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Regulation of glomerular endothelial cell proteoglycans by glucose.

Ha TS, Duraisamy S, Faulkner JL, Kasinath BS - J. Korean Med. Sci. (2004)

Bottom Line: Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted.Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose.Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chungbuk National University, Cheongju, Korea. tsha@med.chungbuk.ac.kr

ABSTRACT
The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.

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Morphology of GEndo undergoing prolonged incubation in control medium containing 5 mM and 30 mM glucose.
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Figure 1: Morphology of GEndo undergoing prolonged incubation in control medium containing 5 mM and 30 mM glucose.

Mentions: Following long term incubation (8 to 14 weeks) in high glucose medium, the net synthesis of 35S-labeled macromolecules was reduced by approximately 53% and 50% (p<0.05) in both the medium and cell layer compartments, respectively. Incubation in 30 mM glucose medium for 7 or 14 days did not affect incorporation of 35S-SO4 into macromolecules (data not shown). Careful attention was paid to possible untoward effects of prolonged incubation periods on GEndo. Incorporation of 35S-SO4 into macromolecules by GEndo incubated in control medium was quantitatively similar to our previous observations with GEndo (12), suggesting that the cells were metabolically active despite prolonged incubation. Protein contents of cell layers did not differ significantly between control and 30 mM glucose treated cell layers at the end of incubation periods, i.e., 399±33.6 µg/well (control, 30 mM glucose) vs. 445±38.5 µg/well (30 mM glucose, Table 1). Finally, morphological examination at the end of prolonged incubation did not reveal any difference in appearance between cells grown in control or the high glucose media, the cells was not noted on careful examination of the monolayers every three days for the duration of incubation. The profile of proteoglycans on molecular sieve chromatography was similar to that reported in pure populations of GEndo (12), further confirming absence of emergence of new cells with a different proteoglycan profile. There was no evidence of excessive cell detachment in cell layers incubated with 30 mM glucose. These observations show that prolonged incubation with 30 mM glucose did not affect the viability of GEndo compared to cell layers incubated for the same duration with medium containing 5 mM glucose (Fig. 1).


Regulation of glomerular endothelial cell proteoglycans by glucose.

Ha TS, Duraisamy S, Faulkner JL, Kasinath BS - J. Korean Med. Sci. (2004)

Morphology of GEndo undergoing prolonged incubation in control medium containing 5 mM and 30 mM glucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822306&req=5

Figure 1: Morphology of GEndo undergoing prolonged incubation in control medium containing 5 mM and 30 mM glucose.
Mentions: Following long term incubation (8 to 14 weeks) in high glucose medium, the net synthesis of 35S-labeled macromolecules was reduced by approximately 53% and 50% (p<0.05) in both the medium and cell layer compartments, respectively. Incubation in 30 mM glucose medium for 7 or 14 days did not affect incorporation of 35S-SO4 into macromolecules (data not shown). Careful attention was paid to possible untoward effects of prolonged incubation periods on GEndo. Incorporation of 35S-SO4 into macromolecules by GEndo incubated in control medium was quantitatively similar to our previous observations with GEndo (12), suggesting that the cells were metabolically active despite prolonged incubation. Protein contents of cell layers did not differ significantly between control and 30 mM glucose treated cell layers at the end of incubation periods, i.e., 399±33.6 µg/well (control, 30 mM glucose) vs. 445±38.5 µg/well (30 mM glucose, Table 1). Finally, morphological examination at the end of prolonged incubation did not reveal any difference in appearance between cells grown in control or the high glucose media, the cells was not noted on careful examination of the monolayers every three days for the duration of incubation. The profile of proteoglycans on molecular sieve chromatography was similar to that reported in pure populations of GEndo (12), further confirming absence of emergence of new cells with a different proteoglycan profile. There was no evidence of excessive cell detachment in cell layers incubated with 30 mM glucose. These observations show that prolonged incubation with 30 mM glucose did not affect the viability of GEndo compared to cell layers incubated for the same duration with medium containing 5 mM glucose (Fig. 1).

Bottom Line: Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted.Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose.Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chungbuk National University, Cheongju, Korea. tsha@med.chungbuk.ac.kr

ABSTRACT
The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.

Show MeSH
Related in: MedlinePlus