Abstract: SA-PO400
Development of a High-Throughput Screening System to Evaluate Nephrin Expression on Plasma Membrane
Session Information
- Genetic Diseases of the Kidney - III
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Kamura, Misato, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
- Kojima, Haruka, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
- Suico, Mary Ann, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
- Shuto, Tsuyoshi, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
- Kai, Hirofumi, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan
Background
Nephrin is an important component of the podocyte slit diaphragm and its dysfunction leads to severe proteinuria and nephrotic syndrome. Most nephrin mutants possibly lack export to the plasma membrane as a result of abnormal folding and post-translational modification. Although normalization of nephrin trafficking is considered as a novel therapeutic target, promising agents have not yet been found, due in part to a lack of suitable screening system for drugs that target nephrin regulation. Here, we established a high-throughput screening (HTS) system for discovery of agents that improve plasma membrane expression of nephrin.
Methods
To establish a high sensitive HTS system, we utilized split Nano-luciferase. NanoLuc fragment (HiBiT)-fusion nephrin was transfected into HEK293T cells and cell surface expression was detected by luminescence upon addition of a nonlytic reagent containing LgBiT fragment and substrate. Treatment with glycosylation inhibitor (Tunicamycin) or chemical chaperone (4-PBA), as well as comparison of clinically reported mutants (15 missense mutants) evaluated the validity of this system.
Results
HiBiT-Nephrin showed remarkable RLU compared to mock (> 200-fold). In addition, the retention of nephrin phosphorylation was confirmed by western blotting. Under this condition, tunicamycin treatment significantly reduced HiBiT-nephrin RLU (< 40%). In contrast, 4-PBA treatment significantly increased RLU (>150%). Furthermore, each of the 15 mutants of HiBiT-Nephrin showed unchanged (5) or reduced (10) expression on plasma membrane, most of which were augmented or recovered by 4-PBA treatment.
Conclusion
In this study, we succeeded in establishing a HTS system that can easily and sensitively quantify the expression of nephrin on the plasma membrane. Although the cell-based assay has limitations, this system reflected the characteristics of nephrin consistent with previous reports. With further optimization, this system will be used to screen for compounds that target nephrin.