heparitin-sulfate and Diabetic-Nephropathies

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

Proteinuria is a hallmark of many glomerular diseases and an independent risk factor for the progression of renal failure. Proteinuria results from damage to the glomerular filtration barrier (GFB), which plays a critical role in size- and charge-selective filtration. The GFB consists of three layers, which is the fenestrated endothelium that is covered by the glycocalyx, the podocytes and the intervening glomerular basement membrane. Defects in one of the three layers in the GFB can lead to the development of proteinuria. Heparan sulphate (HS) is a negatively charged polysaccharide that is abundantly expressed in all layers of the GFB. HS expression in the GFB is reduced in the majority of patients with proteinuria, which is associated with an increased glomerular expression of the HS-degrading enzyme heparanase. The primary role of HS in the development of proteinuria has been challenged after the establishment of several genetically engineered mouse models with an altered HS expression that did not display development of overt proteinuria. However, in a recent study, we showed that heparanase is essential for the development of proteinuria in diabetic nephropathy, which suggests that loss of HS contributes to the development of proteinuria. Recent studies also further highlight the importance of the glomerular endothelial glycocalyx in charge-selective filtration and the development of proteinuria. This review aims to summarize our current knowledge on the role of in particular HS and heparanase in the development of proteinuria.

Topics: Animals; Diabetic Nephropathies; Female; Glomerular Basement Membrane; Glucuronidase; Glycocalyx; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Podocytes; Proteinuria

The physicochemical characteristics of the glomerular capillary filtration membrane restrict the passage of macromolecules on the basis of molecular weight, charge, and shape. The proposed ionic charge permselectivity characteristics of the glomerular basement membrane (GBM) are determined by its chemical composition, primarily the highly sulfated glycosaminoglycan heparan. In diabetic nephropathy, the heparan sulfate content of the GBM is diminished. It has been proposed that decreased GBM heparan sulfate content causes decreased permselectivity to negatively charged macromolecules such as albumin, allowing this protein to leak into the urinary space. One possible explanation for decreased GBM heparan sulfate content in diabetic nephropathy is the observation that heparanase, an enzyme capable of degrading heparan sulfate, is upregulated in the glomerular epithelial cell (GEC) in response to increased glucose. Increased GEC heparanase activity has been demonstrated in glomeruli in diabetic kidneys, and increased urine heparanase has been observed in diabetic nephropathy. In vitro studies have shown that GEC heparanase activity depends on the glucose concentration of the culture medium. GEC heparanase activity can be inhibited by heparin compounds. Sulodexide, an orally active low-molecular weight heparin, has been shown to lower urine albumin excretion. The working hypothesis that has emerged is that sulodexide may be an in vivo heparanase inhibitor that reaches the glomerular capillary wall and prevents heparan sulfate degradation, thus allowing reconstruction of heparan sulfate content and restoration of GBM ionic permselectivity. Two clinical trials are currently being carried out to determine whether sulodexide is renoprotective in diabetic nephropathy.

Topics: Anticoagulants; Basement Membrane; Diabetic Nephropathies; Heparin, Low-Molecular-Weight; Heparinoids; Heparitin Sulfate; Humans; Kidney Glomerulus; Permeability; Urothelium

Diabetic nephropathy can develop in up to one-third of patients with type 1 diabetes and approximately 25% of patients with type 2 diabetes. This complication is important as it not only leads to renal failure but is associated with a high risk of coronary artery disease and other vascular complications. Although hyperglycaemia is necessary for the development of diabetic nephropathy, it is not sufficient, genetic factors also being important. This is evidenced by studies showing that only a subgroup of patients are at risk of nephropathy and that nephropathy clusters in families. The genes involved in susceptibility to diabetic nephropathy have yet to be identified. Most studies to date have been case-control in design, and there have been conflicting results. Genes suggested as having a role include those encoding angiotensin-1 converting enzyme, apolipoprotein E, heparan sulphate and aldose reductase. In order to clarify the role of these and other candidate genes in nephropathy, association studies in families are necessary. Because of the large number required, this will require international collaboration. A genetic marker for nephropathy would enable the earlier detection of this complication, thus facilitating screening and targeted intervention. An understanding of the role of susceptibility genes will ultimately allow the development of novel therapeutic strategies.

Topics: Aldehyde Reductase; Apolipoproteins E; Diabetic Nephropathies; Genetic Predisposition to Disease; Heparitin Sulfate; Humans; Peptidyl-Dipeptidase A; Renin-Angiotensin System

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

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

Basement membranes (BM) are specialized structures of the extracellular matrix. Their composition is of particular importance for the maintenance of normal morphological and functional properties of a multitude of organs and tissue systems and it is thus required for regular homeostasis of body function. Generally, they possess three main functions, i.e. participation in the maintenance of tissue structure, control of fluid and substrate exchange, and regulation of cell growth and differentiation. BMs are made up by various components which are in part specifically localized within the BM zone, or which represent ubiquitous matrix constituents with specific quantitative and/or qualitative differences in their localization. On the basis of a thorough immunohistochemical analysis of normal and diseased tissues, we provide here a concept of "functional morphology/pathomorphology" of the different BM components analyzed: 1.) The ubiquitous BM-constituent collagen IV primarily stabilizes the BM-zone and thus represents the "backbone" of the BM providing mechanical strength. Its loss leads to cystic tissue transformation as it is evidenced from the analysis of polycystic nephropathies. Thus, in other cystic tissue transformations a similar formal pathogenesis may be present. 2.) The specific localization of collagen VII as the main structural component of anchoring fibrils underlines the mechanical anchoring function of this collagenous protein. Defects in this protein lead to hereditary epidermolysis. The rapid re-occurrence of epidermal collagen VII during normal human wound healing indicates a quick reconstitution of the mechanical tensile strength of healing wounds. 3.) The BM-specific heparan sulfate proteoglycan (HSPG, Perlecan) with its highly negative anionic charge can be assumed to exert filter control. This assumption is corroborated by the localizatory findings of a preferential deposition of HSPG in endothelial and particularly in glomerular BM. Similarly, the lack of HSPG in the BM of lymph capillaries can be regarded as the correlate for a free fluid influx into lymphatic capillaries. The relative reduction in HSPG-staining in the developing glomerular BM also explains the still immature filter function. Furthermore, the low content of HSPG in placental chorionic capillaries can be regarded as morphological correlate for the required free fluid exchange between maternal and fetal blood systems. In diabetic glomerulopathy, the loss of HSPG coincid

Topics: Animals; Basement Membrane; Collagen; Diabetic Nephropathies; Extracellular Matrix Proteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Kidney Diseases; Neoplasms; Proteoglycans; Pulmonary Fibrosis; Wound Healing

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

Topics: Amino Acid Sequence; Animals; Basement Membrane; Diabetic Nephropathies; Extracellular Matrix; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Molecular Sequence Data; Muscles; Neoplasms; Promoter Regions, Genetic; Proteoglycans; Sequence Homology, Amino Acid

Diabetic nephropathy develops in many diabetic patients as consequence of glomerulosclerosis. On the basis of a series of recent observations it is suggested that a combination of metabolic and hemodynamic changes is responsible for the pathogenesis of diabetic nephropathy. Since the glomerular filtration unit has been characterized to consist of collagen type IV and minor components like laminin, fibronectin and heparan sulfate proteoglycan, influence of diabetes on basement membrane (BM) components has been studied. Biochemical alterations of glomerular BM consist of an increased nonenzymatic glucosylation of type IV collagen leading to unphysiological crosslinking. This, in turn, may result in alteration of the size selective properties of the glomerular filtration unit. Changes in composition of glomerular BM have recently been described. An increased synthesis of type IV collagen with concomitant decrease of heparan sulfate proteoglycan may lead to alteration of the charge selective barrier and consequently to increased permeability of the glomerular BM. Permanently unbalanced synthesis of BM components finally results in obliteration of the capillary lumen. In late state nephropathy intrinsic basement membrane components are no longer produced. Instead, massive accumulation of PAS positive material occurs.

Topics: Animals; Basement Membrane; Capillary Permeability; Chondroitin Sulfate Proteoglycans; Collagen; Diabetic Nephropathies; Glomerular Filtration Rate; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Proteoglycans

In diabetic nephropathy, heparan sulfate glycosaminoglycan side chains are reduced in glomerular basement membranes proportionally to the degree of proteinuria. Recently, it was demonstrated that additional therapy with danaparoid sodium, a mixture of sulfated glycosaminoglycans with mainly heparan sulfate, lowered proteinuria in type 1 diabetes patients with diabetic nephropathy. A randomized placebo-controlled parallel study was performed with 750 anti-Xa units of danaparoid sodium once daily in type 2 diabetes patients with severe proteinuria. The aim of the study was to evaluate the possible effects of danaparoid sodium on proteinuria, endothelial dysfunction, and hard exudates in the retina and to determine the safety/tolerability of this drug. Twenty-two patients completed the study, and one patient had to stop prematurely after 6 wk of danaparoid sodium treatment because of urticaria at the injection sites. Apart from a small decrease of hemoglobin and minor skin hematomas at the injection site in five patients in the danaparoid sodium group, no other safety parameters showed any clinically or statistically significant difference between and within groups. The relative change in time of both the urinary albumin and protein excretion rate corrected for creatinine did not differ between both treatment arms (P = 0.2 and 0.49, respectively). No retinal complications or changes of hard exudates occurred. von Willebrand factor was elevated in both groups, but was not influenced by either treatment modality. Contrary to the beneficial effects that occurred in type 1 diabetes patients with diabetic nephropathy, treatment for 8 wk with 750 anti-Xa units of danaparoid sodium gave no reduction of proteinuria, hard exudates, and von Willebrand factor.

Topics: Adult; Aged; Analysis of Variance; Anticoagulants; Chondroitin Sulfates; Dermatan Sulfate; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diabetic Retinopathy; Double-Blind Method; Drug Combinations; Endothelium, Vascular; Exudates and Transudates; Female; Follow-Up Studies; Heparitin Sulfate; Humans; Kidney Function Tests; Male; Middle Aged; Proteinuria; Reference Values; Statistics, Nonparametric; von Willebrand Factor

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

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

Topics: Animals; Binding Sites; Calcium Dobesilate; Cell Movement; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endothelial Cells; Heparitin Sulfate; Humans; Kinetics; Mice; Mice, Inbred NOD; Protein Binding; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

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

Glomerular integrity and functions are maintained by growth factor signaling. Heparan sulfate, the major component of glomerular extracellular matrixes, modulates growth factor signaling, but its roles in glomerular homeostasis are unknown. We investigated the roles of heparan sulfate 6-O-endosulfatases, sulfatase (Sulf)1 and Sulf2, in glomerular homeostasis. Both Sulf1 and Sulf2 were expressed in the glomeruli of wild-type (WT) mice. Sulf1 and Sulf2 double-knockout (DKO) mice showed glomerular hypercellularity, matrix accumulation, mesangiolysis, and glomerular basement membrane irregularity. Platelet-derived growth factor (PDGF)-B and PDGF receptor-β were upregulated in Sulf1 and Sulf2 DKO mice compared with WT mice. Glomeruli from Sulf1 and Sulf2 DKO mice in vitro stimulated by either PDGF-B, VEGF, or transforming growth factor-β similarly showed reduction of phospho-Akt, phospho-Erk1/2, and phospho-Smad2/3, respectively. Since glomerular lesions in Sulf1 and Sulf2 DKO mice were reminiscent of diabetic nephropathy, we examined the effects of Sulf1 and Sulf2 gene disruption in streptozotocin-induced diabetes. Diabetic WT mice showed an upregulation of glomerular Sulf1 and Sulf2 mRNA by in situ hybridization. Diabetic DKO mice showed significant increases in albuminuria and serum creatinine and an acceleration of glomerular pathology without glomerular hypertrophy; those were associated with a reduction of glomerular phospho-Akt. In conclusion, Sulf1 and Sulf2 play indispensable roles to maintain glomerular integrity and protective roles in diabetic nephropathy, probably by growth factor modulation.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Extracellular Signal-Regulated MAP Kinases; Genetic Predisposition to Disease; Heparitin Sulfate; Intercellular Signaling Peptides and Proteins; Kidney Glomerulus; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-sis; Receptors, Growth Factor; Signal Transduction; Smad Proteins, Receptor-Regulated; Sulfatases; Sulfotransferases; Time Factors; Tissue Culture Techniques; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Heparanase, a heparan sulfate (HS)--specific endoglucuronidase, mediates the onset of proteinuria and renal damage during experimental diabetic nephropathy. Glomerular heparanase expression is increased in most proteinuric diseases. Herein, we evaluated the role of heparanase in two models of experimental glomerulonephritis, being anti-glomerular basement membrane and lipopolysaccharide-induced glomerulonephritis, in wild-type and heparanase-deficient mice. Induction of experimental glomerulonephritis led to an increased heparanase expression in wild-type mice, which was associated with a decreased glomerular expression of a highly sulfated HS domain, and albuminuria. Albuminuria was reduced in the heparanase-deficient mice in both models of experimental glomerulonephritis, which was accompanied by a better renal function and less renal damage. Notably, glomerular HS expression was preserved in the heparanase-deficient mice. Glomerular leukocyte and macrophage influx was reduced in the heparanase-deficient mice, which was accompanied by a reduced expression of both types 1 and 2 helper T-cell cytokines. In vitro, tumor necrosis factor-α and lipopolysaccharide directly induced heparanase expression and increased transendothelial albumin passage. Our study shows that heparanase contributes to proteinuria and renal damage in experimental glomerulonephritis by decreasing glomerular HS expression, enhancing renal leukocyte and macrophage influx, and affecting the local cytokine milieu.

Topics: Acute Disease; Animals; Diabetic Nephropathies; Glomerular Basement Membrane; Glomerulonephritis; Glucuronidase; Heparitin Sulfate; Mice, Inbred C57BL; Proteinuria; Tumor Necrosis Factor-alpha

The assessment of the clinical significance of chondroitin sulfate in patients with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) for the detection of the relationship between chondroitin sulfate (CS) structure and disease.. Healthy control (n=15), type 2 diabetic patients with normalbuminuria (n=12), and patients with microalbuminuria (n=13) were enrolled in the study. Total sulfated glycosaminoglycans (GAGs) concentration in the first morning urine was evaluated by 1,9-dimethylmethylene blue method and the composition was determined by agarose gel electrophoresis. Urinary chondroitin sulfate was quantified by a combination of treatment with specific lyase digestions and separation of products by SAX-HPLC.. GAGs concentration significantly increased in diabetic patients with microalbuminuria compared to diabetic patients with normalbuminuria. Qualitative analysis of urinary GAGs revealed the presence of chondroitin sulfate, heparan sulfate, and low-sulphated chondroitin sulphate-protein complex (LSC-PG). There was a decrease in CS and an increase in LSC-PG in the urine of patients with diabetes compared to healthy controls. Moreover, in diabetic patients, chondroitin sulfate contains more 6-sulfated disaccharide and less 4-sulfated disaccharide. There was a statistically significant difference in ratio of 6-sulfated disaccharide to 4-sulfated disaccharide among the three groups.. GAGs were significantly increased in diabetic patients with microalbuminuria. The levels of urinary GAGs, ratio of LSC-PG/CS, as well as ratio of 6-sulfated to 4-sulfated disaccharides could be useful markers for diagnosis of patients with diabetic nephropathy.

Topics: Chondroitin Sulfates; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Electrophoresis, Agar Gel; Female; Heparitin Sulfate; Humans; Male; Methylene Blue; Middle Aged

Diabetic nephropathy is one of the main causes of end-stage renal disease, in which the development of tubular damage depends on factors such as high glucose levels, albuminuria and advanced glycation end-product. In this study, we analyzed the involvement of heparanase, a heparan sulfate glycosidase, in the homeostasis of proximal tubular epithelial cells in the diabetic milieu. In vitro studies were performed on a wild-type and stably heparanase-silenced adult tubular line (HK2) and HEK293. Gene and protein expression analyses were performed in the presence and absence of diabetic mediators. Albumin and advanced glycation end-product, but not high glucose levels, increased heparanase expression in adult tubular cells via the AKT/PI3K signaling pathway. This over-expression of heparanase is then responsible for heparan sulfate reduction via its endoglycosidase activity and its capacity to regulate the heparan sulfate-proteoglycans core protein. In fact, heparanase regulates the gene expression of syndecan-1, the most abundant heparan sulfate-proteoglycans in tubular cells. We showed that heparanase is a target gene of the diabetic nephropathy mediators albumin and advanced glycation end-product, so it may be relevant to the progression of diabetic nephropathy. It could take part in several processes, e.g. extracellular-matrix remodeling and cell-cell crosstalk, via its heparan sulfate endoglycosidase activity and capacity to regulate the expression of the heparan sulfate-proteoglycan syndecan-1.

Topics: Albumins; Base Sequence; Cell Line; Diabetic Nephropathies; Glucose; Glucuronidase; Glycated Serum Albumin; Glycation End Products, Advanced; HEK293 Cells; Heparitin Sulfate; Homeostasis; Humans; Kidney Failure, Chronic; Kidney Tubules, Proximal; RNA, Messenger; RNA, Small Interfering; Serum Albumin; Serum Albumin, Bovine; Syndecan-1; Transcriptional Activation

Glycoconjugates in the kidney play an important role in the maintenance of glomerular filtration barrier. Thickening of the glomerular basement membrane (GBM) is well characterized in diabetic nephropathy. Changes in GBM mainly include reduction and undersulfation of heparan sulfate, and laminin with accumulation of type IV collagen leading to kidney dysfunction and there is a need to identify therapies that arrest disease progression to end-stage renal failure. In the present investigation, effect of bitter gourd on streptozotocin-induced diabetic rats with particular emphasis on kidney heparan sulfate (HS) was studied. Earlier, our study showed partial reversal of all the diabetes-induced effects by bitter gourd. Increase in the components of glycoconjugates during diabetes was significantly decreased by bitter gourd feeding. Diabetes associated elevation in the activities of enzymes involved in the synthesis and degradation of glycosaminoglycans (GAGs) were significantly lowered by bitter gourd supplementation. GAGs composition revealed decrease in amino sugar, and uronic acid contents during diabetes and bitter gourd feeding was effective in countering this reduction. Decrease in sulfate content in the GAGs during diabetes was ameliorated by bitter gourd feeding. HS decreased by 43% in diabetic rats while bitter gourd feeding to diabetic rats showed 28% reduction. These results clearly indicate beneficial role of bitter gourd in controlling glycoconjugate and heparan sulfate related kidney complications during diabetes thus prolonging late complications of diabetes.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glycoconjugates; Glycosaminoglycans; Heparitin Sulfate; Kidney; Male; Momordica charantia; Plant Preparations; Rats; Rats, Wistar; Streptozocin

Recent studies suggest that loss of heparan sulphate in the glomerular basement membrane (GBM) of the kidney with diabetic nephropathy is due to the increased production of heparanase, a heparan sulphate-degrading endoglycosidase. Our present study addresses whether heparan sulphate with different modifications is differentially reduced in the GBM and whether heparanase selectively cleaves heparan sulphate with different domain specificities.. The heparan sulphate content of renal biopsies (14 diabetic nephropathy, five normal) were analysed by immunofluorescence staining with four anti-heparan sulphate antibodies: JM403, a monoclonal antibody (mAb) recognising N-unsubstituted glucosamine residues; two phage display-derived single chain antibodies HS4C3 and EW3D10, defining sulphated heparan sulphate domains; and anti-K5 antibody, an mAb recognising unmodified heparan sulphate domains.. We found that modified heparan sulphate domains (JM403, HS4C3 and EW3D10), but not unmodified domains (anti-K5) and agrin core protein were reduced in the GBM of kidneys from patients with diabetic nephropathy, compared with controls. Glomerular heparanase levels were increased in diabetic nephropathy kidneys and inversely correlated with the amounts of modified heparan sulphate domains. Increased heparanase production and loss of JM403 staining in the GBM correlated with the severity of proteinuria. Loss of modified heparan sulphate in the GBM as a result of degradation by heparanase was confirmed by heparan sulphate staining of heparanase-treated normal kidney biopsy specimens.. Our data suggest that loss of modified heparan sulphate in the GBM is mediated by an increased heparanase presence and may play a role in the pathogenesis of diabetes-induced proteinuria.

Topics: Diabetic Nephropathies; Fluorescent Antibody Technique; Glomerular Basement Membrane; Glucuronidase; Heparitin Sulfate; Humans; Immunohistochemistry

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

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

A decrease in glomerular heparan sulfate (HS) proteoglycan (PG), without apparent decrease in HSPG core protein expression, has been reported to occur in diabetic nephropathy (DN). In most studies however, agrin, the major HSPG core protein in the glomerular basement membrane, has not been studied. This prompted us to study the glomerular expression of agrin in parallel to the expression of HS-glycosaminoglycans (GAG) in biopsies of patients with DN. Furthermore, the influence of glucose on agrin production in cultured podocytes and the expression of agrin in fetal kidneys was investigated.. Cryostat sections of renal biopsies from patients with DN (n = 8) and healthy controls (HC, n = 8), were stained for agrin and HS-GAG. Sections of fetal kidneys were double stained for agrin and CD35 or CD31. Stainings were performed by indirect immunofluorescence (IIF). The production of agrin by cultured human podocytes was tested by ELISA and IIF.. The expression of agrin, detected by AS46, was significantly reduced in biopsies from patients with DN compared to HC (p < 0.01). Similar findings were observed when monoclonal antibody JM72 was used (p < 0.05). In addition, a significant reduction in the glomerular expression of HS-GAG was detected with JM403 in these patients (p < 0.01). Agrin is expressed in cultured podocytes, the expression hereof was reduced when the cells were cultured in the presence of 25 mM D-glucose (p < 0.01). In biopsies of human fetal kidneys, glomerular expression of agrin coincided with the expression of CD31. In early stages of glomerular differentiation there was a strong staining for agrin and CD31 while CD35 was only slightly positive.. Our data argue against a selective dysregulation in HSPG sulfation in DN, but suggest a pivotal role for hyperglycemia in the downregulation of agrin core protein production.

Topics: Aged; Agrin; Basement Membrane; Biopsy; Cell Differentiation; Cells, Cultured; Culture Media; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Epithelium; Fetus; Fluorescent Antibody Technique, Indirect; Glomerular Mesangium; Glucose; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Kidney Glomerulus; Male; Membrane Glycoproteins; Middle Aged; Proteoglycans

Heparan sulfate (HS) contributes to the negative charge in the glomerular basement membrane (GBM), which may maintain the GBM charge barrier. Changes in sulfation and/or the concentration of HS may be associated with the development of diabetic nephropathy.. Using two different antibodies specific for HS chains, one that reacts with the N-sulfated sequences in HS chains (10E4) and the other that reacts with neo-epitope of HS, which occurs after heparitinase digestion of HS chains (3G10), we examined the serum and urinary concentrations of HS by enzyme-linked immunosorbent assay and performed immunohistochemical staining of glomeruli in diabetic patients with and without nephropathy.. The level of urinary excretion of 10E4 binding HS/creatinine clearance was significantly reduced in diabetic patients when compared with that in nondiabetic subjects (P < 0.0001), and the level was more decreased in patients with overt nephropathy than in patients without overt nephropathy. No differences or only small differences were found between these groups in serum and urinary 3G10-binding HS and in serum 10E4-binding HS. Immunohistochemical staining with these antibodies was consistent with the findings in the urine.. The results suggest that a decreased HS N-sulfation exists in the urine, which may reflect a structural change or an altered processing of HS within the GBM. Because N-sulfation plays a key role in determining the extent of sulfation within the HS chains, the decreased urinary 10E4-binding HS may have potential implications with regard to the development of diabetic nephropathy.

Topics: Adult; Aged; Antibody Specificity; Basement Membrane; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Epitopes; Extracellular Matrix; Female; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Middle Aged; Polysaccharide-Lyases; Sulfates

The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hyperglycemia but also genetic factors may contribute to the development of diabetic nephropathy Heparan sulfate proteoglycan (HSPG) is thought to play an important role as a component of the charge selectivity barrier in the glomerular basement membrane. Recently, a BamHI restriction fragment length polymorphism (RFLP) in the HSPG gene (HSPG2) was reported to be associated with diabetic nephropathy in Caucasian insulin-dependent diabetes mellitus (IDDM). The aim of the present study was to examine the contribution of the BamHI HSPG2 polymorphism to the development of diabetic nephropathy in Japanese non-insulin-dependent diabetes mellitus (NIDDM). For this purpose, we recruited 102 patients with diabetic nephropathy and 64 age-matched patients without diabetic nephropathy from Japanese NIDDM patients. Since all the subjects had proliferative diabetic retinopathy, it seems likely that they would be exposed to hyperglycemia for a long time. In the present study, the BamHI HSPG2 genotype and allele frequencies were not significantly different between the patients with nephropathy and the patients without nephropathy. Therefore, we conclude that the BamHI HSPG2 polymorphism is not associated with the development of diabetic nephropathy in Japanese NIDDM.

Topics: Case-Control Studies; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Diabetic Retinopathy; Female; Heparitin Sulfate; Humans; Japan; Male; Middle Aged; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Proteoglycans

The primary etiologic factor in diabetic glomerulosclerosis appears to be an overproduction of transforming growth factor-beta by mesangial cells, which in turn reflects a hyperglycemically mediated overactivation of protein kinase C (PKC) throughout the glomerulus. Membrane-active antioxidants, fish oil, and angiotensin-converting enzyme inhibitors can act to down-regulate glomerular PKC activity, via a variety of mechanisms that may include activation of diacylglycerol kinase and suppression of phosphatidate phosphohydrolase, support of endothelial nitric oxide and heparan sulfate production, inhibition of thromboxane and angiotensin synthesis/activity, and correction of glomerular hypertension. The beneficial impact of these measures on vascular endothelial function may be of more general utility in the prevention of diabetic complications such as retinopathy, neuropathy, and atherosclerosis. Adjunctive use of gamma-linolenic acid is indicated for prevention of neuropathy, and it is conceivable that bioactive chromium will have protective activity not solely attributable to improved glycemic control. Re-establishing euglycemia must clearly remain the core strategy for preventing diabetic complications, but when glycemic control remains suboptimal, practical, safe measures are at hand for decreasing risk.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Antioxidants; Diabetic Angiopathies; Diabetic Nephropathies; Enzyme Activation; Fish Oils; Heparitin Sulfate; Humans; Hyperglycemia; Kidney Glomerulus; Lipid Peroxidation; Models, Biological; Nitric Oxide; Protein Kinase C; Thromboxane A2; Transforming Growth Factor beta

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

A decrease in anionic change and the loss of heparan sulfate proteoglycan have previously been observed in the glomerular basement membrane (GBM) during diabetic glomerulosclerosis. We studied the chronological changes in the anionic character and the glycosaminoglycan content in the GBM of WBN/ Kob rats with spontaneous diabetes. Two types of cationic probes were used: polyethyleneimine (PEI) and cationic colloidal gold (CCG). Immunogold labeling was performed with anti-monoclonal-heparan-sulfate-glycosaminoglycan (HS-GAG) and anti-chondroitin-sulfate-glycosaminoglycan (CS-GAG) antibodies. The GBM width, the anionic sites and the GAG sites were investigated in diabetic WBN/Kob rats at 2, 10 and 19 months, compared with control rats. Diabetes was confirmed in WBN/Kob rats after 8 months in this study. The GBM width gradually thickened with age. The PEI anionic sites significantly decreased in the lamina rara externa (LRE) at 19 months (vs. 2 and 10 months). The HS-GAG sites also significantly decreased in the LRE at 10 and 19 months (vs. 2 months). However, the CCG anionic sites and the CS-GAG sites significantly increased in the LRE and the lamina densa at 10 months (vs. 2 months) and, after 19 months, returned to the level seen at 2 months. Results indicate that there is an early transient increase in CS-GAG in the GBM while HS-GAG decreases. We noticed a transient increase in the CCG anionic sites at this early stage of diabetic glomerulosclerosis as well. The increase in CS-GAG may provide a marker for early diabetic changes in the GBM.

Topics: Animals; Anions; Antibodies, Monoclonal; Basement Membrane; Chondroitin Sulfates; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Heparitin Sulfate; Immunohistochemistry; Kidney Glomerulus; Male; Rats; Rats, Wistar

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

In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p < 0.01) and effective renal plasma flow (p < 0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42 +/- 13 vs control rats (C): 0.5 +/- 0.2 mg/24 h, p < 0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20 +/- 0.04 vs C: 0.39 +/- 0.17, p < 0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Glomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152-941) vs C: 262 (244-467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712-2014) vs C: 672 (515-1208) ng/mg glomerulus, p < 0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14-1.16) vs C: 0.39 (0.30-0.47), p < 0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.

Topics: Albumins; Animals; Basement Membrane; Diabetic Nephropathies; Glomerular Filtration Rate; Heparitin Sulfate; Hydroxyproline; Immunoglobulin G; Kidney; Kidney Glomerulus; Longitudinal Studies; Male; Microcirculation; Proteinuria; Rats; Rats, Wistar

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

Previous studies have indicated that administration of glycosaminoglycans can prevent some of the morphological and physiological alterations which occur in experimental diabetic nephropathy. The aims of this study were to further elucidate the effect of these drugs on glomerular basement membrane permeability by dextran clearance studies, to test the ability of glycosaminoglycans to revert established diabetic nephropathy and to examine the effect of glycosaminoglycans on renal extracellular matrix synthesis. Five groups of Sprague-Dawley rats were studied for 12 months: two control groups (treated or untreated non-diabetic), three streptozotocin diabetic animal groups, two of which received a glycosaminoglycan formulation, one from the induction of diabetes and the other after the fifth month of diabetes. At five months the 35S-sulfate glomerular incorporation, albuminuria, glomerular basement membrane thickness and anionic charge density were determined. At 12 months albuminuria, renal collagen IV and perlecan mRNA levels, anionic and neutral dextran clearances, glomerular basement membrane morphometry, and mesangial cell proliferation were evaluated. We demonstrate that long-term administration of glycosaminoglycans prevents renal morphological and functional alterations in diabetic rats and appears to revert established diabetic renal lesions. Glycosaminoglycan administration modified renal matrix composition by the normalization of collagen gene expression and increasing glomerular 35S-sulfate incorporation.

Topics: Animals; Cell Membrane Permeability; Collagen; Dextrans; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glomerular Filtration Rate; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Kidney Glomerulus; Male; Proteoglycans; Rats; Rats, Sprague-Dawley; RNA, Messenger

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

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

Diabetic nephropathy is invariably associated with proteinuria.. To delineate the mechanism(s) of proteinuria in diabetic nephropathy, ultrastructural changes of the glomerular basement membranes (GBMs) were studied by high resolution scanning and immunoelectron microscopy. Acellular glomeruli from diabetic and age-matched control human subjects were prepared by detergent method and subjected to conductive staining, the technique in which the tissues are impregnated with metals rather than surface-coated with metallic alloys for visualization by electron microscopy. Subsequent to conductive staining, the tissues were examined by in-lens field emission scanning electron microscopy.. Thirty glomeruli, each from the control and diabetic groups, were examined by scanning microscopy. In diabetic GBMs, a loose meshwork structure consisting of numerous pores of approximately 8 nm in diameter and distinct strands was observed. In contrast, meshwork structure was not readily discernible in controls and a few pores were observed. Five glomerular capillary loops, each from control and diabetic groups, were examined by immunoelectron microscopy. In controls, heparan sulfate-proteoglycan was localized in the lamina rara interna and externa, and type-IV collagen was distributed throughout the whole width of the GBM. In diabetic GBMs, a relative loss of staining of heparan sulfate-proteoglycan, both in the lamina rara interna and externa of the GBM, was observed. Type IV collagen was distributed in all layers of the thickened GBM, and the absolute number of the immunogold particles was increased. However, immunogold particle density of type IV collagen per unit area was decreased as compared with the control.. These ultrastructural and immunoelectron microscopic changes in the GBM may explain the loss of charge as well as size selectivities of the glomerulus, as observed in diabetic nephropathy associated with proteinuria.

Topics: Aged; Aged, 80 and over; Basement Membrane; Collagen; Diabetic Nephropathies; Extracellular Matrix; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Male; Microscopy, Electron, Scanning; Microscopy, Immunoelectron; Proteoglycans; Reference Values

The pathogenesis of diabetic nephropathy relative to the changes in the glomerular extracellular matrices was investigated. Renal tissues from 10 diabetic patients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This alteration may, in part, be the cause of the proteinuria associated with diabetic nephropathy.

Topics: Adult; Aged; Collagen; Diabetic Nephropathies; Extracellular Matrix; Female; Fibronectins; Fluorescent Antibody Technique; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Laminin; Male; Middle Aged; Proteoglycans

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 effect of streptozotocin-induced diabetes on the structure of heparan sulfate (HS) prepared from rat kidney glycosaminoglycans (GAG) was evaluated. GAG were isolated and purified from the kidneys of diabetic and age-matched control rats by standard procedures. HS was prepared from GAG by digestion with chondroitinase ABC and precipitation with cetylpyridinium chloride. The tissue dry weight of diabetic kidneys was greater than that of the controls. The amounts of protein and DNA per tissue dry weight were decreased in the diabetic group, while GAG and hydroxyproline remained unchanged. The above information indicates that the extracellular components are increased in diabetes. There was no significant difference in the amount of HS to tissue dry weight between the diabetic and control groups. When the molecular weight of the HS from both groups was compared by Sephacryl S-300 HR column chromatography, the HS peak for the diabetic kidney indicated a slightly higher molecular weight and the base of the peak was broader than that for the controls. A reduction in N-sulfate residues was observed in Sephadex G-50 profiles after nitrous acid degradation of the HS. The ratio of glucuronic acid to its epimer, iduronic acid, in diabetic kidney HS was slightly lower than that in the controls. This indicates that diabetes may influence the carbohydrate chain structure of the HS in the kidney. Quantitative and qualitative changes in the kidney HS may contribute to the symptoms associated with diabetic nephropathy.

Topics: Animals; Chromatography, Gel; Chromatography, High Pressure Liquid; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Glycosaminoglycans; Heparitin Sulfate; Kidney Glomerulus; Molecular Structure; Molecular Weight; Rats; Rats, Inbred Strains; Streptozocin

The occurrence of heparan sulfate proteoglycan anionic site staining in the glomerular basement membranes of healthy and streptozotocin-induced diabetic rats was investigated. After 12 weeks diabetes, a marked loss of staining by ruthenium red was observed, compared with the age-matched controls. However, extraction and quantitation of total proteoglycan from the glomerular basement membrane revealed no significant differences between the two groups, in terms of absolute yield of glycosaminoglycan, when the latter is expressed per glomerulus. Glomerular basement membranes from both groups become thicker in an age-dependent manner, although they do so at a faster rate in the diabetic group, and a higher collagen content is found in the diabetic membrane at 12 weeks. Thus when proteoglycan content is expressed per mass protein, relative decreases are observed in diabetes. Similarly, a relative but not absolute decrease in proteoglycan content was observed in glomerular basement membranes from spontaneously diabetic BB rats with diabetes of 12 weeks duration. Glycosaminoglycans isolated from control and diabetic glomerular basement membrane have similar characteristics on ion exchange chromatography, to each other, and to newly sulfated, intact glomerular proteoglycans labelled in vitro. We conclude that loss of anionic site staining with ruthenium red in early diabetes is not a consequence of absolute loss or undersulfation of heparan sulfate proteoglycan but reflects structural or biochemical changes in the diabetic matrix.

Topics: Animals; Anions; Basement Membrane; Chondroitin Sulfate Proteoglycans; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kidney Glomerulus; Male; Membrane Glycoproteins; Proteoglycans; Rats; Rats, Inbred BB; 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