Diabetic Glomerular Basement Membrane Research Articles

Blockade of Vascular Endothelial Growth Factor Signaling Ameliorates Diabetic Albuminuria in Mice

For investigation of how the vascular endothelial growth factor (VEGF) system participates in the pathogenesis of diabetic kidney disease, type 2 diabetic db/db and control db/m mice were treated intraperitoneally with vehicle or 2 mg/kg of a pan-VEGF receptor tyrosine kinase inhibitor, SU5416, twice a week for 8 wk. Efficacy of SU5416 treatment in the kidney was verified by the inhibition of VEGF receptor-1 phosphorylation. Glomerular VEGF immunostaining, normally increased in diabetes, was unaffected by SU5416. Plasma creatinine did not change with diabetes or SU5416 treatment. The primary end point of albuminuria increased approximately four-fold in the diabetic db/db mice but was significantly ameliorated by SU5416. Correlates of albuminuria were investigated. Diabetic glomerular basement membrane thickening was prevented in the SU5416-treated db/db mice, whereas mesangial matrix expansion remained unchanged by treatment. The density of open slit pores between podocyte foot processes was decreased in db/db diabetes but was partly increased toward normal by SU5416. Finally, nephrin protein by immunofluorescence was decreased in the db/db mice but was significantly restored by SU5416. Paradoxically, total nephrin protein by immunoblotting was increased in diabetes, pointing toward a possible dysregulation of nephrin trafficking. Diabetic albuminuria is partially a function of VEGF receptor signaling overactivity. VEGF signaling was found to affect a number of podocyte-driven manifestations such as GBM thickening, slit pore density, and nephrin quantity, all of which are associated with the extent of diabetic albuminuria. By impeding these pathophysiologic processes, VEGF receptor inhibition by SU5416 might become a useful adjunct to anti-albuminuria therapy in diabetic nephropathy.

A specific structural alteration in the heparan sulphate of human glomerular basement membrane in diabetes.

Heparan sulphate proteoglycan is an important component of the glomerular anionic filtration barrier and its reduced amount in diabetes contributes to glomerular dysfunction. The objective of this study was to determine if there is also an alteration in the sulphation pattern of the diabetic heparan sulphate chains. The heparan sulphate in the glomerular basement membrane/mesangial matrix from human diabetic and nondiabetic kidneys obtained at autopsy was fragmented by a hydrazine/nitrous acid procedure and after radiolabelling with NaB[3H]4, the disaccharide products were chromatographically resolved and quantified. Six sulphated disaccharides were identified in both the diabetic and nondiabetic samples and the molar distribution of these was similar, with the notable exception of the iduronic acid-2-O-sulphatectl--> 4glucosamine-3-O-sulphate species which occurred in the diabetic glomeruli in less than half the amount as in the nondiabetic samples (9.0% compared to 18.7% of total sulphated disaccharides, p < 0.005). 3-O-sulphated glucosamine is a rare constituent of heparan sulphate occurring usually in a glucuronic acidbeta1--> 4glucosamine-3-O-sulphate(+/- 6-O-sulphate) sequence within the antithrombin-binding domain. In the glomerular basement membrane where the 3-O-sulphated glucosamine is present in substantial amounts, however, it occurs exclusively in an iduronic acid-containing sequence. It is likely that the recently discovered 3-O-sulphotransferase variant which specifically acts on the iduronic acidalpha1--> 4glucosamine sequence is decreased in human diabetes and moreover that this unusual disaccharide could be a component of a specific heparan sulphate domain which interacts with bioactive proteins.

Differential effects of diabetes and glomerulonephritis on glomerular basement membrane composition.

The hallmark of renal diseases involving the glomerulus is the presence of proteinuria. While the routes of pathogenesis of proteinuria have not been established, alterations in the barrier function of the glomerular basement membrane (GBM) have been implicated. We evaluated the effect of streptozotocin diabetes and passive Heymann nephritis (PHN) over time on the macromolecular composition of rat GBM to determine if changes in composition correlate with proteinuria. Six to twelve rats from each group (control, diabetic, and PHN) were sacrificed 1, 5, 28, 56, or 84 days after induction of disease. Identical amounts of GBM were subjected to a sequential extraction procedure, and type IV collagen, entactin, laminin, fibronectin, and anionic charge content were quantitated in the extracts. Type IV collagen and entactin content did not change with time or disease. Both laminin and fibronectin contents increased with time in GBM in all groups, but this increase was significantly greater in diabetic GBM. A significant decrease in anionic charge content of GBM coincided with the onset of albuminuria at Day 28 in diabetes, but no change was seen in PHN. In diabetic rats, the increase in laminin content over control preceded the onset of albuminuria, while the increase in fibronectin was not apparent until after albuminuria was present. In PHN, no differences in type IV collagen, entactin, laminin, fibronectin, or anionic charge content of GBM were found compared with control, even though profound albuminuria was evident from Day 5 through 84. Thus, while alterations in laminin and fibronectin content may contribute to the loss of glomerular permselectivity in streptozotocin diabetes, such changes apparently are not involved in PHN.

High-resolution ultrastructural study of the rat glomerular basement membrane in long-term experimental diabetes.

The ultrastructure of the glomerular basement membrane of the long-term diabetic and age-matched control rats was studied with the application of advanced high-resolution microscopy. By using the freeze substitution method for the preparation of the renal tissue, it was possible to observe that the glomerular basement membrane in control and diabetic animals is composed on only a single lamina densa without the presence of a lamina lucida interna or externa. High-resolution electron microscopy of the diabetic glomerular basement membrane showed significant alterations in its morphology and ultrastructure. First, the basement membrane in diabetic condition appeared to be split into two halves, endothelial and epithelial. In the epithelial half of the membrane, the network of distinct strands referred to as cords, which were clearly present in the glomerular basement membrane of age-matched control animals, became less distinct and showed a diffused appearance being evenly replaced by thin filaments. The openings of the network were filled with a granular material. In the endothelial half of the membrane, on the other hand, the cord network was variably lost in diabetic condition and, within the resulting vacant spaces, bundles of fibrils 12 nm in width, identified as basotubules, were deposited. Immunolabeling for type IV collagen was found to be enriched in the endothelial half of the basement membrane being associated with the bundles of basotubules. The ultrastructural changes reported by high-resolution microscopy could be related to the molecular alterations of the basement membrane components and to the loss in permselectivity occurring during diabetes.

Loss of sulfated carbohydrate from the glomerular podocyte as a cause of albuminuria in experimental diabetic rats: Ultrastructural histochemical study

Decrease of anionic sites and heparan sulfate proteoglycan has been demonstrated in diabetic glomerular basement membrane (GBM) and causally related to albuminuria. The HID-TCH-SP-PD technique is a sensitive histochemical method for negatively charged sulfated carbohydrates. In this study, we examined the localization of HID-TCH-SP-PD reaction in the glomeruli of diabetic and control rats. In both rats, the reaction was found in assciation with the surface of podocytes and with GBM. In diabetic rats, the former-associated reaction was markedly reduced. By contrast, the latter-associated reaction did not show any differences between both animal groups. Urinary albumin excretion (UAE) increased significantly in alloxanized rats. It is suggested that a decrease of podocyte-associated sulfated carbohydrates, the primary observed change, gives rise to albuminuria in alloxan diabetic rats.

Glomerular basement membrane thickening in streptozotocin diabetic rats despite treatment with an aldose reductase inhibitor

This study concerns the possible prevention of glomerular basement membrane thickening in experimental diabetes by an aldose reductase inhibitor (ARI), Statil ®. ARI added to the chow was given to streptozotocin diabetic rats over a period of 6 months. Reference groups were control rats and diabetic rats on the same chow without ARI. The diabetic rats were given insulin two or three times a week, and blood glucose was measured monthly before insulin injections. There was a marked difference in the occurrence of cataracts between the two diabetic groups. ARI treated rats tended to have lower blood glucose than the diabetic reference group, but the difference was not significant. At the termination of the experiment, the left kidney was perfusion fixed, weighed, and prepared for light and electron microscopy. Systematic random sampling from the entire kidney was performed to obtain light microscopic visual fields and ultrathin sections from two glomeruli. Mean glomerular volume was estimated by light microscopy, and glomerular basement membrane thickness, by electron microscopy. Basement membrane thickness was significantly increased in untreated diabetic rats (174 nm, SD = 4.5 nm) as compared to that of controls (Mean: 154 nm, SD = 11.0 nm), and was even more so in ARI treated rats (187 nm, SD = 18.7 nm,) although the ARI treated rats showed less renal and glomerular hypertrophy than did untreated diabetic rats. In conclusion, the ARI treatment over an experimental period of 6 months attenuated diabetic renal and glomerular hypertrophy, but had no effect at all on diabetic glomerular basement membrane thickening.

Ultrastructural analyses of acellular glomerular basement membranes and mesangial matrix in a spontaneously diabetic rhesus monkey.

Renal changes similar to those considered diagnostic for diabetes in humans are infrequently observed in spontaneous noninsulin-dependent (NID) diabetic monkeys. In the current study, renal cortical tissue blocks are rendered acellular to demonstrate glomerular basement membrane (GBM) and mesangial matrix (MM) changes in a naturally occurring NID diabetic rhesus monkey (Macaca mulatta). Transmission electron micrographs of these specimens show axial MM accumulations with numerous striated collagen fibrils that frequently extend onto internal (endothelial) surfaces of peripheral GBM. The outer (epithelial) component, although compact, appears bilaminar due to folded external surfaces not coinciding with similar irregularities on internal surfaces. By scanning electron microscopy, external surfaces of sclerotic GBMs are extensively wrinkled and, following cryofracture, show congestion and expanded MM. A fenestrated meshwork of MM, which appears less dense and compact than epithelial BM, extends from axial regions onto GBM internal surfaces. The true thickness of randomly sampled peripheral GBM thickness (approximately 400 nm) is approximately double that of normal rhesus GBM. Diabetic GBMs exhibit ruthenium red positivity for surface polyanions with linear site densities not significantly different from normal. These observations indicate that sclerotic GBMs in diabetic rhesus monkeys closely resemble those seen in human end-stage diabetic glomerulopathy and suggest that this nonhuman primate may offer an excellent model for studies of chronic diabetic BM disease.

Undersulfation of glomerular basement membrane heparan sulfate in experimental diabetes and lack of correction with aldose reductase inhibition.

In this study we examined the effect of experimental diabetes and of treatment with an aldose reductase inhibitor on the level of sulfation of glomerular basement membrane (GBM) heparan sulfate, the principal glycosaminoglycan in this extracellular matrix. Glycosaminoglycans were isolated from GBM purified from control, streptozocin-induced diabetic, and sorbinil-treated diabetic rats and analyzed for sulfate and uronate content. Glomerular yields from diabetic kidneys were greater than those from control animals, but the amount of sulfate per glomerulus in diabetic samples, both untreated and sorbinil treated, did not differ significantly from that in control samples. However, the sulfate-to-uronate ratio in heparan sulfate isolated from diabetic GBM (0.34 +/- 0.08) was significantly less than in control samples (0.69 +/- 0.11), and treatment of diabetic rats with an aldose reductase inhibitor did not correct this reduced ratio (0.36 +/- 0.06). The results indicate that there is an undersulfation of heparan sulfate of GBM in experimental diabetes, an abnormality that may contribute to loss of anionic sites and decreased charge selectivity of the glomerular filtration barrier. The findings further suggest that this abnormality results from disturbances in glycosaminoglycan synthesis and/or metabolism in diabetes that are independent of polyol-pathway activation in the renal glomerulus.

Degradation of glomerular basement membrane in diabetes. II. Proteolytic activity of diabetic and nondiabetic glomeruli.

The proteolytic effects of isolated glomeruli of diabetic rats on glomerular basement membrane of nondiabetic rats was investigated at various times after streptozotocin injection. One week after induction of diabetes, proteolytic activity remained unchanged as compared with nondiabetic controls. However, 4 and 10 weeks after streptozotocin injection, glomerular degradation of collagenous (but not noncollagenous) peptides of basement membranes increased (+24% as compared with control experiments). Using diabetic basement membrane as substrate, degradation of collagenous and noncollagenous peptides caused by diabetic glomeruli was 2.6-fold and 1.7-fold higher than in control experiments with nondiabetic glomeruli. The results indicate that the disturbed degradation of glomerular basement membrane in diabetes is not due to a decreased activity of glomerular proteolytic enzymes. In contrast, it can be concluded that the increased resistance of diabetic basement membrane to proteolytic degradation could be partially compensated by quantitative and qualitative changes of the proteolytic potential of diabetic glomeruli.

Disturbances in glomerular basement membrane glycosaminoglycans in experimental diabetes.

Glycosaminoglycans (GAGs) were purified from basement membranes isolated from glomeruli of control and streptozocin-induced diabetic rats and were quantitatively analyzed with a recently described competitive binding assay that is specific for and sensitive to microgram amounts of chondroitin and heparan sulfate. Total GAG content in glomeruli from diabetic rats and in the basement membranes prepared from these samples (17.22 +/- 1.45 and 6.56 +/- 0.49 micrograms/10(5) glomeruli, respectively was significantly less than that found in comparable control preparations (43.71 +/- 3.35 and 16.05 +/- 1.41 micrograms/10(5) glomeruli, respectively). The portion of total GAG in the water-soluble fraction recovered after osmotic lysis of isolated glomeruli was also markedly decreased in diabetic samples (26.11 +/- 4.55 vs. 3.30 +/- 0.32 micrograms/10(5) glomeruli, control vs. diabetic). Treatment of lysed glomeruli with the ionic detergent deoxycholate, required for liberation of the extracellular matrix from plasma membrane lipoproteins and purification of the insoluble glomerular basement membrane (GBM), resulted in solubilization of approximately 10% of the water-insoluble GAG in control samples but greater than 50% in diabetic membranes. Heparan sulfate comprised greater than 90% of the GAGs in both control and diabetic GBM, defined as the water- and detergent-insoluble matrix. The findings clearly demonstrate that the GAG content of GBM is diminished in experimental diabetes and provide evidence that the reduction in GBM anionic sites associated with diabetes derives from a decrease in the constituent GAGs of this extracellular matrix. The results further suggest that the interaction between GBM and populations of GAG associated with the surface of plasma membranes of adjacent cells is disturbed in diabetes.

Studies on macromolecular components of human glomerular basement membrane and alterations in diabetes. Decreased levels of heparan sulfate proteoglycan and laminin.

Treatment of human glomerular basement membrane (GBM) with 4 M guanidine HCl resulted in a preferential extraction of noncollagenous components including laminin, fibronectin, entactin, and heparan sulfate proteoglycan, whereas effective solubilization of type IV collagen required exposure to denaturing solvents in the presence of reducing agents. The guanidine HCl-solubilized constituents were identified by immunochemical procedures after resolution by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography. Two immunologically related heparan sulfate proteoglycans (Mr approximately 350,000 and 210,000) were observed by electrophoresis, with the higher-molecular-weight form being predominant. An examination of the two proteoglycans after heparitinase digestion or chemical deglycosylation indicated that heparan sulfate chains and other carbohydrate units are attached to core proteins with Mr approximately 140,000 and 110,000, respectively. Radioimmunoassays indicated that human diabetic GBM contained significantly lower (P less than .005) amounts of heparan sulfate proteoglycan and laminin with average values that were 30 and 60%, respectively, of nondiabetic controls; the fibronectin content of the diabetic GBM, however, was not significantly different from the normal. These findings, together with previous studies showing increases in GBM collagen, indicate that an alteration in the macromolecular architecture of this basement membrane occurs in diabetes that may be responsible for the filtration defect and the ultimate glomerular occlusion.

Reduced content and abnormal distribution of anionic sites (acid proteoglycans) in the diabetic glomerular basement membrane

Sulfated proteoglycans (fixed anionic sites) on the glomerular basement membrane (GBM) of kidneys from diabetic and nondiabetic patients have been demonstrated by electron microscopy using polycationic dyes (ruthenium red, polyethyleneimine). These substances were used for immersion fixation of renal biopsy specimens. The thickened GBM of diabetics revealed a reduced proteoglycan content within both the narrowed laminae rarae, where normally particles were seen at 60 nm intervals. Proteinuria was observed in all such cases, but no immunopathological alterations of the basement membranes were seen. With both tracer substances anionic sites were also demonstrated in different segments of the thickened lamina densa in diabetics. In polyethyleneimine-treated biopsies some segments of the membrane showed increased anionic moieties at the junction of the basement membrane and the epithelial and endothelial cell membranes. These are probably acid glycoproteins linked to the cell membrane and the synthesis of these basement membrane components may represent a compensatory mechanism seeking to restore normal permeability.

Glomerular membranopathy in adolescents with insulin-dependent diabetes

Kidney biopsy specimens from 15 patients ranging in age from 11 to 19 years with two- to 14-year histories of insulin-dependent diabetes mellitus were evaluated electron microscopically. Although the mean duration of disease was only eight years, the glomerular basement membrane (GBM) in these patients showed a variety of alterations typical of insulin-dependent diabetes mellitus. Saccular glomerular microaneurysms, previously little recognized, were seen in six of the specimens. These lesions, always associated with breaks in the paramesangial BM, were morphologically distinct from the ectatic capillary loops and glomerular capillary aneurysms described previously in diabetic glomerulopathy. All of the patients with such aneurysms also had other severe GBM alterations. Lytic or mechanical injury to the structurally and biochemically altered diabetic GBM may be responsible for the formation of microaneurysms.

Metabolism of glomerular basement membrane in normal, hypophysectomized, and growth-hormone-treated diabetic rats

The in vivo synthesis of the renal glomerular basement membrane (GBM) collagen was studied in normal, hypophysectomized (hypox), diabetic, and growth-hormone (GH)-treated diabetic rats, using tritiated proline ( l[2,3, 3H]proline) as a radioisotopic precursor. After the injection of tritiated proline, all groups of rats were sacrificed at various time intervals and the specific activities of proline and hydroxyproline of GBM were determined, the latter being used as a measure of GBM collagen synthesis. A significant decrease in both proline and hydroxyproline specific activities were observed in GBM of hypox rats at all periods of study. Administration of GH to hypox rats returned the GBM collagen synthesis to normal. Diabetic GBM had higher proline and hydroxyproline specific activities when compared to normal rats. Treatment of diabetic rats with GH for 10 days further increased both proline and hydroxyproline specific activities when compared either to diabetic or normal rats treated with GH. The activity of glucosyltransferase, an enzyme involved in the biosynthesis of the disaccharide unit of GBM collagen was found to be decreased in glomeruli of hypox rats. In contrast, the activity of N-acetyl-β-glucosamini-dase, a glycoprotein-degrading enzyme, was found to be significantly increased in hypox rats. GH treatment restored both enzyme activities to normal. The results of the present study show that GBM collagen synthesis is decreased in hypox rats and increased in diabetic rats. GH treatment not only normalized GBM collagen synthesis in hypox rats but also caused significant increase in diabetic rats. This suggests that the renal GBM metabolism is influenced by GH, and this may be of particular significance in view of GH involvement in diabetic microvascular complications.

Effect of diabetes on in vivo metabolism of [35S]-labeled glomerular basement membrane.

Glomerular basement membrane (GBM) was labeled in vivo by the injection of tracer amounts of [35S]-sulfate into normal and streptozotocin-diabetic rats. The biosynthesis and turnover of sulfated glycosaminoglycans in the GBM was determined from the specific activity of [35S] after pronase digestion of basement membranes purified from glomeruli isolated 1-7 days after injection. Peak radiolabeling of both normal and diabetic GBM occurred 24 h after injection and, when corrected for differences in serum sulfate specific activities, was less in diabetic than in normal samples. The specific activity of GBM sulfate, expressed as cpm/microgram uronic acid, progressively diminished over the ensuing period of study in both normal and diabetic samples. The rate of decrease in specific activity of [35S]-labeled GBM was not significantly different in diabetic preparations compared with that in normal controls. The findings are compatible with diminished sulfation and/or production but normal turnover of glycosaminoglycans in the renal GBM in experimental diabetes.

Glomerular manifestations of diabetes in the BB rat

Morphologic characteristics of the renal glomeruli and tubules of BB rats with spontaneous diabetes mellitus were studied at 30 weeks' duration of diabetes. Whereas the glomerular basement membrane (GBM) was significantly thickened, no changes in the diabetic glomeruli were seen in the peripheral capillary wall area and in the fractional volumes of the mesangial cells or of the mesangial matrix. Light microscopy of the diabetic kidneys were normal, and immunofluorescent examination of diabetic glomeruli showed no increased accumulation of albumin, C3, or IgG. Diabetic rats had increased renal blood flow and glomerular filtration rates. Diabetic rats at 7, 17, and 30 weeks excreted normal amounts of urinary albumin. Thus kidneys of the BB diabetic rat differ from other experimental models of diabetes in that GBM thickening occurs in the absence of mesangial changes and of increased albuminuria. These studies suggest that the mesangium may influence glomerular permeability in diabetes, while thickening of the GBM in diabetes does not necessarily coincide with increased urinary albumin excretion. Furthermore, these results are consonant with the hypothesis that genetic factors may influence the pathological expression of diabetic nephropathy in rats.

Enzymatic reversibility of nonenzymatic glycosylation of the glomerular basement membrane. Is the diabetic glomerulopathy principally reversible?

Recent investigations point to the nonenzymatic glycosylation as a cause of long-term complications in diabetes mellitus. We describe an enzymatic activity that cleaves glucose from the glomerular basement membrane (GBM), present in lysosomal preparations of diabetic lymphocytes. The GBM, nonenzymatically glycosylated or obtained from rats with diabetes, were incubated with enzyme preparations, separated on Sephadex G-25 and applied for glucose measurement on gas chromatography and mass spectroscopy. The lysosomal preparation of diabetic lymphocytes cleaved from rat GBM, which were nonenzymatically glycosylated 300-500 ng glucose/mg GBM protein, from diabetic rat GBM 300 ng glucose/mg GBM protein. A lysosomal preparation of normal lymphocytes failed to do so, indicating enzyme induction in the diabetic state. Control studies with the glycosylated hemoglobin AIc confirmed this finding and showed the specificity of the enzyme, as alpha-glucosidase and beta-glucosidase failed to cleave the N-glycosidic bond between glucose and the protein. The enzymatic activity can be described formally as a N-l-deoxyfructofuranosyl-glucohydrolase, which could be responsible for a potential reversibility of diabetic GBM changes.

In vivo biosynthesis and turnover of glomerular basement membrane in diabetic rats.

Glomerular basement membrane (GBM) was labeled in vivo by the injection of tracer amounts of tritiated proline into normal and streptozotocin-diabetic rats. Basement membrane biosynthesis and turnover were determined from the specific activities of proline and hydroxyproline in samples purified following osmotic lysis of glomeruli isolated 4 h to 12 days after injection. Peak radiolabeling of normal and diabetic GBM occurred within 24-48 h and 48-72 h, respectively, and, when corrected for differences in the serum proline specific activities, [3H]proline incorporation was greater in diabetic than in normal samples. In contrast to the subsequent time-dependent progressive decline in radiolabeling in basement membranes from normal animals, specific activities of proline and hydroxyproline in diabetic glomerular basement membrane did not change significantly over the same period of observation. Renal cortical mass and glomerular basement membrane collagen content were preserved in diabetic animals despite loss of body weight. The findings are compatible with prolongation of glomerular basement membrane turnover in experimental diabetes, and suggest that diminished degradation contributes to the accumulation of glomerular basement membrane that is characteristic of chronic diabetes.

Increased concentration of albumin in kidney basement membranes in diabetes mellitus.

The concentration of albumin was measured by a double-antibody radioimmunoassay in the glomerular basement membrane (GBM) and tubular basement membrane (TBM) of patients with diabetic nephropathy, other kidney diseases, and normal kidneys. The TBM from diabetics contained significantly more albumin (m +/ SD; 2.75 +/- 0.34 microgram/mg) than the TBM from other diseases (1.00 +/- 0.24 microgram/mg) or normals (1.21 +/- 0.26 microgram/mg) (P less than 0.0001). Similarly, diabetic GBM contained more albumin (2.25 +/- 0.59 microgram/mg) than other diseases (1.22 +/- 0.55) or normals (1.31 +/- 0.36 microgram/mg) (P less than 0.01-0.001). No differences were observed between the normal and other disease groups. Although there were no differences in hydroxyproline content, there was a highly significant correlation between the concentration of hydroxyproline and albumin in the diabetic TBM (r = 0.82), diabetic GBM (r = 0.70), and normal TBM (r = 0.79). Elution studies with different buffers on frozen sections of diabetic kidneys suggest that the albumin is relatively firmly bound. Although albumin is present in extracellular membranes of normal and diseased human kidneys, the concentration is higher in diabetes mellitus.

Identification of specific amino acids in diabetic glomerular basement membrane collagen subject to non-enzymatic glucosylation [formula omitted

Glomerular basement membrane was purified from normal and streptozotocin-diabetic rats, and the insoluble collagenous fraction isolated following reduction and alkylation. Amino acid analysis of diabetic samples revealed three unusual peaks eluting near glucosamine. Two of these peaks had respective elution times identical to those of synthetic glucitol-hydroxylysine and glucitol-lysine. These findings identify lysine-derived amino acids in glomerular basement membrane collagen as sites of excess non-enzymatic glucosylation in vivo in hyperglycemic diabetes.