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 4

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Extracellular nephrin-label on transfected HEK293 cells. Scale bars: 100...
Extracellular nephrin-label on transfected HEK293 cells. Scale bars: 100 nm (A), 5 nm (B and C). (A) Small, 5-nm-immunogold particles (in the rectangle and 2 circles) mark nephrin on the cell surface. (The large 10-nm-gold particles are used as coordinates for 3D-reconstruction purposes [39, 67].) Pre-embedding immunolabeling; resin section. (B) Tomogram from reconstructed volume of rectangle in A. The strand with a gold label on its distal end seemingly traverses the cell membrane. Marked extracellular length, measured in 3D, is about 35 nm when the putative anti-nephrin IgG complex (5-nm-gold–anti-rabbit IgG + rabbit anti-nephrin IgG) at the end of the strand (arrowhead) is omitted. Inside the cell, the strand is continuous near the membrane (short arrow) with intracellular strand (ICS). Sigma levels: 0.5 (green and blue) and 0 (white, strand-immunogold complex); 13 (gold particle). (C) A 90–-tilted side view from the direction shown in B (long, bent arrow). Sigma levels: 0.3 (green and blue) and 0 (white). Note minimal volume change in strand between sigma levels in B (from 0.5 to 0) and C (from 0.3 to 0).