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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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Ion exchange chromatography of cell layer 35S-labeled macromolecules. GEndo were incubated in medium containing (A) 5 mM glucose or (B) 30 mM glucose for 8 to 14 weeks and labeled for 24 hr with 35SO4 as in Fig. 2. Fractions containing proteoglycans in peaks B were pooled as indicted by bars.
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Figure 3: Ion exchange chromatography of cell layer 35S-labeled macromolecules. GEndo were incubated in medium containing (A) 5 mM glucose or (B) 30 mM glucose for 8 to 14 weeks and labeled for 24 hr with 35SO4 as in Fig. 2. Fractions containing proteoglycans in peaks B were pooled as indicted by bars.

Mentions: On ion exchange chromatography, 35S-labeled macromolecules in both medium and cell layer of cells incubated with 5 mM glucose resolved into 2 peaks. Peak A appeared at a molarity close to that of start buffer (0.15 M NaC1) whereas peak B eluted at a molarity greater than 0.3 M NaC1 (Fig. 2, 3).


Regulation of glomerular endothelial cell proteoglycans by glucose.

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

Ion exchange chromatography of cell layer 35S-labeled macromolecules. GEndo were incubated in medium containing (A) 5 mM glucose or (B) 30 mM glucose for 8 to 14 weeks and labeled for 24 hr with 35SO4 as in Fig. 2. Fractions containing proteoglycans in peaks B were pooled as indicted by bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Ion exchange chromatography of cell layer 35S-labeled macromolecules. GEndo were incubated in medium containing (A) 5 mM glucose or (B) 30 mM glucose for 8 to 14 weeks and labeled for 24 hr with 35SO4 as in Fig. 2. Fractions containing proteoglycans in peaks B were pooled as indicted by bars.
Mentions: On ion exchange chromatography, 35S-labeled macromolecules in both medium and cell layer of cells incubated with 5 mM glucose resolved into 2 peaks. Peak A appeared at a molarity close to that of start buffer (0.15 M NaC1) whereas peak B eluted at a molarity greater than 0.3 M NaC1 (Fig. 2, 3).

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