Abstract: TH-OR14
Adenine Accumulation Induced by Hypoxia in Kidney Organoids
Session Information
- CKD Mechanisms: Prediction, Propagation, and Prevention
November 02, 2023 | Location: Room 119, Pennsylvania Convention Center
Abstract Time: 04:57 PM - 05:06 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Minakawa, Akihiro, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Singh, Pragya, The University of Texas Health Science Center at San Antonio Division of Nephrology, San Antonio, Texas, United States
- Berthier, Celine C., University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Fischer, Matthew, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- El Saghir, Jamal, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Baek, Judy J., University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- He, Chenchen, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Menon, Rajasree, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Pennathur, Subramaniam, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Sharma, Kumar, The University of Texas Health Science Center at San Antonio Division of Nephrology, San Antonio, Texas, United States
- Kretzler, Matthias, University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
- Harder, Jennifer L., University of Michigan Division of Nephrology, Ann Arbor, Michigan, United States
Background
Kidney disease progression is thought to result from a multitude of metabolic and genetic changes, including hypoxia and adenine. Adenine-rich diet has previously shown to be nephrotoxic in rodent models. Recently, a pathway contributing to endogenous adenine production was identified (manuscript in revision). We explored the association between adenine, kidney hypoxia and kidney injury using a human kidney organoid system with multi-omic analysis.
Methods
Kidney organoids generated from human pluripotent stem cells and exposed to 1% O2 for 24 h were split into 2 treatment groups: group 1 organoids were treated with C13-glucose for the last 3 hours of hypoxic treatment underwent targeted metabolomic analysis (C13 flux study) by LC-MS; group 2 untreated organoids were harvested for IF, qRT-PCR and sc RNA-seq. Organoids of both groups were embedded, sectioned and evaluated by MALDI-mass spectrometry imaging (MSI) using DAN matrix and the METASPACE platform.
Results
IF imaging of hypoxic kidney organoids revealed nuclear accumulation of HIF1α; targeted transcriptional analysis showed that HIF1’s target gene expression, including glycolytic pathway genes, was increased. Single cell transcriptional profiling revealed increased expression of MTAP (methylthioadenosine phosphorylase) and reduced expression of APRT (adenine phosphoribosyltransferase) in proximal tubular cells and podocytes; mitochondrial gene expression was increased in kidney cell types of especially podocytes. Metabolomic analysis showed that the percentage of C13-labeled metabolites was increased in late glycolysis but decreased in TCA cycle. MALDI-MSI of hypoxic kidney organoids revealed an increased intensity of adenine metabolite and decreased intensity of TCA metabolites such as malic acid.
Conclusion
Our hypoxic kidney organoid model successfully recapitulates key transcriptional, protein and functional alterations associated with hypoxic conditions. Under these hypoxic conditions, MTAP pathway was perturbed leading to adenine accumulation in kidney organoid cells, suggesting a potential mechanism of endogenous adenine generation contributing to kidney disease.
Funding
- NIDDK Support – Eli Lilly