Chemical characterization of glomerular and tubular basement membranes of cattle of different ages

Glomerular and tubular basement membranes were isolated from fetal, neonatal, young and adult bovine kidneys.An isolation method with sieves for both glomeruli and tubules from the same kidney was developed. A detergent procedure appeared to give purer glomerular and tubular basement membrane preparations than the generally used sonication method. No large differences were found in the composition of glomerular and tubular basement membrane of adult animals.Glomerular and tubular basement membrane preparations of the four age groups showed an increase with age of hydroxylysine and both 3- and 4-hydroxyproline. The most marked increases appeared at different stages of development, that of tubular basement membrane being between fetal and neonatal stages and glomerular basement membrane between 18 weeks old and adult animals. The ratio of 3- to 4-hydroxyproline increased considerably during development. Total imino acid content was higher for both types of basement membrane from adult than from young animals, while total content of hydroxylysine plus lysine remained fairly constant.The increase in hydroxylation of lysine was accompanied by a corresponding change in glucose and galactose content so that the ratio of galactose to hydroxylysine or glucose to galactose remained constant. Fucose content of both types of basement membranes was the same for all age groups but content of aminosugars and mannose gradually increased with age.     

Self-digestion of erythrocyte membranes of various mammalian species

1. The rate of self-digestion of main membrane proteins, spectrin and band 3 protein, was studied for erythrocyte membranes of eight mammalian species: man, cow, pig, cat, rabbit, hamster, mouse and rat. 2. Spectrin and band 3 protein were most rapidly degraded in human and pig ghosts. The rates of proteolysis for other species were similar. 3. The rates of self-digestion were correlated neither with lifetime of red cells nor lifespan of animals of various species.     

Chemical composition and solubility of human glomerular and tubular basement membranes of adult and senescent men

The effect of aging on the composition of human renal basement membranes was studied in persons aged 22-90 years. The relative proportion of cortex in kidney appears to decrease with aging. Twenty pairs of GBM and TBM preparations were isolated using the detergent method. Protein content of the basement membrane preparations amounts to about 66% and is independent of type of membrane or age. Amino acid and carbohydrate analyses of both GBM and TBM revealed that the extents of hydroxylation of proline at the C4 position and of lysine decrease with aging. In the case of lysine this occurred from the seventh of life onwards. The decreases are probably not caused by a change of the collagen content. The extent of glycosylation of hydroxylysine is similar for all basement membrane preparations. An adapted protein assay is presented for solutions containing SDS and dithiothreitol. In the presence of these two compounds, solubility of GBM and TBM from adult and aged persons is similar and amounts to about 55 and 85% after 10 and 60 min, respectively, of heating at 95 degrees C. In SDS-polyacrylamide gels, no differences were observed for the major peptide bands between the preparations, irrespective of basement membrane type or age.     

Lipids associated with bovine kidney glomerular basement membranes.

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.     

Composition of renal basement membranes isolated under various conditions

GBM isolated from a surgical biopsy directly or after a 22 hr incubation period--to imitate the usual interval between death and isolation--appeared to be nearly identical in amino acid composition. Sonication and detergent procedures for isolation of GBM and TBM lead to preparations of different chemical composition. Phosphorus analysis and electron micrographs indicate the presence of material of supposedly cellular origin in sonicated but not in detergent-treated bovine and human GBM. Detergent-treated bovine and human GBM preparations are more enriched in the collagen-typical amino acids than sonicated samples. SDS-PAGE analyses show a nearly identical polypeptide pattern. Sonicated and detergent-treated bovine TBM preparations are free of cellular material. They show in SDS-PAGE a similar heterogeneous polypeptide pattern, but with lower intensities of three components with molecular weights between 30 and 60 kdalton. Sulfated GAG's are present in higher concentration in sonicated than in detergent-treated GBM and TBM. Collagen is not extracted from glomeruli and tubules by detergent treatment.     

Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes.

1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably.     

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.     

Distribution of glomerular peripolar cells in different mammalian species

Summary Peripolar cells are granulated glomerular epithelial cells that form a cuff around the vascular pole of the glomerulus. Quantitation of these cells in 17 species of mammals (including man, several laboratory animals and a variety of other species) indicated that they were detectable by light microscopy in all but one of the mammals that were examined (the Australian hopping mouse). In adult mammals with detectable peripolar cells, the peripolar cell index (the percentage of randomly sectioned glomeruli that displayed peripolar cells in histological sections of kidney) ranged from 0.15 (for echidna) to 11.86 (for sheep). Newborn lambs and rats showed strikingly high values (23.30 and 10.76, respectively) compared with their adult counterparts. Using electron microscopy, peripolar cells were observed in all species that were examined, including the Australian hopping mouse. Morphologically, peripolar cells were similar in all species although their size and granule population varied. They showed a predominantly outer cortical glomerular distribution and a close anatomical relationship with the renin-containing myoepithelioid cells. These findings indicate that peripolar cells are present in a wide variety of species and support the view that such cells may play a significant role in the regulation of normal renal function.     

Apparent densities of spermatozoa of various mammalian species

The apparent densities of sperm cells in several mammalian species were determined by means of Percoll density-gradient centrifugation. Semen samples were obtained from (1) ejaculates from the bull, human, and rabbit, and (2) from the cauda epididymis of the goat, golden hamster, house musk shrew, mastomys, and mouse. The profiles of sperm distribution showed a single peak after the centrifugation in the first group and goat, whereas two separate main peaks in the second group (except goat). This disparity in sperm distribution profiles may be due mainly to differences in the degree of maturity of the sperm. Highly motile and mature sperm were obtained at higher densities, whereas immotile or immature sperm were found at the apparent densities. Thus, in mammals, the profiles of sperm distribution in the Percoll density gradient are classified into two types, those with a single peak and those with two separate peaks. The apparent density of sperm cells may be of importance in sperm physiology.     

Macromolecular organization of basement membranes. Characterization and comparison of glomerular basement membrane and lens capsule components by immunochemical and lectin affinity procedures

The macromolecular components of bovine glomerular basement membrane (GBM) and lens capsules (anterior and posterior) solubilized by sequential extractions with denaturing agents were quantitated and characterized by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography with the help of immunochemical techniques. Laminin, entactin, fibronectin, and heparan sulfate proteoglycan were primarily recovered (over 80%) from both basement membranes in a guanidine HCl extract which contained only a limited amount of the total protein (10-14%); most of the remainder of these noncollagenous components could be solubilized by the guanidine in the presence of reducing agent. Although a portion of the Type IV collagen could be obtained by these treatments, effective extraction of this protein depended on exposure to sodium dodecyl sulfate under reducing conditions. Immunoblot analysis revealed a remarkably similar pattern for GBM and lens capsule Type IV collagens with prominent bands of Mr = 390,000, 210,000, and 190,000 being evident. Fibronectin was present in much greater amounts in GBM than lens capsule while the reverse was true for entactin. In both GBM and lens capsules, the entactin (Mr = 150,000) exceeded laminin; the latter protein on immunoblotting was found to contain primarily the alpha-subunit (Mr = 200,000). The size of the heparan sulfate proteoglycan from anterior (Mr = 400,000) and posterior lens capsule (Mr greater than 500,000) was substantially larger than that from GBM (Mr = 200,000). During DEAE-cellulose chromatography under nonreducing conditions in a denaturing solvent, a portion of the Type IV collagen coeluted with the proteoglycan from these membranes. Considerable Bandeiraea simplicifolia I binding activity (alpha-D-galactose specific) was observed in GBM and lens capsule extracts and column fractions which could not be accounted for by laminin alone. Several components which reacted with this lectin were seen on transblots and among these Type IV collagen was identified. In contrast to the basement membranes from bovine tissues, the constituents from human GBM did not react with the B. simplicifolia I lectin.     

A comparison of anionic sites in the glomerular basement membranes from different classes of fishes

Summary Cationized ferritin was injected into the circulatory system of teleosts, the sea raven and Atlantic eelpout, and into elasmobranchs, the spiny dogfish and the skate, to determine if the glomerular basement membranes (GBM) from these different groups of fishes possess anionic binding sites similar to those present in the GBM of mammals. The distribution of cationized ferritin was the same in all fishes listed. Cationized ferritin was localized only in the GBM and the mesangial matrix. The regular distribution of cationized ferritin within the laminae rarae (60 nm intervals) was taken as evidence of the presence of anionic binding sites. Cationized ferritin did not bind to the glomerular capillary endothelium, nor was any of it localized at the base of the slit diaphragms of the foot processes of the podocytes. The distribution of binding sites in the GBM of these fishes is similar to that in another teleost, the winter flounder, and in a cyclostome, the hagfish.     

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.