Programmed Cell Death in Cystinosis.

Cystinosis is a lethal autosomal recessive disease that has been known clinically for over 100 years. There are now specific treatments including dialysis, renal transplantation and the orphan drug, cysteamine, which greatly improve the duration and quality of patient life, however, the cellular mechanisms responsible for the phenotype are unknown. One cause, programmed cell death, is clearly involved. Study of extant literature via Pubmed on "programmed cell death" and "apoptosis" forms the basis of this review. Most of such studies involved apoptosis. Numerous model systems and affected tissues in cystinosis have shown an increased rate of apoptosis that can be partially reversed with cysteamine. Proposed mechanisms have included changes in protein signaling pathways, autophagy, gene expression programs, and oxidative stress.

Microvesicle delivery of a lysosomal transport protein to ex vivo rabbit cornea.

Therapeutic use of transmembrane proteins is limited because of irreversible denaturation when away from their native lipid membrane. Mutations in lysosomal membrane transport proteins cause many lethal disorders including cystinosis which results from mutations in CTNS, which codes for the lysosomal cystine transport protein, cystinosin. Cystinosin-deficient fibroblasts, including keratocytes (corneal fibroblasts) accumulate lysosomal cystine. Cystinosis patients develop highly painful corneal cystine crystals, resulting in severe visually debilitating photophobia. The only available therapy is daily treatment with cysteamine eye drops. We have previously shown that microvesicles containing functional cystinosin are spontaneously produced by infecting Spodoptera frugiperda cells (Sf9) with baculovirus containing human wt CTNS. Infecting Sf9 cells for 3 days at a MOI of 1 yields 1011microvesicles /ml with a modal diameter of 90 nm. Addition of these vesicles to cultures of cystinotic fibroblasts produces cystine depletion over the course of 96 h, which persists for 2 weeks. In this paper we show that addition of such microvesicles containing cystinosinGFP to ex vivo rabbit ocular globes yields punctate perinuclear green fluorescence in the corneal keratocytes. These results support potential therapeutic use of these cystinosin containing microvesicles in treating cystinotic corneal keratopathy with the advantage of administering twice/month instead of daily topical administration.

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.

The incidence of atubular glomeruli in nephropathic cystinosis renal biopsies.

Nephropathic cystinosis results from lysosomal cystine storage and, if untreated with cysteamine, results in end-stage renal disease by 10 years of age. The renal Fanconi syndrome occurs in the first year of life and is accompanied by a characteristic "swan neck" deformity of the proximal renal tubule. The linkage between cystine storage, Fanconi syndrome, and renal failure has not been understood. This study reports the presence of substantial numbers of atubular glomeruli (ATG) in end-stage cystinotic renal tissue. Compared to normal renal tissue, cystinotic kidneys at end stage had 69% atubular glomeruli and 30% atrophic glomeruli. Normal renal tissue had 4% ATG and 0% atrophic glomeruli (p<0.0001 for both comparisons). These nonfunctioning nephrons may be the end result of cell loss from the tubules and represent the final stage of the swan neck deformity. The process is consistent with the previously reported increased apoptosis in renal tubule cells due to lysosomal cystine storage.

A review of the role of enhanced apoptosis in the pathophysiology of cystinosis.

The role of lysosomal cystine in development of the phenotype in cystinosis is problematic, in that the cystine is effectively isolated from the rest of cellular metabolism. Several models have been proposed, but most do not provide a mechanism for such an interaction. During early apoptosis the lysosomes are permeablized, providing such access. We have shown that lysosomal cystine enhances apoptosis in cultured normal and cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, that the process occurs via mixed disulfide (cysteinylation) formation, and that PKC delta is involved. Further, the "swan neck" deformity of proximal renal tubules, long a hallmark of cystinosis, is explicable via this model, as is the renal failure that results from progression of tubule cell loss to atubular glomeruli. Modification of this process by other genes may explain the milder forms of the disease.

Increased apoptosis in cystinotic fibroblasts and renal proximal tubule epithelial cells results from cysteinylation of protein kinase Cdelta.

Cystinosis is a rare genetic disease characterized by defective lysosomal cystine transport and increased lysosomal cystine. How lysosomal cystine causes the lethal nephropathic phenotype is unknown. It was shown recently that cultured fibroblasts and renal proximal tubule epithelial cells whose lysosomes are cystine-loaded display a two-fold or greater increase in apoptosis after both intrinsic and extrinsic stimuli. The mechanism for the increased apoptosis is unknown. Protein kinase Cdelta (PKCdelta) is a proapoptotic protein kinase that has been shown in vitro to be activated via cysteinylation. This report now shows that PKCdelta forms disulfide bonds specifically with cystine that is released from lysosomes in cultured fibroblasts and renal proximal tubule epithelial cells during apoptosis. PKCdelta in cystinotic fibroblasts and renal proximal tubule epithelial cells have a four- to six-fold greater association with its substrate, lamin B, and a 2.5-fold increase in specific activity after TNF-alpha exposure. Both RNA inhibition and chemical inhibition of PKCdelta resulted in a significant decrease in apoptosis in cystinotic cells but not in normal cells. It is proposed that abnormally increased apoptosis plays a role in evolution of the cystinotic phenotype.

Lysosomal cystine augments apoptosis and causes the phenotype in cystinosis.

Nephropathic cystinosis is a lethal inborn error of metabolism that destroys kidney function by age 10 years. It is characterized by lysosomal cystine accumulation. How the cystine causes the phenotype is an open question. We propose that during apoptosis, permeablized lysosomes permit cystine to reach the cytosol where mixed disulfide formation occurs, augmenting apoptosis by interaction with a variety of pro-apoptotic proteins.

Potential role of apoptosis in development of the cystinotic phenotype.

Much still remains unclear about the proximal biochemical effects of mutations on development of the phenotype in inborn errors of metabolism. Cystinosis is an example of this phenomenon. We have recently shown that cystinotic cells undergo apoptosis at a two- to fourfold higher rate than controls. Cystinotic cells pre-treated with cysteamine, normalizing cystine content, display a four- to fivefold decrease in apoptosis, while normal cells pre-treated with cystine dimethylester, increasing lysosomal cystine, exhibit a fivefold increase in apoptosis. We speculate that cystine exits the lysosomal compartment during early apoptosis and affects apoptotic proteins in the cytosol, causing an inappropriate commitment to proceed to cell death. The resulting chronic hypocellularity could account for all the characteristics of the nephropathic cystinotic phenotype. The milder variants of cystinosis may result from modifying mutations within an apoptotic protein, ablating the proapoptotic effects of cystine. Failure of the mouse knockout for cystinosis to show renal involvement may be the result of differences in apoptotic processes between man and mouse. Apoptosis is a major final common pathway for many disease states. Therefore, a better understanding of the effect of lysosomal cystine on apoptosis may help to clarify development of other diseases.