Cytotoxic effect of Shiga toxin-1 on human glomerular epithelial cells1  Alisa K. Hughes, Peter K. Stricklett, Doug Schmid, Donald E. Kohan  Kidney International  Volume 57, Issue 6, Pages 2350-2359 (June 2000) DOI: 10.1046/j.1523-1755.2000.00095.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 Cytotoxic effect of Shiga toxin-1 (Stx-1) on glomerular epithelial cells (GECs; N = 12 each data point). Cells were incubated with Stx-1 for (•) 24, (○) 48 and (▪) 72 hours, followed by determination of neutral red uptake. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 Effect of Stx-1 on 3H-leucine incorporation by GECs (N = 3 each data point). Cells were incubated with Stx-1 for (•) 24, (○) 48 and (▪) 72 hours, followed by an addition of 1 μCi/mL 3H-leucine for 20 minutes. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Total globotriaosylceramide (Gb3) content in GEC and Vero cells. A standard curve using purified Gb3 is shown on the left side of the autoradiogram for comparison. Each experimental lane represents a different cell culture preparation (Methods section). Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of IL-1 (A), IL-6 (B), TNF-α (C), LPS (D), or butyrate (mmol/L, E) on Stx-1 (10–8 g/L or 140 fmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.001; #P < 0.01, both vs. Stx-1 alone. Cells were preincubated with inflammatory factors for 24 hours prior to exposure to Stx-1 for 72 hours and subsequent determination of neutral red uptake. Symbols in A are: (▪) 10 U/mL IL-1; () 100 U/mL IL-1; () 1000 IL-1. Symbols in B are: (▪) 0.5 ng/mL IL-6; () 2 ng/mL IL-6; () 5 ng/mL IL-6. Symbols in C are: (▪) 10 U/mL TNF-α; () 100 U/mL TNF-α; () 1000 U/mL TNF-α. Symbols in D are: (▪) 0.1 μg/mL LPS; () 1 μg/mL LPS; () 10 μg/mL LPS. Symbols in E are: (▪) 0.5 mmol/L butyrate; () 2 mmol/L butyrate; () 5 mmol/L butyrate. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Effect of 4 (▪), () 24, and () 48 hours of preincubation with 100 U/mL IL-1, 5 ng/mL IL-6, 100 U/mL TNF-α, 1 μg/mL LPS, or 0.5 to 5 mmol/L butyrate on CHX (10 μmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.05; #P < 0.01, both vs. control. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Effect of 4 (▪), () 24, and () 48 hours of preincubation with 100 U/mL IL-1, 5 ng/mL IL-6, 100 U/mL TNF-α, 1 μg/mL LPS, or 0.5 to 5 mmol/L butyrate on CHX (10 μmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.05; #P < 0.01, both vs. control. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Effect of 4 (▪), () 24, and () 48 hours of preincubation with 100 U/mL IL-1, 5 ng/mL IL-6, 100 U/mL TNF-α, 1 μg/mL LPS, or 0.5 to 5 mmol/L butyrate on CHX (10 μmol/L) cytotoxicity on GECs (N = 8 each data point). *P < 0.05; #P < 0.01, both vs. control. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 6 Effect of 24 hours of incubation with (•) control, (○) 100 U/mL IL-1, (▪) 5 ng/mL IL-6, (□) 100 U/mL TNF-α, (▴) 1 μg/mL LPS, or (▵) 0.5 mmol/L butyrate on binding of 125I-Stx-1 to GECs (N = 6 each data point). Approximately 17,000 cpm of 125I-Stx-1 were added per well for 24 hours at 4°C. Cells were washed and solubilized, and bound cpm were determined. *P < 0.001 for IL-1 and P < 0.005 for LPS and P < 0.001 for butyrate, all vs. control; #P < 0.005 for IL-1 and P < 0.005 for LPS and P < 0.025 for butyrate, all vs. control; fP < 0.005 for IL-1 and P < 0.005 for LPS, both vs. control. Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 7 Effect of 24-hour incubation with 100 U/mL IL-1, 5 ng/mL IL-6, 1000 U/mL TNF, 1 μg/mL LPS, or 0.5 mmol/L butyrate on total Gb3 content in GECs (N = 6 each data point). *P < 0.025; #P < 0.01, both vs. control. Lanes were loaded with an equal weight of total cellular lipids (Methods section). Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 8 Effect of 24-hour incubation with 100 U/mL IL-1, 5 ng/mL IL-6, 100 U/mL TNF, 1 μg/mL LPS, or 0.5 mmol/L butyrate on GalT6 activity in GECs (N = 3 each data point). Control GalT6 activity was 0.36 ± 0.04 densitometry units (Methods section). Kidney International 2000 57, 2350-2359DOI: (10.1046/j.1523-1755.2000.00095.x) Copyright © 2000 International Society of Nephrology Terms and Conditions