Volume 67, Issue 4, Pages 1315-1329 (April 2005) Thrombin stimulates proinflammatory and proliferative responses in primary cultures of human proximal tubule cells  David A. Vesey, Catherine W. Cheung, Wade A. Kruger, Philip Poronnik, Glenda Gobe, David W. Johnson  Kidney International  Volume 67, Issue 4, Pages 1315-1329 (April 2005) DOI: 10.1111/j.1523-1755.2005.00209.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Reverse transcription-polymerase chain reaction (RT-PCR) analysis of protease-activated receptor (PAR) expression by cultured human proximal tubule cells (PTCs). Total RNA was isolated from confluent passage 2, human PTC, reverse transcribed, and amplified with specific primers for PARs and hypoxanthine phosphoribosyltransferase (HPRT). PCR products were separated on 1.8% agarose gels containing ethidium bromide and photographed. (A) Expression of thrombin receptors (PAR-1, PAR-3, and PAR-4), and PAR-2 by PTC. Lane 1, PAR-1 (200 bp); lane 2, PAR-2 (582 bp); lane 3, PAR-3 (382 bp); lane 4, PAR-4, (392 bp); lane 5, HPRT (386 bp); and lane 6, ΦX174 DNA/Hae III markers. (B) Expression of PAR-1 in three different PTC cultures by two different sets of PAR-1 primers. Lane 1, PTC 1; lane 2, PTC 2; lane 3, PTC 3; lane 4, negative control; and lane 5, ΦX174 DNA/Hae III markers. (C) RT-PCR analysis of PAR-1 expression in cells from three different PTC culture in which the RT step is carried out with (RT) or without (NRT) the reverse transcriptase enzyme. Lane 1, PTC 1 RT; lane 2, PTC 1 NRT; lane 3, PTC 2 RT; lane 4, PTC 2 NRT; lane 5, PTC 3 RT; lane 6, PTC 3 NRT; lane 7, negative control; and lane 8, ΦX174 DNA/HaeIII markers. Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 The effects of thrombin and the protease-activated receptor (PAR) peptides on calcium mobilization in human proximal tubular cells (PTCs). Cells were grown to confluence and loaded with Fura-2 AM. They were then exposed to thrombin (5 U/mL), TFLLRN-NH2 (100 μmol/L), SFLLRN-NH2 (100 μmol/L), SLIGKV-NH2 (100 μmol/L), or VKGILS-NH2 (100 μmol/L) and intracellular Ca2+ fluorescence measured. (A) The results are expressed as the change from basal level of the fluorescence ratio (340/380 nm). Each trace is an average response from cells in four different wells and is representative of two different experiments. (B) Results are expressed as a percentage of the SLIGKV-NH2 signal at 24 seconds (17 seconds after agonist addition). Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 The effect of thrombin on human proximal tubule cell (PTCs) DNA synthesis (A), lactate dehydrogenase (LDH) release (B), fibronectin secretion (C), monocyte chemoattractant protein-1 (MCP-1) secretion (D), transforming growth factor-β1 (TGF-β1) secretion (E), and cell protein levels (F). Confluent passage 2 human PTC were treated with or without thrombin for 24 hours and assayed for the above responses as detailed under the Methods section. Data points are the mean ± SEM from five independent experiments, each performed in triplicate. *P < 0.05 vs. control cells. Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Analysis of fibronectin secreted by or associated with human proximal tubular cells (PTCs) treated with thrombin. Confluent, quiescent, passage 2 human PTC cultures were treated with (T) or without (C) thrombin (5 U/mL). After 24 hours, conditioned media and cells lysate were collected and analyzed by electrophoresis and Western blot analysis (A and B) using a specific fibronectin antibody. (A) Conditioned media from four different PTC cultures were analyzed. A single band at 220 kD was observed. No fibronectin degradation products were apparent. Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 The effects of hirudin on thrombin-induced DNA synthesis (A), fibronectin secretion (B and E), monocyte chemoattractant protein-1 (MCP-1) secretion (C), and transforming growth factor-β (TGF-β) secretion (D). Confluent, quiescent, passage 2 human PTC were treated with or without thrombin (5 U/mL) in the presence (□) or absence (▪) of hirudin (5 U/mL) for 24 hours. Each bar represents the mean ± SEM for three independent experiments each performed in triplicate. *P < 0.01 vs. cells treated with thrombin alone; #P < 0.01 vs, control cells. (E) A representative Western blot of fibronectin secreted by PTC treated with either control media (C), thrombin (5 U/mL) (T), or thrombin (5 U/mL plus hirudin 5 U/mL) (T & H). Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 A pan-specific transforming growth factor-β (TGF-β) neutralizing antibody failed to influence thrombin-induced fibronectin secretion. Fibronectin secretion was measured in conditioned media from confluent, quiescent, passage 2 human proximal tubular cells (PTCs) incubated for 24 hours in serum-free media with TGF-β1 (1 ng/mL), or thrombin (5 U/mL) together with a pan-specific TGF-β neutralizing antibody (▪) (15 μg/mL) or nonimmune control rabbit IgG antibody (▪) (15 μg/mL) or no treatment (□) (NIL). Each bar represents the mean ± SEM for three independent experiments each performed in triplicate. *P < 0.05 vs. control treated cells; #P < 0.05 vs. cells treated with thrombin plus nonimmune control IgG. Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 The effects of matrix metalloproteinase (MMP) inhibition and endothelial growth factor (EGF) receptor kinase inhibition on thrombin-induced DNA synthesis, fibronectin secretion and monocyte chemoattractant protein-1 (MCP-1) secretion. Confluent, quiescent, passage 2 human proximal tubular cells (PTCs) were treated with or without thrombin (5 U/mL) in the absence (□) or presence of MMP inhibitor GM6001 (0.1 μmol/L) (▪) or EGF receptor kinase inhibitor AG1478 (0.1 μmol/L) (▪) for 24 hours. Each bar represents the mean ± SEM for three independent experiments each performed in triplicate. *P < 0.05 vs. control-treated cells. #P > 0.05 vs. cells treated with thrombin alone. Kidney International 2005 67, 1315-1329DOI: (10.1111/j.1523-1755.2005.00209.x) Copyright © 2005 International Society of Nephrology Terms and Conditions