Effect of Replicative Senescence on the Expression and Function of Transporters in Human Proximal Renal Tubular Epithelial Cells

Abstract

In the field of cosmetic research, there is a growing interest in alternatives to animal experiments, such as in vitro models using cultured cells. The trend is spreading to the field of food and drugs. Although various types of cells are used as in vitro models, the effect of cellular senescence on the expression and function of transporters in these models is unclear. In the present study, we examined the effect of replicative senescence (by passage culture) on the expression and function of transporters in renal proximal tubular epithelial cells (RPTECs). The increase in senescence-associated-β-galactosidase (SA-β-gal)-positive cells, cell cycle arrest markers, and senescence-associated secretory phenotype (SASP) markers was associated with an increase in passage numbers of RPTECs. Gene expression of various transporters in RPTEC was also altered. The mRNA level of organic cation transporter 2 decreased most rapidly with passage numbers among the transporters. The uptake of fluorescent cationic substrates in SA-β-gal-positive RPTECs was less than that in SA-β-gal-negative RPTECs. However, these changes in the expression of transporters seem to be significantly different from those observed in rodents and human kidneys in many aspects. As cellular senescence is observed in various situations, especially in RPTECs, it may be necessary to exclude it from toxicological and pharmacokinetic evaluations using in vitro models as much as possible. Additionally, when discussing cellular senescence, it is important to note the differences between aging in cells and aging and senescence in individuals.

INTRODUCTION

Cultured cells and animal models have been used for obtaining data on drug safety and efficacy. The concept of 3Rs (replace/reduce/refine) is a long-standing requirement in animal testing.¹⁾ In the European Union, the marketing of cosmetics that use animal testing has been banned since 2009, and testing using animals has been banned since 2013.²⁾ This movement toward utilizing alternatives to animal testing is about to spread not only to the cosmetics industry but also to other fields such as food and drugs.²⁾ Therefore, research and development of in vitro models as an alternative to using animals are currently in progress. For example, research on three dimensional (3D)-culture methods, such as organoids, using cultured cells derived from human tissues or induced pluripotent stem (iPS) cells, and new devices such as organ-on-a-chip and microphysiological systems (MPS) are rapidly advancing.^3,4) However, these in vitro models also have limited applicability in toxicity studies.⁵⁾

Focusing on in vitro models of the renal tubule, various immortalized cells derived from the human kidney (e.g., renal proximal tubular epithelial cell (RPTEC)/telomerase reverse transcriptase immortalized 1, ciPTEC, HK-2, and Nki-2 cells) and animal kidney (e.g., Madin–Darby canine and LLC-PK cells) have been used for screening nephrotoxic compounds or evaluating pharmacokinetics.^6,7) Secretion and reabsorption are important functions of renal tubular cells, mediated by unique transporters, and these functions are closely associated with pharmacokinetic evaluation and nephrotoxicity screening.^3,6–8) Therefore, when immortalized cells are replaced by normal cells or organoids, it remains unclear whether reproducible and stable data, similar to that obtained from the human renal tubule, can be obtained. Further studies are required in this direction to determine the standard of in vitro models as viable animal experiment alternatives, and various matters of concern remain.

It is known that the continuous subculture of primary cells leads to a replicative arrest, which is an effect of cellular senescence.⁹⁾ Therefore, cell division in primary cultured cells is limited, which is considered the cellular lifespan. This limitation is called Hayflick’s limit.⁹⁾ Recent reports suggest that cellular senescence is induced not only by continuous subculture but also by cytotoxic agents, such as cisplatin.^10–12) Senescent cells are characterized by senescence-associated-β-galactosidase (SA-β-gal) staining and an increased expression of p16^INK4A and p21^WAF1/Cip.^13,14) In addition, cytokines, known as senescence-associated secretory phenotype (SASP) factors, are released into the surrounding tissues and cells.^13,15) Research has revealed a strong link between cellular senescence and renal aging.^13,16) However, little is known about the effect of cellular senescence on the function of cultured RPTECs.

The present study shows that long-term passage culture induces cellular senescence and changes the expression and function of transporters in human primary RPTECs.

MATERIALS AND METHODS

Cell Culture and Reagents

Human RPTECs (Lonza, Basel, Switzerland) were grown using the REGM Renal Epithelial Cell Growth Medium BulletKit (Lonza) as previously reported.¹⁷⁾ Cell cultures were maintained in a humidified incubator under 5% CO₂ at 37 °C. Cells were sub-cultured every 4 d, and the medium was changed every other day.

SA-β-Gal Staining

RPTECs were plated in Nunc™ Lab-Tek™ chamber slides (Thermo Fisher Scientific, Waltham, MA, U.S.A.) at a density of 1.5 × 10⁴ cells/well and cultured for 24 h. To detect senescent RPTECs, SA-β-gal staining was conducted using the Cellular Senescence Detection Kit (Cell Biolabs, San Diego, CA, U.S.A.) according to the manufacturer’s instructions.

Co-culture of Senescent RPTECs and Non-senescent RPTECs

RPTECs (passage (P) 4) were plated in 24-well plates (Costar, Corning, NY, U.S.A.) at a density of 2 × 10⁴ cells/well. RPTECs (P4 or P15) were also plated in transwell inserts (pore size, 0.4 µm; Costar) at 4 × 10⁴ cells/well. After incubation for 16 h, the medium was changed. Transwell inserts were then placed in each well of the 24-well plate. RPTECs were co-cultured for 72 h.

Quantitative Real-Time (qRT)-PCR

qRT-PCR analysis was performed as previously reported.¹⁸⁾ Total RNA was extracted from RPTECs of each passage using a Pure link RNA mini kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Using a ReverTra qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., Osaka, Japan), total RNA was reverse-transcribed into cDNA, which served as the template for PCR performed using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). The primer pair sequences are summarized in Supplementary materials (Supplementary Table 1). For detecting cellular senescence, primer pairs for p21^WAF1/Cip, p16^INK4A, and representative SASP factors (interleukin (IL)-6, IL-8, plasminogen activator inhibitor (PAI)-1) were designed.¹³⁾ For measuring the mRNA expression of renal tubular transporters, we selected 16 representative genes.^3,6,8,19)RPL13A, HPRT1, ACTB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured as housekeeping genes. Among these housekeeping genes, HPRT1 was selected as an endogenous control. Amplification and detection were performed using the CFX Connect Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). We used HPRT1 as the endogenous control and quantified mRNA levels using the ^ΔΔCt method (Supplementary Fig. 1). All measurements were performed in triplicates.

Semi-quantitative Uptake Assay

The condition of the uptake assay was determined according to published reports.^19–21) RPTECs (P12) were cultured in Nunc™ Lab-Tek™ chamber slides (Fisher Scientific) for 24 h. After pre-incubation with Hank’s balanced salt solution (HBSS) with N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (Bio Medical Science, Tokyo, Japan) for 15 min, the cells were incubated with fluorescent cationic dye, 100 µM 4-(4-(dimethlamino)styryl)-N-methylpyridinium (ASP⁺) and 200 µM rhodamine 123 (Rh123) for 30 min. These fluorescent cationic dyes are taken up by organic cation transporters (OCTs).^22,23) After incubation with cold HBSS (4 °C), the cells were washed two times with HBSS at 37 °C. SA-β-gal staining was conducted using Cellular Senescence Detection Kit (Cell Biolabs) as per the manufacturer’s protocol, followed by the incubation of cells with 1 µL of 1 mg/mL Hoechst33342 for 30 min. Finally, the cells were observed under an ECLIPSE Ti-S microscope (Nikon Corporation, Tokyo, Japan). Image data were analyzed by ImageJ software.

Statistical Analysis

Data are expressed as the mean ± standard deviation. The heatmap was plotted using the “heatplus” function in R version 4.0.3. Heatmap data are expressed as the fold changes in the mRNA levels of detectable transporters at P9 and P15 (Supplementary Fig. 1) as log₂ values relative to the mRNA levels of transporters at P6, set as the baseline. Each experiment was conducted thrice. After verification that the data were normally distributed, differences were assessed with Welch’s t-test. Multiple comparisons between the control group and the other groups were made by one-way ANOVA with Dunnett’s multiple comparison test. p < 0.05 was considered to indicate a statistically significant difference. These statistical analyses were conducted using SPSS (version 23.0, SPSS Inc., Tokyo, Japan).

RESULTS

Gradual Induction of SA-β-Gal Staining by Long-Term Culture in RPTECs

Figure 1 shows that 14.4 ± 5.0% of SA-β-gal-positive cells at P6. A subsequent increase in SA-β-gal-positive cells at greater passage numbers was observed. In this study, P6 was set as a baseline. Cells displaying the senescent phenotype, like cell-size expansion, were often observed at P15. Nearly all cells were SA-β-gal-positive cells at P18 (96.0 ± 0.7%).

Fig. 1. Gradual Induction of SA-β-Gal-Positive RPTECs by Long-Term Culture

SA-β-gal staining of (A) passage 6 RPTECs and (B) passage 15 RPTECs (scale bar = 200 µm). (C) Quantitative analysis of SA-β-gal-positive cells. Each of the SA-β-gal-positive cells and total cells was counted from three different images. These images were captured using a 10X objective lens. The percentage of SA-β-gal-positive cells is shown. ** p < 0.01 vs. the control group (P6); one-way ANOVA with Dunnett’s multiple comparison test.

Enhancement of Cell Cycle Arrest Markers and SASP Markers

Cellular senescent markers such as cell cycle arrest markers (p21^WAF1/Cip and p16^INK4A) and SASP markers (IL-6, IL-8, and PAI-1) were measured at P6, P9, and P15. Figure 2 shows that the mRNA levels of p21^WAF1/Cip increased significantly by 1.44 ± 0.06 times (p < 0.01). However, no statistically significant differences in the changes in the mRNA levels of p16^INK4A were observed. The mRNA level of IL-6, IL-8, and PAI-1 showed a concomitant increase (Fig. 3). The mRNA levels of IL-6, IL-8, and PAI-1 increased significantly, by 1.26 ± 0.08 times (p < 0.05), by 18.18 ± 0.76 times (p < 0.01), and 2.11 ± 0.10 times (p < 0.01) at P15, respectively.

Fig. 2. Quantitative Analysis of Cell Cycle Arrest Marker Expression in RPTECs under Different Culture Passages

mRNA expression levels of (A) p21^WAF1/Cip and (B) p16^INK4A in RPTECs under different culture passages were analyzed by real-time PCR. * p < 0.05, ** p < 0.01 vs. the control group (P6); one-way ANOVA with Dunnett’s multiple comparison test.

Fig. 3. Quantitative Analysis of SASP Marker Expression in RPTECs under Different Culture Passages

mRNA expression levels of (A) IL-6, (B) IL-8 and (C) PAI-1 in RPTECs under different culture passages were analyzed by real-time PCR. * p < 0.05, ** p < 0.01 vs. the control group (P6); one-way ANOVA with Dunnett’s multiple comparison test.

Late-Passage RPTEC-Derived Secretome Induces Cellular Senescence of RPTECs

Figure 4 shows the number of SA β-gal-positive RPTECs (P4) co-cultured with RPTEC (P4 or P15) using transwell culture conditions. SA-β-gal-positive RPTECs were hardly observed at P4 (approximately 1%). The number of SA-β-gal-positive RPTECs (P4) co-cultured with late-passage RPTECs (P15) increased significantly from 0.52 ± 0.22% to 3.41 ± 0.78% as compared to that of SA-β-gal-positive RPTECs (P4) co-cultured with early-passage RPTECs (P4). This phenomenon is consistent with the characteristics of cellular senescence.

Fig. 4. Late-Passage RPTEC-Derived Secretome Promotes the Cellular Senescence of RPTECs

(A) Illustration of co-culture using transwell and SA-β-gal staining. SA-β-gal staining of (B) passage 4 RPTECs that were co-cultured with passage 4 RPTECs or (C) that were co-cultured with passage 15 RPTECs for 72 h (scale bar = 200 µm). (D) Quantitative analysis of SA-β-gal-positive cells. SA-β-gal-positive cells and total cells were counted from three different images. These images were captured using a 10× objective lens. The percentage of SA-β-gal-positive cells is shown. * p < 0.05; Welch’s t-test.

Long-Term Culture Changes the mRNA Expression of Transporters in RPTECs

The relative mRNA expression of 16 representative transporters present on renal tubular cells was measured (Supplementary Fig. 1). Among these transporters, the mRNA levels of organic anion transporter (OAT)1 and OAT2 were undetectable (Supplementary Fig. 1). In Fig. 5, the heatmap shows the fold changes observed in the mRNA levels of detectable transporters at P9 and P15 expressed as log₂ when the mRNA level of transporters at P6 was set as 0. The most significant change was observed in the mRNA level of OCT2 (p < 0.01). Figure 6 shows the mRNA level of transporters that showed significant alteration with the passage in Fig. 5. The mRNA level of organic anion transporter polypeptide (OATP) 4C1, OCT2, OCT3, multidrug and toxin extrusion (MATE)1, and MATE2K decreased with an increase in passage number. The mRNA levels of OATP4C1, OCT2, OCT3, MATE1, and MATE2K decreased significantly, by 0.69 ± 0.05 times at P9 (p < 0.01) and 0.69 ± 0.06 times at P15 (p < 0.01), 0.38 ± 0.02 times at P9 (p < 0.01) and 0.05 ± 0.00 times at P15 (p < 0.01), 0.36 ± 0.23 times at P15 (p < 0.05), 0.31 ± 0.10 times at P9 (p < 0.01) and 0.27 ± 0.10 times at P15 (p < 0.01), 0.48 ± 0.11 times at P9 (p < 0.05) and 0.43 ± 0.21 times at P15 (p < 0.05), respectively. The levels of multidrug resistance protein (MRP)4, multiple drug resistance (MDR)1, and breast cancer resistance protein (BCRP)1 increased significantly at P15. The mRNA levels of MRP4, MDR1, and BCRP1 increased by 1.20 ± 0.05 times (p < 0.05), 1.77 ± 0.11 times (p < 0.01), and 1.79 ± 0.41 times (p < 0.05) at P15, respectively. No statistically significant changes in the mRNA levels of OAT3, OAT4, MRP2, OCT1, OCTN1, or OCTN2 were observed.

Fig. 5. Replicative Senescence Verifies mRNA Expression of Transporters in RPTECs

Heat map of average mRNA expression levels of detectable transporters under different culture passages.

Fig. 6. Significantly Changed mRNA Expression among Detectable Transporters in RPTECs under Different Culture Passages

The mRNA expression levels of (A) OATP4C1, (B) MRP4, (C) OCT2, (D) OCT3, (E) MATE1, (F) MATE2K, (G) MDR1, and (H) BCRP1 in RPTECs under different culture passages were analyzed by real-time PCR. * p < 0.05, ** p < 0.01 vs. the control group (P6); one-way ANOVA with Dunnett’s multiple comparison test.

The Uptake of Fluorescent Cationic Substrates Decreased with Cellular Senescence in RPTECs

Since cellular senescence appears to be associated with changes in organic cation transporters, the uptake assay using representative fluorescent cationic substrates was conducted. Figure 7 shows the difference in the uptake of fluorescent cationic substrates between SA-β-gal-positive and SA-β-gal-negative cells. ASP⁺ and Rh123 were representative fluorescent cationic substrates in these experiments. The uptake assay using them and SA-β-gal staining were conducted in RPTECs (passages 10–12). The uptake of both ASP⁺ and Rh123 appeared to decrease in SA-β-gal-positive cells. In the ASP⁺-uptake assay, the number of ASP⁺-positive cells in SA-β-gal-positive RPTECs was 22.6 ± 10.1%, but that in SA-β-gal-negative RPTECs was 95.7 ± 0.2%. In the Rh123-uptake assay, the number of Rh123-positive cells in SA-β-gal-positive RPTECs was 17.8 ± 9.0%, but that in SA-β-gal-negative RPTECs was 93.9 ± 2.9%. Additionally, in the uptake experiment using anionic substrate 6-carboxyfluorescein, green fluorescence was hardly observed (data not shown). This may be caused by the low mRNA levels of the organic anion transporters (OAT1, OAT2, and OAT3) in these RPTECs (Supplementary Fig. 1).

Fig. 7. Semi-quantitative Uptake of Fluorescent Cationic Substrates in Senescent or Non-senescent RPTECs

Representative images of SA-β-gal staining after uptake assay of (A) ASP⁺ and (D) Rh123 in passages 10–12 RPTECs (scale bar = 200 µm). (B and E) The images focus on SA-β-gal-positive cells or negative cells, respectively (scale bar = 100 µm). Bright-field microscopic images were turned into gray color and merged with images depicting fluorescent substrates and Hoechst33342. The yellow arrow indicates SA-β-gal-positive cells. These images were captured using a 10X objective lens. The percentage of (C) ASP⁺-positive cells and (F) Rh123-positive cells in SA-β-gal-positive and negative cells. ** p < 0.01; Welch’s t-test.

The differences in the uptake between SA-β-gal-positive and SA-β-gal-negative cells may be the cause of the transcriptional changes in the gene expression of transporters because the uptake of these fluorescent cationic substrates is strongly affected by organic cation transporters.^19–23) However, cationic substrates, such as ASP+ and Rh123, are localized to the mitochondria or DNA of the nucleus, which have a negative electric charge after uptake inside the cells. Therefore, the accumulation of ASP+ and Rh123 in the mitochondria may reduce by a senescence-induced decrease in the mitochondrial membrane potential.²⁴⁾ Thus, a decrease in the mitochondrial membrane potential may partially affect the results of the uptake assay.

DISCUSSION

Long-term culture induced replicative senescence in human primary RPTECs. The transcriptional changes in transporters were largely altered by replicative senescence. The change of uptake of fluorescent substrates was observed in SA-β-gal-positive senescent RPTECs. However, these changes in transporter function seem to be significantly different from that observed in aged rodents and human kidneys. The present study suggests that we must exclude the effect of cellular senescence from the in vitro evaluation as much as possible when primary RPTECs are used for in vitro evaluation of pharmacokinetics and toxicological tests.

First, in human primary RPTECs, long-term passage culture induced cellular senescence as more SA-β-gal-positive RPTECs emerge from long-term passage cultures.¹⁴⁾ The expression of most of the cellular senescent markers also increased. p21^WAF1/Cip and/or p16^INK4A and SASP are often observed in cells undergoing cellular senescence.^15,25,26) p21^WAF1/Cip is reported as a key factor for initiating senescence.²⁵⁾ p16^INK4A belongs to a senescent pathway different from that of p21^WAF1/Cip and is reported as an irreversible cell cycle arrest-induced factor.²⁵⁾ There are multiple phases in senescence. Senescence associated with the p21^WAF1/Cip pathway seems to be an early, reversible phase.²⁵⁾ On the other hand, senescence associated with the p16^INK4A pathway is expressed as late (noon-reversible) senescence.²⁵⁾ SASP is the general term for a variety of pro-inflammatory cytokines secreted by senescent cells.¹⁵⁾ IL-6, IL-8, and PAI-1 are representative SASP factors that contribute to renal senescence and disease.^13,16) In fact, senescent cells can affect surrounding (non-senescent) cells through secretory factors such as SASP.^13,15,25) Meanwhile, the conditional medium of oncogene-induced senescent fibroblasts includes extracellular vesicles (EVs) promoting cellular senescence.²⁶⁾ EVs or SASP factors released from senescent RPTECs might have promoted cellular senescence in this study.

Second, the transcriptional level of each transporter in senescent RPTECs altered with the passage number. However, this change in transporters seems to be bibliographically different from that observed in rodents and human kidney. For example, a study shows age-associated variations of 12 kidney transporters such as OAT1 and 3; OCT1, 2, and 3; OATP4C1; MDR1; MRP2; MRP4; BCRP; MATE1; and MATE2K in male rats.²⁷⁾ The transcriptional level of these transporters was low in fetal kidneys and increased following birth, maturation, and adulthood. After that, the mRNA expression of OCT2, OATP4C1, MDR1, and MATE1 decreased with aging. The transcriptional levels of other transporters remained high with aging. In the present study, the results of OCT2, OATP4C1, and MATE1 appear to be consistent with those of the others, while those of OCT3, MDR1, and MATE2K are inconsistent. Another study shows the protein level of MATE2 in the kidneys of male and female C57BL/6J mice.²⁸⁾ The expression level of MATE2 in 16–17-week-old (young) male mice appear to be slightly high compared to that of MATE2 in 16–17-month-old (aged) male mice. Conversely, the expression level of MATE2 in young female mice was marginally low. The expression level of MATE2 in mice appears to be affected by sex. In the present study, the transcriptional level of MATE2K decreased by long-term passage culturing. In a human kidney cortex sample obtained from 36 Caucasian patients undergoing tumor nephrectomy, the protein level of 12 transporters (Na⁺/K⁺ ATPase, MDR1, MRP1, MRP2, MRP4, OAT1, OAT2, OAT3, OCT2, OCT3, MATE1, and organic cation transporter-like protein (ORCTL)2) was measured by using LC/MS.²⁹⁾ Among these transporters, significant positive correlation was observed between the protein level of MDR1 and ORCTL2 and age. No significant correlation was observed between the protein levels of 12 transporters and sex.²⁹⁾ The mRNA levels of OAT1, OAT2, OCT2, and MATE1 were measured by real-time PCR, but the correlations between the mRNA level of these transporters and age were not significant.²⁹⁾ It should be noted that although these results show the effect of cancer and individual differences, the results hardly match those of our study. To our knowledge, there is no report associated with age-related changes of kidney transporters in healthy people. Additionally, no report associated with age-related changes in MRP4 and BCRP1 levels in the kidney has been published. An investigation of age-related changes of these transporters in the kidney is needed. As mentioned above, the correlation between variations of transporters and age or sex in human kidneys appears to be weaker than that in rodent kidneys. The change in the uptake of fluorescent cationic substrates was observed in SA-β-gal-positive senescent RPTECs. SA-β-gal could be used for quality check of primary culture cells.

The present study reveals the transcriptional and functional changes of transporters associated with replicative senescence in a 2D-cultured RPTEC model. However, as above, the changes in transporters in 2D-cultured human RPTECs are different from that in the kidneys of rodents or humans in many aspects. Thus, at this time, senescent RPTECs would make it difficult to accurately interpret pharmacokinetic and toxicological data. Cellular senescence has been observed not only in RPTECs but also in various cells and tissues such as skin, liver, lung, adipose tissue, and vascular tissue.^9,30–33) Therefore, the maximum limit of the number of passages should be defined for each cell type when primary cultured cells are used to evaluate the pharmacokinetics and toxicity of chemical compounds. In addition, we must also pay attention to other factors promoting cellular senescence. Cellular senescence is also observed under various conditions, apart from primary cultured cells. For example, cisplatin-induced senescence was observed in not only human primary RPTECs (Supplementary Fig. 2) but also in murine-derived RPTECs or immortalized RPTECs.^10–12) With regard to MPS, the interactions via senescence-promoting factors, such as EVs and/or SASP factors, may also affect measurements.²⁶⁾

Senescence and aging are important factors and cannot be excluded from pharmacokinetic and toxicological evaluation, especially cosmetic testing. Lately, complex systems such as co-culture models, 3D models, or microfluidic organ-on-a-chip using cultured cells derived from normal tissues or embryonic stem (ES)/iPS cells are generating valuable data on the cutting edge of animal alternative technology.^4,19,34) Using normal kidney tissues or kidney organoids derived from ES/iPS cells may bring in vitro models closer to ideal models that accurately reflect the biological system, but the effect of cellular senescence on outputs should be closely monitored. The effects of cellular senescence on cellular function and morphogenesis in RPTECs are still not completely understood. The differences in the expression and function of renal transporters between senescence in cultured cells and aging and senescence in individuals were observed in this study. These differences may be observed in other situations. Therefore, focusing on senescence and aging, further studies are required for the development of animal alternative technologies and updating the standard of cell culture methods and cellular conditions during compound measurements. Additionally, results from animal alternative technologies considering differences between senescence in cultured cells and aging and senescence in individuals are warranted.

Acknowledgments

This work was supported in part by the Foundation for Promotion of Cancer Research.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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