Volume 68, Issue 2, Pages 569-583 (August 2005) Synergistic effect of hypoxia and TNF-α on production of PAI-1 in human proximal renal tubular cells  Xuan Li, Hideki Kimura, Kiichi Hirota, Kenji Kasuno, Kunio Torii, Toshiharu Okada, Hisanori Kurooka, Yoshifumi Yokota, Haruyoshi Yoshida  Kidney International  Volume 68, Issue 2, Pages 569-583 (August 2005) DOI: 10.1111/j.1523-1755.2005.00435.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Immunohistochemical analyses of PAI-1 protein expression in renal biopsy samples from patients diagnosed as minor glomerular abnormalities and crescentic glomerulonephritis. (A) In a renal biopsy sample with minor glomerular abnormalities, no specific staining for PAI-1 was observed in tubulointerstitium or glomeruli. (B) In crescentic glomerulonephritis, strong PAI-1 staining was focally observed in proximal tubular cells and in some intraglomerular cells. Original magnification ×100. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Electron micrograph of cultured human proximal tubular epithelial cells. The cells displayed microvilli on the apical membranes (*) and tight junctions on the basolateral membranes (arrow) with clear intracellular polarization. Original magnification ×3000. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Immunoblot analysis of PAI-1 in whole cell lysates of human proximal tubular epithelial cells incubated with DMEM for 24 hours after reaching confluency. Human PAI-1 protein was identified as a single 50-kD band using polyclonal anti-human PAI-1 antibody. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 PAI-1 production by human proximal tubular epithelial cells (HPTECs) during normoxic (□,18% O2) and hypoxic (▪, 1% O2) treatment for up to 48 hours. (A) cDNA was prepared from total cellular RNA extracted from treated HPTECs at the indicated time points, and TaqMan real-time PCR for PAI-1 and β-actin was performed. PAI-1 mRNA amounts were normalized to β-actin. Averaged amounts of PAI-1 mRNA for cells during normoxia at 6 hours were set to 1.0. (B) Supernatants were also harvested at the indicated time points, and assessed for PAI-1 protein by immunoassay. Results were expressed as the mean ± SD of experiments done in triplicate (N = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control cells at each time point, by unpairedt test. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 PAI-1 production by human proximal tubular epithelial cells (HPTECs) during normoxic treatment in the absence (□) or presence (▪) of TNF-α (10ng/mL) for 24 and 48 hours. (A) After 24 hours and 48 hours of treatment with and without TNF-α, cDNA was prepared from total cellular RNA extracted from HPTECs, and TaqMan real-time PCR for PAI-1 and β-actin was performed. PAI-1 mRNA amounts were normalized to β-actin. Averaged amounts of PAI-1 mRNA for cells untreated with TNF-α at 24 hours were set to 1.0. (B) Supernatants were also harvested after 24-hour and 48-hour treatments, and assessed for PAI-1 protein by immunoassay. Results were expressed as the mean ± SD of experiments done in triplicate (N = 6). *P < 0.05 and **P < 0.001 compared with the control cells at each time point, by unpairedt test. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 PAI-1 production by human proximal tubular epithelial cells (HPTECs) during treatment with hypoxia, TNF-α (10ng/mL), or their combination for 24 hours. (A) cDNA was prepared from total cellular RNA extracted from HPTECs after 24-hour treatment, and TaqMan real-time PCR for PAI-1 and β-actin was performed. PAI-1 mRNA amounts were normalized to β-actin. Averaged amounts of PAI-1 mRNA for cells during normoxia without TNF-α treatment were set to 1.0. (B) Supernatants were harvested after 24 hours of treatment, and assessed for PAI-1 protein by immunoassay. Results were expressed as the mean ± SD of experiments done in triplicate (N = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Scheffe's post hoc comparison. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Immunoblot analyses of HIF-1α in human proximal tubular epithelial cells (HPTECs) treated with or without hypoxia (1% O2) in the presence or absence of TNF-α (10ng/mL). (A) Whole cell lysates were prepared from HPTECs cultured with DMEM under normoxic or hypoxic conditions for 16 hours. Lysates of HEK293 cells treated under conditions of hypoxia (1% O2) for 4 hours were used as a positive control. (B) Whole cell lysates were prepared from HPTECs cultured with DMEM under normoxic or hypoxic conditions in the presence or absence of TNF-α (10ng/mL). Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Immunofluorescence of HIF-1α in human proximal tubular epithelial cells (HPTECs) treated under normoxic (A) (18% O2) or hypoxic (B) (1% O2) conditions for 16 hours. (A) Faint HIF-1α staining was observed in the cytoplasm or nucleus of HPTECs. (B) Strong HIF-1α staining was observed predominantly in the nuclei of HPTECs. Original magnification ×400. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Transcriptional activation of human PAI-1 promoter by hypoxia (1% O2), TNF-α (10ng/mL), or their combination for 18 hours. In each experiment, luciferase activity of cells treated with hypoxia and/or TNF-α was determined relative to that of cells incubated under normoxic conditions (18% O2) for 18 hours. Results were expressed as the mean ± SD of experiments done in triplicate (N = 6). *P < 0.05 and **P < 0.01 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Scheffe's post hoc comparison. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 10 HIF-1α dependency of PAI-1 promoter activation in human proximal tubular epithelial cells (HPTECs) during normoxic (□,18% O2) and hypoxic (▪, 1% O2) treatment. HPTECs were cotransfected with expression vectors encoding a constitutively activated form of human HIF-1α (CA), a dominant-negative form of human HIF-1α (DN), or no protein (EV) together with pGL3 PAI-1. After 6 hours, the transfection medium was replaced with DMEM and cells were incubated under normoxic or hypoxic conditions for 18 hours, and then harvested for luciferase assays. Luciferase activity of cells treated was determined relative to that of cells transfected with the empty vector and incubated under normoxic conditions for 18 hours. Results were expressed as the mean ± SD of experiments done in triplicate (N = 6). *P < 0.05, **P < 0.01 and NS (not significant) compared with cells incubated under the indicated conditions, by one-way analysis of variance with Scheffe's post hoc comparison. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 11 Effects of an NFBκ inhibitor, PDTC, on PAI-1 production by human proximal tubular epithelial cells (HPTECs) during normoxic (□, 18% O2) and hypoxic (▪, 1% O2) treatment. Confluent HPTECs were treated with or without PDTC (10 μmol/L) for 2 hours and then incubated under normoxia or hypoxia for up to 48 hours. (A) cDNA was prepared from total cellular RNA extracted from treated cells at 24 hours, and TaqMan real-time PCR for PAI-1 and β-actin was performed. PAI-1 mRNA amounts were normalized to β-actin. Averaged amounts of PAI-1 mRNA for cells during normoxia in the absence of PDTC at 24 hours were set to 1.0. (B) Supernatants were also harvested at 48 hours, and assessed for PAI-1 protein by immunoassay. Results were expressed as the mean ± SD of experiments done in triplicate (N = 6). *P < 0.05, **P < 0.005, and ***P < 0.001, compared with the control cells at each time point, by unpairedt test. Kidney International 2005 68, 569-583DOI: (10.1111/j.1523-1755.2005.00435.x) Copyright © 2005 International Society of Nephrology Terms and Conditions