Abstract: FR-PO564
Kidney Tubuloid Model to Study Aquaporin 2-Mediated Water Transport in the Collecting Duct
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
- Hoenderop, Joost, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands
- Dilmen, Emre, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands
- Olde Hanhof, Charlotte, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands
- Latta, Femke, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands
- Verhaar, Marianne C., University Medical Center Utrecht, Utrecht, Utrecht, Netherlands
- Orhon, Idil, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands
- De Baaij, Jeroen H.F., Radboud Universitair Medisch Centrum, Nijmegen, Netherlands
- Jansen, Jitske, Universitatsklinikum Aachen, Aachen, Nordrhein-Westfalen, Germany
Background
The last segment of the kidney tubules is the collecting duct (CD), where finetuning of sodium and water reabsorption occurs. A dysfunction of this process can lead to nephrogenic diabetes insipidus (NDI) that causes the inability to concentrate urine which can lead to severe dehydration. NDI is caused by dysfunction of the water channel aquaporin 2 (AQP2) or the vasopressin 2 receptor (V2R) in the CD. Despite promising results of pre-clinical experiments, clinical trials often fail due to ineffectiveness or side-effects. Tubuloids are kidney organoid models that are derived from adult stem cells with the promise to study kidney (patho)physiology with increased translational value compared to conventional in vitro research models. By utilizing these tubuloid models, our lab previously demonstrated functional CD-specific sodium regulation. In this study, we show that tubuloids are capable of physiological CD-specific water transport regulation by AQP2 and V2R.
Methods
Tubuloids derived from human kidney tissue were grown were exposed to differentiation medium and differential expression was studies by single cell RNA-seq. The differentially expressed genes were studied by gene ontology analysis. The CD marker and water transporter AQP2 expression and localization was characterized by western blot and immunocytochemistry. Functional water transport assay was performed by measuring swelling over time.
Results
Tubuloids can be differentiated towards the distal tubules, including CD as shown by single cell sequencing. Further experiments revealed that the endogenously expressed AQP2 can be regulated by forskolin and desmopressin shown by significantly increased expression of AQP2 protein. In addition, the translocation towards the apical membrane of AQP2 where it increases water permeability was observed. Finally, we performed 3D swelling assays which confirmed that CD tubuloids are indeed capable of increased water transport and thereby respond to stimuli by swelling.
Conclusion
Human kidney tubuloids endogenously express relevant markers for CD-specific water transport, and AQP2 signaling regulated by physiological stimuli which is measurable in 3D functional assays. These results further confirm that kidney tubuloid models hold great promise to study kidney (patho)physiology, including NDI.