Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography
Jorma Wartiovaara, … , Ulf Skoglund, Karl Tryggvason
Jorma Wartiovaara, … , Ulf Skoglund, Karl Tryggvason
Published November 15, 2004
Citation Information: J Clin Invest. 2004;114(10):1475-1483. https://doi.org/10.1172/JCI22562.
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Article Nephrology

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

Jorma Wartiovaara, Lars-Göran Öfverstedt, Jamshid Khoshnoodi, Jingjing Zhang, Eetu Mäkelä, Sara Sandin, Vesa Ruotsalainen, R. Holland Cheng, Hannu Jalanko, Ulf Skoglund, Karl Tryggvason

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Figure 6

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Comparison of normal and nephrin-deficient glomerular filtration slits. ...
Comparison of normal and nephrin-deficient glomerular filtration slits. Fixations: tannic acid–glutaraldehyde immersion in A and B and glutaraldehyde and osmium in C and D; resin sections. Scale bars: 5 nm (A, B, and D), 100 nm (C). (A) Tomogram of human filtration slit, front view; thick digital section. Globular cross strands, about 35 nm in length (labeled 1 and 2), extend from podocyte membranes. Arrows indicate distal strand ends close to, but not contacting, the opposite membrane. Arrowheads indicate close association of strands at the central density. Contacting short strand at base of cross strand 1. Pore openings are indicated. Surface rendering; sigma level: 0.1. (B) Human podocyte slit, digital section right below the slit diaphragm above the GBM. Short strands (arrows) with up to 5 globules stretch into a narrow region of the filtration slit. This stretching is also sometimes seen in EM. Sigma level: 0.1. (C) Sample from NPHS1 patient 1, homozygous for Fin-major mutation of nephrin; EM of narrow cross-cut filtration slits. Shorter (arrow) or longer (double arrow) stretches of contact are shown. The EM image was taken after a low-dose tilt series and still shows no sign of section contamination. Slit width at arrowheads is about 10 nm. (D) Sample from NPHS1 patient 2, Fin-major homozygote; tomogram of cross-cut slit slightly above GBM. Only short globular strands are now seen between cell membranes in the narrow (about 10–15 nm) filtration slit. Surface rendering; sigma level: 0.2.