Establishing a nanoluciferase-based assay as a high-throughput screening platform for therapeutics in congenital...

Nephrin is crucial for the formation of the glomerular slit diaphragm, which is the final filtration barrier in the kidney. A mutation in the NPHS1 gene that codes for nephrin causes congenital nephrotic syndrome of the Finnish type (CNF). Most missense mutations render nephrin non-functional due to the defective nephrin trafficking to the cell membrane. Pharmacological approaches that induce the expression of nephrin on the cell membrane are feasible, but therapeutic development is hampered by the lack of a high-throughput screening (HTS) system. Here, we developed a nanoluciferase HiBiT-based HTS platform to quantify the cell membrane expression of a nephrin mutant. This evaluation system reflected the previously reported results of various nephrin mutant localization. Using this system, we screened and identified 10 compounds that promoted the expression of the nephrin E725D mutant on the cell membrane. Moreover, the phosphorylation and N-glycosylation of nephrin, which are modifications that indicate its cell surface localization, correlated with the luminescence values of HiBiT-Nephrin in the compound screening. Consequently, this HiBiT-Nephrin evaluation system could be a new platform for predicting the pathogenicity of variants and searching for therapeutic agents for CNF.