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.

Figure 1

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.

Figure 2

Human, rat, and mouse slit diaphragm with double layers. Scale bars: 50 nm (A), 20 nm (B–D). (A) Filtration slit in EM cross section between foot processes of human podocytes (P1, P2) showing double-layered slit diaphragm (arrowheads). Tannic acid–glutaraldehyde and osmium fixation; resin section. (B) Tomogram of same slit as in A. Strands seem to connect the two slit diaphragm layers (arrow). Sigma level: 0.5. (C) Same rat filtration slit as in Figure 1F, tilted 90– around the x axis; showing 2 layers in slit diaphragm. Sigma level: 1.0. (D) Two layers in cross-cut mouse slit diaphragm with connecting strands (arrow); glutaraldehyde and osmium fixation. Sigma level: 1.0.

Figure 3

Localization of nephrin in human slit diaphragm: immuno-cryolabeling of extracellular terminal Ig-domains of nephrin. SD pores (P) are indicated. Scale bars: 40 nm (A and B), 10 nm (C), 5 nm (D). (A) Nephrin label (5-nm gold, arrows) in EM along obliquely cut slit diaphragm. (B) Tomogram of filtration slit bordered by GBM, foot processes, and slit diaphragm. Gold labeling for nephrin appears under the diaphragm at different levels of the digital volume. Sigma levels: 0.05 (tissue), 13 (gold particles). (C) Higher magnification of B (same sigma levels), but visualized from below, through 30-nm-thick digital section encompassing the slit diaphragm. Around 4-nm-wide strands (arrows) extend from the podocyte surface into the diaphragm. (D) Close-up of slit diaphragm cross strand (arrow). Gold label (G) appears near the distal end of cross strand. Note associated globules (sectioned short strands) at the proximal part of cross strand. Only small volume differences in wire frames exist between sigma levels 1.0 (blue and green) and 0.3 (white).

Figure 4

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).

Figure 5

Recombinant nephrin and IgG in vitrified solution in electron tomography. Scale bars: 5 nm. (A) Schematic domain structure of nephrin. C, intracellular carboxyl terminal; filled rectangle, transmembrane domain; hexagon, fibronectin type III–like domain; incomplete circles, Ig repeats with internal disulfide bonds (dashes); S-shaped line, flexible region; N, extracellular amino terminal; SH, regions with free cysteine residues. (B) Schematic drawing of soluble NphHis. Ig domains 1 and 8 and fibronectin type III–like domain (Fn III) are indicated. (C) Tomogram of vitrified solution of recombinant NphHis: solitary (1) and associated (2) structures. Sigma levels: 2.0 (orange) and 1.5 (blue). (D and E) Structures in solution of NphHis and anti-nephrin IgG (against Ig-1 and Ig-2 repeats of nephrin). Y-shaped structures (putative IgG) are associated with the last 2 globules in the long arm of the strands. Arrow indicates putative flexible region of NphHis. Density refinement to a resolution of 25 –. (F) Tomogram of individual reference IgG molecule. Putative Fc region points downward. Refinement and low-pass filtering to 25 –. Volume rendering with contouring levels corresponding to sigma levels 1.5 in D and F and 2.5 in E.

Figure 6

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.