肾脏类器官小管的功能成熟：PIEZO1介导的Ca2+信号

Functional maturation of kidney organoid tubules: PIEZO1-mediated Ca2+ signaling

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY

影响因子:4.7

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

DOI: 10.1152/ajpcell.00288.2022

摘要

在附着基质上培养的肾脏类器官在存在超滤流的条件下，能够形成血管网络，并表现出比静态对照更为成熟的足细胞和小管区（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）。然而，它们的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肾小管内Ca2+浓度（[Ca2+]i）在机械刺激下的变化，采用微灌注或固定在样本腔底部，并加载fura-2以测定[Ca2+]i。当对来自培养超过40天的类器官的微灌注肾小管施加腔内流动时，[Ca2+]i从195.0±22.1 nM（n=9; P ≤ 0.001）迅速增加超过2.5倍。相比之下，来自培养不足30天的肾小管未表现出反应。未灌注的肾小管（41天）在浴流速增加10倍后，亦表现出[Ca2+]i三倍的升高（P< 0.001）。机械敏感的PIEZO1通道在小鼠远端肾小管中的流动诱导[Ca2+]i反应中起作用（Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019）。在培养21天内，定义了管状结构中的极性PIEZO1的免疫检测。基底PIEZO1被认为具有功能性，因为非灌注肾小管暴露于PIEZO1激活剂Yoda 1后，观察到[Ca2+]i增加（P ≤ 0.001），而且峰值随培养天数的增加而升高。这些结果支持在静态培养的肾小管中，流动/拉伸敏感的Ca2+通道（包括PIEZO1）的数量和/或活性逐渐增加，表现出成熟的迹象。

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.