scholarly journals A steady-state labelling approach to the measurement of proteoglycan turnover in vivo and its application to glomerular proteoglycans

1996 ◽  
Vol 320 (1) ◽  
pp. 301-308 ◽  
Author(s):  
Emma L AKUFFO ◽  
Jayne R HUNT ◽  
Jill MOSS ◽  
David WOODROW ◽  
Malcolm DAVIES ◽  
...  
Keyword(s):  
Steady State ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Immunogold Labelling ◽  
Puromycin Aminonucleoside ◽  
Turn Over ◽  
T Values

Rats were implanted with mini osmotic pumps delivering sodium [35S]sulphate and their newly synthesized proteoglycans were labelled over a 146 h period (steady-state labelling). Proteoglycan turnover was measured in vivo using a chase protocol. Glomerular proteoglycans were recovered quantitatively and the perlecan present was isolated by immunoprecipitation. The procedure allows newly synthesized proteoglycans to be quantified in mass units (pmol of glycosaminoglycan sulphate) after labelling and during the chase. Ultrastructural-immunogold experiments identified the location of perlecan as the glomerular basement membrane and mesangial matrix. Perlecan in the basement membrane was quantified using the ultrastructural-immunogold technique. Perlecan comprises about 10% of the total glomerular proteoglycans, which are otherwise associated with glomerular cells and the mesangium. Both the total glomerular heparan sulphate proteoglycans and perlecan turn over rapidly (t½ ∼ 3–4 h and < 3 h respectively). In contrast, turnover of proteoglycans in other tissues was slow, except in the liver where the heparan sulphate and chondroitin sulphate t½ values were 16 h and 9 h respectively. Microalbuminuria was induced with a low-dose regimen of puromycin aminonucleoside. At the onset of microalbuminuria (5 days) there was no change in the level of newly synthesized perlecan, or in perlecan in the glomerular basement membrane detected by immunogold labelling. Newly synthesized perlecan had undergone a minimal change in turnover rate by day 5 in puromycin aminonucleoside-treated rats. In contrast, the total glomerular proteoglycan population showed a dramatic decrease in turnover by day 5. Since there was no evidence of accumulation of glomerular proteoglycans on either day 5 or day 6, it is likely that decreased turnover of cell-associated proteoglycans is accompanied by an equivalent decrease in their synthesis.

scholarly journals Renal glomerular proteoglycans. An investigation of their synthesis in vivo using a technique for fixation in situ

1988 ◽  
Vol 251 (2) ◽  
pp. 411-418 ◽  
Author(s):  
L A Beavan ◽  
M Davies ◽  
R M Mason
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Membrane Fraction ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Basement Membranes ◽  
Chondroitin Sulphate Proteoglycans

Newly synthesized rat glomerular [35S]proteoglycans were labelled in vivo after injecting Na2[35S]SO4 intraperitoneally. At the end of the labelling period (7 h) the kidneys were perfused in situ with 0.01% (w/v) cetylpyridinium chloride. This fixed proteoglycans in the tissue and increased their recovery 2-3-fold during subsequent isolation of glomeruli from the renal cortex. The glomeruli were fractionated by a modified osmotic lysis and detergent extraction procedure [Meezan, Brendel, Hjelle & Carlson (1978) in The Biology and Chemistry of Basement Membranes (Kefalides, N.A., ed.), Academic Press, New York; Kanwar & Farquhar (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4493-4497] to obtain a basement membrane preparation. The proteoglycans released at each stage of the procedure were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC and HNO2 digestion and Sepharose CL-4B gel-permeation chromatography. About 85% of the [35S]proteoglycans synthesized were of the heparan sulphate variety, the remainder being chondroitin sulphate proteoglycans. Three sizes of heparan sulphate proteoglycans were identified. The largest (HS1, Kav. 0.47) accounts for 44% of the total extractable heparan sulphates. About one third of HS1 were extracted from the glomerular basement-membrane fraction with 8 M-urea and 4 M-guanidine hydrochloride but the remainder were released from the glomerulus during preparation of the fraction. The two smaller molecules (HS2, Kav. 0.56 and HS3, Kav. 0.68) accounted for 27% and 28% of the extractable heparan sulphate respectively and were not associated with the basement membrane fraction. HS1, HS2 and HS3 were also isolated from non-fixed glomeruli labelled in vivo but with much lower recovery. In glomeruli labelled in vitro, heparan sulphate accounted for only 35% of the proteoglycans, the remainder being of the chondroitin sulphate type. Proteoglycans similar to HS1, HS2 and HS3 were present in glomeruli labelled in vitro but, in addition, a large, highly charged heparan sulphate (HS1a) was extracted from the glomerular basement-membrane fraction of these glomeruli. It accounted for 6% of the total heparan sulphate.

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

1982 ◽  
Vol 242 (4) ◽  
pp. F385-F389
Author(s):  
M. P. Cohen ◽  
M. L. Surma ◽  
V. Y. Wu
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Diabetic Rats ◽  
Basement Membranes ◽  
Membrane Turnover ◽  
Proline Incorporation ◽  
Basement Membrane Collagen ◽  
Osmotic Lysis ◽  
Specific Activities

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.

scholarly journals Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane

1988 ◽  
Vol 252 (1) ◽  
pp. 301-304 ◽  
Author(s):  
W H Baricos ◽  
Y Zhou ◽  
R W Mason ◽  
A J Barrett
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Kidney ◽  
Ph Optimum ◽  
Dose Dependent ◽  
Hydrolysis Of

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

scholarly journals Reduction of heparan sulphate-associated anionic sites in the glomerular basement membrane of rats with streptozotocin-induced diabetic nephropathy

Diabetologia ◽  
1995 ◽  
Vol 38 (10) ◽  
pp. 1169-1175 ◽  
Author(s):  
J. H. M. Berden ◽  
J. van den Born ◽  
A. A. van Kraats ◽  
M. A. H. Bakker ◽  
K. J. M. Assmann ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Heparan Sulphate ◽  
Anionic Sites

scholarly journals Anti-glomerular basement membrane disease: an update on subgroups, pathogenesis and therapies

2018 ◽  
Vol 34 (11) ◽  
pp. 1826-1832 ◽  
Author(s):  
Mårten Segelmark ◽  
Thomas Hellmark
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
De Novo ◽  
Correct Diagnosis ◽  
Rapidly Progressive Glomerulonephritis ◽  
Human Leukocyte ◽  
Pulmonary Haemorrhage ◽  
Leukocyte Antigen ◽  
Type Iv

Abstract Most patients with anti-glomerular basement membrane (anti-GBM) disease present with rapidly progressive glomerulonephritis with or without pulmonary haemorrhage; however, there are several variants and vigilance is necessary to make a correct diagnosis. Such variants include overlap with anti-neutrophil cytoplasm antibodies-associated vasculitis and membranous nephropathy as well as anti-GBM occurring de novo after renal transplantation. Moreover, patients can present with isolated pulmonary haemorrhage as well as with negative tests for circulating anti-GBM. Virtually all patients with anti-GBM disease have autoantibodies that react with two discrete epitopes on the α3 chain of type IV collagen. Recent evidence suggests that healthy persons have low-affinity natural antibodies reacting with the same epitopes, but most people are protected from developing disease-causing high-affinity autoantibodies by human leukocyte antigen-dependent regulatory T-cells (Tregs). The α3 chain-derived peptides presented by the HLA-DR15 antigen lack the ability to promote the development of such Tregs. The detection of anti-GBM in circulation using the rapid assay test has led to early diagnosis and improved prognosis. However, our present tools to curb the inflammation and to eliminate the assaulting antibodies are insufficient. Only about one-third of all patients survive with functioning native kidneys. More effective therapies need to be developed; agents that inhibit neutrophil recruitment, deplete B cells and cleave immunoglobulin G (IgG) in vivo may become new weapons in the arsenal to combat anti-GBM disease.

Effects of plasma proteins on sieving of tracer macromolecules in glomerular basement membrane

2001 ◽  
Vol 281 (5) ◽  
pp. F860-F868 ◽  
Author(s):  
Matthew J. Lazzara ◽  
William M. Deen
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Plasma Proteins ◽  
Prolate Spheroid ◽  
Osmotic Effect ◽  
Sieving Coefficient ◽  
Total Plasma Protein ◽  
Macromolecular Tracers

It was found previously that the sieving coefficients of Ficoll and Ficoll sulfate across isolated glomerular basement membrane (GBM) were greatly elevated when BSA was present at physiological levels, and it was suggested that most of this increase might have been the result of steric interactions between BSA and the tracers (5). To test this hypothesis, we extended the theory for the sieving of macromolecular tracers to account for the presence of a second, abundant solute. Increasing the concentration of an abundant solute is predicted to increase the equilibrium partition coefficient of a tracer in a porous or fibrous membrane, thereby increasing the sieving coefficient. The magnitude of this partitioning effect depends on solute size and membrane structure. The osmotic reduction in filtrate velocity caused by an abundant, mostly retained solute will also tend to elevate the tracer sieving coefficient. The osmotic effect alone explained only about one-third of the observed increase in the sieving coefficients of Ficoll and Ficoll sulfate, whereas the effect of BSA on tracer partitioning was sufficient to account for the remainder. At physiological concentrations, predictions for tracer sieving in the presence of BSA were found to be insensitive to the assumed shape of the protein (sphere or prolate spheroid). For protein mixtures, the theoretical effect of 6 g/dl BSA on the partitioning of spherical tracers was indistinguishable from that of 3 g/dl BSA and 3 g/dl IgG. This suggests that for partitioning and sieving studies in vitro, a good experimental model for plasma is a BSA solution with a mass concentration matching that of total plasma protein. The effect of plasma proteins on tracer partitioning is expected to influence sieving not only in isolated GBM but also in intact glomerular capillaries in vivo.

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