Genotype-Phenotype Correlations in Congenital Nephrotic Syndrome Associated with Nephrin Gene C-terminal Mutations

Background: Nephrin is a transmembrane protein forming homo- and hetero-dimeric interactions critical to the function of the glomerular slit diaphragm (SD). Homozygous nephrin gene mutations lead to congenital nephrotic syndrome (CNS). Loss of the last 132 aa at the nephrin C-terminal tail (p.R1109X) leads to more severe symptoms than the absence of the last 81 aa (p.R1160X), but the mechanism is not understood. The R1109X nephrin is thought to lack SD localization, while the R1160X mutant retains the SD localization. Studies were performed to test the hypothesis that the sequence between aa 1109 and 1160 in the nephrin C-terminal tail is critical for its plasma membrane (PM) localization. Methods: Immortalized human podocytes were transfected with wild-type (WT) nephrin, R1109X, or R1160X. The nephrin PM localization at steady state was examined by cell surface biotinylation and western blotting. The nephrin PM stability was examined over time in the presence of cycloheximide to inhibit new protein translation. Results: Similar to nephrin R1160X, the R1109X mutant was expressed at the PM, and both had lower PM abundance at steady state, compared to WT nephrin. Nephrin R1160X was more stable at the PM, compared to WT nephrin while the PM stability of nephrin R1109X was similar to WT. The total cellular stability of nephrin mutants was similar to WT nephrin. Conclusion: Unlike the previous report, we demonstrate that nephrin R1109X traffics to the PM in human podocytes, and thus, the C-terminal sequence between aa 1109 and 1160 is not essential to PM localization. The decreased PM abundance and similar PM and total cellular stability suggest that the R1109X truncation decreases nephrin biosynthetic processing but does not alter its PM dynamics or degradation. The decreased abundance and increased stability of nephrin R1160X at PM suggest that R1160X truncation impairs biosynthetic processing and PM dynamics. Our data provide novel insights into the role of the nephrin cytoplasmic tail domain. Ongoing studies focus on examining protein-protein interactions affected by the nephrin truncations to understand their genotype-phenotype correlations in CNS further. Funding: Other NIH Support - National Institutes of Health grant R01-144539, Private Foundation Support