Kidney Week

Abstract: FR-PO572

Downregulation of the Vasopressin-AQP2 Pathway Explains Secondary Nephrogenic Diabetes Insipidus Associated with Cystinosis: In Vivo and In Vitro Evidence

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Ferrulli, Angela, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy

Angela Ferrulli, MS

• Gijsbers, Rik, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium

Rik Gijsbers, PhD

• Di Mise, Annarita, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy

Annarita Di Mise, PhD

• Bokenkamp, Arend, Amsterdam UMC Locatie AMC, Amsterdam, Noord-Holland, Netherlands

Arend Bokenkamp, MD, PhD, FASN

• Cairoli, Sara, Ospedale Pediatrico Bambino Gesu, Roma, Italy

Sara Cairoli, PhD

• Tamma, Grazia, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy

Grazia Tamma, PhD

• Van den Heuvel, Lambertus P.W.J., Katholieke Universiteit Leuven, Leuven, Flanders, Belgium

Lambertus P.W.J. Van den Heuvel, PhD

• Levtchenko, Elena, Amsterdam UMC Locatie AMC, Amsterdam, Noord-Holland, Netherlands

Elena Levtchenko, MD, PhD

• Valenti, Giovanna, Universita degli Studi di Bari Aldo Moro, Bari, Puglia, Italy

Giovanna Valenti, PhD

Background

Cystinosis, an autosomal recessive lysosomal storage disease, is caused by mutations in the CTNS gene, encoding cystinosin, leading to the accumulation of cystine. Kidneys are the first and most affected organs, with severe tubular dysfunction and glomerular damage. Secondary Nephrogenic Diabetes Insipidus (NDI) has been reported in cystinosis. The lack of a collecting duct in vitro model for cystinosis has however limited the research on the mechanisms involved in the impairment of urine concentrating ability. In this study, we established the first mouse collecting duct in vitro model knocked out for CTNS using the CRISPR/Cas9 technology. In parallel, the renal response to vasopressin was tested in four cystinosis patients.

Methods

By employing virus-like particles carrying CRISPR/Cas9 technology, CTNS was efficiently knocked out in MCD4 cells, a mouse renal collecting duct cell line stably expressing the human AQP2 and the vasopressin receptor 2 (V2R). Sanger sequencing, qPCR and mass spectrometry were performed to validate the editing efficiency and assess cystine accumulation. Urinary AQP2 excretion in cystinosis patients was evaluated by ELISA.

Results

The CTNS KO cell line showed a strong reduction of AQP2 expression. In CTNS KO cells, exposure to desmopressin did not increase osmotic water permeability, likely due to reduced AQP2 expression. Interestingly, inhibition of the autophagic pathway by chloroquine treatment resulted in a significant increase in AQP2 expression, indicating that the observed AQP2 reduction in CTNS KO cells is mediated by enhanced autophagic degradation. In cystinosis patients, AQP2 excretion, a biomarker for collecting duct responsiveness to vasopressin, did not significantly increase after desmopressin administration and urine osmolarity remained below 800 mOsm/L.

Conclusion

In conclusion, we established the first collecting duct in vitro model for the study of secondary NDI associated with cystinosis, demonstrating that vasopressin resistance associated with cystinosis depends on reduced expression of AQP2 due to autophagy-mediated degradation. These data together with the in vivo study indicate that secondary NDI in cystinosis is due to a defect in the vasopressin-AQP2 axis.

Funding

• Government Support – Non-U.S.