Cellular mechanisms of renal disease in cystinosis

Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in the CTNS gene, which encodes for the lysosomal cystine transporter cystinosin. The disease is characterized by lysosomal cystine accumulation throughout the body. Although cystinosis is a multi-organ disease, the first and the most severely affected organ is the kidney. Cystinotic patients presentnbsp;generalized proximal tubular dysfunction (renal Fanconi syndrome) and also glomerular proteinuria as a sign of glomerular damage. The disease progression leads to the end-stage renal disease and the need for renal replacement therapy. Current treatment of cystinosis is based on cysteamine therapy. Cysteamine enters the lysosomes and splits cystine molecule into two products that can freely exit into the cytoplasm. Lysosomal storage is therefore prevented if the drug is given regularly. Timely started, cysteamine therapy slows down the disease progression and prevents the development of extra-renal complications. However, it is ineffective against the Fanconi syndrome and eventual glomerular damage associated with the disease. There is therefore a need for therapy improvement, which requires a better understanding of cellular mechanisms of the disease pathogenesis (chapter 1.1). Several lines of evidence indicate that, apart from its transporter function, cystinosin might play a role in cell signalling and the regulation of endo-lysosomal function. Cystinosin-deficient cells are known to accumulate autophagosome markers indicative of a perturbed autophagic flux, which is dependent on correct mTOR signalling. The development of proximal tubular dysfunction is likely to be dependent, at least partly, on impaired endocytosis, which is responsible for protein reabsorption in proximal tubules. Preliminary data indicated the possibility of disturbed Ca2+ signalling in cystinotic cells. In this doctoral project we investigated cellular mechanisms of the pathogenesis of cystinosis and explored the possible roles of cystinosin in cell signalling (including mTOR and Akt kinases), cell motility and cytoskeleton regulation and Ca2+ signalling and homeostasis (chapter 1.2). Study of cystinosis has been hindered by the absence of suitable models. Knock-out murine models have been developed only recently, and they do not fully reproduce the human disease. For instance, renal phenotype in cystinosin-deficient animals was dependent on the genetic background, and even animals with full blown proximal tubular dysfunction had no evidence of glomerular damage. To study the molecular mechanisms of the disease pathogenesis, we used stable lines of human proximal tubular epithelial cells and glomerular podocytes derived from healthy donors and cystinotic patients. We confirmed the obtained results in control cell lines with the CTNS gene silencing using specific siRNA. We have demonstrated that cystinosin-deficient cells had impaired endocytosis and altered lysosomal function (chapter 3.1). Endosomal and lysosomal compartments had abnormal shape and localization, which could not be rescued by treatment of cells with cysteamine. We demonstrated urinary loss of podocytes (podocyturia) in cystinotic patients treated with cysteamine, which could contribute to the development of glomerular damage. Studies of kinase signalling demonstrated that Akt and mTORC1 signalling were deregulated in cystinotic cells. Cystinotic podocytes were characterized by altered actin cytoskeleton, increasednbsp;and impaired adhesion, which corresponds to podocyte damage in in vitro experimental systems. Since Akt kinase is a known regulator of cell motility and adhesion, we tested whether selective Akt inhibition could rescue the pathological phenotype and observed a decrease of cystinotic podocyte motility to the levels comparable with control podocytes, which could not be attained by treatment of cells with cysteamine (chapter 3.2). mTORC1 reactivation in response to growth factors and nutrients was delayed, and Akt phosphorylation was potentiated in cells, deficient for cystinosin (chapter 3.3). Probing for Ca2+ signalling abnormalities did not reveal any significant difference between control and cystinotic proximal tubular cells, except for slight potentiation of Ca2+ release in response to ATP (chapter 3.4).status: publishe