Abstract: PO0375
Modeling Sepsis in Human Kidney Organoids: Cell-Free Hemoglobin-Induced Cytotoxicity Is Attenuated by Ascorbate or Acetaminophen
Session Information
- AKI: Mechanisms of Injury
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Bejoy, Julie, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Farry, Justin M., Vanderbilt University, Nashville, Tennessee, United States
- Ware, Lorraine B., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Bastarache, Julie A., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Woodard, Lauren Elizabeth, Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background
Sepsis-associated acute kidney injury (AKI) is associated with high morbidity and mortality. Cell-free hemoglobin (CFH) is released into the circulation of patients with severe sepsis and higher levels are independently associated with mortality. CFH is toxic to HK-2 cells, suggesting that CFH can directly injure the renal tubular epithelium. Treatment with ascorbate (Vitamin C) or acetaminophen provides protection from CFH-induced toxicity in endothelial cells. Therefore, we sought to generate a model of sepsis-induced AKI in human kidney organoids in which to study CFH-targeted AKI treatments.
Methods
We cultured human kidney organoids from fibroblast-derived human induced pluripotent stem cells (hiPSCs) by the Takasato protocol, with optimization. We then treated mature human kidney organoids with CFH (1 mg/ml) for 48 hours and evaluated cell toxicity, viability, reactive oxygen species (ROS), and mitochondrial fragmentation. To study the protective effects, CFH-exposed organoids were co-treated with ascorbate (200 uM) or acetaminophen (1000 uM) for 48 hours.
Results
The kidney organoids expressed the expected kidney cell type markers ECAD (distal tubule), GATA3 (collecting duct), LTL (proximal tubule) and NEPHRIN (Glomeruli) at Day 21 (n=3). CFH treatment resulted in ROS production in 80% of cells within organoids compared to 5% in control group(n=3). Analysis by Lactate Dehydrogenase showed CFH treatment increased toxicity by 30% in organoids and and analysis by MTT (tetrazolium dye) assays showed reduced viability [by 70% within organoids compared to control group(n=3)(p<.005) . LDH assay also revealed that the addition of ascorbate or acetaminophen attenuated the impact of CFH on organoids by decreasing the toxicity by ~23% within the organoids (p value<.0001).
Conclusion
Human kidney organoids can be used to model sepsis-induced AKI. CFH treatment induced toxicity and reduced viability of the human kidney organoids. Both ascorbate and acetaminophen had protective effects on CFH-induced organoid injury.
Funding
- Private Foundation Support