Application of cationic probes for the ultrastructural localization of proteoglycans in basement membranes

Kuppevelt, T. H. M. S. M. Van; Veerkamp, J.H.

doi:10.1002/jemt.1070280204

Cited by 35 publications

(15 citation statements)

References 124 publications

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“…Presence of GAG in the glomerulus was investigated further by cupromeronic blue staining, which revealed a dense network of fine filaments in the GBM. Each filament represents a PG with the GAG chains collapsed onto the core protein (27). The PG filaments that are associated with the podocytic site of the GBM appeared larger and more electron dense in comparison with the PG filaments at the endothelial site.…”

Section: Glomerular Hs and Neuraminic Acid Are Removed After Heparinamentioning

confidence: 99%

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In Vivo Degradation of Heparan Sulfates in the Glomerular Basement Membrane Does Not Result in Proteinuria

Wijnhoven¹,

Lensen²,

Wismans³

et al. 2007

Journal of the American Society of Nephrology

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Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

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Section: Glomerular Hs and Neuraminic Acid Are Removed After Heparinamentioning

confidence: 99%

“…To visualize GAG, we applied the critical electrolyte concentration method as described previously (27). Kidney tissue was fixed overnight in 25 mM sodium acetate (pH 5.6) that contained 2.5% (vol/vol) glutaraldehyde, 0.2 M MgCl 2 , and 0.2% (wt/vol) cupromeronic blue (Seikagaku, Tokyo, Japan).…”

Section: Electron Microscopymentioning

confidence: 99%

In Vivo Degradation of Heparan Sulfates in the Glomerular Basement Membrane Does Not Result in Proteinuria

Wijnhoven¹,

Lensen²,

Wismans³

et al. 2007

Journal of the American Society of Nephrology

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Section: Hs and Neuraminic Acid Are Removed From The Glomerulus By Hementioning

confidence: 99%

“…This method visualizes sulfated glycosaminoglycans (GAGs) as electron-dense filaments caused by their collapse onto the core protein 26 (Figure 2). 26 There was a strong reduction of GAGs in the GBM after heparinase III ϩ neuraminidase injection. A few short filaments were left, which may be caused by the presence of chondroitin sulfate, which is another class of sulfated GAGs present in small quantities in the GBM.…”

Section: Hs and Neuraminic Acid Are Removed From The Glomerulus By Hementioning

confidence: 99%

“…18 To visualize sulfated GAGs, kidney tissue was fixed in 25 mM sodium acetate buffer (pH 5.6) containing 2.5% (vol/vol) glutaraldehyde, 0.2 M MgCl 2 , and 0.2% (wt/vol) cupromeronic blue (Seikagaku, Tokyo, Japan), according to the critical electrolyte concentration method. 18,26 Ultrathin sections were prepared and studied using a Jeol TEM 1010 microscope (Jeol Ltd., Tokyo, Japan).…”

Section: Electron Microscopymentioning

confidence: 99%

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Removal of Heparan Sulfate from the Glomerular Basement Membrane Blocks Protein Passage

Wijnhoven

Lensen²,

Wismans³

et al. 2007

Journal of the American Society of Nephrology

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Heparan sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example, in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state. We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid-but not HS-from the GBM, and this led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage of proteins through the glomerular capillary wall. The glomerular capillary wall, which consists of fenestrated endothelial cells, the glomerular basement membrane (GBM), and podocytes with foot processes interconnected by slit diaphragms, forms the major filtration barrier of the glomerulus. It excludes plasma proteins with the size of albumin (69 kD, 3.6 nm) and larger from its filtrate. 1,2 Next to size, permeability of the glomerular capillary wall is controlled by charge interactions. Neutral and cationic molecules can more easily penetrate the GBM than anionic molecules, which encounter electrostatic repulsion by HS. In addition, the negative charges of the cell coat (glycocalyx) of the endothelial cells and podocytes may play a role in the charge-dependent glomerular filtration. 3,4 HS contains multiple carboxylic groups and negatively charged N-, 2-O-, 6-O-, and 3-O-sulfate groups. 5,6 Because the majority of HS resides in the GBM, it is assumed that GBM HS is responsible for the charge-selective properties of the glomerular capillary wall. 3,7,8 This is supported by a number of studies. First, a reduction in glomerular HS has been described in a number of human kidney diseases 8 -11 and experimental animal models 8,12-14 characterized by proteinuria. Second, intravenous injection of rats with anti-HS antibody JM403 resulted in acute albuminuria. 15 Third, perfusion of isolated rat kidneys or glomeruli with the HS-degrading enzyme heparinase III resulted in penetration of ferritin and bovine serum albumin into the GBM and passage to the urinary space. 16,17 However, recent observations question the direct role of

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Introduction to the biliary tract, the gallbladder, and gallstones

Gilloteaux

1997

Microsc. Res. Tech.

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Application of cationic probes for the ultrastructural localization of proteoglycans in basement membranes

Cited by 35 publications

References 124 publications

In Vivo Degradation of Heparan Sulfates in the Glomerular Basement Membrane Does Not Result in Proteinuria

In Vivo Degradation of Heparan Sulfates in the Glomerular Basement Membrane Does Not Result in Proteinuria

Removal of Heparan Sulfate from the Glomerular Basement Membrane Blocks Protein Passage

Introduction to the biliary tract, the gallbladder, and gallstones

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