Abstract: TH-PO332
Proteomic Characterization and Plasticity of Human Collecting Duct Principal and Intercalated-Like Cells Derived From Pluripotent Stem Cell-Derived Ureteric Bud Organoids
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders
- 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic
Authors
- Zarei, Mohammad, Brigham and Women's Hospital, Boston, Massachusetts, United States
- McCracken, Kyle, Boston Children's Hospital, Boston, Massachusetts, United States
- Patel, Ankit B., Brigham and Women's Hospital, Boston, Massachusetts, United States
- Brown, Dennis, Massachusetts General Hospital, Boston, Massachusetts, United States
- Eaton, Amity F., Massachusetts General Hospital, Boston, Massachusetts, United States
- Eisert, Robyn J., Harvard Medical School, Boston, Massachusetts, United States
- Kalocsay, Marian, Harvard Medical School, Boston, Massachusetts, United States
- Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background
Using a new protocol to create human ureteric bud organoids we have developed a human collecting duct (hCD) principal cell (hPC) line from ureteric bud organoids differentiated from human pluripotent stem cells. hPC cells form a mitochondria-rich cuboidal epithelium that generates a high transepithelial resistance and ENaC-dependent transepithelial potential (TEP) when grown on transwell filters.
Methods
The hCD cells, including a cell line with stably integrated lentivirus to permit doxycycline-inducible FoxI1 expression were grown under standard conditions and on transwell filters. Tanden-Mass Tag (TMT) Mass Spectroscopy was performed on cells treated with various hormones +/- antagonists for 24 h and compared with untreated controls.
Results
EM revealed a mitochondrial-rich cuboidal hPC epithelium with small apical microvilli. Amiloride-sensitive TEP was responsive to aldosterone, dexamethasone, hydrocortisone, and vasopressin, and these hormones induced partially overlapping but distinct changes in the proteome. The mineralocorticoid antagonists finerenone and spironolactone both decreased the aldosterone-stimulated TEP but showed some differential effects on the proteome. Finerenone and spironolactone decreased the aldosterone-activated amiloride-sensitive TEP. Sixteen different conditions were evaluated, and principal component analysis revealed very good reproducibility of duplicates and good separation of conditions. Conversion of hPCs to intercalated-like cells (hICs) with FoxI1 was associated with the induction of certain carbonic anhydrase isoforms and multiple V-type H+-ATPase subunits (ATP6 [V1] A, B1, D, E1, F, G1, H) and [V0] A4, D2)). Expression of ATP6V1B1 and ATP6V0A4 confirmed by immunofluorescence.
Conclusion
hCD cells exhibit functional and proteomic responsiveness to both glucocorticoid and mineralocorticoid hormone signaling, which induce distinct and overlapping changes in protein expression. The transcription factor FoxI1 is sufficient to induce expression of V-type H+-ATPase subunits to drive the acid secretory phenotype.
Funding
- NIDDK Support