Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: Upregulation of plasminogen activator inhibitor type 1 (PAI-1) as an early endothelial response against podocyte injury. A and B: in NEP25/LMB2 mice, glomerular mRNA expression of the endothelial dysfunction markers VEGF and endothelial nitric oxide synthase (eNOS) was decreased on day 12. *P < 0.05. C: compared with day −1, the thrombosis score was significantly elevated on day 12 in NEP25/LMB2 mice (n = 6). ***P < 0.001. D and E: transforming growth factor (TGF)-β mRNA expression in isolated glomeruli (D) and relative PAI-1 mRNA (E). TGF-β mRNA was increased in NEP25/LMB2 mice on day 1 and further increased on days 8 and 12. PAI-1 mRNA expression in isolated glomeruli was increased on day 1 and further increased on days 8 and 12. n = 5 mice/group for each time point. *P < 0.05; **P < 0.01. F: double immunofluorescence of PAI-1 and CD31 expression in control and NEP25/LMB2 mice in glomeruli on day 1. Magnification: ×600. PAI-1 was absent in control mice, whereas it was expressed and colocalized with CD31 in NEP25/LMB2 mice. G: on day 12, PAI-1 was aberrantly expressed in the podocyte cytoplasm in NEP25/LMB2 mice. H: expression of urokinase plasminogen activator (uPA) receptor (uPAR) in NEP25/LMB2 mice on day 1 as revealed by serial sections. uPAR was not expressed in control mice (top), whereas it was expressed mainly in podocytes in NEP25/LMB2 mice (bottom), as confirmed in synaptopodin staining by serial sections (arrows). I: electron microscopy showed endothelial cell swelling and loss of fenestra (arrows) accompanied with overlaying podocyte foot process effacement in NEP25/LMB2 mice compared with control mice on day 1. Scale bars = 2 μm.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: Expressing, Isolation, Immunofluorescence, Staining, Electron Microscopy

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: PAI-1 inhibitor ameliorated proteinuria and preserved podocyte numbers in NEP25/LMB2 mice. A: expression of PAI-1 was reduced in PAI-1 inhibitor-treated NEP25/LMB2 mice (NEP25/LMB2 + PI group) compared with vehicle-treated NEP25/LMB2 mice (NEP25/LMB2 + VH group). B: proteinuria in NEP25/LMB2 + PI mice (n = 8) was significantly lower than that in NEP25/LMB2 + VH mice (n = 6) on day 12. *P < 0.05. C: the thrombosis score was significantly lower in NEP25/LMB2 + PI mice (n = 8) than in NEP25/LMB2 + VH mice (n = 6). *P < 0.05. D: mean WT-1-positive cells per glomerular profile in NEP25/LMB2 + PI mice (n = 8) were significantly higher than those in NEP25/LMB2 + VH mice (n = 6). ***P < 0.001. E: relative mRNA expression from isolated glomeruli on day 12 in NEP25/LMB2 + PI mice (n = 5) and NEP25/LMB2 + VH mice (n = 5) was compared. Glomeruli were isolated from each mouse, and quantitative real-time PCR was done on each sample. NEP25/LMB2 + PI mice showed significantly higher levels in eNOS and VEGF mRNA but significantly lower levels of desmin and vimentin mRNA compared with NEP25/LMB2 + VH mice.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: PAI-1 inhibitor protected glomerular structure and podocyte injury in NEP25/LMB2 mice. A: histology of the kidney cortex. At lower magnification (×200; scale bars = 15 μm), NEP25/LMB2 + PI mice showed less glomerular injury and tubular dilatation than NEP25/LMB2 + VH mice (left). Higher magnification (×400, scale bars = 15 μm) of periodic acid-silver methenamine staining revealed glomerular epithelial proliferation and thrombi on day 8 (middle left) and glomerulosclerosis on day 12 (middle right) in NEP25/LMB2 + VH mice. Bowman's space cells were interpreted as proliferating parietal epithelial cells and may well represent podocytes undergoing detachment in NEP25/LMB2 + VH mice on day 8. Relatively normal glomeruli were noted in NEP25/LMB2 + PI mice. A transmission EM image of the glomerulus on day 12 is shown on the right. In NEP25/LMB2 + VH mice, thrombotic formation was associated with degeneration in overlaying podocytes (arrow). These features were not observed, and the foot process was preserved in NEP25/LMB2 + PI mice. Scale bars = 2 μm. B: prevalence of glomerulosclerosis in NEP25/LMB2 mice with or without PAI-1 inhibitor. The sclerosis score was significantly lower in NEP25/LMB2 + PI mice (n = 8) than in NEP25/LMB2 + VH mice (n = 6). **P < 0.01.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: Staining, Transmission Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: PAI-1 and the uPA complex induced cell detachment in podocytes in vitro. We evaluated podocyte adhesive capabilities by manual counting cells. All experiments were repeated three times and statistically estimated. The number of podocytes incubated with the PAI-1/uPA complex was significantly reduced compared with those incubated with uPA, PAI-1 alone, and anti-uPAR + PAI-1/uPA. *P < 0.05 vs. uPA, PAI-1, and anti-uPAR + PAI-1/uPA.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: In Vitro, Incubation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: The PAI-1/uPA complex induced translocation of β1-integrin from the podocyte surface to the cytoplasm via a uPAR-dependent mechanism. A: localization of β1-integrin and uPAR was visualized by confocal laser scanning microscopy. Using immunofluorescence microcopy, cultured podocytes treated with the PAI-1/uPA complex expressed β1-integrin in the cytoplasm that colocalized with uPAR, whereas no translocalization occurred in podocytes treated with uPA or PAI-1 alone. β1-Integrin translocalization in response to PAI-1/uPA could be inhibited by the administration of anti-uPAR. Magnification: ×1,000. Scale bars = 10 μm. B: colocalization between β1-integrin and uPAR was significantly augmented in PAI-1/uPA-treated cells (n = 30 cells/group). ***P < 0.001 vs. uPA, PAI-1, and anti-uPAR + PAI-1/uPA. Pearson's correlation coefficient for β1-integrin and uPAR colocalization was calculated using ImageJ and JaCoP software (3, 34). In every experiment using JaCoP software, the red fluorescence indirectly emitted by β1-integrin was primarily detected in Ch1 and the green fluorescence indirectly emitted by uPAR was primarily detected in Ch2. In all calculations, the background was subtracted from the intensity values.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: Translocation Assay, Confocal Laser Scanning Microscopy, Immunofluorescence, Cell Culture, Software, Fluorescence

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: The PAI-1/uPA complex induced β1-integrin and uPAR translocation via an endosomal pathway. A: biotinylated experiments with Western blot analysis of β1-integrin expression revealed a decrease of biotinylated membrane β1-integrin in PAI-1/uPAR complex-treated podocytes. Such changes were not observed in podocytes treated with uPA, PAI-1 alone, or the anti-uPAR + PAI-1/uPA complex. B: β1-integrin was increased in the cytoplasmic fraction in PAI-1/uPAR-treated podocytes. C: double immunogold labeling by immunoelectron microscopy of β1-integrin (5-nm gold particles) and uPAR (10-nm gold particles). For uPA, both β1-integrin and uPAR were predominantly located on the cell surface, whereas PAI-1/uPA showed colocalization of both proteins in the cytoplasm. Note that gold particles of both sizes were associated with endocytotic vesicles (inset). Scale bars = 50 nm. D: under ×40,000 magnification, gold particles of both sizes were counted, and the particle ratio in the cell membrane and total numbers of particles were calculated. Ratios of β1-integrin and uPAR were significantly reduced in podocytes treated with PAI-1/uPA (n = 6) than in those treated with uPA (n = 4). *P < 0.05.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: Translocation Assay, Western Blot, Expressing, Labeling, Immuno-Electron Microscopy

Journal: American Journal of Physiology - Renal Physiology

Article Title: Podocyte injury-driven intracapillary plasminogen activator inhibitor type 1 accelerates podocyte loss via uPAR-mediated β 1 -integrin endocytosis

doi: 10.1152/ajprenal.00616.2014

Figure Lengend Snippet: Schematic demonstration of the podocyte domino effect by PAI-1/uPAR-mediated β1-integrin endocytosis. From the left, damaged podocytes repressed VEGF expression, leading to endothelial dysfunction. Damaged endothelial cells produced and secreted PAI-1 (molecular mass: 45 kDa), which binds to circulating uPA (molecular mass: 31.5 kDa) either in the capillary or on the podocyte surface and then makes a complex with uPAR on the podocyte surface. The complex formation is able to bind β1-integrin, which is known as a strong assembler of podocyte-GBM attachment. With the aid of a large endocytotic receptor, LDL receptor-related protein (LRP), on the podocyte surface (the expression of LRP was detected in vivo and cultured podocytes by real-time PCR in our preliminary experiments), β1-integrin is translocated to the podocyte cytoplasm by endocytosis with the PAI-1/uPA/uPAR complex. Finally, β1-integrin-lost primary healthy podocytes undergo detachment from the GBM.

Article Snippet: Glycosylated mouse PAI-1 (stable mutant) and active two-chain high-molecular-weight mouse uPA were purchased from Molecular Innovations (Novi, MI).

Techniques: Expressing, Produced, In Vivo, Cell Culture, Real-time Polymerase Chain Reaction

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Schematic representation of the promoter region of human plasminogen activator inhibitor type 1 (PAI-1) gene. The top represents the promoter region up to − 800 bp. The arrows indicate the transcription factor binding sites in the promoter region, based on the published sequences (7, 8, 10, 21, 22, 32). D-Box and P-Box contain AP-1-like cis elements. The bottom illustrates major transcription factor binding sites in p549Luc construct. The boxes represent the locations of the transcription factor binding sites, and the sequences are the decoy oligonucleotides (ODNs) that are used in the study. The underlined sequences represent the binding sites of transcription factors (22).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Binding Assay, Construct

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Glutathione (GSH) inhibits transforming growth factor (TGF)-β-induced PAI-1 expression at the transcriptional level. A: NIH/3T3 cells were transiently cotransfected with p800luc reporter gene construct or p19luc control construct and pRL-TK-luciferase (transfection control) and then stimulated with TGF-β (1 ng/ml) for 24 h in the presence or absence of GSH (1–5 mM). B: Mv1Lu cells were treated with 1 ng/ml TGF-β with or without GSH (1–5 mM). The luciferase activity was measured 24 h after TGF-β treatment and normalized with transfection control as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone group (P < 0.05, n = 3).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Expressing, Construct, Luciferase, Transfection, Activity Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Pharmacological inhibitors of JNK and p38 MAPKs block TGF-β-induced PAI-1 expression. 70–80% Confluent, serum-starved NIH/3T3 cells were treated with 1 ng/ml TGF-β in the presence or absence of p38 inhibitor SB-202190 (3 µM) or JNK inhibitor SP-600125 (10 µM) for 24 h. The medium was collected for PAI-1 protein analysis by ELISA (A) while the cells were collected for PAI-1 mRNA measurement by Northern analysis (B) as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone group (P < 0.05, n = 3–4).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Blocking Assay, Expressing, Enzyme-linked Immunosorbent Assay, Northern Blot

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Dominant negative (DN) mutants of p38 or JNK inhibit TGF-β-induced PAI-1 expression. A: DN-p38 and DN-JNK block TGF-β-induced p800luc promoter activity. NIH/3T3 cells were cotransfected with the reporter gene and DN constructs or corresponding vector (pCDNA3.1 for DN-JNK and pIRES2-EGFP for DN-p38) as indicated and then treated with TGF-β (1 ng/ml). The luciferase activity was measured in the cell lysates 24 h after treatment. Renilla luciferase was used to normalize the transfection efficiency. B: DN-p38 and DN-JNK block TGF-β-induced endogenous PAI-1 expression. The experimental conditions were the same as those described for A. The PAI-1 was detected in the media by ELISA as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from the corresponding vector-transfected and TGF-β-treated group (P < 0.05, n = 4–6).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Dominant Negative Mutation, Expressing, Blocking Assay, Activity Assay, Construct, Plasmid Preparation, Luciferase, Transfection, Enzyme-linked Immunosorbent Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Diphenyleneiodonium (DPI) and MnTBaP inhibit TGF-β-induced MAPK phosphorylation and PAI-1 expression. A: DPI and MnTBaP inhibit TGF-β-induced p38 and JNK phosphorylation. 70–80% Confluent NIH/3T3 cells were treated with TGF-β (1.0 ng/ml) in the presence or absence of DPI (2 µM) or MnTBaP (50 µM) for 30 min. The cell lysates were prepared and analyzed by Western blot using specific antibodies for phosphorylated or unphosphorylated p38 and JNK. B: DPI and MnTBaP inhibit TGF-β-induced PAI-1 expression. The cells were pretreated with DPI (2 µM) for 2 h and then with TGF-β (1.0 ng/ml) in the absence of DPI for 24 h or the cells were treated with TGF-β (1.0 ng/ml) in the presence or absence of MnTBaP (50 µM) for 24 h. The media were collected to analyze the PAI-1 protein by ELISA. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone treated group (P < 0.05, n = 4).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: AP-1, SP-1, or Smad decoy ODN abrogates GSH inhibitory effect on TGF-β-induced PAI-1 promoter activity and inhibits TGF-β-induced PAI-1 expression. A: NIH/3T3 cells were cotransfected with luciferase reporter constructs driven by 187 bp, 549 bp, or 800 bp of human PAI-1 promoter region, as shown in Fig. 1, and pRL-TK Renilla luciferase reporter construct (transfection control). After transfection, the cells were treated with TGF-β in the presence or absence of GSH (5 mM) for 24 h. The luciferase activities (firefly luciferase and Renilla luciferase) in the cell lysates were determined using the Dual Luciferase Reporter Assay System (Promega), and the results were normalized based on transfection controls in each experiment. a, Significantly different from the corresponding (transfected with the same reporter construct) untreated control; b, significantly different from the corresponding TGF-β-alone treated cells (P < 0.05, n = 5–9). B: NIH/3T3 cells were transfected with p800luc reporter construct and pRL-TK-luciferase construct (transfection control) as well as specific ODN for AP-1, SP-1, or Smad (final concentration 315 nM) as indicated and then stimulated with TGF-β (1 ng/ml) for 24 h in the presence or absence of 5 mM GSH. The luciferase activities in cell lysates were measured as described above. a, Significantly different from the untreated ODN control group (bar 1); b, significantly different from TGF-β-treated ODN control group (bar 5) (P < 0.05, n = 5–9). C: NIH/3T3 cells were transfected with AP-1, SP-1, or Smad ODN at a final concentration of 315 nM and then treated with 1 ng/ml TGF-β in a serum-free medium for 24 h. The conditioned media were collected, and PAI-1 protein was determined by ELISA. a, Significantly different from the untreated control group; b, significantly different from TGF-β-alone treated group (P < 0.05, n = 4).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Activity Assay, Expressing, Luciferase, Construct, Transfection, Reporter Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: GSH inhibits TGF-β-induced binding of transcription factors to AP-1, SP-1, and Smad cis elements in PAI-1 promoter. The ODNs used for PAI-1 promoter decoy studies were end-labeled with [γ-32P]ATP and incubated with nuclear extracts from NIH/3T3 cells treated with TGF-β with or without GSH for 30 min. The protein DNA complexes were then electrophoresed on native PAGE as described in materials and methods. Tenfold non-radiolabeled (cold) ODNs were used to confirm the specificity of the binding. a, Significantly different from the untreated control; b, significantly different from TGF-β-treated group (P < 0.05, n = 3–4).

Article Snippet: PAI-1 standards (cat. no. MPAI-I91L, Molecular Innovations) ranging from 0 to 5,000 pg/ml were used in the assay.

Techniques: Binding Assay, Labeling, Incubation, Clear Native PAGE