Transgenic human nephrin in Drosophila nephrocytes facilitates variant analysis.

INTRODUCTION Nephrin, the key structural protein of the slit diaphragm, is encoded by NPHS1. Pathogenic variants in this gene are the primary cause of congenital nephrotic syndrome. About 400 variants have been described but functional characterization in vitro is very limited. METHODS Here, we express human nephrin in Drosophila nephrocytes, which possess a molecularly conserved slit diaphragm to facilitate functional studies. RESULTS Immunofluorescence of the human transgene revealed assembly into a complex linear architecture after silencing of sns, the Drosophila nephrin. This pattern suggests lateral clustering of human nephrin into a macromolecular configuration which resembles nephrin in vivo but is absent in cultured cells. In Drosophila nephrocytes, transgenic nephrin colocalized with the endogenous slit diaphragm proteins Pyd and Kirre, indicating a hybrid multi-protein complex. Transmission electron microscopy with pre-embedding immunogold labeling revealed an atypical, tubular ultrastructure. The linear nephrin did not adequately restore membrane invaginations, endocytic function or cellular survival, suggesting that proper signaling function requires additional indispensable co-factors. Murine Neph1 alone was insufficient but associated with transgenic nephrin. Notably, the linear nephrin assembly provided a read-out for investigation of patient-derived variants. This distinct pattern was altered in transgenes reflecting patient variants with milder clinical presentation, including novel variant NPHS1-V1241G. The impact on the pattern largely correlated with the age of onset of nephrotic syndrome of the respective variant, as demonstrated by automated image annotation for quantitative evaluation. CONCLUSION Our findings demonstrate that transgenesis of NPHS1 in nephrocytes is a viable approach for investigation of slit diaphragm formation and precise functional characterization of patient variants.