Regulation of glomerular endothelial cell proteoglycans by glucose

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.

Fig. 1

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

Fig. 2

Ion exchange chromatography of medium ³⁵S-labeled macromolecules. Confluent layers of GEndo were incubated in medium containing (A) 5 mM glucose or (B) 30 mM glucose for 8 to 14 weeks and labeled with 200 µCi/mL of ³⁵SO₄ for 24 hr. Fractions containing proteoglycans in peaks B were pooled as indicated by bars.

Fig. 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.

Fig. 4

Sepharose CL-4B chromatography of medium proteoglycans. Equal amounts (cpm) of ³⁵S-labeled proteoglycans purified on ion exchange chromatography (peaks B in Fig. 1) were subjected to Sepharose CL-4B chromatography using 4 M guanidine-HCI buffer containing protease inhibitors. (A) medium proteoglycans from cells incubated with 5 mM glucose, (B) medium proteoglycans from cells incubated with 30 mM glucose. Peaks M-I and M-II were pooled as indicated by bars for further analysis.

Fig. 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.

Fig. 6

Regulation of basement membrane HSPG, perlecan, mRNA abundance in GEndo by high glucose medium. Top panel shows bands of hybridization with RPD-1, a cDNA for perlecan, basement membrane HSPG. Lower panel shows hybridization bands with GAPDH probe.