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
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ABSTRACT |
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First published July 12, 2001;
10.1152/ajprenal.00025.2001.Peroxisome proliferator-activated
receptor-
(PPAR
) 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 PPAR
in glomeruli in vivo. The purpose of this study
was to determine whether PPAR
is expressed in renal glomeruli. Two
rabbit PPAR
isoforms were cloned. Nuclease protection assays
demonstrate that both PPAR
isoforms are expressed in freshly
isolated glomeruli. Treatment of rabbits with the TZD troglitazone
selectively induced expression of an endogenous PPAR
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
PPAR
expression was detected in cultured rabbit MCs. Endogenous MC PPAR
can also drive PPAR
reporter. Troglitazone and
15-deoxy-
12,14 prostaglandin J2 at low
concentrations reduced mesangial cell [3H]thymidine
incorporation without affecting viability. These data suggest that
constitutive PPAR
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
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INTRODUCTION |
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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 -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 RXR
, 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 PPAR
,
PPAR
/
, and PPAR
, have been cloned from Xenopus
laevis, mouse, rat, and humans. A role for PPAR
in promoting
fat cell formation (i.e., adipogenesis) is well established
(13). Recent reports demonstrate that PPAR
is also
highly expressed in renal epithelial cells (17, 43) as
well as several epithelial cancers (3), where TZD PPAR
ligands induce growth arrest and cell differentiation (16,
29). It has also been reported that PPAR
is highly expressed in monocytes/macrophages, where it may modulate transcription of
inflammatory cytokines (22, 35). In addition, PPAR
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 PPAR
plays diverse roles in cell growth,
differentiation, and extracellular matrix accumulation.
PPAR 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-
in glomeruli from streptozotocin-induced diabetic rats
(21). These effects in insulin-dependent diabetes mellitus
(type I) suggest that PPAR
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 PPAR
ligands.
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MATERIALS AND METHODS |
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5'-Rapid amplification of cDNA ends.
5'-Rapid amplification of cDNA ends (5'-RACE) was used to obtain the
5'-ends of rabbit PPAR1 using a previously cloned fragment of rabbit
PPAR
(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 PPAR 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 PPAR1 and determination of the
tissue distribution by RT-PCR analysis.
Full-length rabbit PPAR
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 PPAR
1 and PPAR
3 isoforms in
mesangial cells, isoform-specific primers were designed for RT-PCR. For
amplification of PPAR
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 PPAR
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 PPAR1 mRNA expression in renal glomeruli,
PPAR
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 PPAR
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 PPAR
1 (456 bp), the PPAR
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 [
-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 PPAR (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 PPAR 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 PPAR
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 PPAR 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 · kg1 · 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 PPAR1 cDNA was cloned into the pRc/CMV2 expression vector
(PPAR
/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 PPAR
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-
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 PPAR 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).
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RESULTS |
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cDNA cloning of full-length rabbit PPAR isoforms and mRNA
expression in rabbit kidney.
A 758-bp fragment of PPAR
was amplified by PCR from rabbit adipose
tissue, yielding a region of rabbit PPAR
with homology to human and
mouse PPAR
as previously reported (17). 5'-RACE yielded
two distinct clones by restriction enzyme digestion. The shorter
clone (rabPPAR
3) comprised 407 bp, whereas the longer clone
(rabPPAR
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 (rabPPAR
3,
146 to
88 bp and rabPPAR
1,
471 to
88 bp) (Fig.
1). No PPAR
2 homolog was found in any of the 5'-RACE clones.
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PPAR expression in native and TZD-treated rabbit glomeruli.
An RNase protection assay was used to examine the expression of
PPAR
1 in native renal glomeruli. PPAR
1 mRNA was detected in
freshly isolated glomeruli (Fig. 2B). Intestinal tissue
served as a positive control. To determine whether endogenous
glomerular PPAR
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 PPAR
and a well-established PPAR
target gene, A-FABP (37). As previous reported
(17), PPAR
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
PPAR
expression, as assessed by in situ hybridization (Fig.
2C).
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PPAR expression in cultured glomerular mesangial cells.
To further characterize the intraglomerular cells expressing PPAR
,
rabbit glomerular mesangial cells were cultured and examined for
PPAR
. Both PPAR
1 and PPAR
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 PPAR
in cultured mesangial cells.
Mesangial cells displayed the expected protected fragment of 316 bp for
PPAR
mRNA. PPAR
protein was also recognized as a ~55-kDa
protein band, using an anti-PPAR
antibody on the immunoblot.
Finally, as observed in the preceding in vivo studies, mesangial
PPAR
mRNA expression appeared to increase after treatment with the
TZD troglitazone (10 µM) for 6 h.
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Induction of A-FABP gene expression by PPAR ligands in cultured
glomerular mesangial cells.
The expression of the PPAR
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 PPAR in mesangial cells.
Endogenous PPAR
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 PPAR
. Cotransfection of PPRE-3x-luciferease with exogenous rabbit PPAR
(PPAR
/CMV) further activated luciferase expression (Fig.
7B), demonstrating that the full-length rabbit PPAR
clone
contains all domains essential for activating target gene
transcription.
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Effects of PPAR ligands on mesangial cell growth and viability.
To examine whether PPAR
activation modulated glomerular mesangial
cell proliferation, [3H]thymidine incorporation was
assessed. As shown in Fig. 8, two structurally distinct PPAR
-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).
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DISCUSSION |
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The present studies provide evidence that PPAR 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
PPAR
. The two PPAR
isoforms identified corresponded to human
PPAR
1 and PPAR
3 (10, 11), and both isoforms were expressed in the rabbit kidney, glomeruli, and mesangial cells. In
humans, the PPAR
gene spans ~100 kb and comprises 9 exons (10, 15). As a result of different transcription start
sites and differential splicing, three isoforms, designated PPAR
1, PPAR
2, and PPAR
3, are produced from this single human gene. PPAR
1 and PPAR
3 mRNAs give rise to an identical protein product, i.e., PPAR
1, whereas PPAR
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 PPAR
1 and PPAR
3 sequences are identical except for the
upstream end of the 5'-UTR. The predicted amino acid sequences for
rabbit PPAR
1 and PPAR
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 PPAR
contains a
putative DNA-binding domain and a ligand-binding domain. Transfection
with the cloned full-length rabbit PPAR
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 PPAR
1. RNase protection assays demonstrate relatively low but
significant expression levels of PPAR
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 PPAR
expression in this preparation. Furthermore, by examining the expression of PPAR
in
kidneys from TZD-treated rabbits, we were able to demonstrate glomerular expression of PPAR
by in situ hybridization. These findings contrast with earlier studies that suggested PPAR
was limited to collecting ducts in human, rabbit, and rat kidney (17, 23). It seems likely that these differences may reflect the induction of PPAR
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 PPAR
in
freshly isolated glomeruli is consistent with the possibility that
antidiabetic TZDs could directly affect glomerular function.
Functional activity of glomerular PPAR in vivo was supported by
demonstrating the capacity of the PPAR
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 PPAR
.
Surprisingly, troglitazone also markedly induced A-FABP expression in
vasa recta of the renal medulla rather than in the collecting ducts,
which abundantly express PPAR
(17). This observation
suggests PPAR
-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 PPAR
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 PPAR
activity is similarly present in glomerular endothelial cells as well.
The presence of PPAR
activity in mesangial cells was further
supported by cell culture studies demonstrating that both PPAR
mRNA
and protein are highly expressed in cultured rabbit mesangial cells
(Fig. 6). Furthermore, two TZD PPAR
ligands, troglitazone and TZD2,
potently upregulated A-FABP expression in cultured mesangial cells.
Transcriptional activity of PPAR
in cultured mesangial cells was
further substantiated by demonstrating that troglitazone and the
putative naturally occurring PPAR
ligand 15-dPGJ2 activated
transcription of a PPAR
luciferase reporter driven by three tandem
PPREs, consistent with the presence of endogenous PPAR
(6). Finally, PPAR
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
PPAR 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 PPAR activity plays an important
role in insulin sensitivity and affects diabetes and atherogenesis
(3, 28). PPAR
ligands, including the thiazolidinedione troglitazone, pioglitazone (Actos), and rosiglitazone (Avandia), have
been proven to be potent antidiabetic drugs in humans (7). These PPAR
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 PPAR
activators,
it is also possible that these drugs exert direct effects on renal
glomerular PPAR
receptors, as demonstrated in the present and other
studies (17, 43). PPAR
ligands also significantly
decrease the expression of
-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 PPAR
activators could be mediated by direct activation of glomerular
PPAR
.
In summary, we report the cloning of the rabbit PPAR and provide
evidence that endogenous PPAR
activity is associated with renal
glomeruli and medullary microvasculature in vivo. We also demonstrate
endogenous PPAR
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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