Developmental expression of mechano-stimulated Ca2+ transport pathways in kidney organoid tubules

Autor: Rolando Carrisoza-Gaytan, Navin Gupta, Ken Hiratsuka, Katharina Kroll, Ryuji Morizane, Jennifer Lewis, Lisa Satlin
Rok vydání: 2023
Předmět:
Zdroj: Physiology. 38
ISSN: 1548-9221
1548-9213
Popis: Kidney organoids derived from human pluripotent stem cells exhibit glomerular- and tubular-like structures. As the technology for culturing organoids advances, the goal of promoting terminal differentiation demands that priority be given to characterization of their physiological function. To begin to functionally phenotype organoids, we examined mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and signaling pathways in tubules isolated from maturing organoids. We found that tubules microdissected from organoids cultured under static conditions and exposed to basolateral Yoda1 (5 μM), an activator of the mechanosensitive Ca2+ permeable channel Piezo1, exhibited an increase in [Ca2+]i of increasing magnitude with advancing days in culture (Carrisoza-Gaytan et al., ASN, 2021). This observation suggests that organoids undergo a developmental increase in the abundance and/or activity of Piezo1 and /or maturation of signal transduction pathways mediating this response with advancing days (d) in culture.To begin to examine these possibilities, transcript expression levels of the putative mechanosensor PIEZO1 and Ca2+ transport proteins TRPV4 and PMCA1 (plasma membrane Ca2+ ATPase) were measured in organoids (n=3-6) cultured for 8, 21, 35 and 49 d by RT-qPCR. ACTB (encoding β-actin) and GAPDH transcript expression were included as housekeeping genes. Abundance of Piezo1 and TRPV4 channel protein expression was studied by immunofluorescence (IF) and confocal microscopy in organoids (n=3) at 21 and 49 d in culture.RT-qPCR demonstrated an increase in PIEZO1 (28.1±2.0 fold, p≤ 0.05), TRPV4 (5.3±0.8 fold, p≤ 0.01) and PMCA1 (1.8±0.1 fold, p≤ 0.05) between 8 and 49 d in culture when normalized to GAPDH expression; we found that ACTB expression also increased with organoid maturation (1.9±0.1 fold, p≤ 0.01) when normalized to GAPDH expression during this interval. IF labeling of organoid cryosections revealed an increase in apical Piezo1 (3.2±0.6 fold, p≤ 0.001) and TRPV4 (1.5±0.1 fold, p≤ 0.05) in tubular structures at 49 vs 21 d in culture.These results demonstrate a developmental increase in molecular expression and cell localization of Ca2+ transport proteins responsible for the transduction of external mechanical stimuli into intracellular Ca2+ signals in organoids cultured over time. UC2DK126023 (JAL, LS, RM); DP2 DK133821 (RM); R01 DK038470 (LMS); R01 DK129285 (LMS); P30 DK079307 (LMS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Databáze: OpenAIRE
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