Organic anion and cation transport assay for kidney organoids (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Catherine Baty

Keywords

[‘organoid’, ‘proximal tubule epithelial cells’, ‘3D imaging’, ‘urinary physiology’]

Subjects

[‘Pharmacology’, ‘Imaging’, ‘Cell biology’, ‘Toxicology’]

Release Date

2022-07-07

Abstract

IPS cell derived kidney organoids are used and function is measured using 6-carboxyfluorescein as the prototypic organic anion (OA) and ethidium bromide as the prototypic organic cation (OC). Live organoids are loaded sequentially with both agents to minimize time involved, and immediate live cell confocal microscopy is used to measure extent of basolateral proximal tubular (PT) uptake and luminal secretion if any. Semiquantitative scoring of at least 10 organoids in replicate experiments have been used to distinguish impaired PT function in response to treatment with known OA/OC inhibitors and toxic agents such as hemin.

Introduction

Organic anion (OA) and cation (OC) transport are important functions of the proximal tubule. This combined assay is relatively efficient and could be used as a semiquantitative functional screening assay for PT function in kidney organoids to help confirm maturation or response to exogenous drugs or toxins.

Reagents

• 6-carboxyfluorescein (6-FAM), C 1360, Invitrogen
• ethidium bromide, (E7637), Sigma-Aldrich
• OptiMEM I reduced serum media, #31985062, ThermoFisher
• human kidney organoids

Equipment

• 37^o^C 5% CO2 incubator with shaker
• Inverted confocal microscope, ideally with environmental chamber and motorized stage
• 30 mm coverglass bottom dish and round coverglass to hold organoids in place for imaging

Procedure

Dye Loading: Make fresh 10 mM stock of 6CF in optiMEM I. Transfer organoids to 1.5 ml Eppendorf tubes for dye loading. Let organoids settle and replace media with optiMEM, let settle and replace optiMEM with fresh aliquot of optiMEM (700 l per tube). Remove excess optiMEM from the top of each of the eppendorfs and add at least 500 l of diluted 6CF (1mM). Place in 37oC 5% CO2 incubator and shake (we use orbital shaker at ~ 146 rpm). Shake for 35 min. In meantime prepare 1mM ethidium bromide solution in optiMEM; the final solution is 10 M but is added at 100x to organoids already loaded with 6CF to maintain final concentration for 10 min of loading with both 6CF and EB. Replace loading solutions with optiMEM (rinse at least twice) and image as soon as possible.

Imaging: We use an inverted confocal microscope with a 25x 0.9 NA water objective and environmental chamber. Transfer organoids into coverslip bottom 30 mm dish with 60 l fresh 37oC optiMEM; addition on another coverglass on top can facilitate imaging. Optimize imaging using conventional laser settings (laser lines: 488 for 6CF and 552 for EB), using sequential between line acquisition to avoid bleed through. Some saturation is acceptable for this semiquantitative assay.

Scoring: We used maximum projection images and score from top and at stack midsection. Tubule number and gross intensity (saturated versus not) have been used to reveal differences in maturation or toxicity.

Timing

• Whole procedure including semiquantative scoring: 5 hr
• Fresh reagent prep time: 15 min
• Loading time: 45 min
• Estimated imaging time: 2 hr

Critical_Steps

Fresh reagents must be made for each assay day.

6-carboxyfluorescein is a pH sensitive dye so selection of buffers and drugs to be tested must take this into account.

It is important to differentiate surface nuclear staining with EB from tubular staining, since EB can also be used for a marker for apoptotic cell death.

Trouble_Shooting

Different optical systems and size of organoids (in different labs) will necessitate different laser settings so a large control assay to generate optimal settings (for each imaging system) and assess batch variability is recommended.

Consider blinded scoring of fluorescent images to initially validate optical settings.

Either portion of the assay (e.g, organic cation transport) can be done separately.

Anticipated_Results

Fluorescent intensity of tubules will range from undetectable to saturated and basolateral uptake will be impacted by organoid size. These variables necessitate a semiquantitative approach. However, we have found significant differences in tubular uptake in organoids treated with conventional OA and OC inhibitors and a dose response to treatment with hemin.

References

Lawrence et al, Scientific Reports 5: 9092 (2015). WK Lee et al. AJP Renal 296: (2009).

Associated_Publications

Przepiorski, A et al. Stem Cell Res Ther 13: (2022)

Acknowledgement

Human iPS cell derived kidney organoids provided by Neil Hukriede lab; thanks especially to Aneta Przepiorski and Eugenel B. Espiritu.

Consortium

(Re)Building a Kidney (RBK) Consortium