Proc. Natl. Acad. Sci. USA
Vol. 76, No. 3, pp.11303-13074, March 1979 Cell Biology
Presence of heparan sulfate in the glomerular basement membrane (glycosaminoglyeans/anionic sites/glomerular filtration)
YASHPAL S. KANWAR AND MARILYN G. FARQUHAR Section of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510
Communicated by George E. Palade, December 22, 1978
ABSTRACT The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. Thefindings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific or heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent ma'or components of these sites since the latter are not affected by digestion with neuraminidase and other gly cosaminoglycan-specific enzymes. Identical findings were o, tained on basement membranes in other locations (Bowman's capsule, tubule epithelium and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.
Physiologic studies have established that the glomerular capillaries function as a size- and charge-selective barrier in the production of the glomerular filtrate and in the retention of plasma proteins in the circulation (1-3). By inference it has been assumed (1-3) that charge selectivity is due to the presence in the glomerulus of fixed negative charges, but their precise nature and location in the capillary wall have not been established. Since macromolecular compounds carrying sialic acid residues have been-up to recently-the only known polyanions present in the glomerulus, they have been considered the most likely candidates responsible for the charge barrier (1, 2, 4-7). Sialyl residues have been found in all three layers of the capillary wall. They have been detected in isolated glomerular basement membranes (GBM) (8-10) and in association with the cell membranes of both the endothelium (11-12) and epithelium (4-7, 11), where they are part of the sialic acid-rich, cell-surface coats. A colloidal iron-stainable, cell-coat material is particularly concentrated on the epithelium. Accordingly, it has been repeatedly suggested (1, 2, 4-7) that this "epithelial polyanion" is responsible for establishing the charge-selective properties of the glomerular filter, and, concomitantly, that its loss leads to proteinuria since loss of epithelial cell-coat staining occurs in several renal diseases (1, 2, 5).
Using cationic probes [lysozyme (13, 14), cationized ferritin (15, 16), and ruthenium red (15, 16)], we have previously demonstrated the existence of a quasiregular network of anionic sites in the laminae rarae (interna and externa) of the GBM that have a higher net negative charge [based on cationized ferritin binding (16)] than the sites in the adjacent epithelium and endothelium. We here report the results of experiments in which we have used specific enzymes to remove the anionic sites in situ (by kidney perfusion) and in vitro (by treatment of isolated GBM). The results obtained indicate that the sites consist of sulfated glycosaminoglycans (GAG) rich in heparan sulfate. The occurrence of these highly negatively charged compounds in the glomerulus can be expected to have broad implications for glomerular function and pathology. MATERIALS AND METHODS Animals. Male Charles River CD rats weighing 100-150 g were used. Materials. Ruthenium red was purchased from Ventron Corp. (Danvers, MA); horse-spleen ferritin (2X crystallized, cadmium free) from Calbiochem; and chondroitin sulfates (types A, B, and C) and heparin (grade I) from Sigma. Chondroitinase ABC (Proteus vulgaris) was obtained from Miles; testicular hyaluronidase from Sigma; leech hyaluronidase from Biotrics, Inc. (Boston, MA); neuraminidase (Clostridium perfringens, NEUA) from Worthington; collagenase form III (Cl. histolyticum) from Advance Biofactures (Lynbrook, NY); and Pronase (Streptomyces griseus, grade B) from Calbiochem. Crude heparinase and purified heparitinase (Flavobacterium heparinium) were the generous gift of Alfred Linker (17).* Neuraminidase and heparitinase were tested by the Azocoll assay (18) and were free of proteolytic activity. Cationized ferritin was prepared as described previously (16). Perfusion Experiments. The left kidney was exposed and perfused as described (16). Initially, the kidney was flushed with 0.15 M NaCI for 1-2 min at a rate of 3-4 ml/min to wash out the blood, after which enzyme solutions or buffers alone were perfused. Perfusion conditions (concentration, buffer, pH, duration, and temperature), taken from work by others, were as follows: chondroitinase ABC (0.5-2.0 units/ml) in Hank's balanced salt solution (pH 7.4) (19); testicular hyaluronidase (2000-6000 units/ml) in 0.1 M NaCl/acetate, pH 5.4 (19); and leech hyaluronidase (0.15-1.5 mg/ml) in citrate/phosphate buffer, pH 5.6, all for 30-60 min at 37'C (19). Heparinase (0.3-1.0 mg/ml) in 0.1 M sodium acetate (pH 7.0) and heparitinase (0.2 mg/ml) in the same buffer were perfused for 30-60 min at 30-350C and 38-40oC, respectively (17, 20). Neuraminidase (0.2-0.5 unit/ml) in 0.1 M NaCl/acetate, pH 5.4, was Abbreviations: GBM, glomerular basement membrane(s); GAG, glycosaminoglycan(s). * Crude heparinase acts on all known GAG except keratan sulfate, whereas purified heparitinase acts only on heparan sulfate and not on heparin or other GAG.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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perfused for 30-60 min at 370C (21), and Pronase (4-6 units/ ml) in balanced salt solution (pH 7.4) for 10-20 min at 30-350C. In each case control specimens were perfused with buffer alone under the same conditions. The enzyme solutions were perfused intermittently, with 3-4 ml given at 8- to 10-min intervals, followed by clamping of the inflow and outflow so that the solution was retained in the vessels. The kidney remained immersed in a 0.15 M NaCl bath maintained at constant temperature during the entire perfusion period. The enzyme concentration and the duration of perfusion were selected so as to achieve removal of specific components while maintaining overall tissue integrity. Bladder fluid was collected at the end of each enzyme perfusion. Nitrous acid [prepared by mixing equal volumes of concentrated (33%) acetic acid and 5% sodium nitrite (22)] was also perfused for 5-10 min; acetate buffer (pH 3.4) served as a control. At the end of the perfusions the kidney was flushed (with 20 ml of 0.15 M NaCl for 5-10 min) and then fixed by perfusion with 25 ml of 0.2% solution of ruthenium red in Karnovsky's aldehyde fixative over 5 min, and further processed as in ref. 16. In some experiments, colloidal iron in acetic acid [4 parts of colloidal iron per 1 part of acetic acid (23)] was perfused (instead of ruthenium red) for the same duration. All experiments were repeated at least three times with the exception of those with leech hyaluronidase, which were performed twice. Experiments with Isolated GBM. Glomeruli were isolated by the technique of Krakower and Greenspon (24) and basement membrane fractions were prepared therefrom by detergent treatment (25). Isolated GBM were then incubated by suspension in different enzyme solutions or buffers under exactly the same conditions as used in perfusion experiments. At the end of the incubation they were washed (by sedimentation and resuspension in 0.15 M NaCl) and then incubated for 15 min at 370C with cationized ferritin (pI 7.3-7.5,50-100,gg/ml) in 0.15 M NaCl to label the sites in the laminae rarae (16). After incubation with cationized ferritin, GBM were washed twice (by sedimentation and resuspension) with 0.15 M NaCl followed by a third wash in 0.15 M cacodylate buffer (pH 7.4). GBM were then pelleted (in a Beckman Microfuge), fixed in aldehyde fixative, postfixed for 3 hr in OS04, and processed as in ref. 16. Analysis of Bladder Fluid. Bladder fluid was collected at the end of each perfusion and 100 ,ul was incubated for 1 hr with appropriate substrates [chondroitin A, B, and C sulfate or heparin sulfate (0.5 ,ug/,ul)] under the same conditions (buffer, temperature) as the tissue in order to ascertain whether or not active enzymes had passed through the glomerular filter. After incubation, 0.5 ,Al of the incubation mixture was electrophoresed on cellulose acetate strips against GAG standards (26) and the strips were stained with 0.1% alcian blue. Fluid collected during neuraminidase perfusion was examined and free sialic acid was determined (27).
RESULTS As described in a previous communication (16) the distribution of anionic sites in the lamina rara interna and externa of the GBM can be visualized in situ by using the cationic dye ruthenium red (Figs. la, 2 a and c, and 3a) and in isolated GBM by labeling with cationized ferritin (Fig. 4 a and b). These sites are distributed at regular (l60 nm) intervals, and after ruthenium red are seen to have a quasiregular lattice-like arrangement (Fig. 2a). It was further shown that the binding of cationized ferritin to isolated GBM is electrostatic in nature since it is displaced by buffers of high ionic strength or pH. In order to gain further information on the nature of these anionic groups, the GBM was exposed to digestion with specific en-
Proc. Nati. Acad. Sci. USA 76 (1979)
zymes either in situ (in perfused glomeruli) or in vitro (in isolated GBM), and the distribution of anionic sites was determined by ruthenium red staining or cationic ferritin binding, respectively. Perfusion Experiments. Perfusion with neuraminidase did not affect the distribution of ruthenium red-stained anionic sites in the laminae rarae of the GBM (Fig. la). Specimens perfused with colloidal iron (or incubated sections) showed no staining of the epithelial cell coat (Fig. lb), indicating that sialic acid was removed by the enzyme treatment. In addition, sialic acid was detected in fluid collected from the urinary bladder, indicating that the enzyme was active and had liberated sialic acid during its passage through the nephron. In contrast, a thick, colloidal iron-stainable cell coat was observed in controls perfused with buffer alone (Fig. lc). The ruthenium red-stained sites were also unaffected by treatment with chondroitinase ABC (Fig. 2a) or testicular and leech hyaluronidase (not shown) at concentrations 4-5 times higher than those used in the past to degrade GAG in the extracellular matrices of several fixed tissues (28-30). Bladder fluid collected after perfusion with these three enzymes degraded the appropriate GAG substrate, indicating that these enzymes passed through the filter and were active during transit across the glomerular capillary. In contrast to the negative results obtained with the enzymes mentioned above, crude heparinase and purified heparitinase used at concentrations comparable to or lower than those required to degrade heparan sulfate in vitro (17) caused loss of the ruthenium red-stained GBM sites (Fig. 2b), whereas the sites were demonstrable in buffer-perfused controls (Fig. 2c). In this case also, as would be expected, the collected bladder fluid degraded heparin. A similar loss of the ruthenium red-stained sites was observed in Bowman's capsule and basement membranes of the tubular epithelium and endothelium of peritubular capillaries. The GBM sites were also susceptible to Pronase (not shown) and to nitrous acid treatment (Fig. 3). The conditions of Pronase perfusion were such that the cellular elements of the glomerulus and the basement membrane remained intact, yet the sites were removed. Experiments with Isolated GBM. The results obtained on isolated GBM incubated with various enzymes after labeling with cationized ferritin were exactly the same as those obtained by perfusion: treatment with neuraminidase (Fig. 4b), chondroitinase ABC (Fig. 4a), and testicular and leech hyaluronidase did not affect the binding of cationized ferritin. Treatment with heparinase, heparitinase (Fig. 4c), or Pronase caused a virtually complete elimination of cationized ferritin binding.
DISCUSSION The results of these experiments provide direct evidence that the anionic sites previously demonstrated with cationic probes in the lamina rara interna and externa of the GBM consist, at least in large part, of heparan sulfate. This is indicated by the fact that the GBM sites are removed by treatment with nitrous acid and by digestion with both crude heparinase and purified heparitinase. The former two procedures do not distinguish between heparin and heparan sulfate, but heparitinase is specific for heparan sulfate (20). The finding that the sites are destroyed by Pronase together with the fact that GAG do not normally occur extracellularly as free polymers (31,32) make it likely that the heparan sulfate in the GBM occurs in the form of proteoglycans or protein-polysaccharide complexes, as in the case of GAG in other connective tissue matrices. The finding that the sites are unaffected by neuraminidase treatment or by other GAG-specific enzymes suggests that neither sialogly-
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FIG. 1. (a) Partially grazing section through a glomerular capillary from a kidney perfused for 30 min with 0.1 M NaCl/acetate, pH 5.4, containingineuraminidase I(0.2-0.5 unit/ml) followedlby perfusion with aldehyde fixative containing ruthenium red. Ruthenium redstained particles (arrows) can be visualized both in the lamina rara interna (in) and externa (ex) of the GBM, indicating they are not removed by the enzyme treatment. En, endothelium; Ep, epithelium; Cap, capillary lumen. (X60,000.) (b and c) (b) Glomerular capillary from a kidney perfused with neuraminidase under identical conditions to that in a, followed by perfusion with colloidal iron. Note the absence of colloidal iron binding to the epithelial membranes around the foot processes (fp). This situation can be contrasted to that in c, which is a control kidney perfused with acetate buffer alone in which there is heavy binding of colloidal iron to the sialic acid-rich coat of the epithelial foot processes (fp). A lesser but still detectable binding to the endothelium and the laminae rarae of the GBM is also present. The colloidal iron deposits on the luminal side of the GBM in b are assumed to represent residues of unfiltered colloidal iron. B, basement membrane. (X60,000.)
coproteins nor other sulfated and neutral GAG form a major component of the sites. The validity of the findings is strengthened by the fact that identical results were obtained when digestion was carried out under different conditions-i.e., on unfixed, intact glomerular capillaries perfused with enzyme solutions in situ and in isolated GBM subjected to enzyme digestion in vitro.
FI(u 2. (a) Partially grazing section of a glomerular capillary from a kidney perfused with chondroitinase ABC (0.5-2.0 units/ml) for 60 min followed by ruthenium red/fixative perfusion. Ruthenium redstained particles were not removed by this treatment since they can be visualized both in the lamina rara interna (in) and externa (ex) of the basement membrane (B). (X60,000.) (b) Small field from a glomerular capillary of a kidney perfused with heparitinase (0.2 mg/ml) for 20 min followed by ruthenium red/fixative perfusion; (c) similar field from a control perfused with buffer only. No ruthenium redstained particles can be visualized in the basement membrane (B) in heparitinase-perfused specimens whereas they can be visualized in the lamina rara interna (in) and externa (ex) of the GBM in the control. Cap, capillary lumen. (X60,000.)
At the time of the original description of the anionic sites in the GBM it was pointed out (13) that these sites could consist of the carboxyl groups of the collagenous or noncollagenous peptides, sialyl groups of glycoproteins, or sulfated groups of GAG. The first two had been found in isolated GBM, whereas the latter had not been detected, but the presence of GAG has been recorded in basement membranes in other locations (28-30). In later experiments in which the sites were stained with ruthenium red, it was noted (16) that the GBM sites and their interconnecting filaments bear a striking resemblance to proteoglycan particles found in other connective tissue matrices (33) or in association with other basement membranes (28-30).
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Cell Biology: Kanwar and Farquhar
Proc. Natl. Acad. Sci. USA 76 (1979) 1.
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FIG. 3. Portions of glomerular capillaries perfused with acetate buffer (pH 3.4) (a) or nitrous acid (b) for 5-10 min followed by ruthenium red/fixative solution. No ruthenium red-stained particles are seen in the nitrous acid-treated specimen whereas they are present in both the lamina rara interna (in) and externa (ex) of the GBM in the buffer control. (a, X90,000; b, X60,000.)
The present experiments, based on removal of the sites with specific enzymes, have allowed us to establish their identity as sulfated GAG rich in heparan sulfate. As far as we are aware, this represents the first description of sulfated GAG in the GBM, and, in addition, the first description of heparan sulfate in any basement membrane. Heparan sulfate has been found, however, in association with the cell surfaces of various different cell types (34), including vascular endothelia (35). The question arises as to why GAG have not been detected heretofore in the GBM either by biochemical assays (8-10) or by histochemical staining procedures (4-7, 11-12). As far as the negative biochemical results are concerned, the explanation may lie in the fact that previous analyses for uronic acid (8, 9) or sulfate groups (8) have been done on isolated GBM prepared by sonication, which, according to our experience, disrupts the anionic sites or, if prolonged, removes them entirely. Previous histochemical studies had the disadvantage that they were done on fixed sections (4-7, 11-12) incubated in basic dyes (with and without prior enzyme digestion). In such preparations it was difficult to make a clear distinction between the charged sites in the laminae rarae and those on the adjacent endothelial and epithelial cell membranes. Sulfated GAG occur widely (as proteoglycan complexes) in connective tissues, where they constitute a main component of the matrix or ground substance. The most extensively studied is the proteoglycan complex of cartilage, which is a complex aggregate consisting of chondroitin sulfate, protein, and hyaluronate (32). Among the well-known physical and chemical properties of GAG are their high net negative charge, high charge density, and gel-like consistency, which results from their ability to bind water. According to Laurent and coworkers (32, 36), who have investigated the physiological functions of connective tissue polysaccharides extensively, proteoglycans are ideally suited as substances influencing the electrochemical properties of tissue since their branched structure represents an efficient way of packing of charges. From their work and that of others, it is quite clear that the GAG serve to retard
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FIG. 4. Loops of isolated GBM subjected to treatment with chondroitinase ABC (a), neuraminidase (b), and heparitinase (c), followed by incubation with cationized ferritin to label the GBM sites. Cationized ferritin molecules are seen binding at regular intervals (arrows) to the chondroitinase- and neuraminidase-treated specimens. Little or no binding is seen to the heparitinase-treated GBM, indicating removal of the sites by this enzyme. Cationized ferritin molecules bind only to the outer or exposed side of the GBM loops because the latter consist of intact, closed tubes, and the tracer does not have access to the inner or unexposed side of the GBM. Disruption of the loops [e.g., by sonication (16)] leads to decoration of the sites on both sides of the GBM. (X60,000.)
transport of macromolecules in the extracellular matrix, with the degree of retardation being dependent on the size of the particle transported and the concentration of GAG. Thus, the presence of proteoglycans in the GBM can be expected to have profound effects on the permeability of the GBM and, accordingly, the GAG become prime candidates for at least one type of polyanion responsible for creating and maintaining the glomerular charge barrier function. Accordingly, it follows that any type of glomerular injury that would affect the biosynthesis or distribution of the GAG could affect adversely the filtration properties of the GBM. A partial loss of anionic sites from the laminae rarae of the GBM was recently demonstrated to occur in aminonucleoside nephrosis (14), a condition in which there
Cell Biology: Kanwar and Farquhar is a partial loss of the glomerular charge barrier res lin albuminuria (1, 2). Besides their expected participation in determining the permeability properties of the glomerulus to plasma proteins, given the known properties of GAG, their presence in the GBM and in basement membranes in general may have other important functional implications. Due to their high net negative charge, GAG have the ability to interact (by electrostatic binding) with various biological macromolecules (see ref. 31). Among the most interesting properties of GAG, viewed in the context of normal glomerular functions, are their antithrombotic effects, ability to induce conformational changes in proteins, including collagen, and ability to interact with fibrillar collagens to influence the deposition of collagen fibrils. Assuming that these last two properties also apply to the nonfibrillar collagenous peptides of basement membranes, the possibility exists that the GAG composition could affect the deposition and the three-dimensional organization (i.e., tertiary and quaternary structure) of these peptides. For fibrillar collagens, GAG are known to bind (at 67-nm intervals) to specific regions in the collagen molecule where basic groups are clustered (37). This raises the possibility that the quasiregular (:t60 nm) distribution of anionic sites we have detected in the laminae rarae of the GBM may be reflected in a complementary concentration of basic groups in the adjacent collagenous peptides. The demonstration of GAG in the GBM provides a new macromolecular component (in addition to collagenous and noncollagenous glycoproteins) to be taken into consideration in explaining the pathogenesis of various glomerular diseases and in contemplating potential mechanisms of glomerular injury. Due to their highly charged and relatively exposed nature, the anionic sites can be expected to bind, and to thereby concentrate, any cationic molecules up to the size of ferritin (t1 1 nm; Mr 480,000). Binding of lysozyme, cationized ferritin, and ruthenium red has already been demonstrated in previous work of our group (13-16) and binding of other cationic compounds [polylysine and polylysine-heparin complexes (38), polyethyleneimine (39), cationic horseradish peroxidase (40), and cytochrome c (40)1 can be inferred from the work of others. Among the molecules of interest suspected or demonstrated to be involved in glomerular injury are various toxic compounds, vasoactive amines, and antigens and antibodies-either individually or as antigen-antibody complexes. In connective tissues, precipitation of antigen-antibody complexes is facilitated by the presence of GAG (41). In short, the existence of anionic sites composed of GAG with a demonstrated ability to bind cationic molecules of varying size raises the possibility that nonspecific trapping of a wide variety of substances could be involved, directly or indirectly, in glomerular injury. We thank Bonnie Peng, Nancy Bull, and Barbara Dannacher for excellent technical assistance, and Lynne Wootton for excellent secretarial and editorial help. We especially thank Dr. Alfred Linker, who provided the crude heparinase and purified heparitinase. This research was supported by U.S. Public Health Service Grant AM 17724. 1. Brenner, B. M., Bohrer, M. P. & Baylis, C. (1977) Kidney Int. 12, 229-237.
Brenner, B. M., Hostetter, T. H. & Humes, H. D. (1978) N. Engl. J. Med. 298, 826-833. 3. Rennke, H. G., Patel, Y. & Venkatachalam, M. A. (1978) Kidney Int. 13,278-288. 2.
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4. Mohos, S. C. & Skoza, L. (1969) Science 164, 1519-1521. 5. Michael, A. F., Blau, E. & Vernier, R. L. (1970) Lab. Invest. 23, 649-657. 6. Blau, E. B. & Haas, J. E. (1973) Lab. Invest. 28,477-481. 7. Latta, H., Johnston, W. H. & Stanley, T. M. (1975) J. Ultrastruct. Res. 51, 354-376. 8. Spiro, R. G. (1972) in Glycoproteins: Their Composition, Structure and Function, ed. Gottschalk, A. (Elsevier, Amsterdam), pp. 964-999. 9. Kefalides, N. A. (1973) Int. Rev. Connect. Tissue Res. 6, 63104. 10. Westberg, N. G. & Michael, A. F. (1970) Biochemistry 9, 3837-3846. 11. Jones, D. B. (1969) Lab. Invest. 21, 119-125. 12. Latta, H. & Johnston, W. H. (1976) J. Ultrastruct. Res. 57, 65-67. 13. Caulfield, J. P. & Farquhar, M. G. (1976) Proc. Natl. Acad. Sci. USA 73, 1646-1650. 14. Caulfield, J. P. & Farquhar, M. G. (1978) Lab. Invest. 39, 502-512. 15. Kanwar, Y. S. & Farquhar, M. G. (1978) J. Cell Biol. 79, 150a. 16. Kanwar, Y. S. & Farquhar, M. G. (1979) J. Cell Biol. 81, in press. 17. Linker, A. & Hovingh, P. (1972) Methods Enzymol. 28, 902911. 18. Mandl, I., MacLennan, J. D. & Howes, E. L. (1953) J. Clin. Invest. 32, 1323-1329. 19. Saito, H., Yamagata, T. & Suzuki, S. (1968) J. Biol. Chem. 243, 1536-1542. 20. Linker, A. & Hovingh, P. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 43-46. 21. Cassidy, J. T., Jourdian, G. W. & Roseman, S. C. (1965) J. Biol. Chem. 240, 3501-3506. 22. Cifonelli, J. A. (1968) Carbohydr. Res. 8,233-242. 23. Rinehart, J. F. & Abul-Haj, S. K. (1951) AMA Arch. Pathol. 52, 189-194. 24. Krakower, C. A. & Greenspon, S. A. (1951) Arch. Pathol. 51, 629-639. 25. Meezan, E., Hjelle, J. T., Brendel, K. & Carlson, E. C. (1975) Life Sci. 17, 1721-1732. 26. Hata, R. & Nagai, Y. (1972) Anal. Biochem. 45,462-468. 27. Warren, L. L. (1959) J. Biol. Chem. 234, 1971-1975. 28. Hay, E. D. & Meier, S. (1974) J. Cell Biol. 62, 889-898. 29. Trelstad, R. L., Hayashi, K. & Toole, B. P. (1974) J. Cell Biol. 62, 815-830. 30. Wight, T. N. & Ross, R. (1975) J. Cell Biol. 67, 660-674. 31. Lindahl, U. & Hook, M. (1978) Annu. Rev. Biochem. 47,385417. 32. Comper, W. D. & Laurent, T. C. (1978) Physiol. Rev. 58, 255-315. 33. Hay, E. D., Hasty, D. L. & Kiehnau, K. L. (1978) in CollagenPlatelet Interaction, eds. Gastpar, H., Kuhn, K. & Marx, R. (Schattauer, Stuttgart), pp. 129-151. 34. Kraemer, P. M. (1971) Biochemistry 10, 1445-1451. 35. Buonassisi, V. & Root, M. (1975) Biochim. Biophys. Acta 385, 1-10. 36. Laurent, T. C. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 24-27. 37. Doyle, B. B., Hukins, D. W. L., Hulmes, D. J. S., Miller, A. & Woodhead-Galloway, J. (1974) J. Mol. Biol. 91, 79-99. 38. Seiler, M. W., Rennke, H. G., Venkatachalam, M. A. & Cotran, R. S. (1977) Lab. Invest. 36, 48-61. 39. Schurer, J. W., Hoedemaeker, Ph. J. & Molenaar, I. (1978) J. Histochem. Cytochem. 26,688-689. 40. Kerjaschki, D., Forster, O., Boltz, G., Scheiner, W. & Albini, B. (1978) in Electron Microscopy 1978, ed. Sturgess, J. M. (Microscopical Society of Canada), pp. 462-463. 41. Hellsing, K. (1969) Biochem. J. 112, 475-481.
Vol. 76, No. 3, pp.11303-13074, March 1979 Cell Biology
Presence of heparan sulfate in the glomerular basement membrane (glycosaminoglyeans/anionic sites/glomerular filtration)
YASHPAL S. KANWAR AND MARILYN G. FARQUHAR Section of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510
Communicated by George E. Palade, December 22, 1978
ABSTRACT The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. Thefindings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific or heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent ma'or components of these sites since the latter are not affected by digestion with neuraminidase and other gly cosaminoglycan-specific enzymes. Identical findings were o, tained on basement membranes in other locations (Bowman's capsule, tubule epithelium and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.
Physiologic studies have established that the glomerular capillaries function as a size- and charge-selective barrier in the production of the glomerular filtrate and in the retention of plasma proteins in the circulation (1-3). By inference it has been assumed (1-3) that charge selectivity is due to the presence in the glomerulus of fixed negative charges, but their precise nature and location in the capillary wall have not been established. Since macromolecular compounds carrying sialic acid residues have been-up to recently-the only known polyanions present in the glomerulus, they have been considered the most likely candidates responsible for the charge barrier (1, 2, 4-7). Sialyl residues have been found in all three layers of the capillary wall. They have been detected in isolated glomerular basement membranes (GBM) (8-10) and in association with the cell membranes of both the endothelium (11-12) and epithelium (4-7, 11), where they are part of the sialic acid-rich, cell-surface coats. A colloidal iron-stainable, cell-coat material is particularly concentrated on the epithelium. Accordingly, it has been repeatedly suggested (1, 2, 4-7) that this "epithelial polyanion" is responsible for establishing the charge-selective properties of the glomerular filter, and, concomitantly, that its loss leads to proteinuria since loss of epithelial cell-coat staining occurs in several renal diseases (1, 2, 5).
Using cationic probes [lysozyme (13, 14), cationized ferritin (15, 16), and ruthenium red (15, 16)], we have previously demonstrated the existence of a quasiregular network of anionic sites in the laminae rarae (interna and externa) of the GBM that have a higher net negative charge [based on cationized ferritin binding (16)] than the sites in the adjacent epithelium and endothelium. We here report the results of experiments in which we have used specific enzymes to remove the anionic sites in situ (by kidney perfusion) and in vitro (by treatment of isolated GBM). The results obtained indicate that the sites consist of sulfated glycosaminoglycans (GAG) rich in heparan sulfate. The occurrence of these highly negatively charged compounds in the glomerulus can be expected to have broad implications for glomerular function and pathology. MATERIALS AND METHODS Animals. Male Charles River CD rats weighing 100-150 g were used. Materials. Ruthenium red was purchased from Ventron Corp. (Danvers, MA); horse-spleen ferritin (2X crystallized, cadmium free) from Calbiochem; and chondroitin sulfates (types A, B, and C) and heparin (grade I) from Sigma. Chondroitinase ABC (Proteus vulgaris) was obtained from Miles; testicular hyaluronidase from Sigma; leech hyaluronidase from Biotrics, Inc. (Boston, MA); neuraminidase (Clostridium perfringens, NEUA) from Worthington; collagenase form III (Cl. histolyticum) from Advance Biofactures (Lynbrook, NY); and Pronase (Streptomyces griseus, grade B) from Calbiochem. Crude heparinase and purified heparitinase (Flavobacterium heparinium) were the generous gift of Alfred Linker (17).* Neuraminidase and heparitinase were tested by the Azocoll assay (18) and were free of proteolytic activity. Cationized ferritin was prepared as described previously (16). Perfusion Experiments. The left kidney was exposed and perfused as described (16). Initially, the kidney was flushed with 0.15 M NaCI for 1-2 min at a rate of 3-4 ml/min to wash out the blood, after which enzyme solutions or buffers alone were perfused. Perfusion conditions (concentration, buffer, pH, duration, and temperature), taken from work by others, were as follows: chondroitinase ABC (0.5-2.0 units/ml) in Hank's balanced salt solution (pH 7.4) (19); testicular hyaluronidase (2000-6000 units/ml) in 0.1 M NaCl/acetate, pH 5.4 (19); and leech hyaluronidase (0.15-1.5 mg/ml) in citrate/phosphate buffer, pH 5.6, all for 30-60 min at 37'C (19). Heparinase (0.3-1.0 mg/ml) in 0.1 M sodium acetate (pH 7.0) and heparitinase (0.2 mg/ml) in the same buffer were perfused for 30-60 min at 30-350C and 38-40oC, respectively (17, 20). Neuraminidase (0.2-0.5 unit/ml) in 0.1 M NaCl/acetate, pH 5.4, was Abbreviations: GBM, glomerular basement membrane(s); GAG, glycosaminoglycan(s). * Crude heparinase acts on all known GAG except keratan sulfate, whereas purified heparitinase acts only on heparan sulfate and not on heparin or other GAG.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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perfused for 30-60 min at 370C (21), and Pronase (4-6 units/ ml) in balanced salt solution (pH 7.4) for 10-20 min at 30-350C. In each case control specimens were perfused with buffer alone under the same conditions. The enzyme solutions were perfused intermittently, with 3-4 ml given at 8- to 10-min intervals, followed by clamping of the inflow and outflow so that the solution was retained in the vessels. The kidney remained immersed in a 0.15 M NaCl bath maintained at constant temperature during the entire perfusion period. The enzyme concentration and the duration of perfusion were selected so as to achieve removal of specific components while maintaining overall tissue integrity. Bladder fluid was collected at the end of each enzyme perfusion. Nitrous acid [prepared by mixing equal volumes of concentrated (33%) acetic acid and 5% sodium nitrite (22)] was also perfused for 5-10 min; acetate buffer (pH 3.4) served as a control. At the end of the perfusions the kidney was flushed (with 20 ml of 0.15 M NaCl for 5-10 min) and then fixed by perfusion with 25 ml of 0.2% solution of ruthenium red in Karnovsky's aldehyde fixative over 5 min, and further processed as in ref. 16. In some experiments, colloidal iron in acetic acid [4 parts of colloidal iron per 1 part of acetic acid (23)] was perfused (instead of ruthenium red) for the same duration. All experiments were repeated at least three times with the exception of those with leech hyaluronidase, which were performed twice. Experiments with Isolated GBM. Glomeruli were isolated by the technique of Krakower and Greenspon (24) and basement membrane fractions were prepared therefrom by detergent treatment (25). Isolated GBM were then incubated by suspension in different enzyme solutions or buffers under exactly the same conditions as used in perfusion experiments. At the end of the incubation they were washed (by sedimentation and resuspension in 0.15 M NaCl) and then incubated for 15 min at 370C with cationized ferritin (pI 7.3-7.5,50-100,gg/ml) in 0.15 M NaCl to label the sites in the laminae rarae (16). After incubation with cationized ferritin, GBM were washed twice (by sedimentation and resuspension) with 0.15 M NaCl followed by a third wash in 0.15 M cacodylate buffer (pH 7.4). GBM were then pelleted (in a Beckman Microfuge), fixed in aldehyde fixative, postfixed for 3 hr in OS04, and processed as in ref. 16. Analysis of Bladder Fluid. Bladder fluid was collected at the end of each perfusion and 100 ,ul was incubated for 1 hr with appropriate substrates [chondroitin A, B, and C sulfate or heparin sulfate (0.5 ,ug/,ul)] under the same conditions (buffer, temperature) as the tissue in order to ascertain whether or not active enzymes had passed through the glomerular filter. After incubation, 0.5 ,Al of the incubation mixture was electrophoresed on cellulose acetate strips against GAG standards (26) and the strips were stained with 0.1% alcian blue. Fluid collected during neuraminidase perfusion was examined and free sialic acid was determined (27).
RESULTS As described in a previous communication (16) the distribution of anionic sites in the lamina rara interna and externa of the GBM can be visualized in situ by using the cationic dye ruthenium red (Figs. la, 2 a and c, and 3a) and in isolated GBM by labeling with cationized ferritin (Fig. 4 a and b). These sites are distributed at regular (l60 nm) intervals, and after ruthenium red are seen to have a quasiregular lattice-like arrangement (Fig. 2a). It was further shown that the binding of cationized ferritin to isolated GBM is electrostatic in nature since it is displaced by buffers of high ionic strength or pH. In order to gain further information on the nature of these anionic groups, the GBM was exposed to digestion with specific en-
Proc. Nati. Acad. Sci. USA 76 (1979)
zymes either in situ (in perfused glomeruli) or in vitro (in isolated GBM), and the distribution of anionic sites was determined by ruthenium red staining or cationic ferritin binding, respectively. Perfusion Experiments. Perfusion with neuraminidase did not affect the distribution of ruthenium red-stained anionic sites in the laminae rarae of the GBM (Fig. la). Specimens perfused with colloidal iron (or incubated sections) showed no staining of the epithelial cell coat (Fig. lb), indicating that sialic acid was removed by the enzyme treatment. In addition, sialic acid was detected in fluid collected from the urinary bladder, indicating that the enzyme was active and had liberated sialic acid during its passage through the nephron. In contrast, a thick, colloidal iron-stainable cell coat was observed in controls perfused with buffer alone (Fig. lc). The ruthenium red-stained sites were also unaffected by treatment with chondroitinase ABC (Fig. 2a) or testicular and leech hyaluronidase (not shown) at concentrations 4-5 times higher than those used in the past to degrade GAG in the extracellular matrices of several fixed tissues (28-30). Bladder fluid collected after perfusion with these three enzymes degraded the appropriate GAG substrate, indicating that these enzymes passed through the filter and were active during transit across the glomerular capillary. In contrast to the negative results obtained with the enzymes mentioned above, crude heparinase and purified heparitinase used at concentrations comparable to or lower than those required to degrade heparan sulfate in vitro (17) caused loss of the ruthenium red-stained GBM sites (Fig. 2b), whereas the sites were demonstrable in buffer-perfused controls (Fig. 2c). In this case also, as would be expected, the collected bladder fluid degraded heparin. A similar loss of the ruthenium red-stained sites was observed in Bowman's capsule and basement membranes of the tubular epithelium and endothelium of peritubular capillaries. The GBM sites were also susceptible to Pronase (not shown) and to nitrous acid treatment (Fig. 3). The conditions of Pronase perfusion were such that the cellular elements of the glomerulus and the basement membrane remained intact, yet the sites were removed. Experiments with Isolated GBM. The results obtained on isolated GBM incubated with various enzymes after labeling with cationized ferritin were exactly the same as those obtained by perfusion: treatment with neuraminidase (Fig. 4b), chondroitinase ABC (Fig. 4a), and testicular and leech hyaluronidase did not affect the binding of cationized ferritin. Treatment with heparinase, heparitinase (Fig. 4c), or Pronase caused a virtually complete elimination of cationized ferritin binding.
DISCUSSION The results of these experiments provide direct evidence that the anionic sites previously demonstrated with cationic probes in the lamina rara interna and externa of the GBM consist, at least in large part, of heparan sulfate. This is indicated by the fact that the GBM sites are removed by treatment with nitrous acid and by digestion with both crude heparinase and purified heparitinase. The former two procedures do not distinguish between heparin and heparan sulfate, but heparitinase is specific for heparan sulfate (20). The finding that the sites are destroyed by Pronase together with the fact that GAG do not normally occur extracellularly as free polymers (31,32) make it likely that the heparan sulfate in the GBM occurs in the form of proteoglycans or protein-polysaccharide complexes, as in the case of GAG in other connective tissue matrices. The finding that the sites are unaffected by neuraminidase treatment or by other GAG-specific enzymes suggests that neither sialogly-
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FIG. 1. (a) Partially grazing section through a glomerular capillary from a kidney perfused for 30 min with 0.1 M NaCl/acetate, pH 5.4, containingineuraminidase I(0.2-0.5 unit/ml) followedlby perfusion with aldehyde fixative containing ruthenium red. Ruthenium redstained particles (arrows) can be visualized both in the lamina rara interna (in) and externa (ex) of the GBM, indicating they are not removed by the enzyme treatment. En, endothelium; Ep, epithelium; Cap, capillary lumen. (X60,000.) (b and c) (b) Glomerular capillary from a kidney perfused with neuraminidase under identical conditions to that in a, followed by perfusion with colloidal iron. Note the absence of colloidal iron binding to the epithelial membranes around the foot processes (fp). This situation can be contrasted to that in c, which is a control kidney perfused with acetate buffer alone in which there is heavy binding of colloidal iron to the sialic acid-rich coat of the epithelial foot processes (fp). A lesser but still detectable binding to the endothelium and the laminae rarae of the GBM is also present. The colloidal iron deposits on the luminal side of the GBM in b are assumed to represent residues of unfiltered colloidal iron. B, basement membrane. (X60,000.)
coproteins nor other sulfated and neutral GAG form a major component of the sites. The validity of the findings is strengthened by the fact that identical results were obtained when digestion was carried out under different conditions-i.e., on unfixed, intact glomerular capillaries perfused with enzyme solutions in situ and in isolated GBM subjected to enzyme digestion in vitro.
FI(u 2. (a) Partially grazing section of a glomerular capillary from a kidney perfused with chondroitinase ABC (0.5-2.0 units/ml) for 60 min followed by ruthenium red/fixative perfusion. Ruthenium redstained particles were not removed by this treatment since they can be visualized both in the lamina rara interna (in) and externa (ex) of the basement membrane (B). (X60,000.) (b) Small field from a glomerular capillary of a kidney perfused with heparitinase (0.2 mg/ml) for 20 min followed by ruthenium red/fixative perfusion; (c) similar field from a control perfused with buffer only. No ruthenium redstained particles can be visualized in the basement membrane (B) in heparitinase-perfused specimens whereas they can be visualized in the lamina rara interna (in) and externa (ex) of the GBM in the control. Cap, capillary lumen. (X60,000.)
At the time of the original description of the anionic sites in the GBM it was pointed out (13) that these sites could consist of the carboxyl groups of the collagenous or noncollagenous peptides, sialyl groups of glycoproteins, or sulfated groups of GAG. The first two had been found in isolated GBM, whereas the latter had not been detected, but the presence of GAG has been recorded in basement membranes in other locations (28-30). In later experiments in which the sites were stained with ruthenium red, it was noted (16) that the GBM sites and their interconnecting filaments bear a striking resemblance to proteoglycan particles found in other connective tissue matrices (33) or in association with other basement membranes (28-30).
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Cell Biology: Kanwar and Farquhar
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FIG. 3. Portions of glomerular capillaries perfused with acetate buffer (pH 3.4) (a) or nitrous acid (b) for 5-10 min followed by ruthenium red/fixative solution. No ruthenium red-stained particles are seen in the nitrous acid-treated specimen whereas they are present in both the lamina rara interna (in) and externa (ex) of the GBM in the buffer control. (a, X90,000; b, X60,000.)
The present experiments, based on removal of the sites with specific enzymes, have allowed us to establish their identity as sulfated GAG rich in heparan sulfate. As far as we are aware, this represents the first description of sulfated GAG in the GBM, and, in addition, the first description of heparan sulfate in any basement membrane. Heparan sulfate has been found, however, in association with the cell surfaces of various different cell types (34), including vascular endothelia (35). The question arises as to why GAG have not been detected heretofore in the GBM either by biochemical assays (8-10) or by histochemical staining procedures (4-7, 11-12). As far as the negative biochemical results are concerned, the explanation may lie in the fact that previous analyses for uronic acid (8, 9) or sulfate groups (8) have been done on isolated GBM prepared by sonication, which, according to our experience, disrupts the anionic sites or, if prolonged, removes them entirely. Previous histochemical studies had the disadvantage that they were done on fixed sections (4-7, 11-12) incubated in basic dyes (with and without prior enzyme digestion). In such preparations it was difficult to make a clear distinction between the charged sites in the laminae rarae and those on the adjacent endothelial and epithelial cell membranes. Sulfated GAG occur widely (as proteoglycan complexes) in connective tissues, where they constitute a main component of the matrix or ground substance. The most extensively studied is the proteoglycan complex of cartilage, which is a complex aggregate consisting of chondroitin sulfate, protein, and hyaluronate (32). Among the well-known physical and chemical properties of GAG are their high net negative charge, high charge density, and gel-like consistency, which results from their ability to bind water. According to Laurent and coworkers (32, 36), who have investigated the physiological functions of connective tissue polysaccharides extensively, proteoglycans are ideally suited as substances influencing the electrochemical properties of tissue since their branched structure represents an efficient way of packing of charges. From their work and that of others, it is quite clear that the GAG serve to retard
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FIG. 4. Loops of isolated GBM subjected to treatment with chondroitinase ABC (a), neuraminidase (b), and heparitinase (c), followed by incubation with cationized ferritin to label the GBM sites. Cationized ferritin molecules are seen binding at regular intervals (arrows) to the chondroitinase- and neuraminidase-treated specimens. Little or no binding is seen to the heparitinase-treated GBM, indicating removal of the sites by this enzyme. Cationized ferritin molecules bind only to the outer or exposed side of the GBM loops because the latter consist of intact, closed tubes, and the tracer does not have access to the inner or unexposed side of the GBM. Disruption of the loops [e.g., by sonication (16)] leads to decoration of the sites on both sides of the GBM. (X60,000.)
transport of macromolecules in the extracellular matrix, with the degree of retardation being dependent on the size of the particle transported and the concentration of GAG. Thus, the presence of proteoglycans in the GBM can be expected to have profound effects on the permeability of the GBM and, accordingly, the GAG become prime candidates for at least one type of polyanion responsible for creating and maintaining the glomerular charge barrier function. Accordingly, it follows that any type of glomerular injury that would affect the biosynthesis or distribution of the GAG could affect adversely the filtration properties of the GBM. A partial loss of anionic sites from the laminae rarae of the GBM was recently demonstrated to occur in aminonucleoside nephrosis (14), a condition in which there
Cell Biology: Kanwar and Farquhar is a partial loss of the glomerular charge barrier res lin albuminuria (1, 2). Besides their expected participation in determining the permeability properties of the glomerulus to plasma proteins, given the known properties of GAG, their presence in the GBM and in basement membranes in general may have other important functional implications. Due to their high net negative charge, GAG have the ability to interact (by electrostatic binding) with various biological macromolecules (see ref. 31). Among the most interesting properties of GAG, viewed in the context of normal glomerular functions, are their antithrombotic effects, ability to induce conformational changes in proteins, including collagen, and ability to interact with fibrillar collagens to influence the deposition of collagen fibrils. Assuming that these last two properties also apply to the nonfibrillar collagenous peptides of basement membranes, the possibility exists that the GAG composition could affect the deposition and the three-dimensional organization (i.e., tertiary and quaternary structure) of these peptides. For fibrillar collagens, GAG are known to bind (at 67-nm intervals) to specific regions in the collagen molecule where basic groups are clustered (37). This raises the possibility that the quasiregular (:t60 nm) distribution of anionic sites we have detected in the laminae rarae of the GBM may be reflected in a complementary concentration of basic groups in the adjacent collagenous peptides. The demonstration of GAG in the GBM provides a new macromolecular component (in addition to collagenous and noncollagenous glycoproteins) to be taken into consideration in explaining the pathogenesis of various glomerular diseases and in contemplating potential mechanisms of glomerular injury. Due to their highly charged and relatively exposed nature, the anionic sites can be expected to bind, and to thereby concentrate, any cationic molecules up to the size of ferritin (t1 1 nm; Mr 480,000). Binding of lysozyme, cationized ferritin, and ruthenium red has already been demonstrated in previous work of our group (13-16) and binding of other cationic compounds [polylysine and polylysine-heparin complexes (38), polyethyleneimine (39), cationic horseradish peroxidase (40), and cytochrome c (40)1 can be inferred from the work of others. Among the molecules of interest suspected or demonstrated to be involved in glomerular injury are various toxic compounds, vasoactive amines, and antigens and antibodies-either individually or as antigen-antibody complexes. In connective tissues, precipitation of antigen-antibody complexes is facilitated by the presence of GAG (41). In short, the existence of anionic sites composed of GAG with a demonstrated ability to bind cationic molecules of varying size raises the possibility that nonspecific trapping of a wide variety of substances could be involved, directly or indirectly, in glomerular injury. We thank Bonnie Peng, Nancy Bull, and Barbara Dannacher for excellent technical assistance, and Lynne Wootton for excellent secretarial and editorial help. We especially thank Dr. Alfred Linker, who provided the crude heparinase and purified heparitinase. This research was supported by U.S. Public Health Service Grant AM 17724. 1. Brenner, B. M., Bohrer, M. P. & Baylis, C. (1977) Kidney Int. 12, 229-237.
Brenner, B. M., Hostetter, T. H. & Humes, H. D. (1978) N. Engl. J. Med. 298, 826-833. 3. Rennke, H. G., Patel, Y. & Venkatachalam, M. A. (1978) Kidney Int. 13,278-288. 2.
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