allele on the distal 17p arm. As deletion of half of 17p is probably not viable, these results suggest mat UPD17 with heterodisomy of 17q and proximal 17p and isodisomy of distal 17p. This is the first demonstration of mat UPD17, in particular of isodisomy 17p, in cystinosis.

Cystinosis, Fanconi syndrome, and odontogenic cysts.

Cystinosis is a multisystemic genetic storage disorder characterized by a mutation in the transporter system of cystine. The disease particularly affects the renal system by causing deposition of cystine crystals leading to, if untreated, Fanconi syndrome and end-stage renal disease. This disease also affects the ocular system, central nervous system, endocrine system, hepatobiliary system, and musculoskeletal system. We present the first case of cystine crystal deposition within an odontogenic cyst, confirmed by the correlation of the clinical, radiographic, and histologic findings on this patient.

Analysis of the CTNS gene in nephropathic cystinosis Mexican patients: report of four novel mutations and identification of a false positive 57-kb deletion genotype with LDM-2/exon 4 multiplex PCR assay.

OBJECTIVE: Identify CTNS gene mutations in nephropathic cystinosis Mexican patients. SUBJECTS AND METHODS: Eleven patients were included, nine presenting infantile nephropathic cystinosis and two siblings with the juvenile phenotype. The common 57-kb deletion was detected by multiplex PCR using large deletion marker-2 (LDM-2)/exon 4 set primers. Those alleles negative for 57-kb deletion were screened by single strand confirmation polymorphism (SSCP) and subsequent direct sequencing. RESULTS: In our sample, five mutations previously reported are identified: 57-kb deletion, EX4_EX5del, c.985_986insA, c.357_360delGACT, and c.537_557del. We detect a false assignation of 57-kb deletion homozygous genotype by using the LDM-2/exon 4 primers. In addition, four novel and severe mutations are identified: c.379delC, c.1090_1093delACCAinsCG, c.986C>G (p.T216R), and c.400+5G>A. CONCLUSIONS: Our sample of Mexican patients display allelic heterogeneity as compared to European or North American cystinosis cases. The identification of novel mutations might suggest the presence of exclusive American CTNS alleles in Mexican population. In order to prevent the false positive assignation of 57-kb deletion genotype, as caused by the presence of another type of intragenic CTNS gross deletion, we propose to analyze a different control CTNS exon to those originally reported in both LDM multiplex PCR assays, especially when parental DNA samples are not available.

Promotion of oxidative stress in kidney of rats loaded with cystine dimethyl ester.

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in most tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are not fully understood. Studies performed in fibroblasts of cystinotic patients and in kidney cells loaded with cystine dimethyl ester (CDME) suggest that apoptosis is enhanced in this disease. Considering that oxidative stress is a known apoptosis inducer, our main objective was to investigate the effects of CDME loading on several parameters of oxidative stress in the kidney of young rats. Animals were injected twice a day with 1.6 micromol/g body weight CDME and/or 0.26 micromol/g body weight cysteamine (CSH) from the 16th to the 20th postpartum day and killed after 1 or 12 h. CDME induced lipoperoxidation and protein carbonylation and stimulated superoxide dismutase, glutathione peroxidase (GPx), and catalase activities, probably through the formation of superoxide anions, hydrogen peroxide, and hydroxyl free radicals. Coadministration of CSH, the drug used to treat cystinotic patients, prevented, at least in part, those effects, possibly acting as a scavenger of free radicals. These results suggest that the induction of oxidative stress might be one of the mechanisms leading to tissue damage in cystinotic patients.

Cystine inhibits creatine kinase activity in pig retina.

BACKGROUND: Cystinosis is an autosomal recessive disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Visual deficit is a possible consequence of cystine accumulation in cornea and retina. Fibroblasts from cystinotic patients present ATP deficit with intact mitochondrial energy-generating capacity by an unknown mechanism. Considering that creatine kinase is a thiol enzyme crucial for energy homeostasis in retina, and disulfides like cystine may alter thiol enzymes, the main objective of the present study was to investigate the effect of cystine and cysteamine, the drug used for treatment of cystinotic patients, on creatine kinase activity in cytosolic and mitochondrial fractions of the retina from adult pigs. METHODS: Retina was isolated from 6-month-old Landrace pigs, homogenized and mitochondrial and cytosolic fractions separated by centrifugation. Cytosolic and mitochondrial creatine kinase activities were determined in the presence of different concentrations of cystine and/or cysteamine. RESULTS: Cystine inhibited the enzyme activity in a dose- and time-dependent manner and cysteamine prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits creatine kinase activity by oxidation of the sulfhydryl groups of the enzyme. CONCLUSIONS: Considering that creatine kinase is a crucial enzyme for retina energy homeostasis, in case cystine leaves lysosome these results provide a possible mechanism for cystine toxicity and also another beneficial effect for the use of cysteamine in patients with cystinosis.

Inhibition of creatine kinase activity by cystine in the kidney of young rats.

Nephropathic cystinosis is a lethal genetic disease caused by a lysosomal transport disorder leading to intralysosomal cystine accumulation in all tissues. Cystinosis is the most common inherited cause of Fanconi syndrome, but the mechanisms by which cystine causes tissue damage are not fully understood. Thiol-containing enzymes are critical for renal energy metabolism and may be altered by disulfides like cystine. Therefore, in the present study our main objective was to investigate the in vivo and in vitro effects of cystine on creatine kinase, which contains critical thiol groups in its structure, in the kidney of young Wistar rats. We observed that cystine inhibited in vivo and in vitro the enzyme activity and that this inhibition was prevented by cysteamine and glutathione. The results suggest oxidation of essential sulfhydryl groups necessary for creatine kinase function by cystine. Considering that creatine kinase and other thiol-containing enzymes are crucial for renal energy metabolism, and programmed cell death occurs in situations of energy deficiency, the enzyme inhibition caused by cystine released from lysosomes might be a mechanism of tissue damage in patients with cystinosis.