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JASN Debates |
Department of Medicine (Nephrology), University of Alberta, Edmonton, Alberta, Canada
Correspondence: Dr. Barbara J. Ballermann, Department of Medicine, University of Alberta, 11-132 CSB, Edmonton, Alberta, Canada. Phone: 780-407-8944; Fax: 780-407-7771; E-mail: barbara.ballermann@ualberta.ca or Dr. Radu V. Stan, Dartmouth Medical School, Department of Pathology, HB 7600, Borwell 502W, 1 Medical Center Drive, Hanover, NH 92093-0651. Phone: 603-650-8781; Fax: 603-650-6120; E-mail: radu.v.stan{at}dartmouth.edu
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The fact that massive proteinuria occurs when specific components of the slit diaphragm between podocytes are absent or defective7,8 and more generally in the presence of any type of podocyte injury9 suggests that the slit diaphragm is critical in maintaining the integrity of the glomerular protein barrier.7 Furthermore, because glomerular endothelial cells are packed with fenestrae apparently lacking a bridging diaphragm,10,11 the view has emerged that endothelia allow free passage of plasma protein, contributing little, if at all, to the protein barrier function of the glomerular capillary wall.
The most significant problem with this line of reasoning is that the glomerular filtration membrane would quickly cease to function if plasma proteins passed freely through glomerular endothelial fenestrae. A model in which slit diaphragms between podocytes allow rapid filtration of water but not plasma proteins coupled with a glomerular endothelial cell layer that provides no barrier would necessarily result in massive convective movement of albumin and other plasma proteins into the glomerular basement membrane (GBM), where they would accumulate at a rate of approximately 5 g/min. Clearly, under such conditions, the glomerular filter would quickly clog.12
In the absence of an effective barrier at the apical surface of the glomerular endothelium, only a high-capacity process of retrograde protein transport back across the endothelia or rapid removal of concentrated plasma proteins from the GBM, possibly into lymphatics or through the mesangium, could prevent plasma protein accumulation in the GBM. Renal pathologists and nephrologists alike know well that linear staining of the GBM with antibodies directed against albumin or Ig does not normally occur. Indeed, using immunolabeling of albumin in conjunction with transmission electron microscopy, Ryan and Karnovsky13 showed early on that albumin does not pass freely through endothelial fenestrae but is retained on the luminal aspect of the glomerular endothelium under physiologic conditions. Furthermore, no evidence exists for retrograde protein transport by glomerular endothelial cells back into the capillary lumen or for its removal by other mechanisms on the scale that would be necessary to maintain the GBM free of plasma proteins. Therefore, it should be readily apparent that free, convective movement of large plasma proteins through endothelial cell fenestrae is a physiologic impossibility in healthy kidneys, and mechanisms preventing protein movement through glomerular endothelium must exist.
Experimental evidence also reveals that the glomerular barrier to protein filtration includes the capillary endothelium. In the late 1980s, Avasthi and Koshy14–16 showed that infused cationic but not anionic or neutral ferritin binds to a thick protein coat that covers the entire glomerular endothelium, including the pores and fenestrae. Rostgaard and Qvortrup17,18 who kept the oxygen tension at physiologic levels during perfusion/fixation and used tannic acid/uranyl nitrate staining, also found evidence for an extensive protein coat on the surface and in the fenestrae of glomerular capillary endothelium. Not only was a glycocalyx visible, but also the glomerular endothelial fenestrae were shown for the first time to be spanned by a proteinaceous diaphragm.18 Hjalmarsson et al.19 extended these observations to show that a glycocalyx not only covers the glomerular endothelium and that glomerular endothelial fenestrae are spanned by a diaphragm but also that an additional, more loosely organized endothelial surface layer (ESL) of glycoproteins extends approximately 200 nm above the apical surface of the glomerular endothelium into the capillary lumen. Functional evidence for the importance of the endothelial cell proteoglycan coat in hindering the passage of albumin also exists. By quantifying a lipid droplet zone of exclusion as a measure of the ESL thickness, Jeansson and Haraldsson20 demonstrated convincingly that an infusion of enzymes that digest glycosaminoglycans reduced the height of the ESL and substantially increased in the flux of albumin across the glomerular capillary barrier. Because the large size of the enzymes confined them largely to the vascular compartment, this evidence is in keeping with a protein barrier function of the ESL. Of note, all of the studies showing the presence of a glomerular endothelial cell glycocalyx or ESL and fenestral diaphragms, as well as the study by Ryan and Karnovsky13 showing that albumin is largely excluded from endothelial fenestrae, used perfusion with oxygenated solutions during labeling and/or fixation. When kidneys were fixed without perfusion, albumin penetrated into the GBM,13 and when kidneys were perfusion/fixed using conventional techniques, the glycoprotein coat and glomerular fenestral diaphragms were not observed.18 Therefore, these structures seem very labile and easy to miss with conventional approaches. Nonetheless, the view that glomerular endothelial fenestrae are wide open is clearly fallacious, because it is based solely on the limitations of tissue preparation for electron microscopy.
It has also been argued that the lack of plasmalemmal vesicle–associated protein–1 (PV–1) in glomerular endothelium21 indicates that these cells must lack fenestral diaphragms. PV1 was discovered as an integral component of diaphragms that span the neck of caveolae and fenestrae in fenestrated endothelial cells of most organs, but it was not found in glomerular or hepatic sinusoidal endothelia,21–23 neither of which was believed to have fenestral diaphragms.10,11 Endothelial fenestrae were thought to form by fusion of caveolar precursors24 and expression of PV–1 implied, a common mechanism for the formation of fenestral and caveolar diaphragms. However, we found that glomerular endothelial cell fenestrae are normal in caveolin-1–deficient mice despite the complete absence of caveolae, leading us to conclude that fenestrae in glomerular endothelial cells do not form from caveolar precursors.25 It therefore seems that diaphragms in glomerular endothelial fenestrae differ in their molecular composition from fenestral diaphragms in most non–glomerular endothelial cells, but this does not prove in any way that glomerular endothelial cells lack fenestral diaphragms.
There also is abundant clinical evidence that injury to glomerular endothelium leads to proteinuria. This is certainly the case in patients with the hemolytic-uremic syndrome with thrombotic thrombocytopenic purpura26 and in patients with preeclampsia.27 In all of these disorders, endothelial cell swelling and loss of fenestrae are the primary renal problems, not overt podocyte injury, and in each case, proteinuria is observed. In preeclampsia, the case for endothelial cell injury leading to proteinuria is underscored by evidence that endogenous inhibitors of the endothelial cell-restricted receptors soluble fms-like tyrosine kinase 1 and soluble Endoglin circulate28,29 and that infusion of such antagonists can mimic both the endotheliosis and proteinuria of preeclampsia in experimental animals.30,31 Clinical nephrologists are also aware that proteinuria, sometimes in the nephrotic range, is a potential adverse effect of infusing anti–vascular endothelial growth factor (anti-VEGF) antibodies into patients who undergo antiangiogenesis treatment for cancer.32 Because VEGF is critical for normal glomerular endothelial cell differentiation,33 a proteinuric response to VEGF inhibition certainly is in keeping with a role of the glomerular endothelium in forming the glomerular protein barrier.
Finally, mutations in the laminin b3 gene that cause the human disorder Herlitz junctional epidermolysis bullosa are associated with profound congenital nephrotic syndrome and lack of glomerular endothelial cell fenestrae without obvious podocyte injury.34 Hence, substantial clinical evidence supports a view that injury or dysfunction in glomerular endothelial cells results in loss of the normal protein barrier function of the glomerular capillary wall.
Should we then conclude that the glomerular endothelium is the key component of the protein barrier in the glomerular capillary wall? Far from it! Neither one nor the other component of the barrier is "the most important" or "the key." Much more attractive is the view proposed by Smithies12 that the glomerular capillary wall is best understood as a gel through which macromolecules move principally by diffusion and not by convection, because spatial constraints and charge in the gel greatly hinder entry of many proteins. It is clear from the elegant mathematical modeling of the glomerular barrier function by Deen and Lazzara3,4,6 that a simplistic view of the glomerular capillary wall is no longer appropriate but that layers of the wall must be viewed as an integrated ultrafilter6 in which one element influences the other, in which mean pore size and density as well as charge define the behavior of macromolecules, and in which the endothelium and its glycocalyx are an integral part of the whole. Loss of the barrier function at the podocyte level certainly influences the behavior of macromolecules upstream, just as a loss of the endothelial component of the barrier would.
Great strides have been made in understanding the molecular nature of podocyte function since the discovery of the unique slit diaphragm protein nephrin.8 Far less is known about the proteins that make up the glycocalyx of glomerular endothelial cells and the proteins that reside within their fenestrae. Whether any of these will be unique or highly restricted to this cell type, as nephrin and podocin are in podocytes, remains unanswered. Furthermore, it is known that podocytes profoundly influence the differentiation of glomerular endothelial cells,35 and glomerular endothelial cells likely influence podocyte function as well. The question of to what degree cross-talk between the cellular compartments of the glomerular capillary wall modifies the barrier to protein filtration35 also remains largely unexplored. Nonetheless, it is clear that glomerular endothelium is not just a passive high-throughput sieve that allows passage of everything except cells. As we move toward the future, its role in forming the intricate structure of the glomerular capillary ultrafilter will hopefully gain more clarity.
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Angiogenesis Research Center, Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire
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.
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