Validating an Ex Vivo 3D Kidney Proximal Tubule

VALIDATING AN EX VIVO 3D KIDNEY PROXIMAL TUBULE MODEL FOR DRUG-INDUCED NEPHROTOXICITY SCREENING by Dorina Eva Lisa Diekjürgen A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pharmaceutics and Pharmaceutical Chemistry The University of Utah August 2017 Copyright © Dorina Eva Lisa Diekjürgen 2017 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Dorina Eva Lisa Diekjürgen has been approved by the following supervisory committee members: David W. Grainger , Chair 5/26/2017 Date Approved James N. Herron , Member 5/26/2017 Date Approved Shawn C. Owen , Member 5/22/2017 Date Approved Tara Lynn Deans , Member 5/25/2017 Date Approved Brenda K. Mann , Member 5/26/2017 Date Approved and by Carol Lim , Chair/Dean of the Department/College/School of Pharmaceutics and Pharmaceutical Chemistry and by David B. Kieda, Dean of The Graduate School. ABSTRACT Kidney toxicity is the second highest cause of new drug candidate failure, after liver toxicity, leading to drug withdrawals from the market and failed clinical trials. Therefore, development of more reliable and accurate in vitro screening systems for assessing drug nephrotoxicity is of high importance. Ideally, future new drug toxicity testing models will be highly sensitive, minimize animal use, and allow for high-throughput screening modalities. Recent technologies emphasize a shift away from monolayer-cultured and immortalized cells on plastic plates to novel three-dimensional (3D) matrix-based culture systems more similar to actual tissue constructs (i.e., organoids, micro- tissues, spheroids and cell/gels). This dissertation focuses on characterization of a recently developed ex vivo 3D murine proximal tubule model and comparison to currently used two-dimensional (2D) kidney cell monocultures which are used for nephrotoxicity drug screening. The overall goal of this dissertation is to establish a scientific basis for justifying this ex vivo 3D model as a more reliable and predictive toxicity testing system for high-throughput drug screening in early drug development stages than currently available. Therefore, expression levels and profiles for key kidney proximal tubule markers and important proximal tubule transporters with known roles in drug transport were obtained from the ex vivo 3D proximal tubule model. These data demonstrate more abundant and enduring apical and basolateral transporter expression than transporters in 2D cell lines, suggesting that they will provide a more reliable response to pharmaceutical compounds in 3D cultures. To test the transporter functional ability and predictive toxicity, the model was further analyzed for its capability to detect nephrotoxicity in early stages using clinically relevant known nephrotoxic and nonnephrotoxic drugs. The obtained assay responses were compared to patient drug toxicity data from the clinic to assess the model’s translational capacity. Data collected during the concentration-response study demonstrate that the model is capable of accurately predicting nephrotoxicity for all tested compounds, producing toxicity responses highly correlated to known human clinical experience with the tested drugs. The 3D murine proximal tubule model’s predictive values were found to be highly improved compared with predictive values from the current industrial “gold standard” for 2D kidney cell cultures still widely used for similar toxicity assessments. Furthermore, this model could be used to gain insight into specific drug structural mechanisms that drive toxicity and facilitate rapid assay of different chemical moieties during structure activity relationship screening. The knowledge gained through this study provides greater understanding of how drug toxicity testing results compare between standard 2D cell assays, 3D assays, and human clinical data. The data underscore the 3D proximal tubule model’s promising future as an improved, more reliable toxicity testing system for high-throughput screening during early phases of drug development. iv TABLE OF CONTENTS ABSTRACT ...................................................................................................................................... iii LIST OF TABLES ........................................................................................................................... vii LIST OF FIGURES ........................................................................................................................ viii ACKNOWLEDGEMENTS ................................................................................................................. x Chapters 1 INTRODUCTION TO THIS DISSERTATION ............................................................................... 1 2 DRUG-INDUCED NEPHROTOXICITY ........................................................................................ 6 Introduction ............................................................................................................................ 6 Pathological mechanisms of drug-induced nephrotoxicity..................................................... 6 Risk factors for nephrotoxicity ................................................................................................ 9 Preventing drug-induced renal impairment .......................................................................... 10 Assessing nephrotoxicity ..................................................................................................... 10 3 POLYSACCHARIDE MATRICES USED IN 3D IN VITRO CELL CULTURE SYSTEMS .......... 14 Abstract ................................................................................................................................ 14 Introduction .......................................................................................................................... 15 In vitro matrix requirements for 3D cultures ......................................................................... 16 Cell-specific polysaccharide binding. ................................................................................... 19 Polysaccharides used in 3D culture systems ...................................................................... 20 Synthetic polysaccharides. .................................................................................................. 41 Perspectives and future outlook. ......................................................................................... 44 4 DRUG TRANSPORTER EXPRESSION PROFILING IN A THREE DIMENSIONAL KIDNEY PROXIMAL TUBULE IN VITRO NEPHROTOXICITY MODEL ..................................................... 53 Abstract ................................................................................................................................ 53 Introduction .......................................................................................................................... 54 Materials and methods ........................................................................................................ 56 Results ................................................................................................................................. 59 Discussion ............................................................................................................................ 60 Acknowledgements .............................................................................................................. 64 5 AN EX VIVO 3D KIDNEY PROXIMAL TUBULE MODEL IMPROVES PREDICTIONS OF CLINICAL DRUG-INDUCED NEPHROTOXICITY ........................................................................ 71 Abstract ................................................................................................................................ 71 Introduction .......................................................................................................................... 72 Materials and methods ........................................................................................................ 74 Results ................................................................................................................................. 79 Discussion ............................................................................................................................ 83 Conclusion ........................................................................................................................... 88 Acknowledgements .............................................................................................................. 88 6 SUMMARY AND FUTURE DIRECTIONS .................................................................................. 95 Summary .............................................................................................................................. 95 Future directions .................................................................................................................. 97 Concluding remarks ........................................................................................................... 102 APPENDIX A RECOMBINANT HEPARIN-BINDING MAJOR AMPULLATE SPIDROIN 2 (MASP2) SILK PROTEIN ............................................................................................................................ 108 REFERENCES ...........................................................................................................................

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