Developing Pro-regenerative Drug Therapies for Acute Kidney Injury

Key Personnel

Neil Hukriede (PI)
University of Pittsburgh

Alan Davidson (PI)
University of Auckland

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. 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.

  2. 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. 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. 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. 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. 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. 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. 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. 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. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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).

  6. 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