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Resolved: Capillary Endothelium Is a Major Contributor to the Glomerular Filtration Barrier

Resolved: Capillary Endothelium Is a Major Contributor to the Glomerular Filtration Barrier

JASN DEBATES www.jasn.org

Resolved: Is a Major Contributor to the Glomerular Filtration Barrier

ABSTRACT There is growing debate as to the primal role of capillary endothelial cells in the barrier function of the glomerular filter. Two experts argue the points for and against. Clearly unresolved is agreement whether glomerular capillary endothelium has fenestrae subtended by diaphragms.

J Am Soc Nephrol 18: 2432–2438, 2007. doi: 10.1681/ASN.2007060687

Pro ment membrane (GBM), where they would accumulate at a rate of approximately 5 g/min. Clearly, under such conditions, the Barbara J. Ballermann glomerular filter would quickly clog.12 Department of Medicine (Nephrology), University of Alberta, In the absence of an effective barrier at the apical surface of Edmonton, Alberta, Canada the glomerular endothelium, only a high-capacity process of retrograde protein transport back across the endothelia or In normal humans, it is estimated that between 0.4 and 9.0 g of rapid removal of concentrated plasma proteins from the GBM, crosses the glomerular capillary wall each day.1,2 This possibly into lymphatics or through the mesangium, could represents a Bowman’s space:plasma albumin ratio of approx- prevent plasma protein accumulation in the GBM. Renal pa- imately 1:500 to 1:1000. Similarly, direct measurement of al- thologists and nephrologists alike know well that linear stain- bumin in rat fluid collected by micropuncture ing of the GBM with antibodies directed against albumin or Ig shows that Ͻ0.1% of the potential filtered load of albumin does not normally occur. Indeed, using immunolabeling of crosses the glomerular capillary wall.3 That the hydraulic con- albumin in conjunction with transmission electron micros- ductivity of the glomerular capillary wall for and small copy, Ryan and Karnovsky13 showed early on that albumin solutes is much greater than its permeability for plasma pro- does not pass freely through endothelial fenestrae but is re- teins is not in doubt,4 and the concept that size, shape, and tained on the luminal aspect of the glomerular endothelium charge determine to what degree plasma proteins pass the glo- under physiologic conditions. Furthermore, no evidence exists merular barrier is widely accepted.3–6 for retrograde protein transport by glomerular endothelial The fact that massive proteinuria occurs when specific cells back into the capillary or for its removal by other components of the slit diaphragm between are mechanisms on the scale that would be necessary to maintain absent or defective7,8 and more generally in the presence of the GBM free of plasma proteins. Therefore, it should be any type of injury9 suggests that the slit diaphragm readily apparent that free, convective movement of large is critical in maintaining the integrity of the glomerular pro- plasma proteins through endothelial cell fenestrae is a physio- tein barrier.7 Furthermore, because glomerular endothelial logic impossibility in healthy kidneys, and mechanisms pre- cells are packed with fenestrae apparently lacking a bridging venting protein movement through glomerular endothelium diaphragm,10,11 the view has emerged that endothelia allow must exist. free passage of plasma protein, contributing little, if at all, to Experimental evidence also reveals that the glomerular bar- 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 func- Published online ahead of print. Publication date available at www.jasn.org. tion if plasma proteins passed freely through glomerular endothe- Correspondence: Dr. Barbara J. Ballermann, Department of Medicine, Univer- lial fenestrae. A model in which slit diaphragms between podo- sity of Alberta, 11-132 CSB, Edmonton, Alberta, Canada. Phone: 780-407-8944; Fax: 780-407-7771; E-mail: [email protected] or Dr. Radu V. Stan, cytes allow rapid filtration of water but not plasma proteins Dartmouth Medical School, Department of Pathology, HB 7600, Borwell 502W, 1 coupled with a glomerular endothelial cell layer that provides no Medical Center Drive, Hanover, NH 92093-0651. Phone: 603-650-8781; Fax: 603- barrier would necessarily result in massive convective movement 650-6120; E-mail: [email protected] of albumin and other plasma proteins into the glomerular base- Copyright © 2007 by the American Society of Nephrology

2432 ISSN : 1046-6673/1809-2432 J Am Soc Nephrol 18: 2432–2438, 2007 www.jasn.org JASN DEBATES rier to protein filtration includes the capillary endothelium. In endothelial cells do not form from caveolar precursors.25 It the late 1980s, Avasthi and Koshy14–16 showed that infused therefore seems that diaphragms in glomerular endothelial cationic but not anionic or neutral ferritin binds to a thick fenestrae differ in their molecular composition from fenestral protein coat that covers the entire glomerular endothelium, diaphragms in most non–glomerular endothelial cells, but this including the pores and fenestrae. Rostgaard and Qvortrup17,18 does not prove in any way that glomerular endothelial cells lack who kept the tension at physiologic levels during per- fenestral diaphragms. fusion/fixation and used tannic acid/uranyl nitrate staining, There also is abundant clinical evidence that injury to glo- also found evidence for an extensive protein coat on the surface merular endothelium leads to proteinuria. This is certainly the and in the fenestrae of glomerular capillary endothelium. Not case in patients with the hemolytic-uremic syndrome with only was a visible, but also the glomerular endothe- thrombotic thrombocytopenic purpura26 and in patients with lial fenestrae were shown for the first time to be spanned by a preeclampsia.27 In all of these disorders, endothelial cell swell- proteinaceous diaphragm.18 Hjalmarsson et al.19 extended ing and loss of fenestrae are the primary renal problems, not these observations to show that a glycocalyx not only covers the overt podocyte injury, and in each case, proteinuria is ob- glomerular endothelium and that glomerular endothelial served. In preeclampsia, the case for endothelial cell injury fenestrae are spanned by a diaphragm but also that an addi- leading to proteinuria is underscored by evidence that endog- tional, more loosely organized endothelial surface layer (ESL) enous inhibitors of the endothelial cell-restricted receptors sol- of extends approximately 200 nm above the api- uble fms-like 1 and soluble circu- cal surface of the glomerular endothelium into the capillary lu- late28,29 and that infusion of such antagonists can mimic both men. Functional evidence for the importance of the endothelial the endotheliosis and proteinuria of preeclampsia in experi- cell proteoglycan coat in hindering the passage of albumin also mental animals.30,31 Clinical nephrologists are also aware that exists. By quantifying a droplet zone of exclusion as a mea- proteinuria, sometimes in the nephrotic range, is a potential sure of the ESL thickness, Jeansson and Haraldsson20 demon- adverse effect of infusing anti–vascular endothelial growth fac- strated convincingly that an infusion of enzymes that digest gly- tor (anti-VEGF) antibodies into patients who undergo antian- cosaminoglycans reduced the height of the ESL and substantially giogenesis treatment for cancer.32 Because VEGF is critical for increased in the flux of albumin across the glomerular capillary normal glomerular endothelial cell differentiation,33 a protein- barrier. Because the large size of the enzymes confined them uric response to VEGF inhibition certainly is in keeping with a largely to the vascular compartment, this evidence is in keeping role of the glomerular endothelium in forming the glomerular with a protein barrier function of the ESL. Of note, all of the protein barrier. studies showing the presence of a glomerular endothelial cell gly- Finally, mutations in the laminin b3 gene that cause the cocalyx or ESL and fenestral diaphragms, as well as the study by human disorder Herlitz junctional epidermolysis bullosa are Ryan and Karnovsky13 showing that albumin is largely excluded associated with profound congenital nephrotic syndrome and from endothelial fenestrae, used with oxygenated solu- lack of glomerular endothelial cell fenestrae without obvious tions during labeling and/or fixation. When kidneys were fixed podocyte injury.34 Hence, substantial clinical evidence sup- without perfusion, albumin penetrated into the GBM,13 and ports a view that injury or dysfunction in glomerular endothe- when kidneys were perfusion/fixed using conventional tech- lial cells results in loss of the normal protein barrier function of niques, the coat and glomerular fenestral dia- the glomerular capillary wall. phragms were not observed.18 Therefore, these structures seem Should we then conclude that the glomerular endothelium is very labile and easy to miss with conventional approaches. None- the key component of the protein barrier in the glomerular capil- theless, the view that glomerular endothelial fenestrae are wide lary wall? Far from it! Neither one nor the other component of the open is clearly fallacious, because it is based solely on the limita- barrier is “the most important” or “the key.” Much more attrac- tions of preparation for electron microscopy. tive is the view proposed by Smithies12 that the glomerular capil- It has also been argued that the lack of plasmalemmal vesi- lary wall is best understood as a gel through which macromole- cle–associated protein–1 (PV–1) in glomerular endothelium21 cules move principally by and not by convection, indicates that these cells must lack fenestral diaphragms. PV1 because spatial constraints and charge in the gel greatly hinder was discovered as an integral component of diaphragms that entry of many proteins. It is clear from the elegant mathematical span the neck of caveolae and fenestrae in fenestrated endothe- modeling of the glomerular barrier function by Deen and Laz- lial cells of most organs, but it was not found in glomerular or zara3,4,6 that a simplistic view of the glomerular capillary wall is no hepatic sinusoidal endothelia,21–23 neither of which was be- longer appropriate but that layers of the wall must be viewed as an lieved to have fenestral diaphragms.10,11 Endothelial fenestrae integrated ultrafilter6 in which one element influences the other, were thought to form by fusion of caveolar precursors24 and in which mean pore size and density as well as charge define the expression of PV–1 implied, a common mechanism for the behavior of macromolecules, and in which the endothelium and formation of fenestral and caveolar diaphragms. However, we its glycocalyx are an integral part of the whole. Loss of the barrier found that glomerular endothelial cell fenestrae are normal in function at the podocyte level certainly influences the behavior of -1–deficient mice despite the complete absence of macromolecules upstream, just as a loss of the endothelial com- caveolae, leading us to conclude that fenestrae in glomerular ponent of the barrier would.

J Am Soc Nephrol 18: 2432–2438, 2007 Capillary Endothelia Contribute to Glomerular Filtration Barrier 2433 JASN DEBATES www.jasn.org

Great strides have been made in understanding the molec- tion. Int 9: 36–45, 1976 ular nature of podocyte function since the discovery of the 14. Avasthi PS, Koshy V: The anionic matrix at the rat glomerular endo- thelial surface. Anat Rec 220: 258–266, 1988 8 unique slit diaphragm protein nephrin. Far less is known 15. Avasthi PS, Koshy V: Glomerular endothelial glycocalyx. Contrib about the proteins that make up the glycocalyx of glomerular Nephrol 68: 104–113, 1988 endothelial cells and the proteins that reside within their 16. Koshy V, Avasthi PS: The anionic sites at luminal surface of peritubular fenestrae. Whether any of these will be unique or highly re- in rats. Kidney Int 31: 52–58, 1987 stricted to this cell type, as nephrin and podocin are in podo- 17. Rostgaard J, Qvortrup K: Sieve plugs in fenestrae of glomerular cap- illaries: Site of the filtration barrier? Cells Tissues Organs 170: 132– cytes, remains unanswered. Furthermore, it is known that 138, 2002 podocytes profoundly influence the differentiation of glomer- 18. Rostgaard J, Qvortrup K: Electron microscopic demonstrations of ular endothelial cells,35 and glomerular endothelial cells likely filamentous molecular sieve plugs in capillary fenestrae. Microvasc Res influence podocyte function as well. The question of to what 53: 1–13, 1997 degree cross-talk between the cellular compartments of the 19. Hjalmarsson C, Johansson BR, Haraldsson B: Electron microscopic glomerular capillary wall modifies the barrier to protein filtra- evaluation of the endothelial surface layer of glomerular capillaries. Microvasc Res 67: 9–17, 2004 35 tion also remains largely unexplored. Nonetheless, it is clear 20. Jeansson M, Haraldsson B: Morphological and functional evidence for that glomerular endothelium is not just a passive high- an important role of the endothelial cell glycocalyx in the glomerular throughput sieve that allows passage of everything except cells. barrier. Am J Physiol Renal Physiol 290: F111–F116, 2006 As we move toward the future, its role in forming the intricate 21. Stan RV, Kubitza M, Palade GE: PV-1 is a component of the fenestral structure of the glomerular capillary ultrafilter will hopefully and stomatal diaphragms in fenestrated endothelia. Proc Natl Acad Sci U S A 96: 13203–13207, 1999 gain more clarity. 22. Stan RV, Tkachenko E, Niesman IR: PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms. Mol Biol Cell 15: 3615–3630, 2004 23. Stan RV, Roberts WG, Predescu D, Ihida K, Saucan L, Ghitescu L, DISCLOSURES Palade GE: Immunoisolation and partial characterization of endothe- None. lial plasmalemmal vesicles (caveolae). Mol Biol Cell 8: 595–605, 1997 24. Chen J, Braet F, Brodsky S, Weinstein T, Romanov V, Noiri E, Goli- gorsky MS: VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells. Am J Physiol Cell Physiol 282: REFERENCES C1053–C1063, 2002 25. So¨rensson J, Fierlbeck W, Heider T, Schwarz K, Park DS, Mundel P, 1. Birn H, Christensen EI: Renal albumin absorption in physiology and Lisanti M, Ballermann BJ: Glomerular endothelial fenestrae in vivo are pathology. Kidney Int 69: 440–449, 2006 not formed from caveolae. J Am Soc Nephrol 13: 2639–2647, 2002 2. Mogensen CE, Solling J:. Studies on renal tubular protein reabsorp- 26. Besbas N, Karpman D, Landau D, Loirat C, Proesmans W, Remuzzi G, tion: Partial and near complete inhibition by certain amino acids. Rizzoni G, Taylor CM, Van de Kar N, Zimmerhackl LB; European Scand J Clin Lab Invest 37: 477–486, 1977 Paediatric Research Group for HUS: A classification of hemolytic ure- 3. Deen WM, Lazzara MJ. Glomerular filtration of albumin: How small is mic syndrome and thrombotic thrombocytopenic purpura and related the sieving coefficient? Kidney Int Suppl S63–S64, 2004 disorders. Kidney Int 70: 423–431, 2006 4. Deen WM, Lazzara MJ, Myers BD: Structural determinants of glomer- 27. Karumanchi SA, Maynard SE, Stillman IE, Epstein FH, Sukhatme VP: ular permeability. Am J Physiol Renal Physiol 281: F579–F596, 2001 Preeclampsia: A renal perspective. Kidney Int 67: 2101–2113, 2005 5. Haraldsson B, Sorensson J: Why do we not all have proteinuria? An 28. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann update of our current understanding of the glomerular barrier. News TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Physiol Sci 19: 7–10, 2004 Karumanchi SA: Excess placental soluble fms-like tyrosine kinase 1 6. Deen WM: Cellular contributions to glomerular size-selectivity. Kidney (sFlt1) may contribute to , , and Int 69: 1295–1297, 2006 proteinuria in preeclampsia. J Clin Invest 111: 649–658, 2003 7. Tryggvason K, Wartiovaara J: How does the kidney filter plasma? 29. Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, Physiology (Bethesda) 20: 96–101, 2005 Bdolah Y, Lim KH, Yuan HT, Libermann TA, Stillman IE, Roberts D, 8. Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala D’Amore PA, Epstein FH, Sellke FW, Romero R, Sukhatme VP, Letarte H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Pel- M, Karumanchi SA: Soluble endoglin contributes to the pathogenesis tonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned of preeclampsia. Nat Med 12: 642–649, 2006 gene for a novel glomerular protein—nephrin—is mutated in congen- 30. Karumanchi SA, Stillman IE: In vivo rat model of preeclampsia. Meth- ital nephrotic syndrome. Mol Cell 1: 575–582, 1998 ods Mol Med 122: 393–399, 2006 9. Shankland SJ: The podocyte’s response to injury: Role in proteinuria 31. Sugimoto H, Hamano Y, Charytan D, Cosgrove D, Kieran M, Sudhakar and glomerulosclerosis. Kidney Int 69: 2131–2147, 2006 A, Kalluri R: Neutralization of circulating vascular endothelial growth 10. Braet F, Wisse E: Structural and functional aspects of sinusoidal factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 endothelial cell fenestrae: A review. Comp Hepatol 1: 1, 2002 (sFlt-1) induces proteinuria. J Biol Chem 278: 12605–12608, 2003 11. Reeves WH, Kanwar YS, Farquhar MG: Assembly of the glomerular 32. Ranieri G, Patruno R, Ruggieri E, Montemurro S, Valerio P, Ribatti D: filtration surface. Differentiation of anionic sites in glomerular capillar- Vascular endothelial (VEGF) as a target of bevacizumab ies of newborn rat kidney. J Cell Biol 85: 735–753, 1980 in cancer: From the biology to the clinic. Curr Med Chem 13: 1845– 12. Smithies O: Why the kidney does not clog: A gel perme- 1857, 2006 ation/diffusion hypothesis of renal function. Proc Natl Acad Sci U S A 33. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, 100: 4108–4113, 2003 Kikkawa Y, Miner JH, Quaggin SE: Glomerular-specific alterations of 13. Ryan GB, Karnovsky MJ: Distribution of endogenous albumin in the rat VEGF-A expression lead to distinct congenital and acquired renal glomerulus: Role of hemodynamic factors in glomerular barrier func- diseases. J Clin Invest 111: 707–716, 2003

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34. Hata D, Miyazaki M, Seto S, Kadota E, Muso E, Takasu K, Nakano A, ing the principal molecular sieve of the capillary wall is known Tamai K, Uitto J, Nagata M, Moriyama K, Miyazaki K: Nephrotic as the glycocalyx junction-break model.3 syndrome and aberrant expression of laminin isoforms in glomerular basement membranes for an infant with Herlitz junctional epidermol- In the glomerulus, ESL components such as heparan sul- ysis bullosa. Pediatrics 116: e601–e607, 2005 fate,17 sialic acid,18,19 chondroitin sulfate,19 and hyaluronan19 35. Eremina V, Baelde HJ, Quaggin SE: Role of the VEGF: A signaling play a role in permselectivity to macromolecules in perfused pathway in the glomerulus—Evidence for crosstalk between compo- kidney. There are inherent difficulties in interpreting experi- nents of the glomerular filtration barrier. Physiol 106: p32– ments that are conducted by perfusion because of loss of the p37, 2007 native circulation in situ. A recent study20 done by intravenous injection of heparitinase, for example, contradicts earlier work Con and found no role for in maintaining glomer- ular barrier functions. An achievement of this work was that Radu V. Stan the parameters of the circulation were disturbed minimally. Research Center, Department of Pathology, Studies with GAG-degrading enzymes also present some cave- Dartmouth Medical School, Hanover, New Hampshire ats, such as possible low-level contaminants in the enzyme preparations and the difficulty in differentiating effects on the Kidneys play a critical role in maintaining the of endothelia from those on GBM. . This is achieved by filtering solutes and water using size Electron microscopic studies of the glycocalyx in glomeru- and charge selective transfer across the glomerular capillary lus21–23 also raise uncertainty regarding the effects of fixation or wall into the urinary space.1,2 The exchange of water and sol- processing on the conformation of GAG chains, the low descrip- utes between and interstitial spaces also suggests tive resolution of probes, and the lack of specificity of the probes. a paracellular path across the endothelial monolayer.3 There Studies addressing the precise role of ESL in glomerular perme- are several morphologic barriers that filtrate must traverse to ability face a daunting task because of molecular and structural reach the urinary space. These barriers include the endothelial complexity and lack of molecular tools dissecting the contribu- cell surface layer (ESL) known as glycocalix and the fenestrae, a tion of individual . Therefore, the precise contribution thicker (300 to 350 nm) glomerular of the endothelial glycocalyx to permselectivity in the glomerulus (GBM), and podocytes with their foot processes bridged by slit as well as in other vascular beds is unclear. diaphragms.4–6The GBM7 and slit diaphragms8 are clearly im- Next one must wonder about endothelial fenestrae in any portant in the filtration of plasma. What about the endothelia? discussion of barrier function. Fenestrae are circular pores The endothelial lining of exchange capillaries and through endothelial cells organized in planar clusters called modulates permeability in all tissues9 and undoubtedly plays sieve plates. The circumference of the fenestral pore is delin- some role in defining the glomerular filtration barrier. The eated by the fenestral rim, which occurs where the luminal contribution of the glomerular endothelial monolayer to aspect of endothelial plasma membrane continues with the permselectivity is suggested by its role in generating GBM, the abluminal one under a sharp angle. There are three discernible presence of fenestrae, and the maintenance of the glycocalyx. I varieties of fenestrae in different types of endothelia. focus on the role of endothelial fenestrae and glycocalix in First, a fenestral diaphragm subtends some fenestrae. This glomerular permselectivity, mostly to stress methodologic is- type occurs in the capillaries of endocrine , digestive sues that preclude assigning a strong role to endothelia as the tract mucosa, and kidney peritubular capillaries. What sets major filtration barrier. these apart is the presence of a diaphragm, and individual The ESL is a complex and poorly understood structure fenestral pores have a remarkably constant diameter (approx- covering the luminal aspect of endothelial cells. ESL pro- imately 62 to 68 nm).24,25 Within the sieve plate, fenestrae oc- vides a negatively charged mesh consisting of polysaccha- cur in ordered, parallel linear arrays with an average distance ride moieties of glycoproteins, glycolipids, and proteogly- between neighboring fenestral centers of approximately 130 cans that are integral to the plasma membrane, as well as nm.24,25 This supramolecular arrangement suggests a role for membrane-associated glycosaminoglycans (GAG). This cytoskeleton in their organization.26,27 Fenestral pores also mesh with gel-like properties interfaces with plasma com- have an octagonal symmetry.28–30 Their diaphragms appear as ponents and water. ESL determines vascular rheology and a thin (5 to 7 nm) protein barrier anchored to the rim by a may be involved in permselectivity, mechanotransduction, central knob or density31 connected by thin radial fibrils,32 and vascular protection. Its putative structure, components, starting at the rim and interweaving into a central mesh.29 Per- and function have been reviewed.3,10–13 fusion fixation of vascular beds with these fenestrated endothe- A role for the ESL in vascular permeability is suggested lia reveal the presence of large (up to 400 nm) tufts, or so-called mostly by studies with degrading enzymes identifying the im- fascinae fenestrae, on the luminal side of these diaphragms.21 portance of sialic acid, heparan sulfate, and hyaluronan in The fibers forming these tufts have been interpreted, although permselectivity. In vascular beds other than glomeruli, this re- not proved, to be heparan sulfate proteoglycans (HSPG). The sults in charge exclusion,14 increased permeability to pro- chemical composition of the fenestral diaphragm have been teins,15 and more hydraulic conductivity.16 Glycocalyx form- investigated with nonspecific probes such as lectins33–36 and

J Am Soc Nephrol 18: 2432–2438, 2007 Capillary Endothelia Contribute to Glomerular Filtration Barrier 2435 JASN DEBATES www.jasn.org cationic molecules.37–41 Fenestral diaphragms on their luminal have a sort of diaphragm not seen unless one uses special fixa- side bind lectins poorly33,34 and have multiple anionic sites tion procedures. This suggests that these diaphragms have a conferred by HSPG.41,42 The presence of the lectin-binding different biochemical makeup than the ones subtended by di- sites41 suggests the presence of other glycoproteins. It is inter- aphragms demonstrated with standard fixation.22 Again, there esting that the HSPG are present only on the luminal side of is no morphometric analysis of how many fenestrae have these fenestral diaphragms,38 which also correlates with data ob- putative diaphragms. tained by electron microscopy.21 The only gene product local- Filamentous plugs reside in glomerular fenestrae, with ized to fenestral diaphragms to date is PV–1,43 a cationic en- fewer found in fenestrae subtended by diaphragms. Is this fix- dothelial glycoprotein encoded by the PLVAP gene.44 PV–1 is ation artifact? It is not entirely clear what contrast-enhancing necessary for fenestrae formation26,27 and integration into di- reagents reveal with microscopy—proteoglycans, glycopro- aphragms.9,45 teins, glycolipids. If so, then which ones, and what is the label- Second, there are other fenestrae in discontinuous endothe- ing efficiency? The thickness of the preparative coat might be lia among the sinusoids of liver and .46,47 These misleading because postfixation was done in hypotonic solu- fenestrae are much larger (100 to 200 nm) and more heteroge- tions that favor the unraveling of GAG chains. As a possible neous in size. They arrange in spiral patterns stemming from a alternative to present interpretation, the filaments seen in the so-called “fenestrae-forming center” with very small pores that fenestral pore might in fact cover the plasmalemma or reside in become progressively larger as they appear farther from the the GBM. The difference in the fenestral tuft thickness between center. These fenestrae do not have diaphragms and do not other fenestrated capillaries and glomerular capillaries is ex- stain with PV–1. tremely significant. High-resolution immunofluorescence Third and finally, fenestrae found in mature glomerular surveys with semithin frozen sections of rat glomeruli also capillaries lack diaphragms. Morphologically, however, they show occasional capillary loops of adult glomeruli staining closely resemble the fenestrae subtended by diaphragms in with PV–1.43 Serial reconstructions of glomeruli by the same terms of regular diameter and organization in linear arrays means reveal that endothelial cells adjacent to the efferent ar- within the sieve plates. This type of fenestrae has been demon- teriole also stain for PV–1 (unpublished observations). This is strated by many laboratories4,48–50 with a clear lack of sub- in line with the concept of endothelial heterogeneity within stance in the glomerular fenestral pores29; they also lack PV–143 and between given vascular segments.52 What the operational as well as fascinae fenestrae.21,22 Filamentous “sieve plugs” are significance of this is is unclear. also found in these fenestrae, but their functional significance Presumably, glomerular endothelial fenestrae have functional is not clear. It is interesting that during glomerular develop- relevance. In the spatial organization of fenestrated capillaries, the ment, diaphragms subtend the fenestrae in emerging glomer- sieve plates containing fenestrae occur always facing the podo- ular capillaries50 but are gradually lost within a week or two cytes, with the cell nuclei and perikaryon facing the center of the after birth. PV–1 is expressed in all of the endothelial cells at glomerulus. This tight spatial control of fenestral position is likely both the S-shaped body and capillary loop stages in the devel- modulated by subjacent epithelia through secretion of specific oping kidney, which correlates with the presence of a dia- matrix components that are characteristic of all fenestrated endo- phragm in these stages. However, only at the capillary loop thelia.9 Therefore, it is reasonable to assume that fenestrae in- stage do endothelial cells have fenestrae, whereas in S-shaped duced by signals from podocytes are a necessary part of the filtra- bodies, there are only caveolae with stomatal diaphragms (H. tion barrier. Loss of fenestrae and switch of endothelial cells to a Kurihara, Juntendo University School of Medicine, Tokyo, continuous phenotype (endotheliosis) result in proteinuria by Japan, personal communication, May 9, 2007). unknown mechanisms. This occurs in human preeclampsia,53 in Although glomerular fenestrae have many identical features rat models of preeclampsia produced by sFlt-1 protein,54 in genet- with fenestrae preserving their diaphragms, do they result ically engineered mice in which glomerular VEGF is reduced by from downregulation of the diaphragm components (such as 50%,55 and after injection of anti-VEGF antibodies.56 However, PV–1 protein), or are they a totally different type of pore bio- when VEGF signaling is repressed, the data are not easy to inter- chemically? This business of whether glomerular fenestrae pret because of poor endothelial cell survival, loss of attachment, have diaphragms has been long in controversy. As discussed, and morphologic modifications of the GBM. although many laboratories agree on the lack of diaphragms in Although some consider fenestrae structurally equivalent to mature glomerular fenestrae, micrographs of glomerular en- large pores, this may not be true. Fenestrated endothelia, more dothelial cells with fenestrae subtended by diaphragms have permeable only to water and small solutes than nonfenestrated been reported in the literature.21–23,32,51 In none of these re- endothelia, have comparable permeability to macromolecules ports, however, is there a careful morphometric analysis eval- such as serum albumin.57 Glomerular fenestrae are also perme- uating heterogeneity or possible impact on function. One ated by plasma proteins as large as native ferritin,5,6 in contrast group first reported that glomerular fenestrae have dia- with fenestrae having diaphragms.24,57,58 This argues for a phragms,21 whereas, in a subsequent article, they reported a lesser role of diaphragm-free glomerular fenestrae in control- lack22 (compare Figures 7a and 1 in the respective articles). In ling permselectivity of macromolecules and strengthens argu- the latter article,22 it was suggested that glomerular fenestrae ments for the barrier role of the ESL and GBM.

2436 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2432–2438, 2007 www.jasn.org JASN DEBATES

From the issues I raise here, a role for podocytes and GBM sion with neuraminidase. Lab Invest 42: 375–384, 1980 as a filtration barrier is far better characterized in molecular 19. Jeansson M, Haraldsson B: Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular and functional terms. Some role for endothelia in mechanisms barrier. Am J Physiol Renal Physiol 290: F111–F116, 2006 of glomerular filtration is reasonable, but as the most impor- 20. Wijnhoven TJ, Lensen JF, Wismans RG, Lamrani M, Monnens LA, tant component of this regulated barrier, such a position is not Wevers RA, Rops AL, van der Vlag J, Berden JH, van den Heuvel LP, established. van Kuppevelt TH: In vivo degradation of heparan sulfates in the glomerular basement membrane does not result in proteinuria JAm Soc Nephrol 18: 823–832, 2007 21. Rostgaard J, Qvortrup K: Electron microscopic demonstrations of DISCLOSURES filamentous molecular sieve plugs in capillary fenestrae. Microvasc Res None. 53: 1–13, 1997 22. Rostgaard J, Qvortrup K: Sieve plugs in fenestrae of glomerular cap- illaries: Site of the filtration barrier? Cells Tissues Organs 170: 132– 138, 2002 REFERENCES 23. Hjalmarsson C, Johansson BR, Haraldsson B: Electron microscopic evaluation of the endothelial surface layer of glomerular capillaries. Microvasc Res 67: 9–17, 2004 1. Smithies O: Why the kidney glomerulus does not clog: A gel perme- 24. Clementi F, Palade GE: Intestinal capillaries. I. Permeability to perox- ation/diffusion hypothesis of renal function. Proc Natl Acad Sci U S A idase and ferritin. J Cell Biol 41: 33–58, 1969 100: 4108–4113, 2003 2. Farquhar MG: The glomerular basement membrane: Not gone, just 25. Simionescu M, Simionescu N, Palade GE: Morphometric data on the forgotten. 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