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In infantile nephropathic cystinosis, variants of the CTNS gene cause accumulation of cystine in lysosomes, causing progressive damage to most organs. Patients usually present before 1 year of age with signs of renal Fanconi syndrome. Cysteamine therapy allows cystine clearance from lysosomes and delays kidney damage but does not prevent progression to end-stage kidney disease, suggesting that pathways unrelated to cystine accumulation are also involved. Among these, impaired autophagy, altered endolysosomal trafficking, and increased apoptosis have emerged in recent years as potential targets for new therapies. We previously showed that luteolin, a flavonoid compound, improves these abnormal pathways in cystinotic cells and in zebrafish models of the disease. Herein, we have investigated if prolonged luteolin treatment ameliorates kidney damage in a murine model of cystinosis. To this end, we have treated Ctns -/- mice from 2 to 8 months with 150 mg/kg/day of luteolin. No significant side effects were observed. Compared to untreated animals, analyses of kidney cortex samples obtained after sacrifice showed that luteolin decreased p62/SQSTM1 levels (p <0.001), improved the number, size, and distribution of LAMP1-positive structures (p <0.02), and decreased tissue expression of cleaved caspase 3 (p <0.001). However, we did not observe improvements in renal Fanconi syndrome and kidney inflammation. Kidney function remained normal during the time of the study. These results indicate that luteolin has positive effects on the apoptosis and endo-lysosomal defects of cystinotic proximal tubular cells. However, these beneficial effects did not translate into improvement of renal Fanconi syndrome. [Display omitted] • Cystinosis is a lysosomal storage disease that causes renal Fanconi syndrome. • Luteolin had a good safety profile in a murine model of cystinosis. • In this model, luteolin improved apoptosis and endo-lysosomal defects. • In this model, luteolin did not ameliorate significantly renal tubular dysfunction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: An infant with a clinical phenotype of early onset hypoaldosteronism has been screened for mutation analysis of the Cyp11b2 gene encoding aldosterone synthase enzyme. We have described a novel nonsense mutation in exon 3 (c.508C>T) that gave rise to a shorter protein (Q170X) and two known concurrent missense mutations (c.594A>C in exon 3 and c.1157T>C in exon 7) that led to substitution of glutamic acid for aspartic acid at amino acid position 198 (E198D) and of valine for alanine at amino acid position 386 (V386A). The father, who carried E198D plus V386A mutations, showed a fractional sodium excretion of 1.25% that was unmodified by dietary salt restriction, suggesting a mild haploinsufficiency. We examined by in silico analysis the effect of the mutations on the secondary and tertiary structures of aldosterone synthase to explain the inefficient enzymatic activity. The Q170X mutation produced a truncated protein, which was consequently associated with a loss of catalytic activity. As predicted by JPred web system and Dock 6.3 software, the concurrent expression of E198D and V386A mutations induced a significant secondary structure rearrangement and a shift of the heme group and the 18-hydroxycorticosterone substrate from their optimal placement. [Copyright &y& Elsevier]

Nephropathic cystinosis (NC) is an autosomal recessive disorder caused by mutations of the CTNS gene that encodes for a cystine transmembrane transporter. Several mutations have been described in the coding and promoter regions of the CTNS gene in affected individuals. We selected three patients with NC from two unrelated families, in whom sequence analysis of the CTNS gene detected only one or no mutations. Total RNA was isolated from peripheral blood mononuclear cells or fibroblasts and CTNS transcripts were analyzed. We observed a skipping of exon 5 (85 bp) in two siblings and an intron 9 retention of 75 bp associated with partial replication of exon 9 in the third patient. Genomic DNA analysis of intron regions surrounding exon 5 showed a point mutation in the hypothetical lariat branch site of intron 4 at position –24 (c.141–24 T > C) in the first two patients and a duplication of 266 bp including a part of exon and intron 9 in the third patient. Analysis of CTNS gene transcripts allowed identification of mutations in patients in whom CTNS mutations could not be detected by traditional DNA sequencing. These results support the hypothesis that cystinosis is a monogenic disorder. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [35S]cys- tine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder. [ABSTRACT FROM AUTHOR]

Bartter syndrome with sensorineural deafness (type IV Bartter syndrome) is a subtype of this tubular disease, and is due to mutations in the BSND gene. Out of a population of 92 patients with Bartter syndrome, five suffered from mild to severe hypoacusia and were selected for mutational screening. A homozygous mutation in the BSND gene was found in two female patients. The first patient was found to have a substitution in intron 1 donor splice site at position +5 (c.420+5G>C), whereas the second patient has a homozygous 3G>A substitution leading to the loss of the start codon for the translation of the BSND mRNA. The clinical courses of these two patients were remarkable for severe polyhydramnios, massive renal salt and water wasting, severe neonatal hypotonia, poor growth and unresponsiveness to prostaglandin inhibitors. The diuretic responses to furosemide and to hydrochlorothiazide were tested under KCl supplementation in one patient. A lack of response to both drugs suggested that inhibition of NaCl reabsorption in type IV Bartter syndrome is not restricted to the thick ascending limb of Henle. In one patient, a combined therapy with indomethacin and captopril was needed to discontinue intravenous fluids and improve weight gain. [ABSTRACT FROM AUTHOR]

Background. Haemolytic uraemic syndrome (HUS) is a disorder characterized by thrombotic microangiopathy, which is caused in ‘typical forms’ by gastrointestinal infections with Escherichia Coli species that produce verotoxins. Several studies have identified negative prognostic factors of the disease, among which prolonged oliguria, neurological involvement and increased leukocytosis have been more consistently reported. We have hypothesized that the genetic background may also predispose to the development of typical forms of HUS and may influence the clinical course of the disease. Methods. Fourteen polymorphisms, known to influence the coagulation pathway or the activity of the renin–angiotensin system, have been selected and studied in 150 Italian children with typical forms of HUS. Two hundred healthy Italian children were used as controls. Results. The risk of developing HUS was strongly associated with the platelet glycoprotein 1bα 145M allele (OR 3.08; CI: 1.62–5.85) (P < 0.001). A significant association was also found with polymorphisms located in the adipocyte-derived leucine aminopeptidase and factor V genes. A longer duration of dialysis was moderately associated with increased leukocytosis and with the 807T allele of the platelet glycoprotein 1a gene. High white blood cell count was also strongly associated with the risk of long-term sequelae (OR 2.91, CI: 1.21–6.98) (P < 0.02), whereas the 1166C allele of the angiotensin II type 1 receptor had a significant protective effect (OR 0.28, CI: 0.09–0.83) (P < 0.02). Conclusions. These results highlight the role of glycoprotein 1bα in the physiopathology of typical forms of HUS and show that the genetic background plays a role in the susceptibility and severity of the disease. [ABSTRACT FROM AUTHOR]

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 wild-type 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 CTNSRed 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nephropathic cystinosis (NC) is a severe disease that is complicated by early-onset chronic renal failure (CRF) and other complications related to cystine deposition in tissue. Since the 1980s, the prognosis of NC has dramatically improved after the introduction of cysteamine treatment. Limited data are available documenting improvement in prognosis. We reviewed our long-term data (follow-up 6.3-27.8 years) on 23 patients followed in the past 26 years. Overall, stage III CRF was reached at 10 years of age in >90% of patients, whereas >80% reached end-stage renal disease before the age of 14 years. Three patients died during the follow-up. Our analysis shows a clear improvement in renal outcome ( p = 0.001) and linear growth ( p = 0.04) in patients treated more recently. Improvement in the evolution of renal function was significantly associated with early initiation of cysteamine ( p = 0.006), with the dose of cysteamine ( p = 0.04), and with the use of angiotensin-converting enzyme inhibitors ( p = 0.01). Nonrenal long-term complications are similar to previously reported data. Of note, 3/23 patients developed rare forms of primary tumors that were successfully treated. In conclusion, our experience shows a significant improvement in the renal and nonrenal complications of cystinosis over the past decades and highlights the importance of early diagnosis in order to initiate cysteamine as soon as possible. [ABSTRACT FROM AUTHOR]

Abstract: The cysteine/cystine (Cys/CySS) couple represents one of the major cell thiol/disulfide systems and is involved in the regulation of several metabolic pathways and the cell redox state. Nephropathic cystinosis (NC) is an autosomal recessive disease characterized by renal cellular dysfunction due to mutations in the CTNS gene, which encodes cystinosin, a CySS lysosomal transporter. To analyze the mechanisms involved in cell damage in NC, we have investigated the effects of CTNS gene overexpression or inhibition on cell thiol/disulfide systems and vice versa. Overexpression of the CTNS gene had no remarkable effect on intracellular Cys/CySS and GSH/GSSG redox state. Silencing the CTNS gene increased cell CySS and Cys and decreased cell GSH and GSSG and increased mildly the redox state of the Cys/CySS-couple. Extracellular CySS and Cys deprivation for 48 h caused an oxidation of the Cys/CySS (73 mV) and GSH/GSSG (100 mV) redox couples and increased CTNS mRNA levels by 1.9±0.2-fold (p <0.001). Conversely, a reduced cell environment associated with a GSH/GSSG reduction from −250.1±3.10 to −330.6±4.70 mV (p <0.001) and a Cys/CySS reduction from −167.0±11.30 to −240.0±8.17 mV (p <0.005) was associated with a 40% decrease in CTNS mRNA levels (p <0.05). By regression analysis, CTNS gene expression was correlated with intracellular Cys level and with Cys/CySS redox state. [Copyright &y& Elsevier]

We employed potent and selective c-Src inhibitors to investigate the functional and molecular consequences of inhibited c-Src tyrosine kinase activity in osteoclasts. These pyrrolopyrimidine derivatives reduced osteoclast numbers and induced osteoclast disruption in vivo. In vitro, they inhibited resorption pit formation and osteoclastogenesis, impaired adhesion ability and actin ring organization, and induced programmed cell death in mature osteoclasts. The cell death receptor Fas and p53 were insensitive to c-Src modulation. The expression of the cyclin-dependent kinase (CDK)-inhibitor p21WAF1/CIP1 was markedly reduced, but neither Bcl-2 nor Bcl-xL or Bax were modulated by c-Src inhibition. Caspase-9, and to a lesser extent caspase-3, but not caspase-8, were transiently cleaved (activated) by treatment with the c-Src inhibitors. c-Src inhibition stabilized p38 mitogen-activated protein kinase (MAPK), whereas the c-Jun N-terminal kinase (JNK) pathway did not appear to be modulated by our compounds. Most interestingly, transient extracellular signal regulated kinase (ERK1/2) dephosphorylation followed by sustained remarkable rephosphorylation overwhelming control levels was observed in response to c-Src inhibition. Blockade of ERK1/2 rephosphorylation by PD98059 reduced osteoclast nuclear disruption, suggesting the involvement of this pathway in apoptosis. Collectively, these data demonstrate that small pyrrolopyrimidine derivatives impair osteoclast function and induce cell damage suggestive of apoptosis in vivo and in vitro, with mechanisms presumably involving selective sustained ERK1/2 phosphorylation. [Copyright &y& Elsevier]