Resolved

Kidneys play a critical role in maintaining the homeostasis of blood. This is achieved by filtering solutes and water using size and charge selective transfer across the glomerular capillary wall into the urinary space.1,2 The exchange of water and solutes between blood plasma and interstitial spaces also suggests a paracellular path across the endothelial monolayer.3 There are several morphologic barriers that filtrate must traverse to reach the urinary space. These barriers include the endothelial cell surface layer (ESL) known as glycocalix and the fenestrae, a thicker (300 to 350 nm) glomerular basement membrane (GBM), and podocytes with their foot processes bridged by slit diaphragms.4–6 The GBM7 and slit diaphragms8 are clearly important in the filtration of plasma. What about the endothelia? The endothelial lining of exchange capillaries and venules modulates permeability in all tissues9 and undoubtedly plays some role in defining the glomerular filtration barrier. The contribution of the glomerular endothelial monolayer to permselectivity is suggested by its role in generating GBM, the presence of fenestrae, and the maintenance of the glycocalyx. I focus on the role of endothelial fenestrae and glycocalix in glomerular permselectivity, mostly to stress methodologic issues that preclude assigning a strong role to endothelia as the major filtration barrier. The ESL is a complex and poorly understood structure covering the luminal aspect of endothelial cells. ESL provides a negatively charged mesh consisting of polysaccharide moieties of glycoproteins, glycolipids, and proteoglycans that are integral to the plasma membrane, as well as membrane-associated glycosaminoglycans (GAG). This mesh with gel-like properties interfaces with plasma components and water. ESL determines vascular rheology and may be involved in permselectivity, mechanotransduction, and vascular protection. Its putative structure, components, and function have been reviewed.3,10–13 A role for the ESL in vascular permeability is suggested mostly by studies with degrading enzymes identifying the importance of sialic acid, heparan sulfate, and hyaluronan in permselectivity. In vascular beds other than glomeruli, this results in charge exclusion,14 increased permeability to proteins,15 and more hydraulic conductivity.16 Glycocalyx forming the principal molecular sieve of the capillary wall is known as the glycocalyx junction-break model.3 In the glomerulus, ESL components such as heparan sulfate,17 sialic acid,18,19 chondroitin sulfate,19 and hyaluronan19 play a role in permselectivity to macromolecules in perfused kidney. There are inherent difficulties in interpreting experiments that are conducted by perfusion because of loss of the native circulation in situ. A recent study20 done by intravenous injection of heparitinase, for example, contradicts earlier work and found no role for heparan sulfate in maintaining glomerular barrier functions. An achievement of this work was that the parameters of the circulation were disturbed minimally. Studies with GAG-degrading enzymes also present some caveats, such as possible low-level contaminants in the enzyme preparations and the difficulty in differentiating effects on the endothelia from those on GBM. Electron microscopic studies of the glycocalyx in glomerulus21–23 also raise uncertainty regarding the effects of fixation or processing on the conformation of GAG chains, the low descriptive resolution of probes, and the lack of specificity of the probes. Studies addressing the precise role of ESL in glomerular permeability face a daunting task because of molecular and structural complexity and lack of molecular tools dissecting the contribution of individual molecules. Therefore, the precise contribution of the endothelial glycocalyx to permselectivity in the glomerulus as well as in other vascular beds is unclear. Next one must wonder about endothelial fenestrae in any discussion of barrier function. Fenestrae are circular pores through endothelial cells organized in planar clusters called sieve plates. The circumference of the fenestral pore is delineated by the fenestral rim, which occurs where the luminal aspect of endothelial plasma membrane continues with the abluminal one under a sharp angle. There are three discernible varieties of fenestrae in different types of endothelia. First, a fenestral diaphragm subtends some fenestrae. This type occurs in the capillaries of endocrine glands, digestive tract mucosa, and kidney peritubular capillaries. What sets these apart is the presence of a diaphragm, and individual fenestral pores have a remarkably constant diameter (approximately 62 to 68 nm).24,25 Within the sieve plate, fenestrae occur in ordered, parallel linear arrays with an average distance between neighboring fenestral centers of approximately 130 nm.24,25 This supramolecular arrangement suggests a role for cytoskeleton in their organization.26,27 Fenestral pores also have an octagonal symmetry.28–30 Their diaphragms appear as a thin (5 to 7 nm) protein barrier anchored to the rim by a central knob or density31 connected by thin radial fibrils,32 starting at the rim and interweaving into a central mesh.29 Perfusion fixation of vascular beds with these fenestrated endothelia reveal the presence of large (up to 400 nm) tufts, or so-called fascinae fenestrae, on the luminal side of these diaphragms.21 The fibers forming these tufts have been interpreted, although not proved, to be heparan sulfate proteoglycans (HSPG). The chemical composition of the fenestral diaphragm have been investigated with nonspecific probes such as lectins33–36 and cationic molecules.37–41 Fenestral diaphragms on their luminal side bind lectins poorly33,34 and have multiple anionic sites conferred by HSPG.41,42 The presence of the lectin-binding sites41 suggests the presence of other glycoproteins. It is interesting that the HSPG are present only on the luminal side of fenestral diaphragms,38 which also correlates with data obtained by electron microscopy.21 The only gene product localized to fenestral diaphragms to date is PV–1,43 a cationic endothelial glycoprotein encoded by the PLVAP gene.44 PV–1 is necessary for fenestrae formation26,27 and integration into diaphragms.9,45 Second, there are other fenestrae in discontinuous endothelia among the sinusoids of liver and bone marrow.46,47 These fenestrae are much larger (100 to 200 nm) and more heterogeneous in size. They arrange in spiral patterns stemming from a so-called "fenestrae-forming center" with very small pores that become progressively larger as they appear farther from the center. These fenestrae do not have diaphragms and do not stain with PV–1. Third and finally, fenestrae found in mature glomerular capillaries lack diaphragms. Morphologically, however, they closely resemble the fenestrae subtended by diaphragms in terms of regular diameter and organization in linear arrays within the sieve plates. This type of fenestrae has been demonstrated by many laboratories4,48–50 with a clear lack of substance in the glomerular fenestral pores29; they also lack PV–143 as well as fascinae fenestrae.21,22 Filamentous "sieve plugs" are also found in these fenestrae, but their functional significance is not clear. It is interesting that during glomerular development, diaphragms subtend the fenestrae in emerging glomerular capillaries50 but are gradually lost within a week or two after birth. PV–1 is expressed in all of the endothelial cells at both the S-shaped body and capillary loop stages in the developing kidney, which correlates with the presence of a diaphragm in these stages. However, only at the capillary loop stage do endothelial cells have fenestrae, whereas in S-shaped bodies, there are only caveolae with stomatal diaphragms (H. Kurihara, Juntendo University School of Medicine, Tokyo, Japan, personal communication, May 9, 2007). Although glomerular fenestrae have many identical features with fenestrae preserving their diaphragms, do they result from downregulation of the diaphragm components (such as PV–1 protein), or are they a totally different type of pore biochemically? This business of whether glomerular fenestrae have diaphragms has been long in controversy. As discussed, although many laboratories agree on the lack of diaphragms in mature glomerular fenestrae, micrographs of glomerular endothelial cells with fenestrae subtended by diaphragms have been reported in the literature.21–23,32,51 In none of these reports, however, is there a careful morphometric analysis evaluating heterogeneity or possible impact on function. One group first reported that glomerular fenestrae have diaphragms,21 whereas, in a subsequent article, they reported a lack22 (compare Figures 7a and 1 in the respective articles). In the latter article,22 it was suggested that glomerular fenestrae have a sort of diaphragm not seen unless one uses special fixation procedures. This suggests that these diaphragms have a different biochemical makeup than the ones subtended by diaphragms demonstrated with standard fixation.22 Again, there is no morphometric analysis of how many fenestrae have these putative diaphragms. Filamentous plugs reside in glomerular fenestrae, with fewer found in fenestrae subtended by diaphragms. Is this fixation artifact? It is not entirely clear what contrast-enhancing reagents reveal with microscopy—proteoglycans, glycoproteins, glycolipids. If so, then which ones, and what is the labeling efficiency? The thickness of the preparative coat might be misleading because postfixation was done in hypotonic solutions that favor the unraveling of GAG chains. As a possible alternative to present interpretation, the filaments seen in the fenestral pore might in fact cover the plasmalemma or reside in the GBM. The difference in the fenestral tuft thickness between other fenestrated capillaries and glomerular capillaries is extremely significant. High-resolution immunofluorescence surveys with semithin frozen sections of rat glomeruli also show occasional capillary loops of adult glomeruli staining with PV–1.43 Serial reconstructions of glomeruli by the same means reveal that endothelial cells adjacent to the efferent arteriole also stain for PV–1 (unpublished observations). This is in line with the concept of endothelial heterogeneity within and between given vascular segments.52 What the operational significance of this is is unclear. Presumably, glomerular endothelial fenestrae have functional relevance. In the spatial organization of fenestrated capillaries, the sieve plates containing fenestrae occur always facing the podocytes, with the cell nuclei and perikaryon facing the center of the glomerulus. This tight spatial control of fenestral position is likely modulated by subjacent epithelia through secretion of specific matrix components that are characteristic of all fenestrated endothelia.9 Therefore, it is reasonable to assume that fenestrae induced by signals from podocytes are a necessary part of the filtration barrier. Loss of fenestrae and switch of endothelial cells to a continuous phenotype (endotheliosis) result in proteinuria by unknown mechanisms. This occurs in human preeclampsia,53 in rat models of preeclampsia produced by sFlt-1 protein,54 in genetically engineered mice in which glomerular VEGF is reduced by 50%,55 and after injection of anti-VEGF antibodies.56 However, when VEGF signaling is repressed, the data are not easy to interpret because of poor endothelial cell survival, loss of attachment, and morphologic modifications of the GBM. Although some consider fenestrae structurally equivalent to large pores, this may not be true. Fenestrated endothelia, more permeable only to water and small solutes than nonfenestrated endothelia, have comparable permeability to macromolecules such as serum albumin.57 Glomerular fenestrae are also permeated by plasma proteins as large as native ferritin,5,6 in contrast with fenestrae having diaphragms.24,57,58 This argues for a lesser role of diaphragm-free glomerular fenestrae in controlling permselectivity of macromolecules and strengthens arguments for the barrier role of the ESL and GBM. From the issues I raise here, a role for podocytes and GBM as a filtration barrier is far better characterized in molecular and functional terms. Some role for endothelia in mechanisms of glomerular filtration is reasonable, but as the most important component of this regulated barrier, such a position is not established. Disclosures None.