Measuring transepithelial resistance and voltage using human pluripotent stem cell-derived novel collecting duct principal and intercalated cells (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

Joseph V Bonventre; Mohammad Zarei; Kyle McCracken; Ankit Patel

Keywords

[‘Collecting Duct Intercalated Cell’, ‘Collecting Duct Principal Cell’, ‘Ion transport measurements’]

Subjects

[‘Cell biology’]

Release Date

2022-07-11

Abstract

Transepithelial voltage (V) and resistance (R) are fundamental properties of epithelia in vivo and represent approaches to characterize transport across kidney epithelia in vitro. Transepithelial voltage and resistance can be measured across monolayers of cells grown on transwell filters. Trans-epithelial current can be measured under short-circuited conditions in Ussing Chamber systems.

Introduction

The collecting duct (CD) epithelium forms a tight barrier to the diffusion of water and ions between formative urine and the surrounding interstitial compartment. Transepithelial electrical resistance (TEER) is a sensitive way of measuring epithelial integrity and permeability. Permeability to ions across an epithelial layer includes movement through cells and through paracellular junctions of a confluent epithelium. We will describe the methods to measure transepithelial voltage and TEER in a two-dimensional system in vitro.

Reagents

DMEM (Dulbecco’s Modified Eagle’s Medium) [+] 4.5 g/L glucose, L-glutamine, sodium pyruvate (#Corning, 10-013-CV), Fetal Bovine Serum (FBS) (Life technologies, 10437028) DMEM/F-12 Medium, (Corning, 10-092-CV), Epidermal Growth Factor (Peprotech, AF-315-09-100 UG), ITS (Sigma, 100X 13146 5ML SLCF9923), and Thyroid hormone L-3,3′,5-triiodothyronine (T3) (Sigma, T6397). Hydrocortisone (Sigma, H0888), Dexamethasone (Sigma D4902), Aldosterone (Sigma A9477), Doxycycline (Sigma D9891), Amiloride hydrochloride (Sigma A3085), Bafilomycin A1(Fisher 196000), and Vasopressin (Sigma V9879).

Equipment

Circulating Ussing chamber & 12 mm Snapwell™ Insert with 0.4 µm Pore Polyester Membrane, Sterile (#Corning, 3801). Epithelial volt/ohm meters 3 (EVOM3) with STX2-PLUS-Modified Electrode-Prototype Type: Silver Chloride Coated & Transwell Permeable Support for 12-well Plate with 0.4 µm Transparent Polyethylene Terephthalate (PET) Membrane, Sterile (#Corning, 353180) or 12 mm TranswellR Permeable Supports, packed in 12 Well Plate, 0.4μm Polycarbonate Membrane Tissue Culture treated, Polystyrene Plates, Sterile (#Costar, 3401). PowerLab 4/25 (No Ref.), -Ag/AgCl electrodes: EK1 (WPI Instruments).

Procedure

a. Electrophysiological measurements in transwell systems

Ureteric bud organoids differentiated to structures containing predominantly collecting duct principal cells were dissociated to single cells and transitioned to 2D transwell culture conditions. The cells are grown at 37oC with 5% CO2 in DMEM/F-12 media supplemented with insulin (5 mg ml-1), apotransferrin (5 mg ml-1), sodium selenite (60 nM) (ITS), triiodothyronine (1 nM), dexamethasone (10 nM), epithelial growth factor (10 ng ml-1), and fetal bovine serum (2% v/v). The cells are serially passaged using Trypsin-EDTA.

For electrophysiology studies, CD cells are seeded on Corning Cell Culture Inserts (12 well format, 0.4 mm pore size PET track-etched membranes) at 100,000 cells per well. A minimum of 1ml media on top (apical) and 1.5 ml media on bottom (basolateral) is provided per transwell. Transepithelial resistance and voltage are monitored using an epithelial volt-ohm meter (World Precision Instruments, EVOM3) with the STX2-plus electrode system. Electrode tips are sterilized in 70% ethanol and then placed in media or buffer for 10 minutes for charge equilibration. Electrodes are then used for transepithelial resistance and voltage measurements. After all measurements are complete, electrode tips are immersed in in 70% ethanol for 5- 10 minutes, rinsed with DI water, and then air dried prior to storing.

In open-circuit conditions, an equivalent current across the epithelia can be calculated using Ohm’s law, Voltage = Current*Resistance. To evaluate ENaC activity, amiloride (10 μM) is added to the apical chamber and transepithelial voltage and resistance measured after 5 minutes to measure amiloride-sensitive voltage and resistance. To evaluate H+-ATPase activity in intercalated cells, which are derived from principal cells by doxycycline-inducible expression of the transcription factor, FOXiI1, bafilomycin-A1 (10 nM) is applied to the apical chamber and TEER and voltage are measured after 5 minutes. Ability to add selectively to the apical or basolateral membranes of the cells allows for evaluation of apical/basal compartment specific treatments on electrophysiologic properties.

b. Electrophysiological measurements including short-circuit current (Isc) using Ussing Chamber systems

CD cells are grown on 12 mM Snapwell inserts with 0.4 μM polycarbonate membranes (Corning) in CD Medium for 7-14 days to ensure confluency. Snapwell inserts were mounted in Ussing Chambers (Physiological Instruments VCC MC8). We have used the facility at the Harvard Digestive Disease Center Core at Boston Children’s Hospital. Bath solutions of 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose are added to the chambers with continuous bubbling of CO2 to maintain a pH of 7.4. Snapwells are continuously clamped at 0 mV and ISC recorded using LabChart software. Amiloride (10-100 μM), bafilomycin-A1 (10-100 nM) or other substances can be added to the apical or basolateral chamber.

Critical_Steps

Temperature, cell culture medium composition, length of time in culture, length of time at confluence, passage number of cells, and shear stress are factors affecting transepithelial voltage and TEER measurements.

Anticipated_Results

These are examples of results obtained using these techniques on human CD cells.

Figure 1: a-b, As CD cells became confluent in transwell plates, there was at first a steep increase and then plateau in the transepithelial resistance, which was then followed by the emergence of a transepithelial voltage. The voltage was entirely suppressible within three minutes of addition of the ENaC antagonist amiloride (10 μM), which also raised the resistance across the epithelium. c, The resulting current generated was calculated using Ohm’s Law, and it was completely ablated immediately following addition of amiloride, confirming the conductance was ENaC-dependent. *p=5.3x10-9, *p=5.7x10-8, *p=4.5x10-6; two-tailed student’s t-test comparing results before and after amiloride; n=6 biological replicates, data representative of 3 independent experiments. d, Under closed-circuit conditions with voltage clamping in Ussing chamber, the current produced by the epithelium was similarly sensitive to amiloride, and it was completely abolished upon increasing the concentration to 100 μM. e-f, Addition of aldosterone for 24 hours led to a dose-dependent increase in transepithelial current measured under closed circuit conditions, and up to a maximum of approximately 2.5-fold increase in amiloride-sensitive current. *p<0.005; two-tailed student’s t-test compared to no aldosterone. From Shi, McCracken et al. Nature Biotechnology In Press

References

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2. Denker, B.M. and E. Sabath, The biology of epithelial cell tight junctions in the kidney. Journal of the american society of nephrology, 2011. 22(4): p. 622-625.
3. Srinivasan, B., et al., TEER measurement techniques for in vitro barrier model systems. SLAS Technology, 2015. 20(2): p. 107-126.
4. Bello-Reuss, E., Electrophysiological identification of cell types in cortical collecting duct monolayers. Kidney and Blood Pressure Research, 1991. 14(1-2): p. 1-11.
5. Pappas, C.A. and B.M. Koeppen, Electrophysiological properties of cultured outer medullary collecting duct cells. American Journal of Physiology-Renal Physiology, 1992. 263(6): p. F1004-F1010.
6. Zheng, H., C. Zou, and T. Yang. Furin-Mediated Modification Is Required For Epithelial Sodium ChannelActivating Activity Of Soluble (Pro) Renin Receptor In Cultured Collecting Duct Cells. in HYPERTENSION. 2021. Lippincott Williams and Wilcons 2 Commerce Square, 2001 Market Street, Philadelphia

Associated_Publications

Shi MM, McCracken KW, Patel AB, Zhang W, Ester E, Valerius MT, and Bonventre JV. Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types. Nature Biotechnology In Press

Consortium

(Re)Building a Kidney (RBK) Consortium