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Kidney Week

Abstract: FR-PO621

A Novel Renal Collecting Duct Model to Study Secondary Nephrogenic Diabetes Insipidus Associated with Cystinosis

Session Information

Category: Genetic Diseases of the Kidneys

  • 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Ferrulli, Angela, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy
  • Gijsbers, Rik, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
  • Di Mise, Annarita, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy
  • Cairoli, Sara, Ospedale Pediatrico Bambino Gesu, Roma, Lazio, Italy
  • Van den heuvel, Lambertus P.W.J., Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
  • Levtchenko, Elena, Universiteit van Amsterdam, Amsterdam, Noord-Holland, Netherlands
  • Valenti, Giovanna, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy
Background

Cystinosis is a severe lysosomal disorder caused by mutations in the CTNS gene, encoding for the lysosomal transporter cystinosin, resulting in the accumulation of cystine throughout the body. It manifests as Renal Fanconi syndrome in the first months of life, and lastly progresses into end stage renal failure. Secondary Nephrogenic Diabetes Insipidus (NDI) has been reported as a secondary complication of cystinosis due to the resistance to vasopressin, a key hormone that, in the collecting duct activates the vasopressin-sensitive water channel Aquaporin-2 (AQP2), regulating water reabsorption. Over the past years, the development of several in vivo and in vitro models of cystinosis has contributed to understand the pathophysiology of this severe disease. Nevertheless, the molecular mechanisms causing the secondary NDI phenotype have not been investigated due to the lack of collecting duct cellular models.

Methods

A CRISPR/Cas9 CTNS knock out model derived from MCD4 cells, a mouse renal collecting duct cell line, stably expressing human AQP2 and vasopressin receptor 2 (V2R), was established and validated by Sanger sequencing, qPCR and mass spectrometry (MS). Osmotic water permeability measurements in presence or absence of Desmopressin (DDAVP), a synthetic vasopressin analog to investigate AQP2 function were carried out.

Results

Sanger sequencing analysis demonstrated that CTNS was efficiently CRISPRed. This result was further confirmed by a significant reduction of CTNS transcript levels, up to 65%, and a significant accumulation of cystine by MS in CRISPRed CTNS KO cells with respect to wt. Preliminary studies on osmotic water permeability indicate that, compared to control, in CTNS CRISPR cells, the osmotic water permeability does not significantly increase in response to DDAVP treatment, consistent with an impairment of the vasopressin-AQP2 pathway.

Conclusion

We provide here the first CTNS KO collecting duct in vitro model useful for the study of secondary NDI in cystinosis.

Funding

  • Government Support – Non-U.S.