Ca²⁺ signal dynamics in maturing ureteric bud (UB) and collecting duct (CD) derived organoid tubules

Mechanical signals sensed by human stem cells are transduced via discrete signaling pathways to modulate developmental phenotype and function. Proximal tubules isolated from nephron lineage-derived kidney organoids undergo a developmental increase in abundance and/or activity of the basolateral mechanosensor PIEZO1 and Ca 2+ signal transduction pathways (Carrisoza-Gaytán et al. AJP Cell Physiol, 2023). Here, we investigate whether human iPSC-derived ureteric bud (UB) and collecting duct (CD) organoid cells exhibit a similar developmental increase in PIEZO1 function. Comparison of cells in tubules microdissected from UB and CD organoids cultured for 34-35 days (d) or 62-65 d showed an: (i) increased [Ca 2+ ] i response to basolateral application of the selective PIEZO1 agonist Yoda1 and (ii) decreased time to peak [Ca 2+ ] i with advancing days in culture. Single cell analyses of the Yoda1-induced [Ca 2+ ] i response revealed 7-15 mHz [Ca 2+ ] i oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) or the plasma membrane Ca 2+ -ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca 2+ ] i oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca 2+ signaling, but not PIEZO1, with advancing days in culture and differentiation. These findings are consistent with a developmental increase in activity of PIEZO1 channels and/or maturation of associated pathways shaping Ca 2+ signaling dynamics in maturing UB and CD organoids. Decoding of [Ca 2+ ] i oscillations may identify molecular mechanisms important in morphologic and functional differentiation of organoid tubules.