Disciplina de Nefrologia, Departamento de Medicina, and 2Disciplina de Biologia Molecular, Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil
Received on May 29, 1999; revised on July 29, 1999; accepted on July 29, 1999.
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Abstract |
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Key words: albuminuria/diabetes mellitus/glycosaminoglycan/kidney/urine
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Introduction |
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Proteoglycans are widely distributed in animal tissues (Dietrich et al., 1976; Cássaro and Dietrich, 1977
) and their biological roles are very diversified, ranging from mechanical support functions to more intricate effects on various cellular processes such as cell adhesion and recognition, motility, and proliferation (Dietrich et al., 1977b
, 1982; Dietrich, 1984
). Proteoglycans may occur in the intracellular compartment (usually in secretory granules), at the cell surface, or in the extracellular matrix (Vogel, 1994
; Iozzo, 1998
). Three major classes of proteoglycans may be distinguished in the extracellular matrix: the large aggregating proteoglycans, which interact with hyaluronic acid, the small leucine-rich proteoglycans, also called "fibrilar proteoglycans" because some of them interact with fibrilar collagens, and the basement membrane proteoglycans. Perlecan is the main basement membrane proteoglycan, present in virtually all vascularized tissues. The name designates a family of multidomain proteins, expressed by different cell types, that most often carry covalently linked heparan sulfate side chains (Iozzo et al., 1994
).
In long term diabetes mellitus, among the most clinically significant complications are the microangiopathies, which are disruption of the normal function of vascular capillary beds. A hallmark of these pathological processes is a significant thickening of microvascular basement membrane, with chronic progression with time. Paradoxically, this thickening of basement membranes is accompanied by a loss of function, allowing charged serum molecules which are normally retained within the circulation to pass across the matrix. This produces systemic effects in skin and muscle, but problems facing investigators are those familiar to matrix researchers: the affected structure in most vascular beds are too diffusely distributed to yield adequate material for biochemical analyses. Most studies therefore focused on the kidney, since the glomerulus provides concentrate and abundant amounts of capillary basement membrane. Furthermore, one of the most marked clinical consequences of diabetes is observed in the renal glomerulus, resulting in diabetic nephropathy (Templeton, 1989). A number of approaches have been developed in the investigation of these complications, many involving experimental models of diabetes which are either induced by chemical destruction of pancreatic islet cells or by the use of strains of animals bred to be genetically susceptible to the disease. Nevertheless, a close examination of the kidney reveals the potential complexity that proteoglycans may have in a very small region. The glomerular capillary wall consists of the epithelial and endothelial coats and glomerular extracellular matrices, which are composed of basement membrane and mesangial matrix. Cell surface proteoglycans as well as extracellular matrix proteoglycans, both in glomerular basement membrane and in mesangial matrix, are present (Kanwar et al., 1983a
,b; Stow and Farquhar, 1985
, 1987; Klein et al., 1986
). The mesangial matrix has been less investigated than the basement membrane, although the two matrices appear to differ with respect to their glycosaminoglycan composition (Kanwar et al., 1983a
,b; Farquhar, 1991
).
A role for heparan sulfate in creating the normal permeability barriers of glomerular basement membrane was suggested by in situ digestion with glycosaminoglycan-degrading enzymes, followed by perfusion with native ferritin (Kanwar et al., 1980).
Both the glomerular basement membrane and the mesangial matrix are increased in area in diabetic nephropathy (Mauer et al., 1992) and this expansion is, possibly, a major contributor to diabetic renal failure. Mesangial cells exposed to high glucose concentrations have shown increased synthesis of extracellular matrix components, such as laminin, fibronectin and type IV collagen (Ayo et al., 1991
). Silbiger et al. (1993)
reported that the total amount of proteoglycans did not change, but a reduction in their charge density was observed. These findings suggest that changes in extracellular matrix organization and metabolism could be involved in the pathogenesis of diabetic nephropathy, with alteration in filtration features. Previous data from our laboratory have shown, in the 12th week after strepzotocin injection, mesangial matrix expansion, a certain degree of hypercellularity and Armani-Ebstein changes at both proximal and distal renal tubules in diabetic Wistar rats. At the same time (12th week post diabetes induction) small increases in plasma creatinine concentration (Ramos, 1988
) and urinary albumin excretion also occurred. Definitive and progressive albuminuria was detected in all animals only 1820 weeks after the streptozotocin injection (Bossolan et al., 1991
). These data indicate the stage of alterations that exist in the kidney at the 12th weeks in the experimental model here used. Since basement membrane thickening and mesangial expansion seem to precede the symptoms of renal dysfunction, such as albuminuria and changes in glomerular filtration rate, the aim of the present study was to investigate whether the urinary excretion of glycosaminoglycans might reflect these biochemical alterations, permitting early diagnosis of diabetic nephropathy.
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Results |
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Discussion |
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We have previously shown that cultured mesangial cells synthesize a mixture of dermatan sulfate and heparan sulfate and most of the dermatan sulfate is released to the culture medium. Mesangial cells from rats which had been diabetic for 4 months incorporate twice the amount of 35S-sulfate in glycosaminoglycans, especially dermatan sulfate, as compared to mesangial cells from normal rats. This increase is proportional to the duration of diabetes, since the mesangial cells from animals that had been diabetic for 5 or 6 months presented higher 35S-incorporations in glycosaminoglycans, particularly dermatan sulfate, than cells from animals which had been diabetic for 4 months (Hadad et al., 1996). The dermatan sulfate was secreted to the medium as two proteoglycans, identified as members of the small matrix proteoglycan decorin/biglycan family (Krusius and Ruoslahti, 1986
; Fisher et al., 1989
; Border et al., 1990
; Harper and Mason, 1994
). The extracellular matrix proteoglycans and glycosaminoglycans are supposed to play a role in the control of cell proliferation (Dietrich, 1984
) and decorin is known to bind collagen (Oldberg and Rouslahti, 1982). So, decorin and other extracellular matrix proteoglycans, such as biglycan, may functionally facilitate the assembly of extracellular matrix (Ruoslahti and Pierschacher, 1987
). The stimulation of proteoglycan production in mesangial cells obtained from diabetic rats may be related to the accumulation of dermatan sulfate and chondroitin sulfate here reported and possibly to the mesangial matrix expansion observed in diabetes mellitus. Taken together, these data suggest morphological and biochemical alterations in renal extracellular matrix.
The present study was designed to investigate whether the urinary excretion of glycosaminoglycans might reflect these biochemical alterations in diabetic nephropathy. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on.
It is apparent from our results that the chronic diabetic state induced by streptozotocin resulted in marked decrease of urinary glycosaminoglycan excretion, on a daily basis. The onset of this feature was a very early event in the development of diabetes. Both heparan sulfate and chondroitin/dermatan sulfate concentrations were decreased. Decreased excretion of glycosaminoglycans have also been observed by others in patients with diabetic nephropathy and other glomerular diseases (Tencer et al., 1997).
The urinary heparan sulfate presents modal molecular weight similar to the kidney heparan sulfate, suggesting that the urinary heparan sulfate could be, at least in part, of renal origin. In contrast, only trace amounts of dermatan sulfate are present in normal rat kidney, while chondroitin sulfate was not detected, suggesting that the urinary chondroitin/dermatan sulfate does not come from the kidney. Although a decrease in glomerular heparan sulfate concentration in diabetic nephropathy has been reported by other authors (Templeton, 1989; Tamsma et al., 1994
; van den Born et al., 1995
), we did not find any changes in the heparan sulfate amounts (per tissue dry weight). Nevertheless, it is possible that the structural organization of the expanded basement membrane and mesangial matrix lead to a looser arrangement of proteoglycans, affecting the tissue filtration properties. Many authors reported the upregulation and overexpression of glomerular extracellular matrix components in diabetic nephropathy and other models of glomerulonephritis (Del Prete et al., 1998
; Kamata et al., 1990
; Mizuno et al., 1999
). Nevertheless, the synthesis of these components may be unbalanced. For instance, the expression of type IV collagen
3(IV),
4(IV), and
5(IV) chains is dissociated from the
1(IV) and
2(IV) expression in the kidney of diabetic mice (Funabiki et al., 1998
). This unbalanced synthesis may affect the type IV collagen assembly and contribute to the basement membrane thickening. This may also be related to the increased urinary excretion of type IV collagen observed in non-insulin-dependent diabetes mellitus (Kado et al., 1996
).
The decreased urinary glycosaminoglycan concentration could also be related to the decreased glycosaminoglycan contents of other tissues in diabetic animals reported by Cechowska-Pasko et al. (1996). Altered urinary excretion of other extracellular matrix macromolecular components, such as laminin, collagen type IV and fibronectin was also reported in diabetic nephropathy (Jaackle-Meyer et al., 1995
).
We believe our results show a potentially important value for the urinary glycosaminoglycan measurement in the early diagnosis of diabetic nephropathy, appearing together with (or even before) the onset of albuminuria.
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Materials and methods |
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Experimental animals
Male Wistar rats, 1214 weeks of age (220260 g body weight), were separated in two groups: "Control" and "Diabetes." Diabetes was induced in 21 rats by a single injection of streptozotocin (50 mg/kg body weight). The drug was dissolved in 300 µl of 10 mM sodium citrate buffer (pH 4.5) and injected into the tail vein. These animals were fed standard laboratory chow and a 5% glucose solution ad libitum, for 48 h. Afterwards, the glucose solution was replaced by water. The glycemia was measured (Glycofilm, Bayer Diagnostics MFG Ltd., Bridgent, Mild Glamorgan, England) and all the diabetic animals presented blood glucose concentrations higher than 200 mg/dl. Twelve age-matched animals that served as controls ("Control" group) were fed standard laboratory chow and water ad libitum. The systolic blood pressure was measured in conscious rats by tail-cuff method. The body weight, and the blood glucose of the animals were measured weekly and every 2 weeks each rat was placed in a metabolic cage (model 6500350, Nalgene Company, Rochester, NJ) for 24 h urine collection. Total volume was measured, the urine was centrifuged to remove debris, and used for the determination of albumin, creatinine, and glycosaminoglycans. Two animals of the "Diabetes" group died during the experimental period and six diabetic animals presented urinary infections and were excluded. All the diabetic rats maintained glycemia higher than 200 mg/dl during all the experimental period.
Quantification of urinary albumin, protein and creatinine
Albumin urinary excretion was determined by the radial immunodiffusion method based on precipitation with rabbit antibodies against rat albumin (Mancini et al., 1965). The diameter of the precipitation halo was measured after 48 h incubation and compared to a standard curve of rat albumin.
Protein was measured by the Coomassie blue method, described by Spector (1978) and urinary creatinine was measured by the picric acid reaction in alkaline conditions (Sigma creatinine kit).
Isolation, identification, and quantification of kidney glycosaminoglycans
At the end of the experiment (12 weeks), the rat kidneys were removed, ground in 10 volumes of acetone and, after standing overnight at room temperature, the fragments were collected by centrifugation and dried under vacuum. The dried material was resuspended in 0.08 M phosphate-cysteine buffer pH 6.5, containing 0.02 M EDTA and 100 mg/l papain (10 ml/g of dry material) and incubated at 50°C overnight. The incubation mixtures were cooled in an ice bath and trichloroacetic acid and NaCl were added to final concentrations of 5% and 1 M, respectively. After standing 15 min in the ice bath, the precipitate formed was removed, the pH of the supernatant was adjusted to 7 and two volumes of ethanol were added. The precipitate formed after 24 h at 20°C was collected by centrifugation and dried. The dried material was resuspended in water and the glycosaminoglycans were identified by a combination of agarose gel electrophoresis in 0.05 M 1,3-diaminopropane-acetate buffer (pH 9), and enzymatic degradation with specific mucopolysaccharidases, as already described (Dietrich et al., 1977a).
The enzymatic degradations with chondroitinases B and AC from Flavobacterium heparinum were carried out as described previously (Petricevich and Michelacci, 1990). Briefly, aliquots of the glycosaminoglycans (50100 µg) were incubated with 2 x 104 units of either chondroitinase B or chondroitinase AC or a mixture of chondroitinases B and AC, in 0.05 M ethylenediamine-acetate buffer, pH 8.0, in a final volume of 20 µl. One unit of enzyme was defined as the amount that degrades 1 µmol of substrate (expressed as disaccharide) per min., at the optimum temperature for each enzyme. After 6 h incubation at either 20°C (for chondroitinase B or the mixture of chondroitinases B + AC) or 37°C (for chondroitinase AC), the incubation mixtures were spotted on Whatman no. 1 paper and chromatographed in isobutyric acid:1.25 M NH4OH (5:3, v/v) for 24 h. The products formed were visualized by silver nitrate staining of the paper chromatograms and quantified by densitometry.
For degradations with heparitinases I and II from Flavobacterium heparinum, aliquots of the glycosaminoglycans (50100 µg) were incubated with 2 x 104 units of either heparitinases I or II or a mixture of both enzymes, in 0.05 M ethylenediamine-acetate buffer, pH 7.0, in a final volume of 20 µl. One unit of enzyme was defined as the amount that degrades 1 µmol of substrate (expressed as disaccharide) per min. After 24 h incubation at 30°C, the incubation mixtures were spotted on Whatman no. 1 paper and chromatographed as above described for chondroitinases. The products formed were visualized by silver nitrate staining of the paper chromatograms and quantified by densitometry.
Isolation, identification, and quantification of urinary glycosaminoglycans
To assess the urinary glycosaminoglycan concentration, samples of urine (10 ml for normal rats and 50 ml for diabetic rats) were incubated with 400 µl of Amberlite IRA 900 at 60°C, under agitation (urine of normal rats was diluted with 10 ml of distilled water). After 1824 h, the resin was collected, exhaustively washed with hot water (60°C) and the glycosaminoglycans were eluted in a step wise fashion with 0.3 M NaCl (1 ml), 0.3 M NaCl (0.5 ml), 1 M NaCl (1 ml), and 1 M NaCl (0.5 ml). To the supernatants, four volumes of ethanol were added and the precipitates formed at 20°C overnight were collected by centrifugation and dried. The dried material was resuspended in water and analyzed as described in the previous section.
Polyacrylamide gel electrophoresis of the glycosaminoglycans was performed in a BRL vertical mini-system. Samples for SDSPAGE were mixed with sample buffer and submitted to electrophoresis as already described (Laemmli, 1970). Aliquots (5 µl) were applied to 6% gels and run for 4560 min at constant power. Gels were stained with toluidine blue.
Statistical analysis
Parametric and nonparametric statistical tests were used. Students two-sided t test for paired samples and the Wilcoxon nonparametric sample rank test for independent groups were used to compare the mean difference in body weight, blood glucose concentration, urinary creatinine concentration, 24 h urine volume, systolic blood pressure, albuminuria, and the urinary glycosaminoglycans of the experimental and control groups. The significance level is indicated in each experiment.
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Acknowledgments |
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Footnotes |
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References |
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Ayo,S.H., Radnik,R.A., Glass,W.F., Garoni,J.A., Rampt,E.R., Appling,D.R. and Kreisberg,J.I. (1991) Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high glucose medium. Am. J. Physiol., 269, F185F191.
Border,W.A., Okuda,S., Languino,L.R. and Ruoslahti,E. (1990) Transforming growth factor-ß regulates production of proteoglycans by mesangial cells. Kidney Int., 37, 689695.[ISI][Medline]
Bossolan,D., Ginoza,M., Santos,A.C., Tavares,A., Marson,O., Ribeiro,A.B. and Kohlmann Jr.,O. (1991) Insulin therapy in streptozotocin (STZ)-induced diabetes melitus (DM): effects upon blood pressure (BP) and microalbuminuria. Hypertension, 17, 461.
Brownlee,M., Cerami,A. and Vlassara,H. (1988) Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N. Engl. J. Med., 318, 13151321.[ISI][Medline]
Cássaro,C.M.F. and Dietrich,C.P. (1977) Distribution of sulfated mucopolysaccharides in invertebrates. J. Biol. Chem., 252, 22542261.[Abstract]
Cechowska-Pasko,M., Palka,J. and Bankowski,E. (1996) Decrease in the glycosaminoglycan content in the skin of diabetic rats. The role of IGF-I, IGF-binding proteins and proteolytic activity. Mol. Cell. Biochem., 154, 18.
Daimon,S. and Koni,I. (1998) Glomerular enlargement in the progression of mesangial proliferative glomerulonephritis. Clin. Nephrol., 49, 145152.[ISI][Medline]
Del Prete,D., Anglani,F., Ceol,M., DAngelo,A., Forino,M., Vianello,D., Baggio,B. and Gambaro,G. (1998) Molecular biology of diabetic glomerulosclerosis. Nephrol. Dial. Transplant., 13 (Suppl 8), 2025.
Dietrich,C.P. (1984) A model for cellcell recognition and control of cell growth mediated by sulfated glycosaminoglycans. Brazilian J. Med. Biol. Res., 17, 515.[ISI][Medline]
Dietrich,C.P. and Nader,H.B. (1974) Fractionation and properties of four heparitin sulfates from beef lung tissue. Isolation and partial characterization of a homogeneous species of heparitin sulfate. Biochim. Biophys. Acta, 343, 3444.[ISI][Medline]
Dietrich,C.P., Sampaio,L.O. and Toledo,O.M.S. (1976) Characteristic distribution of sulfated mucopolysaccharides in different tissues and in their respective mitochondria. Biochem. Biophys. Res. Commun., 71, 110.[ISI][Medline]
Dietrich,C.P., McDuffie,N. and Sampaio,L.O. (1977a) Identification of acidic muco-polysaccharides by agarose gel electrophoresis. J. Chromatogr., 130, 299304.[Medline]
Dietrich,C.P., Sampaio,L.O., Toledo,O.M.S. and Cássaro,C.M.F. (1977b) Cell recognition and adhesiveness. A possible role for the sulfated mucopolysaccharides. Biochem. Biophys. Res. Commun., 75, 329336.[ISI][Medline]
Dietrich,C.P., Armelin,H.A., Nogueira,Y.L., Nader,H.B. and Michelacci,Y.M. (1982) Turnover, change of composition with rate of cell growth and effect of phenylxyloside on synthesis and structure of cell surface sulfated glycosaminoglycans of normal and transformed cells. Biochim. Biophys. Acta, 717, 387397.[ISI][Medline]
Farquhar,M.G. (1991) The glomerular basement membrane: a selective macromolecular filter. In Hay,E.D. (ed.), Cell Biology of Extracellular Matrix, second edition. Plenum, New York, pp. 365418.
Fisher,L.W., Termine,J.D. and Young,M.F. (1989) Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) from several nonconnective tissues in a variety of species. J. Biol. Chem., 264, 45714576.
Funabiki,K., Makita,Y., Yamamoto,M., Shike,T., Fukui,M., Sumiyoshi,Y. and Tomino,Y. (1998) Dissociated expression of collagen type IV subchains in diabetic kidneys of KKAy mice. Nephron, 80, 208213.[ISI][Medline]
Hadad,S.J., Michelacci,Y.M. and Schor,N. (1996) Proteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. Biochim. Biophys. Acta, 1290, 1828.[ISI][Medline]
Harper,K. and Mason,R. (1994) Increased expression of decorin but not of biglycan in hyperglucaemic cultures of human mesangial cells. Biochem. Soc. Trans., 22, 184S.[ISI][Medline]
Iozzo,R.V. (1998) Matrix proteoglycans: from molecular design to cellular function. Annu. Rev. Biochem., 67, 609652.[ISI][Medline]
Iozzo,R.V., Cohen,I.R., Grassel,S. and Murdoch,A.D. (1994) The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem. J., 302, 625639.[ISI][Medline]
Jaackle-Meyer,I., Szukics,B., Neubauer,K., Metze,V., Petzoldt,R. and Stolte,H. (1995) Extracellular matrix proteins as early markers in diabetic nephropathy. Eur. J. Clin. Chem. Clin. Biochem., 33, 211219.[ISI][Medline]
Kado,S., Aoki,A., Wada,S., Katayama,Y., Kugai,N., Yoshizawa,N. and Nagata,N. (1996) Urinary type IV collagen as a marker for early diabetic nephropathy. Diabetes Res. Clin. Pract., 31, 103108.
Kamata,T., Muso,E., Yashiro,M., Kawamura,T., Oyama,A., Matsushima,H., Takeuchi,E., Yoshida,H. and Sasayama,S. (1990) Up-regulation of glomerular extracellular matrix and transforming growth factor-beta expression in RF/J mice. Kidney Int., 55, 864876.
Kanwar,Y.S., Linker,A. and Farquhar,M.G. (1980) Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion. J. Cell Biol., 86, 688693.[Abstract]
Kanwar,Y.S., Jakubowski,M.L. and Rosenzweig,L.J. (1983a) Distribution of sulfated glycosaminoglycans in the glomerular basement membrane and mesangial matrix. Eur. J. Cell Biol., 31, 290295.[ISI][Medline]
Kanwar,Y.S., Rosenzweig,L.J. and Jakubowski,M.L. (1983b) Distribution of de novo synthesized sulfated glycosaminoglycans in the glomerular basement membrane and mesangial matrix. Lab. Invest., 49, 216225.[ISI][Medline]
Klein,D.J., Brown,D.M. and Oegema,T.R., Jr. (1986) Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli. J. Biol. Chem., 261, 1663616652.
Knecht,R., Leber,R. and Hasslacher,C. (1987) Degradation of glomerular basement membrane in diabetes. I. Susceptibility of diabetic and nondiabetic basement membrane to proteolytic degradation of isolated glomeruli. Res. Exp. Med., 187, 323328.[ISI][Medline]
Krusius,T. and Ruoslahti,E. (1986) Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc. Natl Acad. Sci. USA, 83, 76837687.[Abstract]
Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Mancini,G., Carbonara,A.O. and Heremans,J.F. (1965) Immunochemical quantitation of antigens by single radial immunodifusion. Immunochemistry, 2, 235254.[Medline]
Matsumae,T., Jimi,S., Uesugi,N., Takebayashi,S. and Naito,S. (1999) Clinical and morphometrical interrelationships in patients with overt nephropathy induced by non-insulin-dependent diabetes mellitus. A light- and electron-microscopy study. Nephron, 81, 4148.[ISI][Medline]
Mauer,S.N., Lane,P., Hattori,M., Fioretto,P. and Steffes,M.W. (1992) Renal structure and function in insulin-dependent diabetes mellitus and type I membranoproliferative glomerulonephritis in humans. J. Am. Soc. Nephrol., 2, S181S184.[ISI][Medline]
Michelacci,Y.M., Horton,D.S.P.Q. and Población,C.A. (1987) Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum. Biochim. Biophys. Acta, 923, 291301.[ISI][Medline]
Mizuno,S., Mizuno-Horikawa,Y., Yue,B.F., Okamoto,M. and Kurosawa,T. (1999) Nephrotic mice (ICGN strain): a model of diffuse mesangial sclerosis in infantile nephrotic syndrome. Am. J. Nephrol., 19, 7382.[ISI][Medline]
Nader,H.B., Porcionatto,M.A., Tersariol,I.L.S., Pinhal,M.A.S., Oliveira,F.W., Moraes,C.T. and Dietrich,C.P. (1990) Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. J. Biol. Chem., 265, 1680716813.
Oldberg,A. and Ruoslahti,E. (1982) Interaction between chondroitin sulfate proteoglycan, fibronectin and collagen. J. Biol. Chem., 257, 48594863.
Osterby,R., Asplund,J., Bangstad,H.J., Nyberg,G., Rudberg,S., Viberti,G. and Walker,J.D. (1997) Glomerular volume and the glomerular vascular pole area in patients with insulin-dependent diabetes mellitus. Virchows Arch., 431, 351357.[ISI][Medline]
Petricevich,V.L. and Michelacci,Y.M. (1990) Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages. Biochim. Biophys. Acta, 1053, 135143.[ISI][Medline]
Población,C.A. and Michelacci,Y.M. (1986) Structural differences of dermatan sulfates from different origins. Carbohydr. Res., 147, 87100.[ISI][Medline]
Ramos,O.L. (1988) Diabetes mellitus and hypertension. State of Art Lecture. Hypertension, 11 (Suppl I), I14I18.[Medline]
Ruoslahti,E. and Pierschacher,M.D. (1987) New perspectives in cell adhesion: EGD and integrins. Science, 238, 491497.[ISI][Medline]
Silbiger,S., Crowley,S., Shan,Z., Brownlee,M., Satriano,J. and Schlondorff,D. (1993) Non-enzymatic glycation of mesangial matrix and prolonged exposure of mesangial matrix to elevated glucose reduces collagen synthesis and proteoglycan charge. Kidney Int., 43, 853864.[ISI][Medline]
Spector,T. (1978) Refinement of the Coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay of 0.5 to 5 mg of protein. Anal. Biochem., 86, 142145.[ISI][Medline]
Stow,J.L. and Farquhar,M.G. (1985) Basement membrane heparan sulfate proteoglycans are concentrated in the lamina rara and in the podocytes of the rat renal glomerulus. Proc. Natl. Acad. Sci. USA, 82, 32963300.[Abstract]
Stow,J.L. and Farquhar,M.G. (1987) Distinctive populations of basement membrane and cell membrane heparan sulfate proteoglycans are produced by culture cell lines. J. Cell Biol., 105, 529539.[Abstract]
Tamsma,J.T., van den Born,J., Bruijn,J.A., Assmann,K.J.M., Weening,J.J., Berden,J.H.M., Wieslander,J., Schrama,E., Hermans,J., Veerkamp,J.H., Lemkes,H.H.P.J. and van der Woude,F.J. (1994) Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparan sulphate in the glomerular basement membrane. Diabetologogia, 37, 313320.
Templeton,D.M. (1989) Retention of glomerular basement membrane-proteoglycans accompanying loss of anionic site staining in experimental diabetes. Lab. Invest., 61, 202211.[ISI][Medline]
Tencer,J., Torffvit,O., Bjornsson,S., Thysell,H., Grubb,A. and Rippe,B. (1997) Decreased excretion of glycosaminoglycans in patients with primary glomerular diseases. Clin. Nephrol., 48, 212219.[ISI][Medline]
Thomas,G.J., Mason,R.M. and Davies,M. (1991) Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture. Biochem. J., 277, 8188.[ISI][Medline]
Toth,T. and Takebayashi,S. (1998) Glomerular hypertrophy as a prognostic marker in childhood IgA nephropathy. Nephron, 80, 285291.[ISI][Medline]
van den Born,J., van Kraats,A.A., Bakker,M.A.K., Assmann,K.J.M., Dijkman,H.B.P.M., van der Laak,J.A.W.M. and Berden,J.H.M. (1995) Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy. Diabetologia, 38, 11691175.[ISI][Medline]
Vogel,K.G. (1994) Glycosaminoglycans and Proteoglycans. In Extracellular Matrix Assembly and Structure. Academic Press, New York.
Vracko,R. (1974) Basal lamina layering in diabetes mellitus. Diabetes, 23, 94104.[ISI][Medline]