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.

Cystinosin deficiency causes podocyte damage and loss associated with increased cell motility.

The involvement of the glomerulus in the pathogenesis of cystinosis, caused by loss-of-function mutations in cystinosin (CTNS, 17p13), is a matter of controversy. Although patients with cystinosis demonstrate glomerular lesions and high-molecular-weight proteinuria starting from an early age, a mouse model of cystinosis develops only signs of proximal tubular dysfunction. Here we studied podocyte damage in patients with cystinosis by analyzing urinary podocyte excretion and by in vitro studies of podocytes deficient in cystinosin. Urine from patients with cystinosis presented a significantly higher amount of podocytes compared with controls. In culture, cystinotic podocytes accumulated cystine compatible with cystinosin deficiency. The expression of podocyte specific genes CD2AP, podocalyxin, and synaptopodin and of the WT1 protein was evident in all cell lines. Conditionally immortalized podocyte lines of 2 patients with different CTNS mutations had altered cytoskeleton, impaired cell adhesion sites, and increased individual cell motility. Moreover, these cells showed enhanced phosphorylation of both Akt1 and Akt2 (isoforms of protein kinase B). Inhibition of Akt by a specific inhibitor (Akti inhibitor 1/2) resulted in normalization of the hypermotile phenotype. Thus, our study extends the list of genetic disorders causing podocyte damage and provides the evidence of altered cell signaling cascades resulting in impaired cell adhesion and enhanced cell motility in cystinosis.

[Significance of the urine strip test in case of stunted growth]. / Intérêt de l'examen des urines à la bandelette en cas de retard de croissance.

Observation of stunted growth in children usually leads the general practitioner to refer the patient to endocrinologists or gastroenterologists. In most cases, after a complementary check-up, the diagnosis is made and treatment is initiated. However, certain cases remain undiagnosed, particularly renal etiologies, such as proximal tubulopathy. The urine strip test at the initial check-up would be an easy and inexpensive test to avoid delayed diagnosis. The aim of the present paper is to increase general physicians' and pediatricians' awareness of the significance of questioning the parents and using the urine strip test for any child presenting stunted growth. We report a patient case of a 20-month-old child admitted to the emergency department for severe dehydration. He had displayed stunted growth since the age of 5 months and showed a negative etiologic check-up at 9 months of age. Clinical examination at admission confirmed stunted growth with loss of 2 standard deviations and signs of dehydration with persistent diuresis. Skin paleness, ash-blond hair, and signs of rickets were also observed and the urine strip test showed positive pads for glycosuria and proteinuria. Polyuria and polydipsia were also revealed following parents' questioning, suggesting proximal tubulopathy (Fanconi syndrome). Association of stunted growth, rickets, polyuria and polydipsia, glycosuria (without ketonuria and normal glycemia), and proteinuria suggest nephropathic cystinosis. Ophthalmic examination showed cystine deposits in the cornea. The semiotic diagnosis of nephropathic cystinosis was confirmed by leukocyte cystine concentrations and genetic investigations. This case report clearly illustrates the significance of the urine strip test to easily and quickly concentrate the diagnosis of stunted growth on a renal etiology (glycosuria, proteinuria), especially on proximal tubulopathy for which the most frequent cause is nephropathic cystinosis. Specificity of nephropathic cystinosis treatment is that the age of treatment initiation is crucial and determinant for the prognosis of the disease and the onset of final stage renal failure. Therefore, the urine strip test should be included in the systematic check-up of stunted growth to identify any renal etiology.

Novel techniques and newer markers for the evaluation of "proximal tubular dysfunction".

Renal Fanconi syndromes are both clinically challenging and physiologically fascinating. The diagnosis requires a certain index of suspicion to correctly identify the clinical symptomatology and pursue the appropriate laboratory evaluations. The renal Fanconi syndrome (FS) is a defect of proximal tubular function attributable to different rare inherited diseases or acquired disorders caused by a multitude of exogenous agents. It can manifest as complete or incomplete FS, characterized by low molecular weight proteinuria, glucosuria, aminoaciduria, and loss of electrolytes, bicarbonate and lactate. Implementation of new methods and recent findings from urinary proteome pattern in patients with renal FS has led to the identification of new markers for proximal tubular dysfunction. Future combined proteomic and metabonomic studies will provide additional potential biomarkers and may help to gain novel insights in the diagnosis and differentiation of the various forms of FS. Moreover, the observation of poor renal uptake of 99 mTc-DMSA in patients with tubular proteinuria, which is not fully understood yet, may also help to elucidate the individual basis of FS in early stages. This review focuses on the new advances in the evaluation of proximal tubular dysfunction in various forms of Fanconi syndrome.

Successful treatment of the murine model of cystinosis using bone marrow cell transplantation.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. Cystine accumulates in every organ in the body and leads to organ damage and dysfunction, including renal defects. Using the murine model for cystinosis, Ctns(-/-) mice, we performed syngeneic bone marrow cell (BMC), hematopoietic stem cell (HSC), and mesenchymal stem cell transplantation. Organ-specific cystine content was reduced by 57% to 94% in all organs tested in the BMC-treated mice. Confocal microscopy and quantitative polymerase chain reaction revealed a large quantity of transplanted BMC in all organs tested, from 5% to 19% of the total cells. Most of these cells were not from the lymphoid lineage but part of the intrinsic structure of the organ. The natural progression of renal dysfunction was prevented, and deposition of corneal cystine crystals was significantly improved in the BMC-treated mice. HSC had the same therapeutic effect as whole BMC. In contrast, mesenchymal stem cell did not integrate efficiently in any organ. This work is a proof of concept for using HSC transplantation as a therapy for cystinosis and highlights the efficiency of this strategy for a chronic, progressive degenerative disease.

Sedoheptulokinase deficiency due to a 57-kb deletion in cystinosis patients causes urinary accumulation of sedoheptulose: elucidation of the CARKL gene.

The most common mutation in the nephropathic cystinosis (CTNS) gene is a homozygous 57-kb deletion that also includes an adjacent gene carbohydrate kinase-like (CARKL). The latter gene encodes a protein that is predicted to function as a carbohydrate kinase. Cystinosis patients with the common 57-kb deletion had strongly elevated urinary concentrations of sedoheptulose (28-451 mmol/mol creatinine; controls and other cystinosis patients <9) and erythritol (234-1110 mmol/mol creatinine; controls and other cystinosis patients <148). Enzyme studies performed on fibroblast homogenates derived from patients carrying the 57-kb deletion revealed 80% reduction in their sedoheptulose phosphorylating activity compared to cystinosis patients with other mutations and controls. This indicates that the CARKL-encoded protein, sedoheptulokinase (SHK), is responsible for the reaction: sedoheptulose + ATP --> sedoheptulose-7-phosphate + ADP and that deletion of CARKL causes urinary accumulation of sedoheptulose and erythritol.

Exfoliated human proximal tubular cells: a model of cystinosis and Fanconi syndrome.

The renal Fanconi syndrome (FS) is characterised by generalised proximal tubular dysfunction. Cystinosis is the most common genetic cause of the FS and results from defective function of cystinosin, due to mutations of the CTNS gene leading to intralysosomal cystine accumulation. Despite these advances in our understanding of the molecular basis of cystinosis, the mechanisms of proximal tubular cell (PTC) dysfunction are still unknown. We have further characterised an in vitro model of cultured cells exfoliated in patients' urine. We cultured cells from 9 cystinosis children, 4 children with Lowe syndrome and 8 controls. PTC phenotype and homogeneity were studied by lectin staining, immunocytochemistry (using ZO-1 as an epithelial marker) and enzyme cytochemistry (using gamma-glutamyltransferase as a PTC marker). All cultured cells showed PTC phenotype. Cystinosin was stained using anti-cystinosin antibody and co-localised to the lysosomes with LAMP-2 antibody. Additionally, we have demonstrated significantly elevated intracellular cystine levels in cystinotic cell lines (13.8+/-2.3 nmol 1/2 cystine/mg protein, P <0.001) compared with controls. We believe this in vitro model will allow further investigation of cystinosis and other types of the FS.

Negligible urinary cysteamine loss in cystinosis patients with Fanconi syndrome.

Cystinosis is an inborn error of lysosomal cystine transporter, resulting in cystine accumulation in lysosomes of all cells. Renal Fanconi syndrome is an early sign of kidney involvement in cystinosis patients. Cysteamine, a small amino-thiol, depletes intralysosomal cystine content and reduces organ damage. However, it does not reverse renal Fanconi syndrome and only postpones the progression to renal failure. We examined whether cysteamine could be lost in the urine of cystinosis patients with Fanconi syndrome, which may explain the inefficiency of treatment. Urinary cysteamine loss was studied in 6 cystinosis patients with and without Fanconi syndrome and was less than I% of ingested dose in all patients.

Polyuria and proteinuria in cystinosis have no impact on renal transplantation. A report of the North American Pediatric Renal Transplant Cooperative Study.

Because cystinotic patients are polyuric and may have severe proteinuria, each of which is a potential risk factor for graft thrombosis, preemptive transplantation for them is questionable. The objectives of this study were to characterize the changes in urine volume and protein excretion at various stages of cystinosis, determine whether there is serologic evidence of hypercoagulability, and review the clinical experience in renal transplantation in cystinotic children. The records of cystinotic patients followed at the Montreal Children's Hospital between 1992 and 1998 were reviewed. Urinary volume, protein excretion, and coagulation markers were collected to determine the glomerular filtration rate (GFR) >50 ml/min/1.73 m2, <20 ml/min/1.73 m2, before and after starting dialysis. In addition, graft failure and graft thrombosis rates were obtained from the North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) database. Urinary volume and protein excretion remained elevated throughout different phases of the disease. Coagulation factors were within normal limits for all patients. In the NAPRTCS database there were four thromboses among the 114 patients transplanted cystinotic patients. All these occurred in cadaveric grafts and only one occurred after preemptive transplantation. Despite polyuria and severe proteinuria, children with cystinosis do not appear to be at an increased risk of graft failure or graft thrombosis.

Cistinosis nefropática infantil / Nephropathic cystinosis in children

La cistinosis nefropática, rara afección recesiva, se produce por defecto en el transporte lisosomal de cistina, y depósitos de cristales intracelulares en riñón, córnea, y otros tejidos. Constituye la primera causa congénita de síndrome de Fanconi, y evoluciona en la primera década de la vida a insuficiencia renal crónica. El diagnóstico se confirma por una detección de cistina en leucocitos y linfoblastos circulantes. Su tratamiento consiste en la reposición de las pérdidas por la tubolopatía, administración de cisteamina, que depleta cistina y favorece su transporte por la pared lisosomal. El objetivo de la presentación es dar a conocer el primer caso de cistinosis documentado y tratado en Chile. Se presenta el caso de un menor hospitalizado a los quince meses de vida, con desnutrición avanzada, raquitismo clínico, deshidratación severa, acidosis metabólica, hipokalemia e hipofosfemia severas, comprobándose tubulopatía de Fanconi. Se detectó concentración elevada de cistina en polimorfonucleares, confirmando diagnóstico de cistinosis. En tratamiento desde hace dos años con cisteamina oral, muestra excelente evolución pondoestatural y conservación de la función renal, persistiendo la tubulopatía

Medullary nephrocalcinosis in nephropathic cystinosis.

Children with nephropathic cystinosis excrete large amounts of calcium and phosphate due to renal tubular Fanconi syndrome, and also receive substantial supplements of phosphate and alkalinizing agents. Since these constitute risk factors for nephrocalcinosis, we evaluated 41 children age 2 months to 15 years with nephropathic cystinosis and good renal function by performing retroperitoneal ultrasound examinations in a blinded fashion. We also retrospectively analyzed parameters of calcium and phosphate metabolism representing 216 person-years of data on these children. Fifteen children had no evidence of medullary nephrocalcinosis, while 18 had mild nephrocalcinosis, and 8 severe nephrocalcinosis; 5 had renal stones. Mean urine calcium and phosphate concentrations increased from 1.47 mM and 5.30 mM, respectively, in children without nephrocalcinosis to 1.60 mM and 5.69 mM in children with mild nephrocalcinosis to 1.66 mM and 6.19 mM in children with severe nephrocalcinosis. Mean urine pH ranged from 7.5 to 8.1. The mean (+/- SD) age of the 26 patients with nephrocalcinosis was 9.4 +/- 3.8 years compared with 5.1 +/- 3.8 years for those without nephrocalcinosis (P < 0.005). Serum calcium, phosphate, vitamin D, and parathyroid hormone did not correlate with frequency or degree of nephrocalcinosis. We conclude that nephrocalcinosis frequently accompanies nephropathic cystinosis, can be detected by ultrasound examination, and might be managed by reducing oral replacement of phosphate, calcium, vitamin D, and citrate. Consideration should be given to truncating phosphate replacement once bone growth ceases.

Hypercalciuria and ultrasound abnormalities in children with cystinosis.

We noted microscopic haematuria in children with cystinosis. To investigate this we studied urinary calcium excretion and undertook renal ultrasound scans. Most patients had elevated urinary calcium excretion and all had abnormal appearances on ultrasound scan, ranging from increased cortical echogenicity only to those with increased cortical and medullary echogenicity. The ultrasound scan appearance was graded and correlated with laboratory parameters. It remains unclear as to the aetiology of the ultrasound findings and whether they are a consequence of treatment or a hitherto unrecognised feature of the disease.

Culture of renal tubular cells from the urine of patients with nephropathic cystinosis.

Nephropathic cystinosis represents a prototype for lysosomal storage diseases and is the most common cause of renal tubular Fanconi's syndrome. Mechanisms of the tubular transport defects in this disease have not been defined, however, in part because the cells readily cultured from affected patients, leukocytes and fibroblasts, do not express epithelial transport functions. Except for a single autopsy report, renal tubular cells from these patients have not been studied in vitro. In these studies, noninvasive harvesting and culture of renal tubular cells from the urine of patients with cystinosis is described. Cultures of renal tubular cells could be established from over 50% of the isolates which contained viable cells and which remained uncontaminated in vitro. Cells had an epithelial morphology in culture, and the majority of cultured cells expressed proximal tubular brush border marker enzyme. Cultured cells also expressed the storage defect in vitro, containing cystine levels up to 100 times those of normal cells. Cultured cells could be depleted of cystine by using the thiol cysteamine. This in vitro model system should be very useful for studying the mechanisms of renal tubular transport defects in this disease.

Urine glyceraldehyde excretion is elevated in the renal Fanconi syndrome.

We analyzed urinary constituents using GC/MS in 16 children with the renal Fanconi syndrome and 13 normal individuals. Urine glyceraldehyde levels were strikingly elevated in the renal Fanconi syndrome group (mean 5.1 +/- 4.8 mg/mg creatinine) compared to levels in the normal group (mean 0.04 +/- 0.04 mg/mg creatinine, P less than 0.001). Urine lactate levels were also elevated in the renal Fanconi syndrome group (mean 2.3 +/- 2.6 mg/mg creatinine) compared to normals (mean 0.01 +/- 0.01 mg/mg creatinine, P less than 0.003). Only small elevations of glyceraldehyde and lactate were found in urine from children with other renal disorders. Serum levels of glyceraldehyde and lactate were no greater in individuals with the Fanconi syndrome than in the normals. The fractional reabsorption of both glyceraldehyde and lactate was virtually complete in the normals, but was markedly impaired in the Fanconi syndrome patients where, in some cases, glyceraldehyde excretion greatly exceeded the excretion of creatinine. We conclude that marked glyceraldehyde excretion is a previously unrecognized feature of the renal Fanconi syndrome which may result from disordered proximal tubular glycolytic metabolism. Further studies will be required to determine the role of glyceraldehyde loss in the pathogenesis of this generalized disturbance of proximal tubular function.

Urinary excretion of aldosterone metabolite Kelly-M1 in patients with adrenal dysfunction.

Using tetrahydroaldosterone antibody a radioimmunoassay was developed to measure substance Kelly-M1 (K-M1) in human urine. The normal values were lower than observed by Kelly et al. who discovered the catabolite after giving large doses of exogenous aldosterone. While in essential hypertension the excretion of K-M1 was predominantly within the normal range, elevated values were found in most cases of 21-hydroxylase deficiency, both the simple virilizing and salt losing form, primary aldosteronism, renal hypertension and cystinosis. Our findings suggest that K-M1 may be formed from 21-deoxyaldosterone and/or by microbial intervention from aldosterone or its metabolites.