Unraveling the Mechanisms of Glomerular Ultrafiltration: Nephrin, a Key Component of the Slit Diaphragm

Abstract

Glomerular Filtration Barrier Ultrafiltration of plasma during formation of the primary urine in the glomeruli is a major function of the kidney. The glomerulus is a tuft of anastomosing capillaries located within Bowman's capsule. The glomerular capillary wall has a complex morphology that facilitates a unique filtration function. This filtration barrier consists of three layers: a fenestrated endothelium, the glomerular basement membrane (GBM), and the outermost epithelial podocyte foot processes with their interconnecting slit diaphragms. Despite rapidly accumulating knowledge on the molecular nature of the GBM, there has been little information on the nature of the slit diaphragm, the last barrier to glomerular permeability. It is well established that the glomerular filtration barrier behaves as a size-selective filter that restricts the passage of plasma macromolecules based on their size, shape, and charge (1,2,3). Negatively charged molecules are filtered in smaller amounts than neutral molecules of comparable size, and the traversal of positively charged molecules is actually facilitated. The exact locations of the filtration functions within the barrier have been a matter of debate. Farquhar and coworkers demonstrated the presence of anionic sites in the GBM (4,5) and proposed that they contain heparan sulfate (6). The GBM is a molecular scaffold composed of tightly cross-linked type IV collagen, laminin, nidogen, and proteoglycans (7,8). Type IV collagen and laminin form two apparently independent structural networks that are linked through nidogen. In addition to providing strength to the GBM, type IV collagen and laminin probably also have adhesion roles for the endothelial cells and podocytes. The basement membrane-specific heparan sulfate proteoglycans perlecan (9) and agrin (10) are components of the GBM. After perfusion of kidneys with heparinase, which removes all glycosaminoglycans except keratan sulfate, Kanwar et al. (11) showed increased permeability of the GBM to ferritin (molecular weight, 470 kD), and Caulfield and Farquhar (12) have also shown loss of anionic sites from the GBM in aminonucleoside nephrosis. These and similar data from other laboratories support the role of negative glycosaminoglycans as an anionic filtration barrier. Although it is generally acknowledged that the GBM can restrict the traversal of large plasma proteins, there is also evidence that the ultimate barrier for proteins of the size of albumin resides in the slit diaphragm (13,14). However, there is very limited knowledge about the structure and molecular properties of this membrane, which has a remarkably constant width of about 40 nm. Several investigators have attempted to gain insight into the ultrastructure of the slit diaphragm by electron microscopy (15). By analyses of sections obtained in the plane of the diaphragm, Rodewald and Karnovsky in 1974 suggested that it has an isoporous, zipper-like structure (Figure 1) (16). According to their hypothesis, the pores (4 × 14 nm) had a size slightly smaller than that of albumin. Because these studies used thin-sectioning requiring harsh chemical treatments for sample preparation, the zipper structure has been questioned (15). Although most investigators seem to agree that the slit diaphragm is a rigid structure with a fairly constant width, a few electron microscopic studies have indicated that the width may vary between 20 and 50 nm (17,18). It has recently also been suggested that the slit area might increase with increasing intraglomerular pressure (19,20), indicating that the slit diaphragm is partially elastic.Figure 1: . Zipper-like morphologic structure of the glomerular podocyte slit diaphragm as revealed by transmission electron microscopic studies of Rodewald and Karnovsky in 1974 (16). (A) Tannic acid-stained and glutaraldehyde-fixed sections obtained in the plane of the slit diaphragm of a rat glomerulus exhibit a central filament with cross bridges connecting it to the plasma membrane of the foot process. Density of the cytoplasm opposite the points of attachment of the slit diaphragm can be observed. (B) Schematic model of the slit diaphragm predicted based on the results in A (16). The average cross-section dimensions of the pores between the cross bridges are indicated within the rectangle. Reproduced from reference (16).The molecular nature of the slit diaphragm has hitherto been a mystery. Characterization of glomerular proteins conducted for more than two decades has not yielded any slit membrane-specific components. Monoclonal antibodies are often a useful tool for identifying novel proteins, and this approach has also been used to identify proteins in the slit diaphragm. Orikasa et al. (21) generated monoclonal antibodies against glomeruli, one of which (5-1-6) recognized a 51-kD protein based on Western immunoblotting analysis. By using immunoelectron microscopy, this protein was localized exclusively to the slit diaphragm (22). Howe