Identification and Turnover of Glycosaminoglycans in Rat Kidneys

Barry, D. N.; Bowness, J. M.

doi:10.1139/o75-098

Cited by 20 publications

(5 citation statements)

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“…Second, whether anionic sites were rapidly shed from the GBM during turnover i s difficult to evaluate because turnover rates for GBM proteoglycans have not been firmly established. Metabolic labeling studies with 35S in adult rats have suggested that some renal glycosaminoglycans have half-lives of 2-7 d (Barry and Bowness, 1975;Cohen and Surma, 1982), but this time frame is significantly longer than that seen here for CF disappearance in newborns. Third, although CF clearly binds abundantly to GBM anionic sites, including heparan sulfate proteoglycans, this certainly is a n ionic and probably tenuous association, particularly in vivo.…”

Section: Rapid Removal Of Cf From the Gbmcontrasting

confidence: 55%

Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys

Abrahamson

Perry

1986

Anat. Rec.

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To label heparan sulfate proteoglycans and other strong anions within glomerular basement membranes (GBM) during assembly, cationized ferritin (CF), with a narrow isoelectric range of 7.7 to 8.2, was intravenously injected into newborn rats. Kidneys were then fixed and processed for electron microscopy at intervals ranging from 1 to 72 h after CF injection. One hour after injection, CF bound extensively to the lamina rara interna and externa of developing GBM and mesangial matrix and to tubular basement membranes (TBM). In double basement membranes of early stage glomeruli, large amounts of CF were also seen in central areas between the endothelial and epithelial basement membranes. In maturing-stage glomeruli, CF bound throughout interior regions of GBM outpockets projecting into the epithelial side of capillary walls as well as to the laminae rarae. Because in adult rats CF binds only to the laminae rarae, the abundant anionic sites seen here in newborns between double basement membranes and within GBM outpocket interiors may be subsequently neutralized or removed during the GBM assembly process. In addition to basement membranes, CF was also located intracellularly within endocytic vesicles and lysosomes of glomerular endothelial, mesangial, and epithelial cells 1 h postinjection. CF was also present in similar structures within the tubular epithelium. In contrast to these findings, CF was gradually lost from developing GBM 5, 15, and 24 h after injection and was essentially cleared from all GBM, mesangial matrices, and TBM after 48 h. Large CF aggregates were progressively accumulated within mesangial lysosomes, however. The transient binding of CF to GBM anionic sites seen here was most likely due to its endocytic removal by developing glomerular endothelial, mesangial, and epithelial cells. Anions in the circulation probably also competed effectively with the GBM and TBM for bound CF.

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Section: Rapid Removal Of Cf From the Gbmcontrasting

confidence: 55%

Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys

Abrahamson

Perry

1986

Anat. Rec.

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“…As discussed previously (4, 5), GAG (22,23), bovine (29), human (30,31), guinea pig (32), and rabbit (32) (33), who isolated and characterized GAG from renal homogenates (after radiolabeling in mvo). They determined that heparan sulfate is the principal GAG in both cortex and medulla and found, in addition, that the renal heparan sulfate has a half-life comparable to that in other tissues (2-6 days).…”

Section: Discussionmentioning

confidence: 93%

Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes

Kanwar

Farquhar

1979

Proc. Natl. Acad. Sci. U.S.A.

248

105

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Glycosaminoglycans were isolated from purified fractions of Isolation of Glomeruli. Kidneys were obtained from decapitated rats (150-200 g, both male and female), frozen at -20°C in normal saline, and stored for 24 hr to several weeks. Twelve to fifteen kidneys were removed at a time, and glomeruli were isolated therefrom by the technique of Krakower and Greenspon (6), carried out at 4°C. The efficiency of the glomerular isolation was monitored by examining a droplet of the suspension under a dissecting microscope. Any preparation found to contain tubular or interstitial fragments was resuspended in normal saline and centrifuged at 75 X g for 1-2 min, and the sediment was reexamined for contaminating tissue fragments. This process was repeated until all detectable contaminants were removed and the preparation consisted of virtually 100% glomeruli of which -u85% were free of Bowman's capsule and t15% were still encapsulated (Bowman's capsule present). The glomeruli thus isolated were pooled and stored at -20°C in normal saline.Isolation of GBM. Basement membrane fractions were prepared from the isolated glomeruli by the method of Meezan et al. (7) with minor modifications. Briefly, the glomeruli were hypotonically lysed in 0.05% sodium azide for 2 hr, digested with deoxyribonuclease (100 units/ml in 1 M NaCl) for 2 hr, and subsequently treated with 1% deoxycholate for 3 hr, all procedures being carried out at 4°C. The GBM fractions thus obtained were washed twice with distilled water and once with 0.15 M NaCl. An aliquot of each fraction was processed for electron microscopic examination in order to check for contamination by non-GBM (cell) components and to assess the preservation of the anionic sites and of their characteristic distribution pattern by using cationized ferritin (4). The remainder of each GBM fraction was lyophilized.Extraction of GAG from GBM. In general, the method followed was that used for the isolation of GAG from bovine lung by Linker and Hovingh (8,9). Isolated GBM (-50-100 mg) were suspended in 50 ml of 0.1 M acetate buffer (pH 5.5), containing 1 mM EDTA and cysteine. Crystalline papain (10 mg, 10.8 units/mg) was added, and the suspension was incubated at 60°C for 24 hr. The pH was then raised to 7.3 with a few crystals of Tris base, Pronase (50 mg, 89,600 PUK/g) was added, and the digestion was continued for another 24 hr at 370C. The suspension was then centrifuged at 10,000 X g for 15 min. The sediment was saved for electron microscopic exAbbreviations: GAG, glycosaminoglycans; GBM, glomerular basement membrane(s). 4493The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…In the abnormal glomerular basement membrane of diabetic humans, for which decreased levels of heparan sulfate proteoglycan have been demonstrated, chemical and immunochemical techniques were employed [7]; since the very low myocardial proteoglycan levels make their chemical measurement difficult, a comparison of the concentration of these compounds in normal and diabetic hearts would be facilitated by antibodies to purified heart proteoglycans. Previous studies of myocardial proteoglycans have been limited to the identification of the glycosaminoglycans chains present in the tissue, which include in addition to heparan sulfate, hyaluronic acid, chondroitin sulfate and dermatan sulfate [37]. In the present investigation, intact rat myocardial proteoglycans have been characterized by anion exchange chromatography and electrophoresis; of particular interest is the observation that of the three bands seen by SDSpolycrylamide electrophoresis under reducing conditions, most of the label is present in a relatively small molecular weight component (Mr=85,000).…”

Section: Discussionmentioning

confidence: 99%

Sulfate metabolism in the alloxan-diabetic rat: relationship of altered sulfate pools to proteoglycan sulfation in heart and other tissues

1987

Diabetologia

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The incorporation of [35S]sulfate into heart proteoglycans has been studied in normal and alloxan-diabetic rats by perfusion and in vivo administration of the isotope; in the latter situation, comparison was also made of radiolabeled sulfate utilization by several other tissues (kidney, liver, lung, muscle, testes and skin). The radiolabeled products were characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis and anion exchange chromatography, as well as by sizing of the glycosaminoglycan chains by gel filtration both before and after nitrous acid treatment. The most prominent band observed in the heart guanidine extract by electrophoresis had a molecular weight of 85,000 and minor components (Mr = 360,000 and 170,000) were also detected; approximately 20% of the proteoglycan associated [35S]sulfate was present in heparan sulfate chains. After perfusion the pattern, as well as the amount of radioactivity recovered from the diabetic heart, was similar to the normal heart. In contrast, after intraperitoneal injection of the [35S]sulfate, a substantial reduction in incorporation was found not only in heart but in several other tissues studied, although no qualitative differences were noted in the macromolecules formed by the two groups of animals. Measurement of the serum sulfate concentration indicated that the level in the alloxan-diabetic rat (1.23 mmol/l) was significantly less (p less than 0.01) than that of the normal rat (1.67 mmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

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Identification and Turnover of Glycosaminoglycans in Rat Kidneys

Cited by 20 publications

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Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys

Distribution of intravenously injected cationized ferritin within developing glomerular basement membranes of newborn rat kidneys

Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes

Sulfate metabolism in the alloxan-diabetic rat: relationship of altered sulfate pools to proteoglycan sulfation in heart and other tissues

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