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

Ca 2+ signaling in metanephric mesenchymal (MM) cells contributes to key pattern forming events including branching morphogenesis in the embryonic rat kidney (Fontana et al., FASEB J, 2019). We previously reported that basolateral exposure of tubular structures isolated from MM-derived kidney organoids to the PIEZO1 channel agonist Yoda1 elicited a maturation stage-dependent elevation of intracellular Ca 2+ concentration, [Ca 2+ ] i (Carrisoza-Gaytán et al., AJP Cell Physiol, 2023). The aim of our current study is to explore whether tubular cells in human iPSC-derived UB and CD organoids exhibit similar developmental increases in PIEZO1 function.To investigate this, tubular structures from UB and CD organoids cultured for 34-35 or 61-65 d (4 tubules/group; n=38-40 cells/group) are loaded with the Ca 2+ -sensitive fluorophore Fura2-AM to measure [Ca 2+ ] i before and after basolateral exposure to Yoda1 (5 µM). Baseline [Ca 2+ ] i was 108.5±3.6 nM across all tubular cells studied, with no differences detected between UBs and CDs or days in culture. Subsequent exposure to Yoda1 induced an increase in [Ca 2+ ] i and appearance of Ca 2+ oscillations. The Yoda1-induced [Ca 2+ ] i signals of individually identified cells are then subject to spectral analysis using the multitaper method along with a harmonic F-test to identify predominant frequencies in the signal. We found that with advancing days in culture from 34-35 to 61-65 d: (i) Yoda1 induced an increase in [Ca 2+ ] i in UBs from 45.3±18.5 to 275.6±43.7 nM (p≤0.001) and in CDs from 61.7±16.1 to 254.3±56.2 nM (p≤0.001), (ii) the proportion of cells exhibiting Ca 2+ oscillations increased in UBs from 70 to 83% and in CDs from 75 to 84%, and (iii) the predominant oscillatory frequency fell by 50% in UBs and 30% in CDs from 15 mHz. These observations suggest that kidney organoids undergo a developmental increase in PIEZO1 abundance/activity and maturation of Ca 2+ signal transduction pathways, especially those involved in Ca 2+ oscillatory activity. Decoding Ca 2+ oscillations may unveil molecular mechanisms underlying maturation/differentiation. This work was supported by the NIH Re(Building) a Kidney Consortium (NIH UC2DK126023) and the Welcome LEAP Human Organs, Physiology and Engineering (HOPE) program. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.