Aug 15, 2022
Ryuji Morizane’s lab has just published a new paper in Frontiers in Cell and Developmental Biology that calls out work done through the Functional Assays Working Group (check out the link to access the Functional Assays Handbook search):
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. Frontiers in Cell and Developmental Biology. Volume 10. August 15, 2022.
Here is a quote that refers to RBK’s priority to publish functional assays that are accessible to a broad range of researchers:
“The (Re)Building a Kidney (RBK) consortium led by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) hosts ∼20 institutions across the United States and is focused on a wide range of projects related to kidney regeneration and repair. One RBK priority is to develop a data repository for functional assessments that would be accessible to researchers worldwide who wish to functionally phenotype their kidney models including kidney organoids (ReBuilding a Kidney). Functional assessments will determine whether kidney cells and tissues are appropriate for modeling disease and future transplantation. Assays proposed to be included in this repository cover those aimed at measuring glomerular (creatinine clearance, dextran filtration) and tubular (e.g., absorption of water, sodium, glucose, and dextran-albumin; secretion of organic ions) functions (Rizki-Safitri et al., 2021) in kidney models. Some established assays can be modified to cultured cells, yet it is challenging to apply these functional assessments to complex 3D tissues such as 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 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.
View the latest RBK publications here: https://www.rebuildingakidney.org/publications/