Modeling oxidative injury response in human kidney organoids

Aneta Przepiorski,Matthew L Macdonald,Thitinee Vanichapol,Alan J Davidson,Eugenel B Espiritu,Thomas R Kleyman,Michael D Mcdaniels,Lawrence A Vernetti,John A Kellum,Neil A Hukriede,Amanda E Crunk,Catherine J Baty,Dave R Emlet,Ryan Salisbury,Cassandra L Happ,Emily Parasky

doi:10.1186/s13287-022-02752-z

Aneta Przepiorski, Matthew L Macdonald + Show 14 more

Open Access

https://doi.org/10.1186/s13287-022-02752-z

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Abstract

BackgroundHemolysis 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.MethodsTo 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.ResultsWe 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.ConclusionThis 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.

Highlights

Hemolysis occurs in many injury settings and can trigger disease processes
Hemin‐induced injury in kidney organoids models kidney injury Based on a previous studies, on which our organoid induction method is modeled, we hypothesized day 14 would be ideal for initiating hemin treatments, as the organoids are well formed at this stage and proliferation required to establish the nephrons has begun to diminish [26]
We found that organoids contained the segmentation pattern as shown in previous protocols, with Lotus tetragonolobus lectin (LTL) and CDH1 labelling proximal and distal tubule respectively, NPHS1 labelling podocyte clusters and MEIS1/2/3 confirming presence of interstitial cells (Additional file 1: Fig. S1)

Summary

Introduction

Extracellular hemoglobin can induce damage via several mechanisms These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Due to hemoglobin and heme, appears to be multifactorial including cellular membrane damage, oxidative stress imbalance, reaction with other proteins and lipids, and activation of immune responses. Once heme is internalized by the cells, it is catabolized by heme oxygenase 1 (HMOX1) enzyme This reaction is rate limiting and the breakdown of heme produces biliverdin, iron, and carbon monoxide, which are released into the cytosol [9]. Even though carbon monoxide and biliverdin have anti-inflammatory effects, release of iron can trigger Fenton reactions, which generates increases in hydroxyl radicals and oxidative stress that can overwhelm the cellular antioxidant capacity leading to injury [6, 10]

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