Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells

Abstract

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel, the renal outer medullary K⁺ (ROMK, Kir1.1) channel, and the epithelial Na⁺ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BK_Ca channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BK_Ca and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba²⁺-sensitive K⁺ channels. BK_Ca expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K⁺ and Na⁺ channels.

NEW & NOTEWORTHY Our results show that human kidney organoids express key K⁺ and Na⁺ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca²⁺-activated K⁺ channel, renal outer medullary K⁺ channel, and epithelial Na⁺ channel.

INTRODUCTION

Kidney organoids generated as three-dimensional cell aggregates exhibit many of the key structural features of the nephron. They provide a model system to study human kidney development and disease, a tool for in vitro drug screening, and a potential tool for regenerative therapy (1, 2). There have been significant recent advances in protocols to generate differentiated kidney structures from nephron progenitors and to define mRNAs and proteins expressed in these organoids, at a single cell level and in specific structural components (for a review, see Ref. 2). One of the major functions of kidney epithelia is the transport of solutes in a vectorial manner, resulting in either solute absorption or secretion. These functional properties are key for the central role of the kidney in body fluid electrolyte and acid-base homeostasis. At present, there is a paucity of data regarding the functional properties of tubular epithelia within kidney organoids.

Kidneys play a key role in maintaining extracellular fluid volume by regulating the amount of Na⁺, K⁺, and other solutes in urine. In the late distal convoluted tubule, connecting tubule, and collecting duct, Na⁺ exits the urinary space through the epithelial Na⁺ channel (ENaC), a highly Na⁺-selective channel expressed in principal cells of these nephron segments (3). ENaCs are heterotrimeric channels and in the human kidney are composed of structurally related α-, β-, and γ-subunits (4, 5). Following luminal Na⁺ entry into cells via ENaC, Na⁺ is actively transported out of cells across the basolateral membrane by Na⁺-K⁺-ATPase, which also provides the electrochemical driving force for transepithelial Na⁺ transport.

Changes in extracellular K⁺ concentration have major effects on cell and organ function. It is essential that extracellular K⁺ concentration is maintained within a narrow range, and the aldosterone-sensitive distal nephron (ASDN) has a key role in maintaining K⁺ balance through regulated excretion of K⁺ into the urinary space. Under normal or high dietary K⁺ conditions, the content of K⁺ in urine largely reflects its secretion by the distal nephron. K⁺ secretion in the distal nephron is mediated by two apical channels: the renal outer medullary K⁺ (ROMK, Kir1.1) channel and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel. The ROMK channel is a homotetrameric, low-conductance channel that mediates K⁺ secretion in principal cells. BK_Ca channels are expressed in intercalated cells, where they mediate the K⁺ secretion response to increased tubular flow (6, 7). BK_Ca channels are activated by increases in intracellular Ca²⁺ and membrane depolarization (7). They are assembled as tetramers of pore-forming α-subunits together with tissue-specific auxiliary β-subunits (β1–β4), which are typically present in a 1:1 stoichiometry. The BK_Ca α-subunit consists of seven transmembrane spanning domains (S0–S6) with an extracellular NH₂ terminus, P-loop between S5 and S6 domains, and a large intracellular COOH terminus containing several regulatory Ca²⁺-binding sites (8). The β-subunits consist of two transmembrane domains with intracellular NH₂- and COOH-termini and a long extracellular loop. There are also four auxiliary γ-subunits that modulate BK_Ca channel activity (9). These are single-spanning type 1 integral membrane proteins.

In this study, we used the patch-clamp technique to characterize the expression of specific K⁺ and Na⁺ channels in cells dispersed from kidney organoids and in tubular segments isolated from organoids. Our study focused on the characterization of BK_Ca, ROMK, and ENaC present in cells of human induced pluripotent stem cell (hiPSC)-derived kidney organoids, given their key roles in mediating K⁺ secretion and Na⁺ absorption in the ASDN. We also examined the expression and localization of specific channels in organoids by immunoblot analysis and immunofluorescence microscopy. This study provides the first experimental evidence that human kidney organoids express functional BK_Ca, ROMK, and ENaC.

METHODS

Reagents and Antibodies

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Generation of hiPSC-Derived Kidney Organoids

Kidney organoids were generated from hiPSCs using our previously described two-step protocol (10). Briefly, hiPSCs were plated and maintained on 10-cm cell culture dishes coated with Geltrex (ThermoFisher) and mTeSR1 (StemCell Technologies, Inc.) medium and passaged every 3–4 days (70–80% confluency). Differentiation of hiPSC colonies into kidney organoids was induced by 3-day treatment with the small-molecule Wnt agonist CHIR99021, which induces mesoderm formation in the form of spherical embryoid bodies in suspension culture. This was followed by a second stage incubation in DMEM supplemented with 10% KnockOut serum replacement (stage II medium) to drive renal tubule formation. Organoids were maintained in this medium until the experimental time point (10). All experiments were performed in compliance with institutional guidelines and were carried out in a class II biosafety hood with the appropriate personal protective equipment. All reagents were of cell culture grade unless stated otherwise. All cultures were incubated at 37°C in the presence of a 5% CO₂ air atmosphere. The hiPSC lines used to generate this data have been fully characterized and published (11).

RNA Sequencing

RNA-sequencing analysis was performed as previously described (12). Briefly, total RNA from quadruplicate samples of kidney organoids (∼100 organoids/sample) was prepared using TRIzol and Phase Separation Reagent and purified using an Ambion PureLink RNA Mini Kit with in-column RNase-free DNase I (Qiagen) treatment. All sequenced samples contained >1 µg total RNA and had an RNA integrity number value of ≥9.4. Library preparation was done using a TruSeq Stranded mRNA (PolyA+) kit and sequencing on Illumina Sequencing using NextSeq500. Quality control, library preparation, and sequencing were performed by Health Sciences Sequencing Core, UPMC Children’s Hospital of Pittsburgh. Reads were mapped on the human genome, GRCh38.p13 using STAR (13), and counted using feature Counts (14). Expression values were normalized and reported as reads per kilobase of exon per million mapped reads (15). Data were deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus database (GSE182350).

Immunofluorescence Staining of Organoid Sections

Organoids (10–20 organoids/sample) were fixed in 4% paraformaldehyde at room temperature for 30 min, embedded in paraffin, and then sectioned at 4-µm thickness. On the day of staining, sections were deparaffined with xylene and rehydrated with 100%, 90%, and 70% ethanol incubation (5 min each). After being treated with citrate-based antigen-unmasking solution (Cat. No. H3300, Vectorlab), sections were blocked for 1 h at room temperature in CytoVista Blocking Buffer (Cat. No. V11313, ThermoFisher). Anti-γENaC antibody (Cat. No. SPC405D, Stressmarq, final concentration of 10 µg/mL), anti-ROMK antibody (Cat. No. 20201, BiCell, 10 µg/mL), anti-αBK antibody (Cat. No. 75-022, AntibodiesINC, 5 µg/mL), anti-GATA3 antibody (Cat. No. AF2605, R&D Systems, 2.5 µg/mL), or anti-E-cadherin antibody (Cat. No. 610182, BD Biosciences, 1.25 µg/mL) were diluted in CytoVista Antibody Dilution Buffer (Cat. No. V11305, ThermoFisher) and incubated at 4°C overnight. After being washed with PBS, sections were incubated with Alexa Fluor 488 donkey anti-rabbit (Cat. No. 711-545-152, Jackson ImmunoResearch, 2 mg/mL) and Cy3 AffiniPure donkey anti-mouse (Cat. No. 705-165-150, Jackson ImmunoResearch, 0.2 mg/mL) or with Alexa Fluor 488 goat donkey anti-mouse (Cat. No. 711-545-150, Jackson ImmunoResearch, 2 mg/mL) and Cy3 AffiniPure donkey anti-goat (Cat. No. 705-165-147, Jackson ImmunoResearch, 0.2 mg/mL) for 2 h at room temperature. After an extensive wash with PBS, sections were mounted with SlowFade Glass Soft-set Antifade Mountant (Cat. No. S36917, ThermoFisher). Images were obtained on a Leica Stellaris 8 confocal microscope using a ×20 objective (numerical aperture = 1).

Immunofluorescence Staining of Kidney Sections

Male C57BL6 mice were purchased from Jackson Labs and maintained on a standard diet [Prolab Isopro RMH 3000 (LabDiet), containing ∼0.94% K⁺ and 0.23% Na⁺]. Breeding and experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Kidneys were harvested at the age of 9–13 wk and processed for immunofluorescence microscopy (or immunoblot analysis). Briefly, paraffin-embedded mouse kidney sections were deparaffinized and rehydrated as previously described (16). Following antigen retrieval and blockade with 10% horse serum, sections were incubated with anti-ROMK (10 µg/mL) and anti-aquaporin 2 (AQP2; Cat. No. sc9882, Santa Cruz Biotechnology, 0.4 µg/mL). Other sections were incubated with anti-αBK (5 µg/mL) and anti-AQP2 (AQP-002, Alomone, 0.6 µg/mL). All primary antibodies were diluted in CytoVista Antibody Dilution Buffer and incubated overnight at 4°C. Following an extensive wash with PBS, sections were incubated with Alexa Fluor 488 donkey anti-rabbit (1 mg/mL) and Cy3 AffiniPure donkey anti-goat (0.2 mg/mL) or with Alexa Fluor 488 goat anti-mouse IgG2a (Cat. No. A21131, ThermoFisher, 1 mg/mL) and Cy3 AffiniPure anti-rabbit (Cat. No. 705-165-152, Jackson ImmunoResearch, 0.2 mg/mL) for 2 h at room temperature. Kidney sections were mounted with SlowFade Glass Soft-set Antifade Mountant and imaged on a Leica Stellaris 8 confocal microscope using a ×20 objective (numerical aperture = 1).

Whole Mount Immunohistochemistry

Organoids were fixed in 4% paraformaldehyde for 60 min at room temperature and then permeabilized with 0.3% Triton X-100 in PBS for 20 min with gentle shaking. Following 1 h of room temperature block in PBS containing 0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20, and 10% horse serum, organoids were incubated with anti-γENaC antibody (Cat. No. SPC405D, Stressmarq, 10 µg/mL) and an antibody against E-cadherin (Cat. No. 610182, BD Biosciences, 1.25 µg/mL) in blocking buffer overnight at 37°C with gentle shaking. Organoids were then washed with 0.3% Triton X-100 in PBS for 1 h (3 times for 20 min) with gentle shaking and incubated with secondary antibodies (Alexa Fluor 488 donkey anti-rabbit and Cy3 donkey anti-mouse) for 4 h at 37°C with gentle shaking. Optical clearing was performed using a previously established method (17, 18). After a 1-h wash with PBS, organoids were rapidly dehydrated using sequentially higher concentrations of ethanol (30%, 50%, 70%, 90%, 100%, and 100%, 5 min each) at room temperature with gentle agitation. Organoids were then transferred to ethyl cinnamate (Cat. No. 112372, Sigma-Aldrich), covered, and shaken at room temperature overnight. Cleared organoids were then placed in fresh ethyl cinnamate for imaging using a ×25 (numerical aperture = 0.95) objective on a SP8 Leica confocal microscope.

Immunoblot Analysis

Day 14–18 kidney organoids (∼200–500 organoids/sample from 5 different samples) were rinsed with ice-cold PBS and homogenized in CelLytic MT Cell Lysis Reagent (C3228, Sigma) supplemented with 1% protease inhibitor cocktail Set III (Cat. No. 539134, Sigma). Lysates were then incubated for 30 min at 4°C with end-over-end mixing to facilitate protein extraction. Insoluble material was removed by centrifugation at 14,000 rpm for 5 min at 4°C, and the clear supernatant was collected. Kidneys from four adult male C57BL6 mice were processed for immunoblot analysis. A quarter of a kidney was homogenized in 300 µL of lysis buffer supplemented with protease inhibitors. Lysates were then mixed for 30 min at 4°C and centrifuged to remove insoluble tissue, and supernatants were used for immunoblots. Protein concentrations were measured with a Pierce BCA Protein Assay Kit (Cat. No. 23227, ThermoFisher Scientific). Lysates (30–50 µg) of kidney organoids and 10–50 µg lysates of the mouse kidney were denatured in equal volume of 2× Laemmli sample buffer containing 10% β-mercaptoethanol at 37°C for 30 min. Samples were separated on 18-well 4–15% Criterion TGX stain-free gels and transferred to nitrocellulose membranes. After being blocked in 5% nonfat milk for 1 h at room temperature to reduce nonspecific binding, individual membrane strips were incubated overnight with primary antibodies (anti-βENaC and anti-γENaC diluted to 0.5 µg/mL in 1% nonfat milk) at 4°C with gentle rocking. Anti-rabbit IgG horseradish peroxidase (Cat. No. 5220-0336, SeraCare) or anti-mouse IgG horseradish peroxidase (Cat. No. 5450-0011, SeraCare) was diluted to 0.2 µg/mL in 1% nonfat milk and incubated for 2 h at room temperature. Chemiluminescence signal was detected using Clarity Western ECL Substrate (Cat. No. 1705061, Bio-Rad) and imaged with the Bio-Rad ChemiDoc system.

Isolation of Cells From hiPSC-Derived Kidney Organoids

To isolate cells and tubule segments, day 14 kidney organoids were transferred to a clean 15-mL Falcon tube in 2 mL of stage II medium. Kidney organoids were gently triturated with a fire-polished glass pipette, and the cell suspension was centrifuged at 420 g for 5 min. The pellet was resuspended in DMEM supplemented with 10% FCS, 1% penicillin-streptomycin, and 1% minimal essential medium nonessential amino acids (Invitrogen). The centrifugation and resuspension steps were repeated three times. Finally, the pellet was resuspended in 1.5 mL of culture media, and the suspension was seeded on 8-mm-diameter round glass coverslips coated with poly-l-lysine. After an incubation of 2 h at 37°C in an atmosphere with 5% CO₂, 3 mL of warm culture media were added to each well and the tissue culture plate was returned to the incubator. Electrophysiological experiments were performed within 2–10 h of plating.

Electrophysiological Experiments

The electrical activity of cells and tubules isolated from hiPSC-derived kidney organoids was evaluated using the patch-clamp technique as previously described (19). Briefly, glass coverslips containing isolated cells were transferred to a chamber mounted on the stage of a Nikon Ti inverted microscope equipped a Sedat Quad set (Chroma Technology), a Lambda XL light source (Sutter Instruments), and an ORCA-Flash 2.8 camera (Hamamatsu). Micropipettes were pulled from borosilicate glass capillary tubes with a PP-81 puller (Narishige). Fire-polished micropipettes with a tip resistance of 1.5–3 MΩ were used for voltage-clamp recordings. Experiments were performed at room temperature with a PC-505B patch-clamp amplifier (Warner Instruments). Signals were low-pass filtered at 1 kHz (four-pole Bessel filter) and digitized using a Digidata 1440 A (Molecular Devices) at 5 kHz. Cell capacitance was obtained by reading the value for whole cell input capacitance neutralization directly from the amplifier. Command protocols and data acquisition were controlled with pClamp 10 (Molecular Devices).

Cell-Attached Recordings

For cell-attached patch experiments to measure BK channel activity, the recording chamber contained the following solution (in mM): 135 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 glucose, and 10 HEPES, pH 7.4. The pipette solution was composed of (in mM) 145 KCl, 1 MgCl₂, 0.1 CaCl₂, 1 EGTA, and 10 HEPES (pH 7.2). For cell-attached experiments, potentials were expressed in terms of the voltage applied to the pipette (extracellular side of the patch). Currents were evoked by 1-s 10-mV depolarizing steps from −80 to +80 mV. The single channel slope conductance was obtained by fitting a straight line to the linear part of current-voltage curves. To measure ENaC single channel activity in the cell-attached configuration, we used a Li⁺-based pipette solution composed of (in mM) 140 LiCl, 2 MgCl₂, and 10 mM HEPES (pH 7.4) and a bath solution containing (in mM) 150 NaCl, 5 KCl, 1 CaCl₂, 2 MgCl₂, 5 glucose, and 10 HEPES (pH 7.4) (20). Single channel conductance and channel open probability [NP_o; where N is the number of channels present in the patch multiplied by the open probability (P_o)] were obtained from single channel tracings recorded at pipette potentials of −60 mV.

Whole Cell K⁺ Currents

Steady-state conductance-voltage relationships were obtained using the conventional whole cell technique. Cell access was gained by mechanical rupture of the membrane attached to the patch pipette. Tubular segments were patched from the basolateral side. The bath solution was composed of (in mM) 135 NaCl, 5 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 glucose, and 10 HEPES, pH 7.4. The pipette solution was composed of (in mM) 145 KCl, 1 MgCl₂, 1 EGTA, and 10 HEPES (pH 7.2), and free Ca²⁺ was adjusted to 10 µM using MaxChelator software (version 2.4). Whole cell currents were evoked by 0.2-s 10-mV depolarizing steps from −100 to +100 mV. Whole cell slope conductance was estimated by least mean square fitting to the linear portion of the current-voltage curve (+40- to +100-mV range for BK_Ca channels). To define the identity of the K⁺ channels mediating whole cell currents, the following inhibitors were added successively in this order: 1) iberiotoxin (IbTX), 2) VU592, and 3) Ba²⁺. IbTX (Alomone) is a toxin purified from the Eastern Indian red scorpion Buthus tamulus that selectively binds to the BK_Ca α-subunit and inhibits its function by decreasing channel P_o. IbTX was used at a final concentration of 100 nM to define the component of the K⁺ current mediated by BK_Ca (21). VU591, a ROMK-specific inhibitor, was added to the bath solution to obtain a final concentration of 10 µM (22). Ba²⁺ is nonselective K⁺ channel blocker and was added to the bath solution at a final concentration of 5 mM. The voltage of half-maximal activation (V₅₀) was determined by fitting normalized conductance-voltage curves to the following Boltzmann function: G/G_max = 1 + exp[−(V − V₅₀)QF/RT]⁻¹, where G is chord conductance at the command potential voltage (V) assuming a K⁺ potential of −85 mV, G_max is the maximal conductance, Q is the equivalent gating charge (slope of the conductance-voltage relationship or “voltage dependence”), temperature (T) was 20°C, F is Faraday’s constant, and R is the gas constant.

Whole Cell Na⁺ Currents

Cell access was obtained with amphotericin B (perforated patch technique). Patch pipettes were back filled with an intracellular solution containing 120 µg/mL amphotericin B. The bath solution was composed of (in mM) 135 NaCl, 1 MgCl₂, 2.5 CaCl₂, 10 glucose, and 10 HEPES, pH 7.4. The intracellular solution was composed of (in mM) 135 NaCl, 1 MgCl₂, 0.1 CaCl₂, 1 EGTA, and 10 HEPES (pH 7.2). For current recordings, the membrane potential was initially held at −60 mV. Whole cell currents were evoked by 0.2-s 10-mV depolarizing steps from −80 to +80 mV. The single channel slope conductance was obtained by fitting a straight line to the linear part of current-voltage curves. Amiloride, an ENaC blocker, was used at a final concentration of either 10 or 100 µM.

Statistical Analysis

Data are expressed as means ± SD (n), where n equals the number of independent experiments. Parametric or nonparametric tests were used based on the results of normality tests. Comparisons between two groups were performed with a t test (parametric) or Mann-Whitney test (nonparametric). For parametric multiple comparisons, we used ANOVA followed by Tukey’s multiple comparisons test, and for nonparametric multiple comparisons, we used a Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. A P value of <0.05 was considered statistically significant. Fitting and statistical comparisons were performed GraphPad Prism 8 (GraphPad Software) and Clampfit (Molecular Devices).

RESULTS

Kidney Organoids Derived From hiPSCs Express Genes Encoding BK_Ca, ROMK, and ENaC Subunits

With the current protocol, 14-day-old kidney organoids are optimally differentiated with tubular epithelia subdivided into proximal and distal segments (10). By analyzing RNA-sequencing data at this stage, we found expression of genes for specific nephron segments, including the proximal tubule, thick ascending limb, and ASDN. The kidney organoids also expressed genes for subunits of key ion channels expressed in kidney tubules. These included 1) BK_Ca subunits KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4; 2) the ROMK subunit KCNJ1; and 3) ENaC subunits SCNN1A, SCNN1B, SCNN1D, and SCNN1G (see Fig. 1). Lower levels of expression were observed for KCNMA1, KCNMB1, SCNN1B, and SCNN1G.

Figure 1.

RNA-sequencing gene expression analysis of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels (solid black circles), renal outer medullary K⁺ (ROMK) channels (dark gray triangles), and the epithelial Na⁺ channel (ENaC; light gray open circles) in day 14 kidney organoids derived from human induced pluripotent stem cells. Results represent analyses from quadruplicate samples as described in methods (n = 4). Data are presented as reads per kilobase per million mapped reads.

Kidney Organoids Derived From hiPSC Express Functional BK_Ca and ROMK Channels

Figure 2 shows typical BK_Ca channel activity in a cell-attached patch on a cell isolated from kidney organoids. Under these conditions, currents showed a linear relationship in the current-voltage curve, with a reversal potential of −27 ± 3 mV and single channel conductance of 201 ± 25 pS (n = 12; Fig. 2B). In the majority of single channel recordings, one to two channels were observed per patch. The openings were markedly voltage dependent, with P_o increasing sharply with depolarization. We observed an increase in the NP_o at more positive voltages (Fig. 2A), with NP_o = 0.06 ± 0.11 at −60 mV, 0.69 ± 0.31 at 0 mV, and 0.83 ± 0.23 at +60 mV (n = 11). These features are characteristic of BK_Ca channels in kidney epithelia and other cells.

Figure 2.

Kidney organoids express functional large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels. A: representative tracings of cell-attached recordings performed in isolated cells from human induced pluripotent stem cell-derived kidney organoids. Typical single channel current recordings of BK_Ca channels at different membrane voltages are shown (values represent pipette voltage). The dotted lines represent the closed state. B: single channel current-voltage (I–V_m) relationship for BK_Ca channels obtained from 12 membrane patches in a cell-attached configuration (12 cells from 2 different organoid preparations). The unitary current amplitude was determined from the tracings obtained at the different membrane voltages (solid circles). The continuous line corresponds to the experimental data fit. A single channel conductance of 201 ± 25 pS was estimated using the slope fit with a linear function. Data are presented as means ± SD.

The presence of BK_Ca channels in kidney organoids was confirmed using whole cell recordings. From all cells tested, 60% of cells (18/30 cells) exhibited macroscopic currents that were significantly inhibited by the addition of the selective BK_Ca channel inhibitor IbTX (100 nM) to the extracellular medium. The addition of IbTX produced an ∼60% decrease in the outward whole cell current (Fig. 3). IbTX-sensitive whole cell conductance was 2.78 ± 0.85 nS (Fig. 3B). A V₅₀ value of −5.24 ± 6.65 mV was estimated from the fitting of a Boltzman sigmoidal equation to the curve obtained by plotting the normalized conductance (G/G_max) as a function of the holding potential (V). In these cells, subsequent addition of the ROMK channel inhibitor VU591 (10 µM) did not produce a significant further reduction in the whole cell current (Fig. 3C). The addition of 5 mM Ba²⁺ to the extracellular medium produced a further ∼35% inhibition of the macroscopic outward whole cell current (Fig. 3C). In contrast, 30% of the cells evaluated (10 of 30 cells) showed an outward macroscopic current that saturated close to +50 mV (Fig. 4). In these cells, 100 nM IbTX did not display inhibitory activity. However, currents were strongly reduced by the subsequent addition of VU591 (10 µM), a highly selective inhibitor of ROMK channels (22). VU591 produced an ∼70% inhibition of the total outward whole cell current. Subsequent addition of 5 mM Ba²⁺ to the extracellular medium produced an additional ∼25% inhibition of this current (Fig. 4). A small fraction of the cells tested with the patch-clamp technique under whole cell conditions (2 of 30 cells) did not display any significant outward current and were not further studied.

Figure 3.

Effect of inhibitors on whole cell currents recorded in isolated cells from human induced pluripotent stem cell-derived kidney organoids expressing iberiotoxin (IbTX)-sensitive currents [large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels]. A: whole cell currents were evoked by voltage steps from −100 to 100 mV in 10-mV increments from a holding potential of −60 mV. Representative tracings obtained under control (Ctrl) conditions, in the presence of 100 nM IbTX, or in the presence of three inhibitors (100 nM IbTX, 10 µM VU951, and 5 mM Ba²⁺) are shown. B: whole cell current-voltage (I–V_m) relationship for cells exhibiting BK_Ca channel activity. Whole cell currents under control conditions are represented with solid circles, currents after the addition of IbTX are represented by diamonds, currents after the addition of IbTX and VU591 are represented by triangles, and currents after the addition of the three inhibitors (IbTX, VU591, and Ba²⁺) are represented by open circles. Data were obtained from 18 cells from 7 different organoid preparations (mean ± SD). C: summary of the effect of K⁺ channel inhibitors on whole cell currents measured at +40 mV (means ± SD, n = 18, *P < 0.05 and **P < 0.01, ANOVA followed by Tukey’s multiple comparison test).

Figure 4.

Effect of inhibitors on whole cell currents recorded in isolated cells from human induced pluripotent stem cell-derived kidney organoids expressing of VU591-sensitive currents [renal outer medullary K⁺ (ROMK) channels]. A: whole cell currents were evoked by voltage steps from −100 to 100 mV in 10-mV increments from a holding potential of −60 mV. Representative tracings obtained under control (Ctrl) conditions and in the presence of 100 nM iberiotoxin (IbTX) and 10 µM VU591 are shown. These cells did not show an inhibitory effect of 100 nM IbTX. B: whole cell current-voltage (I–V_m) relationship for cells exhibiting ROMK channel activity. Whole cell currents under control conditions are represented by solid circles, currents after the addition of IbTX are represented by diamonds, currents after the addition of IbTX and VU591 are represented by tringles, and currents after the addition of the three inhibitors (IbTX, VU591, and Ba²⁺) are represented by open circles. Data were obtained from 10 cells from 3 different organoid preparations (mean ± SD). C: summary of the effects of serial additions of K⁺ channel inhibitors on whole cell currents measured at +10 mV (means ± SD, n = 10, *P < 0.05 and **P < 0.01, ANOVA followed by Tukey’s multiple comparison test).

To evaluate whether cell dispersion altered the functional expression of BK_ca and ROMK channels, we recorded whole cell currents in isolated tubules from kidney organoids (Fig. 5A). These results confirmed that BK_ca and ROMK channels are expressed in different cell populations. Cells displaying significant IbTX-sensitive currents did not respond to the subsequent addition of VU591 (Fig. 5C). In contrast, cells that exhibited VU591-sensitive outward currents did not exhibit any IbTX-sensitive currents (Fig. 5E). Both cell populations showed significant Ba²⁺-blockable outward currents. In summary, our results indicate that kidney organoids derived from hiPSCs express functional BK_Ca and ROMK channels in different cell populations.

Figure 5.

Effect of K⁺ channel inhibitors on whole cell currents recorded in isolated tubules from human induced pluripotent stem cell-derived kidney organoids. A, left: bright-field micrograph of an isolated tubule from a human induced pluripotent stem cell-derived kidney organoid captured during a patch-clamp experiment. Whole cell currents were evoked by voltage steps from −100 to 100 mV in 10-mV increments from a holding potential of −60 mV. The experimental protocol is shown on the right. B: representative tracings of iberiotoxin (IbTX)-sensitive large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels obtained under control (Ctrl) conditions, in the presence of 100 nM IbTX, or in the presence of the three inhibitors (IbTX, VU951, and Ba²⁺). C: summary of the effect of K⁺ channel inhibitors on whole cell currents in cells expressing IbTX-sensitive BK_Ca channels measured at +40 mV (mean ± SD, n = 5 from 2 different organoid preparations, *P < 0.05 and **P < 0.01, ANOVA followed by Tukey’s multiple comparison test). D: representative tracings of VU591-sensitive renal outer medullary K⁺ (ROMK) channels obtained under control conditions, in the presence of 100 nM IbTX and 10 µM VU591, or in the presence of the three inhibitors. E: summary of the effect of K⁺ channel inhibitors on whole cell currents in cells expressing VU591-sensitive ROMK channels measured at +40 mV (mean ± SD, n = 3 cells from 2 different organoid preparations, *P < 0.05 and **P < 0.01, ANOVA followed by Tukey’s multiple comparison test).

Kidney Organoids Derived From hiPSCs Express Functional Amiloride-Sensitive Na⁺ Channels

To evaluate whether cells isolated from kidney organoids display Na⁺ currents, whole cell currents were studied under Na⁺ symmetrical conditions (i.e., the pipette and extracellular conditions contained the same Na⁺ concentration) using the amphotericin B-perforated patch technique. Consistently, we observed that 50% of the cells tested (8 of 16 cells) displayed significant inward and outward whole cell currents that did not rectify (i.e., linear current-voltage relationship) under these conditions (Fig. 6, A and C). Importantly, the addition of 100 µM amiloride to the extracellular medium significantly inhibited both inward and outward whole cell currents (Fig. 6C). In the presence of extracellular amiloride, inward currents were inhibited by 85% at −60 mV (from −7.7 ± 4.6 pA/pF under control conditions to −1.1 ± 1.5 pA/pF after the addition of 100 µM amiloride, n = 8, P < 0.05, Mann-Whitney nonparametric test). The amiloride-sensitive macroscopic current showed a chord conductance of 140 ± 130 pS and a reversal potential of −3 ± 14 mV. We confirmed that currents at −60 mV were inhibited by 10 µM amiloride in isolated cells (from −12.9 ± 7.6 pA/pF under control conditions to −2.7 ± 2.4 pA/pF after the addition of 10 µM amiloride, n = 5, P < 0.05, t test; Fig. 6B). We also observed the presence of ENaC currents in isolated tubules from kidney organoids where currents at −60 mV were inhibited by 10 µM amiloride (from −11.0 ± 3.8 pA/pF under control conditions to −1.2 ± 0.7 pA/pF after the addition of 10 µM amiloride, n = 5, p < 0.01, t test; Fig. 6D). Single channel currents consistent with ENaC activity were detected in 5 of 46 experiments performed in the cell-attached configuration (Fig. 6E). We used a Li⁺-based pipette solution and a pipette potential of −60 mV. The calculated single channel conductance and NP_o under these conditions were 10.6 ± 3.2 pS (n = 5) and 0.09 ± 0.09 (N = 1.3 ± 0.6 and P_o = 0.06 ± 0.03, n = 3), respectively.

Figure 6.

Effect of amiloride (Amil) on whole cell currents from human induced pluripotent stem cell (hiPSC)-derived kidney organoids. A: patch-clamp experiments were performed with the amphotericin B perforated whole cell technique as described in methods. Whole cell currents were evoked by voltage steps from −80 to 80 mV in 10-mV increments from a holding potential of −60 mV. Representative tracings obtained under control (Ctrl) conditions (left) or after the addition of 100 µM Amil to the extracellular medium are shown. B: summary of the effect of 10 µM Amil on whole cell currents measured at −60 mV (means ± SD, n = 5 cells from 3 different organoid preparations, *P < 0.05, paired t test). C: whole cell current-voltage (I–V_m) relationship under Ctrl conditions (solid circles) or in the presence of extracellular Amil (100 µM, open circles). Data were obtained from 8 cells from 3 different organoid preparations (means ± SD). D: summary of the effect of 10 µM Amil on whole cell currents measured at −60 mV in isolated tubules from hiPSC-derived kidney organoids (means ± SD, n = 5, **P < 0.01, paired t test). E: single channel recordings from hiPSC-derived kidney organoids in the cell-attached mode at −60 mV (negative pipette potential). Pipette and bath solutions contained 140 mM LiCl and 150 mM NaCl, respectively. C and O indicate the closed and open state, respectively. F: representative whole cell current tracings (as described in A) obtained after the application of 2 µg/mL chymotrypsin (CHY) before and after the addition of 100 µM Amil. G: summary of the effect of 100 µM Amil on whole cell currents measured at −60 mV for Ctrl cells or cells treated with 2 µg/mL CHY in the extracellular medium (means ± SD, n = 5–8 cells, P = not significant, Mann–Whitney nonparametric test).

One of the defining characteristics of ENaC is its functional activation by serine and metalloproteases, including chymotrypsin. We examined whether ENaCs in cells isolated from organoids were activated by extracellular chymotrypsin. Interestingly, application of 2 µg/mL chymotrypsin to the extracellular bath, a concentration that activates αβγENaCs expressed on Xenopus oocytes (16), did not affect amiloride-sensitive whole cell Na⁺ currents (Fig. 6, E and F).

Channel Localization and Expression in Kidney Organoids

The expression and localization of epithelial Na⁺ channel subunits in human kidney organoids were assessed by immunofluorescence microscopy and immunoblot analysis. Sections were costained with antibodies against the γ-subunit of ENaC and against E-cadherin, a distal nephron segment marker (23). As shown in Fig. 7A, γENaC localized to the luminal side in E-cadherin-positive tubules as well as intracellularly. In addition, we performed staining on the whole mount and observed γENaC expression within some E-cadherin-positive tubules (Fig. 7B). The intensity of immunostaining staining in whole mount sections was variable, possibly reflecting limited diffusion of antibodies to sites deep within an organoid. Full-length γENaC was also detected by immunoblot analysis of organoid lysates (Fig. 8), whereas both full-length and cleaved γENaC were present in mouse kidney lysates. In contrast, we did not detect luminal βENaC expression in E-cadherin-positive tubules (not shown). βENaC is a ∼100-kDa band on immunoblots and often appears as a doublet where the slower migrating band reflects the presence of processed N-glycans. Although βENaC was detected in the kidney lysate (Fig. 8, arrowhead) along with other presumably nonspecific bands, we only detected a slower migrating βENaC band in organoid lysates.

Figure 7.

γ-Epithelial Na⁺ channel (ENaC) and ROMK expression in human induced pluripotent stem cell-derived kidney organoids. A: sections were costained with antibodies (green) against the γ subunit of ENaC, the renal outer medullary K⁺ (ROMK) channel, and the α-subunit of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel. Distal nephron segments were labeled with E-cadherin (red) or GATA-3 (gray). No signal was detected in the negative control sections where primary antibodies were omitted (No 1° Abs). Expression of ROMK and γENaC is indicated by arrowheads. B: whole mount human kidney organoids were costained with antibodies against the γ-subunit of ENaC (green) and with the distal nephron marker E-cadherin (red). Control sections where primary antibodies were omitted are also shown. C: mouse kidney sections (4 µm) were costained with antibodies against the α-subunit of the BK_Ca channel or the ROMK channel (green) and with the collecting duct marker aquaporin-2 (AQP2; red). Expression of αBK_Ca or ROMK in AQP2-positive tubules is indicated by arrowheads. *Expression of ROMK in AQP2-negative tubules. Scale bars are shown in overlay images. Staining was performed in four different batches of human kidney organoids. Kidney sections were from three individual mice were used immunostaining as positive controls.

Figure 8.

Protein abundance of β- and γ-epithelial Na⁺ channel (ENaC) subunits by immunoblot analyses of human induced pluripotent stem cell-derived kidney organoid (Org) and mouse whole kidney (Kid) tissue lysates. Blots are representative of 5 samples of kidney organoids and 4 samples of mouse kidney lysates (used as positive controls) for each target. Molecular weight markers are shown on the left of each panel, and bands of interests are indicated by arrowheads. Total protein loading was assessed using stain-free gels.

Luminal ROMK localization was noted in E-cadherin-negative tubules in organoids (Fig. 7A). In mouse kidney sections, the ROMK channel was localized to AQP2-positive as well as AQP2-negative tubules (Fig. 7C). We did not detect the BK α-subunit in organoids by immunofluorescence microscopy in human organoid sections (Fig. 7A), whereas the BK α-subunit was detected in AQP2-negative (i.e., intercalated) cells of mouse kidney sections by immunofluorescence microscopy (Fig. 7C).

DISCUSSION

Following the first description of kidney organoids derived from iPSCs in 2015 (24), techniques to derive kidney organoids and studies of the differentiation of specific structures within these organoids continue to evolve. Among these structures are polarized tubular segments, reflecting the differentiation seen in the developing nephron (25–27). Recent advances include the use of ureteric bud progenitors to derive collecting duct structures (28). Kidney organoids have been primarily characterized on the basis of their morphology and expression of specific genes and proteins. Recent nephron progenitor lines expressing fluorescent markers that define specific tubular segments have been developed (29). Progenitors with disease-associated mutations have been used to derive organoids with specific phenotypes, such as cystic structures associated with polycystic kidney disease (28, 30). Kidney organoids are also being used to assess responses to cellular stress associated with specific drugs or toxins (31–33).

One of the key functions of the kidney is the regulated absorption from the ultrafiltrate and secretion into the ultrafiltrate of specific ions and small molecules. Many of the transport processes that occur in kidney epithelia are essential for the regulation of extracellular fluid volume, water homeostasis and cell volume maintenance and K⁺, Na⁺, and acid-base balance (34–37). To date, there have been limited studies investigating the functional properties of channels and other transporters expressed in kidney organoids.

The goal of the present study was to demonstrate that several key cation channels found in the ASDN, including the K⁺ secretory channels BK_Ca and ROMK and the Na⁺ selective channel ENaC, are functionally expressed in cell populations within kidney organoids. RNA-sequencing analysis of 14-day hiPSC-derived organoids demonstrates that they express subunits encoding these channels (Fig. 1). Both BK_Ca channels and ENaC have been shown to be functionally expressed in organoids of different origin. BK_Ca channel expression and activity was detected in brain, inner ear, gastric, and cardiac organoids (38–41), whereas ENaC has been shown to be functional expressed in lung and intestinal organoids (42–47). Both channels have been shown to be coexpressed in pancreatic organoids (48). The presence of SCNN1A (αENaC), SCNN1G (γENaC), and KCNJ1 (ROMK) has been detected by gene expression in kidney organoids (Fig. 1) (28, 49–51). We also found low levels of expression of KCNMA1 (BK α-subunit), SCNN1B (βENaC), and SCNN1G (γENaC) in our organoids (Fig. 1). Expression levels of both SCNN1G and KCNJ1 have been shown to be strongly regulated by aldosterone and vasopressin in kidney organoids (50). Interestingly, removal of both WNT and glial cell-derived neurotrophic factor signaling from the culture medium of kidney organoids induced a shift away from the ureteric epithelium toward a distal nephron phenotype including the expression of SCNN1G (γENaC) (28). However, no functional analyses were performed to characterize these channels in kidney organoids.

We assessed functional expression of BK_Ca, ROMK, and ENaC using whole cell patch clamp. For BK_Ca channels and ENaC, we also used cell-attached patch clamp to assess single channel activity. Although nephron progenitor lines expressing a fluorescent marker (mCherry) for distal segments are available (29), using one of these lines we were unable to detect fluorescent cells with our patch-clamp microscope. However, we frequently found isolated cells as well as cells with isolated tubules expressing these channels. The single channel and whole cell patch clamp characteristics of BK_Ca channels, including voltage-dependent activation, large single channel conductance (∼200 pS), and inhibition by IbTX, have been well described (7). Expression of ROMK channels and ENaC by whole cell patch clamp was defined based on the response of currents to specific ROMK [VU591 (22)] and ENaC [amiloride (52)] inhibitors. We also observed single channel currents consistent with ENaC, using Li⁺ as the charge carrier. Although mature principal cells express both BK_Ca and ROMK channels (7), we did not observe cells that clearly expressed both channels by whole cell patch clamp. The inhibition of whole cell currents by Ba²⁺, observed in the presence of the BK_Ca (IbTX) and ROMK (VU591) inhibitors, suggests that these cells express other K⁺ channels. This is not a surprise, as these cells are likely to express Ba²⁺-inhibitable basolateral K⁺ channels, such as Kir4.1/Kir5.1 (53, 54).

In the kidney, ENaCs are formed by α-, β-, and γ-subunits and are activated by proteases that remove imbedded inhibitory tracts from the α- and γ-subunits (55). Although furin cleaves the α-subunit twice in the biosynthetic pathway releasing an inhibitory tract, the γ-subunit is cleaved only once by furin and requires cleavage by a second protease to release its inhibitory tract, transitioning channels to a high open probability state (55–61). Chymotrypsin and trypsin are two of the proteases that activate ENaC (62). We did not observe activation of amiloride-sensitive whole cell currents by chymotrypsin, suggesting that the channels have released the inhibitory tract from the γ-subunit. Although we did not observe cleavage of the γ-subunit on organoid immunoblots, an apparent lack of ENaC activation by chymotrypsin is consistent with γ-subunit processing by proteases.

A previous study has suggested that δβγ-channels have a high intrinsic open probability and exhibit modest activation by proteases (63). Although the δ-subunit of ENaC is poorly expressed in the human kidney (5), it is well expressed in our human kidney organoids (Fig. 1) and could contribute to functional channels. Although we found low levels of SCNN1B (βENaC) gene expression in kidney organoids, βENaC may also be a component of functional channels. Our results do not allow us to define the subunit composition of the functional channels in kidney organoids.

Perspectives and Significance

We found that hiPSC-derived kidney organoids (day 14) functionally express key functional K⁺ and Na⁺ channels that are expressed on the apical membranes of cells in the ASDN, including BK_Ca, ROMK, and ENaC. In addition, we noted γENaC expression in E-cadherin-positive tubular segments and ROMK channels in E-cadherin-negative tubular segments by immunofluorescence microscopy. Our findings suggest that nephron differentiation in these organoids is associated with the expression of specific distal nephron cation channels that are seen in the early postnatal period of development (64, 65). Furthermore, our work suggests that human kidney organoids provide a novel tool to study the function and regulation of key channels in the nephron as well as the effects of human gene variants on channel functional expression and localization.

GRANTS

This work was supported by National Institutes of Health Grants U01DK107350, R01HL147818, R01DK038470, R01DK069403, R01DK130901, UC2DK126122, T32EB001026, S10OD021627, S10OD028596 and P30DK079307. N.M. was supported by the Urology Care Foundation Research Scholar Award Program, and A.J.P. was supported by an American Society of Nephrology Ben J. Lipps Award.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.M., M.D.C., N.A.H., and T.R.K. conceived and designed research; N.M., A.J.P., S.Shi, S.Sheng, C.J.B., J.C.M., and T.V. performed experiments; N.M., A.J.P., S.Shi, S. Sheng, C.J.B., and T.V. analyzed data; N.M., A.J.P., S.Shi, S. Sheng, C.J.B., M.D.C., T.V., and T.R.K. interpreted results of experiments; N.M. and T.V. prepared figures; N.M. and T.R.K. drafted manuscript; N.M., A.J.P., S.Shi, S. Sheng, C.J.B., M.D.C., T.V., A.J.D., N.A.H., and T.R.K. edited and revised manuscript; N.M., A.J.P., S.Shi, S. Sheng, C.J.B., M.D.C., T.V., A.J.D., N.A.H., and T.R.K. approved final version of manuscript.