Phosphorylation of slit diaphragm proteins such as nephrin plays a critical role in proteinuric kidney diseases. Recently, the presence of anti-nephrin autoantibodies and other anti-slit diaphragm antibodies has been reported in patients with nephrotic syndrome. In our preclinical model of nephrin-immunized mice we could demonstrate that the onset of nephrotic syndrome in immunized mice is accompanied by an increased tyrosine phosphorylation of Y1191, a site known to be implicated in nephrin signaling, actin assembly, cytoskeletal reorganization and nephrin endocytosis. The aims of this study were to i) integrate these novel findings with existing data, ii) investigate how phosphorylation of slit diaphragm proteins affects protein rearrangement, iii) determine how protein subcellular localization changes upon phosphorylation events and iv) identify potential novel binding partners of slit diaphragm proteins affected by phosphorylation. We integrated anti-nephrin phosphoproteomic data with prior phosphoproteomic data, revealing novel serine/threonine phosphorylation motifs of slit diaphragm proteins. In addition to Y1191 our phosphoproteomic analysis of glomeruli from nephrin-immunized mice revealed regulation of these acidophilic phosphorylation motifs. We generated phospho-specific antibodies targeting those novel sites and validated phospho-specificity. Here we present a comprehensive characterization of the acidophilic phosphorylation site S1119 of nephrin (S1105 in human). Electron microscopy indicates a localization of nephrin phosphorylated at S1119 into the cytoplasm, indicating a possible role in nephrin endocytosis. To further interrogate this finding, we created two recombinant proteins of the intracellular domain of nephrin (phosphomimicking (D1119) and phosphoablating (A1119)). Using immunoprecipitation experiments and interaction profiling we aim to discover potential interaction partners of nephrin depending on this phosphorylation site and to examine the influence of the phosphorylation status on other nephrin phosphorylation sites. In conclusion, we present novel evidence suggesting that serine/threonine phosphorylation at acidophilic sites of slit diaphragm proteins may be involved in reorganizing the slit diaphragm in disease. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Wöhner et al. (Fri,) studied this question.