title: Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography.
abstract: Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Jorma
authors.initials: J
authors.lastname: Wartiovaara
authors.forename: Lars-Göran
authors.initials: LG
authors.lastname: Ofverstedt
authors.forename: Jamshid
authors.initials: J
authors.lastname: Khoshnoodi
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhang
authors.forename: Eetu
authors.initials: E
authors.lastname: Mäkelä
authors.forename: Sara
authors.initials: S
authors.lastname: Sandin
authors.forename: Vesa
authors.initials: V
authors.lastname: Ruotsalainen
authors.forename: R Holland
authors.initials: RH
authors.lastname: Cheng
authors.forename: Hannu
authors.initials: H
authors.lastname: Jalanko
authors.forename: Ulf
authors.initials: U
authors.lastname: Skoglund
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
mesh_terms: D000818:Animals; D002196:Capillaries / Q000737:chemistry* / Q000000981:diagnostic imaging / Q000648:ultrastructure; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D014644:Genetic Variation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000098:blood supply* / Q000000981:diagnostic imaging / Q000648:ultrastructure; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014463:Ultrasonography
chemical_list: D004220:Disulfides; D007074:Immunoglobulin G; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; C113267:nephrin