Volume 59, Issue 2, Pages 673-681 (February 2001) Glycated albumin stimulates TGF-βbgr;1 production and protein kinase C activity in glomerular endothelial cells1  Sheldon Chen, Margo P. Cohen, Gregory T. Lautenslager, Clyde W. Shearman, Fuad N. Ziyadeh  Kidney International  Volume 59, Issue 2, Pages 673-681 (February 2001) DOI: 10.1046/j.1523-1755.2001.059002673.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Effect of glycated albumin and protein kinase C (PKC) inhibition on collagen IV production. Glomerular endothelial cells (GEnCs) were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose/2% FCS and no supplement (media), nonglycated albumin (NA), or glycated albumin (GA) at 500 μg/mL, without (□) or with (▪) 500 nmol/L of the PKC inhibitor GF 109203X (GFX). From each condition, media and cell lysates were collected for immunoassay of collagen IV as described in the text. Results represent the mean ± SEM of four independent cultures and are expressed as micrograms of collagen IV normalized to milligrams of protein. *P < 0.05 compared with nonglycated albumin; #P < 0.05 compared with glycated albumin, no GFX. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Protein kinase C (PKC) activity in glomerular endothelial cells (GEnCs). GEnCs were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose/2% FCS and no supplement (media), nonglycated albumin (NA), or glycated albumin (GA) at 500 μg/mL, without or with 500 nmol/L of the PKC inhibitor GF 109203X (GFX). In each lane of the gel, nonphosphorylated substrate is shown on the left and phosphorylated substrate on the right. Negative control (lane A) shows no phosphorylated substrate. Positive control (lane B), obtained with PKC supplied by Promega, shows abundant phosphorylated substrate. Lane C = media; lane D = media + GFX; lane E = nonglycated albumin; lane F = nonglycated albumin + GFX; lane G = glycated albumin; lane H = glycated albumin + GFX. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Effect of glycated albumin and PKC inhibition on PKC activity. GEnCs were cultured and analyzed for PKC activity as described in the legend to Figure 2and in the text. Data were obtained by densitometric scanning of photographs of gels. Results represent the mean ± SEM of four independent cultures and are expressed as the relative ratio of the densitometry of phosphorylated substrate in control cells (media, no GFX), arbitrarily assigned a value of 1. *P < 0.05 compared with nonglycated albumin; #P < 0.05 compared with glycated albumin, no GFX. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 TGF-βbgr;1 production in GEnCs. GEnCs were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose (A) or 25 mmol/L glucose (B) and nonglycated albumin (NA) or glycated albumin (GA) at 500 μg/mL, without (□) or with (▪) 500 nmol/L of the PKC inhibitor GF 109203X (GFX). From each condition, media were collected for immunoassay of TGF-βbgr;1 as described in the text. Results represent the mean ± SEM of four to six independent cultures and are expressed as ng TGF-βbgr;1 normalized to cell number. *P < 0.05 compared with the respective nonglycated albumin; #P < 0.05 compared with the respective glycated albumin, no GFX; †P < 0.05 compared with the respective nonglycated albumin, no GFX. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 TGF-βbgr;1 gene expression in GEnCs. GEnCs were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose/10% FCS and nonglycated albumin (NA) or glycated albumin (GA) at 500 μg/mL. Data were obtained by densitometric scanning of autoradiographs of Northern blots successively probed with TGF-βbgr;1 and mrpL32 cDNA. A representative autoradiograph is shown on top. Results represent the mean ± SEM of four independent cultures and are expressed as the percentage of the densitometric ratio of TGF-βbgr;1 to mrpL32 mRNA in control cells (NA), arbitrarily assigned a value of 100. *P < 0.05 compared with nonglycated albumin. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Effect of glycated albumin and TGF-βbgr; modulation on [3H]-thymidine incorporation. GEnCs were cultured for 48 hours in DMEM containing 10 mmol/L glucose/2% FCS and no supplement (Media), nonglycated albumin (NA), or glycated albumin (GA) at 500 μg/mL. (A) Cells were incubated without (□) or with (▪) 500 nmol/L of the PKC inhibitor GF 109203X (GFX). (B) Cells were incubated without other addition (□) or with 1 ng/mL TGF-βbgr;1 (T, ), 30 μg/mL irrelevant murine IgG (IgG, ▪), or 30 μg/mL neutralizing anti-TGF-βbgr; antibody (αT, ). From each condition, cells were collected for scintillation counting of [3H]-thymidine incorporation as described in the text. Results represent the mean ± SEM of five independent cultures and are expressed as counts per minute (cpm) of [3H] decay. *P < 0.05 compared with the respective Media or nonglycated albumin; #P < 0.05 compared with the respective glycated albumin, no GFX. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Effect of glycated albumin and TGF-βbgr;1 on cell counts. GEnCs were cultured for 48 hours in DMEM containing 10 mmol/L glucose/2% FCS and no supplement (Media), nonglycated albumin (NA), or glycated albumin (GA) at 500 μg/mL, without (□) or with () 1 ng/mL TGF-βbgr;1. From each condition, cells were collected for cell counting as described in the text. Results represent the mean ± SEM of five independent cultures and are expressed as the number of cells per well. *P < 0.05 compared with Media, no TGF-βbgr;1; #P < 0.05 compared with nonglycated albumin. Kidney International 2001 59, 673-681DOI: (10.1046/j.1523-1755.2001.059002673.x) Copyright © 2001 International Society of Nephrology Terms and Conditions