heparitin-sulfate and Diabetes-Mellitus--Type-1

Previous studies and ongoing research indicate the importance of an interaction between a putative receptor on dividing cells in hyperglycemia and the non-reducing end motifs of heparin stored in mast cell secretory granules and how this interaction prevents activation of hyaluronan synthesis in intracellular compartments and subsequent autophagy. This suggests a new role for endosomal heparanase in exposing this cryptic motif present in the initial large heparin chains on serglycin and in the highly sulfated (NS) domains of heparan sulfate.

Topics: Animals; Autophagy; Cell Division; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Endosomes; Extracellular Matrix; Gene Expression; Glucuronidase; Heparin; Heparitin Sulfate; Humans; Hyaluronic Acid; Mast Cells; Mesangial Cells; Proteoglycans; Rats; Receptors, Cell Surface; Secretory Vesicles; Vesicular Transport Proteins

Heparanase (Hpse) is an endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D.

Topics: Animals; Cell Proliferation; Diabetes Mellitus, Type 1; Enzyme Inhibitors; Extracellular Matrix; Free Radicals; Gene Expression Regulation; Glucuronidase; Heparitin Sulfate; Humans; Islets of Langerhans; Mice; Oligosaccharides; Signal Transduction; T-Lymphocytes

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Hemodynamics; Heparitin Sulfate; Humans; Kidney Glomerulus; Kidney Tubules; Molecular Sequence Data

Diabetic nephropathy is a progressive renal disease with thickening of the glomerular basement membrane and mesangial expansion and proliferation as histological hallmarks. The presence of the glycosaminoglycan side chains of heparan sulfate proteoglycan, an important constituent of the glomerular basement membrane, is decreased in diabetic nephropathy proportionally to the degree of proteinuria. Danaparoid sodium is a mixture of sulfated glycosaminoglycans consisting mainly of heparan sulfate. The study presented here involved performing a randomized placebo-controlled crossover study with danaparoid sodium in diabetic patients with overt proteinuria. The aim of the study was to evaluate the effect on proteinuria and safety/tolerability. Nine patients completed the study, without major side effects; the crossover study consisted of two 6-wk periods of treatment with 750 anti-Xa units danaparoid sodium subcutaneously once-daily or placebo. Following danaparoid sodium, significant declines of both albuminuria and proteinuria were found. After danaparoid sodium, the albumin excretion ratio standardized for urinary creatinine reduced with 17% in comparison with an increase of 23% after placebo (95% confidence interval of the difference,-75.9-3.9%; P = 0.03). The percentage change of the urinary protein excretion corrected for urinary creatinine differed at 8 wk significantly between both treatment arms (P = 0.001). Additional parameters for safety as hematological, hemostasis, biochemical parameters, and fundusphotography did not show any clinically significant difference for both groups. Only two patients had minor skin hematomas at the injection site while using danaparoid sodium. In conclusion, the supplementation was found to be feasible and was not associated with side effects. A significant decline of proteinuria was found. More prospective dose-finding and long-term studies must be performed to see whether danaparoid sodium could not only induce a reduction of proteinuria but also halt the progression of renal disease.

Topics: Adult; Chondroitin Sulfates; Cross-Over Studies; Dermatan Sulfate; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Double-Blind Method; Drug Combinations; Female; Heparinoids; Heparitin Sulfate; Humans; Male; Middle Aged; Proteinuria; Treatment Outcome

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

Topics: Adolescent; Adult; Biomarkers; Case-Control Studies; Cells, Cultured; Child; Child, Preschool; Diabetes Mellitus, Type 1; Disease Progression; Female; Heparitin Sulfate; Humans; Infant; Islets of Langerhans; Male; Sensitivity and Specificity; Young Adult

Patients with type 1 diabetes and end-stage renal disease with simultaneous pancreas and kidney (SPK) or kidney transplants alone (KA) were recruited 9-12 years post transplantation. We investigated differences between these groups with regard to inflammatory parameters and long-term structural changes in kidneys.. Blood samples were analyzed by ELISA and multiplex for chemokines, cytokines, growth factors, cell adhesion molecules and matrix metalloproteinases. Kidney graft biopsies were analyzed by electron microscopy for glomerular basement membrane thickness. Heparan- and chondroitin sulfate disaccharide structures were determined by size exclusion chromatography mass-spectrometry.. The SPK and the KA group had average glycated hemoglobin A1c (HbA1c) of 5.8% (40 mmol/mol) and 8.6% (70 mmol/mol) respectively. SPK recipients also had 16.2% lower body mass index (BMI) and 46.4% lower triglyceride levels compared with KA recipients, compatible with an improved metabolic profile in the SPK group. Plasminogen activator inhibitor (PAI-1), C-reactive protein (CRP) and vascular endothelial growth factor (VEGF) were lower in the SPK group. In kidney graft biopsies of the KA-patients an 81.2% increase in average glomerular basement membrane thickness was observed, accompanied by alterations in heparan sulfate proteoglycan structure. In addition to a decrease in 6-O-sulfated disaccharides, an increase in non-N-sulfated disaccharides with a corresponding slight decrease in N-sulfation was found in kidney biopsies from hyperglycemic patients.. Patients with end stage renal disease subjected to KA transplantation showed impaired inflammatory profile, increased thickness of basement membranes and distinct changes in heparan sulfate structures compared with SPK recipients.

Topics: Adult; Cross-Sectional Studies; Diabetes Mellitus, Type 1; Female; Heparitin Sulfate; Humans; Hyperglycemia; Inflammation; Kidney; Kidney Failure, Chronic; Kidney Transplantation; Male; Middle Aged; Pancreas Transplantation; Proteoglycans

Islet beta cells in situ express intracellular heparan sulfate (HS), a property previously shown in vitro to be important for their survival. We report that HS levels inside islet beta cells correlate with the novel intracellular localization of the HSPG core proteins for collagen type XVIII (Col18), a conventional extracellular matrix component. Syndecan-1 (Sdc1) and CD44 core proteins were similarly localized inside beta cells. During isolation, mouse islets selectively lose HS to 11-27% of normal levels but retain their HSPG core proteins. Intra-islet HS failed to recover substantially during culture for 4 days and was not reconstituted in vitro using HS mimetics. In contrast, significant recovery of intra-islet HS to ∼40-50% of normal levels occurred by 5-10 days after isotransplantation. Loss of islet HS during the isolation procedure is independent of heparanase (a HS-degrading endoglycosidase) and due, in part, to oxidative damage. Treatment with antioxidants reduced islet cell death by ∼60% and increased the HS content of isolated islets by ∼twofold compared to untreated islets, preserving intra-islet HS to ∼60% of the normal HS content of islets in situ. These findings suggest that the preservation of islet HS during the islet isolation process may optimize islet survival posttransplant.

Topics: Animals; Biopsy, Needle; Cell Survival; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Flow Cytometry; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

The autoimmune type 1 diabetes (T1D) that arises spontaneously in NOD mice is considered to be a model of T1D in humans. It is characterized by the invasion of pancreatic islets by mononuclear cells (MNCs), which ultimately leads to destruction of insulin-producing β cells. Although T cell dependent, the molecular mechanisms triggering β cell death have not been fully elucidated. Here, we report that a glycosaminoglycan, heparan sulfate (HS), is expressed at extraordinarily high levels within mouse islets and is essential for β cell survival. In vitro, β cells rapidly lost their HS and died. β Cell death was prevented by HS replacement, a treatment that also rendered the β cells resistant to damage from ROS. In vivo, autoimmune destruction of islets in NOD mice was associated with production of catalytically active heparanase, an HS-degrading enzyme, by islet-infiltrating MNCs and loss of islet HS. Furthermore, in vivo treatment with the heparanase inhibitor PI-88 preserved intraislet HS and protected NOD mice from T1D. Our results identified HS as a critical molecular requirement for islet β cell survival and HS degradation as a mechanism for β cell destruction. Our findings suggest that preservation of islet HS could be a therapeutic strategy for preventing T1D.

Topics: Animals; Cell Survival; Diabetes Mellitus, Type 1; Female; Glucuronidase; Heparitin Sulfate; Insulin-Secreting Cells; Leukocytes, Mononuclear; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Reactive Oxygen Species

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

Diabetes -associated hyperlipidemia is generally attributed to reduced clearance of plasma lipoproteins, especially remnant lipoproteins enriched in cholesterol and triglycerides. Hepatic clearance of remnants occurs via low density lipoprotein receptors and the heparan sulfate proteoglycan, syndecan-1. Previous studies have suggested alterations in heparan sulfate proteoglycan metabolism in rat and mouse diabetic models, consistent with the idea that diabetic dyslipidemia might be caused by alterations in proteoglycan expression in the liver. In this study we analyzed the content and composition of liver heparan sulfate in streptozotocin-induced insulin-deficient diabetic mice that displayed fasting hypertriglyceridemia and delayed clearance of dietary triglyceride-rich lipoproteins. No differences between normal and diabetic littermates in liver heparan sulfate content, sulfation, syndecan-1 protein levels, or affinity for heparin-binding ligands, such as apolipoprotein E or fibroblast growth factor-2, were noted. Decreased incorporation of [(35)S]sulfate in insulin-deficient mice in vivo was observed, but the decrease was due to increased plasma inorganic sulfate, which reduced the efficiency of labeling of liver heparan sulfate. These results show that hyperlipidemia in insulin-deficient mice is not due to changes in hepatic heparan sulfate composition.

Topics: Animals; Antibiotics, Antineoplastic; Apolipoproteins E; Blood Glucose; Diabetes Mellitus, Type 1; Fibroblast Growth Factor 2; Heparitin Sulfate; Hypertriglyceridemia; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptozocin; Sulfotransferases; Syndecan-1

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

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

Identification of peptides derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecules has great significance to elucidate the pathogenesis of type 1 diabetes. A bulk culture of Epstein-Barr virus-transformed B-cells, which were established from a 22-year-old type 1 diabetic woman with HLA-DR4 and -DQw8, was pulsed with the homogenate of a human embryonic pancreas-derived cell line 1B2C6, and another culture was not pulsed with antigen. Peptide fractions were obtained by treatment of affinity-purified HLA-DR and -DQ molecules with 0.1% trifluoroacetic acid, and were subjected to reverse-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC profiles of peptides derived from DR molecules revealed three peaks that specifically appeared after pulsing, but no such peaks were obtained from DQ molecules. From one of these three peaks, a peptide that consisted of 14 amino acids (AKSXNHTXXNQXRK, where X represents the undetermined amino acids) was identified. This peptide was derived from heparin/heparan sulfate-interacting protein (HIP). Immunostaining of pancreatic sections using antiserum for HIP peptide revealed exclusive staining of the islets. Thus, HIP was identified as an islet protein naturally processed and presented by HLA-DR4 molecules.

Topics: Alleles; Amino Acids; B-Lymphocytes; Blood Coagulation Factors; Blotting, Western; Cell Line; Cells, Cultured; Chromatography, High Pressure Liquid; Diabetes Mellitus, Type 1; Female; Genes, MHC Class II; Heparin; Heparitin Sulfate; Herpesvirus 4, Human; HLA-DQ Antigens; HLA-DR Antigens; HLA-DR4 Antigen; Humans; Immunohistochemistry; Islets of Langerhans; Leukocytes, Mononuclear; Pancreas; Peptides; Protein Binding; Ribosomal Proteins; RNA-Binding Proteins; Time Factors; Trifluoroacetic Acid

Pathogenesis, frequency, and management of heparin-induced thrombocytopaenia are well-known. They may be related with both unfractioned heparin and low-molecular weight heparin. Suspected heparin must be discontinued as soon as the diagnosis is established. Orgaran (danaparoid sodium) may be used for management of patients with heparin-associated thrombocytopaenia but can itself be associated with a thrombocytopaenia. Our case report allows us to catch in mind such a crossed complication.

Topics: Aged; Angiotensin-Converting Enzyme Inhibitors; Anticoagulants; Captopril; Carotid Stenosis; Chondroitin Sulfates; Dermatan Sulfate; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Drug Combinations; Female; Heparin; Heparitin Sulfate; Humans; Hypertension; Stroke; Thrombocytopenia

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 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

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

Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aged; Basement Membrane; Collagen; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Extracellular Matrix Proteins; Female; Fibronectins; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Middle Aged; Proteoglycans

Topics: Adult; Constriction; Diabetes Mellitus, Type 1; Endothelium, Vascular; Female; Heparitin Sulfate; Humans; Ischemic Attack, Transient; Male; Neoplasms; Veins

Topics: Carcinoma, Squamous Cell; Dermatan Sulfate; Diabetes Mellitus, Type 1; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Keratan Sulfate; Lung Neoplasms; Male; Middle Aged; Protein Binding

Heparan sulfate proteoglycans (HSPG) are negatively charged constituents of the renal extracellular matrix including the glomerular basement membrane (GBM) and mesangial matrix. Biochemical and functional studies of patients with type-1 insulin dependent diabetes mellitus (IDDM) suggest that alterations of HSPG may occur in diabetic nephropathy. We have utilized a specific cytochemical method and electron microscopy to quantitate the distribution of HSPG in the GBM of 10 normal people and in 16 IDDM patients with a spectrum of clinical and structural changes. Enzyme incubation studies of normal infant kidney demonstrated that heparitinase removed 94% of the stainable anionic sites in the lamina rara externa (LRE) and 77% of the sites in the lamina rara interna (LRI) of the GBM. In contrast, incubation in the enzyme chondroitinase ABC did not reduce the number of sites in the LRE but reduced the number of sites in the LRI by 26%. The HSPG anionic sites in normal subjects were distributed in the LRE as 20.9 +/- 1.3, and in the LRI as 13.1 +/- 2.2 per micron GBM length. Anionic sites were slightly reduced (19.6 +/- 1.3, P less than 0.04) in the LRE of IDDM patients with normal urinary albumin excretion rates (UAE), or microalbuminuria, and were reduced in both the LRE and LRI of IDDM patients with clinical proteinuria (13.1 +/- 2.3, P less than 0.001 and 8.9 +/- 2.1, P less than 0.001, respectively). The number of anionic sites in the LRE and LRI, respectively, correlated with UAE (r = +0.78, P less than 0.001, r = +0.58, P less than 0.02), with GBM thickness (LRE, r = +0.81, P less than 0.001; LRI, r = +0.67, P less than 0.01) and with the volume fraction of mesangium (LRE, r = +0.59, P less than 0.02; LRI, r = +0.58, P less than 0.03). These data confirm earlier biochemical findings of a reduction of HSPG in the GBM in advanced diabetic nephropathy but do not provide evidence for the loss of HSPG in the GBM as a mechanism for early microalbuminuria.

Topics: Adolescent; Adult; Aged; Anions; Basement Membrane; Biopsy; Diabetes Mellitus, Type 1; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Histocytochemistry; Humans; Kidney; Kidney Glomerulus; Male; Microscopy, Electron; Middle Aged; Proteoglycans; Reference Values

Anti-single-stranded-DNA antibodies cross-reactive with heparan sulfate were detected in serums of patients with type I (insulin-dependent) diabetes mellitus. The results suggested that heparan sulfate, the major glycosaminoglycan constituent of the glomerular basement membrane, may serve as a target antigen in vivo for cross-reactive anti-DNA antibodies. These polyreactive antibodies, directed toward repeating negatively charged units, may neutralize the heparan sulfate-associated polyanionic sites in the glomerulus, leading to an abnormal permeability of anionic plasma proteins.

Topics: Child; Cross Reactions; Diabetes Mellitus, Type 1; DNA, Single-Stranded; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Heparitin Sulfate; Humans; Immunoglobulin G

Topics: Blood Coagulation; Chondroitin Sulfate Proteoglycans; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Fibrinopeptide A; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Proteoglycans

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

The daily urinary excretions of total polymeric glycosaminoglycans and of polymeric heparan sulfate have been measured in the urine of juvenile-onset and adult-onset diabetics of both sexes and in those of normal controls. The results indicate that diabetic patients excrete more polymeric heparan sulfate than their controls, either in an absolute amount or as a percentage of the total glycosaminoglycans excreted. These results suggest that in the course of diabetes there is an increased degradation of heparan sulfate to large oligosaccharide fragments. These are excreted before being completely degraded to monosaccharides and inorganic sulfate.

Topics: Adolescent; Adult; Child; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Glycosaminoglycans; Heparitin Sulfate; Hexosamines; Humans; Middle Aged; Reference Values; Uronic Acids