Cysteamine-bicalutamide combination treatment restores α-ketoglutarate and corrects proximal tubule phenotype in nephropathic cystinosis

Presented by Amer Jamalpoor, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacology, Utrecht University, The Netherlands

Amer Jamalpoor is the winner of the 2019 Professor Roz Anderson Memorial Prize for Best Scientific Poster and of the Cystinosis Ireland Best Lay Presentation awarded at the 5th Annual Dublin Cystinosis Workshop 2019.

Authors: A Jamalpoor¹, E Zaal², C Berkers², E Levtchenko³, R Masereeuw¹, M Janssen¹

Introduction: Nephropathic cystinosis is a severe genetic disorder caused by mutations in CTNS gene (cystine transporter), leading to the lysosomal accumulation of cystine and progressive organ damage. To date, no appropriate in vitro isogenic cystinotic cell models exist, a pre-requisite to study the link between the CTNS gene and the disease, and to investigate potential therapeutic strategies. Hence, our aim was to generate a cystinosis phenotype in human kidney cells using CRISPR/Cas9 and study cystinosis pathology.

Methods: We selectively knocked-out the CTNS gene in conditionally immortalized proximal tubular epithelial cells (ciPTEC). An untargeted metabolomics approach based on UHPLC-MS/MS was applied for the intra- and extracellular quantification of cystine and other metabolites differentially expressed in knock-out and control cells. Various assays were applied to monitor the lysosomal-autophagy dynamics (TFEB, LC3-II and DQ-BSA) in ciPTEC.

Results: The CTNS-/- isogenic cell line of ciPTEC showed a significant increase in cystine accumulation compared to healthy control cells (6.32 vs. 0.05 nmol/mg protein; p<0.001). Upon treatment with cystine depleting drug cysteamine, CTNS-/- cells showed a significant reduction in cystine levels (0.74 nmol/mg protein; p<0.01). Using metabolomics, we identified that not only cystine but also >25 metabolites and 9 metabolic pathways were affected (p<0.05) in cystinotic cells. CTNS-/- cells demonstrated an abnormally increased autophagy, confirmed by the increased TFEB nuclear translocation (2-fold; p<0.05), increased accumulation of LC3-II (2.3-fold; p<0.05) and decreased lysosomal degradation of DQ-BSA (2-fold; p<0.05). Of note, cysteamine had no effect on the restoration of autophagy, which might explain its limited effect on treating renal Fanconi syndrome. However, a promising registered drug molecule was found to be effective either alone or in combination with cysteamine in resolving cystinotic manifestations.

Conclusion: We developed a genetically engineered cystinotic cell model with isogenic controls. These cells provide a novel versatile tool to study the pathology of cystinosis and develop screens for drugs with the potential to reverse the symptoms. Metabolomics allowed an unbiased analysis of potential new targets for treatment of cystinosis.

Authors’ Affiliations:

¹Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacology, The Netherlands.

²Utrecht University, Biomolecular Mass Spectrometry and Proteomics, Department of Biomolecular Sciences, The Netherlands.

³Department of Paediatric Nephrology & Growth and Regeneration, University Hospitals Leuven KU Leuven, University of Leuven, Leuven, Belgium.