Abstract: PO0448
Establishment and Evaluation of a Primary Human Renal Tubular 3D Spheroid Model for AKI and CKD
Session Information
- AKI: Novel Insights
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 101 AKI: Epidemiology, Risk Factors, and Prevention
Authors
- Wang, Lifeng, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
- Zhai, Yougang, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
- Guo, Lili, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
- Meng, Rong, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
- Ma, Li-Jun, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
- Lee, Seunghun Paul, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
- Pocai, Alessandro, Janssen Research and Development LLC, Spring House, Pennsylvania, United States
Background
The proximal tubule of the nephron is a prime site for tubular injury due to its high energy requirements and its dependence on oxidative metabolism to meet its energy needs. Our understanding of the central role of mitochondrial abnormalities and alterations in metabolism in both acute and chronic kidney injury has steadily improved with potential targets to improve mitochondrial dysfunction that occurs in AKI and in AKI to CKD transition.
Two-dimensional (2D) monolayer cultures and rodent animal models are unable to fully recapitulate clinical drug response, hence 3D models are being developed to provide a physiologically relevant condition.
Methods
Here, we developed a primary human renal tubular 3D spheroid culture and established a cisplatin-injury model for therapeutic target evaluation. Human primary renal tubular cells (RPTEC) seeded in ULA plates showed aggregation after 4 hours and formed initial spheroids after 4 days and the primary cells can be cultured over 5 weeks without major physiological changes.
Results
In 3D setting, gene expression of tubular markers was significantly induced/restored close to the human tissue level comparing to 2D culture (AQP1, OAT, LRP2, PEPT2, SLC12A1, etc.) suggesting a more physiologically relevant condition. As NAD+ level is a critical factor to maintain mitochondria functionality in tubular cells, we evaluated genes involved in de novo NAD synthesis pathway and observed increased expression in 3D- vs. 2D culture (AFMID, KMO, KYNU, HAAO, ACMSD, NMNATs, etc.). Moreover, since TGF-β is activated in AKI and is involved in AKI to CKD transition, we confirmed that TGF-β treatment repressed de novo NAD pathway gene expression, suggesting a possible link to the decrease of de novo NAD+ in the cells producing TGF-β. Lastly, In our cisplatin-induced tubular cell injury model in 3D spheroid, cisplatin induces the dose-dependent increase of cell apoptosis associated with dose-dependent reduction of total cellular NAD+ and GSH.
Conclusion
Taken together, our 3D primary human spheroid model restored key marker gene expression and recapitulated in vivo response to the TGF-β treatment and cisplatin-induced injury, which provide a physiological and pathophysiological relevant tool and translational model to enable the quick screening and evaluation of therapeutic targets for AKI and CKD.