heparitin-sulfate and Albuminuria

The primary filtration of blood occurs in the glomerulus in the kidney. Destruction of any of the layers of the glomerular filtration barrier might result in proteinuric disease. The glomerular endothelial cells and especially its covering layer, the glycocalyx, play a pivotal role in development of albuminuria. One of the main sulfated glycosaminoglycans in the glomerular endothelial glycocalyx is heparan sulfate. The endoglycosidase heparanase degrades heparan sulfate, thereby affecting glomerular barrier function, immune reactivity and inflammation. Increased expression of glomerular heparanase correlates with loss of glomerular heparan sulfate in many glomerular diseases. Most importantly, heparanase knockout in mice prevented the development of albuminuria after induction of experimental diabetic nephropathy and experimental glomerulonephritis. Therefore, heparanase could serve as a pharmacological target for glomerular diseases. Several factors that regulate heparanase expression and activity have been identified and compounds aiming to inhibit heparanase activity are currently explored.

Topics: Albuminuria; Animals; Diabetic Nephropathies; Endothelial Cells; Glomerulonephritis; Glucuronidase; Heparitin Sulfate; Humans; Kidney Diseases; Kidney Glomerulus

Insulin-dependent diabetic patients with increased urinary albumin excretion are characterized by elevated blood pressure and declining kidney function. In addition, such patients have a high risk of atherosclerotic vascular disease, proliferative retinopathy, and cardiomyopathy, suggesting that albuminuria is a marker of widespread vascular dysfunction. Increased transport of macromolecules across the vascular wall, elevated plasma levels of von Willebrand factor, and impaired fibrinolytic capacity have been demonstrated in albuminuric patients. The cause of this vascular vulnerability in susceptible patients is unknown, but increasing evidence has suggested that loss of the proteoglycan heparan sulfate in the vasculature may explain the widespread nature of the disease. Heparan sulfate is important for the glomerular endothelial cell and basement membrane charge densities, the anticoagulant properties of the vessel wall, and the growth regulation of intimal smooth muscle cells. Recent studies have shown that heparin increases the biosynthesis of heparan sulfate in endothelial cell cultures and prevents the characteristic glomerular basement membrane thickening when given to diabetic rats. Moreover, heparin has been shown to reduce albuminuria in patients with incipient diabetic nephropathy. Although increasing evidence supports the hypothesis that loss of heparan sulfate may play a pathophysiological role in the development of diabetic vascular complications, there are still many unresolved problems. What are the mechanisms of action of glycosaminoglycans at the molecular biology level, and how can we select compounds without anticoagulant activity suitable for long-term use in the prevention and treatment of late diabetic complications?

Topics: Albuminuria; Animals; Basement Membrane; Diabetes Mellitus; Diabetic Angiopathies; Diabetic Nephropathies; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Proteoglycans

In summary, the decreased concentration of heparan sulphate within the extracellular matrix of patients with insulin-dependent diabetes mellitus is caused by a combination of genetic factors and poor metabolic control. Decreased concentrations of heparan sulphate are seen in patients with diabetes mellitus and proteinuria and this might be the explanation for the proteinuria as well as the expansion of the mesangium and the intimal dysfunction, including increased permeability of the vessel wall to macromolecules, which is present in such patients. Thus, the effective remodelling of extracellular matrix might explain coincidence of proteinuria, decline in renal function and premature atherosclerosis in patients with diabetes mellitus.

Topics: Albuminuria; Animals; Biomarkers; Cardiovascular Diseases; Diabetic Nephropathies; Heparitin Sulfate; Humans; Models, Biological; Risk Factors

Aldosterone contributes to end-organ damage in heart failure and chronic kidney disease. Mineralocorticoid-receptor inhibitors limit activation of the receptor by aldosterone and slow disease progression, but side effects, including hyperkalemia, limit their clinical use. Damage to the endothelial glycocalyx (a luminal biopolymer layer) has been implicated in the pathogenesis of endothelial dysfunction and albuminuria, but to date no one has investigated whether the glomerular endothelial glycocalyx is affected by aldosterone. In vitro, human glomerular endothelial cells exposed to 0.1 nM aldosterone and 145 mMol NaCl exhibited reduced cell surface glycocalyx components (heparan sulfate and syndecan-4) and disrupted shear sensing consistent with damage of the glycocalyx. In vivo, administration of 0.6 μg/g/d of aldosterone (subcutaneous minipump) and 1% NaCl drinking water increased glomerular matrix metalloproteinase 2 activity, reduced syndecan 4 expression, and caused albuminuria. Intravital multiphoton imaging confirmed that aldosterone caused damage of the glomerular endothelial glycocalyx and increased the glomerular sieving coefficient for albumin. Targeting matrix metalloproteinases 2 and 9 with a specific gelatinase inhibitor preserved the glycocalyx, blocked the rise in glomerular sieving coefficient, and prevented albuminuria. Together these data suggest that preservation of the glomerular endothelial glycocalyx may represent a novel strategy for limiting the pathological effects of aldosterone.

Topics: Albuminuria; Aldosterone; Animals; Cell Line; Disease Models, Animal; Endothelial Cells; Glycocalyx; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Renal Insufficiency, Chronic; Sodium Chloride; Syndecan-4

Glomerular basement membrane (GBM) damage plays a pivotal role in pathogenesis of albuminuria in diabetic nephropathy (DN). Heparan sulfate (HS) degradation induced by podocyte heparanase is the major cause of GBM thickening and abnormal perm-selectivity. In the present study, we aimed to examine the prophylactic effect of hyperoside on proteinuria development and GBM damage in DN mouse model and the cultured mouse podocytes. Pre-treatment with hyperoside (30 mg/kg/d) for four weeks could significantly decrease albuminuria, prevent GBM damage and oxidative stress in diabetes mellitus (DM) mice. Immunofluorescence staining, Real time PCR and Western blot analysis showed that decreased HS contents and increased heparanase expression in DN mice were also significantly improved by hyperoside pre-treatment. Meanwhile, transmission electron microscope imaging showed that hyperoside significantly alleviated GBM thickening in DN mice. In addition, hyperoside pre-treatment inhibited the increased heparanase gene (HPR1) promoter activity and heparanase expression induced by high glucose or reactive oxidative species (ROS) in cultured podocytes. Our data suggested that hyperoside has a prophylactic effect on proteinuria development and GBM damage in DM mice by decreasing podocyte heparanase expression.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Female; Gene Expression Regulation, Enzymologic; Glomerular Basement Membrane; Glucose; Glucuronidase; Heparitin Sulfate; Mice, Inbred C57BL; Podocytes; Promoter Regions, Genetic; Quercetin; Reactive Oxygen Species

We investigated the renoprotective effects of imidapril hydrochloride ((-)-(4 S)-3-[(2 S)-2-[[(1 S)-1-ethoxycarbonyl-3-phenylpropyl] amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril), an angiotensin-converting enzyme inhibitor, in a diabetic animal model. We used BKS.Cg-+Lepr(db)/+Lepr(db) (db/db) mice, a genetic animal model of obese type 2 diabetes. Diabetic db/db mice suffered from glomerular hyperfiltration, albuminuria and hypoalbuminemia. Oral administration of 5 mg/kg/day of imidapril for 3 weeks suppressed renal hyperfiltration, reduced albuminuria and normalized hypoalbuminemia. Imidapril did not influence body weights, blood pressure or blood glucose concentrations in db/db mice. Urinary excretion of heparan sulfate (HS) in non-treated 11-week-old db/db mice was significantly lower than that in age-matched non-diabetic db/+m mice. HS is a component of HS proteoglycans, which are present in glomerular basement membranes and glycocalyx of cell surfaces. Reduced urinary HS excretion indicated glomerular HS loss in db/db mice. Imidapril increased urinary excretion of HS to concentrations observed in db/+m mice, indicating that imidapril prevented the loss of renal HS. These results suggest that imidapril ameliorates renal hyperfiltration and loss of renal contents of HS. Improvement of filtration function and maintenance of HS, which is an important structural component of glomeruli, may contribute to renoprotective effects of imidapril.

Topics: Administration, Oral; Albuminuria; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Glucose; Diabetes Mellitus, Type 2; Heparitin Sulfate; Humans; Hypoalbuminemia; Imidazolidines; Kidney Glomerulus; Mice; Mice, Inbred NOD; Obesity

A reduced heparan sulphate (HS) expression in the glomerular basement membrane of patients with overt diabetic nephropathy is associated with an increased glomerular heparanase expression. We investigated the possible association of urinary heparanase activity with the development of proteinuria in patients with Type 1 diabetes (T1D), Type 2 diabetes (T2D), or membranous glomerulopathy (MGP) as non-diabetic disease controls.. Heparanase activity, albumin, HS and creatinine were measured in the urine of patients with T1D (n=58) or T2D (n=31), in patients with MGP (n=52) and in healthy controls (n=10). Heparanase messenger RNA (mRNA) expression in leukocytes was determined in a subgroup of patients with T1D (n=19).. Urinary heparanase activity was increased in patients with T1D and T2D, which was more prominent in patients with macroalbuminuria, whereas no activity could be detected in healthy controls. Albuminuria levels were associated with increased urinary heparanase activity in diabetic patients (r=0.20; P<0.05) but not in patients with MGP (r=0.11; P=0.43). A lower urinary heparanase activity was observed in diabetic patients treated with inhibitors of the renin-angiotensin-aldosterone system (RAAS), when compared to diabetic patients treated with other anti-hypertensives. Additionally, urinary heparanase activity was associated with age in T1D and MGP. In MGP, heparanase activity and β2-microglobulin excretion correlated. In patients with T1D, no differences in heparanase mRNA expression in leukocytes could be observed.. Urinary heparanase activity is increased in diabetic patients with proteinuria. However, whether increased heparanase activity is a cause or consequence of proteinuria requires additional research.

Topics: Adult; Aged; Albuminuria; Blotting, Western; Case-Control Studies; Diabetes Complications; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Follow-Up Studies; Glomerular Basement Membrane; Glucuronidase; Heparitin Sulfate; Humans; Immunoenzyme Techniques; Male; Middle Aged; Prognosis; Real-Time Polymerase Chain Reaction; Renin-Angiotensin System; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier.

Topics: Albuminuria; Animals; Glycosaminoglycans; Heparitin Sulfate; Mice; Mice, Knockout; N-Acetylglucosaminyltransferases; Phenotype; Podocytes; Proteinuria

One of the major complicating factors in insulin-dependent diabetes mellitus is nephropathy. Several investigators have linked heparan sulfate (HS) alterations in the glomerular basement membrane (GBM) with albuminuria as a marker of abnormal blood filtration and the subsequent progression to renal failure. In this study, we examined the fine structure of HS in the glomerulus and the GBM isolated from the kidneys of rats injected with streptozotocin. Using fluorophore-assisted carbohydrate electrophoresis, we obtained disaccharide composition analyses for HS. In a time course study, we observed that normal rat HS isolated from the GBM becomes more N-sulfated as the glomeruli mature over a period of 8 weeks. Diabetic rats injected with streptozotocin at the beginning of this period showed a reversal of this trend. Using a graded sieve technique, we found that two different sizes of glomeruli could be isolated from the rat kidneys and that there was a significant difference in the HS disaccharide content between these two pools of glomeruli. Only the larger sized glomeruli had less N-sulfation of HS as a result of insulin-dependent diabetes mellitus. This change in the fine structure of HS was localized to the GBM and was not associated with cell surface HS. We also generated oligosaccharides of HS that portray fine structural alterations in the diabetic rats indicative of a loss of the sulfation of N-acetylglucosamine.

Topics: Acetylglucosamine; Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disaccharides; Disease Models, Animal; Heparitin Sulfate; Kidney Glomerulus; Male; Oligosaccharides; Polysaccharide-Lyases; Rats; Rats, Sprague-Dawley; Sulfates

Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

Topics: Albuminuria; Animals; Glomerular Basement Membrane; Glycosaminoglycans; Heparitin Sulfate; Kidney; Male; Microscopy, Electron; Neuraminic Acids; Neuraminidase; Polysaccharide-Lyases; Proteinuria; Rats; Rats, Wistar

Topics: Albuminuria; Animals; Glomerular Basement Membrane; Glycosaminoglycans; Heparitin Sulfate; Mice; Neuraminidase; Polysaccharide-Lyases; Proteinuria; Rats

Recently, we identified specific N- and 6-O-sulphated heparan sulphate (HS) domains on activated glomerular endothelial cells. In this study, we evaluated in lupus nephritis the expression of different HS domains on glomerular endothelium and in the glomerular basement membrane (GBM).. The expression of specific glomerular HS domains and the presence of immunoglobulins (Ig) were determined by immunofluorescence staining of kidney sections of patients with nephritis due to systemic lupus erythematosus (SLE) and MRL/lpr lupus mice. The expression/presence of glomerular HS domains and Ig was also evaluated after eluting Ig from renal sections of lupus mice using two elution methods, and in renal sections of lupus mice treated with heparinoids.. Both MRL/lpr mice and patients with lupus nephritis showed a decreased expression of HS in the GBM. The expression of N- and 6-O-sulphated HS domains on glomerular endothelium was decreased in MRL/lpr mice, but increased in SLE patients. MRL/lpr mice had more extensive glomerular Ig deposits than SLE patients. After elution of Ig, the glomerular endothelial expression of N- and 6-O-sulphated HS domains in MRL/lpr mice was recovered and even increased above normal levels, while the expression of HS in the GBM was restored to normal levels. Treatment with heparinoids prevented Ig deposition and preserved the expression of glomerular HS domains at normal levels in lupus mice.. The expression of specific HS domains on glomerular endothelium and in the GBM is changed during lupus nephritis due to masking by Ig deposits and induction of inflammatory N- and 6-O-sulphated HS domains.

Topics: Adult; Albuminuria; Animals; Basement Membrane; Endothelial Cells; Female; Fluorescent Antibody Technique; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Immunoglobulins; Kidney Glomerulus; Lupus Erythematosus, Systemic; Lupus Nephritis; Male; Mice; Mice, Inbred MRL lpr; Staining and Labeling; Tissue Distribution

Heparan sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example, in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state. We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid--but not HS--from the GBM, and this led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage of proteins through the glomerular capillary wall.

Topics: Albuminuria; Animals; Basement Membrane; Biological Transport; Capillaries; Glycosaminoglycans; Heparitin Sulfate; Kidney Glomerulus; Microscopy, Electron; Models, Biological; Neuraminic Acids; Neuraminidase; Polysaccharide-Lyases; Proteinuria; Rats; Rats, Wistar

Diabetic nephropathy poses an increasing health problem in the Western world, and research to new leads for diagnosis and therapy therefore is warranted. In this respect, heparan sulfates (HSs) offer new possibilities because crude mixtures of these polysaccharides are capable of ameliorating proteinuria. The aim of this study is to immuno(histo)chemically profile HSs from microalbuminuric kidneys from patients with type 1 diabetes and identify specific structural HS alterations associated with early diabetic nephropathy.. Renal cryosections of control subjects and patients with type 1 diabetes were analyzed immunohistochemically by using a set of 10 unique phage display-derived anti-HS antibodies. HS structures defined by relevant antibodies were characterized chemically by means of enzyme-linked immunosorbent assay and probed for growth factor binding and presence in HS/heparin-containing drugs.. In all patients, HS structure defined by the antibody LKIV69 consistently increased in basement membranes of proximal tubules. This structure contained N- and 2-O-sulfates and was involved in fibroblast growth factor 2 binding. It was present in HS/heparin-containing drugs shown to decrease albuminuria in patients with diabetes. The HS structure defined by the antibody HS4C3 increased in the renal mesangium of some patients, especially those who developed macroalbuminuria within 8 to 10 years. This structure contained N- and 6-O-sulfates. For 8 other antibodies, no major differences were observed.. Specific structural alterations in HSs are associated with early diabetic nephropathy and may offer new leads for early diagnosis and the rational design of therapeutic glycomimetics.

Topics: Adolescent; Adult; Albuminuria; Antibodies; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Fibroblast Growth Factor 2; Fluorescent Antibody Technique; Glycosaminoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Kidney; Male

Heparan sulfate (HS) proteoglycans are major anionic glycoconjugates of the glomerular basement membrane and are thought to contribute to the permeability properties of the glomerular capillary wall. In this study we evaluated whether the development of (micro) albuminuria in early human and experimental diabetic nephropathy is related to changes in glomerular HS expression or structure. Using a panel of recently characterized antibodies, glomerular HS expression was studied in kidney biopsies of type I diabetic patients with microalbuminuria or early albuminuria and in rat renal tissue after 5 months diabetes duration. Glomerular staining, however, revealed no differences between control and diabetic specimens. A significant (p < 0.05) approximately 60% increase was found in HS N-deacetylase activity, a key enzyme in HS sulfation reactions, in diabetic glomeruli. Structural analysis of glomerular HS after in vivo and in vitro radiolabeling techniques revealed no changes in HS N-sulfation or charge density. Also HS chain length, protein binding properties, as well as disaccharide composition did not differ between control and diabetic glomerular HS samples. These results indicate that in experimental and early human diabetic nephropathy, increased urinary albumin excretion is not caused by loss of glomerular HS expression or sulfation and suggest other mechanisms to be responsible for increased glomerular albumin permeability.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Rats; Rats, Wistar; Streptozocin

Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. The glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks. At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. The onset of the change in glyco-samino-glycan excretion rate was a very early event, appearing in the second week after diabetes induction. The main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. The heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.

Topics: Albuminuria; Animals; Chondroitin Sulfates; Dermatan Sulfate; Diabetes Mellitus, Experimental; Glycosaminoglycans; Heparitin Sulfate; Kidney; Male; Rats; Rats, Wistar; Reference Values; Time Factors

Forty-nine normoalbuminuric diabetic patients were studied: 22 males and 27 females, in whom urinary heparan sulphate (HS), albuminuria, creatininemia, creatininuria, creatinine clearance, HbA1c and arterial pressure (AP) were determined. Two groups were discerned: group 1, Type 1 DM, diabetic cases (n = 16); and group 2, Type 2 DM diabetic cases (n = 33). Patients were compared with 24 healthy controls: 12 men and 12 women, who showed a mean value +/- SD of 0.36 +/- 0.18 mg/24 h HS with significant differences between males and females (0.43 +/- 0.15 versus 0.28 +/- 0.17, respectively; p = 0.02). The total population of diabetic cases rendered a mean of 0.68 +/- 0.44 and comparison with controls proved highly significant (p < 0.001). Globally, male patients had a mean of 0.82 +/- 0.48 and females 0.54 +/- 0.35, with p < 0.02. Group 1 and 2 values of HS were not significantly different. HS levels failed to correlate either with age, body mass index (BMI), time since onset of diabetes, albuminuria, creatininemia, creatininuria, creatinine clearance, HbA1c or arterial hypertension. To conclude: both normal and diabetic males eliminate a greater quantity of HS than females. Normoalbuminuric diabetic patients of both types eliminate a greater quantity of HS regardless of arterial pressure and time since onset of diabetes.

Topics: Adolescent; Adult; Aged; Albuminuria; Blood Pressure; Case-Control Studies; Diabetes Mellitus, Type 1; Female; Heparitin Sulfate; Humans; Male; Middle Aged; Sex Factors

In a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane, was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria. Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274+/-68 mg/18 h at week 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P = 0.016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (r(s) = -0.82 and r(s) = -0.75, respectively, both mAb P < 0.0001). This decrease in heparan sulfate staining was due to a progressive reduction of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules.

Topics: Albuminuria; Animals; Autoantibodies; Basement Membrane; Capillaries; Complement System Proteins; Female; Free Radicals; Glomerulonephritis; Heparitin Sulfate; Immunoglobulin G; Kidney Glomerulus; Nitrogen; Proteoglycans; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Staining and Labeling

In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the noncollagenous domain of alpha1(collagen IV) and alpha3(collagen IV), against transforming growth factor beta(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy.

Topics: Adult; Aged; Albuminuria; Basement Membrane; Biopsy; Blood Pressure; Cross-Sectional Studies; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Extracellular Matrix; Extracellular Matrix Proteins; Heparitin Sulfate; Humans; Immunohistochemistry; Middle Aged; Reference Values; Renal Replacement Therapy; Skin

To evaluate the effect of interleukin-8 (IL8) on glomerular basement membrane (GBM) sulfated compounds and albuminuria, we infused IL8 in 1% bovine serum albumin (BSA) for 5 days into the left renal artery of Holtzman male rats at the rate of 10 microliters/h using an osmotic pump. Control rats received 1% BSA. A significant increase in urinary albumin/creatinine ratio was seen on the last day of IL8 infusion (0.38 +/- 0.11, mean +/- SEM) when compared with albumin/creatinine ratio prior to infusion (0.19 +/- 0.04, P = 0.04). No significant differences in urinary albumin excretion prior to and after infusion of 1% BSA were observed. On the last day of infusion, rats were injected with 35sulfate (1.0 mCi/200 g body weight) intraperitoneally and killed after 8 h. Glomeruli were isolated and GBM obtained. After 5 days of IL8 administration, there was a significant increase in 35sulfate uptake by GBM of the infused kidney (76 +/- 10 cpm/dry glomerular weight, mean +/- SEM) compared with the uptake seen in the contralateral kidney (53 +/- 9, P = 0.05). The in vivo infusion of IL8 increased the 35sulfate uptake by GBM and augmented the urinary albumin/creatinine ratio, suggesting that IL8 may induce albuminuria by altering the metabolism of the GBM sulfated compounds. This hypothesis needs to be confirmed by studies on glomerular charge selectivity and GBM anionic sites during the course of the infusion. Moreover, the persistence of the effect needs to be evaluated by prolonging the infusion for more than 5 days.

Topics: Albuminuria; Animals; Cell Membrane Permeability; Creatinine; Glomerular Mesangium; Glycosaminoglycans; Heparitin Sulfate; Infusions, Intravenous; Interleukin-8; Kidney Glomerulus; Male; Rats; Renal Artery; Sulfates; Sulfur Radioisotopes

We examined whether blood pressure reduction or good glycemic control equally lower albuminuria by preventing glomerular loss of heparan sulfate and progression of glomerulosclerosis in streptozotocin-induced diabetic rats. We used doxazosin, and alpha 1-adrenergic blocker, to lower systemic blood pressure, and good glycemic control was achieved by insulin treatment. Rats were killed after 20 weeks of treatment. Doxazosin significantly lowered systolic pressure in diabetic rats; however, it had no effect in normal rats. Good glycemic control also lowered systolic pressure. In diabetic rats with good glycemic control, doxazosin had an additive effect on blood pressure. Glomerular heparan sulfate synthesis was significantly lower and urinary albumin excretion higher in diabetic than in normal rats. Both doxazosin treatment and good glycemic control normalized these abnormalities in diabetic rats. Insulin normalized plasma glucose and glycosylated HbA1 concentrations in diabetic rats, as did doxazosin. Significant increases in mesangial area and glomeruloscelerosis were observed in diabetic rats. Only good glycemic control normalized these pathological changes in all diabetic rats. Two-way factorial ANOVA showed an interaction between the effects of doxazosin and insulin on systolic pressure and plasma glucose. The data show that after 20 weeks of doxazosin treatment, albuminuria was reduced by 80%; however, this treatment had no significant effect on mesangial expansion or progression to glomerulosclerosis. Conversely, good glycemic control prevented all three of the preceding sequelae.

Topics: Adrenergic alpha-Antagonists; Albuminuria; Animals; Blood Glucose; Blood Pressure; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Doxazosin; Glomerular Mesangium; Glomerulosclerosis, Focal Segmental; Heparitin Sulfate; Incidence; Insulin; Kidney Glomerulus; Male; Proteinuria; Rats; Rats, Wistar; Systole

Nephropathy is a serious microvascular complication of diabetes mellitus which is preceded by a period of microalbuminura. Increased loss of proteoglycan (PG) from glomerular basement (GBM) has been postulated to alter glomerular charge selectivity which contributes to urinary loss of albumin. In this study we measured the excretion of urinary glycosaminoglycans (GAG), the degradation products of PG, in 82 non-insulin-dependent (NIDDM) (Type 2) diabetic and 34 non-diabetic subjects. We found that diabetic subjects had a significantly higher GAG urinary excretion rate compared to non-diabetic subjects (12.54 +/- 5.67 vs 8.80 +/- 3.99 micrograms glucuronic acid min-1, p = 0.0001). Categorizing for albuminuric status shows that the diabetic normo-, micro- and macroalbuminuric groups have a higher GAG excretion rate than non-diabetic subjects. Heparan sulphate (HS) GAG urinary excretion was measured in 25 samples from diabetic subjects and 18 non-diabetic subjects. Diabetic subjects excreted more HS GAG than controls both as a rate or as a percentage of total GAG (3.70 +/- 1.94 vs 2.38 +/- 1.48 micrograms glucosamine min-1, p = 0.02; 31.6% +/- 12.5 vs 23.1% +/- 10.4, p = 0.02). Categorizing for albuminuric status shows that micro- and macro-albuminuric groups have a significantly higher HS GAG excretion rate than non-diabetic subjects. We conclude that, as in IDDM, excretion of GAG and HS GAG is higher in NIDDM and may precede the development of microalbuminuria.

Topics: Age Factors; Albuminuria; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Middle Aged; Reference Values; Regression Analysis

Recently we found in biopsies of human lupus nephritis a nearly complete loss of heparan sulfate (HS) staining in the glomerular basement membrane (GMB). To clarify the relationship between HS staining and albuminuria in lupus nephritis, we studied MRL/lpr mice with short (< 7 days) or prolonged duration of albuminuria (14-21 days) and compared these with mice of different ages without albuminuria. Kidney sections were stained for mouse immunoglobulin (Ig), HS, heparan sulfate proteoglycan (HSPG)-core protein and laminin in immunofluorescence. In mice with prolonged albuminuria HS staining in the glomerular capillary loops had almost completely disappeared, whereas staining was unaltered in non-albuminuric mice (P = 0.001). In mice with short duration of albuminuria, there was a tendency toward a decrease of HS staining (P = 0.06). The expression of HSPG-core protein and other extra cellular matrix (ECM) components was unaltered in all groups. HS staining correlated inversely with albuminuria (rs = -0.55; P < 0.001) and with staining of Ig deposits in the capillary loops (rs = -0.74; P < 0.001). Despite the nearly complete loss of HS staining in the GBM in mice with prolonged albuminuria, there was no change in glomerular HS content as assessed by agarose electrophoresis and HS inhibition ELISA. We conclude that the development of albuminuria in MRL/lpr mice is accompanied by a loss of HS staining in the GBM, probably due to the masking of HS by deposits of Ig. In vitro studies revealed that autoantibodies complexed to nucleosomal antigens can inhibit the binding of the anti-HS monoclonal antibody to HS. Whether this also occurs in vivo remains to be determined.

Topics: Albuminuria; Animals; Basement Membrane; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Heparitin Sulfate; Kidney; Kidney Glomerulus; Lupus Nephritis; Mice; Mice, Inbred Strains; Staining and Labeling

Topics: Albuminuria; Animals; Basement Membrane; Capillary Permeability; Diabetes Mellitus; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Proteoglycans

Urinary excretion of heparan sulphate proteoglycan (HSPG), the main anionic component of the glomerular basement membrane (GBM), was estimated in 30 adolescents and young adults with insulin dependent diabetes (IDDM), 10 with microalbuminuria and 20 sex matched, diabetic controls of similar age without evidence of microalbuminuria. A further 10 non-diabetic control subjects were also examined. Both groups of patients with diabetes had significantly elevated excretion of HSPG when compared to normal individuals. There was no difference in HSPG excretion between diabetic subjects with and without microalbuminuria.

Topics: Adolescent; Adult; Albuminuria; Child; Creatinine; Diabetes Mellitus, Type 1; Female; Glycated Hemoglobin; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Male; Predictive Value of Tests; Proteoglycans

The effect of short term (8 weeks) sodium (Na+) depletion and its repletion on glomerular synthesis of heparan sulfate and urinary excretions of albumin, total protein, heparan sulfate, Na+ and potassium (K+) was studied in spontaneous hypertensive rats (SHR) and their control normotensive Wistar-Kyoto rats (WKY). Na+ depletion in SHRs significantly increased the synthesis of glomerular heparan sulfate and decreased urinary excretions of albumin, Na+ and heparan sulfate when compared with the Na+ repleted group. In WKY rats, Na+ depletion did not cause any of the above changes. These data suggest that Na+ depletion prevents the urinary loss of protein through preservation of glomerular heparan sulfate only in SHRs.

Topics: Albuminuria; Animals; Blood Pressure; Body Weight; Drinking; Glycosaminoglycans; Heparitin Sulfate; Hypertension; Kidney Glomerulus; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sodium

Heparan sulphate-associated anionic sites in the glomerular basement membrane were studied in rats 8 months after induction of diabetes by streptozotocin and in age- adn sex-matched control rats, employing the cationic dye cuprolinic blue. Morphometric analysis at the ultrastructural level was performed using a computerized image processor. The heparan sulphate specificity of the cuprolinic blue staining was demonstrated by glycosaminoglycan-degrading enzymes, showing that pretreatment of the sections with heparitinase abolished all staining, whereas chondroitinase ABC had no effect. The majority of anionic sites (74% in diabetic and 81% in control rats) were found within the lamina rara externa of the glomerular basement membrane. A minority of anionic sites were scattered throughout the lamina densa and lamina rara interna, and were significantly smaller than those in the lamina rara externa of the glomerular basement membrane (p<0.001 and p<0.01 for diabetic and control rats, respectively). Diabetic rats progressively developed albuminuria reaching 40.3 (32.2-62.0) mg/24 h after 8 months in contrast to the control animals (0.8 (0.2-0.9) mg/24 h, p<0.002). At the same time, the number of heparan sulphate anionic sites and the total anionic site surface (number of anionic sites x mean anionic site surface) in the lamina rara externa of the glomerular basement membrane was reduced by 19% (p<0.021) and by 26% (p<0.02), respectively. Number and total anionic site surface in the remaining part of the glomerular basement membrane (lamina densa and lamina rara interna) were not significantly changed. We conclude that in streptozotocin-diabetic rats with an increased urinary albumin excretion, a reduced heparan sulphate charge barrier/density is found at the lamina rara externa of the glomerular basement membrane.

Topics: Albuminuria; Analysis of Variance; Animals; Anions; Basement Membrane; Blood Glucose; Chondroitin Lyases; Coloring Agents; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Heparitin Sulfate; Kidney Glomerulus; Male; Rats; Rats, Wistar

Examination was made of changes in the anionic sites of the glomerular basement membrane (GBM) in rats with streptozotocin (STZ)-induced diabetes by the immersion method of polyethyleneimine (PEI). PEI particles in GBM of diabetic rats significantly decreased from the 1st through the 8th week. Urinary albumin excretion in diabetic rats significantly increased at the 2nd but not earlier week. Insulin treatment effectively prevented decrease in PEI particles in STZ-injected rats. In rats with STZ-induced diabetes, initial renal alteration was disturbance of the charge barrier, followed by the development of albuminuria. Continued deterioration of anionic sites and possibly additional disturbance of size barrier were considered responsible for the development of albuminuria. Insulin treatment appears to prevent the loss of anionic sites of GBM.

Topics: Albuminuria; Animals; Anions; Basement Membrane; Binding Sites; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Glomerulus; Male; Microscopy, Electron; Molecular Probes; Polyethyleneimine; Proteoglycans; Rats; Rats, Wistar

Decreased glomerular heparan sulfate synthesis could account for the increased albuminuria observed in experimental diabetes. A similar albuminuria has been observed in galactose-fed animals. To test whether heparan sulfate synthesis is also decreased in galactosemic animals we measured the heparan sulfate synthesis in isolated renal glomeruli from streptozotocin diabetic and normal rats fed regular diets or 30% galactose diets for 11-12 weeks. Albumin excretion and glomerular heparan sulfate synthesis were determined at the time of sacrifice. As predicted by this hypothesis glomerular heparan sulfate synthesis was significantly lower in the diabetic group than in normals given regular diets (309 +/- 23 vs 487 +/- 56 dpm/mg glomerular wt.; P < 0.01), and albumin excretion greater in these diabetics than in the normals (20 +/- 4.3 vs 2.0 +/- 0.7 mg/24 hr; P < 0.002). Albumin excretion and glomerular heparan sulfate synthesis were also consistent with the hypothesis, but unpredictably reversed, in the galactose-fed diabetic group. Glomerular heparan sulfate synthesis was significantly increased in this group (713 +/- 67 dpm/mg glomerular wt.) and albumin excretion decreased (10.7 +/- 3.6 mg/24 hr) compared to the diabetic on regular diet. Some possible explanations are discussed, but the galactose effect described here remains phenomenological.

Topics: Albuminuria; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Diet; Galactose; Galactosemias; Heparitin Sulfate; Kidney Glomerulus; Male; Rats; Rats, Sprague-Dawley; Streptozocin

Calcium entry blockers, particularly diltiazem, have been shown to lower not only systemic blood pressure but also improve proteinuria in non-insulin-dependent diabetic patients. The presence of proteinuria is attributed to the loss of glomerular heparan sulfate, which confers a negative charge on the basement membrane. In the present study, we evaluated the efficacy of diltiazem in lowering blood pressure and proteinuria in diabetic rats and also examined the possibility that diltiazem prevents proteinuria through glomerular preservation of heparan sulfate. Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg). One group of diabetic rats was treated with diltiazem (25 mg/L) in drinking water for 20 weeks. Another group of diabetic rats and a group of nondiabetic rats were given tap water only. Systolic blood pressure was measured at 4, 8, 12, and 20 weeks. Urinary excretion of albumin was done at 4, 8, 12, 16, and 20 weeks. At the end of 20 weeks, all rats were killed, kidneys were removed, and glomeruli were isolated. Total glycosaminoglycan and heparan sulfate synthesis were determined by incubating glomeruli in the presence of [35S]sulfate. Diltiazem lowered blood pressure significantly in diabetic rats at 8, 12, and 20 weeks. Diabetic glomeruli synthesized less total glycosaminoglycan and heparan sulfate than glomeruli from normal rats. Characterization of heparan sulfate by ion-exchange chromatography showed that the fraction eluted with 1 M NaCl was significantly lower and the fraction eluted with 1.25 M NaCl significantly higher in diabetic than in normal rats. Diltiazem therapy returned not only glomerular synthesis but also various fractions of heparan sulfate to normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Albuminuria; Animals; Blood Pressure; Diabetes Mellitus, Experimental; Diltiazem; Heparitin Sulfate; Kidney Glomerulus; Male; Rats; Rats, Wistar

To evaluate the specificity of a raised heparan sulphate (HS) excretion previously reported in four children with congenital nephrotic syndrome (CNS), we measured the urinary excretion of HS and chondroitin sulphate (CS) in seven children with Finnish-type congenital nephrotic syndrome (CNSF), seven with diffuse mesangial sclerosis (DMS), nine with focal segmental glomerulosclerosis (FSGS), 14 with steroid-sensitive nephrotic syndrome of whom eight had a biopsy confirming minimal change histology (SSNS), and 17 controls. The urine HS/CS ratio in normal children had a median of 0.36 (observed range 0.21 to 0.68) and was independent of age. HS/CS ratio was significantly greater than controls in CNSF (median 0.80, range 0.43 to 1.28), DMS (median 0.81, range 0.49 to 1.13) and FSGS children (median 0.66, range 0.38 to 1.6), but was not in SSNS (median 0.44, range 0.28 to 0.70). There was a positive correlation between the HS/CS ratio and urine albumin excretion. High HS/CS ratios are not diagnostic of a particular histological variety of CNS.

Topics: Albuminuria; Child; Child, Preschool; Chondroitin Sulfates; Creatinine; Female; Glomerulosclerosis, Focal Segmental; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant; Male; Nephrosis, Lipoid; Nephrotic Syndrome; Steroids

Angiotensin converting enzyme (ACE) inhibitors, particularly enalapril and captopril, have been shown to decrease proteinuria in diabetic animals and human subjects. Since heparan sulfate proteoglycan confers a negative charge on the glomerular basement membrane, and either decreased synthesis or loss of this charge causes albuminuria in diabetic animals, we examined the possibility that enalapril prevents albuminuria through glomerular preservation of heparan sulfate in long-term diabetic rats. A total of 22 male Wistar rats were used in the study. Diabetes was induced in 15 rats by a single intraperitoneal injection of streptozotocin (60 mg/kg). The remaining 7 rats received buffer. One week following induction of diabetes, 8 diabetic rats were allowed to drink tap water containing enalapril at a concentration of 50 mg/liter; the remaining 7 diabetic and 7 nondiabetic rats were given only tap water. The drug treatment was continued for 20 weeks. Systolic blood pressure and 24-hr urinary excretion of albumin were measured at 2, 8, 16, and 20 weeks. At the end of 20 weeks, all rats were killed, kidneys were removed, and glomeruli were isolated by differential sieving technique. Total glycosaminoglycan and heparan sulfate synthesis was determined by incubating glomeruli in the presence of [35S]sulfate. Characterization of heparan sulfate was performed by ion-exchange chromatography. Systolic blood pressures were significantly lower in enalapril-treated diabetic rats compared to untreated diabetic rats. Diabetic glomeruli synthesized less heparan sulfate than glomeruli from nondiabetic rats. Also, glomerular heparan sulfate content of diabetics was significantly lower than that of nondiabetics. Further characterization of heparan sulfate showed that the fraction eluted with 1 M NaCl was significantly lower and the fraction eluted with 1.25 M NaCl significantly higher in diabetic than in normal rats. Enalapril treatment normalized not only glomerular synthesis and content but also various fractions of heparan sulfate in diabetic rats. Diabetic rats excreted increased quantities of heparan sulfate and albumin than nondiabetic rats. Enalapril therapy prevented both these increases in diabetic rats. These data suggest that enalapril treatment improves albuminuria through preservation of glomerular heparan sulfate and prevention of its urinary loss in diabetic rats.

Topics: Albuminuria; Animals; Basement Membrane; Diabetic Nephropathies; Enalapril; Glycosaminoglycans; Heparitin Sulfate; Kidney Glomerulus; Male; Rats; Rats, Inbred Strains

The pathogenesis of clinical nephropathy in Type 1 (insulin-dependent) diabetes was investigated by measuring renal fractional clearances of albumin, total IgG, IgG4 and beta 2-microglobulin, four plasma proteins which differ in size and charge. Seventy patients and eleven control subjects were studied. In diabetic patients with normal urinary albumin excretion (less than 30 mg/24 hr), fractional IgG clearance was two to three times higher than in control subjects, whereas fractional clearance of the anionic plasma proteins IgG4 and albumin was similar to that of control subjects. These alterations indicate an increase in anionic pore charge within the glomerular basement membrane concomitant with an increase in either pore size or impairment of tubular reabsorption. Diabetic patients, whose urinary albumin excretion has started to rise (30 to 100 mg/24 hr), had unchanged fractional IgG compared to patients with normal albumin excretion, while fractional IgG4 and albumin clearances were increased three- to fourfold; indicating unchanged glomerular pore size, but a decrease in anionic pore charge. In patients demonstrating urinary albumin excretion of greater than 100 mg/24 hr fractional IgG clearance increased to the same extent as fractional albumin clearance, indicating an increase in large pore area. Fractional beta 2-microglobulin clearances were similar to that of control subjects in the different patient groups indicating unchanged tubular reabsorption of proteins. Thus, the increase in large pore area seen in patients with clinical nephropathy is preceded by loss of anionic charge in the glomerular basement membrane. It is likely that this loss of anionic charge is due to loss of heparan sulphate-proteoglycan.

Topics: Adult; Albuminuria; Basement Membrane; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Female; Heparitin Sulfate; Humans; Immunoglobulin G; Kidney Glomerulus; Male; Metabolic Clearance Rate