Cytokine-induced expression of tPA is differentially modulated by NO and ROS in rat mesangial cells  Wolfgang Eberhardt, Karl-Friedrich Beck, Josef Pfeilschifter  Kidney International  Volume 61, Issue 1, Pages 20-30 (January 2002) DOI: 10.1046/j.1523-1755.2002.00087.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Effects of exogenous nitric oxide on the expression of the matrix proteases tissue-type plasminogen activator (tPA) and matrix metalloproteinase-9 (MMP-9) and their endogenous inhibitors plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloprotease-1 (TIMP-1) in rat mesangial cells (MCs). (A) Time-course of steady-state levels of mRNA of tPA, PAI-1, MMP-9 and TIMP-1 in MCs following treatment with vehicle (control) or interleukin-1β; (IL-1β;; 2nmol · L-1) with or without S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 500 μmol/L). Twenty micrograms of total cellular RNA were hybridized successively to the corresponding 32P-labeled cDNA inserts as described in the Materials and Methods section. Equivalence of loading was ascertained by rehybridization to a GAPDH probe. The blot is representative for two further independent experiments. Statistical analysis of tPA (B) and PAI-1 (C) mRNA levels after 36 hours of stimulation. Data represent means ± SD (N = 3). **P ≤ 0.01 compared with vehicle, or IL-1β;-stimulated conditions (#P ≤ 0.05; ##P ≤ 0.01). (D) Time-course of tPA levels in the cell culture supernatant following treatment with vehicle (control) or IL-1β; (2nmol/L) with or without SNAP (500 μmol/L) as indicated. Data represent means ± SD (N = 3). *P ≤ 0.05; **P ≤ 0.01 compared with vehicle, or IL-1β;-stimulated conditions (##P ≤ 0.01). Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Dose-dependent modulation of IL-1β;–induced steady-state mRNA level of tPA and PAI-1 by SNAP. MCs were treated for 12 hours (A) and 24 hours (B) with the indicated concentrations of SNAP in the absence (-) or presence (+) of 2nmol · L-1 of IL-1β;. The blots were successively hybridized with the indicated 32P labeled cDNA inserts. The data are representative for two independent experiments with similar results. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Time-course of tPA and PAI-1 mRNA levels in MCs stimulated with IL-1β; or DETA NONOate. MCs were incubated for the indicated time periods with vehicle (control), IL-1β; (2nmol · L-1), DETA-NONOate (DETA-NO, 500 μmol/L) or combinations of IL-1β; plus DETA-NONOate. Twenty micrograms of total cellular RNA were hybridized successively to the corresponding 32P-labeled cDNA inserts. The data shown are representative for two independent experiments giving similar results Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Inhibition of nitric oxide synthase (NOS) leads to a dose-dependent increase of IL-1β;–induced tPA and PAI-1 mRNA levels. Quiescent MCs were cotreated for 48 hours with vehicle (control), IL-1β; (2nmol · L-1) without (-) or with (+) the indicated concentrations of L-NMMA. Twenty micrograms of total cellular RNA were hybridized successively to 32P-labeled cDNA inserts from rat tPA and PAI-1 genes, respectively. The experiment is representative for three experiments giving similar results. The levels of nitrite in the corresponding cell supernatants are shown at the bottom. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 DETA-NONOate increases the half-life of IL-1β;-induced tPA mRNA. (A) Quiescent MCs were treated for 16 hours with IL-1β; (2nmol · L-1) in the presence or absence of DETA NONOate (500 μmol/L) before actinomycin D (10 μg · mL-1) was added to the culture dish (0 hour) and incubated further for the time points indicated. Subsequently, MCs were harvested and extracted for total RNA. Fifteen micrograms of total cellular RNA were hybridized to a 32P-labeled tPA probe. The equal loading of RNA was ascertained by subsequent hybridization to a 32P-labeled 18S rRNA probe. (B) A densitometrical analysis of changes in the half-life of tPA mRNA is shown in the lower panel. The tPA mRNA levels from cells treated with IL-1β; in the presence () or absence (□) of DETA-NONOate, and harvested before the addition of actinomycin D (0 hour) were set as 100%. The data shown are representative for two experiments giving similar results. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 6 Early amplification but not the late suppression of IL-1β;-induced tPA mRNA level by NO is mimicked by cGMP. MCs were stimulated for 12 hours (A) and 24 hours (B) with the indicated concentrations of dibutyryl cGMP in the absence (-) or presence (+) of 2nmol · L-1 of IL-1β;, as indicated. Twenty micrograms of total cellular RNA were successively hybridized to 32P-labeled tPA and GAPDH probes. The data shown are representative of two experiments giving similar results. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 7 (A) Effects of superoxide generators on IL-1β;-induced tPA and PAI-1 mRNA steady-state levels. MCs were incubated for 24 hours with vehicle, 2nmol · L-1 IL-1β;, in the presence (+) or absence (-) of the hypoxanthine/xanthine oxidase (HXXO) system at 8mU · mL-1, or, alternatively, the redox cycler dimethoxy-1,4-naphtoquinone (DMNQ) at 5 μmol/L as indicated. Twenty micrograms of total cellular RNA were successively hybridized to 32P-labeled tPA, PAI-1 and GAPDH probes, respectively. (B) Time-course of amplification of IL-1β;-induced tPA mRNA levels by HXXO. MCs were incubated for the indicated time points with vehicle (control), 2nmol · L-1 IL-1β; in the presence or absence the hypoxanthine/xanthine oxidase system (HXXO) at 8mU · mL-1 as indicated. Twenty micrograms of total cellular RNA was successively hybridized to a 32P-labeled tPA and a probe for 18S ribosomal RNA, respectively. The data are representative for two independent experiments giving similar results. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 8 Effects of HXXO on tPA mRNA levels are not affected by coincubation with SOD, but are mimicked by glucose oxidase. MCs were treated for 24 hours with 2nmol · L-1 IL-1β; in the presence or absence of MnTBAP at 10 and 100 μmol/L, a cell-permeable SOD mimetic, or alternatively with 200 μmol/L H2O2 or 1mmol/L glucose oxidase as indicated. Twenty micrograms of total cellular RNA were successively hybridized to 32P-labeled tPA, PAI-1 and a probe for 18S ribosomal RNA, respectively. The data are representative of two independent experiments giving similar results. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 9 (A) Dose-dependency of H2O2 effects on IL-1β;–stimulated tPA and PAI-1 mRNA levels. MCs were incubated for 24 hours with vehicle, 2nmol · L-1 IL-1β; in the presence of the indicated concentrations of H2O2. Twenty micrograms of total cellular RNA were successively hybridized to 32P-labeled tPA, PAI-1 and GAPDH probes, respectively. The data are representative for two independent experiments giving similar results. (B) Catalase attenuates cytokine-and ROS-mediated induction of tPA and PAI-1 mRNA steady-state level. Quiescent MCs were treated for 24 hours with vehicle (control), IL-1β; or IL-1β; plus HXXO in the presence of the indicated concentrations of catalase. Twenty micrograms of total cellular RNA were hybridized successively to 32P-labeled tPA, PAI-1 and 18S ribosomal RNA probes. The data are representative of two independent experiments giving similar results. Kidney International 2002 61, 20-30DOI: (10.1046/j.1523-1755.2002.00087.x) Copyright © 2002 International Society of Nephrology Terms and Conditions