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NDT Advance Access published online on January 25, 2007
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfl766
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Hypoxia reduces the expression and anti-inflammatory effects of peroxisome proliferator-activated receptor-{gamma} in human proximal renal tubular cells

Xuan Li1, Hideki Kimura1, Kiichi Hirota2, Hidehiro Sugimoto1, Noriyo Kimura1, Naoki Takahashi1, Hiroshi Fujii3 and Haruyoshi Yoshida1

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

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 Matsuokashimoaizuki, Eiheiji-cho, Yoshida, Fukui 910-1193, Japan. Email: hkimura{at}fmsrsa.fukui-med.ac.jp



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Peroxisome proliferator-activated receptor (PPAR)-{gamma} may counteract tissue fibrosis via its anti-inflammatory actions, while hypoxia, a new pro-fibrotic factor, reportedly modifies PPAR-{gamma} expression. However, the effects of hypoxia on the expression and anti-inflammatory actions of PPAR-{gamma} have yet remained to be clarified in renal tubular cells.

Methods. Confluent human proximal renal tubular epithelial cells (HPTECs) were exposed to hypoxia (1% O2) and/or TNF-{alpha} at 10 ng/ml for up to 48 h. The cells were incubated with PPAR-{gamma} agonists, 15d-PGJ2 or pioglitazone, for 30 min before stimulation. Precise amounts of PPAR-{gamma} and MCP-1 mRNA and protein were measured by TaqMan quantitative PCR and immunoblot or ELISA, respectively.

Results. A cDNA array analysis identified PPAR-{gamma} as one of the hypoxia-affected genes in HPTECs. Hypoxia reduced mRNA levels of PPAR-{gamma} at 24 and 48 h and protein levels at 6 and 48 h. Knockout of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) with its dominant negative form did not block the hypoxia-induced reduction in PPAR-{gamma} expression. PPAR-{gamma}'s activation with 15d-PGJ2 or pioglitazone reduced basal and TNF-{alpha}-stimulated MCP-1 expression at mRNA and protein levels at 24 h under normoxia. MCP-1 reduction rates at basal mRNA and protein levels were slightly but significantly lower during hypoxia than normoxia (9 vs 69% and 36 vs 42%, respectively, for 15d-PGJ2, and 0 vs 34% and 12 vs 21%, respectively, for pioglitazone). Finally, a specific inhibitor for PPAR-{gamma}, GW9662, weakened the MCP-1-decreasing effect of 15d-PGJ2 by about 30%, under basal conditions, while it abolished the effect of pioglitazone almost completely.

Conclusions. Hypoxia-induced loss of function of PPAR-{gamma} reduces anti-inflammatory effects of PPAR-{gamma} activation, possibly modulating inflammatory responses in the diseased kidney.

Keywords: HIF-1{alpha}; human proximal tubular cells; hypoxia, MCP-1; PPAR; TNF-{alpha}



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chronic hypoxia has recently been proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various renal diseases regardless of their underlying cause [1]. Reduced post-glomerular circulation due to glomerulosclerosis or an imbalanced constriction of efferent arterioles in glomeruli should cause a substantial reduction in the blood flow and oxygen delivery into the interstitial capillary network and eventually lead to local severe hypoxia in the tubulointerstitium [1]. The cellular response to hypoxia is stringently mediated by the transcription factor, hypoxia-inducible factor 1 (HIF-1), which is a heterodimer protein composed of HIF-1{alpha} and HIF-1ß subunits and activates the transcription of many genes containing a hypoxia response element (HRE) in the promoter region [2]. HIF-1{alpha} is a pO2-sensitive partner, as it is accumulated under hypoxia and unstable under normoxia due to a pO2-dependent degradation involving a ubiqutin-proteasomal pathway [3].

Peroxisome proliferator-activated receptors (PPARs) are a nuclear hormone receptor superfamily of ligand-dependent transcription factors [4]. Three different subtypes designated PPAR-{alpha}, PPAR-ß/{delta} and PPAR-{gamma} exist, characterized by distinct expression patterns, different ligand-binding specificity, and biological functions. These PPARs form heterodimers with the 9-cis retinoic acid receptor, RXR-{alpha}, bind to characteristic DNA sequences termed peroxisome proliferator response elements (PPRE) located in the promoter region of target genes, and exert specific biological actions. PPAR-{gamma} plays a pivotal role in the regulation of adipogenesis and insulin sensitivity, whereas PPAR-{alpha} and PPAR-ß/{delta} are implicated primarily in lipid metabolism and in the control of cell proliferation and differentiation [4]. The transcriptional power of PPARs is selectively promoted by a variety of ligands according to their binding characteristics. PPAR-{alpha} is the molecular target of endogenous long-chain polyunsaturated fatty acids and exogenous hypolipidaemic fibrates. The most potent natural agonist for PPAR-{gamma} is the endogenous prostaglandin D2 metabolite, 15-deoxy-delta12, 14-prostaglandin J2 (15d-PGJ2). Synthetic agonists, anti-diabetic thiazolidinediones (TZDs) including pioglitazone, also activate PPAR-{gamma} by its binding to the receptor with high affinity. As for PPAR-{gamma}'s functions, accumulating evidence indicates that TZDs not only improve insulin sensitivity and glucose metabolism but also possess anti-inflammatory potential that results in a reduction in pro-inflammatory cytokine expression [5]. PPAR-{gamma} agonists were recently shown to reduce tumour necrosis factor (TNF)-{alpha}, interleukin (IL)-1 and IL-6 production in monocytes or vascular endothelial cells [6]. More recently, in several types of cultured renal cells, anti-inflammatory actions of PPAR-{gamma} agonists were also evaluated [7–9]. TZDs and endogenous agonists suppressed the expression of transforming growth factor (TGF)-ß and monocyte chemoattractant peptide (MCP)-1 in human renal fibroblasts or a cell line of human proximal renal tubules [7–9] and antagonized the proinflammatory actions of TGF-ß in mesangial cells [10].

Interestingly, a few recent studies reported a hypoxia-induced reduction in the expression and function of PPARs [11,12]. Hypoxia abolished PPAR-{alpha} expression in intestinal epithelial cells [11], while actual and chemical hypoxia inhibited PPAR-{gamma} induction and adipogenesis in embryonic fibroblasts and pre-adipocytes receiving adipogenic stimulation [12]. Taking these earlier findings into account, we can hypothesize that hypoxia, an unavoidable milieu occurring during renal fibrosis progression, may influence PPAR expression and then modulate the response to inflammation in renal tubular cells. However, no studies have so far been conducted on the hypoxic effects on PPAR expression and function, especially its anti-inflammatory effect, in cultured renal cells.

In the current study, we investigated how hypoxia regulates the expression and anti-inflammatory actions of PPAR-{gamma} in cultured human proximal renal tubular epithelial cells (HPTECs).



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Tubular cell cultures
HPTECs were purchased as twice-passaged cells from Clonetics, Inc. (San Diego, CA, USA). The cells were grown in 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). HPTECs (passage 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 (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. O2 concentrations in the chamber were kept at ~1% for 48 h, which was confirmed by an oximeter placed in the chamber. There was no significant difference in LDH release into the medium between the cells treated with hypoxia and those untreated with hypoxia for 48 h (8.6 ± 1.0 vs 8.0 ± 1.2 U/l, NS, n = 6). There was no significant difference in values of pH of the cell culture media between treatments with normoxia and hypoxia for 48 h (data not shown).

To study the effects of proinflammatory cytokines on MCP-1 expression, recombinant human TNF-{alpha} (Invitrogen Corp.) as a representative inflammatory cytokine was added to the medium at a final concentration of 10 ng/ml for various periods of time. To assess the effects of free radical oxygen on PPAR-{gamma} expression, a free radical scavenger, 4-hydroxyl tetramethylpiperidine-1-oxyl (Tempol, Calbiochem, San Diego, CA, USA) was used [13]. Growth-arrested confluent HPTECs were treated with Tempol (0.5 mM) for 6 h before being stimulated and during 24 h of exposure to hypoxia. To study the effect of the activation of PPAR-{gamma} on MCP-1 expression, PPAR-{gamma} agonists, 15d-PGJ2 (Calbiochem, San Diego, CA, USA) and pioglitazone (a gift from Takeda Chemical Industries, Osaka, Japan), were used. Growth-arrested confluent HPTECs were treated with 15d-PGJ2 (5 or 10 µM) or pioglitazone (3 µM) for 30 min before being stimulated and during 24 h of exposure to hypoxia and/or TNF-{alpha}. To study the actual PPAR-{gamma}-dependence of the MCP-1-modulating effects induced by the PPAR-{gamma} agonists during normoxia, growth-arrested cells were pre-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 the agonists. As 15d-PGJ2, pioglitazone and GW9662, were dissolved in 0.1% dimethyl sulfoxide (DMSO), the vehicle (0.1% DMSO) was added to control samples. Tempol was dissolved in distilled water. Treatment with Tempol (0.5 mM), 15d-PGJ2 (up to 10 µM), pioglitazone (3 µM), GW9662 (2.5 µM), or the vehicle (up to 0.1% DMSO) had no harmful influence on cell viability.

cDNA array analysis
The cDNA array analysis was performed as reported previously [14], using a commercially available kit, the GeneNavigator cDNA Array System (Toyobo Co., Osaka, Japan), according to the manufacturer's instructions. Briefly, HPTECs grown in T-175 flasks were incubated in DMEM with 0.5% FBS under normoxic (18% O2) or hypoxic (1% O2) conditions for 48 h. Then, mRNA was prepared for hybridization to a cDNA array filter, human immunology (Toyobo Co.). The cDNA probes synthesized from the mRNAs were labelled with biotin by the incorporation of biotin-16-deoxyuracil triphosphate (dUTP) during the amplification of cDNA. The biotin-labelled probes were hybridized to the cDNA array filters at 68°C overnight. After a standard post-hybridization wash, specific signals on the filters were detected with an Imaging high Chemiluminescence detection kit (Toyobo) according to the manufacturer's recommendations. The filter was scanned with the FAS 1000 system (Toyobo) and the resulting signals were quantified by measuring their intensity using an Array-Pro Analyzer (Media Cybernetics, MA, USA). A relative intensity greater than the averaged values for the pUC and luciferase signals was considered to be suitable for statistical analysis. A change in gene expression was considered significant only if the ratio between hypoxic and normoxic samples at 48 h was >3.0 or less than one-third.

Determination of human MCP-1 antigen concentrations
Concentrations of MCP-1 in cell culture supernatants were measured by immunoassay using a commercially available ELISA kit (Quantikine Human MCP-1; R&D systems. Inc., MN, USA). Intra-assay and inter-assay coefficients of variation for same samples were below 4.9 and 4.6% according to the manufacturer's instructions.

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. 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 MCP-1 gene (Assay ID: Hs00234140_m1), human PPAR-{gamma} gene (Assay ID: Hs00234592_m1), and human glucose transporter 1 (Glut 1) gene (Assay ID: Hs00197884_m1). A TaqMan human ß-actin MGB (6-FAM dye-labelled) control reagent kit (Applied Biosystems, Accession No: NM_001101 [GenBank] ) was used to detect human ß-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. MCP-1, PPAR-{gamma} or Glut 1, and ß-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 MCP-1, PPAR-{gamma}, or Glut 1 cDNA content of each sample was normalized to the levels of ß-actin as the housekeeping gene. All TaqMan real-time RT-PCR data was 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 were separated on an 8% SDS- polyacrylamide gel (PAGE) and then electrophoretically transferred to nitrocellulose membranes (Trans-Blot SD; BioRad, Hercules, CA, USA). 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 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 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). Signal intensities for specific bands were quantified with a densitometer (Fluorochem; Alpha Innotech Corp. CA, USA).

For detection of MCP-1 antigen, the supernatants from HPTECs stimulated with TNF-{alpha} (10 ng/ml) for 24 h were 20-fold concentrated on a Centrifugal Filter Unit, Microcon YM-3 (Millipore Corp. MA, USA). A 15-µl portion of the concentrated samples was electrophoresed on a 10% SDS-PAGE. The primary monoclonal mouse antibody against human MCP-1 (R&D systems. Inc., MN, USA) and the secondary polyclonal rabbit anti- mouse immunoglobulins antibody conjugated horseradish peroxidase (Dako) were used in a 1:25 dilution and a 1:500 dilution, respectively. Recombinant human MCP-1 (R&D systems) was used as a positive control.

Transient transfection with an expression vector coding a dominant negative form of HIF-1{alpha}
To limit HIF-1{alpha} function, an expression vector encoding a dominant negative (DN) form of HIF-1{alpha} was used [15]. The DN form of HIF-1{alpha} can generate a biological inactive heterodimer with HIF-1ß which cannot bind HRE nor activate transcription, and therefore can compete with endogenous HIF-1{alpha} and HIF-2{alpha} for heterodimerization with HIF-1ß. HPTECs were seeded in a 12-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 an expression vector encoding a dominant negative (DN) form of HIF-1{alpha} [15] and 100 ng of an empty vector encoding no protein using lipofect-amine 2000 (Invitrogen Corp.) and OPTI-MEN (Invitrogen Corp.). Several hours later, the transfection medium was replaced with DMEM. For hypoxic treatment, the transfected cells were transferred to a MIC-101 modular incubator chamber (Billups-Rosenberg) which was flushed with 1% O2—5% CO2—94% N2, sealed and placed at 37.0°C for 24 h. For normoxic treatment, the cells were placed in a humidified atmosphere of 5% CO2 and 95% air at 37°C for 24 h. Then, the cells were harvested for quantification of PPAR-{gamma} or Glut 1 mRNA. To study transfection efficiency, the cells were transfected with an expression vector encoding green fluorescence protein (GFP) (Invitrogen Corp.).

Statistic analyses
All samples were run in triplicate, and the results are presented as the mean and standard deviation (±SD). Experiments in triplicate were performed at least two times, and results from typical experiments are shown in the figures. The unpaired t-test was used to evaluate the significance of differences between two groups of experiments. The analysis of covariance with Scheffe's post hoc test was used for multiple comparisons. A two-tailed P-value <0.05 was considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
cDNA array analysis for hypoxia-modulated gene expression of nuclear hormone receptors
First, we attempted to examine the hypoxia-induced changes to nuclear hormone receptor genes in HPTECs using a microarray. We used the human immunology filter (621 genes) containing retinoid X receptor (RXR) and farnesoid X receptor (FXR) genes as well as PPAR genes, because PPARs must form heterodimers with RXR-{alpha} to function as a transcription factor, and FXR has recently been reported to be a strong inducer for PPAR-{gamma} [16]. This cDNA array analysis identified PPAR-{gamma} as one of the genes whose expression is markedly down-regulated during hypoxia. After 48 h of hypoxia, PPAR-{gamma} gene expression was significantly and markedly decreased by 71% compared with that under normoxia, while PPAR-{alpha} and ß expression was reduced by only 30% (Table 1). RXR-{alpha} expression was also reduced by 32% and FXR expression was unchanged during hypoxia. The complete achievement of hypoxic conditions in the cultured cells was verified by a marked induction of glucose transporter-1 (Glut-1), a representative of the hypoxia-induced genes (Table 1). These findings encouraged us to focus next on detailed experiments clarifying how hypoxia reduces the expression and modulates the function of PPAR-{gamma}.


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  		Table 1. Hypoxia-induced changes in gene expression of nuclear hormone receptors in human proximal renal tubular epithelial cellsa

 
Effects of hypoxia on PPAR-{gamma} expression in HPTECs
Amounts of PPAR-{gamma} mRNA in the cells under hypoxia were significantly decreased by 57% at 24 h and by 80% at 48 h compared with those in the normoxic control cells at 24 h, while the amounts during normoxia at 48 h were increased by only 8% (Figure 1A). Immunoblot analysis revealed that the proteins reactive to the specific antibody for PPAR-{gamma} migrated as a single band with a molecular mass of 50 kDa, suggesting that the immunoreacted protein corresponded to PPAR-{gamma}1. Amounts of PPAR-{gamma} protein in the lysates from hypoxic cells were decreased by about 30% at 24 and 48 h compared with those of normoxic cells at the corresponding time points (Figure 1B).


Figure 1
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  		Fig. 1. PPAR-{gamma} production by HPTECs under normoxia (18% O2) or hypoxia (1% O2) for up to 48 h. (A) cDNA was prepared from total cellular RNA extracted from treated HPTECs at the indicated time points, and TaqMan real-time PCR for PPAR-{gamma} and ß-actin was performed. PPAR-{gamma} mRNA amounts were normalized to ß-actin levels. Averaged amounts of PPAR-{gamma} mRNA for cells during normoxia at 24 h were set to 1.0. Results were expressed as the mean ± SD of experiments done in triplicate (n = 6). *P < 0.01 and **P < 0.005 compared with the normoxic cells at each time point, by unpaired t-test. (B) Whole cell lysates were prepared from HPTECs under normoxia or hypoxia at the indicated time points, and assessed for PPAR-{gamma} protein by immunoblotting. Results shown are from one experiment representative of two performed.

 
Effects of a scavenger for oxygen-free radicals on hypoxic reduction of PPAR-{gamma} expression in HPTECs
A cell-permeable free radical scavenger, Tempol, was used to clarify the involvement of oxygen free radicals in the hypoxic reduction of PPAR-{gamma} expression. There were no significant differences in amounts of PPAR-{gamma} mRNA between the cells treated with and without Tempol under normoxia (1.0 ± 0.30 vs 0.87 ± 0.12%, NS) or under hypoxia (0.42 ± 0.19 vs 0.32 ± 0.08%, NS).

Effects of TNF-{alpha} and 15d-PGJ2 on PPAR-{gamma} expression in HPTECs
In order to examine how TNF-{alpha} and 15d-PGJ2 influence PPAR-{gamma} expression, the cells thus treated were analyzed for PPAR-{gamma} mRNA using real-time PCR. mRNA amounts after 24 h of treatment with TNF-{alpha} under normoxia were similar to those of untreated controls (102 ± 24 vs 100 ± 9%, NS). PPAR-{gamma} mRNA amounts after 24 h of treatment with 15d-PGJ2 (10 µM) under normoxia were also similar to those of untreated controls (94 ± 12 vs 100 ± 13%, NS).

Overexpression of a dominant negative HIF-1{alpha} form and its effect on PPAR-{gamma} expression in HPTECs
In order to study the relationship between HIF-1{alpha} and PPAR-{gamma} expression, the HPTECs were transfected with an empty vector encoding no protein (EV) or an expression vector encoding a DN form of HIF-1{alpha} (DN-V) that can inhibit the abilities of HIF-1{alpha} and HIF-2{alpha} to activate transcription by competing with them for heterodimerization with HIF-ß. Transfection efficiency measured by GFP-transfection of the cells was approximately 30% under normoxia (Figure not shown) or hypoxia (Figure 2). Therefore, the effect of DN-V transfection on PPAR-{gamma} expression was directly assessed by comparing amounts of PPAR-{gamma} mRNA between HPTECs transfected with EV and those transected with DN-V. The DN-V transfection, namely knockout of HIF-1{alpha} function, did not inhibit hypoxia-induced reduction of PPAR-{gamma} mRNA compared with the EV transfection (Figure 3A), while it significantly suppressed hypoxia-induced expression of glucose transporter 1 (Glut 1) mRNA compared with the EV transfection (Figure 3B). These results suggest that a non-HIF-1 pathway activated by hypoxia contributes partly to the hypoxia-induced reduction of PPAR-{gamma} mRNA expression.


Figure 2
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  		Fig. 2. A phase-contrast image (A, x20) and a green fluorescent image (B, x20) of HPTECs transfected with a GFP-expressing vector in the same field. HPTECs were transfected with an expression vector encoding GFP. After 6 h, the transfection medium was replaced with DMEM, and the cells were incubated under hypoxia for 24 h. Images were recorded on an Inverted System Microscope Olympus IX70 (Olympus, Tokyo, Japan).

 

Figure 3
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  		Fig. 3. Effect of a dominant negative form of HIF-1{alpha} on PPAR-{gamma} and Glut 1 mRNA expression in HPTECs under normoxia (18% O2) or hypoxia (1% O2). HPTECs were transfected with expression vectors encoding a dominant negative form of human HIF-1{alpha} (DN) or no protein (EV). After 6 h, the transfection medium was replaced with DMEM, and the cells were incubated under normoxia or hypoxia for 24 h and then harvested for the preparation of cDNA. TaqMan real-time PCR for PPAR-{gamma}, Glut 1 and ß-actin was performed. PPAR-{gamma} (A) and Glut 1 (B) mRNA amounts were normalized to ß-actin levels. Averaged amounts of PPAR-{gamma} or Glut 1 mRNA for cells during normoxia were set to 1.0. Results are expressed as the mean ± SD of experiments done in triplicate (n = 6). *P < 0.05 and NS (not significant) compared with cells incubated under the indicated conditions with the unpaired t-test.

 
Inhibitory effects of 15d-PGJ2 and pioglitazone on constitutive and TNF-{alpha}–stimulated expression of MCP-1 in HPTECs under normoxia
Immunoblot analysis of the concentrated supernatants from TNF-{alpha}-stimulated HPTECs proved that the cells produced and secreted MCP-1 proteins actually (Figure 4). Therefore, MCP-1 expression was evaluated by TaqMan real-time PCR assay for MCP-1 mRNA amounts and by ELISA for MCP-1 antigen amounts in the supernatants. A PPAR-{gamma} agonist, 15d-PGJ2, decreased basal MCP-1 expression at the mRNA and protein levels at 24 h under normoxia in a dose-dependent manner compared with untreated controls (Figure 5A and B). PPAR-{gamma} activation with 15d-PGJ2 (10 µM) also reduced TNF-{alpha}-stimulated MCP-1 expression at mRNA and protein levels to 40 ± 10% (P < 0.05) and 50 ± 8% (P < 0.05), respectively, of corresponding control values at 24 h during normoxia. A more specific PPAR-{gamma} agonist, pioglitazone (3 µM), decreased basal MCP-1 expression at mRNA and protein levels to 66 ± 3% (P < 0.05) and 80 ± 2% (P < 0.05), respectively, of control values at 24 h. Pioglitazone treatment for 24 h also caused a considerable decrease in TNF-{alpha}-stimulated MCP-1 mRNA levels to 55 ± 7% (P < 0.05) of control values, while it produced a slight and insignificant decrease in TNF-{alpha}-stimulated MCP-1 protein levels (96 ± 6% of control values). These findings proved that both PPAR-{gamma} activators, 15d-PGJ2 and pioglitazone, possessed an anti-inflammatory effect of decreasing basal and stimulated MCP-1 expression in HPTECs.


Figure 4
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  		Fig. 4. Immunoblot analysis of MCP-1 in supernatants of HPTECs. As a positive control, recombinant human MCP-1 of 9 kDa (Lane 1, 0.5 ng; Lane 2, 5 ng; Lane 3, 50 ng) was used. Supernatants from HPTECs stimulated with TNF-{alpha} (10 ng/ml) for 24 h were 20-fold concentrated. A 15-µl portion of the concentrated samples (Lanes 4 and 5) was subjected to immunoblot analysis. Two isoforms of MCP-1 were detected in the supernatant, the larger one possibly being MCP-1 with post-translational modification. Results shown are from one experiment representative of two performed.

 

Figure 5
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  		Fig. 5. Inhibitory effects of 15-d PGJ2 on MCP-1 production by (HPTECs) under normoxia. (A) HPTECs were incubated with DMEM containing no 15d-PGJ2 (control) or different concentrations of 15d-PGJ2 during 24 h of normoxic treatment. cDNA was prepared from total cellular RNA extracted from the cells at 24 h, and TaqMan real-time PCR for MCP-1 and ß-actin was performed. MCP-1 mRNA amounts were normalized to ß-actin levels. (B) Supernatants were also harvested at 24 h, and assessed for MCP-1 protein by immunoassay. Averaged amounts of MCP-1 mRNA and protein for the control cells were set to 100%. Results are expressed as the mean ± SD of experiments done in triplicate (n = 6). *P < 0.05, **P < 0.01 and ***P < 0.005 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Scheffe's post hoc comparison.

 
Influence of hypoxia on MCP-1-decreasing effects of 15d-PGJ2 or pioglitazone
Since we observed hypoxia-reduced PPAR-{gamma} expression, we further examined whether or not hypoxia suppressed the MCP-1-decreasing effect of the PPAR-{gamma} agonists, 15d-PGJ2 and pioglitazone in HPTECs. In each condition, normoxia and hypoxia, the effects of the agonists were evaluated as the rates of decrease in MCP-1 mRNA and protein amounts relative to the values in cells untreated with the agonists and were regarded as an anti-inflammatory effect of the agonists. The rate of reduction in MCP-1 mRNA and protein amounts were expressed as a percentage of the untreated control value (n = 3 in one experiment) and then compared between normoxia and hypoxia. Concerning 15d-PGJ2 (10 µM), the rates of reduction during hypoxia were significantly lower than those during normoxia, under basal conditions (10 ± 14 vs 68 ± 10%, P < 0.001 for mRNA levels and 33 ± 2 vs 42 ± 5%, P < 0.05 for protein levels) and TNF-{alpha}-stimulated conditions (39 ± 8 vs 60 ± 10%, P < 0.05 for mRNA levels and 33 ± 4 vs 57 ± 4%, P < 0.001 for protein levels). Hypoxia also diminished the MCP-1-decreasing effects of pioglitazone. The rates of reduction induced by pioglitazone during hypoxia were significantly lower than those during normoxia, under basal conditions (3 ± 10 vs 34 ± 6%, P < 0.005 for mRNA levels and 12 ± 10 vs 21 ± 10%, P < 0.05 for protein levels). These findings indicated that hypoxia not only reduced the expression but also weakened the anti-inflammatory effect of PPAR-{gamma} activated by the agonists.

PPAR-{gamma}-dependence of MCP-1-decreasing effects produced by 15d-PGJ2 or pioglitazone
Considering the earlier reports that not all of 15d-PGJ2's effects were mediated through the activation of PPAR-{gamma} [17,18], we attempted to clarify the extent to which the MCP-1-decreasing effects of 15d-PGJ2 and pioglitazone depend on the specific actions of PPAR-{gamma}. The effects of 15d-PGJ2 or pioglitazone were measured under basal conditions during normoxia in the absence or presence of a 6-h pre-incubation with a specific inhibitor for PPAR-{gamma}, GW9662 (2.5 µM). In the absence of GW9662, 15d-PGJ2 (5 µM) reduced MCP-1 mRNA and protein levels by 49 and 19%, respectively, compared with those of untreated cells, while in the presence of GW9662, the agonist reduced the mRNA and protein levels by 36% and 14%, respectively, compared with those of untreated cells (Figure 6A and B). Therefore, GW9662 treatment weakened the MCP-1-decreasing effect of 15d-PGJ2 by 27% at the mRNA level and by 26% at the protein level. Furthermore, GW9662 also diminished the MCP-1-decreasing effect of pioglitazone (3 µM) by 100% at the mRNA level and by 80% at the protein level (Figure 7A and B). In the absence of GW9662, pioglitazone reduced MCP-1 mRNA and protein levels by 34 and 20%, respectively (Figure 7A), while in the presence of GW9662, the agonist produced no reduction but a slight induction of the mRNA expression and only a 4% reduction in the protein levels (Figure 7B). These findings proved that the MCP-1-decreasing effects of 15d-PGJ2 and pioglitazone were PPAR-{gamma}-dependent at a rate of up to 30% and over 80%, respectively, under basal conditions during normoxia.


Figure 6
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  		Fig. 6. GW9662 mildly weakens MCP-1-decreasing effects of 15-d PGJ2 in HPTECs. HPTECs were pre-incubated with DMEM containing no GW9662 or 2.5 µM GW9662 for 6 hours, and incubated with no 15d-PGJ2 or 5 µM PGJ2 during a 24 h normoxic treatment. (A) cDNA was prepared from total cellular RNA extracted from the cells at 24 h, and subjected to TaqMan real-time PCR. MCP-1 mRNA amounts were normalized to ß-actin levels. (B) Supernatants were also harvested at 24 h and assessed for MCP-1 protein by immunoassay. Averaged amounts of MCP-1 mRNA and protein for control cells treated with no GW9662 or 15d-PGJ2 were set to 100%. Results are expressed as the mean ± SD of experiments done in triplicate (n = 6). *P < 0.1, **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.

 

Figure 7
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  		Fig. 7. GW9662 markedly weakens MCP-1-decreasing effects of pioglitazone on production in HPTECs. HPTECs were pre-incubated with DMEM containing no GW9662 or 2.5 µM GW9662 for 6 h, and incubated with no pioglitazone or 3 µM pioglitazone during a 24 h normoxic treatment. (A) cDNA was prepared from total cellular RNA extracted from the cells at 24 h, and subjected to TaqMan real-time PCR. MCP-1 mRNA amounts were normalized to ß-actin levels. (B) Supernatants were also harvested at 24 h and assessed for MCP-1 protein by immunoassay. Averaged amounts of MCP-1 mRNA and protein for control cells treated with no GW9662 or pioglitazone were set to 100%. Results are expressed as the mean ± SD of experiments done in triplicate (n = 6). *P < 0.05 and **P < 0.001 compared with cells incubated under the indicated conditions, by one-way analysis of variance with Scheffe's post hoc comparison.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
Here, we reported for the first time that hypoxia reduced PPAR-{gamma} expression at the mRNA and protein levels and weakened its anti-inflammatory effect of decreasing MCP-1 expression, activated by PPAR-{gamma} agonists in cultured HPTECs. We found that hypoxia-activated signals other than the HIF-1 pathway might be partly associated with the reduced PPAR-{gamma} expression in HPTECs. We also provided evidence that an endogenous PPAR-{gamma} agonist, 15d-PGJ2, and a synthetic specific PPAR-{gamma} agonist, pioglitazone, can exert anti-inflammatory effects in HPTECs, suggesting that these agonists can play a protective role in the inflamed kidney.

In the kidney, PPAR-{gamma} is selectively expressed in medullary collecting ducts and the pelvic urothelium [4], while PPAR-{alpha} is predominantly expressed in proximal tubular cells and PPAR-ß is expressed ubiquitously throughout the nephron [4]. Although it remains to be clarified whether PPAR-{gamma} is newly expressed in cortical portions, proximal tubules and glomeruli, under pathological conditions, the PPAR-{gamma} mRNA and protein expression has also been clearly identified in several types of cultured renal cells such as proximal tubular cells, cortical fibroblasts, and mesangial cells [7–9,19]. In human cultured proximal tubular epithelial cells, considerable amounts of PPAR-{gamma} mRNA and protein were detected (Figure 1). The regulation of PPAR-{gamma} expression has been extensively investigated in vitro. In adipocytes, insulin and corticosteroids synergistically induce PPAR-{gamma} expression [20], while TNF-{alpha} and retinoic acid suppress PPAR-{gamma} levels in adipocytes [21,22]. In an immortalized cell line of human renal proximal tubules (HK-2), high glucose and PPAR-{gamma} agonists increased PPAR-{gamma} mRNA and protein levels by 1.5- to 5-fold [8]. In our study, TNF-{alpha} and a PPAR-{gamma} agonist, 15d-PGJ2, had no effect on PPAR-{gamma} expression in HPTECs, not supporting the earlier findings. Most recently, an FXR agonist has been reported to induce PPAR-{gamma} expression markedly in hepatic stellate cells [16]. Since FXR is evidently expressed in the kidney [23] and the mRNA was detected in HPTECs (Table 1), FXR may be an important regulator for PPAR-{gamma} expression in the kidney, although the receptor has so far remained to be closely investigated in the organ.

One of the most notable findings in our study is the hypoxia-induced reduction of PPAR-{gamma} expression at the mRNA and protein levels in HPTECs. Very few studies have so far been performed in order to clarify how hypoxia regulates PPAR gene expression. In in-vitro experiments with fibroblasts, hypoxia induced a loss of PPAR-{gamma}2 and inhibited PPAR-{gamma}-promoted adipogenesis [12]. In an elegant study using two intestinal epithelial cell lines and microvascular endothelial cells, the expression of PPAR-{alpha} but not PPAR-{gamma} was down-regulated by hypoxia in all the cells [11]. These findings together with our results indicate that the reduction in the expression of PPAR genes is very probably a common response of cultured cells to hypoxia, although which of the PPAR genes is down-regulated by hypoxia depends on the cell type. The mechanism underlying the hypoxic loss of PPAR-{gamma}2 has been closely studied in cultured fibroblasts [12,24]. Hypoxia induced the expression of an HIF-1-regulated gene, DEC1/Stra13, a member of the Drosophila hairy/Enhancer of split transcription repressor family in several types of cultured fibroblasts [12]. DEC1/Stra13 repressed PPAR-{gamma}2 promoter activation and protein production in a HDAC (Histone Deacetylase)-independent manner [12,24]. Hypoxic down-regulation of PPAR-{alpha} gene expression was also reported to be mediated by HIF-1 [11]. In our study, the knockout of HIF-1{alpha} with its dominant negative form did not weaken the hypoxia-induced reduction in PPAR-{gamma} expression, suggesting that a non-HIF pathway is involved in the hypoxic loss of PPAR-{gamma}. Other repressors are known as an inhibitory factor of PPAR-g2 expression. The zinc finger family transcription factors, GATA-2 and GATA-3, suppress PPAR-{gamma}2 expression [25]. From the results of our preliminary microarray analysis, expression of the GATA-2 and GATA-3 genes was unchanged under hypoxia (data not shown). Furthermore, we also studied an impact of oxygen free radicals on the hypoxic reduction of PPAR-{gamma} expression using a free radical scavenger, Tempol, because hypoxic conditions induce reactive oxygen species [26]. Treatment with Tempol did not inhibit the hypoxic reduction, suggesting little association of oxidative stress with the PPAR-{gamma} expression. Considering these conjectural findings in our study, further detailed experiments will be required to clarify the precise mechanism for the hypoxia-induced PPAR-{gamma} loss in HPTECs.

We were able to confirm the anti-inflammatory effect of PPAR-{gamma}'s activation in HPTECs, supporting the results of earlier reports using renal tubular cell lines [8,9,27,28]. In HK-2 cells, {omega}-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), an unselective PPAR-{gamma} agonist, reduced LPS-induced NF-{kappa}B activation and MCP-1 expression in a PPAR-{gamma}-dependent pathway [9]. More specific PPAR-{gamma} agonists, pioglitazone and L805645, diminished high glucose-induced AP-1 activation and attenuated an increase in MCP-1, TGF-ß1, and extracellular matrix proteins in the same cell line [8,27]. In opossum kidney proximal tubular cells, pioglitazone reduced MCP-1 and TGF-ß1 expression induced by low-density lipoprotein, independent of NF-{kappa}B [28]. These anti-inflammatory actions of PPAR-{gamma} protein could be explained in part by its binding to PPAR-{gamma} agonists and subsequent enhancement of its own ability to antagonize the transcription factors, AP-1, STAT and NF-{kappa}B [29]. PPAR-{gamma} agonists are generally considered to exert anti-inflammatory effects in both PPAR-{gamma}-dependent and independent pathways [18]. The specific PPAR-{gamma} agonists function mainly in a PPAR-{gamma}-dependent manner [9,30,31], while 15d-PGJ2 serves in both ways with the independent pathway predominant [17,18]. A possible PPAR-{gamma}-independent function of 15d-PGJ2 is the inhibition of NF-{kappa}B's activation via the suppression of I{kappa}B kinase [17,18]. In our study, treatment with GW9662, a specific inhibitor for PPAR-{gamma} [32], proved that the inhibitory effect of pioglitazone was largely dependent on PPAR-{gamma}, while the effect of 15d-PGJ2 was dependent partly on PPAR-{gamma}. Our finding was in complete agreement with these earlier results [9,17,18,30,31].

It is also worth noting that hypoxia diminished the anti-inflammatory effect of PPAR-{gamma} in HPTECs. We observed that hypoxia produced about a 30% decrease in PPAR-{gamma} protein amounts at 24 h compared with normoxia (Figure 1B). We also found significant reductions in MCP-1-decreasing effects (MCP-1 reduction rates) of PPAR-{gamma} agonists during hypoxia compared with those during normoxia. Therefore, it seems reasonable that the hypoxic loss of PPAR-{gamma} protein could account for the hypoxia-induced attenuation of the MCP-1-decreasing effect of PPAR-{gamma} agonists. There are other possible explanations for the hypoxic reduction of PPAR-{gamma} agonist's effects. Hypoxia also might weaken PPAR-{gamma}'s function independent of protein amounts. Activation of PPAR-{gamma} by the agonists can be depressed by mitogen-activated protein kinase (MAPK)-mediated phosphorylation of the N-terminal A/B domain and ensuing reduction of its agonist (ligand)-binding affinity [33]. Considering that MAPK can be activated by hypoxia [34,35], hypoxia may induce production of phosphorylated PPAR-{gamma} with low agonist-binding affinity and may reduce the pharmacological action of PPAR-{gamma} agonists. Furthermore, MCP-1 expression status, modulated by hypoxia itself [36–38], may influence the anti-inflammatory actions of PPAR-{gamma} agonists. In our study, hypoxia decreased MCP-1 expression by about 50% at the mRNA level and by about 20% at the protein level, which was similar to our previous finding [37]. Some repressors for MCP-1 expression, induced by hypoxia also may interact with PPAR-{gamma} and suppress its function. These possible modifications of PPAR-{gamma} under hypoxia will need to be analysed in order to clarify the precise mechanism for hypoxia-reduced PPAR-{gamma} function.

Several limitations exist in the current study. Because sample sizes (n = 3) were small in some experiments, there may have been some ambiguity about statistic significances calculated when only small changes in mRNA and protein amounts were observed. It would have been preferable to use RNase protection assay for quantification of mRNA.

In conclusion, hypoxia repressed PPAR-{gamma} expression at the mRNA and protein levels and inhibited the anti-inflammatory effects of PPAR-{gamma} in HPTECs. These results suggest that the local O2 concentration may be an important regulator for inflammation, altering PPAR-{gamma} function in the renal tubulointerstitium. The reduced PPAR-{gamma} expression might be explained partly by hypoxia-activated signals other than the HIF-1 pathway.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by a Grant-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, Philadelphia, Pennsylvania, USA, November 2005.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Fine LG, Bandyopadhay D, Norman JT. (2000) Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia. Kidney Int Suppl 75:S22–S26.[CrossRef][Medline]
  2. Semenza GL. (2000) HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88:1474–1480.[Abstract/Free Full Text]
  3. Jaakkola P, Mole DR, Tian YM, et al. (2001) Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472.[Abstract/Free Full Text]
  4. Guan Y and Breyer MD. (2001) Peroxisome proliferator-activated receptors (PPARs): novel therapeutic targets in renal disease. Kidney Int 60:14–30.[CrossRef][Web of Science][Medline]
  5. Guan Y. (2004) Peroxisome proliferator-activated receptor family and its relationship to renal complications of the metabolic syndrome. J Am Soc Nephrol 15:2801–2815.[Abstract/Free Full Text]
  6. Jiang C, Ting AT, Seed B. (1998) PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82–86.[CrossRef][Medline]
  7. Zafiriou S, Stanners SR, Saad S, Polhill TS, Poronnik P, Pollock CA. (2005) Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts. J Am Soc Nephrol 16:638–645.[Abstract/Free Full Text]
  8. Panchapakesan U, Pollock CA, Chen XM. (2004) The effect of high glucose and PPAR-gamma agonists on PPAR-gamma expression and function in HK-2 cells. Am J Physiol Renal Physiol 287:F528–F534.[Abstract/Free Full Text]
  9. Li H, Ruan XZ, Powis SH, et al. (2005) EPA and DHA reduce LPS-induced inflammation responses in HK-2 cells: evidence for a PPAR-gamma-dependent mechanism. Kidney Int 67:867–874.[CrossRef][Web of Science][Medline]
  10. Li Y, Wen X, Spataro BC, Hu K, Dai C, Liu Y. (2006) Hepatocyte growth factor is a downstream effector that mediates the antifibrotic action of peroxisome proliferator-activated receptor-{gamma} agonists. J Am Soc Nephrol 17:54–65.[Abstract/Free Full Text]
  11. Narravula S and Colgan SP. (2001) Hypoxia-inducible factor 1-mediated inhibition of peroxisome proliferator-activated receptor alpha expression during hypoxia. J Immunol 166:7543–7548.[Abstract/Free Full Text]
  12. Yun Z, Maecker HL, Johnson RS, Giaccia AJ. (2002) Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia. Dev Cell 2:331–341.[CrossRef][Web of Science][Medline]
  13. Kwan J, Wang H, Munk S, Xia L, Goldberg HJ, Whiteside CI. (2005) In high glucose protein kinase C-zeta activation is required for mesangial cell generation of reactive oxygen species. Kidney Int 68:2526–2541.[CrossRef][Web of Science][Medline]
  14. Li X, Kimura H, Hirota K, et al. (2005) Synergistic effect of hypoxia and tumor necrosis factor-{alpha} on production of plasminogen activator inhibitor-1 in human proximal renal tubular cells. Kidney Int 68:569–583.[CrossRef][Web of Science][Medline]
  15. Kasuno K, Takabuchi S, Fukuda K, et al. (2004) Nitric oxide induces hypoxia-inducible factor 1 activation that is dependent on MAPK and phosphatidylinositol 3-kinase signaling. J Biol Chem 279:2550–2558.[Abstract/Free Full Text]
  16. Fiorucci S, Rizzo G, Antonelli E, et al. (2005) Cross-talk between farnesoid-X-receptor (FXR) and peroxisome proliferator-activated receptor gamma contributes to the antifibrotic activity of FXR ligands in rodent models of liver cirrhosis. J Pharmacol Exp Ther 315:58–68.[Abstract/Free Full Text]
  17. Rovin BH, Lu L, Cosio A. (2001) Cyclopentenone prostaglandins inhibit cytokine-induced nf-kappab activation and chemokine production by human mesangial cells. J Am Soc Nephrol 12:1659–1667.[Abstract/Free Full Text]
  18. Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, Evans RM. (2001) PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med 7:48–52.[CrossRef][Web of Science][Medline]
  19. Asano T, Wakisaka M, Yoshinari M, et al. (2000) Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expresses in rat mesangial cells and PPARgamma agonists modulate its differentiation. Biochim Biophys Acta 1497:148–154.[Medline]
  20. Vidal-Puig AJ, Considine RV, Jimenez-Linan M, et al. (1997) Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids. J Clin Invest 99:2416–2422.[Web of Science][Medline]
  21. Schwarz EJ, Reginato MJ, Shao D, Krakow SL, Lazar MA. (1997) Retinoic acid blocks adipogenesis by inhibiting C/EBPbeta-mediated transcription. Mol Cell Biol 17:1552–1561.[Abstract]
  22. Zhang B, Berger J, Hu E, et al. (1996) Negative regulation of peroxisome proliferator-activated receptor-gamma gene expression contributes to the antiadipogenic effects of tumor necrosis factor-alpha. Mol Endocrinol 10:1457–1466.[Abstract/Free Full Text]
  23. Zhang Y, Kast-Woelbern HR, Edwards PA. (2003) Natural structural variants of the nuclear receptor farnesoid X receptor affect transcriptional activation. J Biol Chem 278:104–110.[Abstract/Free Full Text]
  24. Kim KH, Song MJ, Chung J, Park H, Kim JB. (2005) Hypoxia inhibits adipocyte differentiation in a HDAC-independent manner. Biochem Biophys Res Commun 333:1178–1184.[CrossRef][Web of Science][Medline]
  25. Tong Q, Dalgin G, Xu H, Ting CN, Leiden JM, Hotamisligil GS. (2000) Function of GATA transcription factors in preadipocyte-adipocyte transition. Science 290:134–138.[Abstract/Free Full Text]
  26. Schafer M, Schafer C, Ewald N, Piper HM, Noll T. (2003) Role of redox signaling in the autonomous proliferative response of endothelial cells to hypoxia. Circ Res 92:1010–1015.[Abstract/Free Full Text]
  27. Panchapakesan U, Sumual S, Pollock CA, Chen X. (2005) PPARgamma agonists exert antifibrotic effects in renal tubular cells exposed to high glucose. Am J Physiol Renal Physiol 289:F1153–F1158.[Abstract/Free Full Text]
  28. Zafiriou S, Stanners SR, Polhill TS, Poronnik P, Pollock CA. (2004) Pioglitazone increases renal tubular cell albumin uptake but limits proinflammatory and fibrotic responses. Kidney Int 65:1647–1653.[CrossRef][Web of Science][Medline]
  29. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. (1998) The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391:79–82.[CrossRef][Medline]
  30. Al-Rasheed NM, Chana RS, Baines RJ, Willars GB, Brunskill NJ. (2004) Ligand-independent activation of peroxisome proliferator-activated receptor-gamma by insulin and C-peptide in kidney proximal tubular cells: dependent on phosphatidylinositol 3-kinase activity. J Biol Chem 279:49747–49754.[Abstract/Free Full Text]
  31. Seimandi M, Lemaire G, Pillon A, et al. (2005) Differential responses of PPARalpha, PPARdelta and PPARgamma reporter cell lines to selective PPAR synthetic ligands. Anal Biochem 344:8–15.[CrossRef][Web of Science][Medline]
  32. Leesnitzer LM, Parks DJ, Bledsoe RK, et al. (2002) Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry 41:6640–6650.[CrossRef][Medline]
  33. Shao D, Rangwala SM, Bailey ST, Krakow SL, Reginato MJ, Lazar MA. (1998) Interdomain communication regulating ligand binding by PPAR-gamma. Nature 396:377–380.[CrossRef][Medline]
  34. Koong AC, Chen EY, Mivechi NF, Denko NC, Stambrook P, Giaccia AJ. (1994) Hypoxic activation of nuclear factor-kappa B is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2). Cancer Res 54:5273–5279.[Abstract/Free Full Text]
  35. Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y. (1997) Hypoxia and hypoxia/reoxygenation activate p65PAK, p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun 239:840–844.[CrossRef][Web of Science][Medline]
  36. Bosco MC, Puppo M, Pastorino S, et al. (2004) Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages. J Immunol 172:1681–1690.[Abstract/Free Full Text]
  37. Li X, Kimura H, Hirota K, Sugimoto H, Yoshida H. (2005) Hypoxia reduces constitutive and TNF-alpha-induced expression of monocyte chemoattractant protein-1 in human proximal renal tubular cells. Biochem Biophys Res Commun 335:1026–1034.[Web of Science][Medline]
  38. Safronova O, Nakahama K, Onodera M, Muneta T, Morita I. (2003) Effect of hypoxia on monocyte chemotactic protein-1 (MCP-1) gene expression induced by Interleukin-1beta in human synovial fibroblasts. Inflamm Res 52:480–486.[CrossRef][Web of Science][Medline]
Received for publication: 6. 5.06
Accepted in revised form: 23.11.06


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