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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

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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 1

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Glomerular filtration slit in EM and electron tomography. Resin sections...
Glomerular filtration slit in EM and electron tomography. Resin sections after standard embedding (A–G) and high-pressure freezing–freeze-substitution embedding (H). Wire-frame (E) and surface (F–H) rendering. Scale bars: 200 nm (A), 40 nm (B), 20 nm (C), 10 nm (D–H). (A) Human glomerular capillary wall; 2.5% glutaraldehyde and osmium fixation. Arrowheads indicate the level of slit diaphragm in the cross-section. S, filtration slit; FP, foot process; E, capillary endothelium. (B) Mouse filtration slit; tannic acid–glutaraldehyde and osmium fixation. The arrows indicate the so-called central dot in the nearly cross-cut slit diaphragm above the GBM. (C) Front view of human slit diaphragm; tannic acid–glutaraldehyde and osmium fixation. SD, slit diaphragm. (D) Higher magnification of box in C. Lateral pores (P) are indicated. Black arrows, central filament; white arrows, staggered slit diaphragm cross strands from wall. (E) Human slit, electron tomography front-view; glutaraldehyde fixation. Arrowheads indicate intracellular strands from undulating cell membrane (M) opposite slit diaphragm cross strands (short arrows). CD, central density. Sigma level: 1.0. (F) Rat slit, slanted front view; tannic acid–glutaraldehyde perfusion; thick digital section. The image reveals a double ladder–type slit-diaphragm structure with merged cross strands (arrows) bordering lateral pores. Arrowheads, pore with multiple slit diaphragm layers. Sigma level: 0.5. (G) Mouse slit, front-view; glutaraldehyde and osmium fixation. Zipperlike slit diaphragm. Staggered cross strands (arrows) border large lateral and small central pores. Sigma level: 1.0. (H) Mouse slit; glutaraldehyde fixation. The image was tilted 30– around the y axis to demonstrate the path of the pores (arrows). Sigma level: 0.1. For comparison with pore size, a space-filled model of the crystal structure of albumin molecule (Alb) is superimposed.
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