Reduced number of anionic sites is associated with glomerular basement membrane thickening in the diabetic BB-rat

Summary Glomerular basement membranes of diabetic and age- and sex-matched non-diabetic BB rats were studied morphometrically and ultrastructurally using a quantitative histochemical technique employing the cationic dye cuprolinic blue. Six months of diabetes resulted in a significant reduction in the density of anionic sites associated with increased thickness of the glomerular basement membrane. These findings suggest that loss of anionic sites may be an important mechanism in the genesis of glomerular basement membrane dysfunction in diabetes.     

Alterations of glomerular basement membrane charge and structure in diabetic nephropathy

Summary We examined glomerular basement membrane anionic site distribution identified by cationic gold in seven patients with insulin-dependent and four patients with non-insulin-dependent diabetes mellitus, presenting a spectrum of clinical and glomerular changes. Anionic sites were investigated by pretreatment of tissue with glycosaminoglycan-degrading enzymes prior to cationic gold staining. The distribution of chondroitin sulphate proteoglycans — a previously unrecognized glomerular basement membrane component — and type IV collagen was examined by immunoelectron microscopy to identify structural changes in the basement membrane. Findings were compared with those of non-diabetic patients showing minor proteinuria and morphologically normal glomerular basement membranes. Two patients, originally diagnosed as having diabetic nephropathy were also examined at 19 weeks and 5 years after renal transplantation. Characteristic redistribution of type IV collagen and chondroitin sulphate proteoglycans was noted in thickened glomerular basement membrane segments (>400 nm) of diabetic patients and those with renal transplants. Extension of anionic sites deep into the glomerular basement membrane at pH 2.5, together with loss of interna sites at pH 5.8 is unique to diabetic nephropathy. Reduced charge density was apparent in some patients due to thickening of the glomerular basement membrane, although the number of anionic sites per unit length of membrane was actually increased. Thus, charge aberration in diabetic nephropathy is due to displacement rather than loss of anionic sites. Removal of more than 90% of these sites by heparitinase, confirms their association with heparan sulphate proteoglycans. Similar derangement of anionic sites in all patients with diabetic nephropathy irrespective of the degree of proteinuria, suggests that a heparan sulphate proteoglycan-related charge barrier plays a minor role in controlling permeability of the diabetic glomerular basement membrane.Abbreviations BSA Bovine serum albumin - CG cationic gold - CSPG chondroitin sulphate proteoglycans - GAG glycosaminoglycan - GBM glomerular basement membrane - HSPG heparan sulphate proteoglycans - LRE lamina rara externa - LRI lamina rara interna - PCI protein:creatinine index     

Muscle magnesium and capillary basement membrane thickness in diabetes mellitus

To evaluate a possible relationship between Mg deficiency and the development of microvascular disease in diabetes mellitus, quadriceps muscle biopsies for estimating Mg content and capillary basement membrane thickness, were studied in 16 patients with type I diabetes. The diabetic individuals had a slightly but significantly reduced muscle Mg content as compared with 13 healthy controls. There was a significant, positive correlation between capillary basement membrane width and age in the diabetic group, but no relationship between membrane thickness and muscle or serum concentration of Mg. However, diabetic patients with retinopathy (n = 6) showed a nonsignificant inverse correlation between basement membrane thickness and Mg parameters. The opposite tendency was found in patients without retinal lesions.     

Anionic sites in basement membranes. Differences in their electrostatic properties in continuous and fenestrated capillaries

We have used ruthenium red, a cationic dye, to detect at the electron microscopic level the presence of anionic sites in various murine basement membranes, with particular emphasis on those of the microvasculature. We have observed anionic sites in all continuous and fenestrated capillaries examined. Terminal lymphatics, which have a discontinuous basement membrane, have sites only where the basement membrane is present. Anionic sites are not present beneath sinusoidal lining cells of the liver which lack a basement membrane. Basement membranes of epithelial cells and those surrounding striated and smooth muscle cells, pericytes, fat cells, and Schwann cells also exhibit anionic sites. We compared the electrostatic properties of anionic sites in basement membranes of continuous and fenestrated capillaries by determining the salt concentration (critical electrolyte concentration, Scott and Dorling, 1965) required to displace ruthenium red from the sites. A concentration of 0.5 M Na⁺ was required to displace ruthenium red from the basement membrane of continuous capillaries of muscle whereas 1.3 M Na⁺ was required to displace ruthenium red from the basement membrane of fenestrated peritubular capillaries of the renal cortex. Our results suggest that anionic sites in the basement membrane of fenestrated peritubular capillaries are more strongly negatively charged than those in the basement membrane of continuous capillaries of muscle. We conclude from this study, first, that anionic sites are a general property of vascular, epithelial, and pericellular basement membranes and, second, that the electrostatic properties of the sites differ in different vascular basement membranes. We speculate that the anionic sites in vascular basement membranes and the variation in their electrostatic properties in different types of capillaries may have important implications for exchange of substances across the capillary wall.     

Basement membrane thickness,insulin antibodies and HLA-antigens in long standing insulin dependent diabetics with and without severe retinopathy

The study was designed to show whether there was any relation between muscle capillary basement membrane thickness, HLA-antigens, anti-insulin antibodies and proliferative retinopathy. Electron microscopic measurements of muscle capillary basement membrane thickness were performed on muscle biopsies from 15 insulin-dependent diabetics and severe proliferative retinopathy, 24 insulin-dependent diabetics with minimal retinopathy and 18 age- and sex matched non-diabetics. All the patients had had diabetes for 20 years or more. None had biochemical or clinical evidence of diabetic nephropathy. Basement membrane thickness was measured according to the methods of Siperstein and Williamson. Muscle capillary basement membrane thickening occurred in 32 of 39 diabetics, using the Siperstein method, but patients with proliferative retinopathy did not exhibit thicker basement membranes than patients with no or minimal changes in the retina. There were apparent differences in HLA-antigens between diabetics with and without proliferative retinopathy, but they did not reach statistical significance. There was no correlation between muscle capillary basement membrane thickness and the quantity of insulin antibodies. The results indicate that factors other than basement membrane thickening and genetic factors in the HLA-region, are responsible for the development of proliferative retinopathy.     

Pancreatic islet allograft prevents basement membrane thickening in the diabetic rat retina

Summary Progressive basement membrane thickening is a characteristic structural abnormality in diabetic tissues in cluding the retina. We examined the effect of pancreatic islet allotransplantation on basement membrane thickening and irregularities in retinal capillaries of the streptozotocin-diabetic rat. Diabetic animals received intraportal or intracerebral pancreatic islet allografts. Animals with functioning allografts demonstrated euglycaemia and a normal body weight gain during the 400-day post-transplantation period. The characteristic thickening of capillary basement membranes was completely prevented in animals with successful transplantation. The present findings suggest that islet allotransplantation may be a rational therapeutic approach in the treatment of diabetes mellitus and the prevention of ensuing secondary complications.     

Diabetic glomerulosclerosis without glucose intolerance.

The pathophysiology of the microangiopathy of diabetes mellitus is poorly understood, and the relevance of carbohydrate intolerance remains uncertain. Four patients are presented with renal abnormalities suggestive of diffuse diabetic glomeruloscierosis. These patients have no evidence of carbohydrate intolerance by standard clinical technics. A familial incidence of diabetes mellitus and delayed insulin response to an oral glucose load support a classification of prediabetes or suspected diabetes mellitus for these patients. Early intercapillary nodule formation was seen in only two of the four patients. In the absence of this infrequent pathognomonic finding, an alternate approach to the diagnosis of diabetic glomerulosclerosis is suggested. Diffuse glomerular capillary basement membrane thickening, consistently present with diabetic glomerulosclerosis, is demonstrated by measurements utilizing the latex microsphere technic. The mean glomerular capillary basement membrane thickness of these patients was 4,403 A, compared with the control value of 3.098 A (P less than 0.001). Other pathologic findings suggestive of diabetic nephropathy include efferent arteriolosclerosis and linear immunofluorescence without electron dense deposits or inflammation. Skeletal muscle capillary basement membranes of all four patients also demonstrated significant thickening. The mean value for the patients was 1,510 A, as compared with a control value of 961 A (P less than 0.001). The importance of this muscle capillary basement membrane thickening to the diagnosis of diabetic microangiopathy is discussed. The pathologic alterations in the renal biopsy specimens and the demonstration of muscle capillary basement membrane thickening strongly suggest that diabetic glomerulosclerosis may occur in the absence of overt clinical carbohydrate intolerance.     

The choriocapillaris in spontaneously diabetic rats.

During diabetes in rats, the choroid of the eye shows increased permeability to albumin, basement membrane thickening, and decreased anionic charge sites on the abluminal surfaces of the choriocapillary microvessels. In other microvascular beds, permeability differences are correlated with differences in luminal membrane microdomains as indicated by the distribution of luminal membrane anionic charge. To see whether luminal surface charge distribution or other structural features of the choroidal microvasculature become altered during diabetes, we studied spontaneously diabetic and control rats using ultrastructural tracers and morphometric techniques. Rats were injected with horseradish peroxidase and perfused with aldehydes, and then retina-choroid tissue sections were incubated with cationized ferritin, reacted to visualize peroxidase, and prepared for electron microscopic study. The most striking alterations in the diabetic rats were vascular debris and migrating cells resembling vascular cells in the choriocapillaris stroma, suggesting an increase in capillary turnover. In addition, extracellular matrix material was increased, and peroxidase uptake and ferritin binding were low in some vessels of the diabetic rats compared with the controls. Variability was large in the diabetic animals, however, and other vessels remained apparently normal.     

Modification of anionic sites in myocardial capillary basal laminae of diabetic rats

Capillary basal laminar thickening is a distinctive feature of diabetic microangiopathy; however, the mechanism responsible for this abnormality remains to be clarified. Recent reports have described a reduction in the distribution of anionic sites in diabetic glomerular basement membranes, with the suggestion that this reduction may generate a compensatory synthesis of basal laminar constituents, causing laminar thickening. In order to provide additional information, the character and distribution of the basal laminar anionic profile were examined in the myocardium of diabetic rats. Diabetes mellitus was induced in 14 rats by injection with streptozotocin, ip; 6 rats served as controls. Myocardial tissue was subjected to Charonis' procedure for the demonstration of anionic sites with the cationic electron-dense dye, ruthenium red, following the sacrifice of the animals at intervals up to 11 months after the induction of the diabetes. The tissues were then processed routinely for electron microscopic examination. A total of 20 electron micrographs, at magnifications of 13,000x and 33,000x, were obtained from each rat for the quantitation of anionic sites. A length measuring 6 micron along each basal lamina was utilized for determining the number of anionic loci. Results of this study show that (1) the number and size of anionic sites in myocardial basal laminae is reduced in diabetic rats, (2) this decrease becomes more pronounced with prolongation of the diabetes, (3) it is detectable prior to the demonstration of basal laminar thickening by electron microscopy, and (4) enzyme digestion treatments indicated that heparan sulfate proteoglycan is the essential stainable component of the anionic sites. These findings provide evidence that the laminar anionic profile is altered in the diabetic myocardium and support the view that this abnormality constitutes a significant initial event in the pathogenesis of basal laminar thickening.     

Binding of charged ferritin to alveolar wall components and charge selectivity of macromolecular transport in permeability pulmonary edema in rats

Rat lungs were inflated and incubated in either anionic or cationic ferritin, and alveolar and capillary basement membranes were examined by electron microscopy. Cationic ferritin bound to heparan sulfate proteoglycans on the external surface of the alveolar basement membrane, whereas cationic ferritin bound to the lamina densa of the capillary basement membranes. Anionic and cationic ferritin was also perfused through the pulmonary circulation of lungs isolated from control rats and rats previously injected with alpha-naphthylthiourea, which produces permeability pulmonary edema. Neither anionic nor cationic ferritin leaked from the pulmonary capillaries in perfused controls; cationic, but not anionic, ferritin adhered to endothelial cell surfaces. In lungs with alpha-naphthylthiourea pulmonary edema, perfused for 2-15 minutes, anionic ferritin leaked from pulmonary capillaries into the alveolar interstitium and alveolar space, while cationic ferritin remained within the capillary lumen. Five times as much anionic ferritin appeared in the capillary basement membranes on the thick side of the alveolar wall, as in the alveolar basement membranes on the thin side of the alveolar wall. In alpha-naphthylthiourea lungs perfused for 45-60 minutes, cationic ferritin also leaked through the injured endothelium and bound twice as much to the alveolar as the capillary basement membranes. The negatively charged pulmonary capillary endothelium, the positively charged capillary basement membranes, and the negatively charged alveolar basement membranes may influence the transport of macromolecules from the pulmonary circulation in permeability pulmonary edema.     

Improved diabetes control reduces skeletal muscle capillary basement membrane width in insulin-dependent diabetes mellitus

We studied the relationship between the control of blood glucose and the width of skeletal muscle capillary basement membrane in 54 insulin-dependent diabetic patients. After initial measurement of levels of glycosylated hemoglobin and the width of skeletal muscle capillary basement membrane, the patients were divided into two groups: an intensive treatment group of 30 patients who were treated with continuous subcutaneous insulin infusion and a control group of 24 patients who continued to receive conventional treatment, usually two daily injections of insulin. Both groups have been followed prospectively for periods of time up to 4 years. Within 1 year the intensive treatment group had a significant decrease in glycosylated hemoglobin levels as compared to baseline values reflecting improved control of blood glucose. This level of glycosylated hemoglobin was stable over the remainder of the follow-up period. This group also had a significant reduction in the width of skeletal muscle capillary basement membrane within 1 year and it persisted for the 4 years of observation. The control group of patients had no significant change in their level of glycosylated hemoglobin and the width of the skeletal muscle capillary basement membrane tended to increase with time. It this result in skeletal muscle capillaries applies to those of retinal and renal tissue, meticulous diabetic control for a prolonged period of time may be beneficial in preventing the progression of the microvascular complications of diabetes mellitus.     

Alterations of ultrastructures and anionic sites in basement membranes of myocardial cells and capillaries in patients with cyanotic congenital heart disease due to tetralogy of Fallot

Electron microscopic cytochemical studies of the basement membranes of myocardial cells and capillaries were performed in 13 patients with tetralogy of Fallot who were divided into 2 groups. Group 1 included 7 patients in the early stage of the disease, ranging in age from 7 months to 5 years. Group 2 consisted of 6 patients in the far advanced stage of the disease, ranging in age from 30 to 46 years. The operatively excised infundibular muscles of the right ventricle were prepared for conventional electron microscopy and electron microscopic cytochemistry. The anionic sites in the basement membranes were characterized by cationic polyethyleneimine. The basement membrane ultrastructures of the myocardial cells and capillaries in the early stage of tetralogy of Fallot showed no apparent alterations with regular distribution of anionic sites, particularly in the external lamina of the basement membranes. In contrast, irregular thickening, wide splitting and lamination of the basement membranes of myocardial cells and capillaries, always associated with derangement and focal loss of anionic sites in the membranes were consistently observed in the far advanced stage of tetralogy of Fallot. The aforementioned results suggest that altered surface membrane integrity of myocardial cells and capillaries resulting from pathologic changes of the basement membranes are an important pathogenetic mechanism responsible for progressive degeneration of infundibular muscle cells and myocardial dysfunction in the course of tetralogy of Fallot.     

Pericyte degeneration and acellular capillaries are increased in the feet of human diabetic patients

Summary Ultrastractural morphometry was used to quantify capillary basement membrane width, pericyte coverage of capillaries, pericyte degeneration, and the extent of acellular capillaries in skeletal muscle obtained at autopsy from neck, thigh, calf and foot of five male and four female diabetic subjects and an equal number of sex- and age-matched nondiabetic subjects. Within diabetic or nondiabetic subjects, the trend for all four parameters to increase in frequency or magnitude in the order neck < thigh < calf was highly significant; the only statistically significant difference between calf and foot muscles for any of the four parameters was capillary basement membrane width for nondiabetic subjects, which was significantly thinner in foot than in calf muscle (t=2.45;p<0.05). Pericyte coverage of capillaries did not differ between diabetic and nondiabetic subjects for each muscle examined; however, capillary basement membrane width, the frequency of pericyte debris and acellular capillaries were increased significantly in the lower extremity muscles of diabetic compared to nondiabetic subjects, and the magnitude of the difference between these two groups increased in the order thigh < calf < foot. The observations that pericyte degeneration and acellular capillaries are present in skeletal muscle as well as in retinal microvessels suggest that common pathophysiological mechanisms may contribute to vascular disease in these two very different tissues. The additional finding that relative differences between diabetic and nondiabetic subjects, in the frequency and magnitude of these changes, increase in the order neck < calf < foot is consistent with the marked increase in peripheral vascular disease and gangrene in the lower extremities of diabetic patients.     

The effect of aldose reductase inhibition with ponalrestat on the width of the capillary basement membrane in diabetes mellitus

There is evidence to suggest that hyperglycemia is required for the development of the microvascular complications of diabetes. However, the precise mechanism by which hyperglycemia might cause diabetic complications is not completely clear. One possibility is the increased activity of the polyol pathway. Capillary basement membrane thickness is a hallmark histological finding in diabetic microangiopathy. Previous studies in experimental models of diabetes have related the polyol pathway with the thickness of basement membrane in retinal capillaries. To study the effect of aldose reductase inhibition with ponalrestat on the width of the skeletal muscle capillary basement membrane in subjects with diabetes, we measured the capillary basement membrane width in 55 subjects with diabetes in a double masked, placebo controlled randomized trial over a period of 18 months. Twenty-nine patients received ponalrestat (two 300 mg tablets daily) and twenty-six received placebo tablets. The age, sex distribution, type and duration of diabetes were similar in both groups. The glycosylated hemoglobin remained at a constant level throughout the study in both groups. The baseline capillary basement membrane width of the ponalrestat group was 3134 +/- 146 A, it was 3074 +/- 226 A at month 12 and 2548 +/- 182 A at month 18 (P less than 0.001 vs baseline value). The placebo group also had a significant reduction in the width of the capillary basement membrane, from a baseline value of 3026 +/- 147 A to 2818 +/- 144 A at month 12 and 2618 +/- 156 A at month 18 (P less than 0.001 vs baseline value). There was no statistical difference in the capillary basement membrane width between the two groups at any time point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endoneurial localisation of microvascular damage in human diabetic neuropathy

Summary Twenty diabetic patients with neuropathy underwent clinical and neurophysiological evaluation together with a detailed morphometric assessment of capillary pathology in endoneurial and epineurial microvascular beds of the sural nerve. Morphological data were compared with ten non-diabetic control subjects. There were no significant differences in control subjects between basement membrane area, endothelial cell area, endothelial cell profile number or luminal area of endoneurial when compared with epineurial capillaries. In contrast, when compared with epineurial capillaries, endoneurial capillaries from diabetic patients demonstrated a significant increase in basement membrane (p<0.001) and endothelial cell (p<0.001) area and a significant reduction in luminal area (p<0.001). There was no significant difference in endothelial cell profile number between endoneurial and epineurial capillaries amongst diabetic patients. Previous studies have demonstrated a good correlation between the degree of microangiopathy and measures of neuropathic severity. In the present study increased endoneurial capillary basement membrane area was significantly related to reduced peroneal nerve conduction velocity (p<0.001), myelinated fibre density (p<0.001) and elevated vibration (p<0.05) and thermal (p<0.001) perception. Increased endothelial cell area and reduced luminal size were related to a reduced peroneal nerve conduction (p<0.05, p<0.01, respectively), reduced myelinated fibre density (p<0.05, p<0.01) and elevated thermal perception (p<0.05, p<0.001). Epineurial capillary basement membrane, endothelial cell and luminal area failed to relate to measures of neuropathic severity. This study has demonstrated more advanced microangiopathy and a more significant relationship to neuropathic severity in endoneurial compared with epineurial capillaries, thus providing further support for the role of microangiopathy in the pathogenesis of human diabetic neuropathy.     

Early loss of arteriolar smooth muscle cells: more than just a pericyte loss in diabetic retinopathy.

Incipient diabetic retinopathy is characterized by increased capillary permeability and progressive capillary occlusion. The earliest structural change is the loss of pericytes (PC) from the retinal capillaries. With the availability of the XLacZ mouse, which expresses the LacZ reporter in a PC/vascular smooth muscle cell (vSMC) specific fashion, we quantitatively assessed the temporal dynamics of smooth muscle cells in arterioles under hyperglycemic conditions. We induced stable hyperglycemia in XLacZ mice. After 4, 8, and 12 weeks of diabetes retinae were isolated and beta-galactosidase/lectin stained. The numbers of smooth muscle cells were counted in retinal whole mounts, and diameters of retinal radial and branching arterioles and venules were analyzed at different distances apart from the center of the retina. After eight weeks of diabetes, the numbers of vSMCs were significantly reduced in radial arterioles 1000 microm distant from the optic disc. At proximal sites of branching arterioles (400 microm distant from the center), and at distal sites (1000 microm), vSMC were significantly reduced already after 4 weeks (to a maximum of 31 %). These changes were not associated with any measurable variation in vessel diameters. These data indicate quantitatively that hyperglycemia not only causes pericyte loss, but also loss of vSMCs in the retinal vasculature. Our data suggest that arteriolar vSMC in the eye underlie similar regulations which induce early pericyte loss in the diabetic retina.     

The retinal microvasculature of spontaneously diabetic BB rats: structure and luminal surface properties

Endothelial cell permeability and luminal surface anionic sites were studied in the retinal microvasculature of spontaneously diabetic rats. Horseradish peroxidase (HRP) was used as a tracer of pinocytotic transport, and cationized ferritin (CF) was used as a marker of luminal surface anionic sites. Diabetic and control rats were injected with HRP, and their retinas were fixed. Retinal tissue sections were then incubated in CF, reacted to visualize HRP, and prepared for quantitative electron microscopic analysis. In both control and diabetic rats treated with serotonin and histamine antagonists to prevent HRP-induced vascular changes, the endothelium formed a barrier to the tracer. Pinocytotic uptake was relatively low in most vessels. Reaction product was restricted to pinocytotic vesicles, tubular cisternae, and multivesticular bodies. HRP uptake appeared high in some of the deep capillaries of the diabetic retinas as compared with that of the controls, but the difference was not statistically significant. HRP-induced transendothelial permeability was observed in both control and diabetic rats when serotonin and histamine antagonist pretreatment was omitted. CF studies showed anionic sites in four luminal surface microdomains in control and diabetic endothelial cells. CF-binding, anionic sites were present on the plasma membrane, on all coated vesicles, on some uncoated vesicles, and on most diaphragms of uncoated vesicles. Plasma membrane binding was sparse and patchy in some diabetic vessels, especially in the deep vessels of rats that were not treated with the serotonin and histamine antagonists. However, statistical analysis showed similar numbers of plasma membrane binding sites in diabetic and control rats pretreated with serotonin and histamine antagonists. Our data suggest that the retinal microvasculature in diabetic rats remains normal in terms of permeability and luminal membrane anionic charge.     

The kidney of spiny mice (Acomys cahirinus): Electron microscopy of glomerular changes associated with ageing and tubular glycogen accumulation during hyperglycemia

Summary Glomeruli and tubules of the kidney of normoglycemic and diabetic spiny mice were studied with the electron microscope. Progressive thickening of the basement membranes of glomerular capillaries with a concomitant increase in the deposition of basement-membrane-like mesangial matrix occurred with age. Focal hemispherical thickenings of the basement membrane on its epithelial side were observed with increasing frequency in older animals. The plasma membrane of adjacent foot processes exhibited features suggestive of pinocytosis. Collagen fibers in electronlucent areas surrounded by mesangial matrix were regularly seen in animals beyond the age of twelve months. In diabetic animals, the alterations of the glomerular capillary basement membranes and of the mesangial region appeared to be more pronounced but no specific lesions were observed. This negative finding may be related to the relatively short duration of the diabetic state. Measurements of the thickness of glomerular capillary basement membranes showed a significant age-dependent increase in basement membrane width and indicated that this process may be accelerated in diabetic animals. Examination of the renal tubules of diabetic animals showed a characteristic segmental pattern of glycogen storage in epithelial cells with considerable variations in the degree of glycogen infiltration between different segments but also between individual cells of a given segment. The most surprising feature associated with glycogen storage was the occurrence of lysosomes filled with glycogen. The mechanisms responsible for the accumulation of glycogen within lysosomes are unknown but may be related to an increase in glycogen turnover in cells actively involved in the reabsorption of glucose or to an impairment of lysosomal function secondary to diabetes.Supported in part by the Fonds National Suisse de la Recherche Scientifique, Berne, Switzerland (Grants No. 5344 and 4848.3), and by a grant-in-aid through Zyma S.A., Nyon, Switzerland.     

Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy

Summary 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- and 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 × 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.Abbreviations HS Heparan sulphate - GBM glomerular basement membrane - HSPG heparan sulphate proteoglycan - STZ streptozotocin - LRE lamina rara externa - LD lamina densa - LD + LRI lamina densa + lamina rara interna - ANOVA analysis of variance