Developing Pro-regenerative Drug Therapies for Acute Kidney Injury

Key Personnel

Project Description

Acute kidney injury (AKI) is a major health problem and there are currently no effective treatments. We have identified a novel class of compounds (PTBA) that reduce AKI injury when administered days after the initiating injury. However, how PTBA prevents kidney injury remains unclear. Our goal is to use human kidney organoids generated from induced pluripotent stem cells (iPSCs) as a tool to understand the pro-regenerative mechanism-of-action of PTBA analogs during kidney injury and repair, and to further validate the kidney organoid system as a pre-clinical drug screening platform.

Publications

1. Abstract

   Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium

   Naved, Bilal A.; Bonventre, Joseph V.; Hubbell, Jeffrey A.; Hukriede, Neil A.; Humphreys, Benjamin D.; Kesselman, Carl; Valerius, M. Todd; McMahon, Andrew P.; Shankland, Stuart J.; Wertheim, Jason A.; White, Michael J.V.; de Caestecker, Mark P.; Drummond, Iain A. Kidney International . March 2022.

   Acute kidney injury impacts ∼13.3 million individuals and causes ∼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK’s open access data hub for accessing, downloading, and further analyzing data sets.

2. Abstract

   Validation of HDAC8 Inhibitors as Drug Discovery Starting Points to Treat Acute Kidney Injury

   Long, Keith; Vaughn, Zoe; McDaniels, Michael David; Joyasawal, Sipak; Przepiorski, Aneta; Parasky, Emily; Sander, Veronika; Close, David; Johnston, Paul A.; Davidson, Alan J.; de Caestecker, Mark; Hukriede, Neil A.; Huryn, Donna M.. ACS Pharmacology & Translational Science . March 2022.

   Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting. Using biochemical assays, zebrafish AKI phenotypic assays, and human kidney organoid assays, we show that selective HDAC8 inhibitors can lead to efficacy in increasingly stringent models. One of these, PCI-34051, was efficacious in a rodent model of AKI, further supporting the potential for HDAC8 inhibitors and, in particular, this scaffold as a therapeutic approach to AKI.

3. Abstract

   Experimental models of acute kidney injury for translational research

   Hukriede, Neil A.; Soranno, Danielle E.; Sander, Veronika; Perreau, Tayla; Starr, Michelle C.; Yuen, Peter S. T.; Siskind, Leah J.; Hutchens, Michael P.; Davidson, Alan J.; Burmeister, David M.; Faubel, Sarah; de Caestecker, Mark P.. Nature Reviews Nephrology . February 2022.

   Preclinical models of human disease provide powerful tools for therapeutic discovery but have limitations. This problem is especially apparent in the field of acute kidney injury (AKI), in which clinical trial failures have been attributed to inaccurate modelling performed largely in rodents. Multidisciplinary efforts such as the Kidney Precision Medicine Project are now starting to identify molecular subtypes of human AKI. In addition, over the past decade, there have been developments in human pluripotent stem cell-derived kidney organoids as well as zebrafish, rodent and large animal models of AKI. These organoid and AKI models are being deployed at different stages of preclinical therapeutic development. However, the traditionally siloed, preclinical investigator-driven approaches that have been used to evaluate AKI therapeutics to date rarely account for the limitations of the model systems used and have given rise to false expectations of clinical efficacy in patients with different AKI pathophysiologies. To address this problem, there is a need to develop more flexible and integrated approaches, involving teams of investigators with expertise in a range of different model systems, working closely with clinical investigators, to develop robust preclinical evidence to support more focused interventions in patients with AKI.

4. Abstract

   Modeling oxidative injury response in human kidney organoids

   Przepiorski, Aneta; Vanichapol, Thitinee; Espiritu, Eugenel B.; Crunk, Amanda E.; Parasky, Emily; McDaniels, Michael D.; Emlet, Dave R.; Salisbury, Ryan; Happ, Cassandra L.; Vernetti, Lawrence A.; MacDonald, Matthew L.; Kellum, John A.; Kleyman, Thomas R.; Baty, Catherine J.; Davidson, Alan J.; Hukriede, Neil A.. Stem Cell Research & Therapy . 13(1):76. February 2022.

   Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

5. Abstract

   Chapter 32: Small molecules in regeneration

   Crunk, Amanda E.; Przepiorski, Aneta; Hukriede, Neil A.. Regenerative Nephrology. 2022.

   Acute kidney injury (AKI) is a major clinical and economic problem worldwide and currently there are no effective treatments. Due to the numerous renal insults that lead to AKI and the complexity of injury, many pathways are dysregulated leading to maladaptive repair, which can lead to chronic kidney disease. The focus of this chapter is to illustrate the pathways that play a role in AKI and how small molecules for therapeutic intervention can be utilized to modulate their activity and enhance productive repair. Given the large number of pathways that could be therapeutic targets, we will focus on only a limited number that have shown promising results in the treatment of AKI.

6. Abstract

   A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

   AU - Przepiorski, Aneta; AU - Crunk, Amanda E.; AU - Holm, Teresa M.; AU - Sander, Veronika; AU - Davidson, Alan J.; AU - Hukriede, Neil A.. JoVE . April 2021.

   Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

7. Abstract

   Protocol for Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells.

   Sander, Veronika; Przepiorski, Aneta; Crunk, Amanda E.; Hukriede, Neil A.; Holm, Teresa M.; Davidson, Alan J.. STAR protocols . 1(3):100150. December 2020.

   Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).

8. Abstract

   Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish.

   Brilli Skvarca, Lauren; Han, Hwa In; Espiritu, Eugenel B.; Missinato, Maria A.; Rochon, Elizabeth R.; McDaniels, Michael D.; Bais, Abha S.; Roman, Beth L.; Waxman, Joshua S.; Watkins, Simon C.; Davidson, Alan J.; Tsang, Michael; Hukriede, Neil A. Dis Model Mech . 12(4). April 2019.

   Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified

9. Abstract

   Wnt signaling mediates new nephron formation during zebrafish kidney regeneration

   Kamei, Caramai N.; Gallegos, Thomas F.; Liu, Yan; Hukriede, Neil; Drummond, Iain A. Development . 146(8). April 2019.

   Zebrafish kidneys use resident kidney stem cells to replace damaged tubules with new nephrons: the filtration units of the kidney. What stimulates kidney progenitor cells to form new nephrons is not known. Here, we show that wnt9a and wnt9b are induced in the injured kidney at sites where frizzled9b- and lef1-expressing progenitor cells form new nephrons. New nephron aggregates are patterned by Wnt signaling, with high canonical Wnt-signaling cells forming a single cell thick rosette that demarcates: domains of cell proliferation in the elongating nephron; and tubule fusion where the new nephron plumbs into the distal tubule and establishes blood filtrate drainage. Pharmacological blockade of canonical Wnt signaling inhibited new nephron formation after injury by inhibiting cell proliferation, and resulted in loss of polarized rosette structures in the aggregates. Mutation in frizzled9b reduced total kidney nephron number, caused defects in tubule morphology and reduced regeneration of new nephrons after injury. Our results demonstrate an essential role for Wnt/frizzled signaling in adult zebrafish kidney development and regeneration, highlighting conserved mechanisms underlying both mammalian kidney development and kidney stem cell-directed neonephrogenesis in zebrafish.

1. Abstract

   Access and use of the GUDMAP database of genitourinary development

   Davies, JA; Little, MH; Aronow, B; Armstrong, J; Brennan, J; Lloyd-MacGilp, S; Armit, C; Harding, S; Piu, X; Roochun, Y; Haggarty, B; Houghton, D; Davidson, D; Baldock, R. Methods Mol Biol . 886:185–201. 2012.

   The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene expression profiling (from laser-dissected and FACS-sorted samples) and in situ hybridization at both low (whole-mount) and high (section) resolutions. Data are annotated to a published, high-resolution anatomical ontology and can be accessed using a variety of search interfaces. Here, we explain how to run typical queries on the database, by gene or anatomical location, how to view data, how to perform complex queries, and how to submit data.

2. Abstract

   The GUDMAP database–an online resource for genitourinary research

   Harding, SD; Armit, C; Armstrong, J; Brennan, J; Cheng, Y; Haggarty, B; Houghton, D; Lloyd-MacGilp, S; Pi, X; Roochun, Y; Sharghi, M; Tindal, C; McMahon, AP; Gottesman, B; Little, MH; Georgas, K; Aronow, B; Potter, SS; Brunskill, EW; Southard-Smith, EM; Mendelsohn, C; Baldock, RA; Davies, JA; Davidson, D. Development . 138(13):2845–53. July 2011.

   The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.

3. Abstract

   GUDMAP: the genitourinary developmental molecular anatomy project

   McMahon, AP; Aronow, B; Davidson, DR; Davies, JA; Gaido, KW; Grimmond, SM; Lessard, JL; Little, MH; Potter, SS; Wilder, EL; Zhang, P; GUDMAP, Project. J Am Soc Nephrol . 19(4):667–71. April 2008.

   In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.

4. Abstract

   A high-resolution anatomical ontology of the developing murine genitourinary tract

   Little, MH; Brennan, J; Georgas, K; Davies, JA; Davidson, DR; Baldock, RA; Beverdam, A; Bertram, JF; Capel, B; Chiu, HS; Clements, D; Cullen-McEwen, L; Fleming, J; Gilbert, T; Herzlinger, D; Houghton, D; Kaufman, MH; Kleymenova, E; Koopman, PA; Lewis, AG; McMahon, AP; Mendelsohn, C; Mitchell, EK; Rumballe, BA; Sweeney, DE; Valerius, MT; Yamada, G; Yang, Y; Yu, J. Gene Expr Patterns . 7(6):680–99. June 2007.

   Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.

5. Abstract

   Validation of HDAC8 Inhibitors as Drug Discovery Starting Points to Treat Acute Kidney Injury

   Long, Keith; Vaughn, Zoe; McDaniels, Michael David; Joyasawal, Sipak; Przepiorski, Aneta; Parasky, Emily; Sander, Veronika; Close, David; Johnston, Paul A.; Davidson, Alan J.; de Caestecker, Mark; Hukriede, Neil A.; Huryn, Donna M.. ACS Pharmacology & Translational Science . March 2022.

   Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting. Using biochemical assays, zebrafish AKI phenotypic assays, and human kidney organoid assays, we show that selective HDAC8 inhibitors can lead to efficacy in increasingly stringent models. One of these, PCI-34051, was efficacious in a rodent model of AKI, further supporting the potential for HDAC8 inhibitors and, in particular, this scaffold as a therapeutic approach to AKI.

6. Abstract

   Experimental models of acute kidney injury for translational research

   Hukriede, Neil A.; Soranno, Danielle E.; Sander, Veronika; Perreau, Tayla; Starr, Michelle C.; Yuen, Peter S. T.; Siskind, Leah J.; Hutchens, Michael P.; Davidson, Alan J.; Burmeister, David M.; Faubel, Sarah; de Caestecker, Mark P.. Nature Reviews Nephrology . February 2022.

   Preclinical models of human disease provide powerful tools for therapeutic discovery but have limitations. This problem is especially apparent in the field of acute kidney injury (AKI), in which clinical trial failures have been attributed to inaccurate modelling performed largely in rodents. Multidisciplinary efforts such as the Kidney Precision Medicine Project are now starting to identify molecular subtypes of human AKI. In addition, over the past decade, there have been developments in human pluripotent stem cell-derived kidney organoids as well as zebrafish, rodent and large animal models of AKI. These organoid and AKI models are being deployed at different stages of preclinical therapeutic development. However, the traditionally siloed, preclinical investigator-driven approaches that have been used to evaluate AKI therapeutics to date rarely account for the limitations of the model systems used and have given rise to false expectations of clinical efficacy in patients with different AKI pathophysiologies. To address this problem, there is a need to develop more flexible and integrated approaches, involving teams of investigators with expertise in a range of different model systems, working closely with clinical investigators, to develop robust preclinical evidence to support more focused interventions in patients with AKI.

7. Abstract

   Modeling oxidative injury response in human kidney organoids

   Przepiorski, Aneta; Vanichapol, Thitinee; Espiritu, Eugenel B.; Crunk, Amanda E.; Parasky, Emily; McDaniels, Michael D.; Emlet, Dave R.; Salisbury, Ryan; Happ, Cassandra L.; Vernetti, Lawrence A.; MacDonald, Matthew L.; Kellum, John A.; Kleyman, Thomas R.; Baty, Catherine J.; Davidson, Alan J.; Hukriede, Neil A.. Stem Cell Research & Therapy . 13(1):76. February 2022.

   Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

8. Abstract

   A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

   AU - Przepiorski, Aneta; AU - Crunk, Amanda E.; AU - Holm, Teresa M.; AU - Sander, Veronika; AU - Davidson, Alan J.; AU - Hukriede, Neil A.. JoVE . April 2021.

   Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

9. Abstract

   Protocol for Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells.

   Sander, Veronika; Przepiorski, Aneta; Crunk, Amanda E.; Hukriede, Neil A.; Holm, Teresa M.; Davidson, Alan J.. STAR protocols . 1(3):100150. December 2020.

   Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).

10. Abstract

    Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish.

    Brilli Skvarca, Lauren; Han, Hwa In; Espiritu, Eugenel B.; Missinato, Maria A.; Rochon, Elizabeth R.; McDaniels, Michael D.; Bais, Abha S.; Roman, Beth L.; Waxman, Joshua S.; Watkins, Simon C.; Davidson, Alan J.; Tsang, Michael; Hukriede, Neil A. Dis Model Mech . 12(4). April 2019.

    Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified