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Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids

Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity...

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Autores principales: Rizki-Safitri, Astia, Gupta, Navin, Hiratsuka, Ken, Kobayashi, Kenichi, Zhang, Chengcheng, Ida, Kazumi, Satlin, Lisa M., Morizane, Ryuji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9420851/
https://www.ncbi.nlm.nih.gov/pubmed/36046340
http://dx.doi.org/10.3389/fcell.2022.978888
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author Rizki-Safitri, Astia
Gupta, Navin
Hiratsuka, Ken
Kobayashi, Kenichi
Zhang, Chengcheng
Ida, Kazumi
Satlin, Lisa M.
Morizane, Ryuji
author_facet Rizki-Safitri, Astia
Gupta, Navin
Hiratsuka, Ken
Kobayashi, Kenichi
Zhang, Chengcheng
Ida, Kazumi
Satlin, Lisa M.
Morizane, Ryuji
author_sort Rizki-Safitri, Astia
collection PubMed
description Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity of these organoid structures. Transcriptomic analyses provide insight into maturation and transporter activities that represent kidney functions. However, functional assays in organoids are necessary to demonstrate the activity of these transport proteins in live tissues. The three-dimensional (3D) architecture adds complexity to real-time assays in kidney organoids. Here, we develop a functional assay using live imaging to assess transepithelial transport of rhodamine 123 (Rh123), a fluorescent substrate of P-glycoprotein (P-gp), in organoids affixed to coverslip culture plates for accurate real-time observation. The identity of organoid structures was probed using Lotus Tetragonolobus Lectin (LTL), which binds to glycoproteins present on the surface of proximal tubules. Within 20 min of the addition of Rh123 to culture media, Rh123 accumulated in the tubular lumen of organoids. Basolateral-to-apical accumulation of the dye/marker was reduced by pharmacologic inhibition of MDR1 or OCT2, and OCT2 inhibition reduced the Rh123 uptake. The magnitude of Rh123 transport was maturation-dependent, consistent with MDR1 expression levels assessed by RNA-seq and immunohistochemistry. Specifically, organoids on day 21 exhibit less accumulation of Rh123 in the lumen unlike later-stage organoids from day 30 of differentiation. Our work establishes a live functional assessment in 3D kidney organoids, enabling the functional phenotyping of organoids in health and disease.
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spelling pubmed-94208512022-08-30 Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids Rizki-Safitri, Astia Gupta, Navin Hiratsuka, Ken Kobayashi, Kenichi Zhang, Chengcheng Ida, Kazumi Satlin, Lisa M. Morizane, Ryuji Front Cell Dev Biol Cell and Developmental Biology Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity of these organoid structures. Transcriptomic analyses provide insight into maturation and transporter activities that represent kidney functions. However, functional assays in organoids are necessary to demonstrate the activity of these transport proteins in live tissues. The three-dimensional (3D) architecture adds complexity to real-time assays in kidney organoids. Here, we develop a functional assay using live imaging to assess transepithelial transport of rhodamine 123 (Rh123), a fluorescent substrate of P-glycoprotein (P-gp), in organoids affixed to coverslip culture plates for accurate real-time observation. The identity of organoid structures was probed using Lotus Tetragonolobus Lectin (LTL), which binds to glycoproteins present on the surface of proximal tubules. Within 20 min of the addition of Rh123 to culture media, Rh123 accumulated in the tubular lumen of organoids. Basolateral-to-apical accumulation of the dye/marker was reduced by pharmacologic inhibition of MDR1 or OCT2, and OCT2 inhibition reduced the Rh123 uptake. The magnitude of Rh123 transport was maturation-dependent, consistent with MDR1 expression levels assessed by RNA-seq and immunohistochemistry. Specifically, organoids on day 21 exhibit less accumulation of Rh123 in the lumen unlike later-stage organoids from day 30 of differentiation. Our work establishes a live functional assessment in 3D kidney organoids, enabling the functional phenotyping of organoids in health and disease. Frontiers Media S.A. 2022-08-15 /pmc/articles/PMC9420851/ /pubmed/36046340 http://dx.doi.org/10.3389/fcell.2022.978888 Text en Copyright © 2022 Rizki-Safitri, Gupta, Hiratsuka, Kobayashi, Zhang, Ida, Satlin and Morizane. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Rizki-Safitri, Astia
Gupta, Navin
Hiratsuka, Ken
Kobayashi, Kenichi
Zhang, Chengcheng
Ida, Kazumi
Satlin, Lisa M.
Morizane, Ryuji
Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
title Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
title_full Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
title_fullStr Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
title_full_unstemmed Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
title_short Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
title_sort live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9420851/
https://www.ncbi.nlm.nih.gov/pubmed/36046340
http://dx.doi.org/10.3389/fcell.2022.978888
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