2 Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction 33 Discussion The last common ancestor of humans and zebrafish was a marine vertebrate that lived approximately 450 million years ago; however, 70% of protein-coding human genes are related to genes found in the zebrafish and 84% of genes known to be associated with human disease have a zebrafish counterpart40,41. In the current study we established and characterized actns‒/‒zebrafish mutant and uncovered many important aspects of the renal pathophysiology resulting in the functional abnormalities of the early larval stage of the ctns‒/‒ zebrafish. The key-findings of the study are: 1) significant cystine accumulation, increased mortality and increased rate of apoptosis in the ctns‒/‒ zebrafish which are partially responsive to cysteamine treatment. 2) early impairment of pronephros affecting both glomerular and proximal tubular function. Significant accumulation of cystine, the main pathologic landmark of nephropathic cystinosis, validated our model and confirmed the pathogenic nature of the mutation. Both morphant and ctns‒/‒ zebrafish larvae showed comparable phenotypes. We preferred to proceed with embryos and larvae of the genetic ctns‒/‒ model, as it is well known that the phenotype severity and toxicity of morphant models are dependent on the morpholino dose injected42, which is not an issue in the genetic model. Although cystinosin in humans is ubiquitously expressed, the expression is especially high in the kidneys43. Interestingly, the adult ctns‒/‒zebrafish kidneys accumulated the highest concentrations of cystine (>50 times the wt) contrasting the murine model of cystinosis in which the highest cystine accumulations were observed in adult liver and spleen10,44. Interestingly, similar to our data in zebrafish homogenates, Wilmer et al., 2011 detected a significant increase in GSSG in cystinotic PTECs compared to wt without alterations in GSH levels45. In their in vitro study PTECs in culture responded to the antioxidant cysteamine treatment by decreasing GSSG and increasing GSH45 while in vivo in fish only a significant fall in GSSG was observed. Cystine accumulation has been shown to cause increased apoptosis rate inhuman tissues and in the mouse model of cystinosis7,11,12. The suggested mechanism is enhanced cysteinylation of pro-apoptotic enzyme protein kinase C delta due to the lysosomal overload and increased lysosomal membrane permeability46. Moreover, oxidative mitochondrial stress and ER stress have been attributed to enhanced apoptosis in cystinotic cells7,12.Similarly, in our zebrafish model the AO rapid screening technique revealed increased number of apoptotic signals in whole larvae. These data were confirmed by caspase-3 immunohistochemistry and enzyme activity in homogenates of the ctns‒/‒ larvae. Importantly, AO apoptotic signals were significantly reduced by therapeutic doses
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