scholarly journals Degradation of glomerular basement membrane by purified mammalian metalloproteinases

1988 ◽  
Vol 254 (2) ◽  
pp. 609-612 ◽  
Author(s):  
W H Baricos ◽  
G Murphy ◽  
Y W Zhou ◽  
H H Nguyen ◽  
S V Shah
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Degradation Products ◽  
Gel Chromatography ◽  
Collagen Types ◽  
Percentage Release ◽  
Dose Dependent

Neutral metalloproteinases degrade components of the extracellular matrix, including collagen types I-V, fibronectin, laminin and proteoglycan. However, their ability to degrade intact glomerular basement membrane (GBM) has not previously been investigated. Incubation of [3H]GBM (50,000 c.p.m.; pH 7.5; 24 h at 37 degrees C) with purified gelatinase or stromelysin (2 units) resulted in significant GBM degradation: gelatinase, 46 +/- 2.2; stromelysin, 59 +/- 5.8 (means +/- S.E.M.; percentage release of non-sedimentable radioactivity; n = 4). In contrast, 2 units of collagenase released only 5.6 +/- 0.52% (n = 3) of the [3H]GBM radioactivity compared with 2.0 +/- 0.15% (n = 7) released from [3H]GBM incubated alone. Sephadex G-200 gel chromatography of supernatants obtained from incubations of [3H]GBM with either gelatinase or stromelysin confirmed the ability of these enzymes to degrade GBM and revealed both high-(800,000) and relatively low-(less than 20,000) Mr degradation products for both enzymes. GBM degradation by gelatinase and stromelysin was dose-dependent (range 0.02-2.0 units), near maximal between pH 6.0 and 8.6, and was completely inhibited (greater than 95%) by 2 mM-o-phenanthroline. Collagenase (2 units) did not enhance the degradation of GBM by either gelatinase (0.02 or 0.2 unit) or stromelysin (0.02 or 0.2 unit). Our results indicate that metalloproteinase-mediated GBM degradation by neutrophils and glomeruli may be attributable to gelatinase (neutrophils) and/or stromelysin (glomeruli) and suggest an important role for these proteinases in glomerular pathophysiology.

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 Localization of α-Dystroglycan on the Podocyte: from Top to Toe

2005 ◽  
Vol 53 (11) ◽  
pp. 1345-1353 ◽  
Author(s):  
Nils P.J. Vogtländer ◽  
Henry Dijkman ◽  
Marinka A.H. Bakker ◽  
Kevin P. Campbell ◽  
Johan van der Vlag ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Confocal Microscopy ◽  
Cell Membrane ◽  
Basement Membrane ◽  
Immunoelectron Microscopy ◽  
Glomerular Basement Membrane ◽  
Human Kidney ◽  
Charged Membrane ◽  
Apical Localization

α-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that α-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against α-DG. Therefore, we studied the exact localization of glomerular α-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of α-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that α-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that α-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.

scholarly journals Resolution and limitations of the immunoperoxidase procedure in the localization of extracellular matrix antigens.

1983 ◽  
Vol 31 (7) ◽  
pp. 945-951 ◽  
Author(s):  
P J Courtoy ◽  
D H Picton ◽  
M G Farquhar
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Cationized Ferritin ◽  
Anionic Sites ◽  
Double Labeling ◽  
Extracellular Matrix Components ◽  
Matrix Components ◽  
Indirect Immunoperoxidase ◽  
Labeling System

A double labeling system was used to test the resolution of the indirect immunoperoxidase procedure in the localization of extracellular matrix components. A recognizable antigen, cationized ferritin, was first implanted at specific anionic sites (approximately 60 nm periodicity) in the lamina rara interna and externa of the glomerular basement membrane (GBM) and subsequently localized by immunoperoxidase. The coincidence between the location of reaction product and the ferritin clusters was assessed. When the amount of immunoadsorbed peroxidase and time of exposure to the 3,3'-diaminobenzidine (DAB)-containing medium were limited, discrete deposits of reaction product were observed around individual ferritin clusters. When immunolabeling was increased, the whole GBM was stained, and DAB staining was also found along the endothelial plasmalemma and the epithelial plasmalemma at the base of the foot processes at some distance (greater than 100 nm) from the ferritin clusters in the laminae rarae. These findings indicate that oxidized DAB reaction product can diffuse over long distances and be reabsorbed onto cell membranes. Even under limited incubation conditions some diffusion of DAB reaction product was encountered. The value and limitations of the DAB-peroxidase procedures are discussed.

The molecular basis of glomerular basement membrane disorders

2018 ◽  
Author(s):  
Rachel Lennon ◽  
Neil Turner
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Heparan Sulphate ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Glomerular Disease ◽  
The Body ◽  
Monogenic Disorders ◽  
Filtration Barrier

The glomerular basement membrane (GBM) is a condensed network of extracellular matrix molecules which provides a scaffold and niche to support the function of the overlying glomerular cells. Within the glomerulus, the GBM separates the fenestrated endothelial cells, which line capillary walls from the epithelial cells or podocytes, which cover the outer aspect of the capillaries. In common with basement membranes throughout the body, the GBM contains core components including collagen IV, laminins, nidogens, and heparan sulphate proteoglycans. However, specific isoforms of these proteins are required to maintain the integrity of the glomerular filtration barrier.Across the spectrum of glomerular disease there is alteration in glomerular extracellular matrix (ECM) and a number of histological patterns are recognized. The GBM can be thickened, expanded, split, and irregular; the mesangial matrix may be expanded and glomerulosclerosis represents a widespread accumulation of ECM proteins associated with loss of glomerular function. Whilst histological patterns may follow a sequence or provide diagnostic clues, there remains limited understanding about the mechanisms of ECM regulation and how this tight control is lost in glomerular disease. Monogenic disorders of the GBM including Alport and Pierson syndromes have highlighted the importance of both collagen IV and laminin isoforms and these observations provide important insights into mechanisms of glomerular disease.

scholarly journals Oral Administration of Glomerular Basement Membrane Prevents the Development of Experimental Autoimmune Glomerulonephritis in the WKY Rat

2001 ◽  
Vol 12 (1) ◽  
pp. 61-70
Author(s):  
JOHN REYNOLDS ◽  
CHARLES D. PUSEY
Keyword(s):  
T Cells ◽  
Basement Membrane ◽  
Oral Administration ◽  
Glomerular Basement Membrane ◽  
Crescent Formation ◽  
Necrotizing Glomerulonephritis ◽  
Wistar Kyoto ◽  
Dose Dependent ◽  
Experimental Autoimmune

Abstract. Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by a single injection of collagenase-solubilized rat glomerular basement membrane (GBM) in adjuvant. EAG is characterized by circulating and deposited anti-GBM antibodies, accompanied by focal necrotizing glomerulonephritis with crescent formation. The inhibitory effect of orally administered antigens has been reported in various animal models of autoimmunity but not in EAG in the rat. The effects of feeding rat GBM by gavage, at total doses of 0.5, 2.5, or 5 mg, before immunization were examined. A dose-dependent effect was observed on the development of EAG. A dose of 0.5 mg of GBM had no effect on disease, 2.5 mg resulted in a moderate reduction in the severity of nephritis but no change in anti-GBM antibody production, and 5 mg resulted in a marked reduction in circulating and deposited anti-GBM antibodies, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of infiltrating T cells and macrophages. Animals that were fed 5 mg of GBM showed a significant reduction in IgG2a but not IgG1, anti-GBM antibody levels, suggesting downregulation of Th1 responses. There was also a dose-dependent reduction in the proliferative responses of splenic T cells from treated animals to GBM antigen in vitro. These results clearly demonstrate that mucosal tolerance can be induced by oral administration of GBM antigen and that this approach is effective in preventing EAG.

Extracellular matrix synthesis by coal dust-exposed type II epithelial cells

1994 ◽  
Vol 267 (4) ◽  
pp. L365-L374
Author(s):  
Y. C. Lee ◽  
R. Hogg ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Coal Dust ◽  
Alveolar Epithelium ◽  
Dust Exposure ◽  
Type Ii ◽  
Direct Effects ◽  
Matrix Synthesis ◽  
Ecm Proteins ◽  
Dose Dependent

Although integrity of the alveolar basement membrane may influence progression of lung injury induced by inhaled particulates, little is known about direct effects of coal dusts on the alveolar epithelium or its extracellular matrix (ECM). Effects of dust on synthesis of cell and ECM proteins by type II cells (T2P) was thus investigated. Three coal dusts (anthracite no. 867; bituminous no. 1451 and no. 1361) and mine dust MIT-3, of respirable size, were studied as a function of dose and time over 3 days of primary T2P culture. On day 1, 750 micrograms/ml of 867 were required to increase relative synthesis of ECM proteins (ECM/cell). MIT-3, 1451, and 1361 were without effect. By day 3, 867 or MIT-3 increased ECM/cell 60-100% at 300 micrograms/ml; 1451 produced modest, dose-dependent stimulation, whereas 1361 remained without effect. None of the dusts caused significant cytotoxicity. The results show dose- and time-dependent effects of well-characterized coal and mind dusts to modify partitioning of newly synthesized proteins into the ECM and suggest that coal dust exposure may modulate structure or function of the subepithelial basement membrane.

scholarly journals Structure-function relationships associated with extracellular matrix alterations in diabetic glomerulopathy.

1994 ◽  
Vol 5 (5) ◽  
pp. 1165-1172
Author(s):  
S Adler
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Matrix Elements ◽  
Type Iv ◽  
Physico Chemical ◽  
Basement Membrane Thickening ◽  
Matrix Expansion

Proteinuria and progressive renal insufficiency are the primary manifestations of diabetic nephropathy. Accumulating evidence suggests that these clinical features can be linked, at least in part, to pathologic changes in the glomerular extracellular matrix. Most evidence suggests that glomerular basement membrane thickening and mesangial matrix expansion consist of at least three elements. These are (1) an accumulation of normal extracellular components; (2) an increase in the novel peptide chains of the normal components of Type IV collagen; and (3) an increase in matrix elements not normally expressed in the glomerulus. The pathogenetic features underlying these changes include increased synthesis and decreased degradation of matrix. Abnormal physico-chemical interactions among these matrix elements likely contribute to alterations in three-dimensional structure, leading to proteinuria and loss of glomerular basement membrane filtering surface area. Many of these changes may be explained in whole or in part by direct or secondary effects of hyperglycemia, as well as by hemodynamic changes.

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