Organoid > Volume 2; 2022 > Article
Kim, Lim, Jung, and Kang: Generation of proximal tubule spheroids for nephrotoxicity assessment

Abstract

Background

To date, nephrotoxicity in new drug development has been evaluated through two-dimensional culture of representative cell lines, such as HK-2 and human proximal tubule epithelial cells (hPTECs). Approximately 20% of new drugs that were safe in preclinical studies were withdrawn from clinical trials due to nephrotoxicity, which means the current renal cell lines used in preclinical trials have limitations for the accurate detection of nephrotoxicity.

Methods and Results

Here, we established proximal tubule cell lines from immortalized mixed primary renal cells and generated functional proximal tubule cell spheroids, which expressed all apical basolateral transporters and showed epithelial polarity. Moreover, they showed a more sensitive drug response than hPTECs, which have been commonly used as in vitro kidney models.

Conclusion

Taken together, the proximal tubule cells described in this study provide a more stable, reproducible, and accurate in vitro kidney model for predicting nephrotoxicity, which could help early compound development.

Introduction

The kidney consists of a complex basic functional unit called the nephron, which is composed of the glomerulus, Bowman's capsule, proximal tubule, loop of Henle, distal tubule, and collecting duct [1]. The main functions of the kidney are to filter metabolites and waste products and excrete them into the urine. The blood flow to the kidneys receives 20% to 25% of cardiac output and passes waste products for excretion. Among the various segments of the nephron, the proximal tubule is responsible for most of the reabsorption process, including glucose and amino acids, sodium ions, chlorine ions, water, and urea [2]. In the process of concentrating and reabsorbing glomerular filtrates that have passed through the glomerulus, the concentration of various toxic substances increases, and the proximal tubule is first exposed to the toxicity of concentrated drugs [3]. Moreover, the proximal tubule has a high energy requirement with large amounts of mitochondria, which are prone to cell damage, dedifferentiation, and cell death [4]. Toxic drugs can cause proximal tubular damage by direct tubular cell toxicity, an inflammatory response, oxidative stress, diminished mitochondrial function, and restricted tubular transporter activity [5]. For example, drugs such as immunosuppressive agents, chemotherapeutics, and antimicrobials disrupt tubular cell polarity, which is critical for renal cell function, resulting in the dislocation of apical and basolateral transporters and a leaky epithelium [6]. On the contrary, a reliable in vitro model system to study drug effects or toxicity should necessarily have proximal tubule cell polarity and critical transporter expression.
Human proximal tubule epithelial cells (hPTECs) are the most commonly used in vitro kidney model for nephrotoxicity testing [7,8]. However, these cells have limited growth capacity and tend to lose their original phenotypes, such as epithelial polarity and drug transporter expression and activity over time [8-10]. In a previous study, we established stable and reproducible renal primary cells from patient kidney samples that can be differentiated into functional spheroids for in vitro drug-induced nephrotoxicity assessments [11]. Using the mixed immortalized cell lines described in the previous study, in this study, we isolated and established immortalized primary renal proximal tubule cells (iRPTCs), and tried to generate functional spheroids for in vitro drug-induced nephrotoxicity studies. Compared to hPTECs, iRPTCs in our culture system showed enhanced functionality such as epithelial polarity, transporter expression, and enzyme activity and response, as well as sensitive responsiveness to drug-induced toxicity.

Materials and methods

1. Antibodies and reagents

CD13 (Sorting and IF, #ab52461; Abcam, Cambridge, UK), lotus tetragonolobus lectin (LTL) (IF, #FL-1321; Vector Labs, Newark, CA, USA), aquaporin 1 (IF, #sc32737; Santa Cruz, Dallas, TX, USA), Dulbecco's modified Eagle’s medium with nutrient mixture F-12 (DMEM/F12) (Gibco, Waltham, MA, USA), FBS (Gibco), epidermal growth factor (EGF) (PeproTech, Waltham, MA, USA), basic fibroblast growth factor (PeproTech), insulin-transferrin-selenium (ITS, Gibco), 1α,25-dihydroxyvitamin D3 (Sigma, St. Louis, MO, USA), all-trans-retinoic acid (ATRA) (Sigma), and the RNAeasy kit (Qiagen, Valencia, CA, USA).

2. Immortalized primary cells and sorted proximal tubule cell culture

Human kidneys were collected from patients who underwent surgery for nephrectomy at Chungnam National University Human Resources Bank (Daejeon, Korea) in accordance with the relevant guidelines and regulations. Informed consent was obtained from patients for the use of specimens for research purposes only. We selected healthy and normal kidneys (glomerular filtration rate above 65 mL/min/1.73 m2) for cell isolation. Cells were isolated by 2 mg/mL collagenase I digestion for 30 minutes at 37°C with gentle stirring. The cells were then filtered through a 100-μm mesh to isolate single cells. Cell suspensions were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 20 ng/mL EGF, and 1% penicillin/streptomycin at 5% CO2 and 37°C. After 2 to 6 hours, cells were incubated with human telomerase reverse transcriptase and SV40 lentiviruses and 4 μg/mL polybrene. To isolate proximal tubule cells, cells were sorted using a fluorescein isothiocyanate-conjugated anti-CD13 antibody. Cells were detached using 0.1% trypsin-ethylenediaminetetraacetic acid at 80% confluence and passaged. To determine the half maximal inhibitory concentration, human immortalized primary cells (2×103 cells) were seeded into 96-well plates and incubated in DMEM/F12 containing serially diluted drugs for 24 hours. Cell viability was measured using the CytoX cell viability assay kit (LPS Solution, Daejeon, Korea) according to the manufacturer’s instructions.

3. Ethics

All experimental protocols were approved by the Institutional Ethics Committee/IRB of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) (No. P01-202004-31-005).

4. Quantitative real-time polymerase chain reaction

RNA was isolated using the RNAeasy Mini kit, 1 μg was reverse transcribed using a cDNA archival kit (Life Technologies, Gaithersburg, MD, USA), and quantitative polymerase chain reaction was performed according to the manufacturer’s instructions (Applied Biosystems, Waltham, MA, USA, and Agilent Technologies, Santa Clara, CA, USA) using the SYBRGreen Master Mix. The data were normalized and analyzed using the ΔΔCT method. The primers used are listed in Table S1.

5. Generation of kidney spheroids using mouse and human renal primary cell lines

Immortalized renal cells were suspended in a 2:1 mixture of extracellular matrix (ECM) gel (Sigma) and Matrigel, and 50-μL droplets at 1 to 2×104 cells/μL were deposited on the inverted lid of a culture dish. The lid was then placed onto a phosphate-buffered saline-filled dish, and after gelation (3-6 hours later), cells in the matrix were transferred to a spheroid-forming unit filled with DMEM/F12 supplemented with 10% FBS, 1× ITS, 20 ng/mL EGF, 100 nM dexamethasone, 20 μM 1α,25-dihydroxyvitamin D3 and 5 μM ATRA for 6 hours. Cells were then cultured for 2 to 5 additional days without ATRA. For nephrotoxicity tests, kidney spheroids were treated with various concentrations of drugs in serum-free DMEM/F12 for 24 hours, and for transporter activity assays, spheroids were pretreated overnight with cimetidine or verapamil before drug treatment.

6. Immunofluorescence and immunocytochemical analysis

Spheroids embedded in 2% agar were fixed with 10% formalin, embedded in paraffin, and cut to a 5-μm thickness. The sections were incubated in 2% bovine serum albumin with 0.2% fish skin gelatin at room temperature for 1 hour to block nonspecific binding. The sections were incubated with primary antibodies overnight at 4°C, and then secondary antibodies at room temperature for 1 hour. Nuclei were counterstained with 4',6-diamidino-2-phenylindole.

7. Gamma-glutamyl transferase activity

Gamma-glutamyl transferase (GGT) activity was measured by following the release of para-nitroanilide from gamma-glutamyl-p-nitroanilide using a GGT activity colorimetric assay kit (BioVision, Milpitas, CA, USA). Cell and kidney spheroids were homogenized in 200 μL of ice-cold GGT assay buffer, and 10-μL aliquots were combined with 90 μL of GGT substrate solution and added to the assay plate for a 5-hour incubation. Absorbance changes at 418 nm were measured every 30 minutes at 37°C.

8. Response to parathyroid hormone

Parathyroid hormone (PTH) was obtained from Prospec (New Brunswick, NJ, USA). After overnight incubation with 0.1 mM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, cells were treated with 100 nM PTH for 30 minutes. Intracellular cyclic adenosine monophosphate (cAMP) was measured using a cAMP direct enzyme immunoassay kit.

9. Statistical analysis

Data are displayed as the mean ± standard error of the mean and were analyzed with Microsoft Excel (Microsoft Corp., Redmond, WA, USA). Statistical significance was determined using the two-tailed Student t-test. A p-value <0.05 was considered significant.

Results

1. Generation of functional renal proximal tubule spheroids using established primary proximal tubule cell lines

To isolate the proximal tubule cells from mixed renal primary cells, we used CD13, specific markers with extracellular epitopes expressed in the brush border of proximal tubule cells (Fig. 1A). About 70% of the mixed primary cells expressed CD13, and when cultured after sorting, it was confirmed that almost all cells expressed CD13 (Fig. 1B). We established three different cell lines using primary cells from three different donors, and these cell lines maintained epithelial cell morphology throughout the culture period (Fig. 1C). Moreover, they showed higher GGT activity than hPTECs (Fig. 1D). To generate in vitro functional proximal tubule spheroids, we used a combination of the hanging-drop seeding and rotation methods with ECM gel, as previously described [11]. iRPTC-spheroids showed a tubular structure with AQP1 (a proximal tubule marker) and LTL (an apical glycoprotein in the brush border of the proximal tubule) expression (Fig. 2A). In contrast, hPTEC-spheroids cultured under the same conditions did not show a tubular structure or LTL expression, even though the cells expressed AQP1 (Fig. 2A). The expression levels of major uptake and efflux drug transporters were increased in iRPTC-spheroids generated with the proximal tubule cell lines described in this study compared to hPTEC-spheroids (Fig. 2B). GGT activity and the magnitude of the cAMP response to PTH were significantly higher in iRPTC-spheroids than in hPTEC-spheroids, suggesting that spheroids generated with primary proximal tubule cells have normal functions related to drug and xenobiotic detoxification and a mature PTH receptor expression and cellular response (Fig. 2C and 2D). These data suggest that the iRPTC-spheroids were structurally and functionally matured compared to those formed from hPTECs, which is the most commonly used in vitro renal cell model.

2. Proximal tubule cell spheroids can be used for in vitro nephrotoxicity assessments

To examine the response to the toxic effects of nephrotoxic drugs, a cell viability assay was conducted in iRPTCs and hPTECs treated with four different compounds: cisplatin, cyclosporin A, digoxin, and doxorubicin (Fig. 3). iRPTCs were exposed to these drugs for 48 hours at different concentrations, and the IC50 values were 18.3 μM for cisplatin, 24.1 μM for cyclosporin A, and 2.8 μM for doxorubicin, respectively (Fig. 3). Cell toxicity was not detected in the spheroids treated with digoxin. However, the IC50 values of hPTECs were 58.4 μM for cisplatin, 99.81 μM for cyclosporin A, 97.3 μM for digoxin and 7.8 μM for doxorubin, respectively (Fig. 3). These data suggest that the iRPTCs were more sensitive to the nephrotoxic drugs than the hPTECs. Moreover, the absence of cytotoxic effects from digoxin indicated that the efflux transporter properly worked in the iRPTCs. Next, we investigated the apoptosis profile in spheroids generated with iRPTCs and hPTECs after treatment with 50 μM cisplatin and 5 μM doxorubicin for 48 hours using the MUSE analyzer. The apoptosis analysis showed that ~75% of cells were apoptotic (annexin V positive cells) in 50 μM cisplatin, and ~62% of cells were apoptotic cells in 5 μM doxorubicin in the spheroids generated with iRPTCs (Fig. 4A). However, in hPTEC-spheroids, the number of dead cells was dramatically decreased after the same concentration of cisplatin or doxorubicin treatment, indicating that spheroids using iRPTCs showed greater sensitivity to nephrotoxic compounds, as shown by the IC50 results. Kidney injury markers such as the expression of HAVCR1, LCN2, CLUSTERIN, and CASPASE-3 were significantly higher in iRPTC-spheroids than in hPTEC-spheroids (Fig. 4B). These results demonstrate that the proximal tubule spheroids described in this study are a more reliable and sensitive in vitro kidney model for nephrotoxicity assessments.

Discussion

In drug development, nephrotoxicity is one of the most frequent issues, along with cardiac toxicity and liver toxicity. In particular, renal toxicity events were observed in 2% of the preclinical studies, but their frequency showed a 10-fold increase to about 20% in clinical trials due to the absence of a reliable in vivo mimic kidney model [12-15]. Primary cells from the kidney are used for in vitro nephrotoxic models since they mimic the physiological state of in vivo cells; however, they have some limitations, such as a short life span, and tend to lose their specific characteristics in the epithelial mesenchymal transition [16]. To overcome these limitations of primary cells, immortalized cells could be used, but they also have changed characteristics, which could affect their functions and structure during the immortalization procedure or culture period. Indeed, immortalized proximal tubule cells did not express the key renal uptake or efflux transporter related to drug response [10,17,18].
Over the past decade, several research groups have developed differentiation protocols for pluripotent stem cells towards renal lineages by mimicking in vivo kidney development, which has provided an unlimited renal cell source to overcome the limitations of primary cells, such as poor availability and accessibility [19-23]. Although kidney organoid differentiation protocols have been developed to a remarkable extent, there are still challenges to be solved, such as maturation, off-target populations, and reproducibility [24-26]. Regarding maturity, the absence of the ureteric bud and collecting duct, the polarity of the cells in the tubular structure, and the lack of lumen formation are representative. Single-cell transcriptomics of kidney organoids identified up to 20% of the cell population to be non-renal cells such as neurons, muscle cells, and melanoma cells [22,27]. In addition, there is also the possibility of a different cell composition due to off-target populations in the organoid. So far, many efforts to solve the hurdles blocking a broader range of applications of kidney organoids have been tried, such as control of the tubular patterning and reduction of batch variations between organoids [28-30]. It is clear that matured and functional kidney organoids constitute promising tools for successful drug development. However, before the appearance of effective and reliable protocols, an improved, more stable, easy to prepare, and reproducible in vitro model for accurate prediction of nephrotoxicity could help early compound development.
In this study, we established stable proximal tubule cell lines that can generate matured functional proximal tubule cell spheroids that showed enriched transporter expression, enzyme activity, and mature cell polarity. In addition, they showed more sensitive reactivity to nephrotoxic drugs than hPTECs, which are commonly used for in vitro nephrotoxicity tests. These data indicate that these new renal proximal tubule cells might provide a useful in vitro model for the prediction of nephrotoxicity in new drug development.

Supplementary Information

Supplementary materials are presented online (available at https://doi.org/10.51335/organoid.2022.2.e30).

Table S1.

Sequences of oligonucleotide primers used for quantitative polymerase chain reaction
organoid-2022-2-e30-suppl.pdf

NOTES

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Funding

This research was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM4722122), and the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2019R1C1C1006822).

Author contributions

Conceptualization: HMK, CRJ; Data curation: HMK; Formal analysis: DHK, JHL; Funding acquisition: HMK, CRJ; Investigation: HMK, DHK, JHL; Methodology: HMK, DHK; Project administration: HMK; Resources: DHK; Writing-original draft: KHM; Writing-review & editing: KHM.

Fig. 1.
Establishment and characterization of proximal tubule cell lines from mixed primary cells. (A) Schematic diagram of the isolation of proximal tubule cells from immortalized mixed primary cells. (B) Expression of the CD13 profile in the mixed primary cells and immortalized renal proximal tubule cells (iRPTCs). (C) Morphology of three different iRPTCs during in vitro culture. (D) Gamma-glutamyl transferase (GGT) activity of iRPTCs compared to human proximal tubule epithelial cells (hPTECs). n=6 for iRPTCs and hPTECs, respectively. All data are shown as the means±standard error of the mean. cAMP, cyclic adenosine monophosphate. *p<0.05 compared with hPTECs by the unpaired Student t-test. Scale bar, 100 μM.
organoid-2022-2-e30f1.jpg
Fig. 2.
Generation of functional proximal tubule spheroids from immortalized renal proximal tubule cells. (A) Representative images of spheroids generated with immortalized renal proximal tubule cells (iRPTCs) and human proximal tubule epithelial cells (hPTECs) stained with hematoxylin and eosin (H&E) or immunostained for the proximal tubule-specific markers, aquaporin 1 (AQP1) and lotus tetragonolobus lectin (LTL). A schematic diagram of the isolation of proximal tubule cells from immortalized mixed primary cells. (B, C) Relative mRNA expression of (B) apical and (C) basolateral transporters in iRPTC-spheroids compared with those of hPTECs. n=6 for iRPTC-spheroids and n=3 for hPTEC-spheroids. (D) Gamma-glutamyl transferase (GGT) activity of iRPTC-spheroids compared to hPTEC-spheroids. n=4 for iRPTCs and hPTECs, respectively. (E) Response to parathyroid hormone (PTH) of iRPTC-spheroids compared with hPTEC-spheroids. n=4 for iRPTCs and hPTECs, respectively. All data are shown as the means±standard error of the mean. *p<0.05 compared with hPTECs by the unpaired Student t-test. Scale bar, 100 μM.
organoid-2022-2-e30f2.jpg
Fig. 3.
In vitro nephrotoxicity assessment of immortalized renal proximal tubule cells. (A-D) Half maximal inhibitory concentration (IC50) of immortalized renal proximal tubule cells (iRPTCs) and human proximal tubule epithelial cells (hPTECs) after 48 hours of treatment with the indicated concentrations of (A) cisplatin, (B) cyclosporin A, (C) digoxin, and (D) doxorubicin. n=3 for iRPTCs and hPTECs, respectively. All data are shown as the means±standard error of the mean. *p<0.05 compared with hPTECs by the unpaired Student t-test.
organoid-2022-2-e30f3.jpg
Fig. 4.
In vitro nephrotoxicity assessment of proximal tubule spheroids. (A) Apoptosis profile (performed using a MUSE analyzer) in immortalized renal proximal tubule cell (iRPTC) spheroids (upper) and human proximal tubule epithelial cells (hPTEC) spheroids (bottom) incubated with 50 μM cisplatin (CPT) or 5 μM doxorubicin (DOX) for 24 hours. The results were obtained with the MUSE Count & Viability software module and statistics were shown for the percentage of viable cells and annexin V positive apoptotic cells. (B) Relative mRNA expression of kidney injury markers such as HAVCR1, LCN2, clusterin (CLU), and caspase-3 (CASP3) in the iRPTC-spheroids after nephrotoxicant treatment. n=4 for iRPTC-spheroids and hPTEC-spheroids, respectively. All data are shown as the mean±standard error of the mean. *p<0.05 compared with hPTECs by the unpaired Student t-test.
organoid-2022-2-e30f4.jpg

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