Ca ²⁺ signal dynamics in maturing ureteric bud- and collecting duct-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 Ca²⁺ signal transduction pathways (Carrisoza-Gaytan R, Kroll KT, Hiratsuka K, Gupta NR, Morizane R, Lewis JA, Satlin LM. <i>Am J Physiol Cell Physiol</i> 324: C757-C768, 2023). Here, we investigate whether human induced pluripotent stem cell (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 or 62-65 days showed <i>1</i>) increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to basolateral application of the selective PIEZO1 agonist Yoda1 and <i>2</i>) decreased time to peak [Ca²⁺]_i with advancing days in culture. Single-cell analyses of the Yoda1-induced [Ca²⁺]_i response revealed 7- to 15-mHz [Ca²⁺]_i oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) or the plasma membrane Ca²⁺-ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca²⁺]_i oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca²⁺ signaling, but not <i>PIEZO1</i>, 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 Ca²⁺ signaling dynamics in maturing UB and CD organoids. Decoding of [Ca²⁺]_i oscillations may identify molecular mechanisms important in morphological and functional differentiation of organoid tubules.<b>NEW & NOTEWORTHY</b> This investigation, focused on analyzing the role of PIEZO1 mechanotransduction in maturing human induced pluripotent stem cell (iPSC)-derived ureteric bud (UB) and collecting duct (CD) kidney organoids, unexpectedly reveals developmentally regulated Ca²⁺ oscillations and begins to dissect their mechanistic underpinnings. Specifically, transcriptomic analysis reveals that with time in culture and differentiation from UB to CD, organoids progressively acquire the molecular machinery necessary for complex Ca²⁺ signaling dynamics. These results lead us to speculate that information encoded in oscillatory signals drives renal epithelial differentiation.