Thomas Kleyman (PI)
University of Pittsburgh
Proposed studies will determine specific functional properties of tubular epithelia in human kidney organoids. Key transporters, cytoskeletal elements, and other relevant proteins will be localized in epithelia within developing kidney organoids. Key physiological properties of epithelia within distinct tubular segments in organoids will be defined. The uptake of fluorescent albumin and dextran in proximal tubules will be examined to demonstrate cell specific receptor-mediated and/or fluid phase endocytosis. H+ secretion by specific epithelial H+ transporters in specific cells and tubular segments will be determined. Functional expression of K+ and Na+ channels in specific cells and tubular segments will be determined. Na+ and K+ transport in isolated, microperfused tubules will be determined.
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.