Podocin oligomers regulate the ordering of nephrin chains, providing the molecular basis of NPHS2 interallelic interactions

The most frequent causative genes in podocytopathies are NPHS1 and NPHS2 that encode the main slit diaphragm components nephrin and podocin, respectively. The significance of the nephrin–podocin interaction has remained elusive. The NPHS2 R229Q variant is unique in human genetics as it is only pathogenic when trans -associated to specific 3’ variants causing R229Q podocin to form distorted heterooligomers. To examine the effect of podocin on the distance between nephrin molecules, nephrin constructs labeled with YPet or mCherry/mRuby were transitionally coexpressed in HEK-293 cells. We assessed the nephrin–nephrin distance based on the Förster Resonance Energy Transfer (FRET) efficiency between YPet and mCherry/mRuby in living cells. Podocin markedly increased the FRET efficiency between nephrin molecules, reflecting reduced and ordered nephrin–nephrin distances. Its effect was abolished by pathogenic podocin variants. Pathogenic R229Q heterooligomers exhibited increased FRET efficiency between their PHB domains that correlated with a larger nephrin–nephrin distance, thereby explaining the associated mild phenotype. The effect of pathogenic and benign R229Q heterooligomers separated sharply in their effect on nephrin–nephrin spacing ( P = 1.19E-33). Based on an intermediate effect on the nephrin–nephrin distance and five families with late-onset FSGS, we reconsider the R229Q-R286Tfs*17 association as pathogenic, but via a mechanism different from other R229Q associations and with incomplete penetrance. In conclusion, podocin provides regularly spaced intracellular anchor points for nephrin chains. Podocin homo-oligomerization affects the nephrin–nephrin spacing, thereby providing an explanation for albuminuria in patients with podocin dysfunction and for the interallelic interactions of R229Q.