A New and Rapid LC-MS/MS Method for the Determination of Cysteamine Plasma Levels in Cystinosis Patients.

Cystinosis is a rare lysosomal storage disorder caused by autosomal recessive mutations in the CTNS gene that encodes for the cystine transporter cystinosin, which is expressed on the lysosomal membrane mediating the efflux of cystine. Cysteamine bitartrate is a cystine-depleting aminothiol agent approved for the treatment of cystinosis in children and adults. In this study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cysteamine levels in plasma samples. This LC-MS/MS method was validated according to the European Medicines Agency (EMA)'s guidelines for bioanalytical method validation. An ultra-performance liquid chromatograph (UPLC) coupled with a 6470 mass spectrometry system was used for cysteamine determination. Our validated method was applied to plasma samples from n = 8 cystinosis patients (median, interquartile range (IQR) = 20.5, 8.5-26.0 years). The samples were collected before cysteamine oral administration (pre-dose) and 1 h after (post-dose). Our bioanalytical method fulfilled the regulatory guidelines for method validation. The cysteamine plasma levels in pre-dose samples were 2.57 and 1.50-3.31 µM (median and IQR, respectively), whereas the post-dose samples reported a cysteamine median concentration of 28.00 µM (IQR: 17.60-36.61). Our method allows the rapid determination of cysteamine plasma levels. This method was successfully used in cystinosis patients and, therefore, could be a useful tool for the evaluation of therapy adherence and for future pharmacokinetic (PK) studies involving a higher number of subjects.

Nlrp2 deletion ameliorates kidney damage in a mouse model of cystinosis.

Cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene that encodes cystinosin, a ubiquitous lysosomal cystine/H+ antiporter. The hallmark of the disease is progressive accumulation of cystine and cystine crystals in virtually all tissues. At the kidney level, human cystinosis is characterized by the development of renal Fanconi syndrome and progressive glomerular and interstitial damage leading to end-stage kidney disease in the second or third decade of life. The exact molecular mechanisms involved in the pathogenesis of renal disease in cystinosis are incompletely elucidated. We have previously shown upregulation of NLRP2 in human cystinotic proximal tubular epithelial cells and its role in promoting inflammatory and profibrotic responses. Herein, we have investigated the role of NLRP2 in vivo using a mouse model of cystinosis in which we have confirmed upregulation of Nlrp2 in the renal parenchyma. Our studies show that double knock out Ctns-/- Nlrp2-/- animals exhibit delayed development of Fanconi syndrome and kidney tissue damage. Specifically, we observed at 4-6 months of age that animals had less glucosuria and calciuria and markedly preserved renal tissue, as assessed by significantly lower levels of inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis. Also, the mRNA expression of some inflammatory mediators (Cxcl1 and Saa1) and the rate of apoptosis were significantly decreased in 4-6-month old kidneys harvested from Ctns-/- Nlrp2-/- mice compared to those obtained from Ctns-/-mice. At 12-14 months of age, renal histological was markedly altered in both genetic models, although double KO animals had lower degree of polyuria and low molecular weight proteinuria and decreased mRNA expression levels of Il6 and Mcp1. Altogether, these data indicate that Nlrp2 is a potential pharmacological target for delaying progression of kidney disease in cystinosis.

Genistein improves renal disease in a mouse model of nephropathic cystinosis: a comparison study with cysteamine.

Cysteamine is currently the only therapy for nephropathic cystinosis. It significantly improves life expectancy and delays progression to end-stage kidney disease; however, it cannot prevent it. Unfortunately, compliance to therapy is often weak, particularly during adolescence. Therefore, finding better treatments is a priority in the field of cystinosis. Previously, we found that genistein, an isoflavone particularly enriched in soy, can revert part of the cystinotic cellular phenotype that is not sensitive to cysteamine in vitro. To test the effects of genistein in vivo, we fed 2-month-old wild-type and Ctns-/- female mice with either a control diet, a genistein-containing diet or a cysteamine-containing diet for 14 months. Genistein (160 mg/kg/day) did not affect the growth of the mice or hepatic functionality. Compared with untreated mice at 16 months, Ctns-/- mice fed with genistein had lower cystine concentrations in their kidneys, reduced formation of cystine crystals, a smaller number of LAMP1-positive structures and an overall better-preserved parenchymal architecture. Cysteamine (400 mg/kg/day) was efficient in reverting the lysosomal phenotype and in preventing the development of renal lesions. These preclinical data indicate that genistein ameliorates kidney injury resulting from cystinosis with no side effects. Genistein therapy represents a potential treatment to improve the outcome for patients with cystinosis.

Benefits and Toxicity of Disulfiram in Preclinical Models of Nephropathic Cystinosis.

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns-/- mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns-/- mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells.

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis.

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis.

BACKGROUND: Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis. METHODS: To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish. RESULTS: Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy-lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin. CONCLUSIONS: Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.

Prohibitins: A Critical Role in Mitochondrial Functions and Implication in Diseases.

Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are proteins that are ubiquitously expressed, and are present in the nucleus, cytosol, and mitochondria. Depending on the cellular localization, PHB1 and PHB2 have distinctive functions, but more evidence suggests a critical role within mitochondria. In fact, PHB proteins are highly expressed in cells that heavily depend on mitochondrial function. In mitochondria, these two proteins assemble at the inner membrane to form a supra-macromolecular structure, which works as a scaffold for proteins and lipids regulating mitochondrial metabolism, including bioenergetics, biogenesis, and dynamics in order to determine the cell fate, death, or life. PHB alterations have been found in aging and cancer, as well as neurodegenerative, cardiac, and kidney diseases, in which significant mitochondrial impairments have been observed. The molecular mechanisms by which prohibitins regulate mitochondrial function and their role in pathology are reviewed and discussed herein.

Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis.

Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in CTNS gene leading to Fanconi syndrome. Independent studies reported defective clearance of damaged mitochondria and mitochondrial fragmentation in cystinosis. Proteins involved in the mitochondrial dynamics and the mitochondrial ultrastructure were analyzed in CTNS-/- cells treated with cysteamine, the only drug currently used in the therapy for cystinosis but ineffective to treat Fanconi syndrome. CTNS-/- cells showed an overexpression of parkin associated with deregulation of ubiquitination of mitofusin 2 and fission 1 proteins, an altered proteolytic processing of optic atrophy 1 (OPA1), and a decreased OPA1 oligomerization. According to molecular findings, the analysis of electron microscopy images showed a decrease of mitochondrial cristae number and an increase of cristae lumen and cristae junction width. Cysteamine treatment restored the fission 1 ubiquitination, the mitochondrial size, number and lumen of cristae, but had no effect on cristae junction width, making CTNS-/- tubular cells more susceptible to apoptotic stimuli.

Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis.

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/- by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

Administrative data underestimate acute ischemic stroke events and thrombolysis treatments: Data from a multicenter validation survey in Italy.

BACKGROUND: Informing health systems and monitoring hospital performances using administrative data sets, mainly hospital discharge data coded according to International-Classification-Diseases-9edition-Clinical-Modifiers (ICD9-CM), is now commonplace in several countries, but the reliability of diagnostic coding of acute ischemic stroke in the routine practice is uncertain. This study aimed at estimating accuracy of ICD9-CM codes for the identification of acute ischemic stroke and the use of thrombolysis treatment comparing hospital discharge data with medical record review in all the six hospitals of the Florence Area, Italy, through 2015. METHODS: We reviewed the medical records of all the 3915 potential acute stroke events during 2015 across the six hospitals of the Florence Area, Italy. We then estimated sensitivity and Positive Predictive Value of ICD9-CM code-groups 433*1, 434*1 and thrombolysis code 99.10 against medical record review with clinical adjudication. For each false-positive case we obtained the actual diagnosis. For each false-negative case we obtained the primary and secondary ICD9-CM diagnoses. RESULTS: The medical record review identified 1273 acute ischemic stroke events. The hospital discharge records identified 898 among those (true-positive cases),but missed 375 events (false-negative cases), and identified 104 events that were not eventually confirmed as acute ischemic events (false-positive cases). Code-group specific Positive Predictive Value was 85.7% (95%CI,74.6-93.3) for 433*1 and 89.9% (95%CI, 87.8-91.7) for 434*1 codes. Thrombolysis treatment, as identified by ICD9-CM code 99.10, was only documented in 6.0% of acute ischemic stroke events, but was 13.6% in medical record review. CONCLUSIONS: Hospital discharge data were found to be fairly specific but insensitive in the reporting of acute ischemic stroke and thrombolysis, providing misleading indications about both quantity and quality of acute ischemic stroke hospital care. Efforts to improve coding accuracy should precede the use of hospital discharge data to measure hospital performances in acute ischemic stroke care.

Inhibition of Drp1-mediated mitochondrial fission improves mitochondrial dynamics and bioenergetics stimulating neurogenesis in hippocampal progenitor cells from a Down syndrome mouse model.

Functional and structural damages to mitochondria have been critically associated with the pathogenesis of Down syndrome (DS), a human multifactorial disease caused by trisomy of chromosome 21 and associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, we demonstrated in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice -a widely used model of DS - a severe impairment of mitochondrial bioenergetics and biogenesis and reduced NPC proliferation. Here we further investigated the origin of mitochondrial dysfunction in DS and explored a possible mechanistic link among alteration of mitochondrial dynamics, mitochondrial dysfunctions and defective neurogenesis in DS. We first analyzed mitochondrial network and structure by both confocal and transmission electron microscopy as well as by evaluating the levels of key proteins involved in the fission and fusion machinery. We found a fragmentation of mitochondria due to an increase in mitochondrial fission associated with an up-regulation of dynamin-related protein 1 (Drp1), and a decrease in mitochondrial fusion associated with a down-regulation of mitofusin 2 (Mnf2) and increased proteolysis of optic atrophy 1 (Opa1). Next, using the well-known neuroprotective agent mitochondrial division inhibitor 1 (Mdivi-1), we assessed whether the inhibition of mitochondrial fission might reverse alteration of mitochondrial dynamics and mitochondrial dysfunctions in DS neural progenitors cells. We demonstrate here for the first time, that Mdivi-1 restores mitochondrial network organization, mitochondrial energy production and ultimately improves proliferation and neuronal differentiation of NPCs. This research paves the way for the discovery of new therapeutic tools in managing some DS-associated clinical manifestations.

Uptake of cervical cancer screening among the migrant population of Prato Province, Italy.

OBJECTIVE: To determine the level of participation in cervical cancer screening among the migrant population of Prato Province, Italy. METHODS: A retrospective cross-sectional study was conducted using data for women aged 25-64 years who were resident in one of the municipalities of Prato Province and had received at least one invitation to undergo a cervical cancer screening test. Data were extracted from both the Local Health Unit Serviceable Registry and cervical cancer screening archives for the period July 1, 2004, to June 30, 2007. RESULTS: Of the 69 459 residents eligible for cervical cancer screening, 7339 (10.6%) did not have Italian citizenship. Adherence with cervical cancer screening among the migrant population was lower than that of the Italian resident population: uptake increased from 52.4% in 2004 to 57.3% in 2007 among the Italian resident population, but decreased from 31.4% to 28.2% among migrants. CONCLUSION: The migrant population of Prato Province has decreased adherence with cervical cancer screening.

Cystinosin-LKG rescues cystine accumulation and decreases apoptosis rate in cystinotic proximal tubular epithelial cells.

BACKGROUND: Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. METHODS: We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. RESULTS: Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immune-electron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα- and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. CONCLUSION: Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.

Carboxyl-Terminal SSLKG Motif of the Human Cystinosin-LKG Plays an Important Role in Plasma Membrane Sorting.

Cystinosin mediates an ATP-dependent cystine efflux from lysosomes and causes, if mutated, nephropathic cystinosis, a rare inherited lysosomal storage disease. Alternative splicing of the last exon of the cystinosin sequence produces the cystinosin-LKG isoform that is characterized by a different C-terminal region causing changes in the subcellular distribution of the protein. We have constructed RFP-tagged proteins and demonstrated by site-directed mutagenesis that the carboxyl-terminal SSLKG sequence of cystinosin-LKG is an important sorting motif that is required for efficient targeting the protein to the plasma membrane, where it can mediate H+ coupled cystine transport. Deletion of the SSLKG sequence reduced cystinosin-LKG expression in the plasma membrane and cystine transport by approximately 30%, and induced significant accumulation of the protein in the Golgi apparatus and in lysosomes. Cystinosin-LKG, unlike the canonical isoform, also moves to the lysosomes by the indirect pathway, after endocytic retrieval from the plasma membrane, mainly by a clathrin-mediated endocytosis. Nevertheless, silencing of AP-2 triggers the clathrin-independent endocytosis, showing the complex adaptability of cystinosin-LKG trafficking.

Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis.

Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

BACKGROUND: Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H(+) symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis. METHODS: Morphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs). RESULTS: Despite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 µM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs. CONCLUSIONS: Our results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

Gender-related effects on urine L-cystine metastability.

Cystinuria is an autosomal recessive disease that causes L-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare L-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with L-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the L-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence L-cystine solubility. L-cystine solubility Z score was +0.44 ± 1.1 and -0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the L-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro L-cystine precipitation assays confirmed that these molecules induce higher rates of L-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate L-cystine solubility and may represent new targets for therapy in cystinuria.

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

Distribution of cystinosin-LKG in human tissues.

Nephropathic cystinosis is multisystemic progressive disorder caused by mutations of CTNS gene that encodes for the lysosomal cystine co-transporter cystinosin, and for a less abundant isoform termed cystinosin-LKG, which is expressed in not only lysosomes but also other cell compartments. To overcome the absence of high-quality antibodies against cystinosin, we have obtained a rabbit antiserum against cystinosin-LKG and have analyzed in human tissues the expression of the two known cystinosin isoforms by RT-PCR, and the expression of cystinosin-LKG by immunohistochemistry. In most tissues, CTNS-LKG represents 5-20 % of CTNS transcripts, with the exception of the testis that expresses both isoforms in equal proportions. Cystinosin-LKG was found to be highly expressed in renal tubular cells, pancreatic islets of Langerhans, Leydig cells of the testis, mucoserous glands of the bronchial wall, melanocytes and keratinocytes. These results are parallel with many features of cystinosis, such as early onset Fanconi syndrome, male infertility, diabetes mellitus and hypopigmentation. Intermediate expression levels were of the LKG isoform observed in the gastro-intestinal tract and thyroid glands; low levels of expression were observed in the brain, skeletal and cardiac muscles.

Transcriptional and posttranscriptional regulation of the CTNS gene.

Cell cysteine (Cys) levels and/or the [Cys/CySS] redox potential have been shown to regulate mRNA levels of the CTNS gene, which encodes for a lysosomal cystine (CySS) carrier that is defective in cystinosis. To investigate the mechanisms involved CTNS mRNA regulation, different portions of the CTNS promotor were cloned into a luciferase vector and transfected in HK2 cells. A 1.5-2.4-fold increase in luciferase activity was observed when cells were incubated in culture medium containing low CySS concentrations. Conversely, CTNS mRNA levels decreased by 47-56% in the presence of N-acetyl-L-cysteine (NAC). Chase experiments with actinomycin D (ActD) demonstrated a 3-fold stabilization of the CTNS mRNA when cells were cultured in low CySS medium for 48 h. Treatment of control cells with cyclohexamide (CHX) increased CTNS mRNA levels, suggesting that CHX blocked the synthesis of proteins involved in mRNA degradation or in repression of the CTNS gene. Finally, in vitro binding assays showed increased binding (30-110%) of the Sp-1 transcription factor to two regions of the CTNS promotor when cells were incubated in low CySS medium. These results indicate that the CTNS gene is actively regulated at the transcriptional and posttranscriptional levels and suggest that CTNS plays a pivotal role in regulating cell thiol concentrations.