Effect of puromycin aminonucleoside on HSPG core protein content of glomerular epithelial cells

1988 ◽  
Vol 255 (4) ◽  
pp. F590-F596 ◽  
Author(s):  
B. S. Kasinath ◽  
A. K. Singh ◽  
Y. S. Kanwar ◽  
E. J. Lewis
Keyword(s):  
Protein Synthesis ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Heparan Sulfate ◽  
Protein Content ◽  
Glomerular Basement Membrane ◽  
Core Protein ◽  
Puromycin Aminonucleoside ◽  
The Core ◽  
Glomerular Epithelial Cells

It has been suggested that the glomerular basement membrane heparan sulfate proteoglycan (HSPG) is an important determinant of the glomerular permselectivity barrier. Derangements in the content of basement membrane heparan sulfate have been implicated in alterations in glomerular permselectivity seen in many glomerular diseases such as aminonucleoside nephrosis. The cellular origin and metabolism of the glomerular basement membrane HSPG have not been studied in detail. We have detected the expression of the proteoglycan by cloned glomerular visceral epithelial cells of the rat by employing a specific antibody against the core protein of HSPG isolated from the rat glomerular basement membrane. These findings suggest that in the rat in vivo glomerular visceral epithelial cells are one source of heparan sulfate present in the glomerular basement membrane. The effect of puromycin aminonucleoside (PAN) on the HSPG core protein content of the cloned glomerular epithelial cells was studied. By a quantitative immunoperoxidase method, the aminonucleoside caused a 28% reduction in the core protein content of the epithelial cells (P less than 0.01) following 72 h of incubation. However, the content of Heymann nephritis-related antigen, Fx1A was unchanged. Studies employing [3H]leucine incorporation showed that PAN was a weak inhibitor of de novo protein synthesis at 24 h of incubation, with complete recovery at 48 and 72 h. These data suggest that PAN effect on heparan sulfate core protein cannot be attributed to generalized inhibition of protein synthesis. The precise mechanism underlying the aminonucleoside effect on heparan sulfate core protein remains to be elucidated.

scholarly journals Puromycin Aminonucleoside Suppresses Integrin Expression in Cultured Glomerular Epithelial Cells

2001 ◽  
Vol 12 (4) ◽  
pp. 758-766 ◽  
Author(s):  
UMA KRISHNAMURTI ◽  
BING ZHOU ◽  
WEI-WEI FAN ◽  
EFFIE TSILIBARY ◽  
ELIZABETH WAYNER ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Type Iv Collagen ◽  
Cell Detachment ◽  
Puromycin Aminonucleoside ◽  
Glomerular Epithelial Cell ◽  
Type Iv ◽  
Glomerular Epithelial Cells

Abstract. Puromycin aminonucleoside (PAN)-induced nephrosis is a well-described model of human idiopathic nephrotic syndrome, but the mechanism of PAN's effect is not completely understood. Because PAN injection into rats results in retraction of glomerular epithelial cell foot processes and glomerular epithelial cell detachment, it was hypothesized that PAN might alter the contacts between these cells and the glomerular basement membrane. The major integrin expressed by glomerular epithelial cells is α3β1, which mediates attachment of these cells to extracellular matrix proteins including type IV collagen. T-SV 40 immortalized human glomerular epithelial cells were used to study PAN's effects on α3β1 expression, as well as that of podocalyxin and the slit diaphragm component ZO-1. Glomerular epithelial cells were seeded into plastic flasks and allowed to attach and proliferate for 48 h. The cells were then incubated for another 48 h in media containing 0, 0.5, or 5.0 μg/ml PAN. PAN exposure resulted in dose-dependent decreases in α3 and β1 expression, both at the protein level and at the mRNA level. This was accompanied by a significant decrease in the adhesion of glomerular epithelial cells to type IV collagen. PAN did not affect ZO-1 protein expression. Treatment with PAN increased the expression of podocalyxin at the protein and mRNA levels. Reduced glomerular epithelial cell expression of α3β1 integrins and impaired adhesion to type IV collagen may contribute to the glomerular epithelial cell detachment from glomerular basement membrane seen in the PAN nephrosis model.

SM22α: The Novel Phenotype Marker of Injured Glomerular Epithelial Cells in Anti-Glomerular Basement Membrane Nephritis

2007 ◽  
Vol 106 (3) ◽  
pp. e77-e87 ◽  
Author(s):  
Asa Ogawa ◽  
Minoru Sakatsume ◽  
Xingzhi Wang ◽  
Yunichi Sakamaki ◽  
Yutaka Tsubata ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
The Novel ◽  
Glomerular Epithelial Cells

scholarly journals Glomerular basement membrane proteoglycans are derived from a large precursor.

1988 ◽  
Vol 106 (3) ◽  
pp. 963-970 ◽  
Author(s):  
D J Klein ◽  
D M Brown ◽  
T R Oegema ◽  
P E Brenchley ◽  
J C Anderson ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Core Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Immunological Methods ◽  
Core Proteins ◽  
Single Precursor ◽  
Species Being

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.

scholarly journals Reduction in Glomerular Heparan Sulfate Correlates with Complement Deposition and Albuminuria in Active Heymann Nephritis

1999 ◽  
Vol 10 (8) ◽  
pp. 1689-1699 ◽  
Author(s):  
C. J. ILSE RAATS ◽  
MARIKEN E. LUCA ◽  
MARINKA A. H. BAKKER ◽  
ANNEMIEKE VAN DER WAL ◽  
PETER HEERINGA ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Core Protein ◽  
Control Level ◽  
Staining Intensity ◽  
Capillary Wall ◽  
Glomerular Capillary Wall ◽  
Glomerular Capillary ◽  
Heymann Nephritis

Abstract. In a time-study of active Heymann nephritis, the expression of agrin, the main heparan sulfate proteoglycan in the glomerular basement membrane, was analyzed in relation to deposition of IgG and complement in the glomerular capillary wall and the development of albuminuria. Binding of IgG autoantibodies to the glomerular capillary wall could be detected from 2 wk onward, followed by activation of complement after 6 wk. Progressive albuminuria developed from 6 wk onward to a level of 274 ± 68 mg/18 h at week 12. The staining intensity for the agrin core protein decreased slightly, and the staining intensity for the heparan sulfate stubs that were still attached to the core protein after heparitinase digestion remained normal. From week 6 onward, however, a progressive decrease was seen in the staining of two monoclonal antibodies (mAb) directed against different epitopes on the heparan sulfate polysaccharide side chain of agrin (to 35 and 30% of the control level, respectively, at week 12, both mAb P = 0.016). Moreover, albuminuria was inversely correlated with heparan sulfate staining as revealed by these antibodies (rs = -0.82 and rs = -0.75, respectively, both mAb P < 0.0001). This decrease in heparan sulfate staining was due to a progressive reduction of glomerular heparan sulfate content to 46 and 32% of control level at week 10 and week 12 of the disease, respectively, as measured biochemically. It is speculated that the observed decrease in glomerular heparan sulfate in active Heymann nephritis is due to complement-mediated cleavage of heparan sulfate, resulting in an increased permeability of the glomerular basement membrane to macromolecules.

scholarly journals Glomerular epithelial cells secrete a glomerular basement membrane-degrading metalloproteinase.

1992 ◽  
Vol 2 (9) ◽  
pp. 1388-1397
Author(s):  
R Johnson ◽  
H Yamabe ◽  
Y P Chen ◽  
C Campbell ◽  
K Gordon ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Type Iv Collagen ◽  
Dependent Manner ◽  
Type Iv Collagenase ◽  
Glomerular Diseases ◽  
Major Band ◽  
Type Iv ◽  
Glomerular Epithelial Cells

Cultured rat glomerular epithelial cells (GEC) were examined for their ability to release extracellular matrix-degrading proteinases with [3H]gelatin as substrate. GEC-conditioned media, under serum-free conditions, contained modest amounts of gelatinase activity (1 to 10 U/mg of protein); the activity was maximal at neutral pH, was inhibited by zinc chelators, was not inhibited by tissue inhibitor of metalloproteinase-2, and could not be further activated by trypsin or organomercurials. Gelatin substrate sodium dodecyl sulfate-polyacrylamide gels of GEC-conditioned medium revealed several zones of lysis, with molecular sizes of 150 kd (major band), and 220, 86 to 93, and 52 to 54 kd (minor bands). Northern blot analysis demonstrated that the GEC metalloproteinase(s) were distinct from the 68- to 72-kd type IV collagenase/gelatinase present in mesangial cells or the 92-kd type IV collagenase present in neutrophils. The GEC gelatinolytic activity also degraded insoluble type IV collagen in glomerular basement membrane in a dose-dependent manner. The major metalloproteinase activity responsible for the type IV collagen degradation has a molecular size of 150 kd with a type IV collagen substrate gel. Thus, GEC produce several neutral metalloproteinases, which, by virtue of their substrate specificity, may play an important role in glomerular basement membrane remodeling and in glomerular diseases characterized by alterations in basement membrane permeability.

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