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V Y Wu

Publications and source records attributed to V Y Wu.

35 records · Page 2Linked to original sources
PubMed1988

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.

Aldehyde Reductase↗
PubMed1987

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.

Animals↗
PubMed1985

Quantitative analysis of glomerular basement membrane glycosaminoglycans and evidence for their binding to adjacent cell membrane lipids.

Glycosaminoglycans (GAG) were isolated from rat renal glomerular basement membranes subjected to extraction with detergents, and were quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to microgram amounts of chondroitin and heparan sulfate. Whereas crude membranes prepared by osmotic lysis contained only 6 micrograms/mg dry wt, subsequent treatment with Triton X-100 or deoxycholate (DOC) increased measureable GAG to about 17 and 34 micrograms/mg, respectively. Repeated freezing and thawing of isolated glomeruli also augmented measurable GAG content in subsequently osmotically lysed membranes to levels observed in Triton-treated membranes. DOC solubilized approximately equal to 15-20% of membrane-associated GAG. Chondroitin sulfate comprised approximately equal to 30% of total GAG, and all of the chondroitin sulfate but only 10% of the heparan sulfate was extracted from the insoluble matrix by DOC. The findings indicate that GAG content of glomerular basement membrane is several-fold higher than previously estimated, and that a substantial portion is bound to cell membrane lipids. The results further suggest two populations of GAG in basement membrane; one that is intercalated with adjacent cell membranes, and another that remains as an integral component of the insoluble matrix after detergent extraction.

Animals↗
PubMed1984

A competitive binding assay for measurement of heparan sulfate in tissue digests.

Glycosaminoglycans complex with constituents of normal human serum, a finding that was exploited to develop a competitive binding assay for these substances. Heparan sulfate was isolated from renal cortex and radiolabeled with tritiated borohydride. The elution pattern of the radiolabeled material on Sephadex G-25, Bio-Gel P-30, and AG- 1X8 resin was identical to that of unlabeled heparan sulfate. The tritiated heparan sulfate formed radiolabeled precipitates when incubated with serum and zinc acetate. Binding was dose dependent and saturable. Heparin, heparan sulfate, and the chondroitin sulfates, but not hyaluronate or keratan sulfate, competed with the radiolabeled heparan sulfate for binding in a dose-dependent manner. The assay is specific for heparin polysaccharides in chondroitinase ABC-treated samples and is sensitive to microgram quantities.

Binding, Competitive↗
PubMed1984

Platelet factor 4 binding to glomerular microvascular matrix.

Platelet factor 4, a unique peptide released during platelet aggregation, can bind to natural sulfated glycosaminoglycans from human renal cortex. The glycosaminoglycan isolate contained components sensitive to hyaluronidase, chondroitinase ABC and nitrous acid. Binding was demonstrated by the change in electrophoretic mobility of platelet factor 4 applied in combination with glycosaminoglycan compared to either applied alone. This effect, which occurred at physiologic pH but not at acidic pH or with high ionic strength, was preserved in samples subjected to prior hyaluronidase and chondroitinase digestion. Further demonstration that platelet factor 4 can interact with heparan sulfate anionic sites in the glomerular microvascular matrix was obtained by incubating radiolabeled platelet factor 4 with isolated rat glomeruli, and with purified human and rat glomerular basement membrane, followed by displacement with heparin. Total binding and heparin-released binding were decreased in glomerular basement membrane prepared from diabetic patients and from rats with streptozotocin-diabetes compared to control samples. These findings implicate platelet factor 4 in the pathogenesis of the altered capillary integrity associated with diabetes, and provide novel evidence that heparan sulfate anionic sites in glomerular basement membrane are diminished in human and experimental diabetes.

Animals↗
PubMed1983

Intermolecular reducible cross-links in rat glomerular basement membrane.

Glomerular basement membrane (GBM) was purified from adult rats and treated with tritiated borohydride for analysis of the reducible cross-links. After acid hydrolysis, samples were subjected, with and without prior gel filtration on Biogel P-2, to a chromatographic system standardized with known cross-links. The major peak of radioactivity co-eluted with authentic di-hydroxylysinonorleucine (di-OHLNL) standard. Derivation of this cross-link from lysine was corroborated by demonstrating its presence in GBM purified after incubation of isolated glomeruli with [14c]-lysine and after in vivo injection of [14C]-lysine. GBM labeled in vitro with radioactive lysine also contained peaks co-eluting with hydroxylysinonorleucine and lysinonorleucine standards, as well as at unidentified positions in the chromatogram. The findings indicate that rat GBM contains lysine-derived cross-links, of which di-OHLNL is the major reduced form.

Amino Acids↗
PubMed1982

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.

Animals↗
PubMed1981

Non-collagen protein and proteoglycan in renal glomerular basement membrane.

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8-12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [35S]sulfate into basement membrane from which this non-collagenous 35S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (Mr greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [35S]sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.

Amino Acids↗
PubMed1981

Nonenzymatic glycosylation of glomerular basement membrane.

To test the hypothesis that increased nonenzymaticc glycosylation contributes to the microangiopathic sequelae of diabetes, we examined the interaction of glucose with basement membranes in vivo and in vitro. The level of nonenzymatic glycosylation, determined by hydroxymethylfurfuraldehyde (HMF) generation, in glomerular basement membrane (GMB) collagen purified from rats with streptozotocin diabetes was significantly greater than that of nondiabetic GMB. Incubation of purified rat GBM and lens capsule basement membrane with [14C]-glucose in vitro resulted in the incorporation of [14C] into acid precipitable radioactivity in a reaction that was dependent on time, temperature, and glucose concentration. Glucose-dependent nonenzymatic glycosylation of GBM in vitro was documented by measurement of HMF generation after incubation with unlabeled glucose. These results provide direct experimental evidence that hyperglycemia, via nonenzymatic glycosylation, modifies the chemistry of proteins specifically involved in diabetic glomerulosclerosis.

Amino Acids↗
PubMed1981

Biochemistry of glomerular basement membrane components purified by nondisruptive techniques.

In contrast to the fragmentation and loss of spatial arrangement seen in preparations of basement membrane isolated after sonic disruption, the architectural integrity of glomerular basement membrane is presented with the described technique of osmotic lysis and sequential detergent treatment. The biochemistry of components solubilized without pepsin digestion from GBM prepared with this technique is described.

Amino Acids↗
PubMed1981

Deposition of basement membrane in vitro by normal and diabetic rat renal glomeruli.

Glomeruli isolated from rat renal cortex were incubated with radioactive precursor to study basement membrane production in vitro. Following incubation, basement membranes were purified with a novel technique of osmotic lysis followed by selective solubilization with detergents that leaves the membranes architecturally intact and results in their quantitative isolation. Incorporation of [14C]-proline into basement membrane increased in a linear fashion during the 3 h of incubation studied, with a progressive increase in the specific activity of hydroxy-[14C]-proline in isolated glomerular basement membrane. With identical incubational conditions, glomeruli from streptozotocin diabetic rats incorporated significantly greater amounts of precursor into the extracellular matrix than did glomeruli from nondiabetic animals.

Animals↗
PubMed1977

Purification and characterization of human and bovine platelet factor 4.

Platelet antiheparin, platelet factor 4, was isolated from freeze-thaw lysates of fresh bovine and outdated human platelet concentrates by a single step affinity chromatographic procedure. The yields of PF4 were 93 microgram and 142 microgram/ml of human and bovine platelets respectively. Antiheparin activity of the products were 558 units/mg for the bovine isolate and 489 units/mg for the human material. The bovine product is a single chain polypeptide with an apparent molecular weight of 12,300. Amino acid composition indicates 107-109 residues compared to the smaller human product which has an apparent molecular weight of 8,000 for a 70 residue polypeptide. The intact polypeptide was resistant to enzymatic hydrolysis as opposed to the reduced-alkylated derivative which was susceptible to hydrolysis in the presence and absence of heparin.

Amino Acids↗