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Recessive mutations in the CTNS gene encoding the lysosomal transporter cystinosin cause cystinosis, a lysosomal storage disease leading to kidney failure and multisystem manifestations. A Ctns knockout mouse model recapitulates features of cystinosis, but the delayed onset of kidney manifestations, phenotype variability and strain effects limit its use for mechanistic and drug development studies. To provide a better model for cystinosis, we generated a Ctns knockout rat model using CRISPR/Cas9 technology. The Ctns−/− rats display progressive cystine accumulation and crystal formation in multiple tissues including kidney, liver and thyroid. They show an early onset and progressive loss of urinary solutes, indicating generalized proximal tubule dysfunction, with development of typical swan-neck lesions, tubulointerstitial fibrosis and kidney failure, and decreased survival. The Ctns−/− rats also present crystals in the cornea, and bone and liver defects, as observed in patients. Mechanistically, the loss of cystinosin induces a phenotype switch associating abnormal proliferation and dedifferentiation, loss of apical receptors and transporters, and defective lysosomal activity and autophagy in the cells. Primary cultures of proximal tubule cells derived from the Ctns−/− rat kidneys confirmed the key changes caused by cystine overload, including reduced endocytic uptake, increased proliferation and defective lysosomal dynamics and autophagy. The novel Ctns−/− rat model and derived proximal tubule cell system provide invaluable tools to investigate the pathogenesis of cystinosis and to accelerate drug discovery.

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. ispartof: CELLS vol:10 issue:12 ispartof: location:Switzerland status: published

Study question:Is azoospermia in male cystinosis patients obstructive or nonobstructive in origin?Summary answer: Azoospermia in male cystinosis patients is obstructive in origin.What is known already: Cystinosis is a rare autosomal recessive metabolic disorder caused by mutations in the lysosomal membrane protein cystinosin (CTNS), which leads to intracellular lysosomal cystine accumulation. Depending on the disease severity, three forms are distinguished: infantile (most severe), juvenile (intermediate), and ocular (benign) form. The main clinical presentation in infantile cystinosis is the renal Fanconi syndrome, leading to end stage renal disease (ESRD) by the age of ten, if the patient is left untreated. Cysteamine, a cystine-depleting agent, beside renal replacement therapy, has improved the life expectancy of cystinosis patients. In contrast to females, male infantile cystinosis patients suffer from infertility.Study design, size, duration: Three groups were included in a prospective case control study; adult cystinosis patients (three infantile, two juvenile, and one ocular male patients), control group (seven adult fertile male subjects), and a positive control group for obstructive azoospermia (ten adult vasectomized males who underwent vasectomy 4-8 weeks prior to enrollment). In addition, clinical data and testicular sections for five adult infantile male cystinosis patients were retrospectively included in the study (in total, eight infantile cystinosis patients).Participants/materials, setting,methods:Scrotal ultrasound for screening for soft biomarkers of obstruction and semen analysis were performed in all prospectively included subjects, while sexual hormonal levels were evaluated only in the cystinosis patients. In addition, clinical data, semen analysis results, percutaneous epididymal sperm aspiration (PESA) results, sexual hormonal profile, and testicular sections from the retrospectively included cystinosis patients were analyzed. For the testicular sections, morphological evaluation and immunohistochemistry were performed.Main results and the role of chance:All testicular sections taken from three infantile cystinosis patients (3/3) showed the presence of testicular sperm and normal spermatogenesis, with a Johnsen’s score of 7 to 9. Besides, epididymal sperm was present in two other infantile cystinosis patients (2/2), obtained by PESA procedure, which was only performed in those two patients. Hence, all of the five investigated infantile cystinosis patients showed either testicular or epididymal sperm (5/5 or 100%), indicating that testicular function was preserved in those patients. In contrast, seven out of the total eight infantile cystinosis patients underwent semen analysis, and they all presented with azoospermia (7/7 or 100%), including four out of the five patients that showed testicular or epididymal sperm, while a semen sample from one infantile patient, who underwent testicular biopsy, could not be retrieved. Meanwhile, the two juvenile cystinosis patients (2/2) showed a reduced sperm count (15.9 and 6.4 million/ml), and the ocular cystinosis patient (1/1) showed normal sperm count (71.6 million/ml). Remarkably, the scrotal ultrasound results revealed a significant higher epididymis caput diameter (normalized to the testis volume) in infantile cystinosis group compared with healthy controls (1.63±0.66 mm/cm3 for infantile cystinosis vs 0.50±0.18 mm/cm3 for controls, p

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.