Sequential effects of high glucose on mesangial cell transforming growth factor-β1 and fibronectin synthesis Jong Hoon Oh, Hunjoo Ha, Mi Ra Yu, Hi Bahl.

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Sequential effects of high glucose on mesangial cell transforming growth factor-β1 and fibronectin synthesis Jong Hoon Oh, Hunjoo Ha, Mi Ra Yu, Hi Bahl Lee Kidney International Volume 54, Issue 6, Pages 1872-1878 (January 1998) DOI: 10.1046/j.1523-1755.1998.00193.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Representative Northern blot analysis of mesangial cell expression of TGF-β1 and fibronectin mRNAs. After incubation of quiescent mesangial cells with control (C; 5.6mm) or high (H; 30mm) glucose for the given period, total RNA was isolated, electrophoresed and transferred onto nylon membranes. Northern blots were hybridized with [32P]-labeled cDNA probe for TGF-β1, fibronectin, and GAPDH. Autoradiographs were developed by exposing blots to X-ray films. Mesangial cell TGF-β1 mRNA expression increased significantly at six hours and continued to increase until 48hours in response to high glucose. Compared to TGF-β1, the increase in fibronectin mRNA in response to high glucose was delayed, significant only at 24 and 48hours. Kidney International 1998 54, 1872-1878DOI: (10.1046/j.1523-1755.1998.00193.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Effects of high glucose on mesangial cell TGF-β1 bioactivity. After incubation of quiescent mesangial cells with control (5.6mm; □) or high (30mm; ▪) glucose for the given period, aliquots of mesangial cell conditioned media were analyzed by a mink lung epithelial cell (MLEC) growth inhibition assay. Values are expressed as mean ± se from three separate experiments. *P < 0.05 compared to control glucose. Heat-activated conditioned media showed time-dependent increase in TGF-β1 protein in both control and high glucose conditions. High glucose significantly increased TGF-β1 secretion at 24 and 48hours when compared to control glucose. Kidney International 1998 54, 1872-1878DOI: (10.1046/j.1523-1755.1998.00193.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Immunoblot analysis of fibronectin production by mesangial cell. After incubation of quiescent mesangial cells with control (C; 5.6mm; □) or high (H; 30mm ▪) glucose for the given period, aliquots of mesangial cell conditioned media were electrophoresed under reducing conditions and transferred onto nitrocellulose membranes. Western blots were performed as described in the text. Values are expressed as mean ± se of three experiments. *P < 0.05 compared to control glucose. The inset Western blot is a representative of three individual experiments. Shown here is cumulative amount of fibronectin produced by mesangial cells that progressively increased with time in both control and high glucose. High glucose induced significantly more fibronectin synthesis than control glucose at 48hours. Kidney International 1998 54, 1872-1878DOI: (10.1046/j.1523-1755.1998.00193.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Effects of anti-TGF-β antibody on high glucose-induced mesangial cell fibronectin production. Quiescent mesangial cells were cultured for 48hours in serum-free medium containing 5.6 or 30mm glucose in the presence or absence of 25 μg/ml anti-TGF-β antibody. After the incubation period, aliquots of mesangial cell conditioned media were electrophoresed under reducing conditions and transferred onto nitrocellulose membranes. Western blots using ECL detection system were performed as described in the text. Values are expressed as means ± se of three experiments. *P < 0.05 compared to 5.6mm glucose in the absence of anti-TGF-β antibody. #P < 0.05 compared to 30mm glucose in the absence of anti-TGF-β antibody. The inset Western blot is a representative of three individual experiments. Anti-TGF-β antibody completely abolished high glucose-induced fibronectin production without significant change in basal production of fibronectin in control glucose. Kidney International 1998 54, 1872-1878DOI: (10.1046/j.1523-1755.1998.00193.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Kidney International 1998 54, 1872-1878DOI: (10. 1046/j. 1523-1755 Kidney International 1998 54, 1872-1878DOI: (10.1046/j.1523-1755.1998.00193.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Kidney International 1998 54, 1872-1878DOI: (10. 1046/j. 1523-1755 Kidney International 1998 54, 1872-1878DOI: (10.1046/j.1523-1755.1998.00193.x) Copyright © 1998 International Society of Nephrology Terms and Conditions