The Study on the Mechanism Regulating the Production of Extracellular Matrix in Renal Tubular Epithelial Cells Cultured in High Glucose Concentration

Hwang, Jae Ha; Yoo, Tae Hyun; Song, Hyun Yong; Kim, Joo Seong; Song, Young Su; Chong, Deug Lim; Kim, Kyung Sup; Kang, Shin Wook; Lee, Ho Yung; Han, Dae Suk; Choi, Kyu Hun

Abstract

Thickening of tubular basement membrane and progressive tubulointerstitial fibrosis has been reported as important components of diabetic nephropathy, In order to investigate the mechanisms of tubulinterstitial changes in diabetic nephropathy, we evaluated the effects of a high concentration of glucose(25mM; 450mg/dL) on glucose transporter GLUT1 level, fibronectin production and tissue inhibitors of metalloproteinases (TIMP)-1 concentration in renal tubular(LLC-PK1) cells. As the effect of high glucose-induced alteration in LLC-PK1 cells, the expression of facilitative glueose transporter, GLUT1 was decreased after longer than 24-hours exposure to 25mM glucose, compared to control(5.6mM). The administration of protein kinase C (PKC) inhibitor GF109203X(10 microM) did not show significant effect on high glucose-induced decrease of GLUT1 level. On western blot analysis of fibronectin production, The exposure of LLC-PK cells to 25mM glucose for 48 hours significantly increasc4 fibro- nectin production, dose-dependently. The addition of GF102903X at the concentration of 10pM induced the significant increase of fibronectin level in LLC-PK1cells under glucose-free condition, whereas there was no significant effect on the high glucose-induced increase of fibronectin production. The addition of anti-TGF-beta antibody at 30 microgram/mL partly inhibited the high glucose-induced increase of fibronectin production. Concerning the changes of tissue inhibitor of metallo-proteinase(TIMP)-1 levels in the presence of high glucose, the exposure to high glucose for 24 and 43 hours increased TIMP-1 levels in culture supernatant of LLC-PK1 cells, dose-dependently. The TIMP-1 levels of 48-hour exposure to 15 and 25mM glucose were also significantly higher than those of 24-hourexposure. The treatment with 10 microM GF102903X or 30 microgram/mL anti-TGF-Beta antibody had no significant effects on TIMP-1 levels measured under the high glucose culture condition. In conclusion, the expression of facilitative glucose transporter, GLUT1 is inhibited and the production of fibronectin is increased in renal tubular cells cultured in the presence of high concentration of glucose, which is partly mediated by TGF-beta. The TIMP-1 level is also increased under high glucose culture condition. The enhanced productions of fibronectin and TIMP-1 of renal tubular cells under high glucose concentration may contribute to tubulointerstitial fibrosis that occurs in diabetic nephropathy.