Its intracellular domain is rich in serine and tyrosine residues (7) that can be phosphorylated (19). 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. Introduction Knowledge about the molecular mechanisms of plasma filtration in the renal glomeruli and mechanisms of proteinuria is still limited. The filtration barrier consists of 3 layers: a fenestrated capillary endothelium, a glomerular basement membrane (GBM), and a podocyte layer. The extracellular slit diaphragm bridges the filtration slit as a thin continuous band between podocyte foot processes covering the capillary surface in an interdigitating manner. Molecules traversing the filtration barrier are selected according to size, shape, and charge (1). Ample data suggest that the slit diaphragm forms the ultimate barrier for macromolecular permeability (2C4). Defects in the slit diaphragm lead to proteinuria, a hallmark of numerous acquired and Goat polyclonal to IgG (H+L)(HRPO) genetic kidney diseases. Based on EM of perfusion-fixed rodent kidneys, Rodewald and Karnovsky (5) originally proposed an isoporous zipperlike structure model for the slit diaphragm. In that model, staggered cross-bridges extend from the slit walls to a longitudinal central filament, thus forming rectangular pores in the diaphragm. This model was later questioned, particularly in light of results from freeze-etching studies with unfixed tissue using deep-etching of quick-frozen samples, which suggested a sheet-like, rather than zipperlike, substructure for the diaphragm (6). Until recently, the molecular nature of the slit diaphragm remained obscure. Nephrin (7) was the first molecule to be localized to the slit diaphragm area (8C10). Nephrin is essential for the development and function of the normal glomerular filter, as seen in congenital nephrotic syndrome of the Finnish type (NPHS1), where the nephrin gene is mutated (7, 11). The NPHS1 disorder (12), as well as inactivation of the mouse nephrin gene (13), lead to Vancomycin deleterious proteinuria and absence of the slit diaphragm. For proteins of the size of albumin molecules or larger, nephrin is a decisive determinant for glomerular filtration, as seen in 3 genetic mouse models for glomerular protein leakage (13, 14). In addition to nephrin, P-cadherin (15), the nephrin homolog Neph1 (16, 17), and the large cadherin-like protein FAT (human homologue to the tumor suppressor fat) (18) have been localized extracellularly to the slit diaphragm region. Nephrin is a type I transmembrane protein with both structural and signaling functions. Its intracellular domain is rich in serine and tyrosine residues (7) that can be phosphorylated (19). Intracellularly, nephrin apparently associates with podocin, CD2-associated protein, and Neph1 (20C25). Extracellularly, nephrin molecules may interact across the filtration slit (3, 10). Evidence for extracellular homophilic interaction of nephrin and heterophilic interactions of nephrin and Neph1 has recently been obtained (16, 25C27). Therefore, nephrin, along with the other proteins, probably contributes to the slit diaphragm structure. In this study, we have used electron tomography to reconstruct the 3D structure of the slit diaphragm, elucidate nephrin location therein, and examine slit diaphragm changes following nephrin absence. Electron tomography has provided new possibilities for visualization of cellular macromolecules and structures (28C30). It is currently the only 3D reconstruction method that can reveal individual cellular and molecular entities without the need for population-wide averaging. It makes imaging (31C33) and identification (34, 35) of individual macromolecular structures possible in their native context, usually at a resolution of up to 5C10 nm. Here, we Vancomycin present electron tomographic evidence that the slit diaphragm is a uniformly Vancomycin wide organized network of winding strands. The complex network contains, among shorter strands, a class of 35-nm-long cross strands, which border lateral pores smaller than albumin molecules and can be decorated with nephrin immunogold labeling. In contrast, NPHS1 patients and nephrin-knockout mice have narrow slits lacking the slit diaphragm and 35-nm strands. In addition, individual immunolabeled molecules on nephrin-transfected cells and recombinant nephrin in vitrified solution appear in electron tomography as convoluted strands, similar to those in native slit diaphragm. Based on these findings, a structural model is proposed for the slit diaphragm with a role for nephrin in constituting its porous scaffold. Results Electron tomography reveals a porous network of strands in the slit diaphragm. In Vancomycin thin-section EM of normal kidney glomeruli, podocyte foot processes were separated by an about 30- to 40-nm-wide filtration slit (Figure ?(Figure1,1, ACD). In cross section (Figure ?(Figure1,1, A.