Peroxisome proliferator-activated receptor-gamma activity is associated with renal microvasculature

Youfei Guan1, Yahua Zhang1, André Schneider1, Linda Davis1, Richard M. Breyer2, and Matthew D. Breyer1,3

1 Division of Nephrology, Department of Medicine, and Departments of 3 Molecular Physiology and Biophysics and 2 Pharmarcology, Vanderbilt University Medical Center, Nashville, Tennessee 37212


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

First published July 12, 2001; 10.1152/ajprenal.00025.2001.---Peroxisome proliferator-activated receptor-gamma (PPARgamma ) is a nuclear transcription factor and the pharmacological target for antidiabetic thiazolidinediones (TZDs). TZDs ameliorate diabetic nephropathy and have direct effects on cultured mesangial cells (MCs); however, in situ hybridization failed to detect expression of PPARgamma in glomeruli in vivo. The purpose of this study was to determine whether PPARgamma is expressed in renal glomeruli. Two rabbit PPARgamma isoforms were cloned. Nuclease protection assays demonstrate that both PPARgamma isoforms are expressed in freshly isolated glomeruli. Treatment of rabbits with the TZD troglitazone selectively induced expression of an endogenous PPARgamma target gene, adipocyte fatty acid-binding protein (A-FABP), in renal glomerular cells and renal medullary microvascular endothelial cells, demonstrated by both in situ hybridization and immunostain. Troglitazone also dramatically increased A-FABP expression in cultured MCs. Constitutive PPARgamma expression was detected in cultured rabbit MCs. Endogenous MC PPARgamma can also drive PPARgamma reporter. Troglitazone and 15-deoxy-Delta 12,14 prostaglandin J2 at low concentrations reduced mesangial cell [3H]thymidine incorporation without affecting viability. These data suggest that constitutive PPARgamma activity exists in renal glomeruli in vivo and could provide a pharmacological target to directly modulate glomerular injury.

glomeruli; mesangial cell; adipocyte fatty acid-binding protein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PEROXISOME PROLIFERATORS ARE composed of a group of structurally diverse compounds, including hypolipidemic fibrates (e.g., clofibrate) and antidiabetic thiazolidinediones (TZDs; e.g., rosiglitazone and troglitazone), that induce proliferation of peroxisomes and modulate the expression of genes involved in lipid beta -oxidation, cell differentiation, and inflammation (3). These effects are mediated, at least in part, by a family of nuclear receptors designated peroxisome proliferator-activated receptors (PPARs). PPARs form heterodimers with the 9-cis retinoic acid receptor RXRalpha , which bind to characteristic DNA sequences termed peroxisome proliferator response elements (PPREs) located in the 5'-flanking region of target genes (30). Three PPAR isoforms, designated PPARalpha , PPARbeta /delta , and PPARgamma , have been cloned from Xenopus laevis, mouse, rat, and humans. A role for PPARgamma in promoting fat cell formation (i.e., adipogenesis) is well established (13). Recent reports demonstrate that PPARgamma is also highly expressed in renal epithelial cells (17, 43) as well as several epithelial cancers (3), where TZD PPARgamma ligands induce growth arrest and cell differentiation (16, 29). It has also been reported that PPARgamma is highly expressed in monocytes/macrophages, where it may modulate transcription of inflammatory cytokines (22, 35). In addition, PPARgamma activation abrogated gene expression of metalloproteinase 9 in murine macrophages (34, 35) and secretion of metalloproteinase 9 in THP-1 cells, a human monocytic cell line (38). These findings suggest that PPARgamma plays diverse roles in cell growth, differentiation, and extracellular matrix accumulation.

PPARgamma ligands, including the TZDs troglitazone, pioglitazone, and rosiglitazone, have recently become clinically available for treating insulin-resistant (type II) diabetes mellitus (1, 12, 26). Accumulating evidence suggests that these drugs not only significantly improve insulin sensitivity but also may reduce microalbuminuria and diabetic nephropathy in genetically obese diabetic rodents and patients with type II diabetes (6, 14, 20, 27, 45). Moreover, troglitazone reduced expression of extracellular matrix proteins (fibronectin and type IV collagen) and transforming growth factor-beta in glomeruli from streptozotocin-induced diabetic rats (21). These effects in insulin-dependent diabetes mellitus (type I) suggest that PPARgamma ligands might have a direct, beneficial renal effect, independent of their capacity to improve glucose tolerance (21). The present studies were aimed at further elucidating cellular targets in the kidney that might exert direct, beneficial effects of PPARgamma ligands.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

5'-Rapid amplification of cDNA ends. 5'-Rapid amplification of cDNA ends (5'-RACE) was used to obtain the 5'-ends of rabbit PPARgamma 1 using a previously cloned fragment of rabbit PPARgamma (17). Three gene-specific antisense primers were designed: primer 1 (5'-CGG TGA TTT GTC TGT CGT CTT-3'), nested primer 2 (5'-TCG GCA GAC TCG GGG TTC AGC-3'), and nested primer 3 (5'-TCC CCA CAG CAA GGC ATT TCT-3'). One microgram of total RNA pretreated with RNase-free DNase I (Promega) was used as a template for the first-strand cDNA synthesis with primer 1 and 200 U of SuperScript II RT according to the manufacturer's description (GIBCO BRL). The first-strand cDNA was purified and tailed with dCTP using terminal deoxynucleotidyl transferase. Amplification of the 5'-terminal cDNA sequence was performed with primer 2 and the abridged anchor primer, and the product was used in a second nested PCR with the internal primer 3 and the abridged universal amplification primer (94°C for 30 s, 57°C for 1 min, 72°C for 2 min for 35 cycles). PCR products were subcloned into the pCR II 2.1 vector (Invitrogen).

3'-RACE. Two gene-specific sense primers were designed for amplification of a sequence downstream of the known PPARgamma cDNA: a 3'-primer (primer 4, 21 mer: 5'-CAG GAA AGA CGA CAG ACA AAT-3') and a nested primer (primer 5, 21 mer: 5'-CTT TGT GAG CCT TGA CTT GAA-3'). One microgram of total RNA was reverse-transcribed with the adapter primer and 200 U of SuperScript II RT at 42°C (3'-RACE system, GIBCO BRL) according to the manufacturer's protocol. Two microliters of cDNA were used for the first round of PCR amplification with the abridged universal amplification primer and primer 4. A nested PCR was performed with internal primer 5 and the universal amplification primer (94°C for 30 s, 57°C for 40 s, and 72°C for 3 min for 35 cycles). A single 3'-RACE product was purified (Qiaquick gel extraction kit, Qiagen, Valencia, CA) and subcloned for sequencing.

Construction of full-length PPARgamma 1 and determination of the tissue distribution by RT-PCR analysis. Full-length rabbit PPARgamma 1 was obtained by RT-PCR using primer pairs located in the common region of the 5'-untranslated region (UTR; 5'-AGC AAC AGT CAT CCA TAA AAG-3') and the 3'-UTR (5'-TC CCT CAA AAT AAT AGT GC-3') and subcloned into the pRc/CMV2 vector (Invitrogen). To investigate the expression of PPARgamma 1 and PPARgamma 3 isoforms in mesangial cells, isoform-specific primers were designed for RT-PCR. For amplification of PPARgamma 3 cDNA, the following pair of primers was synthesized: primer 6 (sense), 5'-CTG GGC TCC TCC TGT CGC CTC-3', and primer 7 (antisense), 5'-CGC CGC AGA CAC GAC ACT CAA T-3'. For rabbit PPARgamma 1, the sense primer (primer 8) was 5'-TAG GGA CAG ATG CGA TGA GTT-3', and the antisense (primer 9) was 5'-ACA TCC CCA CAG CAA GGC ATT TCT-3'. One microgram of total RNA from rabbit mesangial cells was subjected to RT-PCR as above.

Solution hybridization/RNase protection assays. To determine the level of PPARgamma 1 mRNA expression in renal glomeruli, PPARgamma 1 (456 bp, -450 to +6 bp) was subcloned into pBluescript SK(-) vector, and isoform-specific riboprobes were synthesized for mapping the mRNA expression of PPARgamma 1 in whole kidney and freshly isolated glomeruli. RNase protection assays were performed as previously described (17). Briefly, the plasmids containing genes of interest were linearized with appropriate restriction enzymes. Radiolabeled riboprobes were synthesized from 1 µg of linearized plasmid containing PPARgamma 1 (456 bp), the PPARgamma coding region (316 bp of an EcoRI fragment), and adipocyte fatty acid-binding protein (A-FABP; 339 bp) in vitro using a MAXIscript kit (Ambion) for 1 h at 37°C in a total volume of 20 µl. The reaction buffer contained (in mM) 10 dithiothreitol (DTT), 0.5 ATP, 0.5 CTP, 0.5 GTP, and 2.5 UTP as well as 5 µl of 800 Ci/mmol [alpha -32P]UTP at 10 mCi/ml (DuPont-NEN). The hybridization buffer included 80% deionized formamide, 100 mM sodium citrate, pH 6.4, and 1 mM EDTA (RPA II, Ambion). Thirty micrograms of total RNA from freshly isolated rabbit glomeruli, intestine, and cultured mesangial cells were incubated at 45°C for 12 h in hybridization buffer with 5 × 104 cpm-labeled riboprobes (where cpm is counts/min). After hybridization, RNAse digestion was carried out at 37°C for 30 min, and protected fragments were precipitated and separated on a 6% polyacrylamide gel at 200 V for 4 h. The gel was exposed to Kodak XAR-5 film overnight at -80°C with intensifying screens.

Northern blot and hybridization. Total RNA was extracted from peritoneal white adipose tissue using TRIzol reagent (GIBCO BRL). RNA (20 µg) was fractionated by 1.0% agarose-formaldehyde gel electrophoresis in 1× MOPS buffer and transferred to a nylon membrane. The membrane was prehybridized in 6× standard sodium citrate (SSC), 5× Denhardt's, 1% SDS, and 150 µg/ml of freshly denatured salmon sperm DNA for 4 h and then hybridized overnight in the same buffer with 1 × 106 cpm/ml of a 32P-labeled rabbit PPARgamma (761-bp) cDNA probe at 62°C. After hybridization, the blot was washed once in 2× SSC, 0.1% SDS for 30 min at room temperature followed by two washes in 0.1× SSC, 0.1% SDS at 62°C for 30 min and exposed to X-ray film at -70°C overnight.

Immunoblot analysis of PPARgamma expression. Mesangial cells were lysed in SDS-PAGE sample buffer (120 mM Tris · HCl, pH 6.5, 4% SDS, 5 mM DTT, and 20% glycerol) followed by repetitive aspiration. The lysate was boiled for 3 min, and protein concentration was measured by BCA protein assay (Pierce, Rockford, IL). Ten micrograms of each protein sample were loaded onto 10% SDS-PAGE minigels and run at 100 V. Proteins were transferred to a nitrocellulose membrane at 22 V, overnight at 4°C. The nitrocellulose membranes were washed three times with PBS and incubated in blocking buffer (Tris-buffered saline, which contained 150 mM NaCl, 50 mM Tris, 0.05% Tween 20 detergent, and 5% nonfat dry milk, pH 7.5) for 1 h at room temperature. The membranes were then incubated in rabbit anti-mouse PPARgamma 1,2 polyclonal antibody (BioMol) diluted 1:2,000 in blocking buffer for 2 h at room temperature. After three washes in blocking buffer, the membranes were incubated with biotinylated anti-rabbit IgG antibody (1:2,000, Vector Labs, Burlingame, CA) for 1 h, followed by three 15-min washings. Antibody labeling was visualized by adding chemiluminescence reagent (DuPont-NEN) and exposing the membrane to Kodak XAR-5 film.

Preparation of rabbit glomeruli and culture of glomerular mesangial cells. Glomeruli were isolated by fractional sieving of minced cortex using 125- and 75-µm mesh wire. Rabbit glomerular mesangial cells were cultured according to a previous report (25). Cells were cultured in 10% fetal bovine serum (FBS)-supplemented RPMI-1640 medium (GIBCO BRL). To test whether PPARgamma is constitutively expressed in mesangial cells, cells were cultured in RPMI-1640 medium supplemented with 10% FBS until complete confluence and then made quiescent by being cultured in serum-free medium for 24 h. Quiescent mesangial cells were treated with phorbol 12-myristate 13-acetate (1 µM) and troglitazone (10 µM) for 6 h.

Amplification of a rabbit A-FABP cDNA fragment. A fragment of rabbit A-FABP was amplified by RT-PCR using total RNA, with adipose tissue as a template. Two primers were chosen from a mouse A-FABP cDNA sequence as reported by Hunt et al. (19). The sense oligomer (5'-CTG GAA GCT TGT CTC CAG TGA-3') and antisense oligomer (5'-CAT AAC ACA TTC CAC CAC CAG-3') were used to amplify 337 bp of rabbit A-FABP. The PCR product was gel purified, cloned into TOPO vector (Invitrogen), and sequenced. Sequencing confirmed homology to mouse A-FABP. This fragment was used for an RNase protection assay and in situ hybridization.

In situ hybridization of A-FABP. Female New Zealand White rabbits were treated orally with troglitazone (200 mg · kg-1 · day-1) for 2 wk. An 35S-labeled antisense riboprobe probe was generated from a 337-bp PCR fragment of A-FABP (see above) hybridized to the section and then washed as previously described (16). Slides were dehydrated with graded ethanol containing 300 mM ammonium acetate, dipped in emulsion (Ilford K5, Knutsford, Cheshire, UK), and exposed for 4-5 days at 4°C. After development in Kodak D-19, slides were counterstained with hematoxylin. Photomicrographs were taken using a Zeiss Axioskop microscope and either darkfield or brightfield optics.

Immunohistochemistry. The primary anti-AFABP antibody was a gift from Dr. David Bernlohr (Dept. of Biochemistry, Univ. of Minnesota). For detection of A-FABP immunoreactive protein, the polyclonal anti-A-FABP (1:200 dilution) was preincubated with an anti-rabbit horseradish peroxidase-coupled antibody in PBS, followed by addition of an excess of rabbit IgG (10% rabbit serum) to adsorb the unreacted secondary antibody. Sections were cut at 7-µm thickness, deparaffinized in xylene, and incubated for 30 min in methanol containing 0.3% H2O2 to block endogenous peroxidase activity. The anti-A-FABP+ secondary antibody complex was added to the sections and incubated for 2 h. Antibody labeling was detected using a Vectastain ABC kit. The specificity of A-FABP antibody staining was demonstrated by the following control experiments: 1) omission of the primary antibody resulted in unstained sections; 2) strong staining in white fat tissues; and 3) similar localization in the kidney and fat tissue of wild-type mice but absence of immunoreactivity in fat tissue and kidneys from A-FABP (aP2)-knockout mice (tissue provided by Dr. MacRae Linton, Div. of Cardiology, Vanderbilt Univ.).

Chemical reagents. Troglitazone (provided by Warner Lambert/Parke-Davis Pharmaceutical Research, Ann Arbor, MI) and an experimental TZD, 5-{4-[2-(5-methyl-2-phenyl-4-oxazoly)-2-hydroxyethoxy] benzyl-2,4-thiazolidinone}, henceforth referred to as TZD2 (Merck Pharmaceutics), were dissolved in DMSO at a concentration of 30 mM. Phorbol 12-myristate 13-acetate and other reagents were purchased from Sigma (St. Louis, MO). Troglitazone was mixed with rabbit chow (0.35% wt/wt, Purina Diet, Indianapolis, IN) and compressed into pellets by the manufacturer.

Transient transfections and luciferase reporter assays. Rabbit PPARgamma 1 cDNA was cloned into the pRc/CMV2 expression vector (PPARgamma /CMV). A PPAR reporter was provided by Dr. Raymond N. DuBois (Vanderbilt Univ.) (16). Mesangial cells were transfected with a mixture of PPRE3-tk-luciferase, pRL-SV40, and PPARgamma expression vector using Effectene Transfection Reagent as recommended by the supplier (Qiagen) for 8 h. The transfection mixture was replaced with complete media containing either DMSO or troglitazone and 15-deoxy-Delta 12,14 prostaglandin J2 (15dPGJ2). After 14 h, cells were harvested in 1× luciferase lysis buffer (Dual Luciferase Kit, Promega), and relative light units were determined using a luminometer (Mono light 2010, Analytical Luminescence Laboratory, San Diego, CA).

Measurement of DNA synthesis and cell viability. [3H]thymidine incorporation was measured to determine the effect of PPARgamma ligands troglitazone and 15dPGJ2 on DNA synthesis. The rabbit mesangial cells were maintained in RPMI-1640 medium supplemented with 10% (vol/vol) FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 200 mg/l L-glutamine. Cells were cultured in a humidified atmosphere of 5% CO2 at 37oC. After achieving confluence, cells were cultured in serum-depleted medium for 24 h. Cells were incubated in the presence or absence of troglitazone and 15dPGJ2 (1-20 µM) for 24 h. [3H]thymidine (1 mCi/ml) was added to the cells for the last hour of treatment. [3H]thymidine incorporation was detected by scintillation counting. A modified colorimetric assay based on the selective ability of living cells to reduce the yellow salt 3-[4,5-dimethylthiozole]-2,5-diphynyl tetrazolium bromide (MTT) to formazan was used to quantify cell viability (16, 24).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA cloning of full-length rabbit PPARgamma isoforms and mRNA expression in rabbit kidney. A 758-bp fragment of PPARgamma was amplified by PCR from rabbit adipose tissue, yielding a region of rabbit PPARgamma with homology to human and mouse PPARgamma as previously reported (17). 5'-RACE yielded two distinct clones by restriction enzyme digestion. The shorter clone (rabPPARgamma 3) comprised 407 bp, whereas the longer clone (rabPPARgamma 1) included 732 bp. These clones were identical in the 5'-coding region (amino acids 1-87) and the proximal portion of the 5'-UTR (nucleotide -87 to -1 bp), differing only in the upstream portion of the 5'-UTR (rabPPARgamma 3, -146 to -88 bp and rabPPARgamma 1, -471 to -88 bp) (Fig. 1). No PPARgamma 2 homolog was found in any of the 5'-RACE clones.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 1.   Nucleotide and deduced amino acid sequence of the rabbit peroxisome proliferator-activated receptor-gamma (PPARgamma )1 and PPARgamma 3. DNA binding domain (DBD) and ligand binding domains (LBD) are underlined, and polyadenylation sequence is shown in italics (bottom).

3'-RACE yielded a single 744-bp fragment. This product overlaps the original 758-bp PCR product by 157 bp and extends the rabbit PPARgamma 3'-sequence by 587 bp. The sequence includes a termination codon TAG in the open reading frame, as well as a 224-bp 3'-UTR with a poly (A) tail of 24 A nucleotides and one putative polyadenylation signal. This is consistent with amplification of a full-length 3'-UTR for rabbit PPARgamma 1 and PPARgamma 3. Interestingly, the 3'-UTR sequence possesses a total of 5 AU-rich domains, suggesting rapid turnover of PPARgamma 1 mRNA in vivo.

Assembly of the RACE product sequences yielded a full-length rabbit PPARgamma 1 consisting of 2,123 bp (Fig. 1). This sequence encodes a polypeptide of 475 amino acids with an estimated molecular mass of 54.1 kDa (Fig. 1). Comparison of the amino acid sequence of rabbit PPARgamma 1 with human and mouse homologs revealed 95 and 94% identity, respectively. As expected, a putative DNA-binding domain (amino acids 109-173), ligand-binding domain (amino acids 285-444), and two zinc finger regions (amino acids 109-125 and 125-140) are among the conserved regions. The size of full-length rabbit PPARgamma mRNA was further confirmed by the detection of a major transcript migrating at 1.8-2.1 kb on a Northern blot (Fig. 2A), consistent with the 1,798 bp of PPARgamma 3 and 2,123 bp of PPARgamma 1 mRNA.


View larger version (90K):
[in this window]
[in a new window]
 
Fig. 2.   Determination of transcript size and expression of PPARgamma 1 in adipose tissue and renal glomeruli. A: Northern hybridization analysis of 20 µg of total RNA from rabbit white fat tissue was performed by using 758-bp RT-PCR fragment from the middle coding region of PPARgamma 1 as hybridization probe. B: RNase protection assay indicating expression of PPARgamma 1 (456 bp) mRNA in freshly isolated glomeruli. C: darkfield illuminated photomicrograph showing an in situ hybridization for PPARgamma mRNA expression in normal (left) rabbit kidneys and kidneys from a rabbit treated with the PPARgamma ligand 5-{4-[2-(5-methyl-2-phenyl-4-oxazoly)-2-hydroxyethoxy] benzyl-2,4-thiazolidinone} (TZD2; right). White grains indicate hybridization signals of PPARgamma mRNA over glomeruli (Glom; arrow).

PPARgamma expression in native and TZD-treated rabbit glomeruli. An RNase protection assay was used to examine the expression of PPARgamma 1 in native renal glomeruli. PPARgamma 1 mRNA was detected in freshly isolated glomeruli (Fig. 2B). Intestinal tissue served as a positive control. To determine whether endogenous glomerular PPARgamma was transcriptionally active, we examined the effect of oral troglitazone (200 mg · kg-1 · day-1 for 2 wk) or TZD2 (3 mg · kg-1 · day-1) treatment on renal expression of PPARgamma and a well-established PPARgamma target gene, A-FABP (37). As previous reported (17), PPARgamma was not detected by in situ hybridization in glomeruli of kidneys from untreated rabbits. However, the kidneys of rabbits treated with TZD2 for 2 wk displayed a marked increase in PPARgamma expression, as assessed by in situ hybridization (Fig. 2C).

To assess the in vivo transcriptional activity of PPARgamma , the expression of its downstream target gene, A-FABP, was determined. A fragment of rabbit A-FABP was amplified by RT-PCR. This product was 92% identical to human A-FABP. As with PPARgamma , no A-FABP expression was observed by in situ hybridization in renal parenchyma from untreated rabbits (Fig. 3A). In contrast, treatment with the PPARgamma ligand troglitazone markedly induced A-FABP mRNA expression in both renal cortex and medulla (Fig. 3B). In the renal cortex, A-FABP mRNA was restricted to glomeruli, consistent with the presence of transcriptionally active PPARgamma in glomeruli (Fig. 4). Moreover, consistent with this localization of A-FABP mRNA, immunostaining showed that A-FABP was highly expressed in the mesangial compartment in glomeruli from troglitazone-treated rabbits (Fig. 5, left).


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 3.   In situ hybridization showing an induction of A-FABP mRNA level in rabbit glomeruli and vasa recta after rabbit was treated with troglitazone (200 mg · kg-1 · day-1 for 2 wk). A: autoradiogram showing the intrarenal localization of adipocyte fatty acid binding protein (A-FABP) mRNA in the kidneys of rabbits treated with (right) or without troglitazone (left). B: ×10 darkfield illumination of rabbit kidney. The white areas indicate grains from radiolabeling of glomeruli in renal cortex, vasa recta in renal medulla, urothelial cell layer and surrounding perirenal fat tissue.



View larger version (121K):
[in this window]
[in a new window]
 
Fig. 4.   Brightfield and darkfield illuminated photomicrograph showing that in situ hybridization with A-FABP mRNA expression was detected in rabbit glomeruli and vasa recta of renal medulla. Left: ×200 rabbit cortex (A) and medulla (C). White grains indicate hybridization signals of A-FABP over glomeruli (Glom; A, arrows) and vasa recta (C). B: brightfield ×400 photomicrograph of rabbit cortex. Brown grains indicate A-FABP mRNA in the mesangium of glomeruli.



View larger version (91K):
[in this window]
[in a new window]
 
Fig. 5.   Immunostaining for the A-FABP protein in the kidney of rabbits treated with troglitazone. A-FABP immunoreactivity (indicated by the brown reaction product) is expressed predominantly in mesangial cells (arrowhead) and endothelial cells (arrow) of renal glomeruli (left) as well as endothelial cells of vasa recta (arrow, right). Note the blood cells evident in vasa recta (right).

In the renal medulla, intense expression of A-FABP mRNA was detected in vasa recta (Fig. 4), and immunostaining for A-FAPB suggested that this expression was greatest in the capillary endothelial cells of vasa recta (Fig. 5, right). Interestingly, no signal of A-FABP mRNA or immunoactivity were detected in medullary collecting ducts, where previous studies had shown high levels of PPARgamma expression (17).

PPARgamma expression in cultured glomerular mesangial cells. To further characterize the intraglomerular cells expressing PPARgamma , rabbit glomerular mesangial cells were cultured and examined for PPARgamma . Both PPARgamma 1 and PPARgamma 3 were detected by RT-PCR in cultured mesangial cells (data not shown). RNase protection analysis and immunoblot assays (Fig. 6A) confirmed the expression of PPARgamma in cultured mesangial cells. Mesangial cells displayed the expected protected fragment of 316 bp for PPARgamma mRNA. PPARgamma protein was also recognized as a ~55-kDa protein band, using an anti-PPARgamma antibody on the immunoblot. Finally, as observed in the preceding in vivo studies, mesangial PPARgamma mRNA expression appeared to increase after treatment with the TZD troglitazone (10 µM) for 6 h.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   A: expression and regulation of PPARgamma mRNA and protein in cultured rabbit mesangial cells. Top: RNase protection assay demonstrating the constitutive expression of PPARgamma in rabbit mesangial cells. Messenger RNA for PPARgamma was induced by troglitazone. The protected fragments were electrophoretically separated on a 6% agarose-7 M urea gel. The film was exposed for 36 h. Bottom: immunoblot assays using an anti-PPARgamma 1 and -PPARgamma 2 polyclonal antibody showing immunoreactivity of the expected molecular size (~55 kDa). PPARgamma 1 immunoreactivity did not change after treatment with 1 µM phorbol 12-myristate 13-acetate (PMA) and was slightly enhanced by treatment with 10 µM troglitazone for 6 h. B: PPARgamma ligands induce A-FABP gene expression in cultured mesangial cells. Rabbit mesangial cells were treated with troglitazone (10 µM) and TZD2 (1 µM) for 3 days. A RNase protection assay was performed to measure mRNA level of A-FABP. Ten micrograms of total RNA were used for analysis, and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was the control for the amount of the loaded RNA.

Induction of A-FABP gene expression by PPARgamma ligands in cultured glomerular mesangial cells. The expression of the PPARgamma target gene A-FABP was studied in cultured mesangial cells. A-FABP mRNA was not detected in quiescent mesangial cells; however, after 3 days of treatment with troglitazone (5 µM) or TZD2 (1 µM), A-FABP gene expression was dramatically induced (Fig. 6B). Consistent with their pharmacological properties, TZD2 was more potent than troglitazone in inducing A-FABP gene expression (9).

Functional transcriptional activity of PPARgamma in mesangial cells. Endogenous PPARgamma transcriptional activity in rabbit mesangial cells was confirmed by using a luciferase reporter construct (PPRE3x-luciferase; Fig. 7A). Mesangial cells transfected with PPRE3X-luciferase alone exhibit a significant induction of luciferase activity after treatment with troglitazone (5 µM) or 15dPGJ2 (1 µM) (Fig. 7B), consistent with transcriptionally active PPARgamma . Cotransfection of PPRE-3x-luciferease with exogenous rabbit PPARgamma (PPARgamma /CMV) further activated luciferase expression (Fig. 7B), demonstrating that the full-length rabbit PPARgamma clone contains all domains essential for activating target gene transcription.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Luciferase reporter activity demonstrating PPARgamma activity in cultured rabbit mesangial cells. Mesangial cells were cotransfected with peroxisome proliferator response element (PPRE)3X-luciferase reporter plasmid, control vector (pRc/CMV2), or rabbit PPARgamma expression vector (PPARgamma /CMV). Eight hours after transfection, cells were treated with troglitazone (5 µM) and 15-deoxy-Delta 12,14 prostaglandin J2 (15dPGJ2; 1 µM) for 14 h. Values are means ± SE of 4 wells in a single experiment representative of 3 independent experiments (n = 4). *P < 0.05. **P < 0.01.

Effects of PPARgamma ligands on mesangial cell growth and viability. To examine whether PPARgamma activation modulated glomerular mesangial cell proliferation, [3H]thymidine incorporation was assessed. As shown in Fig. 8, two structurally distinct PPARgamma -specific ligands, troglitazone and 15 dPGJ2, significantly inhibited [3H]thymidine incorporation in a concentration-dependent manner. At higher concentrations, troglitazone (>15 µM) or 15 dPGJ2 (>10 µM) also decreased cell viability as measured by an MTT assay (Fig. 8).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   PPARgamma ligands troglitazone and 15dPGJ2 inhibit [3H]thymidine incorporation (A) and decrease cell viability (B) of rabbit mesangial cells. Quiescent mesangial cells were treated with troglitazone and 15dPGJ2 at concentrations of 1, 5, 10, 15, and 20 µM for 24 h. Cell growth was assessed by [3H]thymidine incorporation, and cell viability was measured by 3-[4,5-dimethylthiozole]-2,5-diphynyl tetrazolium bromide (MTT) assay. Values are means ± SE from a single experiment, represent 2 independent experiments, and are expressed as percent inhibition compared with untreated cells (n = 4). *P < 0.05. #P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies provide evidence that PPARgamma is both present and functionally active in vivo in renal glomeruli and in vitro in cultured mesangial cells. To facilitate these studies, we cloned rabbit PPARgamma . The two PPARgamma isoforms identified corresponded to human PPARgamma 1 and PPARgamma 3 (10, 11), and both isoforms were expressed in the rabbit kidney, glomeruli, and mesangial cells. In humans, the PPARgamma gene spans ~100 kb and comprises 9 exons (10, 15). As a result of different transcription start sites and differential splicing, three isoforms, designated PPARgamma 1, PPARgamma 2, and PPARgamma 3, are produced from this single human gene. PPARgamma 1 and PPARgamma 3 mRNAs give rise to an identical protein product, i.e., PPARgamma 1, whereas PPARgamma 2 mRNA encodes a protein that contains an additional NH2 terminus with 28 additional amino acids and appears to be relatively selective for adipocytes (42). Similar to reports in humans (10, 11), rabbit PPARgamma 1 and PPARgamma 3 sequences are identical except for the upstream end of the 5'-UTR. The predicted amino acid sequences for rabbit PPARgamma 1 and PPARgamma 3 are highly conserved compared with those in humans, cows, pigs, mice, and rats, with 95-98% identities for all species. As with other nuclear receptors, rabbit PPARgamma contains a putative DNA-binding domain and a ligand-binding domain. Transfection with the cloned full-length rabbit PPARgamma 1 further activated luciferase expression, thereby documenting functional activity of this clone. Northern blot analysis of total RNA from rabbit peritoneal white adipose tissue revealed a major mRNA transcript of ~1.8 kb, consistent with the predicted cDNA sequence and the size of the cloned rabbit PPARgamma 1. RNase protection assays demonstrate relatively low but significant expression levels of PPARgamma mRNAs in the kidney (17). The present studies used a glomerular isolation technique to provide sufficient material to perform a highly specific nuclease protection assay confirming PPARgamma expression in this preparation. Furthermore, by examining the expression of PPARgamma in kidneys from TZD-treated rabbits, we were able to demonstrate glomerular expression of PPARgamma by in situ hybridization. These findings contrast with earlier studies that suggested PPARgamma was limited to collecting ducts in human, rabbit, and rat kidney (17, 23). It seems likely that these differences may reflect the induction of PPARgamma mRNA by TZD ligands as well as the limitations of in situ hybridization in detecting lower and diffuse mRNA expression levels in kidneys from untreated rabbits. The expression of PPARgamma in freshly isolated glomeruli is consistent with the possibility that antidiabetic TZDs could directly affect glomerular function.

Functional activity of glomerular PPARgamma in vivo was supported by demonstrating the capacity of the PPARgamma ligand troglitazone to induce expression of a downstream target gene, A-FABP (also known as aP2) (37, 41). The present studies were facilitated by cloning a fragment of rabbit A-FABP cDNA (GenBank accession no. AF136241), which was 92 and 89% homologous with human and mouse A-FABP, respectively (4, 5). As expected, in situ hybridization showed the highest endogenous A-FABP expression in perirenal fat adipocytes (Fig. 3). Treatment with troglitazone dramatically upregulated A-FABP expression in rabbit renal parenchyma. In the renal cortex, both A-FABP mRNA and immunoreactivity were selectively induced in glomeruli, consistent with functionally active glomerular PPARgamma . Surprisingly, troglitazone also markedly induced A-FABP expression in vasa recta of the renal medulla rather than in the collecting ducts, which abundantly express PPARgamma (17). This observation suggests PPARgamma -driven A-FABP expression is tissue specific and is consistent with the possibility that the A-FABP promoter may be suppressed in collecting ducts. These phenomena might be explained by the fact that in certain cells PPARgamma activity depends on the availability and accessibility of several cofactors, including SRC-1, PBP, CBP, PGC-1, and ARA70 (18). It remains to be determined whether these cofactors are differentially expressed in glomeruli and collecting ducts.

In the medulla, A-FABP expression was present in endothelial cells of the vasa recta. Glomerular A-FABP immunoreactivity was localized predominantly in the mesangium. It remains uncertain whether PPARgamma activity is similarly present in glomerular endothelial cells as well. The presence of PPARgamma activity in mesangial cells was further supported by cell culture studies demonstrating that both PPARgamma mRNA and protein are highly expressed in cultured rabbit mesangial cells (Fig. 6). Furthermore, two TZD PPARgamma ligands, troglitazone and TZD2, potently upregulated A-FABP expression in cultured mesangial cells. Transcriptional activity of PPARgamma in cultured mesangial cells was further substantiated by demonstrating that troglitazone and the putative naturally occurring PPARgamma ligand 15-dPGJ2 activated transcription of a PPARgamma luciferase reporter driven by three tandem PPREs, consistent with the presence of endogenous PPARgamma (6). Finally, PPARgamma ligands dose dependently reduced mesangial cell thymidine incorporation, suggesting that these ligands could inhibit mesangial proliferation.

A-FABP belongs to a homologous family of proteins called intracellular lipid-binding proteins (iLBPs) and is involved in storage, trafficking, and solubilization of fatty acids (33, 40). Previous studies had suggested that A-FABP expression was relatively restricted to adipocytes (37). More recently, A-FABP has been recognized in macrophages and monocytes (32). The present studies now identified a subpopulation of cells associated with renal microvasculature that also expresses A-FABP. Cell culture studies suggested that the mesangial cell could be one cell type expressing A-FABP. Heart fatty acid-binding protein (H-FABP), another member of the iLBP family, has also been identified in rat mesangial cells (23). These fatty acid-binding proteins have been suggested to act as differentiation factors in muscle and mammary epithelial cells (36, 39, 44). Loss of A-FABP has also been associated with dedifferentiation of human bladder transitional cell carcinomas (8). The induction of A-FABP by TZD PPARgamma ligands in glomeruli may reflect enhanced mesangial cell differentiation and correspond to mesangial growth inhibition in culture. The full complement of cells expressing A-FABP in glomeruli and vasa recta remains to be established, especially the question of whether these cells include resident marcorphage, microvascular endothelial cells, or associated pericytes (31).

Accumulating evidence suggests that PPARgamma activity plays an important role in insulin sensitivity and affects diabetes and atherogenesis (3, 28). PPARgamma ligands, including the thiazolidinedione troglitazone, pioglitazone (Actos), and rosiglitazone (Avandia), have been proven to be potent antidiabetic drugs in humans (7). These PPARgamma activators significantly lower blood glucose levels and may protect against diabetic nephropathy (6, 45). In humans, troglitazone has been shown to decrease microalbuminuria in patients with diabetic nephropathy (20). Although the renal protective action may be a result of the hypoglycemic, antihypertensive and antihyperlipidemic effects of PPARgamma activators, it is also possible that these drugs exert direct effects on renal glomerular PPARgamma receptors, as demonstrated in the present and other studies (17, 43). PPARgamma ligands also significantly decrease the expression of alpha -smooth muscle actin in cultured rat mesangial cells, a marker of the dedifferentiation marker that is upregulated in diabetic nephropathy (2). This observation further supports the idea that the beneficial effects of PPARgamma activators could be mediated by direct activation of glomerular PPARgamma .

In summary, we report the cloning of the rabbit PPARgamma and provide evidence that endogenous PPARgamma activity is associated with renal glomeruli and medullary microvasculature in vivo. We also demonstrate endogenous PPARgamma activity in cultured glomerular mesangial cells. These results suggest that the renal glomeruli or, more specifically, glomerular mesangial cells could be a direct target for TZD-mediated amelioration of diabetic nephropathy.


    ACKNOWLEDGEMENTS

We thank Dr. Wick Johnson at Warner Lambert, Ann Arbor, MI, for troglitazone and Dr. David Bernlohr for supplying the A-FABP antibody. We also thank Dr. Rajnish A. Gupta for technical assistance in establishing the PPRE luciferase reporter assay.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant PPG P01-DK-38226 (to M. D. Breyer), Diabetes Research Training Grant P60-DK-20593 (to Y. Guan), an American Heart Association Beginning Grant-in-Aid (0160200B to Y. Guan), and a pilot grant from Merck Pharmaceutics (to M. D. Breyer and Y. Guan).

Address for reprint requests and other correspondence: Y. Guan, Div. of Nephrology, S-3223 MCN, Vandebilt Univ. Medical Ctr., Nashville, TN 37232-2372 (E-mail: youfei.guan{at}mcmail.vanderbilt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published July 12, 2001;10.1152/ajprenal.00025.2001

Received 1 January 2001; accepted in final form 10 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aronoff, S, Rosenblatt S, Braithwaite S, Egan JW, Mathisen AL, and Schneider RL. Pioglitazone hydrochloride monotherapy improves glycemic control in the treatment of patients with type 2 diabetes: a 6-month randomized placebo-controlled dose-response study. The Pioglitazone 001 Study Group. Diabetes Care 23: 1605-1611, 2000[Abstract].

2.   Asano, T, Wakisaka M, Yoshinari M, Iino K, Sonoki K, Iwase M, and Fujishima M. Peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expresses in rat mesangial cells and PPARgamma agonists modulate its differentiation. Biochim Biophys Acta 1497: 148-154, 2000[ISI][Medline].

3.   Auwerx, J. PPARgamma , the ultimate thrifty gene. Diabetologia 42: 1033-1049, 1999[ISI][Medline].

4.   Baxa, CA, Sha RS, Buelt MK, Smith AJ, Matarese V, Chinander LL, Boundy KL, and Bernlohr DA. Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA. Biochemistry 28: 8683-8690, 1989[ISI][Medline].

5.   Bernlohr, DA, Angus CW, Lane MD, Bolanowski MA, and Kelly TJ, Jr. Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein. Proc Natl Acad Sci USA 81: 5468-5472, 1984[Abstract].

6.   Buckingham, RE, Al-Barazanji KA, Toseland CDN, Slaughter M, Connor SC, West A, Bond B, Turner NC, and Clapham JC. Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats. Diabetes 47: 1326-1334, 1998[Abstract].

7.   Campbell, IW. Antidiabetic drugs present and future: will improving insulin resistance benefit cardiovascular risk in type 2 diabetes mellitus? Drugs 60: 1017-1028, 2000[ISI][Medline].

8.   Celis, JE, Ostergaard M, Basse B, Celis A, Lauridsen JB, Ratz GP, Andersen I, Hein B, Wolf H, Orntoft TF, and Rasmussen HH. Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res 56: 4782-4790, 1996[Abstract].

9.   Elbrecht, A, Chen Y, Cullinan CA, Hayes N, Leibowitz M, Moller DE, and Berger J. Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma 1 and gamma 2. Biochem Biophys Res Commun 224: 431-437, 1996[ISI][Medline].

10.   Fajas, L, Auboeuf D, Raspé E, Schoonjans K, Lefebvre AM, Saladin R, Najib J, Laville M, Fruchart JC, Deeb S, Vidal-Puig A, Flier J, Briggs MR, Staels B, Vidal H, and Auwerx J. The organization, promoter analysis, and expression of the human PPARgamma gene. J Biol Chem 272: 18779-18789, 1997[Abstract/Free Full Text].

11.   Fajas, L, Fruchart JC, and Auwerx J. PPARgamma 3 mRNA: a distinct PPARgamma mRNA subtype transcribed from an independent promoter. FEBS Lett 438: 55-60, 1998[ISI][Medline].

12.   Fonseca, VA, Valiquett TR, Huang SM, Ghazzi MN, and Whitcomb RW. Troglitazone monotherapy improves glycemic control in patients with type 2 diabetes mellitus: a randomized, controlled study. The Troglitazone Study Group. J Clin Endocrinol Metab 83: 3169-3176, 1998[Abstract/Free Full Text].

13.   Forman, B, Tontonoz P, Chen J, Brun R, Spiegelman B, and Evans R. 15-Deoxy-Delta 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR-gamma. Cell 83: 803-812, 1995[ISI][Medline].

14.   Fujii, M, Takemura R, Yamaguchi M, Hasegawa G, Shigeta H, Nakano K, and Kondo M. Troglitazone (CS-045) ameliorates albuminuria in streptozotocin-induced diabetic rats. Metabolism 46: 981-983, 1997[ISI][Medline].

15.   Greene, ME, Blumberg B, McBride OW, Yi HF, Kronquist K, Kwan K, Hsieh L, Greene G, and Nimer SD. Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping. Gene Expression 4: 281-299, 1995[Medline].

16.   Guan, Y, Zhang Y, Breyer RM, Davis L, and Breyer MD. Expression of peroxisome proliferator-activated receptor gamma  (PPARgamma ) in human transitional bladder cancer and its role in inducing cell death. Neoplasia 1: 330-339, 1999[Medline].

17.   Guan, Y, Zhang Y, Davis L, and Breyer MD. Expression of peroxisome proliferator-activated receptors in urinary tract of rabbits and humans. Am J Physiol Renal Physiol 273: F1013-F1022, 1997[Abstract/Free Full Text].

18.   Heinlein, CA, Ting HJ, Yeh S, and Chang C. Identification of ARA70 as a ligand-enhanced coactivator for the peroxisome proliferator-activated receptor gamma. J Biol Chem 274: 16147-16152, 1999[Abstract/Free Full Text].

19.   Hunt, CR, Ro JH, Dobson DE, Min HY, and Spiegelman BM. Adipocyte P2 gene: developmental expression and homology of 5'-flanking sequences among fat cell-specific genes. Proc Natl Acad Sci USA 83: 3786-3790, 1986[Abstract].

20.   Imano, E, Kanda T, Nakatani Y, Nishida T, Arai K, Motomura M, Kajimoto Y, Yamasaki Y, and Hori M. Effect of troglitazone on microalbuminuria in patients with incipient diabetic nephropathy. Diabetes Care 21: 2135-2139, 1998[Abstract].

21.   Isshiki, K, Haneda M, Koya D, Maeda S, Sugimoto T, and Kikkawa R. Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats. Diabetes 49: 1022-1032, 2000[Abstract].

22.   Jiang, C, Ting AT, and Seed B. PPARgamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82-86, 1998[ISI][Medline].

23.   Kimura, H, Fujii H, Suzuki S, Ono T, Arakawa M, and Gejyo F. Lipid-binding proteins in rat and human kidney. Kidney Int Suppl 71: S159-S162, 1999[Medline].

24.   Law, RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, and Faxon D. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest 98: 1897-1905, 1996[Abstract/Free Full Text].

25.   Macconi, D, Benigni A, Morigi M, Ubiali A, Orisio S, Livio M, Perico N, Bertani T, Remuzzi G, and Patrono C. Enhanced glomerular thromboxane A2 mediates some pathophysiologic effect of platelet-activating factor in rabbit nephrotoxic nephritis: evidence from biochemical measurements and inhibitor trials. J Lab Clin Med 113: 549-560, 1989[ISI][Medline].

26.   Malinowski, JM, and Bolesta S. Rosiglitazone in the treatment of type 2 diabetes mellitus: a critical review. Clin Ther 22: 1149-1168, 2000[ISI].

27.   McCarthy, KJ, Routh RE, Shaw W, Walsh K, Welbourne TC, and Johnson JH. Troglitazone halts diabetic glomerulosclerosis by blockade of mesangial expansion. Kidney Int 58: 2341-2350, 2000[ISI][Medline].

28.   Michalik, L, and Wahli W. Peroxisome proliferator-activated receptors: three isotypes for a multitude of functions. Curr Opin Biotechnol 10: 564-570, 1999[ISI][Medline].

29.   Murphy, GJ, and Holder JC. PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol Sci 21: 469-474, 2000[ISI][Medline].

30.   Olefsky, JM, and Saltiel AR. PPARgamma and the treatment of insulin resistance. Trends Endocrinol Metab 11: 362-368, 2000[ISI][Medline].

31.   Park, F, Mattson DL, Roberts LA, and Cowley AW, Jr. Evidence for the presence of smooth muscle alpha -actin within pericytes of the renal medulla. Am J Physiol Regulatory Integrative Comp Physiol 273: R1742-R1748, 1997[Abstract/Free Full Text].

32.   Pelton, PD, Zhou L, Demarest KT, and Burris TP. PPARgamma activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes. Biochem Biophys Res Commun 261: 456-458, 1999[ISI][Medline].

33.   Reese-Wagoner, A, Thompson J, and Banaszak L. Structural properties of the adipocyte lipid binding protein. Biochim Biophys Acta 1441: 106-116, 1999[ISI][Medline].

34.   Ricote, M, Huang J, Fajas L, Li A, Welch J, Najib J, Witztum JL, Auwerx J, Palinski W, and Glass CK. Expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci USA 95: 7614-7619, 1998[Abstract/Free Full Text].

35.   Ricote, M, Li AC, Willson TM, Kelly CJ, and Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391: 79-82, 1998[ISI][Medline].

36.   Rump, R, Buhlmann C, Borchers T, and Spener F. Differentiation-dependent expression of heart type fatty acid-binding protein in C2C12 muscle cells. Eur J Cell Biol 69: 135-142, 1996[ISI][Medline].

37.   Sandouk, T, Reda D, and Hofmann C. Antidiabetic agent pioglitazone enhances adipocyte differentiation of 3T3-F442A cells. Am J Physiol Cell Physiol 264: C1600-C1608, 1993[Abstract/Free Full Text].

38.   Shu, H, Wong B, Zhou G, Li Y, Berger J, Woods JW, Wright SD, and Cai TQ. Activation of PPARalpha or gamma  reduces secretion of matrix metalloproteinase 9 but not interleukin 8 from human monocytic THP-1 cells. Biochem Biophys Res Commun 267: 345-349, 2000[ISI][Medline].

39.   Specht, B, Bartetzko N, Hohoff C, Kuhl H, Franke R, Borchers T, and Spener F. Mammary derived growth inhibitor is not a distinct protein but a mix of heart-type and adipocyte-type fatty acid-binding protein. J Biol Chem 271: 19943-19949, 1996[Abstract/Free Full Text].

40.   Storch, J, and Thumser AE. The fatty acid transport function of fatty acid-binding proteins. Biochim Biophys Acta 1486: 28-44, 2000[ISI][Medline].

41.   Thuillier, P, Baillie R, Sha X, and Clarke SD. Cytosolic and nuclear distribution of PPARgamma2 in differentiating 3T3-L1 preadipocytes. J Lipid Res 39: 2329-2338, 1998[Abstract/Free Full Text].

42.   Tontonoz, P, Hu E, Graves RA, Budavari AI, and Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8: 1224-1234, 1994[Abstract].

43.   Yang, T, Michele DE, Park J, Smart AM, Lin Z, Brosius FC, III, Schnermann JB, and Briggs JP. Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney. Am J Physiol Renal Physiol 277: F966-F973, 1999[Abstract/Free Full Text].

44.   Yang, Y, Spitzer E, Kenney N, Zschiesche W, Li M, Kromminga A, Muller T, Spener F, Lezius A, Veerkamp JH, Smith GH, Salomon DS, and Grosse R. Members of the fatty acid binding protein family are differentiation factors for the mammary gland. J Cell Biol 127: 1097-1109, 1994[Abstract].

45.   Yoshimoto, T, Naruse M, Nishikawa M, Naruse K, Tanabe A, Seki T, Imaki T, Demura R, Aikawa E, and Demura H. Antihypertensive and vasculo- and renoprotective effects of pioglitazone in genetically obese diabetic rats. Am J Physiol Endocrinol Metab 272: E989-E996, 1997[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1036-F1046
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society