Upregulation of type I collagen by TGF-beta in mesangial cells is blocked by PPARgamma activation

Feng Zheng1, Alessia Fornoni1, Sharon J. Elliot1, Youfei Guan2, Matthew D. Breyer2, Liliane J. Striker1, and Gary E. Striker1

1 Vascular Biology Institute and Division of Nephrology, Department of Medicine, University of Miami School of Medicine, Miami, Florida 33136; and 2 Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Vanderbilt, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We found that peroxisome proliferator-activated receptor-gamma (PPARgamma ) mRNA was reduced by 77% in glomeruli of diabetic mice. Because mesangial cells play an important role in diabetic nephropathy, we examined regulation of type I collagen expression by PPARgamma and transforming growth factor-beta 1 (TGF-beta 1) in mouse mesangial cells in the presence of 6 and 25 mM glucose. Mesangial cells contained functionally active PPARgamma . Exposure to 25 mM glucose resulted in reduced PPARgamma expression and transcriptional activity, accompanied by increased type I collagen expression. Restoration of PPARgamma activity to normal levels in cells cultured in 25 mM glucose, by transfection with a PPARgamma expression construct and treatment with the PPARgamma agonist troglitazone, returned type I collagen levels toward normal values. Activation of PPARgamma by troglitazone also decreased type I collagen mRNA and blocked TGF-beta 1-mediated upregulation of type I collagen mRNA and protein. Moreover, PPARgamma activation suppressed basal and activated TGF-beta 1 responses in mesangial cells. This action was blocked by transfection of cells with a dominant-negative PPARgamma construct. In summary, PPARgamma suppresses the increased type I collagen mRNA and protein expression mediated by TGF-beta 1 in mesangial cells.

peroxisome proliferator-activated receptor-gamma ; transforming growth factor-beta 1; diabetic nephropathy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PEROXISOME proliferator-activated receptor (PPAR) is a family of ligand-activated transcription factors (19). Three PPAR subtypes (alpha , beta , and gamma ) have been identified and cloned. PPARs regulate gene transcription by directly binding to repeat-1 sites [peroxisome proliferator response elements (PPRE)] in targeted gene promoters or upstream enhancer regions as heterodimeric complexes with the retinoid X receptor (RXR). PPARalpha is expressed at high levels in the liver, kidney, and heart and at lower levels in the retina and pancreatic islets (5, 19). PPARalpha activation results in proliferation of peroxisomes and induction of hepatic genes involved in fatty acid beta -oxidation (5). PPARbeta is ubiquitously expressed in many tissues, but its function and the genes it regulates have not been completely elucidated (5). PPARgamma is expressed at high levels in adipose tissue and is a key regulator of adipocyte differentiation. In addition, PPARgamma has also been found to play an important role in regulation of systemic insulin sensitivity. Severe insulin resistance and diabetes mellitus have been shown in patients with PPARgamma mutations (2). Thiazolidinediones (TZD), insulin-sensitizing agents, have been recognized as ligands for PPARgamma (20). Recently, PPARgamma was also found in a variety of nonadipose tissues and cells, including skeletal muscle, heart, kidney, bone marrow stromal cells, neutrophils, lymphocytes, and macrophages (8, 9, 17, 37, 39).

PPARgamma expression in macrophages and lymphocytes regulates inflammatory responses in a negative fashion (17, 39), suggesting a function of this receptor distinct from its metabolic activity. Further evidence for these nonmetabolic functions was obtained in studies of diabetic rats, where it was found that troglitazone, one of the TZD family of PPARgamma ligands, prevented albuminuria and increased glomerular extracellular matrix (ECM) gene expression (14). These effects were obtained in the absence of changes in systemic blood glucose levels. A similar improvement in glomerular lesions was also found in Zucker rats, a model of non-insulin-dependent diabetic glomerulosclerosis, and in 5/6 nephrectomized rats (3, 23, 24). Taken together, these data indicate that TZD may have direct effects on glomeruli by mechanisms other than their insulin-sensitizing activity. The finding that PPARgamma and RXR were expressed in rat glomerular mesangial cells further supports this possibility (1, 28).

The pathological hallmark of diabetic glomerular lesions is ECM accumulation, including type I collagen (7, 35). Although endothelial, epithelial, and mesangial cells are involved in glomerular ECM turnover, mesangial cells appear to be the major source of pathological glomerular ECM (21). An imbalance between ECM synthesis and degradation by mesangial cells contributes directly to the development and progression of diabetic glomerular lesions (22). Nuclear receptors, including estrogen receptors, the retinoic acid receptor, and RXR, are known to regulate expression of ECM and metalloproteinase genes in various tissues and cells, including mesangial cells (27, 29, 32). Although a PPARgamma response element (PPRE) has not been found upstream of ECM genes, there is evidence suggesting that PPARgamma may participate in regulation of ECM genes, especially type I collagen. First, peroxisome proliferator reduced type I collagen and elastin gene expression in lung fibroblasts (25). Second, ligand-induced activation of PPARgamma inhibited type I collagen promoter activity and decreased alpha 1 type I collagen mRNA levels in hepatic stellate cells (26). Finally, TZD treatment decreased glomerular ECM gene expression and protein accumulation in diabetic rats and in 5/6 nephrectomized rats (14, 23). Mesangial cells, fibroblasts, and activated stellate cells share several aspects of ECM turnover (18). Therefore, we hypothesized that one of the functions of PPARgamma in mesangial cells was to regulate type I collagen expression.

Transforming growth factor-beta (TGF-beta ) is one of the key regulators of ECM genes in mesangial cells. Elevated glomerular TGF-beta 1 levels have been shown to make a significant contribution to the pathogenesis of diabetic glomerular lesions, including accumulation of type I collagen (15, 34). Recently, glomerular TGF-beta 1 expression was found to be decreased in diabetic rats and 5/6 nephrectomized rats treated with troglitazone (14, 23). We postulated that the effect of PPARgamma on type I collagen expression might be mediated by its inhibition of TGF-beta 1.

We found that PPARgamma expression was reduced in mesangial cells in conditions associated with an increase in type I collagen expression, namely, cells cultured in 25 mM glucose and glomeruli isolated from animals with diabetic nephropathy. In addition, we found that activation of PPARgamma resulted in suppression of type I collagen expression. Thus one possible mechanism by which PPARgamma could suppress type I collagen expression in mesangial cells is via inhibition of TGF-beta responses.


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

Mesangial cell culture. Mesangial cells from C57 mice were isolated and characterized as previously described (6). Cells were grown in DMEM-Ham's F-12 mixture supplemented with 20% fetal bovine serum (FBS), 100 U/ml streptomycin, and 100 U/ml penicillin. Cells were maintained in culture medium containing 6 or 25 mM glucose. To test the effect of a PPARgamma ligand on collagen I expression, cells were switched to DMEM-Ham's F-12 mixture containing 0.1% BSA and 2-8 µM troglitazone. Because 20 µM troglitazone was associated with mesangial cell toxicity (20% loss of adherence), we chose 8 µM troglitazone for most experiments. At this concentration, there was no increase in cellular trypan blue staining compared with cells treated with vehicle (0.1% DMSO).

Transfection and reporter gene assay. Transient transfection was performed in cultured mesangial cells using Transfast according to the manufacturer's instructions (Promega, Madison, WI). To examine whether PPARgamma was functionally active in mesangial cells, cells were transfected with a PPARgamma reporter construct containing three copies of the PPRE from the acyl-CoA oxidase gene linked to the thymidine kinase promoter and luciferase vector or an RL-TK empty vector in the presence or absence of the PPARgamma ligand troglitazone. Cotransfection with a beta -galactosidase vector served as an internal control. Cells were harvested 24-48 h after transfection. Luciferase and beta -galactosidase activities were measured as previously described (32). Overexpression of PPARgamma was achieved by transfecting the full-length rabbit PPARgamma obtained from 5'- and 3'-rapid amplification of cDNA ends and cloned into the pcDNA3 expression vector. Mouse dominant-negative PPARgamma was constructed by mutation of highly conserved hydrophobic and charged residues (L466/L467) in helix 12 of the ligand-binding domain to alanine (L466A/L467A), as previously reported in human PPARgamma dominant-negative mutation (2, 10). To test the effect of our dominant-negative PPARgamma construct on basal and ligand-activated PPARgamma transcriptional activity in mesangial cells, cells were transfected with a mouse dominant-negative PPARgamma construct together with a PPAR reporter construct in the presence or absence of the PPARgamma ligand troglitazone. We further examined the dominant-negative function of this construct by cotransfecting it with a full-length PPARgamma cDNA expression vector. Cells transfected with empty pcDNA3 vector served as a control. To determine the role of PPAR activation/inhibition on TGF-beta responses in mesangial cells, the TGF-beta reporter construct p3TP-Lux [a gift from Dr. J. Massague (38)] was introduced into mesangial cells together with a PPARgamma expression vector, a mouse dominant-negative PPARgamma vector, or a control vector in the presence or absence of PPARgamma ligands and recombinant human TGF-beta 1 (4 ng/ml; R&D Systems, Minneapolis, MN). The same amount of DNA was used for each transfection throughout the experiments. The transfection efficiency remained at 28-32%, as determined by transfection of C57 mesangial cells with beta -galactosidase.

Mouse model of diabetes. We reported that diabetic C57 mice had mild glomerular lesions, while diabetic C57 Os/+ mice, in which the number of nephrons was reduced 50% by the Os mutation, developed moderate glomerulosclerosis (40). Therefore, C57 Os/+ mice were selected for this study. Diabetes was induced within 2 wk by 7-14 intraperitoneal injections of streptozotocin (25-50 µg/g body wt) or vehicle (citrate buffer) (40). Mice were weighed, and blood glucose levels were determined at baseline and weekly thereafter. Mice with stable hyperglycemia between 300 and 400 mg/dl were included in the study. Mice were kept without insulin treatment and allowed free access to food and water. After 12 wk, mice were killed according to University of Miami School of Medicine-approved procedures. The left kidney was perfused with a solution containing collagenase and RNase inhibitors for glomerular microdissection (30, 40). The right kidney was perfused with fixative containing 4% freshly prepared paraformaldehyde and was used for microscopic studies.

RT-PCR. Total RNA was extracted from cells or microdissected glomeruli using TriReagent (MRC, Cincinnati, OH). Reverse transcription was performed on 0.3-2 µg of total RNA with oligo(dT)15 primer and avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, IN). Expression of PPARgamma in mesangial cells and diabetic glomeruli was determined by PCR using a specific set of primers: 5'-GACATCCAAGACAACCTGCTG (sense) and 5'-GCAATCAATAGAAGGAACACG (antisense). The amplified samples were separated on a 3% agarose gel and analyzed by densitometry (NIH Image). To determine the linear range of the PCR, the intensity of amplified products was plotted against the cycle number as previously described (data not shown). The optimum number of PCR cycles for PPARgamma was 32 for the mesangial cells and 35 for the glomeruli. Because we found that the glomerular expression of beta -actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) remained stable in diabetic mice compared with nondiabetic mice, beta -actin or GAPDH was used as a housekeeping gene control. Expression levels of alpha 1 type I collagen in mesangial cells were quantitated by competitive PCR using the primers 5'-GTGAACCTGGCAAACAAGGT (sense) and 5'-CTGGAGACCAGAGAAGCCAC (antisense). The alpha 1 type I collagen mutant was created by deletion of a 140-bp fragment from the original PCR product using the primers 5'-GAATCTGGACGTGAAAGAATGGCGATCG (sense) and 5'-CGATCGCCATTCTTTCACGTCCAGATTC (antisense). Competitive PCR was performed by addition of decreasing amounts of mutant to sample tubes, as previously described (30). Data are expressed as the ratio of type I collagen to GAPDH mRNA.

Western blots. Mesangial cells were grown to 80% confluence in 10-cm petri dishes. Cells were scraped off the plates and lysed in ice-cold buffer (20 mM Tris, 140 mM NaCl, 3 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM NaVO4, 10% glycerol, pH 7.4, 1% Triton X-100) with protease inhibitors (1.5 µM aprotinin, 20 µM leupeptin, 50 µM phenylmethylsulfonyl fluoride, 1.5 µM benzamidine). The insoluble material was removed by centrifugation at 20,000 g for 30 min at 40°C. Samples containing equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After incubation with rabbit anti-PPARgamma antibody (Santa Cruz Biotechnology, Santa Cruz, CA), the blots were washed and incubated with peroxidase-conjugated secondary antibody, and protein bands were analyzed using a chemiluminescence kit (Santa Cruz Biotechnology).

ELISA. Mesangial cells (3 × 104) were plated in 24-well plates in medium supplemented with 20% FBS. Cells were transfected with a PPARgamma expression vector, a dominant-negative PPARgamma vector, or a control vector. After 2 h, medium containing 10% FCS and 8 µM troglitazone or 0.1% DMSO was added. TGF-beta 1 (4 ng/ml) or vehicle (4 mM HCl and 1 mg/ml BSA) was added 12 h later. After 24 h, the medium was replaced with medium containing 0.1% FBS, and beta -aminopropionitrile (80 µg/ml) was added. Supernatants were collected, and cells were lysed in 0.5 ml of 5 M guanidine-0.1 M Tris · HCl (pH 8.6) at 48 h in the presence of 20 µM phenylmethylsulfonyl fluoride, 2.5 µM EDTA, and 10 µM N-ethylmaleimide. The amount of type I collagen was determined in the supernatants and cell layers by ELISA (16). Briefly, 96-well ELISA plates were coated with 100 µl of sample or mouse type I collagen standards and incubated at 37°C for 2 h. After they were blocked with PBS + 0.25% BSA + 0.05% Tween 20, the plates were incubated with rabbit anti-type I collagen diluted 1:2,000 (Biodesign International, Kennebunk, ME) at 4°C overnight. A 1:2,000 dilution of a biotinylated goat anti-rabbit IgG was used as secondary antibody (Biosource International, Camarillo, CA). Final values were normalized for cell number.

Statistics. Values are means ± SE. ANOVA and Student's t-test were used. P < 0.05 was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Diminished PPARgamma expression in glomeruli of diabetic mice. Diabetic C57 Os/+ mice developed moderate glomerulosclerosis after 3 mo of untreated diabetes (40). PPARgamma expression was examined by RT-PCR in glomeruli that had been individually microdissected (30) from control and diabetic C57 Os/+ mice. The diabetic mice had persistently elevated glucose levels, which averaged 333 ± 32.66 mg/dl. PPARgamma mRNA levels were conspicuously reduced in diabetic glomeruli (77% reduction compared with nondiabetic controls; Fig. 1). Functionally active PPARgamma is expressed in mesangial cells. To determine whether mesangial cells express PPARgamma , total RNA from C57 mouse mesangial cells was examined by RT-PCR. A 258-bp PPARgamma fragment was obtained by PCR (Fig. 2A). The specificity of the PCR amplification was confirmed by sequence analysis (data not shown). Western blot analysis confirmed the presence of PPARgamma in mesangial cells (Fig. 2B).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Peroxisome proliferator-activated receptor (PPAR)-gamma mRNA levels are reduced in glomeruli isolated from diabetic mice. A: glomeruli were microdissected from C57 Os/+ diabetic and nondiabetic mice and subjected to reverse transcription in situ, and PPARgamma and beta -actin mRNA levels were determined by semiquantitative PCR. Lanes 1-6, glomeruli from 6 diabetic mice; lanes 7-12, glomerular samples from 6 nondiabetic mice. B: results expressed as PPARgamma -to-beta -actin ratio. ** P < 0.01 vs. control.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   PPARgamma is expressed and functionally active in mesangial cells. A: PPARgamma mRNA was demonstrated in 2 mesangial cell lines (lanes 1 and 2) by assessment of total RNA by RT-PCR. beta -Actin was amplified as an internal control. B: bands identified as PPARgamma protein are present in 2 separate mesangial cell lines (lanes 1 and 2) by Western blot analysis using a polyclonal rabbit antibody against human PPARgamma that cross-reacts with mouse PPARgamma . C: PPARgamma is functionally active in mesangial cells. Basal and ligand-mediated activation of PPARgamma was assessed in mesangial cells transfected with 150 ng of a peroxisome proliferator response element (PPRE)-containing reporter construct and 150 ng of a Rous sarcoma virus (RSV) beta -galactosidase plasmid in the presence or absence of a PPARgamma agonist (2-16 µM troglitazone). Basal PPRE activity is arbitrarily defined as 1. Troglitazone-induced transcriptional activity is expressed as a function of basal activity. Values are means ± SE of quadruplicate measurements from 3 independent experiments. * P < 0.05 vs. basal activity. D: exogenous PPARgamma increases PPAR reporter activity. Cells were transfected with 200 ng of pcDNA3 PPARgamma expression vector and 150 ng of PPRE reporter construct in the presence or absence of 8 µM troglitazone. ** P < 0.01 vs. basal activity. E: suppression of basal and ligand-mediated activation of PPARgamma by a dominant-negative PPARgamma (DNPPARgamma ) construct. Cells were transfected with 200 ng of an L466A/L467A PPARgamma (DNPPARgamma ) expression vector and 150 ng of a PPRE reporter construct in the presence or absence of 8 µM troglitazone. ** P < 0.01 vs. basal activity. F: inhibition of exogenous PPARgamma stimulates PPAR transcriptional activity by DNPPARgamma . Cells were transfected with 200 ng of an L466A/L467A DNPPARgamma expression vector, 200 ng of a wild-type PPARgamma expression vector or a pcDNA3 empty vector, and 150 ng of a PPRE reporter construct. ** P < 0.01 vs. cells transfected with a pcDNA3 empty vector; # P < 0.05 vs. cells transfected with a wild-type PPARgamma expression vector or a wild-type PPARgamma expression vector and a pcDNA3 empty vector.

To test whether PPARgamma in mesangial cells is functionally active, mesangial cells were transfected with a PPAR reporter vector in the presence or absence of troglitazone. As shown in Fig. 2C, the PPAR-dependent PPRE-driven promoter was activated by addition of the PPARgamma agonist troglitazone (2-fold), suggesting the presence of a functionally active PPAR response. As expected, cotransfection with a PPARgamma expression vector significantly increased (3.1-fold) the reporter activity (Fig. 2D), especially in the presence of troglitazone (9.6-fold). Transfection of cells with the empty pcDNA3 vector did not affect basal or troglitazone-activated PPARgamma transcriptional responses (data not shown).

Cotransfection with a mouse dominant-negative PPARgamma construct significantly suppressed the reporter activity (67% inhibition; Fig. 2E) and also decreased the transcriptional response to troglitazone (25% elevation; P > 0.05 vs. untreated cell; Fig. 2E). The dominant-negative function of the L466A/L467A PPARgamma cDNA construct was further tested by cotransfecting the mutant construct with the wild-type PPARgamma cDNA expression vector. As shown in Fig. 2F, dominant-negative PPARgamma significantly inhibited (44%) the increase of PPARgamma transcriptional responses induced by PPARgamma overexpression.

Diminished PPARgamma expression in mesangial cells exposed to 25 mM glucose. PPARgamma mRNA levels were significantly reduced in mesangial cells cultured in 25 mM glucose (Fig. 3, A and B). Similarly, Western blot analysis revealed that the PPARgamma protein levels were reduced in mesangial cells cultured in 25 mM glucose (Fig. 3C). To test whether the decrease of PPARgamma expression was associated with diminished PPARgamma responses in cells cultured in 25 mM glucose, cells were transfected with the PPAR reporter construct or the empty RL-TK vector and studied in the presence or absence of troglitazone. In agreement with PPARgamma mRNA and protein levels, the PPARgamma response was decreased (65%) in mesangial cells cultured in 25 mM glucose (Fig. 3D). Although troglitazone still induced a PPARgamma response in cells cultured in 25 mM glucose, the levels did not reach that in unstimulated cells cultured in 6 mM glucose (Fig. 3D). Transfection of cells cultured in 25 mM glucose with a PPARgamma expression vector restored PPARgamma transcription to levels comparable to that in cells cultured in 6 mM glucose, and this was further activated by troglitazone (Fig. 3D). PPARgamma transcriptional activity remained low in cells cultured in 25 mM glucose that had been transfected with the empty pcDNA3 vector (data not shown).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of glucose levels on PPARgamma levels. A and B: PPARgamma expression is decreased in mesangial cells cultured in 25 mM glucose. PPARgamma mRNA expression was measured by semiquantitative RT-PCR in cells cultured with 6 mM (lanes 1-3) or 25 mM (lanes 4-6) glucose for 4 wk. * P < 0.05 vs. cells cultured in 6 mM glucose. C: PPARgamma protein bands are less intense in mesangial cells cultured in 25 mM glucose. PPARgamma protein expression was determined by Western blot analysis in cells cultured in 6 mM (lanes 1 and 2) and 25 mM (lanes 3 and 4) glucose. D: mesangial cells cultured in 25 mM glucose exhibit a 60% reduction in basal PPARgamma transcriptional activity. Mesangial cells were transfected with 150 ng of a PPRE reporter construct, 150 ng of an RSV beta -galactosidase plasmid, and 200 ng of PPARgamma expression vector or DNPPARgamma in the presence or absence of 8 µM troglitazone. Basal PPARgamma transcriptional activity in mesangial cells cultured in 6 mM glucose is arbitrarily defined as 1. PPARgamma activity was partially restored by introduction of a PPARgamma expression vector and was further increased by troglitazone. DNPPARgamma completely blocked reporter activity, even when a PPARgamma agonist was added. ** P < 0.01 vs. basal activity in 6 mM glucose; # P < 0.05 and ## P < 0.01 vs. basal activity in 25 mM glucose.

Decreased type I collagen expression by PPARgamma activation. To determine the effects of PPARgamma activation on type I collagen expression, 1 × 105 mesangial cells cultured in six-well plates were collected 6-24 h after addition of troglitazone or DMSO. Type I collagen mRNA levels were determined by competitive PCR. Addition of a PPARgamma agonist for 24 h inhibited type I collagen mRNA expression in a dose-dependent fashion (Fig. 4, A and B). To determine whether the suppressive effect of the PPARgamma agonist on type I collagen expression in mesangial cells was mediated via the PPARgamma pathway, mesangial cells were transfected with a dominant-negative PPARgamma expression vector in the presence or absence of troglitazone. The suppressive effect of troglitazone on mesangial cell type I collagen expression was blocked by the transfected dominant-negative PPARgamma expression vector (Fig. 4C). Furthermore, we found that type I collagen expression was significantly increased in mesangial cells transfected with dominant-negative PPARgamma expression vector (Fig. 4, C and D). Transfection of cells with an empty pcDNA3 vector had no effect on type I collagen expression (data not shown). These data indicated that inhibition of mesangial cell type I collagen expression with troglitazone was mediated via the PPARgamma transcriptional pathway.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Activation of the PPARgamma pathway by PPARgamma agonist suppresses type I collagen expression. alpha 1 Type I collagen and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were assessed by competitive RT-PCR using total mesangial cell RNA. Results are expressed as the ratio of alpha 1 type I collagen to GAPDH mRNA. A: PPARgamma activation reduces alpha 1 type I collagen mRNA as shown by representative competitive PCR for alpha 1 type I collagen mRNA levels in mesangial cells treated with vehicle (0.1% DMSO) or 8 µM troglitazone. cDNA (wild-type) from different-treated cells was amplified with decreasing amounts of competing mutant (from left to right: 0.1-0.00125 amol). Type I collagen expression is reduced by addition of troglitazone. B: alpha 1 type I collagen mRNA expression is inhibited in a dose-dependent manner by a PPARgamma agonist. * P < 0.05 and ** P < 0.01 vs. untreated cells. C: DNPPARgamma increases alpha 1 type I collagen mRNA expression. alpha 1 Type I collagen mRNA levels were assessed by competitive PCR in mesangial cells treated with DNPPARgamma or vehicle (0.1% DMSO). DNPPARgamma was introduced into mesangial cells as described in MATERIALS AND METHODS. Cells transfected with DNPPARgamma exhibited increased alpha 1 type I collagen mRNA expression at baseline. D: DNPPARgamma blocks PPARgamma agonist-induced suppression of alpha 1 type I collagen mRNA expression. Troglitazone did not suppress alpha 1 type I collagen mRNA expression in DNPPARgamma -transfected mesangial cells. Moreover, alpha 1 type I collagen mRNA expression was increased at baseline in cells transfected with the DNPPARgamma construct. ** P < 0.01 vs. untreated cells.

Decreased type I collagen expression by restoration of PPARgamma expression in mesangial cells cultured in 25 mM glucose. To examine the effect of a decrease in PPARgamma transcription on type I collagen expression, total RNA extracted from cells cultured in 25 mM glucose was analyzed by competitive PCR. Levels of alpha 1 type I collagen mRNA were significantly increased in cells cultured in 25 mM glucose compared with cells cultured in 6 mM glucose (Fig. 5, A and B). Type I collagen accumulation was also significantly increased in cells cultured in 25 mM glucose: 8.8 ± 0.76 vs. 4.9 ± 0.3 ng/104 cells (P < 0.01; Fig. 5C). Interestingly, the addition of troglitazone did not decrease type I collagen expression in the presence of 25 mM glucose (Fig. 5, B and C). To determine whether the lack of suppression of type I collagen expression by troglitazone was due to low levels of PPARgamma expression, cells were transfected with a PPARgamma expression vector. This expression vector induces increased PPARgamma transcriptional activity, especially in the presence of troglitazone. As shown in Fig. 5, B and C, restoration of PPARgamma transcriptional responses in these cells significantly reduced the increased type I collagen expression.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of glucose and PPARgamma expression levels on type I collagen. A: restoration of PPARgamma expression inhibits increased alpha 1 type I collagen mRNA levels in mesangial cells cultured in 25 mM glucose. Representative competitive PCR is shown for alpha 1 type I collagen mRNA levels in cells cultured in 25 mM glucose and cells containing a PPARgamma expression vector and exposed to a PPARgamma agonist (from left to right: mutant levels of 0.5-0.015 amol). B: transfection of mesangial cells with a PPARgamma expression vector partially restores ability of PPARgamma agonist to suppress elevated levels of alpha 1 type I collagen mRNA in mesangial cells cultured in 25 mM glucose. alpha 1 Type I collagen mRNA levels were increased in cells cultured in 25 mM glucose compared with cells cultured in 6 mM glucose. ** P < 0.01; # P < 0.01 vs. cells cultured in 25 mM glucose alone. C: similar to its effect on alpha 1 type I collagen mRNA, transfection of mesangial cells with a PPARgamma expression vector partially restores ability of PPARgamma agonist to suppress elevated levels of type I collagen production in mesangial cells cultured in 25 mM glucose. Type I collagen production was increased in mesangial cells cultured in 25 mM glucose. ** P < 0.01 and # P < 0.05 vs. cells cultured in normal glucose. Transfection with pcDNA3 vector or treatment with troglitazone had no effect on type I collagen production.

Inhibition of TGF-beta 1-mediated type I collagen expression by PPARgamma activation. Because TGF-beta has been shown to stimulate type I collagen expression in mesangial cells, TGF-beta 1 (4 ng/ml) was added to the mesangial cells in the presence or absence of troglitazone. As shown in Fig. 6, A and B, TGF-beta 1 induced a twofold increase in type I collagen expression, while troglitazone abrogated this effect. Type I collagen production was also stimulated by TGF-beta 1, and this was also inhibited by troglitazone (Fig. 6C).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Type I collagen. A: representative competitive PCR showing baseline levels of alpha 1 type I collagen mRNA in control mesangial cells and cells treated with transforming growth factor-beta 1 (TGF-beta 1, 4 ng/ml) and TGF-beta 1 (4 ng/ml) + PPARgamma agonist (8 µM troglitazone). Mesangial cell cDNA was amplified with decreasing amounts of a competing mutant (from left to right: 0.5-0.015 amol). alpha 1 Type I collagen mRNA levels were corrected to GAPDH mRNA levels. B: activation of PPARgamma suppresses TGF-beta 1-mediated type I collagen expression. Troglitazone, a PPARgamma agonist, suppressed the TGF-beta 1-mediated increase in alpha 1 type I collagen mRNA expression. ## P < 0.01 vs. untreated cells; ** P < 0.01 vs. cells treated with TGF-beta 1 alone. C: TGF-beta 1 induced a 2.2-fold increase in type I collagen production. Increased PPARgamma transcriptional activity, induced by the PPARgamma agonist troglitazone, significantly inhibited basal and TGF-beta 1-mediated type I collagen production. # P < 0.05 and ## P < 0.01 vs. untreated cells; ** P < 0.01 vs. cells treated with TGF-beta 1 alone.

Inhibition of a TGF-beta response element by PPARgamma activation. To further examine the effect of PPARgamma activation on TGF-beta 1 responses, mesangial cells were transfected with the TGF-beta reporter construct p3TP-Lux. TGF-beta 1 (4 ng/ml) or vehicle was added to transfected cells in the presence or absence of a PPARgamma agonist. Troglitazone significantly inhibited the basal TGF-beta responses (Fig. 7A). TGF-beta 1 induced a 9.7-fold increase in reporter activity, and troglitazone caused a 50% reduction in this activity. To test whether the effect of troglitazone on the TGF-beta responses was mediated via the PPAR pathway, a dominant-negative PPARgamma expression vector or a pcDNA3 empty vector was cotransfected with the TGF-beta reporter construct, and the responses were examined in the presence or absence of troglitazone. Transfection with a dominant-negative PPARgamma abrogated the ability of troglitazone to suppress TGF-beta responses (Fig. 7B). Furthermore, we found that mesangial cells transfected with a dominant-negative PPARgamma expression vector exhibited significantly increased TGF-beta responses at baseline. Transfecting cells with a pcDNA3 empty vector did not change the TGF-beta responses (8.9-fold increase of luciferase activity in the presence of TGF-beta 1) or the inhibition of TGF-beta responses (47% inhibition) by troglitazone. These data suggest that inhibition of mesangial cell TGF-beta responses by troglitazone was mediated via the PPARgamma transcriptional pathway.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 7.   Activation of PPARgamma inhibits responses to TGF-beta . A: mesangial cells were transfected with 150 ng of a TGF-beta reporter construct, p3TP-Lux, and 150 ng of an RSV beta -galactosidase plasmid and examined in the presence or absence of TGF-beta 1 (4 ng/ml) and/or troglitazone (8 µM). Transcriptional activity in response to added TGF-beta 1 is expressed relative to basal activity. PPARgamma agonist reduced basal transcriptional activity (41%), and TGF-beta 1 stimulated activity (50%). ## P < 0.01 vs. basal activity; ** P < 0.01 vs. cells treated with TGF-beta 1 alone. B: mesangial cells were transfected with 150 ng of p3TP-Lux, 150 ng of RSV beta -galactosidase plasmid, and 200 ng of DNPPARgamma or pcDNA3. Transfected mesangial cells were examined in the presence or absence of TGF-beta 1 (4 ng/ml) and/or troglitazone (8 µM). The suppressive effect of PPARgamma agonist on TGF-beta responses was blocked by DNPPARgamma . Moreover, DNPPARgamma increased the TGF-beta response in mesangial cells. ## P < 0.01 vs. basal activity. * P <0.05 and ** P < 0.01 vs. DNPPARgamma alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We found that mouse glomerular mesangial cells express functionally active PPARgamma . PPARgamma mRNA and protein were expressed by mesangial cells. Treatment with the PPARgamma agonist troglitazone increased the PPAR transcriptional responses. In addition, we found that overexpression of PPARgamma increased PPAR responses in mouse mesangial cells. A dominant-negative PPARgamma expression vector, developed by mutating the highly conserved hydrophobic and charged residues L466/L467 in helix 12 of the ligand-binding domain to alanine (L466A/L467A), significantly reduced basal and ligand-activated PPAR responses. Furthermore, transfection with the dominant-negative PPARgamma expression vector inhibited the increase of PPARgamma transcriptional activity induced by PPARgamma overexpression. Gurnell et al. (10) also found decreased basal and ligand-activated PPARgamma responses in 293 cells transiently transfected with human L468A/E471A dominant-negative PPARgamma . The effects of dominant-negative PPARgamma may be related to its recruitment of corepressors, delay of ligand-dependent corepressor release, or impairment of the recruitment of coactivators (10). Nevertheless, expression of PPARgamma in mesangial cells, together with expression of 9-cis retinoic acid receptor, may play an important role in mesangial cell gene regulation (1, 28). This speculation is supported by the finding by Nicholas et al. (28) that the angiotensin II-mediated increase in plasminogen activator inhibitor-1 (PAI-1) expression in mesangial cells was inhibited by the PPARgamma ligand troglitazone.

Mesangial cells are key players in the accumulation of ECM in diabetic nephropathy (21, 22, 36). An imbalance between matrix synthesis and degradation by mesangial cells has been thought to directly contribute to the pathogenesis of diabetic glomerulosclerosis. Recently, Isshiki et al. (14) found that troglitazone, one of the PPAR ligands, prevented increased glomerular ECM expression in streptozotocin-induced diabetic rats without lowering blood glucose levels. In addition, troglitazone treatment reduced glomerulosclerosis in Zucker fatty rats and 5/6 nephrectomized rats (3, 23, 24). These data suggest that PPARgamma agonists may have direct effects on glomerular extracellular turnover, other than through their insulin-sensitizing and metabolic activities.

We found that activation of PPARgamma by troglitazone decreased type I collagen expression in mouse mesangial cells. Inasmuch as troglitazone has also been shown to have antioxidant properties (12), it was necessary to determine whether this response was directly related to its activity as a PPARgamma ligand. This was assessed by transfecting mesangial cells with a mouse dominant-negative PPARgamma vector. We found that this dominant-negative PPARgamma vector completely abrogated the suppressive effect of troglitazone on mesangial cell type I collagen expression, suggesting that the inhibition was mediated through the PPARgamma pathway. Our observations also showed that the reduction of basal PPAR response levels by the dominant-negative PPARgamma was associated with an increase in type I collagen expression. This indicates that one function of PPARgamma may be to downregulate type I collagen expression in mesangial cells.

The mechanism by which PPARgamma controls type I collagen gene transcription is unknown. Because a typical direct repeat-1 PPAR binding site is not present in type I collagen gene promoter, it is likely that this effect of PPARgamma is mediated indirectly through other pathway(s). It is well established that TGF-beta is one of the key mediators of mesangial cell ECM accumulation (31). Because we found that activation of PPARgamma by troglitazone suppressed TGF-beta 1-mediated type I collagen expression in mesangial cells, we speculated that one of the mechanisms by which PPARgamma might regulate mesangial cell type I collagen gene expression could be via inhibition of TGF-beta 1. The findings that increased PPARgamma activity in mesangial cells inhibited TGF-beta responses, while decreased PPARgamma activity led to increased TGF-beta responses, provided further support for this possibility. Therefore, the expression levels of type I collagen in mesangial cells may be determined, at least in part, by the balance between TGF-beta and PPARgamma levels. Our observation that reducing basal PPARgamma activity by transfecting mesangial cells with a dominant-negative PPARgamma vector was associated with increased TGF-beta responses and type I collagen expression provides further evidence for this balance in determining net cellular responses. The mechanism by which PPARgamma inhibited TGF-beta responses is not clear. Because the TGF-beta reporter construct p3TP-Lux, used in our study, contains activator protein-1 (AP-1) and PAI-1 promoter sites, we speculate that the action of PPARgamma may be via its interaction with AP-1 and other transcription factors. This speculation is supported by the findings that the activation of PPARgamma decreased AP-1 transcriptional activity and attenuated angiotensin II-mediated upregulation of PAI-1 (4, 28).

It has been found that TGF-beta 1 and type I collagen expression is increased in diabetic glomeruli and mesangial cells cultured in 25 mM glucose (11, 15, 34). We found that this increase was associated with decreased PPARgamma expression. These data contradict the previous report that PPARgamma expression was increased in glomeruli of diabetic rats (28). This difference may be caused by the use of different species and different types and degrees of glomerular lesions. Furthermore, our in vitro data on mouse mesangial cells agree with our in vivo data. Namely, mesangial cells exposed to 25 mM glucose had decreased PPARgamma expression and PPAR responses, which were associated with increased type I collagen expression. Similarly, others have found that elevated glucose levels lead to decreased PPARgamma expression in cultured macrophages (33). Interestingly, the macrophages isolated from type II diabetic patients also showed decreased PPARgamma expression (33). When a PPARgamma expression vector was transfected into mesangial cells cultured in 25 mM glucose, PPARgamma activity was restored to levels comparable to those in cells cultured in 6 mM glucose. Furthermore, the addition of troglitazone resulted in significantly reduced levels of type I collagen.

Mesangial cells cultured in 25 mM glucose were previously found to have increased TGF-beta 1 expression and TGF-beta responses (13). These data, together with our findings, support the postulate that PPARgamma and TGF-beta may play an important role in regulation of type I collagen expression. The fact that long-term treatment of diabetic rats and 5/6 nephrectomized rats with troglitazone decreased TGF-beta 1 and ECM gene expression in glomeruli, thereby preventing development and progression of glomerulosclerosis, further supports the importance of counterbalancing interaction(s) between PPARgamma and TGF-beta in vivo (14, 23).

Thus activation of PPARgamma in mesangial cells may be a useful way to inhibit the prosclerotic actions of TGF-beta in these cells. However, because PPARgamma levels were significantly decreased in mouse mesangial cells cultured in high glucose and in diabetic mouse glomeruli with moderate sclerotic lesions, a higher level of TZDs or a stronger PPARgamma ligand may be required to achieve this therapeutic effect.


    FOOTNOTES

Address for reprint requests and other correspondence: F. Zheng, 1600 NW 10th Ave., Rm. 1009B (R-104), Miami, FL 33136 (E-mail: fzheng{at}med.miami.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.

10.1152/ajprenal.00189.2001

Received 22 June 2001; accepted in final form 28 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Asano, T, Wakisaka M, Yoshinari M, Iino K, Sonoki K, Iwase M, and Fujishima M. Peroxisome proliferator-activated receptor gamma 1 (PPARgamma 1) expression in rat mesangial cells and PPARgamma agonists modulate its differentiation. Biochim Biophys Acta 1497: 148-154, 2000[ISI][Medline].

2.   Barroso, I, Gurnell M, Crowley VE, Agostini M, Schwabe JW, Soos MA, Maslen GL, Williams TD, Lewis H, Schafer AJ, Chatterjee VK, and O'Rahilly S. Dominant-negative mutations in human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 402: 880-883, 1999[ISI][Medline].

3.   Buckingham, RE, Al-Barazanji KA, Toseland CD, 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].

4.   Delerive, P, Martin-Nizard F, Chinetti G, Trottein F, Fruchart JC, Najib J, Duriez P, and Staels B. Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway. Circ Res 85: 394-402, 1999[Abstract/Free Full Text].

5.   Escher, P, and Wahli W. Peroxisome proliferator-activated receptors: insight into multiple cellular functions. Mutat Res 448: 121-138, 2000[ISI][Medline].

6.   Fornoni, A, Lenz O, Tack I, Potier M, Elliot SJ, Striker LJ, and Striker GE. Matrix accumulation in mesangial cells exposed to cyclosporine A requires a permissive genetic background. Transplantation 70: 587-593, 2000[ISI][Medline].

7.   Glick, AD, Jacobson HR, and Haralson MA. Mesangial deposition of type I collagen in human glomerulosclerosis. Hum Pathol 23: 1373-1379, 1992[ISI][Medline].

8.   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 Expr 4: 281-299, 1995[Medline].

9.   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].

10.   Gurnell, M, Wentworth JM, Agostini M, Adams M, Collingwood TN, Provenzano C, Browne PO, Rajanayagam O, Burris TP, Schwabe JW, Lazar MA, and Chatterjee VK. A dominant-negative peroxisome proliferator-activated receptor-gamma (PPARgamma ) mutant is a constitutive repressor and inhibits PPARgamma -mediated adipogenesis. J Biol Chem 275: 5754-5759, 2000[Abstract/Free Full Text].

11.   Hoffman, BB, Sharma K, Zhu Y, and Ziyadeh FN. Transcriptional activation of transforming growth factor-beta 1 in mesangial cell culture by high glucose concentration. Kidney Int 54: 1107-1116, 1998[ISI][Medline].

12.   Inoue, I, Katayama S, Takahashi K, Negishi K, Miyazaki T, Sonoda M, and Komoda T. Troglitazone has a scavenging effect on reactive oxygen species. Biochem Biophys Res Commun 235: 113-116, 1997[ISI][Medline].

13.   Isono, M, Cruz MC, Chen S, Hong SW, and Ziyadeh FN. Extracellular signal-regulated kinase mediates stimulation of TGF-beta 1 and matrix by high glucose in mesangial cells. J Am Soc Nephrol 11: 2222-2230, 2000[Abstract/Free Full Text].

14.   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].

15.   Iwano, M, Kubo A, Nishino T, Sato H, Nishioka H, Akai Y, Kurioka H, Fujii Y, Kanauchi M, Shiiki H, and Dohi K. Quantification of glomerular TGF-beta 1 mRNA in patients with diabetes mellitus. Kidney Int 49: 1120-1126, 1996[ISI][Medline].

16.   Jacot, TA, Striker GE, Stetler-Stevenson WG, and Striker LJ. Mesangial cells from transgenic mice with progressive glomerulosclerosis exhibit stable, phenotypic changes including undetectable MMP-9 and increased type IV collagen. Lab Invest 75: 791-799, 1996[ISI][Medline].

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

18.   Johnson, RJ, Floege J, Yoshimura A, Iida H, Couser WG, and Alpers CE. The activated mesangial cell: a glomerular "myofibroblast"? J Am Soc Nephrol 2: S190-S197, 1992[Abstract].

19.   Kersten, S, Desvergne B, and Wahli W. Roles of PPARs in health and disease. Nature 405: 421-424, 2000[ISI][Medline].

20.   Lehmann, JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, and Kliewer SA. An antidiabetic thiazolidinedione is a high-affinity ligand for peroxisome proliferator-activated receptor-gamma (PPARgamma ). J Biol Chem 270: 12953-12956, 1995[Abstract/Free Full Text].

21.   Lenz, O, Striker LJ, Jacot TA, Elliot SJ, Killen PD, and Striker GE. Glomerular endothelial cells synthesize collagens but little gelatinase A and B. J Am Soc Nephrol 9: 2040-2047, 1998[Abstract].

22.   Lupia, E, Elliot SJ, Lenz O, Zheng F, Hattori M, Striker GE, and Striker LJ. IGF-1 decreases collagen degradation in diabetic NOD mesangial cells: implications for diabetic nephropathy. Diabetes 48: 1638-1644, 1999[Abstract].

23.   Ma, LJ, Marcantoni C, Linton MF, Fazio S, and Fogo AB. Peroxisome proliferator-activated receptor-gamma agonist troglitazone protects against nondiabetic glomerulosclerosis in rats. Kidney Int 59: 1899-1910, 2001[ISI][Medline].

24.   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].

25.   McGowan, SE, Jackson SK, Doro MM, and Olson PJ. Peroxisome proliferators alter lipid acquisition and elastin gene expression in neonatal rat lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 273: L1249-L1257, 1997[Abstract/Free Full Text].

26.   Miyahara, T, Schrum L, Rippe R, Xiong S, Yee HFJ, Motomura K, Anania FA, Willson TM, and Tsukamoto H. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem 275: 35715-35722, 2000[Abstract/Free Full Text].

27.   Neugarten, J, Acharya A, Lei J, and Silbiger S. Selective estrogen receptor modulators suppress mesangial cell collagen synthesis. Am J Physiol Renal Physiol 279: F309-F318, 2000[Abstract/Free Full Text].

28.   Nicholas, SB, Kawano Y, Wakino S, Collins AR, and Hsueh WA. Expression and function of peroxisome proliferator-activated receptor-gamma in mesangial cells. Hypertension 2 37: 722-727, 2001[Abstract/Free Full Text].

29.   Pan, L, Eckhoff C, and Brinckerhoff CE. Suppression of collagenase gene expression by all-trans and 9-cis retinoic acid is ligand dependent and requires both RARs and RXRs. J Cell Biochem 57: 575-589, 1995[ISI][Medline].

30.   Peten, EP, Garcia-Perez A, Terada Y, Woodrow D, Martin BM, Striker GE, and Striker LJ. Age-related changes in alpha 1- and alpha 2-chain type IV collagen mRNAs in adult mouse glomeruli: competitive PCR. Am J Physiol Renal Fluid Electrolyte Physiol 263: F951-F957, 1992[Abstract/Free Full Text].

31.   Poncelet, AC, and Schnaper HW. Regulation of human mesangial cell collagen expression by transforming growth factor-beta 1. Am J Physiol Renal Physiol 275: F458-F466, 1998[Abstract/Free Full Text].

32.   Potier, M, Elliot SJ, Tack I, Lenz O, Striker GE, Striker LJ, and Karl M. Expression and regulation of estrogen receptors in mesangial cells: influence on matrix metalloproteinase-9. J Am Soc Nephrol 12: 241-251, 2001[Abstract/Free Full Text].

33.   Sartippour, MR, and Renier G. Differential regulation of macrophage peroxisome proliferator-activated receptor expression by glucose: role of peroxisome proliferator-activated receptors in lipoprotein lipase gene expression. Arterioscler Thromb Vasc Biol 20: 104-110, 2000[Abstract/Free Full Text].

34.   Sharma, K, Ziyadeh FN, Alzahabi B, McGowan TA, Kapoor S, Kurnik BR, Kurnik PB, and Weisberg LS. Increased renal production of transforming growth factor-beta 1 in patients with type II diabetes. Diabetes 46: 854-859, 1997[Abstract].

35.   Striker, GE, Peten EP, Carome MA, Pesce CM, Schmidt K, Yang CW, Elliot SJ, and Striker LJ. The kidney disease of diabetes mellitus (KDDM): a cell and molecular biology approach. Diabetes Metab Rev 9: 37-56, 1993[ISI][Medline].

36.   Striker, LJ, Doi T, Conti F, and Striker GE. Role of mesangial cells in glomerulosclerosis. J Diabetes Complications 5: 60-61, 1991.

37.   Vidal-Puig, AJ, Considine RV, Jimenez-Linan M, Werman A, Pories WJ, Caro JF, and Flier JS. 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, 1997[Abstract/Free Full Text].

38.   Wrana, JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang XF, and Massague J. TGF-beta signals through a heteromeric protein kinase receptor complex. Cell 71: 1003-1014, 1992[ISI][Medline].

39.   Yang, XY, Wang LH, Chen T, Hodge DR, Resau JH, DaSilva L, and Farrar WL. Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor-gamma (PPARgamma ) agonists. PPARgamma co-association with transcription factor NFAT. J Biol Chem 275: 4541-4544, 2000[Abstract/Free Full Text].

40.   Zheng, F, Striker GE, Esposito C, Lupia E, and Striker LJ. Strain differences, rather than hyperglycemia, determine the severity of glomerulosclerosis in mice. Kidney Int 54: 1999-2007, 1998[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 282(4):F639-F648
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society