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NDT Advance Access published online on March 26, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn139
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


A natural PPAR-{gamma} agonist, 15-deoxy-delta 12,14-prostaglandin J2, may act as an enhancer of PAI-1 in human proximal renal tubular cells under hypoxic and inflammatory conditions

Hideki Kimura1, Xuan Li1, Kunio Torii1, Toshiharu Okada1, Naoki Takahashi1, Hiroshi Fujii2, Shunji Ishihara3 and Haruyoshi Yoshida1

1 Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui 2 Department of Biochemistry, School of Medicine, Niigata University, Niigata 3 Department of Gastroenterology and Hepatology, Shimane University School of Medicine, Shimane, Japan

Correspondence and offprint requests to: Hideki Kimura, Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, 23 Matsuoka-shimoaizuki, Eiheiji-cho, Yoshida, Fukui 910-1193, Japan. Tel: +81-776-61-3111 (Ext. 3361); Fax: +81-776-61-8120; E-mail: hkimura{at}u-fukui.ac.jp, kimura.hideki{at}maroon.plala.or.jp



   Abstract
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 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Hypoxia and inflammation, an unavoidable milieu for renal tubular cells during the development of renal fibrosis, reportedly up-regulate production of plasminogen activator inhibitor-1 (PAI-1), a promoter of tissue fibrosis. Peroxisome proliferator-activated receptor (PPAR)-{gamma} agonists may modulate renal fibrosis progression via their anti-inflammatory effects in a PPAR-{gamma}-dependent or -independent manner. However, no information is known about the effects of PPAR-{gamma} agonists on PAI-1 expression in human proximal renal tubular cells (HPTECs) under hypoxia and/or inflammation.

Methods. Confluent HPTECs were exposed to normoxia (18% O2), hypoxia (1% O2) and/or TNF-{alpha} at 10 ng/mL for up to 48 h. The cells were incubated with two PPAR-{gamma} ago- nists, 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) and pioglitazone. Precise amounts of PAI-1 mRNA and protein were measured by TaqMan quantitative PCR and immunoassay, respectively. PPAR response element (PPRE) activity induced by 15d-PGJ2 was measured by transfection with PPRE-luciferase construct.

Results. Basal PAI-1 was significantly increased, in a dose-dependent manner, by 15d-PGJ2. It also enhanced hypoxia-, TNF-{alpha}- and hypoxia plus TNF-{alpha}-stimulated PAI-1 expression at the mRNA and protein levels. Pioglitazone had no influence on PAI-1 protein production. Although 15d-PGJ2 enhanced PPRE activity significantly in the HPTECs expressing PPAR-{gamma}, a specific inhibitor for PPAR-{gamma}, GW9662, did not diminish 15d-PGJ2-induced PAI-1 expression. In contrast, a non-selective tyrosine kinase (TK) inhibitor, genisteine or a MEK1 (MAPK kinase) inhibitor, PD98059, inhibited 15d-PGJ2-induced PAI-1 production completely.

Conclusions. The endogenous PPAR-{gamma} agonist, 15d-PGJ2, increased PAI-1 expression independently of PPAR-{gamma} via the activation of TK or MAP kinase in HPTECs and may act as an enhancer of PAI-1 production in the kidney under hypoxic and inflammatory conditions.

Keywords: 15d-PGJ2; human proximal tubular cells; hypoxia; PAI-1; PPAR-{gamma}



   Introduction
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 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of plasminogen activators in vivo and generally recognized to induce the intrarenal accumulation of extracellular matrix (ECM) by preventing the generation of plasmin and matrix metalloproteinase which can degrade ECM proteins such as fibronectin and collagenous proteins [1]. While PAI-1 is essentially undetectable in normal kidney [2,3], PAI-1 overexpression is frequently found near or within fibrotic lesions in various models of renal injury [2,4] and in damaged glomerular or tubular cells and infiltrating macrophages in many human kidney diseases [3,5,6]. Furthermore, in several mouse models of renal fibrosis, a deficiency of PAI-1 attenuated the fibrogenic response compared with that in the wild-type mice [7,8]. As major inducers for PAI-1 production, growth factors such as tumour necrosis factor-{alpha} (TNF-{alpha}) [9,10] and transforming growth factor-β (TGF-β) [10] and metabolic factors such as glucose [11] have been reported in many different cultured cells including renal tubular cells. More recently, we identified hypoxia as an enhancer of the basal and TNF-{alpha}-stimulated PAI-1 expression in human proximal tubular cells [12]. During the progression of chronic kidney disease, occasional inflammation occurs in the glomeruli and interstitium due to exacerbation of underlying immunological or metabolic disorders, and the ensuing expansion of renal fibrosis causes hypoxia of renal tubular cells [13]. Therefore, PAI-1 production by tubular cells thus stimulated may play an indispensable part as a vicious factor in the progression of renal fibrosis.

Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a representative member of a nuclear hormone receptor superfamily of ligand-dependent transcription factors [14]. PPAR-{gamma} plays a pivotal role in the regulation of adipogenesis and insulin sensitivity. It forms heterodimers with the 9-cis retinoic acid receptor, RXR-{alpha}, binds to characteristic DNA sequences termed peroxisome proliferator response elements (PPREs) located in the promoter region of target genes, and then exerts transcriptional power enhanced by specific lipid ligands (agonists). The most potent natural agonist for PPAR-{gamma} is 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2), a naturally occurring prostaglandin containing a cyclopentenone ring [15,16]. Synthetic agonists, anti-diabetic thiazolidinediones (TZDs), including pioglitazone, also activate PPAR-{gamma} by binding to the receptor with high affinity. Recently, accumulating evidence has indicated that 15d-PGJ2 and TZDs not only improve insulin sensitivity and glucose metabolism but also possess anti-inflammatory potential that results in a reduction in the expression of pro-inflammatory cytokines and chemokines [17]. As for 15d-PGJ2, its anti-inflammatory action was shown to be both dependent on and independent of PPAR-{gamma} [15,18]. It not only enhances PPAR-{gamma}'s action of antagonizing the transcriptional activity of NF-kB [18], but also suppresses NF-kB's activation by inhibiting I-kB's degradation and DNA binding in a PPAR-{gamma}-independent manner in mesangial cells [15].

Effects of PPAR-{gamma} agonists on PAI-1 production have been investigated in various types of cultured cells and appear to be not uniform but agonist- and cell-specific [19–24] although a down-regulation of production is expected in view of the anti-inflammatory effects of the agonists. In human endothelial cells, 15d-PGJ2 increased PAI-1 expression [19,20] while pioglitazone decreased it [21]. In adipocytes, troglitazone decreased PAI-1 secretion [22] but pioglitazone had no effect [23]. In human and rat mesangial cells, troglitazone and 15d-PGJ2 were reported to reduce PAI-1 expression [24]. However, it has so far remained to be clarified how PPAR-{gamma} agonists regulate PAI-1 expression in renal tubular cells. More recently, hypoxia and inflammation were reported to increase 15d-PGJ2 production in several types of cells [25,26]. Therefore, it seems more intriguing to investigate the effects of PPAR-{gamma} agonists, especially 15d-PGJ2, on the enhanced PAI-1 production in renal tubular cells stimulated with hypoxia and/or inflammatory factors, corresponding to cellular conditions in vivo during the progression of kidney disease.

In the current study, we first investigated how 15d-PGJ2 and pioglitazone regulate basal PAI-1 production and then clarified how 15d-PGJ2 modifies PAI-1 expression stimulated by hypoxia, TNF-{alpha}, and a combination of hypoxia and TNF-{alpha} in cultured human proximal renal tubular epithelial cells (HPTECs). We also examined whether or not the PAI-1-modulating effect of 15d-PGJ2 is PPAR-{gamma}- dependent.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Tubular cell cultures
HPTECs were purchased as twice-passaged cells from Clonetics, Inc. (San Diego, CA, USA). The cells were grown in a renal epithelial cell growth medium (REGM) with 5% CO2 and 18% O2 in a humidified atmosphere at 37.0°C. REGM was supplemented with 0.5% FBS, EGF (10 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/mL), epinephrine (0.5 µg/mL), triiodothyronine (6.5 ng/mL), transferrin (10 µg/mL), gentamicin (10 µg/mL) and amphotericin-B (50 ng/mL). The specific outgrowth of HPTECs was characterized as we previously described [12]. HPTECs (passages 3 through 5) were seeded in 12-well plates (3.8 cm2 with 2 mL of medium) at a density of 5 x 104 cells/well. The REGM was renewed every 2 days until confluence was achieved. Confluent cells were growth-arrested in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen Corp., Carlsbad, CA, USA) with 0.5% FBS (Invitrogen Corp.) for 24 h, and the DMEM was renewed immediately before the stimulation experiment. For hypoxic treatment, the cells were transferred to a MIC-101 modular incubator chamber (Billups-Rosenberg, Del Mar, CA, USA), which was flushed with 1% O2–5% CO2–94% N2, sealed and placed at 37.0°C for periods of up to 48 h. To study the effects of proinflammatory or pro-fibrotic cytokines on PAI-1 expression, recombinant human TNF-{alpha} (Invitrogen Corp.) as a representative inflammatory cytokine and human recombinant TGF-β (R&D systems, Inc., MN, USA) as a pro-fibrotic cytokine were added to the medium at a final concentration of 10 ng/mL and 5 ng/mL, respectively, for 24 h.

PPAR-{gamma} agonists, 15d-PGJ2 (Calbiochem, San Diego, CA, USA) and pioglitazone (a gift from Takeda Chemical Industries, Osaka, Japan), were used to study their effects on PAI-1 expression. Growth-arrested confluent HPTECs were treated with 15d-PGJ2 (0.05, 0.1, 1 or 5 µM) or pioglitazone (3 µM) for 30 min before being stimulated and during 24 to 48 h of exposure to hypoxia and/or TNF-{alpha}. To examine whether or not the PAI-1-modulating effect of 15d-PGJ2 is PPAR-{gamma}-dependent, growth-arrested cells were treated with GW9662 (2.5 µM), a specific inhibitor for PPAR-{gamma} (Alexis Biochemicals Corp., San Diego, CA, USA), for 6 h before and during 24 h of treatment with 15d-PGJ2. The GW9662 concentration of 2.5 µM was previously reported to be effective to block PPAR-{gamma} activation in HPTECs [27] and several types of tumour cells [28].

To study the effects of inhibiting tyrosine kinase (TK) or mitogen-activated protein (MAP) kinase on 15d-PGJ2-induced PAI-1 production, genistein (a TK inhibitor) and PD98059 (an MAP kinase kinase (MEK1) inhibitor) were used, respectively. Growth-arrested confluent HPTECs were treated with genistein (25 µM) or PD98059 (25 µM) for 2 h and 30 min, respectively, and during 24 h of treatment with 15d-PGJ2. As 15d-PGJ2, pioglitazone, GW9662, genistein and PD98059 were dissolved in 0.1% dimethyl sulfoxide (DMSO), the vehicle (0.1% DMSO) was added to control samples. Treatment with 15d-PGJ2 (up to 5 µM), pioglitazone (3 µM), GW9662 (2.5 µM), PD98059 (25 µM), genistein (25 µM) or the vehicle (up to 0.1% DMSO) had no harmful influence on cell viability.

Determination of human PAI-1 antigen concentrations
Total concentrations of PAI-1 in cell culture supernatants were measured by immunoassay using a commercial analyser (LPIA 200; Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) and a commercially available kit (LPIA-tPAI TEST; Mitsubishi Kagaku Iatron Inc.) according to a method reported recently [29]. This method had a detection limit of 1.5 ng/mL. The intra- and inter-assay coefficients of variation were 1.4% and 5.8%, respectively.

TaqMan real-time PCR assay
Total RNA was extracted from cultured cells using Trizol Reagent (Invitrogen Corp.) and phenol/chloroform according to the manufacturer's instructions. The cDNA synthesis was performed with a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA) following the protocol supplied. Reverse transcription was carried out in a 60-µl volume containing about 2 µg of purified total RNA, 6 µl of 10 x RT buffer, 2.4 µl of 25 x NTPs, 6 µl of 10 x random primers and 150 U of MultiScribe reverse transcriptase at 25°C for 10 min and then at 37°C for 2 h. The TaqMan real-time RT-PCR was performed with a TaqMan ABI 7000 sequence detection system (Applied Biosystems) using TaqMan universal PCR master mix (Applied Biosystems). Unlabelled specific primers and the TaqMan MGB probes (6-FAM dye-labelled) were purchased from Applied Biosystems for detecting the human PAI-1 gene (Assay ID: Hs00167155_m1), and human PPAR-{gamma} gene (Assay ID: Hs00234592_m1). A TaqMan human β-actin MGB (6-FAM dye-labelled) control reagent kit (Applied Biosystems, Accession No: NM_001101 [GenBank] ) was used to detect human β-actin. After an initial 2 min at 50°C and 10 min at 95°C, the samples were cycled 40 times at 95°C for 15 s and 60°C for 1 min. PAI-1, or PPAR-{gamma}, and β-actin cDNA templates were quantified separately using standard curves of diluted standard cDNA. Here, the threshold cycle of each sample corresponded to a dilution of the standard cDNA (arbitrary units). For quantitative analysis, the PAI-1 or PPAR-{gamma} cDNA content of each sample was normalized to the levels of β-actin as the housekeeping gene. All TaqMan real-time RT-PCR data were captured using Sequence Detector Software (SDS version 1.1, Applied Biosystems).

Immunoblot analysis
Cells were lysed in the lysis buffer (50 mM Tris–HCl pH 6.8, and 2% SDS). Twenty micrograms of protein was separated on an 8% SDS–PAGE gel and then electrophoretically transferred to nitrocellulose membranes (Trans-Blot SD; BioRad, Hercules, CA, USA). The membranes were blocked overnight with 5% skim milk powder in Tris-buffered saline, pH 8.0, containing 0.05% Tween-20 (TBS-T) and then incubated in a 1:100 dilution (5% skim milk in TBS-T) of a primary polyclonal rabbit antibody against human PPAR-{gamma} (Santa Cruz Laboratories Inc., Carlsbad, CA, USA) for 2 h at room temperature. Next, the membranes were incubated for 20 min at 37°C in a 1:1000 dilution (5% skim milk in TBS-T) of a goat anti-rabbit immunoglobulin antibody conjugated with horseradish peroxidase (Dako, Glostrup, Denmark). Finally, the secondary antibody was visualized using enhanced chemiluminescence (Amersham Life Science, Buckinghamshire, England).

Transient transfection of a PPAR response element-luciferase expression vector
PPAR-{gamma}-induced transcriptional activity was evaluated by transient transfection of an expression vector containing four copies of a consensus PPAR response element (PPRE) placed upstream from the TK-luciferase reporter (pPPRE-TK-Luc), which was generated previously [28]. HPTECs were seeded in a 24-well plate at a density of 5 x 104 cells/well and incubated in REGM without antibiotics. The next day, the cells were transfected with 100 ng of pRL-TK control vector and 700 ng of pPPRE-TK-Luc using lipofectamine 2000 (Invitrogen Corp.) and OPTI-MEN (Invitrogen Corp.). Six hours later, the transfection medium was replaced with DMEM containing no PGJ2 or 5 µM PGJ2, and then placed in a humidified atmosphere of 5% CO2 and 95% air at 37°C for 24 h. Finally, the cells were lysed in the lysis buffer, and firefly luciferase and renilla luciferase activities in the lysates were determined with a luminometer using the dual-luciferase reporter assay kit (Promega Corp., WI, USA).

Statistical analyses
All samples were run in triplicate, and the results were presented as the mean and standard deviation (± SD). Experiments in triplicate were performed three or four times unless otherwise stated. The unpaired t-test was used to evaluate the significance of differences between two groups of experiments. The analysis of covariance with Fischer's post hoc test was used for multiple comparisons. A two-tailed P value <0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Stimulatory effects of 15d-PGJ2, but not piogolitazone, on PAI-1 production in HPTECs
First, we examined the effect of 15d-PGJ2 on PAI-1 production in HPTECs under normoxia for 24 h (basal conditions). The treatment increased PAI-1 mRNA amounts by 1.4-fold at 1 µM 15d-PGJ2 and by 2.1-fold at 5 µM 15d-PGJ2 compared with no treatment (Figure 1A). It also increased amounts of secreted PAI-1 protein by 1.4-fold at 1 µM 15d-PGJ2 and by 1.6-fold at 5 µM 15d-PGJ2 (Figure 1B). The treatment with 0.05 or 0.1 µM 15d-PGJ2 did not significantly increase PAI-1 expression at mRNA or protein levels (data for 0.05 µM not shown, data for 0.1 µM shown in Figure 1). Figure 2 shows the time course of PAI-1 expression during the treatment with PGJ2 (5 µM) for up to 48 h. The PAI-1 mRNA levels in the 15d-PGJ2-treated cells were increased by 1.8-fold at 6 h, by 3.8-fold at 24 h and by 2.8-fold at 48 h compared with those in the untreated cells at 6 h (Figure 2A). The PAI-1 protein secretion in the treated cells was increased by 1.2-fold at 6 h, by 4.4-fold at 24 h and by 7.1-fold at 48 h compared with that in the untreated cells at 6 h (Figure 2B). Treatment with pioglitazone for 24 h had no influence on PAI-1 at the mRNA (1.1 ± 0.2 versus 1.0 ± 0.2, NS) or protein (19.9 ± 0.8 versus 19.0 ± 1.0 ng/mL, NS) level compared with no treatment. Additionally, treatment with 15d-PGJ2 (up to 5 µM) or pioglitazone (3 µM) for up to 48 h had no harmful influence on HPTECs as assessed based on morphological appearance and the amount of LDH released in the medium. These findings indicated that 15d-PGJ2 increased basal PAI-1 production in HPTECs in a dose-dependent manner while pioglitazone had no effect on PAI-1 production.


Figure 1
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  		Fig. 1 Production of PAI-1 is increased by 15d-PGJ2 in a dose-dependent manner in human proximal tubular epithelial cells (HPTECs) under normoxia. HPTECs were incubated with DMEM containing no 15d-PGJ2 (control), 0.1 µM 15d-PGJ2, 1 µM 15d-PGJ2 or 5 µM 15d-PGJ2 for 24 h under normoxic conditions. (A) The cDNA was prepared from total cellular RNA extracted from treated HPTECs, and TaqMan real-time PCR for PAI-1 and β-actin was performed. PAI-1 mRNA amounts were normalized to β-actin levels. Averaged amounts of PAI-1 mRNA for the control cells at 24 h were set to 1.0. (B) Supernatants were also harvested at 24 h, and assessed for PAI-1 protein by immunoassay. Results were expressed as the mean ± SD of three experiments done in triplicate (n = 3). *P < 0.05 and **P < 0.001 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Fischer's post hoc comparison.

 

Figure 2
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  		Fig. 2 PAI-1 production by human proximal tubular epithelial cells (HPTECs) under normoxic conditions for 6, 24 and 48 h in the absence ({square}) or presence (Figure 2) of 15d-PGJ2 (5 µM). (A) The 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 levels. Averaged amounts of PAI-1 mRNA for the normoxic cells untreated at 6 h 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 three experiments done in triplicate (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.005 compared with cells under the indicated conditions, by the unpaired t-test.

 
Potentiating effects of 15d-PGJ2 on hypoxia-stimulated, TNF-{alpha}-stimulated, TGF-β-stimulated and hypoxia plus TNF-{alpha} -stimulated PAI-1 production in HPTECs
We previously reported that hypoxia or TNF-{alpha} alone increased PAI-1 expression in HPTECs and that their combination produced a further increase in PAI-1 production [12]. Therefore, we next examined whether or not 15d-PGJ2 can enhance the PAI-1-increasing effects of hypoxia and TNF-{alpha} in HPTECs. Under normoxic conditions or under hypoxic conditions, HPTECs were treated with 5 µM 15d-PGJ2 for 24 h in the presence or absence of TNF-{alpha}. The data for PAI-1 expression under all the conditions are shown in Figures 3 and 4. This experiment clearly reconfirmed the PAI-1-increasing effects of hypoxia, TNF-{alpha}, and their combination found in our previous study [12]. As a new remarkable finding, 15d-PGJ2 (5 µM) further increased TNF-{alpha}-stimulated PAI-1 production by 1.4-fold at the mRNA level and by 1.3-fold at the protein level under normoxic conditions (Figures 3 and 4). Even under hypoxic conditions, 15d-PGJ2 significantly enhanced hypoxia-stimulated PAI-1 production by 1.9-fold at the mRNA level and by 1.3-fold at the protein level. Moreover, it was also proved that 15d-PGJ2 further augmented PAI-1 production stimulated by a combination of hypoxia and TNF-{alpha}, by 1.9-fold at the mRNA level and by 1.4-fold at the protein level (Figures 3 and 4). Additionally, we also examined the effects of 15d-PGJ2 on TGF-β-modulated PAI-1 production (Figure 5). PAI-1 protein production was significantly increased by TGF-β (5 ng/mL). Also, 15d-PGJ2 further increased the TGF-β-induced PAI-1 production by 1.4-fold (Figure 5). These novel findings indicated that the endogenously occurring PPAR agonist, 15d-PGJ2, was able to further potentiate PAI-1 production stimulated in HPTECs under hypoxic and/or inflammatory conditions.


Figure 3
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  		Fig. 3 In human proximal cells (HPTECs), 15d-PGJ2 potentiates hypoxia-stimulated, TNF-{alpha}-stimulated and hypoxia plus TNF-{alpha}-stimulated PAI-1 mRNA expression in human proximal tubular epithelial cells (HPTECs). HPTECs were incubated with DMEM containing no 15d-PGJ2 or 5 µM 15d-PGJ2 in the absence or presence of TNF-{alpha} (10 ng/mL) under normoxia or hypoxia for 24 h. The cDNA was prepared from total cellular RNA extracted from the cells at 24 h, and TaqMan real-time PCR for PAI-1 and β-actin was performed. PAI-1 mRNA amounts were normalized to β-actin levels. Average amounts of PAI-1 mRNA in the untreated normoxic cells were set to 1.0. Results were expressed as the mean ± SD of four experiments done in triplicate (n = 4). *P < 0.01, **P < 0.005 and ***P < 0.001 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Fischer's post hoc comparison. +P < 0.001 compared with cells under the same conditions at normoxia, by the unpaired t-test.

 

Figure 4
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  		Fig. 4 In human proximal tubular epithelial cells, 15d-PGJ2 potentiates hypoxia-stimulated, TNF-{alpha}-stimulated and hypoxia plus TNF-{alpha}-stimulated PAI-1 protein production in human proximal tubular epithelial cells (HPTECs). HPTECs were incubated with DMEM containing no 15d-PGJ2 or 5 µM 15d-PGJ2 in the absence or presence of TNF-{alpha} (10 ng/mL) under normoxia or hypoxia for 24 h. Supernatants were harvested at 24 h and assessed for PAI-1 protein by immunoassay. Average amounts of PAI-1 protein in the untreated normoxic cells were set to 1.0. Results are expressed as the mean ± SD of four experiments done in triplicate (n = 4). *P < 0.01 and **P < 0.001 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Fischer's post hoc comparison. +P < 0.001 compared with cells under the same conditions at normoxia, by the unpaired t-test.

 

Figure 5
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  		Fig. 5 In human proximal tubular epithelial cells, 15d-PGJ2 potentiates TNF-β-stimulated PAI-1 protein production in human proximal tubular epithelial cells (HPTECs). HPTECs were incubated with DMEM containing no 15d-PGJ2 or 5 µM 15d-PGJ2 in the absence or presence of TNF-β (5 ng/mL) under normoxia for 24 h. Supernatants were harvested at 24 h and assessed for PAI-1 protein by immunoassay. Average amounts of PAI-1 protein in the untreated cells were set to 1.0. Results are expressed as the mean ± SD of three experiments done in triplicate (n = 3). *P < 0.01 and **P < 0.001 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Fischer's post hoc comparison.

 
Activation of PPAR-{gamma} by 15d-PGJ2 in HPTECs
In order to examine whether or not 15d-PGJ2 can actually activate PPAR-{gamma} in HPTECs, the cells were first analysed for PPAR-{gamma} mRNA and protein. No significant differences in the amounts of PPAR-{gamma} mRNA and protein were found between HPTECs treated with and without 15d-PGJ2 for 24 h (Figure 6A and B). However, considerable amounts of PPAR-{gamma} protein (about 50 kDa) were observed in the cell lysates with or without 15d-PGJ2 treatment (Figure 6B). Next, HPTECs were transfected with the PPRE-TK-Luc expression vector, treated with 15d-PGJ2 (5 µM) for 24 h, and then analysed for luciferase activity as PPRE-mediated transcriptional activity, i.e. specific PPAR-{gamma} activity. As shown in Figure 6C, treatment with 15d-PGJ2 enhanced PPRE-mediated transcriptional activity by 3.1-fold compared with that without 15d-PGJ2 treatment. From these findings, we were able to confirm the presence of a functioning PPAR-{gamma} protein and its actual activation by 15d-PGJ2 in HPTECs.


Figure 6
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  		Fig. 6 PPAR-{gamma}-dependent transcription is activated by 15d-PGJ2 in human proximal tubular epithelial cells (HPTECs) under normoxia. (A) HPTECs were treated with no 15d-PGJ2 or 5 µM 15d-PGJ2 for 24 h. The cDNA was prepared from total cellular RNA extracted from the cells at 24 h, and TaqMan real-time PCR for PPAR-{gamma} and β-actin was performed. PPAR-{gamma} mRNA amounts were normalized to β-actin levels. Averaged amounts of PPAR-{gamma} mRNA in the untreated cells were set to 1.0. Results are expressed as the mean ± SD of three experiments done in triplicate (n = 3). NS, not significant, compared with the untreated cells, by the unpaired t-test. (B) HPTECs were treated with no 15d-PGJ2 or 5 µM 15d-PGJ2 for 24 h. Whole cell lysates were prepared from HPTECs at 24 h and assessed for PPAR-{gamma} protein by immunoblotting. Results shown are from one experiment representative of two performed. (C) HPTECs were transfected with PPRE-TK-Luc. The cells then were incubated without or with 5 µM 15d-PGJ2 for 24 h. Firefly luciferase activities were determined relative to those of the untreated cells. Results are expressed as the mean ± SD of three experiments done in triplicate (n = 3). *P < 0.001 compared with the untreated cells by the unpaired t-test.

 
PPAR-{gamma} independence of 15d-PGJ2-induced PAI-1 production
Considering the earlier reports that 15d-PGJ2's effects were mediated not only in a PPAR-{gamma}-dependent manner but also in an independent one, we attempted to clarify the mechanism underlying the 15d-PGJ2-induced PAI-1 production by pre-treatment with a specific inhibitor for PPAR-{gamma}, GW9662 (2.5 µM), a non-selective TK inhibitor, genistein (25 µM) or an MEK1 (MAPK kinase) inhibitor, PD98059 (25 µM). The effects of 15d-PGJ2 on PAI-1 production were evaluated under normoxic conditions in the absence or presence of these inhibitors. In the absence of GW9662, 15d-PGJ2 (5 µM) increased PAI-1 protein secretion by 2.1-fold compared with that of untreated cells, while in the presence of GW9662, it also increased PAI-1 protein secretion by 2.1-fold (Figure 7A). Therefore, GW9662 treatment had no influence on the PAI-1-increasing effect of 15d-PGJ2. In contrast, treatment with genistein (Figure 7B) or PD98059 (data not shown) abolished the PAI-1-increasing effect of 15d-PGJ2 completely. These findings indicated that 15d-PGJ2 increased PAI-1 production through activation of TK or MAPK kinase in a PPAR-{gamma}-independent manner.


Figure 7
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  		Fig. 7 Genistein, but not GW9662 at all, inhibits the PAI-1-increasing effects of 15d-PGJ2 completely in human proximal tubular epithelial cells (HPTECs). HPTECs were incubated with DMEM containing no 15d-PGJ2 or 5 µM 15d-PGJ2 for 24 h. (A) The cells were treated without GW9662 or 2.5 µM GW9662 for 6 h before and during the 15d-PGJ2 treatment. (B) The cells were treated without genistein or 25-µM genistein for 2 h before and during the 15d-PGJ2 treatment. Supernatants were harvested at 24 h and assessed for PAI-1 protein by immunoassay. Averaged amounts of PAI-1 protein for the untreated cells were set to 100%. Results are expressed as the mean ± SD of three experiments done in triplicate (n = 3). NS, not significant, *P < 0.001 compared with cells incubated under the indicated conditions, by the unpaired t-test.

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Here, we reported for the first time that the naturally occurring PPAR-{gamma} agonist, 15d-PGJ2, not only increased basal PAI-1 production but also augmented hypoxia-stimulated, TNF-{alpha}-stimulated and hypoxia plus TNF-{alpha}-stimulated PAI-1 production in HPTECs. We also found that 15d-PGJ2 increased PAI-1 production independently of PPAR-{gamma} probably through TK and MAPK kinase pathways in HPTECs although 15d-PGJ2 was able to enhance PPRE-mediated transcriptional activity in the cells.

Local overproduction of PAI-1, a major in vivo inhibitor of plasminogen activators, prevents plasmin from being generated in the neighbouring area of renal tissues [1]. This reduction in the level of plasmin probably results in an accumulation of intraglomerular and interstitial ECM, leading to renal fibrosis, because plasmin not only degrades the matrix proteins, fibronectin and type IV collagen but also activates metalloproteinases (MMPs) that enzymatically cleave collagenous proteins including type I and III collagen [1]. Recently, new possible roles for PAI-1 in renal fibrosis have been proposed in several experiments using PAI-1 knockout mice [7,8,30]. PAI-1 may serve as an inducer of monocyte recruitment [7,30] and a stimulator of TGF-β expression [8] as well as an inhibitor for ECM degradation. Considering the close association between PAI-1 production and renal fibrosis, it is worth mentioning that 15d-PGJ2, a naturally occurring prostaglandin, increases PAI-1 production in HPTECs as a main component of renal parenchyma.

There have so far been only a few studies investigating the effect of 15d-PGJ2 on PAI-1 expression in cultured cells [19,20,24,31]. The effect on PAI-1 production appears to be cell specific and bidirectional. In human endothelial cells and fibroblasts, 15d-PGJ2 up-regulates PAI-1 mRNA and protein expression [19,20,31] while it down-regulates PAI-1 expression in human and mouse mesangial cells [24]. The effect of 15d-PGJ2 in human fibroblasts was dependent on PPAR-{gamma} expression [19], but that in the endothelial cells still remains to be analysed for PPAR-{gamma} dependence [20,31]. The reduction caused by 15d-PGJ2 in mesangial cells was attributable to a PPAR-{gamma}-dependent expression of hepatocyte growth factor [24]. In the current study, we found that the PAI-1-increasing effect of 15d-PGJ2 in HPTECs occurred independently of PPAR-{gamma} and probably via the activation of TK or MAPK kinase (Figure 7) although a functioning PPAR-{gamma} was actually expressed and 15d-PGJ2 was capable of activating PPAR-{gamma} in the cells (Figure 6). A recent study on vascular smooth muscle cells showed that 15d-PGJ2 activated MAPK (ERK) kinase and ensuing c-fos expression [32]. Furthermore, TK or MAPK kinase was reported to mediate PAI-1's induction by TGF-β [33] or hypoxia [12,34]. Taking these recent findings into consideration, it seems highly plausible that 15d-PGJ2-induced PAI-1 production in HPTECs is mediated via activation of TK or MAPK kinase although further detailed experiments are required to confirm the actual activation of signalling proteins in each kinase pathway.

PPAR-{gamma}-independent PAI-1 induction by 15d-PGJ2 may be explained by direct chemical modification of proteins by its cyclopentenone ring which can covalently bind to bioactive proteins via its electrophilic carbon [15,16]. In our preliminary study, however, another cyclopentenone prostaglandin, PGA2, which can also activate PPAR-{gamma} [15,35,36], did not induce PAI-1 expression significantly. As for the effect of PPAR-{gamma}'s activation on PAI-1 production in HPTECs, pioglitazone had little influence on the production, supporting an earlier report regarding human adipocytes that specific PPAR-{gamma} activators, thiazolidinediones including pioglitazone, had no effect on PAI-1 production [23]. In human proximal renal tubular cell line (HK-2 cells), pioglitazone was reported to reduce TGF-β-stimulated PAI-1 production [37], while in the current study, 15d-PGJ2 further enhanced TGF-β-stimulated PAI-1 production in HPTECs. These findings also suggest that the PAI-1-increasing effects of 15d-PGJ2 in HPTECs may be independent of PPAR-{gamma} although involvement of a cyclopentenone ring in the PAI-1's production is not yet confirmed in the current study.

One of the most notable findings in the current study is that 15d-PGJ2 further enhances PAI-1 production stimulated by hypoxia, TNF-{alpha} and their combination. We previously reported the synergistic effect of hypoxia and TNF-{alpha} on PAI-1 production in HPTECs [12]. Hypoxia-stimulated PAI-1 production was mediated in part by induction of hypoxia-inducible factor-1{alpha} and activation of TK [12]. Chronic hypoxia has recently been proposed as a common mechanism of renal fibrosis [13]. In the early phase of renal injury, relative hypoxia may occur in renal tubular cells due to decreased peritubular capillary blood flow as a result of imbalance of vasoactive substances and increased tubular metabolic demand, whereas in the advanced phase, absolute hypoxia in tubular cells may be induced by loss of peritubular capillary and decreased oxygen diffusion due to renal fibrosis. Local inflammation and recruitment of macrophages, a major producer of TNF-{alpha}, also occur frequently in damaged and hypoxic tissues [13,38]. Therefore, hypoxia and inflammatory reactions must coexist during the advancement of kidney disease. More recently, hypoxia and inflammation were reported to increase 15d-PGJ2 production in microvascular pericytes [25] and Kupffer cells [26]. These results raise the possibility that 15d-PGJ2 may increase in amount and may advance renal fibrosis as an enhancer of PAI-1 in the diseased kidney under hypoxic and inflammatory conditions. Detailed in vivo studies are, however, needed to clarify the precise in vivo role of 15d-PGJ2 in the progression of renal injury, a pro-fibrotic or anti-fibrotic role as a whole, since 15d-PGJ2 is well known to exert an anti-inflammatory effect in renal cells [15,17,27].

Finally, it should be considered that the PGJ2 concentrations required to enhance PAI-1 production in HPTECs were 1 µM or more, which appeared to be much higher than physical levels recently reported in liquid samples of humans [39]. Covalent binding of 15d-PGJ2 to proteins may cause the lower concentrations of unbound and free 15d-PGJ2 in the fluid samples. As mentioned above, hypoxia and inflammation may increase 15d-PGJ2 production. However, it is essential to clarify precise actual amounts of 15d-PGJ2 produced in normal or injured local tissues.

In conclusion, the naturally occurring PPAR-{gamma} agonist, 15d-PGJ2 increased PAI-1 production independently of PPAR-{gamma} probably through TK and MAP kinase in HPTECs although 15d-PGJ2 was actually able to activate PPAR-{gamma} in the cells. Also, 15d-PGJ2 augmented PAI-1 production stimulated by hypoxia, TNF-{alpha} and their combination in HPTECs. These results suggest that 15d-PGJ2 may serve as an enhancer of PAI-1 in the diseased kidney under hypoxic and inflammatory conditions.



   Acknowledgments
 
This study was supported by Grants-in-Aid for Scientific Research (c) (no. 17590823, no. 17590487 and no. 18570104) from the Japan Society for the Promotion of Science. Portions of this study were presented at the American Society of Nephrology Meeting, San Diego, CA, USA, November 2006. The authors are very thankful to Ms Kazuko Kamiyama (Division of Nephrology, University of Fukui) for greatly valued technical assistance.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 1. 7.07
Accepted in revised form: 20. 2.08


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