Abstract: SA-PO648
2,8-Dihydroxyadenine Impacts Kidney Cell Health and Barrier Integrity in an In Vitro Model of Adenine Phosphoribosyltransferase (APRT) Deficiency
Session Information
- Genetic Kidney Diseases: Models, Mechanisms, and Therapies
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1202 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Helgudottir, Hildur Run, Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland
- Palsson, Runolfur, Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland
- Edvardsson, Vidar O., Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland
- Gudjonsson, Thorarinn, Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland
Background
Adenine phosphoribosyltransferase (APRT) deficiency is a rare genetic disorder that results in excessive generation and excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stones and crystal nephropathy. This condition causes inflammation and fibrosis of the kidneys. Although the effects of other types of crystal nephropathies and kidney stone diseases have been studied in cell culture models, the mechanism of DHA-mediated kidney injury is not well understood. This study aimed to establish a comprehensive cell culture model to investigate DHA crystal-induced kidney injury and identify therapeutic targets for clinical intervention.
Methods
Three kidney cell lines, MDCK, HK-2, and HEK293, were tested in both monolayer and 3D cultures, including liquid-liquid interface (LLI) and "on-top" of Matrigel methods. Cells were exposed to DHA at concentrations similar to those found in the urine of APRT-deficient patients. Cell viability and migration assays, RT-PCR, western blotting, phase-contrast microscopy, and immunostaining were used as read-out assays. Immunohistochemistry (IHC) was used to analyze kidney tissue samples from APRT deficiency patients and healthy controls obtained from Landspitali University Hospital Biobank.
Results
DHA exposure decreased kidney cell viability and migration across all cell lines. Rising DHA concentrations increased CD44 expression, suggesting enhanced crystal-binding potential of the kidney cells. In a 3D environment “on-top” of Matrigel, MDCK cells maintained polarized structures despite DHA accumulation and did not show an increase in the EMT phenotype compared with TGFβ-treated cells. MDCK cells formed a polarized cell layer grown on Transwell polyester membranes and demonstrated trans-epithelial electrical resistance (TEER) in LLI, which decreased when the cells were treated with DHA. Analysis of patient kidney tissue samples revealed increased collagen I and III expression, well-known markers of fibrosis, in APRT deficiency patients.
Conclusion
Our findings demonstrate the deleterious effects of DHA on kidney cell health, barrier function, and its potential role in promoting fibrosis in APRT deficiency. Future studies in 3D cultures will focus on further exploring the phenotypic changes in kidney cells after exposure to DHA both at the cellular and molecular level.
Funding
- Government Support – Non-U.S.