肾脏类器官管道的功能成熟：PIEZO1介导的Ca2+信号通路
Functional maturation of kidney organoid tubules: PIEZO1-mediated Ca2+ signaling
影响因子:4.70000
分区:生物学3区 / 细胞生物学3区 生理学3区
发表日期:2023 Mar 01
作者:
Rolando Carrisoza-Gaytan, Katharina T Kroll, Ken Hiratsuka, Navin R Gupta, Ryuji Morizane, Jennifer A Lewis, Lisa M Satlin
摘要
在附着基质上培养的肾脏类器官，伴有血管网络生成，显示出比静态对照更为成熟的足细胞和管道部分（Homan KA等，Nat Methods 2019; Takasato M等，Nature 2015）。然而，其生理功能尚未系统性研究。本研究测量了从培养21至64天的类器官中分离的管道在体内微灌流或固定于样品室底部时的机械诱导的细胞内Ca2+浓度变化（[Ca2+]i），使用fura-2染料进行测定。在对超过40天培养的类器官管道进行腔内流体流动时，观察到[Ca2+]i从195.0 ± 22.1 nM基线迅速增加超过2.5倍（n=9，P ≤ 0.001）。而在未超过30天培养的类器官管道中未检测到反应。未灌流的管道（41天）在浴流速提高10倍后，也表现出[Ca2+]i的三倍增加（P < 0.001）。机械敏感的PIEZO1通道在小鼠远端管中参与流动诱导的[Ca2+]i反应（Carrisoza-Gaytan R等，FASEB J 2019）。在培养21天的管道结构中，检测到极性膜的PIEZO1。基底暴露于PIEZO1激活剂Yoda 1可增加[Ca2+]i（P ≤ 0.001），在培养超过30天的类器官段中，峰值[Ca2+]i随培养天数增加。这些结果支持静态培养中的管道通过PIEZO1等流动/拉伸敏感的Ca2+通道数量或活性的逐步增加而趋于成熟。
Abstract
Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.