Mo032podocin Regulates the Size of the Glomerular Pore

Abstract

Background and Aims Podocin is a major component of the glomerular slit diaphragm. It is encoded by NPHS2, the most frequently mutated gene in steroid-resistant nephrotic syndrome. We previously showed that podocin oligomerization occurs exclusively through the C-terminal helical region and is responsible for mediating interallelic interactions of NPHS2. Podocin anchors the main slit diaphragm component nephrin to the podocyte cell membrane. The shortest dimension of the glomerular pore (3,5-4nm) is determined by the distance between two neighboring nephrin molecules in cis position and – according to our structural model – corresponds perfectly to the distance between the nephrin binding domains of two podocin molecules in an oligomer. We therefore hypothesized that podocin regulates the distance between nephrin molecules, thus affecting the size of the glomerular pore. Here we aimed to explore the effects of podocin variants on the distance between neighboring nephrin molecules. Method Wild type nephrin was tagged with C-terminal YPet or mRuby3 fluorescent proteins and the two constructs were transiently coexpressed in HEK293 cells, together with podocin variants of different oligomer-forming capacity. We verified the plasmamembrane localization of the fluorescently labelled nephrin molecules by confocal microscopy. Förster resonance energy transfer (FRET) was measured between the fluorescently labelled nephrin molecules in living cells. In a second set of experiments the fluorescent tags (Ypet and mCherry) were inserted in the extracellular part of nephrin at a distance of ten amino acids from the N-terminal end of the transmembrane domain. The fluorescence decay curves measured by time-correlated single photon counting (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. Nonparametric tests were used to analyze the data. Results Similar results were obtained with the extracellular and the intracellular tags. FRET efficiency was increased in the presence of the wild type podocin (p<0.0005 with both extracellular and intracellular tags), but remained unaltered in the presence of the monomer-forming R286Tfs*17 podocin. Similar to R286Tfs*17, the other pathogenic podocin variants (R138Q, A284V and F344Lfs*4) did not alter the FRET efficiency, irrespective of the different effect of these mutations on podocin oligomerization and localization. On the other hand, the coexpression of the R229Q podocin variant, which is benign in the homozygous state, resulted in a similarly high FRET efficiency as the wild type podocin. The FRET efficiency measured between extracellularly labelled nephrin molecules in the presence of the V290M podocin variant correlated with its clinically hypomorphic effect: it fell between that of the wild type and the R286Tfs*17 podocin, being statistically different from both. Conclusion Wild type podocin significantly reduces the distance between the nephrin molecules in cis: the shortest dimension of the glomerular pore. Several pathogenic podocin variants do not. The FRET efficiency between the nephrin molecules correlates with the clinical effect of the podocin variants. We thus suggest that a major function of podocin is the regulation of the critical dimension of the glomerular pore.