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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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Related in: MedlinePlus

Sepharose CL-4B chromatography of cell layer proteoglycans. Equal amounts (cpm) of 35S-labeled proteoglycans purified on ion exchange chromatography (peaks B in Fig. 2) were subjected to Sepharose CL-4B chromatography using 4 M guanidine-HCI buffer containing protease inhibitors. (A) cell layer proteoglycans from cells incubated with 30 mM glucose. Peaks C-I and C-II were pooled as indicated by bars for further analysis.
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Figure 5: Sepharose CL-4B chromatography of cell layer proteoglycans. Equal amounts (cpm) of 35S-labeled proteoglycans purified on ion exchange chromatography (peaks B in Fig. 2) were subjected to Sepharose CL-4B chromatography using 4 M guanidine-HCI buffer containing protease inhibitors. (A) cell layer proteoglycans from cells incubated with 30 mM glucose. Peaks C-I and C-II were pooled as indicated by bars for further analysis.

Mentions: Cell layer proteoglycans of GEndo incubated with control medium also resolved into two distinct peaks of Kav 0.24 and 0.67, respectively (Fig. 5). Peak C-I of Kav 0.24 accounted for 18% of all cell layer proteoglycans (Table 2). GAG analysis showed that heparan sulfate and chondroitin/dermatan sulfate accounted for 54% and 46% of GAGs in peak C-I (Kav 0.24), respectively, whereas peak C-II was composed mostly of chondroitin/dermatan sulfate (73%). Incubation with high glucose medium did not alter the hydrodynamic size or the relative proportion of the proteoglycan peaks (Fig. 5). There was a reduction of approximately 26% in the synthesis of peak C-I proteoglycans, which did not reach statistical significance (Table 2). High glucose medium decreased the synthesis of peak C-II proteoglycans significantly by 53% (31,550±5,260 vs. 14,970±4,720, p<0.05) (Table 2). Incubation with high glucose medium did not change the GAG constituent profile of either peak.


Regulation of glomerular endothelial cell proteoglycans by glucose.

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

Sepharose CL-4B chromatography of cell layer proteoglycans. Equal amounts (cpm) of 35S-labeled proteoglycans purified on ion exchange chromatography (peaks B in Fig. 2) were subjected to Sepharose CL-4B chromatography using 4 M guanidine-HCI buffer containing protease inhibitors. (A) cell layer proteoglycans from cells incubated with 30 mM glucose. Peaks C-I and C-II were pooled as indicated by bars for further analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Sepharose CL-4B chromatography of cell layer proteoglycans. Equal amounts (cpm) of 35S-labeled proteoglycans purified on ion exchange chromatography (peaks B in Fig. 2) were subjected to Sepharose CL-4B chromatography using 4 M guanidine-HCI buffer containing protease inhibitors. (A) cell layer proteoglycans from cells incubated with 30 mM glucose. Peaks C-I and C-II were pooled as indicated by bars for further analysis.
Mentions: Cell layer proteoglycans of GEndo incubated with control medium also resolved into two distinct peaks of Kav 0.24 and 0.67, respectively (Fig. 5). Peak C-I of Kav 0.24 accounted for 18% of all cell layer proteoglycans (Table 2). GAG analysis showed that heparan sulfate and chondroitin/dermatan sulfate accounted for 54% and 46% of GAGs in peak C-I (Kav 0.24), respectively, whereas peak C-II was composed mostly of chondroitin/dermatan sulfate (73%). Incubation with high glucose medium did not alter the hydrodynamic size or the relative proportion of the proteoglycan peaks (Fig. 5). There was a reduction of approximately 26% in the synthesis of peak C-I proteoglycans, which did not reach statistical significance (Table 2). High glucose medium decreased the synthesis of peak C-II proteoglycans significantly by 53% (31,550±5,260 vs. 14,970±4,720, p<0.05) (Table 2). Incubation with high glucose medium did not change the GAG constituent profile of either peak.

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