 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cell Biology |
* Department of Medicine, University of Sydney, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales, Australia; and School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland, Australia
Address correspondence to: Prof. Carol A. Pollock, Kolling Institute, Department of Medicine, Level 3, Wallace Freeborn Professorial Block, Royal North Shore Hospital, St. Leonards, Sydney, New South Wales 2065, Australia. Phone: 61-2-9926-7126; Fax: 61-2-9436-3719; E-mail: carpol{at}med.usyd.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(PPAR-) agonists are increasingly used in patients with diabetes, and small studies have suggested a beneficial effect on renal function, but their effects on extracellular matrix (ECM) turnover are unknown. The aims of this study were to investigate the effects of the PPAR- agonist pioglitazone on growth and matrix production in human cortical fibroblasts (CF). Cell growth and ECM production and turnover were measured in human CF in the presence and absence of 1 and 3 µM pioglitazone. Exposure of CF to pioglitazone caused an antiproliferative (P < 0.0001) and hypertrophic (P < 0.0001) effect; reduced type IV collagen secretion (P < 0.01), fibronectin secretion (P < 0.0001), and proline incorporation (P < 0.0001); decreased MMP-9 activity (P < 0.05); and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 secretion (P < 0.001 and P < 0.0001, respectively). These effects were independent of TGF-
1. A reduction in ECM production was similarly observed when CF were exposed to a selective PPAR- agonist (L-805645) in concentrations that caused no toxicity, confirming the antifibrotic effects of pioglitazone were mediated through a PPAR-–dependent mechanism. Exposure of CF to high glucose conditions induced an increase in the expression of collagen IV (P < 0.05), which was reversed both in the presence of pioglitazone (1 and 3 µM) and by L-805645. In summary, exposure of human CF to pioglitazone causes an antiproliferative effect and reduces ECM production through mechanisms that include reducing TIMP activity, independent of TGF-1. These studies suggest that the PPAR- agonists may have a specific role in ameliorating the course of progressive tubulointerstitial fibrosis under both normoglycemic and hyperglycemic states. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A new class of antidiabetic drugs, the thiazolidinediones (TZD), targeted to improve insulin sensitivity in patients with diabetes, has been used clinically relatively recently for the treatment of type 2 diabetes. These agents have been shown to act primarily by stimulating a member of the nuclear receptor hormone family, peroxisome proliferator–activated receptor- (PPAR-). Hence, TZD are agonists for PPAR- and when bound to PPAR- lead to modulation of expression of specific genes. Accumulating evidence suggests that these drugs not only significantly improve insulin sensitivity but also may reduce microalbuminuria and progression to diabetic nephropathy in genetically obese rodents and also in humans with type 2 diabetes (2–6). However, the mechanism for a potential renoprotective effect is lacking
We recently demonstrated that in the opossum kidney model of proximal tubular cells, pioglitazone reduces proteinuria by increasing tubular uptake of albumin but concomitantly reduces tubular cell production of the inflammatory and profibrotic cytokines monocyte chemoattractant protein-1 and TGF-1 (7). Troglitazone has been demonstrated to reduce the expression of ECM proteins (fibronectin and type IV collagen) and TGF-1 in glomeruli from streptozotocin-induced diabetic rats (8). In addition, the renal microvasculature in rabbit models of diabetes is now recognized as being associated with PPAR- activity (9). However, it is clearly recognized that it is tubulointerstitial disease, not glomerular pathology, that is predictive of long-term renal outcome, and the cellular entity most linked to fibrosis in the human kidney is the cortical fibroblast (CF). The presence of PPAR- receptors in the human renal CF has not been established. These studies were aimed at determining whether the human CF is an additional cellular target in the kidney that acts as a ligand for a synthetic PPAR- agonist. Studies were performed using a commercially available PPAR- agonist but with PPAR-
effects observed in pharmacologic concentrations. The specificity of observed effects for PPAR- activation were confirmed by additional experiments in the presence of the selective PPAR- agonist L-805645 (Merck Laboratories, Rahway, NJ) (10). Subsequent experiments then were performed to determine the direct effects of PPAR- activation on key ECM proteins in human CF under normal and high glucose conditions
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental Protocol
The methods for primary culture of CF from human renal cortex are described in detail elsewhere (11). All experiments were carried out on quiescent, confluent CF, at passage 2. The cells were exposed to media that contained 5 mM d-glucose alone (control) or 5 mM d-glucose with pioglitazone as detailed below for 72 h before study. A dose-response curve was initially undertaken to determine the effects of PPAR- activation on CF growth and matrix parameters; cells were exposed to pioglitazone (0.01, 0.1, 1, 3, and 10 µM) for 72 h. In all cases, exposure to 0.01 or 0.1 µM pioglitazone had no effect on cell growth or matrix production or turnover. Exposure to both 1 and 3 µM had a dose-related effect on these parameters, but concentrations beyond 3 µM pioglitazone increasingly induced cell death. Hence, exposure to 1 to 3 µM was chosen for future experiments as this range approximates serum concentrations in patients in clinical treatment programs (12). Media were changed every 48 h
Reverse Transcription–PCR for PPAR-
PPAR- expression in CF was confirmed by reverse transcription–PCR (RT-PCR). Total RNA was isolated from human CF that were grown in 35-mm Petri dishes using TRIZOL (Invitrogen, Life Technologies, Carlsbad, CA) as per the manufacturer’s instructions. Briefly, 1 µg of RNA was reverse transcribed to cDNA using SuperScript II RNase H Reverse Transcriptase (Life Technologies). Amplification of the resulting cDNA was performed using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany) in a 25-µl reaction volume with 2.5 mM MgCl, 200 µM dNTP, and 50 nM forward and reverse primers. The thermal profile consisted of denaturation at 94°C for 40 s, annealing at 60°C for 50 s, and extension at 72°C for 50 s, for 40 cycles. The primers used were as follows: PPAR- sense primer 5`-TCTCTCCGTAATGGAAGACC-3` and antisense primer 5`-GCATTATGAGACATCCCCAC-3` (13); and for -actin, sense primer 5`-CATGTACGTTGCTATCCAG-3` and antisense primer 5`-CGCAACTAAGTCATAGTCC-3`. The primer sets yielded 474- and 966-bp products, respectively. PCR products were run on a 2% agarose gel in TAE buffer (40 mM Tris acetate and 1 mM EDTA) and visualized by ethidium bromide staining
Western Blot Analysis for PPAR-, TIMP, Collagen IV, and Fibronectin
For PPAR- determination, CF were exposed for 72 h to 5 mM d-glucose. For extracting protein from cell lysates, cells first were washed twice with PBS, then lysed in lysis buffer that contained 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 20 mM sodium orthovanadate, and 0.5% Triton-X 100 with proteinase inhibitor cocktail "Complete" (Roche Applied Sciences, Indianapolis, IN) for 20 min, followed by centrifugation at 10,000 x g for 10 min at 4°C. Fifty micrograms of protein from cell lysates was separated under reduced conditions by 15% SDS-PAGE, transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences UK, Little Chalfont, Buckinghamshire, England), and then blocked overnight at 4°C in 5% skim milk in Tris-buffered saline (TBS). The membranes were incubated with 200 ng/ml mouse anti–PPAR- antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature, followed by incubation with an anti-mouse IgG horseradish peroxidase–conjugated antibody (1:1600; Amersham Biosciences) for 1 h at room temperature. Protein was detected using ECL Western blotting analysis system (Amersham Biosciences)
For TIMP, collagen IV, and fibronectin determination, confluent cells that were grown in 96-well plates were exposed to either no pioglitazone (control) or pioglitazone 1 and 3 µM for 72 h. The culture supernatants then were harvested and mixed with Laemmli buffer, and equal volumes then were separated under reducing conditions on SDS-polyacrylamide gels (15% for TIMP, 7.5% for collagen and fibronectin). Western blotting was performed as described above. The membranes were probed with either mouse anti–TIMP-1 (1:300) and anti–TIMP-2 (1:200) primary antibodies (Oncogene Research Products, Cambridge, MA) and anti-mouse peroxidase-linked secondary antibodies (1:1600) or goat anti–type IV collagen (1:200; Chemicon International, Temecula, CA) and mouse antifibronectin (1:100; Neomarkers, Fremont, CA) primary antibodies and anti-goat (1:10,000) and anti-mouse (1:1600) peroxidase-linked secondary antibodies, respectively. Blots were developed using ECL and quantified using densitometry. Densitometric results were corrected for the protein loading
Assessment of Cell Growth and Apoptosis
For determining the effects of PPAR- activation on CF growth parameters, cells were exposed to pioglitazone at concentrations of 0.01 to 10 µM for 72 h. The total protein content of cells was determined as a marker of cellular hypertrophy, as described previously (14). Manual cell counts were performed on trypsin-dispersed cells using a hemocytometer
Cell death was assessed by measuring lactate dehydrogenase (LDH) release in the supernatants of CF that were exposed to various concentrations of pioglitazone for 72 h using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). For determining the extent to which apoptosis contributed to a reduction in cell number, the pre-G1 peak was determined by flow cytometric analysis, as described previously (15)
Gelatin Zymography
Confluent cells that were grown in 96-well plates (Nunclon, Roskide, Denmark) were exposed to 1 and 3 µM pioglitazone for 72 h. The culture supernatants were harvested at 72 h and mixed with gel sample buffer, and equal volumes were loaded onto a 10% SDS polyacrylamide gel that contained 1 mg/ml gelatin and subjected to electrophoresis. After electrophoresis, the gels were washed in 50 mM Tris buffer that contained 2.5% Triton X-100. The gels were incubated for an additional 24 h in 50 mM Tris buffer (pH 7.6) that contained 5 mM CaCl2, after which the gels were stained with Coomassie Blue and subsequently destained in methanol/acetic acid/H2O. Gels were scanned, and density analysis of the bands was performed using NIH Image. Densitometric results were corrected for protein loading
Active TGF-1 Production
Supernatants were harvested from cells that were grown on 96-well plates and exposed to pioglitazone 1 and 3 µM for 72 h. For activating latent TGF-1, the samples were acid-treated with 1 N HCl followed by neutralization with 1.2 N NaOH/0.5 mM HEPES. Active TGF-1 levels were assayed using a colorimetric ELISA according to the manufacturer’s instructions (Promega). Results were corrected for cell protein
[3H]Proline Incorporation
Because collagen is proline rich, incorporation of proline was used as an additional relative estimate of collagen production (16). Confluent CF in 96-well plates were exposed to 1 and 3 µM pioglitazone for 72 h. A total of 1 µCi of l[2,3,4,5-3H]proline (Amersham Pharmacia, Piscataway, NJ) was added to the cells in the final 24 h of treatment. The cells then were washed four times with PBS and solubilized in 0.2 M NaOH, and the samples counted in a liquid scintillation counter (LKB-Wallac, Turku, Finland). Results were corrected for cell protein
Antifibrotic Effects of Pioglitazone Are PPAR- Dependent
For determining whether the antifibrotic effects were specific to upregulation of PPAR-, selected experiments were undertaken using the PPAR-–specific agonist L-805645. Cells were exposed to L-805645 for 72 h in concentrations of 0.1, 1, 3, and 10 µM, and measures of LDH release as a marker of cell toxicity, cell count, cell protein content, and selective markers of ECM accumulation (collagen IV and proline incorporation) were undertaken as detailed above
Antifibrotic Effects of PPAR- Agonists under High Glucose Conditions
As PPAR- agonists are largely used in the treatment of patients with diabetes, additional experiments assessing the expression of collagen IV were undertaken in cells that were exposed to normal (5 mM) or high (25 mM) glucose in the presence or absence of pioglitazone or L-805645 at the concentrations described above
Statistical Analyses
Unless otherwise stated, all experiments were replicated three to eight times. All results are expressed as a percentage of the control value (100%). Results are expressed as mean ± SEM. Statistical comparisons between groups were made by the unpaired t test. Analyses were performed using the software package StatView, version 5.01 (Abacus Concepts, Berkeley, CA). P < 0.05 was considered significant
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in CF. There was a strong band at 474 bp using PPAR-–specific primers, indicating that primary cultures of human CF do express the mRNA for PPAR- (Figure 1A). To determine whether PPAR- is expressed at a protein level, we performed Western blot on Triton X-100 soluble fractions of the cells. The PPAR- antibody (E-8) used in this study recognizes PPAR- isoforms 1 and 2. PPAR-1 has a molecular weight of 50 kD. PPAR-2 is a splice variant and has an additional 30 amino acids at the N-terminus (17). In CF, a doublet was demonstrated, indicating that PPAR-1 and PPAR-2 both are expressed (Figure 1B), and this was confirmed using a PPAR-2–specific antibody on Western blot (Affinity BioReagents, Golden, CO)
|
|
|
|
|
|
|
|
1 Production1 levels compared with control. This suggested that the observed reduction in matrix protein was not due to alteration in autocrine TGF-1 production (Figure 9).
|
DependentCells that were exposed to 3 and 10 µM L-805645 demonstrated a significant reduction in collagen IV and proline incorporation. Collagen IV expression was reduced to 56 ± 16% (P = 0.05) and to 20 ± 12% (P < 0.01) when CF were exposed to L-805645 at 3 and 10 µM, respectively. Proline incorporation was reduced to 91 ± 3% (P < 0.05) and 73 ± 5% (P < 0.0001) when CF were exposed to L-805645 at 3 and 10 µM, respectively
Antifibrotic Effects of PPAR- Agonists under High Glucose Conditions
Exposure to 25 mM glucose resulted in an increase in collagen IV expression to 117 ± 6% (P < 0.05). The presence of pioglitazone decreased the expression of collagen IV to 68 ± 2% (P < 0.001) and to 53 ± 10% (P < 0.01) of control values at 1 and 3 µM, respectively (Figure 10). Similarly, L-805645 also decreased the glucose-induced increase in collagen IV to 65 ± 6% (P < 0.001) and to 92 ± 5% (P < 0.05) of control values at 3 and 10 µM, respectively
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
mRNA in human renal CF. This study importantly demonstrates that PPAR- agonists have a direct effect on CF, resulting in a reduced accumulation of both collagen- and non–collagen-containing matrix protein. Our results suggest that this is associated with a reduction in the proteins that normally inhibit matrix degradation, both TIMP-1 and -2. Although TGF-1 is recognized as a key profibrotic cytokine in the renal tubulointerstitium, no alteration in the autocrine production of TGF-1 was observed. These studies collectively demonstrate that PPAR- agonists are likely to protect against the progression of chronic kidney disease in both normal and high glucose conditions by mitigating against tubulointerstitial disease
Our results contrast to initial studies in which expression of PPAR- gene and protein in the human kidney was not detected (18,19). However, subsequently, PPAR- has been confirmed in human kidney (20,21), and several groups have since identified the presence of PPAR- message and protein in major components of the glomerulus, in mesangial cells, and in endothelial cells in animal models (9,22,23). Studies in our laboratory have also confirmed that in several in vitro proximal tubular cell models, these cells also express PPAR- (7,15). This study additionally confirms that PPAR- is functionally expressed in the human CF
Previous studies from our laboratory have also suggested a renoprotective effect of pioglitazone on proximal tubular cells, as pioglitazone increased tubular albumin absorption without a concomitant increase in inflammatory or profibrotic markers. It further attenuated the increase in monocyte chemoattractant protein-1 and TGF-1 in response to exposure of the tubules to LDL (7). The results of this study clearly demonstrate that the reduction in matrix accumulation by the CF was not mediated by reduced autocrine production of TGF-1. It is widely known that PPAR- agonists may alter transcription of various nuclear factors other than those that regulate TGF-1, for example, NF-
B, which transduces fibrotic responses through initial upregulation of inflammatory cytokines. However, it is also possible that in our previous studies in tubular cells, TGF-1 was upregulated by exposure to LDL, whereas in the current study, the cells at baseline were in an unstimulated state. Hence, downregulation of TGF-1 was not possible. In the in vivo state, it is possible that downregulation of TGF-1 by proximal tubular cells in response to PPAR- agonists would further mitigate against a profibrotic response
No previous study has specifically addressed the role of PPAR- agonists in limiting renal tubulointerstitial matrix deposition; in particular, there are no available data on PPAR- stimulation on the key modulators of matrix degradation, namely the MMP and TIMP system. Galli et al. (24) previously studied collagen deposition in a rat model of liver fibrosis using two thiazolidinedione PPAR- agonists, pioglitazone and rosiglitazone. PPAR-–specific DNA binding was significantly impaired in nuclear extracts of hepatic stellate cells that were isolated from rats with liver fibrosis compared with those that were isolated from control rats. Administration of either thiazolidinedione restored PPAR- DNA binding in hepatic stellate cell nuclei. Subsequent in vitro studies using these cells demonstrated that thiazolidinedione-induced PPAR- activation inhibited collagen and fibronectin synthesis induced by TGF-1. These results are consistent with the antifibrotic effects of pioglitazone observed in our study. However, in contrast to our findings, thiazolidinediones reduced the TGF-1–induced activity (24). Our results identify MMP-9 as being downregulated but both TIMP-1 and TIMP-2 being further downregulated by PPAR- agonist activity. Clearly, these modulators of matrix degradation play key roles in determining net matrix accumulation, and the observed modification of function by PPAR- agonists in this study is likely to be reflected in reduced matrix accumulation
Our studies also demonstrated an antiproliferative effect of pioglitazone in the CF, which clearly would additionally reduce the fibrotic response in the tubulointerstitium. Although some of this effect could be attributed to cell toxicity, higher concentrations of the selective PPAR- agonist L-805645 similarly induced an antiproliferative effect in the absence of observed toxicity. Parameswaran et al. (25) additionally demonstrated the apoptotic and negative growth effects of ciglitazone on a rat kidney interstitial fibroblast cell line. Clearly, these agents are used clinically in circumstances in which hyperglycemic conditions prevail. This study confirms that commercially available PPAR- agonists are likely to reduce matrix accumulation under these conditions
In conclusion, the results of this study establish the presence of PPAR- in the human CF and highlight the potential ability of PPAR- agonist drugs to modify matrix parameters in the human tubulointerstitium, potentially in conditions associated with both normal and high glucose conditions. On the basis of these findings, we speculate that the PPAR- agonists have the potential to prevent or retard the progressive tubulointerstitial changes that characterize chronic kidney disease and in doing so delay the progression to end-stage kidney failure
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.
Kidney Int 62
: 901
–913, 2002[CrossRef][Medline]
gene.
J Biol Chem 272
: 18779
–18789, 19972 versus PPAR1 and activation with retinoid X receptor agonists and antagonists.
J Biol Chem 272
: 8071
–8076, 19971 (PPAR1) expresses in rat mesangial cells and PPAR agonists modulate its differentiation.
Biochim Biophys Acta 1497
: 148
–154, 2000[Medline]
This article has been cited by other articles:
 |
|
 |
|
  S. Saad, D. J. Agapiou, X.-M. Chen, V. Stevens, and C. A. Pollock The role of Sgk-1 in the upregulation of transport proteins by PPAR-{gamma} agonists in human proximal tubule cells Nephrol. Dial. Transplant., November 8, 2008; (2008) gfn614v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Ruan, F. Zheng, and Y. Guan PPARs and the kidney in metabolic syndrome Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1032 - F1047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Boor, K. Sebekova, T. Ostendorf, and J. Floege Treatment targets in renal fibrosis Nephrol. Dial. Transplant., December 1, 2007; 22(12): 3391 - 3407. [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xu, S. R. Farmer, and B. D. Smith Peroxisome Proliferator-activated Receptor {gamma} Interacts with CIITA{middle dot}RFX5 Complex to Repress Type I Collagen Gene Expression J. Biol. Chem., September 7, 2007; 282(36): 26046 - 26056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Li, H. Kimura, K. Hirota, H. Sugimoto, N. Kimura, N. Takahashi, H. Fujii, and H. Yoshida Hypoxia reduces the expression and anti-inflammatory effects of peroxisome proliferator-activated receptor-{gamma} in human proximal renal tubular cells Nephrol. Dial. Transplant., April 1, 2007; 22(4): 1041 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Crossno Jr., C. V. Garat, J. E. B. Reusch, K. G. Morris, E. C. Dempsey, I. F. McMurtry, K. R. Stenmark, and D. J. Klemm Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L885 - L897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Turturro, R. Oliver III, E. Friday, I. Nissim, and T. Welbourne Troglitazone and pioglitazone interactions via PPAR-{gamma}-independent and -dependent pathways in regulating physiological responses in renal tubule-derived cell lines Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1137 - C1146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Calkin, S. Giunti, K. A. Jandeleit-Dahm, T. J. Allen, M. E. Cooper, and M. C. Thomas PPAR-{alpha} and -{gamma} agonists attenuate diabetic kidney disease in the apolipoprotein E knockout mouse Nephrol. Dial. Transplant., September 1, 2006; 21(9): 2399 - 2405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, X. Wen, B. C. Spataro, K. Hu, C. Dai, and Y. Liu Hepatocyte Growth Factor Is a Downstream Effector that Mediates the Antifibrotic Action of Peroxisome Proliferator-Activated Receptor-{gamma} Agonists J. Am. Soc. Nephrol., January 1, 2006; 17(1): 54 - 65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Panchapakesan, S. Sumual, C. A. Pollock, and X. Chen PPAR{gamma} agonists exert antifibrotic effects in renal tubular cells exposed to high glucose Am J Physiol Renal Physiol, November 1, 2005; 289(5): F1153 - F1158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Han, J. D. Ritzenthaler, H. N. Rivera, and J. Roman Peroxisome proliferator-activated receptor-{gamma} ligands suppress fibronectin gene expression in human lung carcinoma cells: involvement of both CRE and Sp1 Am J Physiol Lung Cell Mol Physiol, September 1, 2005; 289(3): L419 - L428. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
