No abstract available.
Glomerular Basement Membrane* ; Heparan Sulfate Proteoglycans* ; Heparitin Sulfate*

HSPG, a component of size-and charge-selective barrier of glomerular basement membrane, is one of important matrix proteins which has been known to be reduced in the kidney of diabetic patients or animals. To examine the effects of glucose and AGE on the HSPG production by cultured GEC, we cultured rat GEC on the AGE- or BSA-coated plate under normal(5mM) and high glucose.(30mM) conditions and measured the change of HSPG production by sandwich-ELISA assay and northern blot analysis at 2 days and one week incubation periods. There was no difference in proliferation between 2 different conditions of culture plate surface. We measured the relative amount of the extracted HSPC and observed significant decreases in high glucose condition at one week incubation, and particularly on the AGE-coated surface as compared to the results of BSA-coated condition, by 22% and 5%, respectively. The expression of mRNA for perlecan promoter was decreased in condition of high glucose and AGE-coated surface by 20Yo at 2 days and 61i at one week. Even in normal glucose condition, the expression of mRNA was reduced by 30Yo at one week if the plate was coated with AGE. In conclusion, both high glucose and AGE have reducing effects on the production of HSPG by GEC in vitro. Their effects seem to be additive, however, the role of AGE is greater than that of glucose, This means that the effort to inhibit AGE formation is more important than short-term glucose control for the prevention of diabetic proteinuria.
Animals ; Blotting, Northern ; Diabetic Nephropathies ; Glomerular Basement Membrane ; Glucose* ; Heparan Sulfate Proteoglycans ; Heparitin Sulfate* ; Humans ; Kidney ; Proteinuria ; Rats* ; RNA, Messenger

Orientia tsutsugamushi is obligate intracellular bacterium that grows within the cytoplasm of the eukaryotic host cells. Therefore capability of the attachment, entry into the host cell and intracellular survival should be critical process for oriential infection. In this study we investigated the cellular invasion mechanism of Orientia tsutsugamushi and the role of transmembrane heparan sulfate proteoglycan, which binds diverse components at the cellular microenvironment and is implicated as host cell receptors for a variety of microbial pathogens. First of all Orientia tsutsugamushi can invade a wide range of nonprofessional phagocytic cells including fibroblast, epithelial cells a#nd endothelial cells of various host species, including B and T lymphocytes. Thus, it was postulated that the attachment of O. tsutsugamushi requires the recognition of ubiquitous surface structures of many kinds of host cells. Treatments with heparan sulfate and heparin inhibited the infection of Orientia tsutsugamushi in dose-dependent manner for L cell, mouse fibroblast, whereas other glycosaminoglycans such as chondroitin sulfate had no effect. Collectively, these findings provide strong evidence that initial interaction with heparan sulfate proteoglycan is required for the oriential invasion into host cells.
Animals ; Cellular Microenvironment ; Chondroitin Sulfates ; Cytoplasm ; Endothelial Cells ; Epithelial Cells ; Eukaryotic Cells* ; Fibroblasts ; Glycosaminoglycans ; Heparan Sulfate Proteoglycans ; Heparin ; Heparitin Sulfate ; Mice ; Orientia tsutsugamushi* ; Phagocytes ; T-Lymphocytes

It is well known that the glomerular basement membrane heparan sulfate proteoglycan(GBM HSPG) synthesized by glomerular epithelial cell(GEC) has an important role in the permeability of glomerular basement membrane and cyclic AMP(cAMP) is involved in regulation of a wide variety of genes maybe including GBM HSPG gene. The direct effect of cAMP on GBM HSPG gene expression and metabolism was not evaluated as yet. Proteinuria represents an impairment of permselectivity function of glomerular basement membrane regulated by GBM HSPG and could be associated with increased glomerular level of cAMP in nephrotic syndrome of diverse causes. RPD-I(rat GBM HSPG core protein domain-I) detected a >9.5kb transcript of GBM HSPG in RNA of rat GEC. Emp1oying a riboprobe synthesized from RPD-I in RNase protection assay, we examined whether cAMP regulated perlecan expression in the GEC. At l, 6, 24 and 48 hrs of incubation, l mM cAMP caused 43%, 32%, 47% and 40% reduction in mRNA expression of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 51%, 70% and 68% in the synthesis of 35SO4 labeled GBM HSPG by the GEC fol1owing l2, 24 and 48 hrs of incubation with cAMP. Our results show that decrease in GBM HSPG gene expression and synthesis by cAMP may be of relevance to proteinuric states characterized by activation of these mediators.
Animals ; Cyclic AMP* ; Epithelial Cells* ; Gene Expression ; Glomerular Basement Membrane* ; Heparan Sulfate Proteoglycans* ; Heparitin Sulfate* ; Immunoprecipitation ; Metabolism ; Nephrotic Syndrome ; Permeability ; Proteinuria ; Rats* ; Ribonucleases ; RNA ; RNA, Messenger

PURPOSE: We describe the changes of rat glomerular epithelial cells when exposed to high levels of glucose and advanced glycosylation endproducts(AGE) in the in vitro diabetic condition. We expect morphological alteration of glomerular epithelial cells and permeability changes experimentally and we may correlate the results with a mechanism of proteinuria in DM. METHODS: We made 0.2 M glucose-6-phsphate solution mixed with PBS(pH 7.4) containing 50 mg/mL BSA and protease inhibitor for preparation of AGE. As control, we used BSA. We manufactured and symbolized five culture dishes as follows; B5 - normal glucose(5 mM) + BSA, B30 - high glucose(30 mM) + BSA, A5 - normal glucose(5 mM) + AGE, A30 - high glucose(30 mM) + AGE, A/B 25 - normal glucose(5 mM) + 25 mM of mannitol(osmotic control). After the incubation period of both two days and seven days, we measured the amount of heparan sulfate proteoglycan(HSPG) in each dish by ELISA and compared them with the B5 dish at 2nd and 7th incubation days. We observed the morphological changes of epithelial cells in each culture dish using scanning electron microscopy(SEM). We tried the permeability assay of glomerular epithelial cells using cellulose semi-permeable membrane measuring the amount of filtered BSA through the apical chamber for 2 hours by sandwich ELISA. RESULTS: On the 2nd incubation day, there was no significant difference in the amount of HSPG between the 5 culture dishes. But on the 7th incubation day, the amount of HSPG increased by 10% compared with the B5 dish on the 2nd day except the A30 dish(P<0.05). Compared with the B5 dish on the 7th day the amount of HSPG in A30 and B30 dish decreased to 77.8% and 95.3% of baseline, respectively(P>0.05). In the osmotic control group (A/B 25) no significant correlation was observed. On the SEM, we could see the separated intercellular junction and fused microvilli of glomerular epithelial cells in the culture dishes where AGE was added. The permeability of BSA increased by 19% only in the A30 dish on the 7th day compared with B5 dish on the 7th day in the permeability assay(P<0.05). CONCLUSION: We observed not only the role of a high level of glucose and AGE in decreasing the production of HSPG of glomerular epithelial cells in vitro, but also their additive effect. However, the role of AGE is greater than that of glucose. These results seems to correlate with the defects in charge selective barrier. Morphological changes of the disruption of intercellular junction and fused microvilli of glomerular epithelial cells seem to correlate with the defects in size-selective barrier. Therefore, we can explain the increased permeability of glomerular epithelial units in the in vitro diabetic condition.
Animals ; Cellulose ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Glucose* ; Glycosylation* ; Heparan Sulfate Proteoglycans ; Heparitin Sulfate ; Intercellular Junctions ; Membranes ; Microvilli ; Permeability* ; Protease Inhibitors ; Proteinuria ; Rats

No Abstract Available.
Heparitin Sulfate* ; Orientia tsutsugamushi*

BACKGROUND: Minimal Change Nephrotic Syndrome (MCNS) is one of the most common primary nephrotic syndromes in children and its pathogenesis has not been exactly known. Interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha ) may be involved in the pathogenesis of this disese, because they are increased in blood and/or urine during relapse. This study was conducted to see changes of IL-8 and TNF-alpha in children with MCNS and to see the effects of IL-8 and TNF-alpha on the abundance of HSPG mRNA in rats glomerular epithelial cells (GECs). METHODS: Study patients consisted of 19 biopsy-proven MCNS children aged 2-15 years old. Ten age-matched healthy children were used as controls. Both blood and urinary IL-8 and TNF-alpha were measured using ELISA kit. GECs were cultured until confluent. IL-8 or TNF-alpha were added at various concentrations. Total RNA was extracted at 12 or 24 hours after adding IL-8 or TNF-alpha . RT-PCR using HSPG-specific primers and beta-actin as internal controls was done. Densities and areas of GBM HSPG corresponding bands to beta-actin bands were measured. RESULTS: Values of urinary IL-8 (ng/mg'cr) were 13,996+/-2,811, 2,811+/-3,734, and 5,331+/-6,403, during relapse, remission, and in control, respectively. Urinary IL-8 'during relapse' was significantly measured increased compared to 'during remission' and in controls (p<0.05). Values of urinary TNF-alpha (ng/mg'cr) were 364.4+/-512.1, 155.3+/-208.0, and 36.0+/-45.0, during relapse, remission, and in control, respectively. Urinary TNF-alpha during relapse was also significantly increased compared to 'during remission' and 'control' (p<0.05). Values of Blood IL-8 (ng/mL) were 1.19+/-1.23, 0.51+/-0.84, and 0.77+/-0.62, during relapse, remission, and in control, respectively. Blood IL-8 during relapse was significantly increased compared to 'during remission' and 'in control' (p<0.05). No significant change was seen in blood TNF-alpha. And no significant difference was seen in abdundance of HSPG mRNA after adding various concentraons IL-8 or TNF-alpha into rats GEC. CONCLUSION: The values of plasma and urinary IL-8 and TNF-alpha during relapse were increased compared to those of remission period and in control. but neither IL-8 nor TNF-alpha affect the abundance of HSPG-mRNA in rats GECs at various concentraions. So, it seems that both IL-8 and TNF-alpha do not play a direct role in GBM permeability and the elevated values of these cytokines in plasma and/or urine are secondary effects.
Actins ; Animals ; Child* ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Heparan Sulfate Proteoglycans* ; Heparitin Sulfate* ; Humans ; Interleukin-8* ; Nephrosis, Lipoid* ; Nephrotic Syndrome ; Permeability ; Plasma ; Rats* ; Recurrence ; RNA ; RNA, Messenger ; Tumor Necrosis Factor-alpha*

Minimal Change Nephrotic Syndrome(MCNS) reflects a disorder of T-lymphocytes. These T-cells are thought to release a vascular permeability factor (UPF) that injures the glomerular epithelial cells (GECs). Glomerular epithelial cellular damage may lead to proteinuria in MCNS by decreasing the synthesis of polyanions such as heparan sulfate proteoglycan(HSPG) : these polyanions constitute most of the normal charge barrier to glomerular filtration of macromolecules such as albumin. This study evaluates the direct effect of supernatant of culture media of peripheral blood mono- nuclear cells(PBMC) which was isolated from children with MCNS to GBM HSPG mRNA expression in rats GEC. GEC were cultured until confluent. Supernatant of culture media of PBMC from each group of 3 chilren with MCNS, IgA nephropathy or normal healthy were added. Total RNA was extracted at 12, 24 and 72hrs after adding supernatant. RT-PCR using Rat Perlecan Domain-I(RPD- I) specific primers and beta-actin as internal controls was done. Densities and areas of GRM HSPG corresponding bands to beta-actin bands were measured. At 24 hrs, supernatant of culture media of PBMC from 3 children with MCNS caused 62, 70, and 75Vo reductions, respectvely, in GEC's GBM HSPG mRNA expression compared to normal children. However, supernatant of culture media of PRMC from 3 children with IgA nephropathy did not. In addition, reductions of GEC's GBM HSK' mRNA expressions caused by supernatant of culture media of PBMC from 3 children with MCNS were restored upto levels of normal children at 72hrs after adding supernatant. Mutant cDNA was synthesized as primers for competitive PCR to quantify GBM HSPG mRNA expression. Mutant template was 212 base pairs shorter than RPD-I, 497 base pairs. In conclusion, we found that supernatant of culture media of PBMC from children with MCNS reversibly suppressed GBM HSPG mRNA expression in rats GEC. This study suggests cytokines of PBMC from children with MCNS directly injures GEC and leads to decrease in synthesis of GBM HSPG by GEC in the pathogenesis of MCNS.
Actins ; Animals ; Base Pairing ; Child* ; Culture Media ; Cytokines ; DNA, Complementary ; Epithelial Cells* ; Filtration ; Glomerulonephritis, IGA ; Heparan Sulfate Proteoglycans ; Heparitin Sulfate* ; Humans ; Polymerase Chain Reaction* ; Proteinuria ; Rats* ; RNA ; RNA, Messenger* ; T-Lymphocytes ; Vascular Endothelial Growth Factor A

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.
Adult ; Agrin/genetics ; Antigens, CD44/genetics ; Base Sequence ; DNA Primers/genetics ; Gene Expression/radiation effects ; Glucuronidase/genetics ; Heparan Sulfate Proteoglycans/genetics/*metabolism ; Heparitin Sulfate/metabolism ; Humans ; Male ; Matrix Metalloproteinase 1/genetics ; N-Acetylglucosaminyltransferases/genetics ; RNA, Messenger/genetics/metabolism ; Skin/*metabolism/*radiation effects ; Skin Aging/genetics/physiology ; Syndecan-1/genetics ; Syndecan-4/genetics ; Ultraviolet Rays/*adverse effects ; Young Adult

Heparan sulfate (HS) is ubiquitously expressed on the surfaces and in the extracellular matrix of virtually all cell types, making it an ideal receptor for viral infection. Compared with wild-type viruses, cell culture-adapted laboratory strains exhibit more efficient binding to cellular HS receptors. HS-binding viruses are typically cleared faster from the circulation and cause lower viremia than their non-HS-binding counterparts, suggesting that the HS-binding phenotype is a tissue culture adaptation that lowers virus fitness in vivo. However, when inoculated intracranially, efficient cell attachment through HS binding can contribute to viral neurovirulence. The primary aim of this review is to discuss the roles of HS binding in viral pathogenicity, including peripheral virulence and neurovirulence. Understanding how heparan sulfate functions during virus infection in vivo may prove critical for elucidating the molecular mechanism of viral pathogenesis, and may contribute to the development of therapeutics targeting HS.
Heparitin Sulfate ; physiology ; Humans ; Receptors, Virus ; physiology ; Virulence ; Viruses ; pathogenicity