Podocin Regulates the Nephrin-Nephrin Distance in the Glomerular Pore

Podocin, encoded by NPHS2, is an integral protein of the glomerular slit diaphragm. Mutations of NPHS2 cause autosomal recessive nephrotic syndrome. Previously we have shown that its most frequent missense variant, R229Q, is pathogenic only when trans-associated with specific C-terminal podocin mutations secondary to an altered C-terminal hetero-oligomerization. Podocin anchors the main slit diaphragm component nephrin to the podocyte cell membrane. Here we explored the effect of podocin oligomerization on the oligomerization of nephrin. YPet- and mRuby-tagged wild-type nephrin and podocin variants with different oligomer-forming capacity were transiently expressed in HEK293 cells. FRET was measured by fluorescence spectroscopy between YPet and mRuby in living cells. The fluorescence decay curves measured by TCSPC (Chronos BH, ISS Inc) were decomposed into lifetime components, and the longest lifetime population – most suitable for long-range FRET – was used for further calculations. We found a significant reduction in the lifetime of the donor YPet fluorescence in the presence of wt podocin, but not with the monomer-forming R286Tfs∗17, indicating that podocin oligomerization reduces the distance between neighboring nephrin molecules. While R229Q podocin had a similar effect as the wt, pathogenic podocin variants failed to significantly reduce donor fluorescence lifetime. Thus, podocin oligomerization significantly reduces the nephrin-nephrin distance: the shortest dimension of the glomerular pore. We thus suggest that a major function of podocin is the regulation of the glomerular pore size. The study was supported by the MTA-SE Lendulet Research Grant (LP2015-11/2015) of the Hungarian Academy of Sciences and the NKFIA/OTKA K109718, KH125566 grants (KT) and NKFIA/NVKP-16-1-2016-0017 (MK,GS) Additionally, the research was financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the therapeutic thematic programme of the Semmelweis University.