Genetically Modified Human Renal Proximal Tubule Epithelial Cells (RPTEC/TERT1) A New Model for Drug Toxicity Studies

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1 Genetically Modified Human Renal Proximal Tubule Epithelial Cells (RPTEC/TERT1) A New Model for Drug Toxicity Studies Chaozhong Zou, Ph.D. Senior Scientist, ATCC

About ATCC Founded in 1925, ATCC is a non-profit

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researchers and scientists ATCC collaborates with and

industry-standard biological products and innovative

2 About ATCC Founded in 1925, ATCC is a non-profit organization with headquarters in Manassas, VA World s premiere biological materials resource and standards development organization Established partner to global researchers and scientists ATCC collaborates with and supports the scientific community with industry-standard biological products and innovative solutions Strong team of 400+ employees; over one third with advanced degrees 2

3 Agenda Renal transport Current renal models Generation of RPTEC renal uptake models Application data Summary 3

bidirectional Expression and activities at the basolateral and apical side of transporting epithelia are significant determinants for: Drug

4 Renal transport proteins Play important roles for drug: Absorption Distribution Elimination Can be divided into 2 classes: The ATP-binding cassette (ABC) family, most are efflux transporters The solute carrier (SLC) family, most are influx transporters, some are efflux and bidirectional Expression and activities at the basolateral and apical side of transporting epithelia are significant determinants for: Drug disposition Drug-drug interactions Variability in drug response and toxicity Kidney epithelial cells recapitulate in vivo tubule formation, image courtesy of Moe Mahjoub 4

Toxicologically important transport proteins Transporter/alias OATP1B1/OATP-C, OATP2, LST-1 OATP1B3/OATP-8 (SLCO1B3) OAT1 (SLC22A6) OAT3 (SLC22A8) OCT2 (SLC22A2) Organs/cells Hepatocytes

5 Toxicologically important transport proteins Transporter/alias OATP1B1/OATP-C, OATP2, LST-1 OATP1B3/OATP-8 (SLCO1B3) OAT1 (SLC22A6) OAT3 (SLC22A8) OCT2 (SLC22A2) Organs/cells Hepatocytes (sinusoidal) Hepatocytes (sinusoidal) Kidney proximal tubule, placenta Kidney proximal tubule, choroid plexus, blood brain barrier Kidney proximal tubule, neurons RPTEC/TERT1-OCT2 (ATCC CRL-4031-OCT2 ) 5

Enzyme- and Transporter-Based Drug Drug Interactions. DOI 10.

6 Renal transport protein substrates and inhibitors OAT1 Substrates Cipro Methotrexate Acyclovir Tenofovir OAT3 NSAIDS Cefaclor, Ceftizoxime Furosemide Bumetanide OCT2 Pindolol Amiloride Oxalliplatin Varenicline Pang Ks, et al. Enzyme- and Transporter-Based Drug Drug Interactions. DOI / _2,C Am Assoc Pharmaceut Sci 2010 Inhibitors OAT1 Probenecid Novobiocin OAT3 Probenecid Novobiocin OCT2 Cimetidine Pilsialnide Etrizine Testosterone Quinidine 6

7 Focus on transporters in new regulatory documents FDA guidance 2006 ITC Recommendations 2010 FDA Guidance 2012 EMA Guidelines 2010 Tissue P-gp/MDR Multi BCRP + + Multi BSEP + Liver OATP1B1 + + Liver OATP1B3 + + Liver OCT1 + Liver OAT1 + + Kidney OAT3 + + Kidney OCT2 + + Kidney New (draft) regulatory documents published by FDA, EMA, and ITC recommended evaluate NME as substrate and drug interaction on the most important membrane transporters expressed in liver, intestine, and kidney 7

8 Agenda Renal transport Current renal models Generation of RPTEC renal uptake models Application data Summary 8

CCL-34 ), image couresty of Christopher Chin Do not have the human kidney tissue origination

9 Cell line-based models Current cell line-based models are available: MDCK (ATCC CCL-34 ) CHO-K1 (ATCC CCL-61 ) U-2 OS (ATCC HTB-96 ) Others Problems with these lines: MDCK cells (ATCC CCL-34 ), image couresty of Christopher Chin Do not have the human kidney tissue origination The cell line itself is a cancer line Therefore, the clinical predictability is greatly compromised 9

Primary cell-derived models Problems with primary kidney cell models: Primary Renal

kidney comprises 15 cell types The nephron comprises 20 cell types Homogeneous

OAT1, OCT2, and OAT3 expression in culture Transiently expressing transporters in

10 Primary cell-derived models Problems with primary kidney cell models: Primary Renal Cortical Cells (ATCC PCS ) Obtaining primary cultures is difficult The kidney comprises 15 cell types The nephron comprises 20 cell types Homogeneous cultures retaining physiological functions are hard to obtain Primary RPTEC lose OAT1, OCT2, and OAT3 expression in culture Transiently expressing transporters in primary RPTEC show large variations between production lots Makes the data hard to interpret 10

11 Renal cell lines Cell Line ATCC No. Species of origin Nephron segment of origin LLC-PK1 CL-101 Yorkshire Pig Proximal nephron OK CRL-1840 North American Opossum Proximal nephron JTC-12 N/A Monkey Proximal nephron MDCK CCL-34 Dog Collecting duct A6 CCL-102 Xenopus laevis Distal tubule HK-2 CRL-2190 Human HPV16-transformed, Proximal/Distal? Caki-1 HTB-46 Human Kidney carcinoma HEK293/OAT1 CRL-11268G-1 Human Embryonic None of the continuous renal epithelial cell lines fully recapitulate the functions of the parental cells in vivo 11

12 Pros and cons of different cell models for tissuerelevant functional studies Mimic in vivo tissue phenotype Genotypic stability Proliferative capacity Primary cells htert immortalized Oncogene, virally immortalized Cancer cell lines Diploid Diploid / Near diploid Near diploid / Aneuploid Aneuploid Supply Inter-experimental reproducibility Low Good Good Good Cost High Medium Low Low Ease-of-use

htert-immortalized cells provide unique tools htert-immortalized cells combine: The in vivo nature of primary cells The ability to be cultured continuously

13 htert-immortalized cells provide unique tools htert-immortalized cells combine: The in vivo nature of primary cells The ability to be cultured continuously htert-immortalized cells avoid the limitations of primary cells and continuous cell lines while still reaping their benefits RPTEC/TERT1-OCT2 (ATCC CRL-4031-OCT2 ) 13

RPTEC/TERT1 exhibit: Uniform expression of E-cadherin and CD13

14 The parental cell RPTEC/TERT1 (ATCC CRL-4031 ) An epithelial cell line Isolated from human renal proximal tubes Immortalized by htert only RPTEC/TERT1 exhibit: Uniform expression of E-cadherin and CD13 (aminopeptidase N) Dome-like structures Stabilized TEER RPTEC/TERT1 (ATCC CRL-4031 ) 14

15 Agenda Renal transport Current renal models Generation of RPTEC renal uptake models Application data Summary 15

16 Stable cell line generation OAT1, OCT2, and OAT3 delivery RPTEC/ TERT1 cells Surviving RPTEC/ TERT1 cells Clonal selection, Validation, and expansion RPTEC/TERT1- OAT1, OCT2, and OAT3 clonal cells Antibiotic selection 16

17 RPTEC/TERT1-OAT1 75 kda anti-oat1 50 kda 50 kda anti-tubulin RT-PCR Western Blot Sequencing: no mutation 17

18 OAT1 correctly localizes to the cell membrane in RPTEC/TERT1 OAT1 Merged with DAPI RPTEC/TERT1-OAT1 RPTEC/TERT1 Scale bar: 100 µm 18

19 RPTEC/TERT1-OCT2 A. B. 100 kda 75 kda anti-oct2 50 kda 50 kda anti-tubulin RT-PCR Western Blot C. Sequencing: No mutation 19

20 OCT2 correctly localizes to cell membrane in RPTEC/TERT1 OCT2 Merged with DAPI Scale bar: 100µm RPTEC/TERT1-OCT2 RPTEC/TERT1 Scale bar: 100 µm 20

21 Growth characteristics of stably transfected RPTEC/TERT1 RPTEC/TERT1 RPTEC/TERT1-OAT1 RPTEC/TERT1-OCT2 High density Low density Scale bar: 400 µm 21

22 RPTEC/TERT1 renal uptake key marker staining CD13 Merged with DAPI E-cadherin Merged with DAPI RPTEC/TERT1 OAT1 RPTEC/TERT1 OCT2 Scale bar: 100 µm RPTEC/TERT1 22

23 Dome formation RPTEC/TERT1 RPTEC/TERT1-OAT1 RPTEC/TERT1-OCT2 Scale bar: 100 µm 23

24 Agenda Renal transport Current renal models Generation of RPTEC renal uptake models Application data Summary 24

25 RPTEC/TERT1-OAT1 drug kinetic profile RPTEC/TERT1-OAT1 5-CF uptake uptake(rfu) RPTEC/TERT1 RPTEC/TERT1-OAT1 EC 50 =89.21µ M CF concentration, µ M 25

26 Known OAT1 inhibitors block the RPTEC/TERT1- OAT1 5-CF uptake Probenecid Inhibits RPTEC/TERT1 OAT1 5-CF uptake Percentage of rem aining activities 120 IC 50 =4.48µ M Probenecid Concentration, Log(M) Novobiocin Inhibits RPTEC/TERT1 OAT1 5-CF uptake Percentage of rem aining activities 120 IC50=77.63µ M Novobiocin Concentration, Log(M) 26

27 RPTEC/TERT1-OCT2 drug kinetic profile RPTEC/TERT1-OCT2 uptake assay RPTEC/TERT RPTEC/TERT1-OCT2 OCT2 EC 50 =15.43µ M 1200 uptake(rfu) ASP + concentration, µ M 27

28 Known OCT2 inhibitors block the RPTEC/TERT1- OCT2 Asp+ uptake Percentage of rem aining activities Cimetidine inhibits OCT2 Asp+ uptake 120 IC50=138.9µ M Cim etidine concentration, Log(M) Percentage of rem aining activities Quinitin inhibits OCT2 ASP + uptake IC50=55.14µ M Quinitin concentration, log(m) 28

29 Agenda Renal transport Current renal models Generation of RPTEC renal uptake models Application data Summary 29

keep the original characteristics of the RPTEC/TERT1 cells The performance of these stable cells are well

30 Summary We generated clonal RPTEC/TERT1 renal uptake cell models by stably expressing OAT1 and OCT2 proteins Expression has been confirmed by: PCR Western blot Immunocytochemistry The clonal stable cells keep the original characteristics of the RPTEC/TERT1 cells The performance of these stable cells are well characterized by: 5-CF uptake assays ASP uptake assays Inhibitor assays RPTEC/TERT1-OCT2 (ATCC CRL-4031-OCT2 ) 30

31 More toxicology resources Are you going to the SOT 56 th Annual Meeting and ToxExpo in Baltimore, MD? Stop by booth #2246 to learn about ATCC's new toxicology tools Visit our poster session: Development of Solute Carrier Transporter Kidney Cell Models Using htert-immortalized Renal Proximal Tubule Epithelial Cells Tuesday, March 14, 9:30 AM 12:45 PM Abstract #1667, Board #P129 We will release the new htert/rptecs in April We can notify you by once the products are available RPTEC/TERT1-OCT2 (ATCC CRL-4031-OCT2 ) 31

the American Type Culture Collection unless indicated otherwise.

32 Disclaimers American Type Culture Collection. The ATCC trademark and trade name, and any other trademarks listed in this publication are trademarks owned by the American Type Culture Collection unless indicated otherwise. ToxExpo is a trademark of the Society of Toxicology (SOT). The htertimmortalized cells are distributed under the terms of the ATCC Material Transfer Agreement and Addendum for TERT products. RPTEC/TERT1-OAT1 (ATCC CRL-4031-OAT1 ) 32

org/webinars Learn more about transfection at www.atcc.

33 Thank you for joining today! Register for more ATCC webinars at Learn more about transfection at For more information about cell health visit Please additional questions to: 33

In Vitro Transporter Testing Alignment with Regulatory Agency Guidelines and ITC Recommendations. Elke S. Perloff, Ph.D.

In Vitro Transporter Testing Alignment with Regulatory Agency Guidelines and ITC Recommendations Elke S. Perloff, Ph.D. BD Biosciences February 23, 2011 Presentation Overview Guidance documents and position