JASN Express. Published on February 14, 2007 as doi: 10.1681/ASN.2007010086 Editorial Breaking Down the Barrier: Evidence against a Role for Heparan Sulfate in Glomerular Permselectivity Scott J. Harvey and Jeffrey H. Miner Renal Division, Washington University School of Medicine, St. Louis, Missouri J Am Soc Nephrol 18: 672–674, 2007. doi: 10.1681/ASN.2007010086 he glomerular capillary wall is thought to function as Charge barrier dysfunction has long been touted as an un- both a size- and charge-selective barrier. The concept of derlying cause of human glomerular disease (10–12). This may T charge selectivity emerged from a series of now classic be brought about by decreased expression or undersulfation of studies that used tracers such as dextran, peroxidase, and fer- GBM-HSPG (13,14). Segmental or global loss of GBM-HS has ritin to evaluate the influence of molecular charge on glomer- been reported in human membranous nephritis, lupus nephri- ular filtration (1–5). The permeability of anionic derivatives of tis, minimal change disease, and diabetic nephropathy (13,15), each tracer was lower than their neutral counterparts of com- as well as in rat models of adriamycin and Heymann nephritis parable size, whereas the permeability of cationic forms was (16,17). The intensity of GBM labeling inversely correlates with enhanced. This led to the theory that the passage of endoge- severity of disease, which supports the theory that reductions nous circulating polyanions, notably albumin, would likewise in GBM-HS contribute directly to a loss of barrier function. be impeded by the “fixed” or intrinsic negative charge of the However, in a recent study, GBM-HS was reported to be nor- glomerular capillary wall. These types of studies have since mal in diabetic humans and rats with microalbuminuria, and it been challenged on many fronts, particularly on the basis of was concluded that its loss may be a secondary event occurring deformation, degradation, or selective uptake of the tracers. at advanced stages of this disease (18). Whether charge selectivity actually exists and is important for In this issue of JASN, Wijnhoven and colleagues provide glomerular function is a subject of intense debate (6). Despite evidence that challenges the notion that HS is an important the controversy, the concept remains a cornerstone of renal constituent of the glomerular filtration barrier (19). Here they physiology. report on a short-term study of renal function in rats following Considerable work has gone into localizing and defining the intravenous administration of heparinase to degrade glomeru- biochemical nature of the charge barrier. Glomerular anionic lar HS. As a control, rats were injected with neuraminidase, sites can be detected on the basis of their affinity for cationic which cleaves neuraminic acid from glomerular sialoproteins probes (e.g., polyethyleneimine, cationized ferritin, lysozyme) such as podocalyxin and causes proteinuria. Careful analysis of and have been found in association with each layer of the the specificity and efficiency of deglycosylation was performed capillary wall. The anionic glycocalyx of podocytes and endo- using lectin histochemistry and immunostaining with a panel thelial cells, formed largely by the sialoprotein podocalyxin (7), of well-defined HS antibodies. It is this approach, combined may contribute to the barrier. Indeed, podocalyxin was shown with the simple determination of albumin excretion (as op- to play a critical role in dictating podocyte foot process archi- posed to the use of a surrogate tracer), that distinguishes this tecture, presumably through charge-related repulsive effects study. Injection of heparinase was followed quickly by urinary (8). However, the glomerular basement membrane (GBM) is excretion of HS. After 24 h there was complete loss of glomer- generally considered to be of primary importance for the ular HS as assessed by immunostaining, which indicated there charge barrier. GBM charge is imparted by the sulfated glycos- was no regeneration of HS over the course of the experiment. aminoglycan (GAG) side chains of proteoglycans, and to a Staining for the core protein agrin, the major GBM-HSPG, was lesser extent by carboxyl and sialyl groups of glycoproteins. not disrupted. Labeling with the cationic probe cuprolinic blue, The former were classified as heparan sulfate (HS) proteogly- which detects proteoglycans with their anionic GAG side cans (HSPG) in an influential study in which in vivo perfusion chains as a filamentous network in the GBM, was virtually of heparinase, but not other GAG-degrading enzymes, led to a abolished after heparinase injection, yet glomerular and tubular dramatic increase in the permeability of rat GBM to native architecture was otherwise normal. The key finding of this (anionic) ferritin (9). Other sulfated GAG are not found in the study is that the glomerular filtration barrier of heparinase- GBM in significant amounts, but hyaluronic acid is present. treated rats was functionally intact, despite the loss of HS and a disruption of GBM charge. The authors conclude that the loss Published online ahead of print. Publication date available at www.jasn.org. of GBM-HS does not lead to increased permeability to protein. Their findings are in accordance with recent work using iso- Address correspondence to: Dr. Jeffrey H. Miner, Washington University School of Medicine, Renal Division, Box 8126, 660 S. Euclid Avenue, Street Louis, MO lated perfused kidneys that have pointed to other GAG (chon- 63110. Phone: 314-362-8235; Fax: 314-362-8237; E-mail: [email protected] droitin sulfate and hyaluronic acid) present in the endothelial Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1803-0672 J Am Soc Nephrol 18: 672–674, 2007 Evidence against a Role for HS in Glomerular Permselectivity 673 glycocalyx as determinants of glomerular permselectivity 2. Bohrer M, Baylis C, Humes H, Glassock R, Robertson C, (20,21). The nature of the study by Wijnhoven et al. leaves open Brenner B: Permselectivity of the glomerular capillary wall. questions about the possibility of compensation by tubular Facilitated filtration of circulating polycations. J Clin Invest resorption and whether there might be long-term consequences 61: 72–78, 1978 arising from the loss of glomerular HS. Nevertheless, their 3. Rennke H, Patel Y, Venkatachalam M: Glomerular filtra- important finding highlights the fact that it is time to revisit tion of proteins: Clearance of anionic, neutral and cationic horseradish peroxidase in the rat. Kidney Int 13: 278–288, (and likely revise) the concept of the glomerular charge barrier. 1978 The study by Wijnhoven et al. complements recent work by 4. Rennke H, Cotran R, Venkatachalam M: Role of molecular others that have turned to knockout mouse models to address charge in glomerular permeability. Tracer studies with these questions by targeting HS-bearing core proteins. Three cationized ferritins. J Cell Biol 67: 638–646, 1975 genetically distinct GBM-HSPG are recognized: perlecan, colla- 5. Rennke H, Venkatachalam M: Glomerular permeability: In gen XVIII, and agrin. Mice that lack the HS attachment sites on vivo tracer studies with polyanionic and polycationic fer- perlecan have a normal glomerular ultrastructure and no evi- ritins. Kidney Int 11: 44–53, 1977 dence of renal disease, but show increased susceptibility to 6. Comper W, Glascow E: Charge selectivity in kidney ultra- protein-overload proteinuria (22,23). Collagen XVIII mutants filtration. Kidney Int 47: 1242–1251, 1995 have mild mesangial expansion and only slightly elevated se- 7. Kerjaschki D, Sharkey D, Farquhar M: Identification and rum creatinine levels (24). Our own work shows that podocyte- characterization of podocalyxin-the major sialoprotein of specific agrin-knockout mice lack GBM-HS and have a signifi- the renal glomerular epithelial cell. J Cell Biol 98: 1591–1596, cant GBM charge defect, but kidney function is normal (25). 1984 Others have taken an alternative approach by targeting synthe- 8. Doyonnas R, Kershaw D, Duhme C, Merkens H, Chelliah sis of the GAG chains themselves, thereby disrupting all forms S, Graf T, McNagny K: Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podoca- of HS linked to both GBM and cell-associated proteoglycans lyxin. J Exp Med 194: 13–27, 2001 (e.g., syndecans and glypicans). Transgenic overexpression of 9. Kanwar Y, Linker A, Farquhar M: Increased permeability heparanase leads to changes in podocyte ultrastructure and a of the glomerular basement membrane to ferritin after slight increase in urinary protein excretion (26), whereas dis- removal of glycosaminoglycans (heparan sulfate) by en- ruption of HS biosynthesis specifically in podocytes was very zyme digestion. J Cell Biol 86: 688–693, 1980 recently reported to cause profound structural and functional 10. Myers B, Okarma T, Friedman S, Bridges C, Ross J, Asseff defects (27). S, Deen W: Mechanisms of proteinuria in human glomer- Finally, the findings of Wijnhoven et al. should be of interest ulonephritis. J Clin Invest 70: 732–746, 1982 to clinical nephrologists, as unraveling this aspect of glomerular 11. Guasch A, Deen W, Myers B: Charge selectivity of the biology will hopefully advance our understanding of the cause glomerular filtration barrier in healthy and nephrotic hu- and treatment of kidney disease. Increased heparanase activity mans. J Clin Invest 92: 2274–2282, 1993 has been implicated in the pathogenesis of experimental Hey- 12. Carrie B, Salyer W, Myers B: Minimal change nephropathy: mann nephritis, and its inhibition in this model prevents the loss An electrochemical disorder of the glomerular membrane. of glomerular HS and reduces proteinuria (28,29). Heparanase has Am J Med 70: 262–268, 1981 13. Van den Born J, van den Heuvel L, Bakker M, Veerkamp J, also been associated with the pathogenesis of human diabetic Assmann K, Weening J, Berden J: Distribution of GBM nephropathy (30,31). With this report, Wijnhoven and colleagues heparan sulfate proteoglycan core protein and side chains raise important questions about the significance of this finding in in human glomerular disease.