Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes

Abstract

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.

Preventive effect of cerivastatin on diabetic nephropathy through suppression of glomerular macrophage recruitment in a rat model

“…These sites consist of glycosaminoglycans, which are rich in heparan sulphate proteoglycan (HSPG) [34]. HSPG molecules on the GBM are synthesized by mesangial cells as well as by glomerular epithelial cells.…”

Glycosaminoglycans from renal brush border membranes of rabbits.

“…The present data indicate that the renal brush border membrane contains chondroitin sulfate A/C and dermatan sulfate, but that hyaluronic acid and heparan sulfate are absent. Heparan sulfate has been shown to be the predominant GAG in kidneys (Murata 1983), and Kanwar and Farquhar (1979) reported that heparan sulfate is associated with the basement membrane. In our previous study (Munakata and Yosizawa,1980b), we isolated a sulfated glycoprotein from intestinal brush borders, histological appearance of which resembles that of the renal brush border.…”

“…There has been, however, little information concerning the distribution of individual GAG in specific renal structural components, although proteoheparan sulfate and heparan sulfate have been isolated from the glomerular basement membrane (Kanwar and Farquhar 1979;Stow et al 1983).…”

Alteration in the distribution of type IV collagen in glomerular basal laminae in diabetic rats as revealed by immunocytochemistry and morphometrical approach

“…Application of various immunocytochemical techniques has allowed for the localization of these different components in basal laminae of tissues from normal animals [16][17][18][19]. Although the distribution and three dimensional arrangement of basal lamina components may constitute the basis for the selectivity of capillary permeability [20,21], they have been little investigated in tissues from diabetic animals. Recently however, some studies have detected biochemical alterations in the proteoglycan component of 'diabetic' basal laminae [22][23][24].…”